Title of Invention

A PHARMACEUTICAL FORMULATION OF A HUMAN GROWTH HORMONE (hGH) VARIANT

Abstract Formulations of modified human growth hormone polypeptides are provided.
Full Text

Formulations of Human Growth Hormone Comprising a Non-NaturallyμlEncoded Amino Acid
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims priority to U.S. provisional patent application Serial No. 60/638,616 filedμlDecember 22, 2004, U.S. provisional patent application Serial No. 60/680,617, filed May 13, 2005,μland U.S. provisional patent application entitled 60/728,035, filed October 17, 2005, theμlspecifications of which are incorporated herein in their entirety.
FIELD OF THE INVENTION
This invention relates to stabilized human growth hormone (hGH) formulations of hGH polypeptideμlcomprising a non-natural amino acid covalently linked to poly(ethylene glycol) (PEG).
BACKGROUND OF THE INVENTION
[01] Human growth hormone participates in much of the regulation of normal human
growth and development This naturally-occurring single-chain pituitary hormone consists of 191μlamino acid residues and has a molecular weight of approximately 22 kDa. hGH exhibits aμlmultitude of biological effects, including linear growth (somatogenesis), lactation, activation ofμlmacrophages, and insulin-like and diabetogenic effects, among others (Chawla, R., et al, Ann. Rev.μlMed. 34:519-547 (1983); Isaksson, O., et al, Ann. Rev. Physiol, 47:483-499 (1985); Hughes, J. andμlFriesen, H., Ann. Rev. Physiol, 47:469-482 (1985)).
[02] The structure of hGH is well known (Goeddel, D., et al, Nature 281:544-548
(1979)), and the three-dimensional structure of hGH has been solved by X-ray crystallography (deμlVos, A., et al, Science 255:306-312 (1992)). The protein has a compact globular structure,μlcomprising four amphipathic alpha helical bundles, termed A-D beginning from the N-terminus,μlwhich are joined by loops. Further discussion of hGH including its receptor and variants, and otherμlGH superfamily members is provided in U.S. Patent Application No. 11/046,432 entitled "ModifedμlHuman Growth Hormone Polypeptides and Their Uses" and PCT International Patent Application

No. PCTYUS05/03537 entitled "Modified Human Four Helical Bundle Polypeptides and TheirμlUses," which are incorporated by reference in their entirety.
[03] Recombinant hGH is used as a therapeutic and has been approved for the treatment of
a number of indications. hGH deficiency leads to dwarfism, for example, which has beenμlsuccessfully treated for more than a decade by exogenous administration of the hormone. Inμladdition to hGH deficiency, hGH has also been approved for the treatment of renal failure (inμlchildren), Turner's Syndrome, and cachexia in AIDS patients. Recently, the Food and DrugμlAdministration (FDA) has approved hGH for the treatment of non-GH-dependent short stature.μlhGH is also currently under investigation for the treatment of aging, frailty in the elderly, shortμlbowel syndrome, and congestive heart failure. Target populations for hGH treatment includeμlchildren with idiopathic short stature (ISS) and adults with GHD-like symptoms.μl[04] Recombinant hGH is currently sold as a daily injectable product, with five major
products currently on the market: Humatrope™ (Eli Lilly & Co.), Nutropin™ (Genentech),μlNorditropin™ (Novo-Nordisk), Genotropin™ (Pfizer) and Saizen/Serostim™ (Serono). Aμlsignificant challenge to using growth hormone as a therapeutic, however, is that the protein has a .μlshort in vivo half-life and, therefore, it must be administered by daily subcutaneous injection forμlmaximum effectiveness (MacGillivray, et al9 J. Clin. Endocrinol Metab. 81: 1806-1809 (1996)).μlConsiderable effort is focused on means to improve the administration of hGH agonists and .μlantagonists, by lowering the cost of production, making administration easier for the patient, -μlimproving efficacy and safety profile, and creating other properties that would provide competitiveμladvantages. For example, Genentech and Alkermes formerly marketed Nutropin Depot™, a depotμlformulation of hGH, for pediatric growth hormone deficiency. While the depot permits lessμlfrequent administration (once every 2-3 weeks rather than once daily), it is also associated withμlundesirable side effects, such as decreased bioavailability and pain at the injection site and wasμlwithdrawn from the market in 2004. Another product, Pegvisomant™ (Pfizer), has also recentlyμlbeen approved by the FDA. Pegvisomant™ is a genetically-engineered analogue of hGH thatμlfunctions as a highly selective growth hormone receptor antagonist indicated for the treatment ofμlacromegaly (van der Lely, et al9 The Lancet 358: 1754-1759 (2001). Although several of theμlamino acid side chain residues in Pegvisomant*™ are derivatized with polyethylene glycol (PEG)μlpolymers, the product is still administered once-daily, indicating that the pharmaceutical propertiesμlare not optimal. In addition to PEGylation and depot formulations, other administration routes,

including inhaled and oral dosage forms of hGH, are under early-stage pre-clinical and clinicalμldevelopment and none have yet received approval from the FDA. Accordingly, there is a need for aμlpolypeptide that exhibits growth hormone activity but that also provides a longer serum half-lifeμland, therefore, more optimal therapeutic levels of hGH and an increased therapeutic half-life.μl[05] Covalent attachment of the hydrophilic polymer poly(ethylene glycol), abbreviated
PEG, is a method of increasing water solubility, bioavailability, increasing serum half-life,μlincreasing therapeutic half-life, modulating immunogenicity, modulating biological activity, orμlextending the circulation time of many biologically active molecules, including proteins, peptides,μland particularly hydrophobic molecules. PEG has been used extensively in pharmaceuticals, onμlartificial implants, and in other applications where biocompatibility, lack of toxicity, and lack ofμlimmunogenicity are of importance. In order to maximize the desired properties of PEG, the totalμlmolecular weight and hydration state of the PEG polymer or polymers attached to the biologicallyμlactive molecule must be sufficiently high to impart the advantageous characteristics typicallyμlassociated with PEG polymer attachment, such as increased water solubility and circulating half '.μllife, while not adversely impacting the bioactivity of the parent molecule.
[06] Recently, an entirely new technology in the protein sciences has been reported,
which promises to overcome many of the limitations associated with site-specific modifications ofμlproteins. Specifically, new components have been added to the protein biosynthetic machinery ofμlthe prokaryote Escherichia coli (E. colt) (e.g., L. Wang, et al., (2001), Science 292:498-500) and ;μlthe eukaryote Sacchromyces cerevisiae (S. cerevisiae) (e.g., J. Chin et al., Science 301:964-7μl(2003)), which has enabled the incorporation of non-genetically encoded amino acids to proteins inμlvivo. A number of new amino acids with novel chemical, physical or biological properties,μlincluding photoaffinity labels and photoisomerizable amino acids, photocrosslinking amino acidsμl(see, e.g.t Chin, J. W., et al. (2002) Proc. Natl. Acad. Sci. U. S. A. 99:11020-11024; and, Chin, J.μlW., et al., (2002) J. Am. Chem. Soc. 124:9026-9027), keto amino acids, heavy atom containingμlamino acids, and glycosylated amino acids have been incorporated efficiently and with high fidelityμlinto proteins in E. coli and in yeast in response to the amber codon, TAG, using this methodology.μlSee, e.g., J. W. Chin et al., (2002), Journal of the American Chemical Society 124:9026-9027; J. W.μlChin, & P. G. Schultz, (2002), ChemBioChem 3(11):1135-1137; J. W. Chin, et al., (2002), PNASμlUnited States of America 99:11020-11024; and, L. Wang, & P. G. Schultz, (2002), Chem. Comm.,μl1:1-11. All references are incorporated by reference in their entirety. These studies have

demonstrated that it is possible to selectively and routinely introduce chemical functional groups,μlsuch as ketone groups, alkyne groups and azide moieties, that are not found in proteins, that areμlchemically inert to all of the functional groups found in the 20 common, genetically-encoded aminoμlacids and that may be used to react efficiently and selectively to form stable covalent linkages.μl[07] The ability to incorporate non-genetically encoded amino acids into proteins permits
the introduction of chemical functional groups that could provide valuable alternatives to theμlnaturally-occurring functional groups, such as the epsilon -NH2 of lysine, the sulfhydryl -SH ofμlcysteine, the imino group of histidine, etc.
[08] Human growth hormone formulations may be lyophilized preparations requiring
reconstitution or aqueous formulations. Per vial, Protropin hGH consists of 5 mg hGH, 40 mgμlmannitol, 0.1 mg monobasic sodium phosphate, 1.6 mg dibasic sodium phosphate, reconstituted toμlpH 7.8 (Physician's Desk Reference, Medical Economics Co., Orawell, NJ., p. 1049, 1992). Per
...
vial, Humatrope hGH consists of 5 mg hGH, 25 mg mannitol, 5 mg glycine, 1.13 mg dibasic
sodium phosphate, reconstituted to pH 7.5 (Physician's Desk Reference, p. 1266, 1992). Examples .
of aqueous human growth hormone formulations are described in U.S. Patent Nos. 5,763,394;
5,981,485; 6,448,225; and U.S. Patent Publication No. 2003/0013653, each of which are
incorporated by reference herein.
[09] For a general review for growth hormone formulations, see Pearlman et al., Current
Communications in Molecular Biology, eds. D. Marshak and D. Liu, pp. 23-30, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., 1989, which is incorporated by reference herein. Other
publications of interest regarding stabilization of proteins are as follows.
[10] U.S. Pat. No. 4,297,344, which is incorporated by reference herein, discloses
stabilization of coagulation factors II and VIE, antithrombin Hμand plasminogen against heat by
adding selected amino acids such as glycine, alanine, hydroxyproline, glutamine, and aminobutyric
acid, and a carbohydrate such as a monosaccharide, an oligosaccharide, or a sugar alcohol.
[11] U.S. Pat. No. 4,783,441, which is incorporated by reference herein, discloses a
method for the prevention of denaturation of proteins such as insulin in aqueous solution at
interfaces by the addition of up to 500 ppm surface-active substances comprising a chain of
alternating, weakly hydrophilic and weakly hydrophobic zones at pH 6.8-8.0.
[12] U.S. Pat No. 4,812,557, which is incorporated by reference herein, discloses a
method of stabilization of interleukin-2 using human serum albumin.

[13] European Patent Application Publication No. 0 303 746, which is incorporated by
reference herein, discloses stabilization of growth promoting hormones with polyols consisting of
non-reducing sugars, sugar alcohols, sugar acids, pentaerythritol, lactose, water-soluble dextrans,
and Ficoll, amino acids, polymers of amino acids having a charged side group at physiological pH,
and choline salts.
[14] European Patent Application Publication No. 0 211 601, which is incorporated by
reference herein, discloses the stabilization of growth promoting hormones in a gel matrix formed
by a block copolymer containing polyoxyethylene-polyoxypxopylene units and having an average
molecular weight of about 1,100 to about 40,000.
[15] European Patent Application Publication No. 0 193 917, which is incorporated by
reference herein, discloses a biologically active composition for slow release characterized by a
water solution of a complex between a protein and a carbohydrate.
[16] International Patent Publication No. WO 89/09614 and Australian patent application
No. 30771/89, which are incorporated by reference herein, disclose a stable pharmaceutical
formulation containing human growth hormone, glycine, and mannitol. Such a preparation shows
improved stability during normal processing and storage in a lyophilized state as well as in the
period of use after the reconstitution.
[17] U.S. Pat. No. 5,096,885, which is incorporated by reference herein, discloses a
formulation of hGH for lyophilization containing glycine, mannitol, a non-ionic surfactant, and a
buffer.
[18] U.S. Pat. No. 4,876,568, which is incorporated by reference herein, discloses that
animal growth hormone may be stabilized with various stabilizers to give decreased formation of
insolubles and preservation of the soluble activity in aqueous environments. Such stabilizers
including certain polyols, amino acids, polymers of amino acids having a charged side group at
physiological pH, and choline salts. Polyols are selected from the group consisting of non-reducing
sugars, sugar alcohols, sugar acids, pentaerythritol, lactose, water-soluble dextrans and Ficoll;
amino acids are selected from the group consisting of glycine, sarcosine, lysine or salts thereof,
serine, arginine or salts thereof, betaine, N,N,-dimethyl-glycine, aspartic acid or salts thereof,
glutamic acid or salts thereof; a polymer of an amino acid having a charged side group at
physiological pH may be selected from polylysine, polyaspartic acid, polyglutamic acid,
polyarginine, polyhistidine, polyornithine and salts thereof; and choline derivatives are selected

from the group consisting of choline chloride, choline dihydrogen citrate, choline bitartrate, cholineμlbicarbonate, tricholine citrate, choline ascorbate, choline borate, choline gluconate, cholineμlphosphate, di(choline)sulphate and dicholine mucate. U.S. Pat. No. 4,876,568, which isμlincorporated by reference herein, notes that polyhistidine can be used as a potential stabilizer forμlanimal growth hormone but there is no indication whether it stabilizes an animal growth hormone orμlhuman growth hormone. Furthermore, U.S. Pat. No. 4,876,568 mentions that poly-DL-lysine HBrμlis preferred.
[19] EP 374120, which is incorporated by reference herein, discloses a stabilized
preparation of growth hormone comprising a buffered polyol excipient comprising a polyol havingμlthree hydroxy groups and a buffer to achieve a pH in a range in which the growth hormone retainsμlits bioactivity for a sufficient period of time. Histidine is mentioned as a buffer for a polyol havingμlthree hydroxy groups. Specifically, EP 374120 teaches that histidine hydrochloride may be used asμla buffer for buffering a polyol having three hydroxy groups for improving the stability of a growthμlhormone preparation in the form of a solution comprising a high concentration of growth hormoneμland a polyol as stabilizer. Furthermore, histidine hydrochloride must be added in an amount of,μlabout 3% by weight of the solution corresponding-to a concentration of -0.15M solution ofμlhistidine hydrochloride. EP 374120 also teaches that histidine alone does not impart chemical andμlphysical stability to a growth hormone preparation.
[20] Sorensen et al., WO 93/12812, which is incorporated by reference herein, teaches
that growth hormone can be stabilized by the presence of histidine or a histidine derivative. If theμlgrowth hormone is lyophilized, the composition can also comprise a bulking agent, i.e. sugarμlalcohols, disaccharides, and mixtures thereof. Sorensen et al., U.S. Pat. No. 5,849,704, which isμlincorporated by reference herein, discloses a pharmaceutical formulation comprising a growthμlhormone and histidine or a derivative of histidine as an additive or buffering substance added toμlprovide stability against deamidation, oxidation or cleavage of the peptide bonds in the growthμlhormone. Also disclosed is that crystallization of growth hormone in the presence of histidine or aμlderivative thereof gives rise to a higher yield of crystals having higher purity than known methods.μlFormulations of human growth hormone variants have been described in U.S. Patent No. 6,136,563μland 5,849,535, which are incorporated by reference herein.
[21] hGH undergoes breakdown via several degradative pathways, including
deamidation, aggregation, clipping of the peptide backbone, and oxidation of methionine residues.

Additional products result from degradation of conjugates of hGH covalently attached to a waterμlsoluble polymer such PEG. A pharmaceutical formulation of hGH that provides acceptable controlμlof degradation products, has maintained stability of hGH over a prolonged period of time, and isμlstable to vigorous agitation (which induces aggregation) would be particularly advantageous.μlBRIEF SUMMARY OF THE INVENTION
[22] This invention provides formulations of hGH polypeptides comprising one or more
non-naturally encoded amino acids.
[23] In some embodiments, the hGH polypeptide comprises one or -more post-
translational modifications. In some embodiments, the hGH polypeptide is linked to a linker,
polymer, or biologically active molecule. In some embodiments, the hGH polypeptide is linked to a
bifunctional polymer, bifunctional linker, or at least one additional hGH polypeptide.
[24] In some embodiments, the non-naturally encoded amino acid is linked to a water
soluble polymer. In some embodiments, the water soluble polymer comprises a polyethylene
glycol) moiety. In some embodiments, the non-naturally encoded amino acid is linked to the water
soluble polymer with a linker or bonded to a water soluble polymer. In some embodiments, the .
water soluble polymer comprises a polyethylene glycol) moiety. in some embodiments, the
invention is a single-dose lyophilized formulation of hGH polypeptide. In some embodiments, the,
invention is a liquid formulation of hGH polypeptide.
[25] In some embodiments, the poly(ethylene glycol) molecule has a molecular weight of
between about 0.1 kDa and about 100 kDa. In some embodiments, the poly(ethylene glycol)
molecule has a molecular weight of between 0.1 kDa and 50 kDa.
[26] In some embodiments, the poly(ethylene glycol) molecule is a branched polymer. Li
some embodiments, each branch of the poly(ethylene glycol) branched polymer has a molecular
weight of between 1 kDa and 100 kDa, or between 1 kDa and 50 kDa.
[27] In one embodiment, the pharmaceutical formulation of hGH polypeptide comprising
one or more non-naturally encoded amino acids comprises a buffer, at least one a carrier, excipient,
or stabilizer, and a pharmaceutical quantity of human growth hormone (hGH).
In another embodiment, the at least one a carrier, excipient, or stabilizer is selected from the group
consisting of an antioxidant, an amino acid, a carbohydrate, a chelating agent, a sugar alcohol, a
salt-forming counter ion, and a non-ionic surfactant. The present invention also provides methods

of treating a patient having a disorder modulated by hGH with an effective amount of the
formulation of a hGH molecule of the present invention.
[28] In one embodiment of the present invention, formulations of hGH polypeptide
comprising a non-naturally encoded amino acid that minimize formation of undesirable aggregated
species or cause chemical changes that reduce biological activity or alter receptor recognition are
provided Such formulations are capable of maintaining activity for appropriate storage times, are
readily formulated, and are acceptable for administration to patients.
[29] In one embodiment, the formulation of hGH polypeptide, including but not limited to
PEGylated hGH, comprising one or more non-naturally encoded amino acids is a lyophilized
formulation that is reconstituted prior to use for subcutaneous injection. The formulations of the
present invention may be pharmaceutical formulations, in particular, formulations for subcutaneous
administration.
[30] In another embodiment, the invention provides a method for the treatment,
prophylactic or therapeutic, of a disorder treatable by the protein formulated, including but not '
limited to, PEGylated hGH, using the formulations disclosed herein. Such formulations are '
particularly useful for subcutaneous administration.
[31] Also provided is an article of manufacture comprising a container enclosing a
formulation disclosed herein, as well as pre-filled syringes.
BRIEF DESCRIPTION OF THE DRAWINGS
[32] Figure 1 shows the pH stability analysis of formulation buffers after six weeks at
4°C.
[33] Figure 2 shows SDS-PAGE reducing (Fig. 2C and 2D) and non-reducing gels (Fig.
2A and 2B) of Met Y35pAF hGH. Gels in Fig. 2A and 2B are loaded as follows: Lane 1: MW,
Lane 2: WHO hGH, Lane 3: WHO hGH 1%; Lane 4: Al; Lane 5: A2; Lane 6: A3; Lane 7: A4;
Lane 8: A5; Lane 9: A6; Lane 10: A7; Lane 11: A8; Lane 12: MW. Gels in Fig. 2C and 2D are
loaded as follows: Lane 1: MW, Lane 2: WHO hGH, Lane 3: WHO hGH 1%; Lane 4: Bl; Lane 5:
B2; Lane 6: B3; Lane 7: B4; Lane 8: B5; Lane 9: B6; Lane 10: B7; Lane 11: B8; Lane 12: MW.
[34] Figure 3A-F shows Differential Scanning Calorimetry (DSC) thermoprofiles of
formulation groups A-F.
[35] Figure 4 shows DSC thermoprofiles from groups B7 (Fig. 4A) and F2 (Fig. 4B) of
the matrix.

[36] Figure 5 provides a table summarizing the DSC melting temperatures and changes to
Tm for the full matrix.
[37] Figure 6 shows a summary of the DSC melting temperatures for the full matrix.
[38] Figure 7 shows an analysis of the ratio of AHV/AH for the formulation groups.
[39] Figure 8 shows an analysis of the change in enthalpy (AUC) of each sample.
[40] Figure 9A-D shows RP-HPLC datasets of samples in Group B, C, E, and F compared
to the WHO hGH standard.
[41] Figure 10A shows an analysis of the different Y35pAF peaks for Group E5 (Relative
percent of all peaks were plotted at each time point to visualize any and all changes to the main
MetY35pAF hGH peak), and Figure 10B shows a zoom of the primary deamination/oxidation peak
(Zoomed in Deamidation Peak - increase in deamidation over time (t ~ 0 to 4weeks). RP-HPLC
Analysis ws performed using Agilent Chemstation Software.
[42] Figure 11 shows an analysis of the primary deamidation/oxidation peak across the
matrix. (Analysis of the Met Y35pAF hGH Over 4 weeks @ 4°C)
[43] Figure 12 shows an analysis of the secondary deamidation/oxidation peak with the
formulation groups. (Analysis of the Met Y35pAF hGH Over 4 weeks @ 4°C)
[44] Figure 13 shows an analysis of the main GH peak for the formulation groups. .
(Analysis of the Met Y35pAF hGH Over 4 weeks @ 4°C)
[45] Figure 14 shows RP-HPLC analysis of the primary deamidation/oxidation peak in
samples that had undergone freeze-thaw cycles with formulation groups B and C. (Analysis of MetμlY35pAF hGH Peak over 5 Freeze/Thaw Cycles)
[46] Figure 15 shows RP-HPLC analysis of the secondary deamidation peak in samples
that had undergone freeze-thaw cycles with formulation groups B and C. (Analysis of Met Y35pAFμlhGH over 5 Freeze/Thaw Cycles)
[47] Figure 16 shows RP-HPLC analysis of the main GH peak in samples that had
undergone freeze-thaw cycles with formulation groups B and C. (Analysis of Met Y35pAF hGHμlover 5 Freeze/Thaw Cycles)
[48] Figure 17 shows results from an analysis by RP-HPLC.
[49] Figure 18 shows results from an analysis by SEC-HPLC.

[50] Figure 19 shows results from an analysis by cEEX-HPLC.
[51] Figure 20 shows an example of SEC-HPLC integration.
[52] Figure 21 shows an example of RP-HPLC integration.
[53] Figure 22 shows an SDS-PAGE analysis (reduced) of control samples in a
formulation study (t=0). Lane 1: Mark 12; Lane 2: Ref. Std; Lane 3: P6MT; Lane 4: S4MT; Lane 5:
S5MT; Lane 6: S5GT; Lane 7: H6MT; Lane 8: P6GT; Lane 9: P6MA; Lane 10: P6MS.
[54] Figure 23 shows an SDS-PAGE analysis (reduced) of control samples in a
formulation study (t=0). Lane 1: Mark 12; Lane 2: Ref. Std; Lane 3: P6MTMet; Lane 4: P7MT;μlLane 5: P7GT; Lane 6: P6MGT; Lane 7: P6MGT-P; Lane 8: P6MT-P; Lane 9: P6GT-P.
[55] Figure 24 shows an SDS-PAGE analysis (non-reduced) of control samples in a
formulation study (t=0). For Figure 24, panel A, Lane 1: Mark 12 Standard; Lane 2: PEG-hGHμlStandard; Lane 3: P6MT;Lane4: S4MT; Lane 5: S5MT;Lane6: S5GT;Lane7: H6MT; Lane 8:μlP6GT; Lane 9: P6MA; Lane 10: P6MS. Tor Figure 24, panel B, Lane 1: Mark 12 Standard; Lane ,μl2: PEG-hGH Standard; Lane 3: P6MTMet; Lane 4: P7MT; Lane 5: P7GT; Lane 6: P6MGT; ,.μlLane 7: P6MGT-P; Lane 8: P6MT-P; Lane 9: P6GT-P.
[56] Figure 25 shows an SDS-PAGE analysis (non-reduced) of samples stored at 4°C for
1 week. For Figure 25, panel A, Lane 1: Mark 12 Standard; Lane 2: PEG-hGH Standard; Lane 3: μlP6MT; Lane 4: S4MT; Lane 5: S5MT; Lane 6: S5GT; Lane 7: H6MT; Lane 8: P6GT; Lane 9: .:μlP6MA; Lane 10: P6MS. For Figure 25, panel B, Lane 1: Mark 12 Standard; Lane 2: PEG-hGHμlStandard; Lane 3: P6MTMet; Lane 4: P7MT; Lane 5: P7GT; Lane 6: P6MGT; Lane 7: P6MGT-μlP; Lane 8: P6MT-P; Lane 9: P6GT-P.
[57] Figure 26 shows an SDS-PAGE analysis (reduced) of samples stored at 4°C for 1
week. For Figure 26, panel A, Lane 1: Mark 12 Standard; Lane 2: PEG-hGH Standard; Lane 3:μlP6MT; Lane 4: S4MT; Lane 5: S5MT; Lane 6: S5GT; Lane 7: H6MT; Lane 8: P6GT; Lane 9:μlP6MA; Lane 10: P6MS. For Figure 26, panel B, Lane 1: Mark 12 Standard; Lane 2: PEG-hGHμlStandard; Lane 3: P6MTMet; Lane 4: P7MT;Lane5: P7GT;Lane6: P6MGT;Lane7: P6MGT-μlP; Lane 8: P6MT-P; Lane 9: P6GT-P.
[58] Figure 27 shows an SDS-PAGE analysis (non-reduced) of samples stored at 4°C for
2 weeks. For Figure 27, panel A, Lane 1: Mark 12 Standard; Lane 2: PEG-hGH Standard; Lane 3:

P6MT; Lane 4: S4MT; Lane 5: S5MT; Lane 6: S5GT; Lane 7: H6MT; Lane 8: P6GT; Lane 9:μlP6MA; Lane 10: P6MS. For Figure 27, panel B, Lane 1: Mark 12 Standard; Lane 2: PEG-hGHμlStandard; Lane 3: P6MTMet; Lane 4: P7MT; Lane 5: P7GT; Lane 6: P6MGT; Lane 7: P6MGT-μlP; Lane 8: P6MT-P; Lane 9: P6GT-P.
[59] Figure 28 shows an SDS-PAGE analysis (reduced) of samples stored at 4°C for 2
weeks. For Figure 28, panel A, Lane 1: Mark 12 Standard; Lane 2: PEG-hGH Standard; Lane 3:μlP6MT;Lane4: S4MT; Lane 5: S5MT;Lane6: S5GT;Lane7: H6MT; Lane 8: P6GT; Lane 9:μlP6MA; Lane 10: P6MS. For Figure 28, panel B, Lane 1: Mark 12 Standard; Lane 2: PEG-hGHμlStandard; Lane 3: P6MTMet; Lane 4: P7MT; Lane 5: P7GT; Lane 6: P6MGT; Lane 7: P6MGT-μlP; Lane 8: P6MT-P; Lane 9: P6GT-P.
[60] Figure 29 shows an SDS-PAGE analysis (non-reduced) of samples stored at 4°C for
4 weeks. For Figure 29, panel A, Lane 1: Mark 12 Standard; Lane 2: PEG-hGH Standard; Lane 3: μlP6MT;Lane4: S4MT; Lane 5: S5MT; Lane 6: S5GT; Lane 7: H6MT; Lane 8: P6GT; Lane 9: .μlP6MA; Lane 10: P6MS. For Figure 29, panel B, Lane 1: Mark 12 Standard; Lane 2: PEG-hGHμlStandard; Lane 3: P6MTMet; Lane 4: P7MT; Lane 5: P7GT;Lane6: P6MGT; Lane 7: P6MGT-μlP; Lane 8: P6MT-P; Lane 9: P6GT-P.
[61] Figure 30 shows an SDS-PAGE analysis (reduced) of samples stored at 4°C for 4
weeks. For Figure 30, panel A, Lane 1: Mark 12 Standard; Lane 2: PEG-hGH Standard; Lane 3,: 'μlP6MT; Lane 4: S4MT; Lane 5: S5MT; Lane 6: S5GT; Lane 7: H6MT; Lane 8: P6GT; Lane 9:μlP6MA; Lane 10: P6MS. For Figure 30, panel B, Lane 1: Mark 12 Standard; Lane 2: PEG-hGHμlStandard; Lane 3: P6MTMet; Lane 4: P7MT; Lane 5: P7GT;Lane6: P6MGT;Lane7: P6MGT-μlP; Lane 8: P6MT-P; Lane 9: P6GT-P.
[62] Figure 31 shows an SDS-PAGE analysis (non-reduced) of samples stored at 4°C for
6 weeks. Lane 1: PEG-hGH Standard; Lane 3: P6MT; Lane 4: S5MT; Lane 5: S5GT; Lane 6:μlH6MT; Lane 7: P6GT; Lane 8: P6MS; Lane 9: P6MTMet; Lane 10: P6MT; Lane 11: P7GT;μlLane 12: P6MGT; Lane 13: P6MGT-P; Lane 14: P6MT-P; Lane 15: P6GT-P.
[63] Figure 32 shows an SDS-PAGE analysis (reduced) of samples stored at 4°C for 6
weeks. Lane 1: PEG-hGH Standard; Lane 3: P6MT; Lane 4: S5MT; Lane 5: S5GT; Lane 6:

H6MT;Lane7: P6GT; Lane 8: P6MS; Lane 9: P6MTMet; Lane 10: P6MT; Lane 11: P7GT;μlLane 12: P6MGT; Lane 13: P6MGT-P; Lane 14: P6MT-P; Lane 15: P6GT-P.
[64] Figure 33 shows an SDS-PAGE analysis (non-reduced) of samples stored at 4°C for
2 months. Lane 1: PEG-hGH Standard; Lane 3: hGH (1 ug); Lane 5: P6MT; Lane 6: H6MT; Laneμl7: P6GT; Lane 8: P6MS; Lane 9: P6MTMet; Lane 10: P6MT; Lane 11: P7GT; Lane 12:μlP6MGT; Lane 13: P6MGT-P; Lane 14: P6MT-P; Lane 15: P6GT-P.
[65] Figure 34 shows an SDS-PAGE analysis (reduced) of samples stored at 4°C for 2
months. Lane 1: PEG-hGH Standard; Lane 3: hGH (1 ug); Lane 5: P6MT; Lane 6: H6MT; Laneμl7: P6GT; Lane 8: P6MS; Lane 9: P6MTMet; Lane 10: P6MT; Lane 11: P7GT; Lane 12:μlP6MGT; Lane 13: P6MGT-P; Lane 14: P6MT-P; Lane 15: P6GT-P.
[66] Figure 35 shows an SDS-PAGE analysis (non-reduced) of samples stored at 4°C for
3 months. Lane 1: PEG-hGH Standard; Lane 3: hGH (lug); Lane 5: P6MT; Lane 6: H6MT; Lane 'μl7: P6GT; Lane 8: P6MS; Lane 9: P6MTMet; Lane 10: P6MT; Lane 11: P7GT; Lane 12:μlP6MGT; Lane 13: P6MGT-P; Lane 14: P6MT-P; Lane 15: P6GT-P.
[67] Figure 36 shows an SDS-PAGE analysis (reduced) of samples stored at 4°C for 3
months. Lane 1: PEG-hGH Standard; Lane 3: hGH (1 ug); Lane 5: P6MT; Lane 6: H6MT; Laneμl7: P6GT; Lane 8: P6MS; Lane 9: P6MTMet (contamination); Lane 10: P6MT; Lane 11: P7GT; μlLane 12: P6MGT; Lane 13: P6MGT-P; Lane 14: P6MT-P; Lane 15: P6GT-P.
[68] Figure 37 shows an SDS-PAGE analysis (non-reduced) of samples stored at 25°C for
1 week. For Figure 37, panel A, Lane 1: Mark 12 Standard; Lane 2: PEG-hGH Standard; Lane 3:μlP6MT; Lane 4: S4MT; Lane 5: S5MT; Lane 6: S5GT; Lane 7: H6MT; Lane 8: P6GT; Lane 9:μlP6MA; Lane 10: P6MS. For Figure 37, panel B, Lane 1: Mark 12 Standard; Lane 2: PEG-hGHμlStandard; Lane 3: P6MTMet; Lane 4: P7MT; Lane 5: P7GT;Lane6: P6MGT; Lane 7: P6MGT-μlP; Lane 8: P6MT-P; Lane 9: P6GT-P.
[69] Figure 38 shows an SDS-PAGE analysis (reduced) of samples stored at 25°C for 1
week. For Figure 38, panel A, Lane 1: Mark 12 Standard; Lane 2: PEG-hGH Standard; Lane 3:μlP6MT;Lane4: S4MT; Lane 5: S5MT; Lane 6: S5GT; Lane 7: H6MT; Lane 8: P6GT; Lane 9:μlP6MA; Lane 10: P6MS. For Figure 38, panel B, Lane 1: Mark 12 Standard; Lane 2: PEG-hGH

Standard; Lane J: P6MTMet; Lane 4: P7MT; Lane 5: P7GT; Lane 6: P6MGT; Lane 7: P6MGT-μlP; Lane 8: P6MT-P; Lane 9: P6GT-P.
[70] Figure 39 shows an SDS-PAGE analysis (non-reduced) of samples stored at 25°C for
2 weeks. For Figure 39, panel A, Lane 1: Mark 12 Standard; Lane 2: PEG-hGH Standard; Lane 3:μlP6MT;Lane4: S4MT; Lane 5: S5MT; Lane 6: S5GT; Lane 7: H6MT; Lane 8: P6GT; Lane 9:μlP6MA; Lane 10: P6MS. For Figure 39, panel B, Lane 1: Mark 12 Standard; Lane 2: PEG-hGHμlStandard; Lane 3: P6MTMet; Lane 4: P7MT; Lane 5: P7GT; Lane 6: P6MGT;Lane7: P6MGT-μlP; Lane 8: P6MT-P; Lane 9: P6GT-P.
[71] Figure 40 shows an SDS-PAGE analysis (reduced) of samples stored at 25°C for 2
weeks. For Figure 40, panel A, Lane 1: Mark 12 Standard; Lane 2: PEG-hGH Standard; Lane 3:μlP6MT;Lane4: S4MT; Lane 5: S5MT; Lane 6: S5GT; Lane 7: H6MT; Lane 8: P6GT; Lane 9:μlP6MA; Lane 10: P6MS. For Figure 40, panel B, Lane 1: Mark 12 Standard; Lane 2: PEG-hGHμlStandard; Lane 3: P6MTMet; Lane 4: P7MT; Lane 5: P7GT; Lane 6: P6MGT; Lane 7: P6MGT-,μlP; Lane 8: P6MT-P; Lane 9: P6GT-P.
[72] Figure 41 shows an SDS-PAGE analysis (non-reduced) of samples stored at 25°C for
4 weeks. For Figure 41, panel A, Lane 1: Mark 12 Standard; Lane 2: PEG-hGH Standard; Lane 3:μlP6MT; Lane 4: S4MT; Lane 5: S5MT; Lane 6: S5GT; Lane 7: H6MT; Lane 8: P6GT; Lane 9:μlP6MA; Lane 10: P6MS. For Figure 41, panel B, Lane 1: Mark 12 Standard; Lane 2: PEG-hGHμlStandard; Lane 3: P6MTMet; Lane 4: P7MT; Lane 5: P7GT; Lane 6: P6MGT; Lane 7: P6MGT-μlP; Lane 8: P6MT-P; Lane 9: P6GT-P.
[73] Figure 42 shows an SDS-PAGE analysis (reduced) of samples stored at 25°C for 4
weeks. For Figure 42, panel A, Lane 1: Mark 12 Standard; Lane 2: PEG-hGH Standard; Lane 3:μlP6MT;Lane4: S4MT; Lane 5: S5MT; Lane 6: S5GT; Lane 7: H6MT; Lane 8: P6GT;Lane9:μlP6MA;LanelO: P6MS. For Figure 42, panel B, Lane 1: Mark 12 Standard; Lane 2: PEG-hGHμlStandard; Lane 3: P6MTMet; Lane 4: P7MT;Lane5: P7GT;Lane6: P6MGT;Lane7: P6MGT-μlP; Lane 8: P6MT-P; Lane 9: P6GT-P.
[74] Figure 43 shows an SDS-PAGE analysis (non-reduced) of samples stored at 25 C for
6 weeks. Lane 1: PEG-hGH Standard; Lane 3: P6MT; Lane 4: S5MT; Lane 5: S5GT; Lane 6:

H6MT; Lane 7: P6GT; Lane 8: P6MS; Lane 9: P6MTMet; Lane 10: P6MT; Lane 11: P7GT;μlLane 12: P6MGT; Lane 13: P6MGT-P; Lane 14: P6MT-P; Lane 15: P6GT-P.
[75] Figure 44 shows an SDS-PAGE analysis (reduced) of samples stored at 25°C for 6
weeks. Lane 1: PEG-hGH Standard; Lane 3: P6MT; Lane 4: S5MT; Lane 5: S5GT; Lane 6:μlH6MT; Lane 7: P6GT; Lane 8: P6MS; Lane 9: P6MTMet; Lane 10: P6MT; Lane 11: P7GT;μlLane 12: P6MGT; Lane 13: P6MGT-P; Lane 14: P6MT-P; Lane 15: P6GT-P.
[76] Figure 45 shows an SDS-PAGE analysis (non-reduced) of samples stored at 25°C for
2 months. Lane 1: PEG-hGH Standard; Lane 3: hGH (1 ug); Lane 5: P6MT; Lane 6: H6MT; Laneμl7: P6GT; Lane 8: P6MS; Lane 9: P6MTMet; Lane 10: P6MT; Lane 11: P7GT; Lane 12:μlP6MGT; Lane 13: P6MGT-P; Lane 14: P6MT-P; Lane 15: P6GT-P.
[77] Figure 46 shows an SDS-PAGE analysis (reduced) of samples stored at 25°C for 2
months. Lane 1: PEG-hGH Standard; Lane 3: hGH (1 ug); Lane 5: P6MT; Lane 6: H6MT; Lane 7:μlP6GT;Lane8: P6MS; Lane 9: P6MTMet; Lane 10: P6MT; Lane 11: P7GT; Lane 12: P6MGT; μlLane 13: P6MGT-P; Lane 14: P6MT-P; Lane 15: P6GT-P. !
[78] Figure 47 shows an SDS-PAGE analysis (non-reduced) of samples stored at 25°C for
3 months. Lane 1: PEG-hGH Standard; Lane 3: hGH (1 ug); Lane 5: P6MT; Lane 6: H6MT; Laneμl7: P6GT; Lane 8: P6MS; Lane 9: P6MTMet; Lane 10: P6MT; Lane 11: P7GT; Lane 12:μlP6MGT; Lane 13: P6MGT-P; Lane 14: P6MT-P; Lane 15: P6GT-P.
[79] Figure 48 shows an SDS-PAGE analysis (reduced) of samples stored at 25°C for 3
months. Lane 1: PEG-hGH Standard; Lane 3: hGH (1 ug); Lane 5: P6MT; Lane 6: H6MT; Laneμl7: P6GT; Lane 8: P6MS; Lane 9: P6MTMet; Lane 10: P6MT; Lane 11: P7GT; Lane 12:μlP6MGT; Lane 13: P6MGT-P; Lane 14: P6MT-P; Lane 15: P6GT-P.
[80] Figure 49 shows an SDS-PAGE analysis (non-reduced) of samples stored at 40°C for
1 week. For Figure 49, panel A, Lane 1: Mark 12 Standard; Lane 2: PEG-hGH Standard; Lane 3:μlP6MT;Lane4: S4MT; Lane 5: S5MT; Lane 6: S5GT;Lane7: H6MT; Lane 8: P6GT; Lane 9:μlP6MA; Lane 10: P6MS. For Figure 49, panel B, Lane 1: Mark 12 Standard; Lane 2: PEG-hGHμlStandard; Lane 3: P6MTMet; Lane 4: P7MT; Lane 5: P7GT; Lane 6: P6MGT;Lane7: P6MGT-μlP; Lane 8: P6MT-P; Lane 9: P6GT-P.

[81] Figure 50 shows an SDS-PAGE analysis (reduced) of samples stored at 40°C for 1
week. For Figure 50, panel A, Lane 1: Mark 12 Standard; Lane 2: PEG-hGH Standard; Lane 3:μlP6MT; Lane 4: S4MT; Lane 5: S5MT; Lane 6: S5GT; Lane 7: H6MT; Lane 8: P6GT; Lane 9:μlP6MA; Lane 10: P6MS. For Figure 50, panel B, Lane 1: Mark 12 Standard; Lane 2: PEG-hGHμlStandard; Lane 3: P6MTMet; Lane 4: P7MT; Lane 5: P7GT; Lane 6: P6MGT; Lane 7: P6MGT-μlP; Lane 8: P6MT-P; Lane 9: P6GT-P.
[82] Figure 51 shows an SDS-PAGE analysis (non-reduced) of samples stored at 40°C for
2 weeks. For Figure 51, panel A, Lane 1: Mark 12 Standard; Lane 2: PEG-hGH Standard; Lane 3:μlP6MT;Lane4: S4MT; Lane 5: S5MT; Lane 6: S5GT;Lane7: H6MT; Lane 8: P6GT; Lane 9:μlP6MA; Lane 10: P6MS. For Figure 51, panel B, Lane 1: Mark 12 Standard; Lane 2: PEG-hGHμlStandard; Lane 3: P6MTMet; Lane 4: P7MT; Lane 5: P7GT; Lane 6: P6MGT; Lane 7: P6MGT-μlP; Lane 8: P6MT-P; Lane 9: P6GT-P.
[83] Figure 52 shows an SDS-PAGE analysis (reduced) of samples stored at 40°C for 2
weeks. For Figure 52, panel A, Lane 1: Mark 12 Standard; Lane 2: PEG-hGH Standard; Lane 3:μlP6MT;Lane4: S4MT; Lane 5: S5MT; Lane 6: S5GT; Lane 7: H6MT; Lane 8: P6GT; Lane 9:μlP6MA;Lanel0: P6MS. For Figure 52, panel B, Lane 1: Mark 12 Standard; Lane 2: PEG-hGHμlStandard; Lane 3: P6MTMet; Lane 4: P7MT;Lane5: P7GT;Lane6: P6MGT;Lane7: P6MGT-μlP; Lane 8: P6MT-P; Lane 9: P6GT-P.
[84] Figure 53 shows an SDS-PAGE analysis (non-reduced) of samples stored at 40°C for
4 weeks. For Figure 53, panel A, Lane 1: Mark 12 Standard; Lane 2: PEG-hGH Standard; Lane 3:μlP6MT;Lane4: S4MT; Lane 5: S5MT; Lane 6: S5GT;Lane7: H6MT; Lane 8: P6GT; Lane 9:μlP6MA; Lane 10: P6MS. For Figure 53, panel B, Lane 1: Mark 12 Standard; Lane 2: PEG-hGHμlStandard; Lane 3: P6MTMet; Lane 4: P7MT; Lane 5: P7GT;Lane6: P6MGT;Lane7: P6MGT-μlP; Lane 8: P6MT-P; Lane 9: P6GT-P.
[85] Figure 54 shows an SDS-PAGE analysis (reduced) of samples stored at 40°C for 4
weeks. For Figure 54, panel A, Lane 1: Mark 12 Standard; Lane 2: PEG-hGH Standard; Lane 3:μlP6MT; Lane 4: S4MT; Lane 5: S5MT; Lane 6: S5GT; Lane 7: H6MT; Lane 8: P6GT; Lane 9:μlP6MA;LanelO: P6MS. For Figure 54, panel B, Lane 1: Mark 12 Standard; Lane 2: PEG-hGH

Standard; Lane 3: P6MTMet; Lane 4: P7MT; Lane 5: P7GT; Lane 6: P6MGT; Lane 7: P6MGT-μlP; Lane 8: P6MT-P; Lane 9: P6GT-P.
[86] Figure 55 shows an SDS-PAGE analysis (non-reduced) of samples stored at 40°C for
6 weeks. Lane 1: PEG-hGH Standard; Lane 3: P6MT; Lane 4: S5MT; Lane 5: S5GT; Lane 6:μlH6MT;Lane7: P6GT; Lane 8: P6MS; Lane 9: P6MTMet; Lane 10: P6MT; Lane 11: P7GT;μlLane .12: P6MGT; Lane 13: P6MGT-P; Lane 14: P6MT-P; Lane 15: P6GT-P.
[87] Figure 56 shows an SDS-PAGE analysis (reduced) of samples stored at 40°C for 6
weeks. Lane 1: PEG-hGH Standard; Lane 3: P6MT; Lane 4: S5MT; Lane 5: S5GT; Lane 6:μlH6MT; Lane 7: P6GT; Lane 8: P6MS; Lane 9: P6MTMet; Lane 10: P6MT; Lane 11: P7GT;μlLane 12: P6MGT; Lane 13: P6MGT-P; Lane 14: P6MT-P; Lane 15: P6GT-P.
[88] Figure 57 shows an SDS-PAGE analysis (non-reduced) of samples stored at 40°C for
2 months. Lane 1: PEG-hGH Standard; Lane 3: hGH (μg); Lane 5: P6MT; Lane 6: H6MT; Lane-μl7: P6GT; Lane 8: P6MS; Lane 9: P6MTMet; Lane 10: P6MT; Lane 11: P7GT; Lane 12μlP6MGT; Lane 13: P6MGT-P; Lane 14: P6MT-P; Lane 15: P6GT-P.
[89] Figure 58 shows an SDS-PAGE analysis (reduced) of samples stored at 40°C for 2
months. Lane 1: PEG-hGH Standard; Lane 3: hGH (μg); Lane 5: P6MT; Lane 6: H6MT; Lane 7:..μlP6GT; Lane 8: P6MS; Lane 9: P6MTMet; Lane 10: P6MT; Lane 11: P7GT; Lane 12: P6MGT;,.μlLane 13: P6MGT-P; Lane 14: P6MT-P; Lane 15: P6GT-P.
[90] Figure 59 shows an SDS-PAGE analysis (non-reduced) of reconstituted samples
stored for week at 4°C. For Figure 59, panel A, Lane 1: PEG-hGH Standard; Lane 3: P6MT;μlLane 4: S4MT; Lane 5: S5MT; Lane 6: S5GT; Lane 7: H6MT; Lane 8: P6GT; Lane 9: P6MA;μlLane 10: P6MS. For Figure 59, panel B, Lane 1: PEG-hGH Standard; Lane 3: P6MTMet; Lane 4:μlP7MT; Lane 5: P7GT; Lane 6: P6MGT; Lane 7: P6MGT-P; Lane 8: P6MT-P; Lane 9: P6GT-P.
[91] Figure 60 shows an SDS-PAGE analysis (reduced) of reconstituted samples stored
for 1 week at 4°C. For Figure 60, panel A, Lane 1: PEG-hGH Standard; Lane 3: P6MT; Lane 4:μlS4MT;Lane5: S5MT; Lane 6: S5GT;Lane7: H6MT;Lane8: P6GT;Lane9: P6MA; Lane 10:μlP6MS. For Figure 60, panel B, Lane 1: PEG-hGH Standard; Lane 3: P6MTMet; Lane 4: P7MT;μlLane 5: P7GT; Lane 6: P6MGT; Lane 7: P6MGT-P; Lane 8: P6MT-P; Lane 9: P6GT-P.

[92] Figure 61 shows an SDS-PAGE analysis (non-reduced) of control samples in a
formulation study (agitation/UV controls). For Figure 61, panel A, Lane 1: PEG-hGH Standard;μlLane 3: P6MT;Lane4: S4MT; Lane 5: S5MT; Lane 6: S5GT;Lane7: H6MT; Lane 8: P6GT;μlLane 9: P6MA; Lane 10: P6MS. For Figure 61, panel B, Lane 1: PEG-hGH Standard; Lane 3:μlP6MTMet; Lane 4: P7MT;Lane5: P7GT; Lane 6: P6MGT; Lane 7: P6MGT-P; Lane 8: P6MT-μlP; Lane 9: P6GT-P.
[93] Figure 62 shows an SDS-PAGE analysis (reduced) of control samples in a
formulation study (agitation/UV controls). For Figure 62, panel A, Lane 1: PEG-hGH Standard;μlLane 3: P6MT; Lane 4: S4MT; Lane 5: S5MT; Lane 6: S5GT;Lane7: H6MT; Lane 8: P6GT;μlLane 9: P6MA; Lane 10: P6MS. For Figure 62, panel B, Lane 1: PEG-hGH Standard; Lane 3:μlP6MTMet; Lane 4: P7MT; Lane 5: P7GT; Lane 6: P6MGT; Lane 7: P6MGT-P; Lane 8: P6MT-μlP; Lane 9: P6GT-P.
[94] Figure 63 shows an SDS-PAGE analysis (non-reduced) of samples agitated for 4
hours at ambient room temperature. For Figure 63, panel A, Lane 1: PEG-hGH Standard; Lane 3:μlP6MT; Lane 4: S4MT; Lane 5: S5MT; Lane 6: S5GT; Lane 7: H6MT; Lane 8: P6GT; Lane 9:..μlP6MA; Lane 10: P6MS. For Figure 63, Panel B, Lane 1: PEG-hGH Standard; Lane 3: P6MTMet;μlLane 4: P7MT; Lane 5: P7GT; Lane 6: P6MGT; Lane 7: P6MGT-P; Lane 8: P6MT-P; Lane 9:μlP6GT-P.
[95] Figure 64 shows an SDS-PAGE analysis (reduced) of samples agitated for 4 hours at
ambient room temperature. For Figure 64, panel A, Lane 1: PEG-hGH Standard; Lane 3: P6MT;μlLane 4: S4MT; Lane 5: S5MT; Lane 6: S5GT; Lane 7: H6MT; Lane 8: P6GT; Lane 9: P6MA;μlLane 10: P6MS. For Figure 64, panel B, Lane 1: PEG-hGH Standard; Lane 3: P6MTMet; Lane 4:μlP7MT;Lane5: P7GT;Lane6: P6MGT;Lane7: P6MGT-P; Lane 8: P6MT-P; Lane 9: P6GT-P.
[96] Figure 65 shows an SDS-PAGE analysis (non-reduced) of samples exposed to UV
light for 4 hours at ambient temperature. For Figure 65, panel A, Lane 1: PEG-hGH Standard; Laneμl3: P6MT;Lane4: S4MT; Lane 5: S5MT; Lane 6: S5GT;Lane7: H6MT; Lane 8: P6GT; Laneμl9: P6MA; Lane 10: P6MS. For Figure 65, panel B, Lane 1: PEG-hGH Standard; Lane 3:μlP6MTMet; Lane 4: P7MT; Lane 5: P7GT; Lane 6: P6MGT;Lane7: P6MGT-P; Lane 8: P6MT-μlP; Lane 9: P6GT-P.

[97] Figure 66 shows an SDS-PAGE analysis (reduced) of samples exposed to UV light
for 4 hours at ambient temperature. For Figure 66, panel A, Lane 1: PEG-hGH Standard; Lane 3:μlP6MT;Lane4: S4MT; Lane 5: S5MT; Lane 6: S5GT; Lane 7: H6MT; Lane 8: P6GT; Lane 9:μlP6MA; Lane 10: P6MS. For Figure 66, panel B, Lane 1: PEG-hGH Standard; Lane 3: P6MTMet;μlLane 4: P7MT; Lane 5: P7GT; Lane 6: P6MGT; Lane 7: P6MGT-P; Lane 8: P6MT-P; Lane 9:μlP6GT-P.
[98] Figure 67 shows an SDS-PAGE analysis (non-reduced) of samples that were subject
to freeze/thaw conditions (H7MT-P). Lane 1: PEG-hGH Standard; Lane 2: hGH (μg); Lane 3:μlH7MT-P 8 mg/mL t = 0; Lane 4: H7MT-P 8 mg/mL F/T 1; Lane 5: H7MT-P 8 mg/mL F/T 2; Laneμl6: H7MT-P 8 mg/mL F/T 3; Lane 7: H7MT-P 8 mg/mL F/T 4; Lane 8: H7MT-P 8 mg/mL F/T 5;μlLane 10: H7MT-P 14 mg/mL t = 0; Lane 11: H7MT-P 14 mg/mL F/T 1; Lane 12: H7MT-P 14μlmg/mL F/T 2; Lane 13: H7MT-P 14 mg/mL F/T 3; Lane 14: H7MT-P 14 mg/mL F/T 4; Lane 15:μlH7MT-P 14 mg/mL F/T 5.
[99] Figure 68 shows an SDS-PAGE analysis (reduced) of samples that were subject to ;
freeze/thaw conditions (H7MT-P). Lane 1: PEG-hGH Standard; Lane 2: hGH (μg); Lane 3:μlH7MT-P 8 mg/mL t = 0; Lane 4: H7MT-P 8 mg/mL F/T 1; Lane 5: H7MT-P 8 mg/mL F/T 2; Laneμl6: H7MT-P 8 mg/mL F/T 3; Lane 7: H7MT-P 8 mg/mL.F/T 4; Lane 8: H7MT-P 8 mg/mL F/T 5;μlLane 10: H7MT-P 14 mg/mL t = 0; Lane 11: H7MT-P 14 mg/mL F/T 1; Lane 12: H7MT-P 14μlmg/mL F/T 2; Lane 13: H7MT-P 14 mg/mL F/T 3; Lane 14: H7MT-P 14 mg/mL F/T 4; Lane 15:μlH7MT-P 14 mg/mL F/T 5.
[100] Figure 69 shows an SDS-PAGE analysis (non-reduced) of samples that were subject
to freeze/thaw conditions (H7MGT-P). Lane 1: PEG-hGH Standard; Lane 2: hGH (μg); Lane 3:μlH7MGT-P 8 mg/mL t = 0; Lane 4: H7MGT-P 8 mg/mL F/T 1; Lane 5: H7MGT-P 8 mg/mL FAT 2;μlLane 6: H7MGT-P 8 mg/mL F/T 3; Lane 7: H7MGT-P 8 mg/mL F/T 4; Lane 8: H7MGT-P 8μlmg/mL ¥/T 5; Lane 10: H7MGT-P 14 mg/mL t = 0; Lane 11: H7MGT-P 14 mg/mL F/T 1; Lane 12:μlH7MGT-P 14 mg/mL F/T 2; Lane 13: H7MGT-P 14 mg/mL F/T 3; Lane 14: H7MGT-P 14 mg/mLμl¥fT 4; Lane 15: H7MGT-P 14 mg/mL F/T 5.
[101] Figure 70 shows an SDS-PAGE analysis (reduced) of samples that were subject to
freeze/thaw conditions (H7MGT-P). Lane 1: PEG-hGH Standard; Lane 2: hGH (1 |ig); Lane 3:

H7MGT-P 8 mg/mL t = 0; Lane 4: H7MGT-P 8 mg/mL F/T 1; Lane 5: H7MGT-P 8 mg/mL F/T 2;μlLane 6: H7MGT-P 8 mg/mL F/T 3; Lane 7: H7MGT-P 8 mg/mL F/T 4; Lane 8: H7MGT-P 8μlmg/mL F/T 5; Lane 10: H7MGT-P 14 mg/mL t = 0; Lane 11: H7MGT-P 14 mg/mL F/T 1; Lane 12:μlH7MGT-P 14 mg/mL F/T 2; Lane 13: H7MGT-P 14 mg/mL F/T 3; Lane 14: H7MGT-P 14 mg/mLμlF/T 4; Lane 15: H7MGT-P 14 mg/mL F/T 5.
[102] Figure 71 shows an SDS-PAGE analysis (non-reduced) of control samples and
samples that were subject to agitation for 6 hours and UV light for 4 hours. Lane 1: PEG-hGHμlStandard; Lane 2: hGH (μg); Lane 4: Vortex/UV Control H7MT-P 8 mg/mL; Lane 5: Vortex/UVμlControl H7MT-P 14 mg/mL; Lane 6: Vortex/UV Control H7MGT-P 8 mg/mL; Lane 7: Vortex/UVμlControl H7MGT-P 14 mg/mL; Lane 8: Vortex H7MT-P 8 mg/mL; Lane 9: Vortex H7MT-P 14μlmg/mL; Lane 10: Vortex H7MGT-P 8 mg/mL; Lane 11: Vortex H7MGT-P 14 mg/mL; Lane 12:μlUV H7MT-P 8 mg/mL; Lane 13: UV H7MT-P 14 mg/mL; Lane 14: UV H7MGT-P 8 mg/mL; Lane μl15: UV H7MGT-P 14mg/mL.
[103] Figure 72 shows an SDS-PAGE analysis (reduced) of control samples and samples
that were subject to agitation for 6 hours and UV light for 4 hours. Lane 1: PEG-hGH Standard;μlLane 2: hGH (μg); Lane 4: Vortex/UV Control H7MT-P 8 mg/mL; Lane 5: Vortex/UV ControlμlH7MT-P 14 mg/mL; Lane 6: Vortex/UV Control H7MGT-P 8 mg/mL; Lane 7: Vortex/UV Control ,μlH7MGT-P 14 mg/mL; Lane 8: Vortex H7MT-P 8 mg/mL; Lane 9: Vortex H7MT-P 14 mg/mL; !μlLane 10: Vortex H7MGT-P 8 mg/mL (contaminated); Lane 11: Vortex H7MGT-P 14 mg/mL;μlLane 12: UV H7MT-P 8 mg/mL; Lane 13: UV H7MT-P 14 mg/mL; Lane 14: UV H7MGT-P 8μlmg/mL; Lane 15: UV H7MGT-P 14mg/mL.
[104] Figure 73 shows an SDS-PAGE analysis (non-reduced) of samples stored at 4°C or
40°C for 1 week and samples that were exposed to thermal unfolding conditions. Lane 1: PEG-μlhGH Standard; Lane 2: hGH (μg); Lane 4: H7MT-P 8 mg/mL 4°C; Lane 5: H7MT-P 14 mg/mLμl4°C; Lane 6: H7MGT-P 8 mg/mL 4°C; Lane 7: H7MGT-P 14 mg/mL 4°C; Lane 8: H7MT-P 8μlmg/mL 40°C; Lane 9: H7MT-P 14 mg/mL 40°C; Lane 10: H7MGT-P 8 mg/mL 40°C; Lane 11:μlH7MGT-P 14 mg/mL 40°C; Lane 12: H7MT-P 8 mg/mL Thermal-unfolding; Lane 13: H7MT-P 14μlmg/mL Thermal-unfolding; Lane 14: H7MGT-P 8 mg/mL Thermal-unfolding; Lane 15: H7MGT-Pμl14 mg/mL Thermal-unfolding.

(105] Figure 74 shows an SDS-PAGE analysis (reduced) of samples stored at 4°C or 40°C
for 1 week and samples that were exposed to thermal unfolding conditions. Lane 1: PEG-hGHμlStandard; Lane 2: hGH (1 fig); Lane 4: H7MT-P 8 mg/mL 4°C; Lane 5: H7MT-P 14 mg/mL 4°C;μlLane 6: H7MGT-P 8 mg/mL 4°C; Lane 7: H7MGT-P 14 mg/mL 4°C; Lane 8: H7MT-P 8 mg/mLμl40°C; Lane 9: H7MT-P 14 mg/mL 40°C; Lane 10: H7MGT-P 8 mg/mL 40°C; Lane 11: H7MGT-Pμl14 mg/mL 40°C; Lane 12: H7MT-P 8 mg/mL Thermal-unfolding; Lane 13: H7MT-P 14 mg/mLμlThermal-unfolding; Lane 14: H7MGT-P 8 mg/mL Thermal-unfolding; Lane 15: H7MGT-P 14μlmg/mL Thermal-unfolding.
[106] Figure 75 shows an SDS-PAGE analysis (non-reduced) of samples stored at 4°C or
40°C for 2 weeks. Lane 1: PEG-hGH Standard; Lane 2: hGH (μg); Lane 3: H7MT-P 8 mg/mLμl4°C; Lane 4: H7MT-P 14 mg/mL 4°C; Lane 5: H7MGT-P 8 mg/mL 4°C; Lane 6: H7MGT-P 14μlmg/mL 4°C; Lane 7: H7MT-P 8 mg/mL 40°C; Lane 8: H7MT-P 14 mg/mL 40°C; Lane 9: H7MGT-μlP 8 mg/mL 40°C; Lane 10: H7MGT-P 14 mg/mL 40°C.
[107] Figure 76 shows an SDS-PAGE analysis (reduced) of samples stored at 4°C or 40°C
for 2'weeks. Lane 1: PEG-hGH Standard; Lane 2: hGH (μg); Lane 3: H7MT-P 8 mg/mL 4°C; .μlLane 4: H7MT-P 14 mg/mL 4°C; Lane 5: H7MGT-P 8 mg/mL 4°C; Lane 6: H7MGT-P 14 mg/mLμl4°C; Lane 7: H7MT-P 8 mg/mL 40°C; Lane 8: H7MT-P 14 mg/mL 40°C; Lane 9: H7MGT-P 8μlmg/mL 40°C; Lane 10: H7MGT-P 14 mg/mL 40°C.
[108] Figure 77 shows an SDS-PAGE analysis (non-reduced) of samples stored at 4°C for
4 months. Lane 1: PEG-hGH Standard; Lane 2: hGH (μg); Lane 4: P6MT; Lane 5: H6MT; Laneμl6: P6GT; Lane 7: P6MS; Lane 8: P6MTMet; Lane 9: P6MT; Lane 10: P7GT; Lane 11: P6MGT;μlLane 12: P6MGT-P; Lane 13: P6MT-P; Lane 14: P6GT-P.
[109] Figure 78 shoes an SDS-PAGE analysis (reduced) of samples stored at 4°C for 4
months. Lane 1: PEG-hGH Standard; Lane 2: hGH (μg); Lane 4: P6MT; Lane 5: H6MT; Lane 6:μlP6GT; Lane 7: P6MS; Lane 8: P6MTMet; Lane 9: P6MT; Lane 10: P7GT; Lane 11: P6MGT; Laneμl12: P6MGT-P; Lane 13: P6MT-P; Lane 14: P6GT-P.
[110] Figure 79 shows an SDS-PAGE analysis (non-reduced) of samples stored at 25°C for
4 months. Lane 1: PEG-hGH Standard; Lane 2: hGH (μg); Lane 3: P6MT; Lane 4: H6MT; Lane

5: P6GT; Lane 6: P6MS; Lane 7: P6MTMet; Lane 8: P6MT; Lane 9: P7GT; Lane 10: P6MGT;μlLane 11: P6MGT-P; Lane 12: P6MT-P; Lane 13: P6GT-P.
[Ill] Figure 80 shows an SDS-PAGE analysis (reduced) of samples stored at 25°C for 4
months. Lane 1: PEG-hGH Standard; Lane 2: hGH (1 ig); Lane 3: P6MT; Lane 4: H6MT; Lane 5:μlP6GT; Lane 6: P6MS; Lane 7: P6MTMet; Lane 8: P6MT; Lane 9: P7GT; Lane 10: P6MGT; Laneμl11: P6MGT-P; Lane 12: P6MT-P; Lane 13: P6GT-P.
[112] Figure 81 shows an SDS-PAGE analysis (non-reduced) of samples stored at 4°C or
40°C for 4 weeks. Lane 1: PEG-hGH Standard; Lane 2: hGH (0.5 ^ig); Lane 3: H7MT-P 8 mg/mLμl4°C; Lane 4: H7MT-P 14 mg/mL 4°C; Lane 5: H7MGT-P 8 mg/mL 4°C; Lane 6: H7MGT-P 14μlmg/mL 4°C; Lane 7: H7MT-P 8 mg/mL 40°C; Lane 8: H7MT-P 14 mg/mL 40°C; Lane 9: H7MGT-μlP 8 mg/mL 40°C; Lane 10: H7MGT-P 14 mg/mL 40°C.
[113] Figure 82 shows an SDS-PAGE analysis (non-reduced) of samples stored at 4°C or .
40°C for 4 weeks. Lane 1: PEG-hGH Standard; Lane 2: hGH (0.5 jig); Lane 3: H7MT-P 8 mg/mL μl4°C; Lane 4: H7MT-P 14 mg/mL 4°C; Lane 5: H7MGT-P 8 mg/mL 4°C; Lane 6: H7MGT-P 14'μlmg/mL 4°C; Lane 7: H7MT-P 8 mg/mL 40°C; Lane 8: H7MT-P 14 mg/mL 40°C; Lane 9: H7MGT- 'μlP 8 mg/mL 40°C; Lane 10: H7MGT-P 14 mg/mL 40°C.
[114] Figure 83 shows an SDS-PAGE analysis of samples. Lane 1: PEG-hGH Standard
(Batch 2); Lane 2: hGH (1 p.g); Lane 4: 39.9 mg/mL Non-reduced; Lane 5: 24.3 mg/mL Non-μlreduced; Lane 6: 1.1 mg/mL Non-reduced; Lane 8: 39.9 mg/mL Reduced; Lane 9: 24.3 mg/mLμlReduced; Lane 10: 1.1 mg/mL Reduced.
DEFINITIONS
[115] It is to be understood that this invention is not limited to the particular methodology,
protocols, cell lines, constructs, and reagents described herein and as such may vary. It is also to be
understood that the terminology used herein is for the purpose of describing particular embodiments
only, and is not intended to limit the scope of the present invention, which will be limited only by
the appended claims.
[116] As used herein and in the appended claims, the singular forms "a," "an," and "the"
include plural reference unless the context clearly indicates otherwise. Thus, for example,

reference to a "hGH" is a reference to one or more such proteins and includes equivalents thereofμlknown to those of ordinary skill in the art, and so forth.
[117] Unless defined otherwise, all technical and scientific terms used herein have the
same meaning as commonly understood to one of ordinary skill in the art to which this inventionμlbelongs. Although any methods, devices, and materials similar or equivalent to those describedμlherein can be used in the practice or testing of the invention, the preferred methods, devices andμlmaterials are now described.
[118] All publications and patents mentioned herein are incorporated herein by reference
for the purpose of describing and disclosing, for example, the constructs and methodologies that areμldescribed in the publications, which might be used in connection with the presently describedμl.invention. The publications discussed herein are provided solely for their disclosure prior to theμlfiling date of the present application. Nothing herein is to be construed as an admission that theμlinventors are not entitled to antedate such disclosure by virtue of prior invention or for any other .μlreason.
[119] U.S. Patent Application Serial No. 11/046,432 is incorporated by reference in its .
entirety. Thus, the disclosures provided in paragraphs numbered 79-153, in U.S. PatentμlApplication Serial No. 11/046,432 apply fully to the methods, compositions, techniques andμlstrategies for making, purifying, characterizing, and using non-natural amino acids, non-natural μlamino acid hGH polypeptides and modified non-natural amino acid hGH polypeptides describedμlherein to the same extent as if such disclosures were fully presented herein.
[120] As used herein, "growth hormone" or "GH" shall include those polypeptides and
proteins that have at least ondT biological activity of a human growth hormone, as well as GHμlanalogs, GH isoforms, GH mimetics, GH fragments, hybrid GH proteins, fusion proteins, oligomersμland multimers, homologues, glycosylation pattern variants, variants, splice variants, and muteins,μlthereof, regardless of the biological activity of same, and further regardless of the method ofμlsynthesis or manufacture thereof including, but not limited to, recombinant (whether produced fromμlcDNA, genomic DNA, synthetic DNA or other form of nucleic acid), in vitro, in vivo, byμlmicroinjection of nucleic acid molecules, synthetic, transgenic, and gene activated methods. Theμlterm "hGH polypeptide" encompasses hGH polypeptides comprising one or more amino acidμlsubstitutions, additions or deletions.

[121] For the complete full-length naturally-occurring GH amino acid sequence as well as
the mature naturally-occurring GH amino acid sequence and naturally occurring mutant, see SEQμlID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively, in U.S. Patent Application No.μl11/046,432, which is incorporated by reference herein. In some embodiments, hGH polypeptides ofμlthe invention are substantially identical to these sequences or any other sequence of a growthμlhormone polypeptide.
[122J The term "hGH polypeptide" also includes the phaimaceutically acceptable salts and
prodrugs, and prodrugs of the salts, polymorphs, hydrates, solvates, biologically-active ftagments,μlbiologically active variants and stereoisomers of the naturally-occurring hGH as well as agonist,μlmimetic, and antagonist variants of the naturally-occurring hGH and polypeptide fusions thereof.μlFusions comprising additional amino acids at the amino terminus, carboxyl terminus, or both, areμlencompassed by the term "hGH polypeptide." Exemplary fusions include, but are not limited to,μle.g., methionyl growth hormone in which a methionine is linked to the N-terminus of hGH resultingμlfrom the recombinant expression, fusions for the purpose of purification (including, but not limited μlto, to poly-histidine or affinity epitopes), fusions with serum albumin binding peptides and fusionsμlwith serum proteins such as serum albumin. U.S. Patent No. 5,750,373, which is incorporated byμlreference herein, describes a method for selecting novel proteins such as growth hormone andμlantibody fragment variants having altered binding properties for their respective receptor molecules. ;μlThe method comprises fusing a gene encoding a protein of interest to the carboxy terminal domain μlof the gene IQ coat protein of the filamentous phage M13.
[123] Various references disclose modification of polypeptides by polymer conjugation or
glycosylation. The term "hGH polypeptide" includes polypeptides conjugated to a polymer such asμlPEG and may be comprised of one or more additional derivitizations of cysteine, lysine, or otherμlresidues. In addition, the hGH polypeptide may comprise a linker or polymer, wherein the aminoμlacid to which the linker or polymer is conjugated may be a non-natural amino acid according to theμlpresent invention, or may be conjugated to a naturally encoded amino acid utilizing techniquesμlknown in the art such as coupling to lysine or cysteine.
[124] Polymer conjugation of hGH polypeptides has been reported. See, e.g. U.S. Pat.
Nos. 5,849,535, 6,136,563 and 6,608,183, which are incorporated by reference herein. U.S. Pat.μlNo. 4,904,584 discloses PEGylated lysine depleted polypeptides, wherein at least one lysine residueμlhas been deleted or replaced with any other amino acid residue. WO 99/67291 discloses a process

for conjugating a protein with PEG, wherein at least one amino acid residue on the protein isμldeleted and the protein is contacted with PEG under conditions sufficient to achieve conjugation toμlthe protein. WO 99/03887 discloses PEGylated variants of polypeptides belonging to the growthμlhormone superfamily, wherein a cysteine residue has been substituted with a non-essential aminoμlacid residue located in a specified region of the polypeptide. WO 00/26354 discloses a method ofμlproducing a glycosylated polypeptide variant with reduced allergenicity, which as compared to aμlcorresponding parent polypeptide comprises at least one additional glycosylation site. U.S. Pat. No.μl5,218,092, which is incorporated by reference herein, discloses modification of granulocyte colonyμlstimulating factor (G-CSF) and other polypeptides so as to introduce at least one additionalμlcarbohydrate chain as compared to the native polypeptide.
[125] The term "hGH polypeptide" also includes glycosylated hGH, such as but not limited
to, polypeptides glycosylated at any amino acid position, N-linked or O-linked glycosylated formsμlof the polypeptide. Variants containing single nucleotide changes are also considered asμlbiologically active variants of hGH polypeptide. In addition, splice variants are also included. Theμlterm "hGH polypeptide" also includes hGH polypeptide heterodimers, homodimers, ;iμlheteromultimers, or homomultimers of any one or more hGH polypeptides or any other polypeptide,μlprotein, carbohydrate, polymer, small molecule, linker, ligand, or other biologically active moleculeμlof any type, linked by chemical means or expressed as a fusion protein, as well as polypeptide .«.μlanalogues containing, for example, specific deletions or other modifications yet maintain biological _:μlactivity.
[126] All references to amino acid positions in hGH described herein are based on the
position in SEQ ID NO: 2 as listed in U.S. Patent Application No. 11/046,432, entitled "ModifedμlHuman Growth Hormone Polypeptides and Their Uses," which is incorporated by reference herein,μlunless otherwise specified (i.e., when it is stated that the comparison is based on another hGHμlsequence such as SEQ ID NO: 1, 3). Those of skill in the art will appreciate that amino acidμlpositions corresponding to positions in SEQ ID NO: 1, 2, 3 listed in U.S. Patent Application No.μl11/046,432, which is incorporated by reference herein, or any other GH sequence can be readilyμlidentified in any other hGH molecule such as hGH fusions, variants, fragments, etc. For example,μlsequence alignment programs such as BLAST can be used to align and identify a particular positionμlin a protein that corresponds with a position in SEQ ED NO: 1, 2, 3 of U.S. Patent Application No.μl11/046,432, which is incorporated by reference herein, or other GH sequence. Substitutions,

deletions or additions of amino acids described herein in reference to SEQ ID NO: 1, 2, 3 of U.S.μlPatent Application No. 11/046,432, which is incorporated by reference herein, or other GHμlsequence are intended to also refer to substitutions, deletions or additions in corresponding positionsμlin any other hGH molecule such as hGH fusions, variants, fragments, etc. described herein orμlknown in the art and are expressly encompassed by the present invention.
[127] The term "hGH polypeptide" or "hGH" encompasses hGH polypeptides comprising
one or more amino acid substitutions, additions or deletions. hGH polypeptides of the presentμlinvention may be comprised of modifications with one or more natural amino acids in conjunctionμlwith one or more non-natural amino acid modification. Exemplary substitutions in a wide varietyμlof amino acid positions in naturally-occurring hGH polypeptides have been described, including butμlnot limited to substitutions that modulate one or more of the biological activities of the hGHμlpolypeptide, such as but not limited to, increase agonist activity, increase solubility of theμlpolypeptide, decrease protease susceptibility, convert the polypeptide into an antagonist, etc. andμlare encompassed by the term " hGH polypeptide."
[128] In some embodiments, the hGH polypeptides further comprise an addition, "
substitution or deletion that modulates biological activity of the hGH polypeptide. For example, feeμladditions, substitutions or deletions may modulate one or more properties or activities of hGH. Forμlexample, the additions, substitutions or deletions may modulate affinity for the hGH polypeptideμlreceptor, modulate (including but not limited to, increases or decreases) receptor dimerization,μlstabilize receptor dimers, modulate circulating half-life, modulate therapeutic half-life, modulate μlstability of the polypeptide, modulate cleavage by proteases, modulate dose, modulate release orμlbio-availability, facilitate purification, or improve or alter a particular route of administration.μlSimilarly, hGH polypeptides may comprise protease cleavage sequences, secretion signalμlsequences, reactive groups, antibody-binding domains (including but not limited to, FLAG or poly-μlHis) or other affinity based sequences (including but not limited to, FLAG, poly-His, GST, etc.) orμllinked molecules (including but not limited to, biotin) that improve detection (including but notμllimited to, GFP), purification or other traits of the polypeptide.
[129] The term "hGH polypeptide" also encompasses homodimers, heterodimers,
homomultimers, and heteromultimers that are linked, including but not limited to those linkedμldirectly via non-naturally encoded amino acid side chains, either to the same or different non-μlnaturally encoded amino acid side chains, to naturally-encoded amino acid side chains, or indirectly

via a linker. Exemplary linkers including but are not limited to, small organic compounds, waterμlsoluble polymers of a variety of lengths such as poly(ethylene glycol) or polydextran, orμlpolypeptides of various lengths.
[130] A "non-naturally encoded amino acid" refers to an amino acid that is not one of the
20 common amino acids or pyxrolysine or selenocysteine. Other terms that may be usedμlsynonymously with the term "non-naturally encoded amino acid" are "non-natural amino acid,"μl"unnatural amino acid," "non-naturally-occurring amino acid," and variously hyphenated and non-μlhyphenated versions thereof. The term "non-naturally encoded amino acid" also includes, but is notμllimited to, amino acids that occur by modification (e.g. post-translational modifications) of aμlnaturally encoded amino acid (including but not limited to, the 20 common amino acids orμlpyrrolysine and selenocysteine) but are not themselves naturally incorporated into a growingμlpolypeptide chain by the translation complex. Examples of such non-naturally-occurring aminoμlacids include, but are not limited to, iV^acetylglucosaminyl-L-serine, JV"-acetylglucosaminyl-L-μlthreonine, and O-phosphotyrosine.
[131] An "amino terminus modification group" refers to any molecule that can be attached
to the amino terminus of a polypeptide. Similarly, a "carboxy terminus modification group" refersμlto any molecule that can be attached to the carboxy terminus of a polypeptide. Terminusμlmodification groups include, but are not limited to, various water soluble polymers, peptides orμlproteins such as serum albumin, or other moieties that increase serum half-life of peptides.μl[132] The terms "functional group", "active moiety", "activating group", "leaving group",
"reactive site", "chemically reactive group" and "chemically reactive moiety" are used in the art andμlherein to refer to distinct, definable portions or units of a molecule. The terms are somewhatμlsynonymous in the chemical arts and are used herein to indicate the portions of molecules thatμlperform some function or activity and are reactive with other molecules.
[133] The term "linkage" or 'linker" is used herein to refer to groups or bonds that
normally are formed as the result of a chemical reaction and typically are covalent linkages.μlHydrolytically stable linkages means that the linkages are substantially stable in water and do notμlreact with water at useful pH values, including but not limited to, under physiological conditions forμlan extended period of time, perhaps even indefinitely. Hydrolytically unstable or degradableμllinkages mean that the linkages are degradable in water or in aqueous solutions, including forμlexample, blood. Enzymatically unstable or degradable linkages mean that the linkage can be

degraded by one or more enzymes. As understood in the art, PEG and related polymers may includeμldegradable linkages in the polymer backbone or in the linker group between the polymer backboneμland one or more of the terminal functional groups of the polymer molecule. For example, esterμllinkages formed by the reaction of PEG carboxylic acids or activated PEG carboxylic acids withμlalcohol groups on a biologically active agent generally hydrolyze under physiological conditions toμlrelease the agent. Other hydrolytically degradable linkages include, but are not limited to, carbonateμllinkages; imine linkages resulted from reaction of an amine and an aldehyde; phosphate esterμllinkages formed by reacting an alcohol with a phosphate group; hydrazone linkages which areμlreaction product of a hydrazide and an aldehyde; acetal linkages that are the reaction product of anμlaldehyde and an alcohol; orthoester linkages that are the reaction product of a formate and anμlalcohol; peptide linkages formed by an amine group, including but not limited to, at an end of aμlpolymer such as PEG, and a carboxyl group of a peptide; and oligonucleotide linkages formed by aμlphosphoramidite group, including but not limited to, at the end of a polymer, and a 5' hydroxyl;μlgroup of an oligonucleotide.
[134] The term "biologically active molecule", "biologically active moiety" or ;
"biologically active agent" when used herein means any substance which can affect any physical orμlbiochemical properties of a biological system, pathway, molecule, or interaction relating to anμlorganism, including but not limited to, viruses, bacteria, bacteriophage, transposon, prion, insects,μlfungμplants, animals, and humans. In particular, as used herein, biologically active moleculesμlinclude, but are not limited to, any substance intended for diagnosis, cure, mitigation, treatment, orμlprevention of disease in humans or other animals, or to otherwise enhance physical or mental well-μlbeing of humans or animals. Examples of biologically active molecules include, but are not limitedμlto, peptides, proteins, enzymes, small molecule drugs, hard drugs, soft drugs, carbohydrates,μlinorganic atoms or molecules, dyes, lipids, nucleosides, radionuclides, oligonucleotides, toxins,μlcells, viruses, liposomes, microparticles and micelles. Classes of biologically active agents that areμlsuitable for use with the invention include, but are not limited to, drugs, prodrugs, radionuclides,μlimaging agents, polymers, antibiotics, fungicides, anti-viral agents, anti-inflammatory agents, antiμltumor agents, cardiovascular agents, anti-anxiety agents, hormones, growth factors, steroidal agents,μlmicrobially derived toxins, and the like.
[135J A "bifunctional polymer" refers to a polymer comprising two discrete functional
groups that are capable of reacting specifically with other moieties (including but not limited to,

amino acid side groups) to form covalent or non-covalent linkages. A bifunctional linker havingμlone functional group reactive with a group on a particular biologically active component, andμlanother group reactive with a group on a second biological component, may be used to form aμlconjugate that includes the first biologically active component, the bifunctional linker and theμlsecond biologically active component. Many procedures and linker molecules for attachment ofμlvarious compounds to peptides are known. See, e.g., European Patent Application No. 188,256;μlU.S. Patent Nos. 4,671,958, 4,659,839, 4,414,148, 4,699,784; 4,680,338; and 4,569,789 which areμlincorporated by reference herein. A "multi-functional polymer" refers to a polymer comprising twoμlor more discrete functional groups that are capable of reacting specifically with other moietiesμl(including but not limited to, amino acid side groups) to form covalent or non-covalent linkages. Aμlbi-functional polymer or multifunctional polymer may be any desired length or molecular weigjit,μland may be selected to provide a particular desired spacing or conformation between one or moreμlmolecules linked to the GH, e.g., hGH, molecule.
[136] As used herein, the term "water soluble polymer" refers to any polymer that is
soluble in aqueous solvents. Linkage of water soluble polymers to hGH polypeptides can result inμlchanges including, but not limited to, increased or modulated serum half-life, or increased orμlmodulated therapeutic half-life relative to the unmodified form, modulated immunogenicity, ,.μlmodulated physical association characteristics such as aggregation and multimer formation, altered ';μlreceptor binding and altered receptor dimerization or multimerization. The water soluble polymer .μlmay or may not have its own biological activity. Suitable polymers include, but are not limited to,μlpolyethylene glycol, polyethylene glycol propionaldehyde, mono C1-C10 alkoxy or aryloxyμlderivatives thereof (described in U.S. Patent No. 5,252,714 which is incorporated by referenceμlherein), monomethoxy-polyethylene glycol, polyvinyl pyrrolidone, polyvinyl alcohol, polyaminoμlacids, divinylether maleic anhydride, iV-(2-Hydroxypropyl)-methacrylamide, dextran, dextranμlderivatives including dextran sulfate, polypropylene glycol, polypropylene oxide/ethylene oxideμlcopolymer, polyoxyethylated polyol, heparin, heparin fragments, polysaccharides, oligosaccharides,μlglycans, cellulose and cellulose derivatives, including but not limited to methylcellulose andμlcarboxymethyl cellulose, starch and starch derivatives, polypeptides, polyalkylene glycol andμlderivatives thereof, copolymers of polyalkylene glycols and derivatives thereof, polyvinyl ethylμlethers, and alpha-beta-poly[(2-hydroxyethyl)-DL-aspartamide, and the like, or mixtures thereof.

Examples of such water soluble polymers include, but are not limited to, polyethylene glycol andμlserum albumin.
[137] As used herein, the term "polyalkylene glycol" or "poly(aIkene glycol)" refers to
polyethylene glycol (poly(ethylene glycol)), polypropylene glycol, polybutylene glycol, andμlderivatives thereof. The term "polyalkylene glycol" encompasses both linear and branchedμlpolymers and average molecular weights of between 0.1 kDa and 100 kDa. Other exemplaryμlembodiments are listed, for example, in commercial supplier catalogs, such as ShearwaterμlCorporation's catalog "Polyethylene Glycol and Derivatives for Biomedical Applications" (2001).μl[138] As used herein, the term "modulated serum half-life" means the positive or negative
change in circulating half-life of a modified hGH relative to its non-modified form. Serum half-lifeμlis measured by taking blood samples at various time points after administration of hGH, andμldetermining the concentration of that molecule in each sample. Correlation of the serumμlconcentration with time allows calculation of the serum half-life. Increased serum half-life ,-μldesirably has at least about two-fold, but a smaller increase may be useful, for example where it μlenables a satisfactory dosing regimen or avoids a toxic effect. In some embodiments, the increase is ;μlat least about three-fold, at least about five-fold, or at least about ten-fold.
[139] The term "modulated therapeutic half-life" as used herein means the positive or .
negative change in the half-life of the therapeutically effective amount of hGH, relative to its non- .;μlmodified form. Therapeutic half-life is measured by measuring pharmacokinetic and/or .μlpharmacodynamic properties of the molecule at various time points after administration. Increasedμltherapeutic half-life desirably enables a particular beneficial dosing regimen, a particular beneficialμltotal dose, or avoids an undesired'effect In some embodiments, the increased therapeutic half-lifeμlresults from increased potency, increased or decreased binding of the modified molecule to itsμltarget, increased or decreased breakdown of the molecule by enzymes such as proteases, or anμlincrease or decrease in another parameter or mechanism of action of the non-modified molecule,μl[140] The term "substantially purified" refers to a hGH polypeptide that may be
substantially or essentially free of components that normally accompany or interact with the proteinμlas found in its naturally occurring environment, i.e. a native cell, or host cell in the case ofμlrecombinantly produced hGH polypeptides. hGH polypeptide that may be substantially free ofμlcellular material includes preparations of protein having less than about 30%, less than about 25%,μlless than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about

4%, less than about 3%, less than about 2%3 or less than about 1% (by dry weight) of contaminatingμlprotein. When the hGH polypeptide or variant thereof is recombinantly produced by the host cells,μlthe protein may be present at about 30%, about 25%, about 20%, about 15%, about 10%, about 5%,μlabout 4%, about 3%, about 2%, or about 1% or less of the dry weight of the cells. When the hGHμlpolypeptide or variant thereof is recombinantly produced by the host cells, the protein may beμlpresent in the culture medium at about 5g/L, about 4g/L, about 3g/L, about 2g/L, about Ig/L, aboutμl750mg/L, about 500mg/L, about 250mg/L, about lOOmg/L, about 50mg/L, about lOmg/L, or aboutμllmg/L or less of the dry weight of the cells. Thus, "substantially purified" hGH polypeptide asμlproduced by the methods of the present invention may have a purity level of at least about 30%, atμlleast about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, atμlleast about 60%, at least about 65%, at least about 70%, specifically, a purity level of at least aboutμl75%, 80%, 85%, and more specifically, a purity level of at least about 90%, a purity level of at leastμlabout 95%, a purity level of at least about 99% or greater as determined by appropriate methods μlsuch as SDS/PAGE analysis, RP-HPLC, SEC, and capillary electrophoresis.
[141] The term "isolated," when applied to a nucleic acid or protein, denotes that the ,/
nucleic acid or protein is free of at least some of the cellular components with which it is associatedμlin the natural state, or that the nucleic acid or protein has been concentrated to a level greater thanμlthe concentration of its in vivo or in vitro production. It can be in a homogeneous state. Isolatedμlsubstances can be in either a dry or semi-dry state, or in solution, including but not limited to, an μlaqueous solution. It can be a component of a pharmaceutical composition that comprises additionalμlpharmaceutically acceptable carriers and/or excipients. Purity and homogeneity are typicallyμldetermined using analytical chemistry techniques such as polyacrylamide gel electrophoresis orμlhigh performance liquid chromatography. A protein which is the predominant species present in aμlpreparation is substantially purified. In particular, an isolated gene is separated from open readingμlframes which flank the gene and encode a protein other than the gene of interest. The termμl"purified" denotes that a nucleic acid or protein gives rise to substantially one band in anμlelectrophoretic gel. Particularly, it may mean that the nucleic acid or protein is at least 85% pure, atμlleast 90% pure, at least 95% pure, at least 99% or greater pure.
[142] The term "subject" as used herein, refers to an animal, in some embodiments a
mammal, and in other embodiments a human, who is the object of treatment, observation orμlexperiment

J -
[143] The term "effective amount" as used herein refers to that amount of the modified
non-natural amino acid polypeptide being administered which will relieve to some extent one orμlmore of the symptoms of the disease, condition or disorder being treated. Compositions containingμlthe modified non-natural amino acid polypeptide described herein can be administered forμlprophylactic, enhancing, and/or therapeutic treatments.
[144] The terms "enhance" or "enhancing" means to increase or prolong either in potency
or duration a desired effect. Thus, in regard to enhancing the effect of therapeutic agents, the termμl"enhancing" refers to the ability to increase or prolong, either in potency or duration, the effect ofμlother therapeutic agents on a system. An "enhancing-effective amount," as used herein, refers to anμlamount adequate to enhance the effect of another therapeutic agent in a desired system. When usedμlin a patient, amounts effective for this use will depend on the severity and course of the disease,μldisorder or condition, previous therapy, the patient's health status and response to the drugs, and theμljudgment of the treating physician.
[145] The term "modified," as used herein refers to any changes made to a given
polypeptide, such as changes to the length of the polypeptide, the amino acid sequence, chemical μlstructure, co-translational modification, or post-translational modification of a polypeptide. Theμlform "(modified)" term means that the polypeptides being discussed are optionally modified, that is,μlthe polypeptides under discussion can be modified or unmodified. ±
[146] The term "post-translationally modified" refers to any modification of a natural or
non-natural amino acid that occurs to such an amino acid after it has been incorporated into aμlpolypeptide chain. The term encompasses, by way of example only, co-translational in vivoμlmodifications, co-translational in vitro modifications (such as in a cell-free translation system),μlpost-translational in vivo modifications, and post-translational in vitro modifications.μl[147] hi prophylactic applications, compositions containing the modified non-natural
amino acid polypeptide are administered to a patient susceptible to or otherwise at risk of aμlparticular disease, disorder or condition. Such an amount is defined to be a "prophylacticallyμleffective amount" hi this use, the precise amounts also depend on the patient's state of health,μlweight, and the like. It is considered well within the skill of the art for one to determine suchμlprophylactically effective amounts by routine experimentation (e.g., a dose escalation clinical trial).μl[148] In therapeutic applications, compositions containing the modified non-natural amino
acid polypeptide are administered to a patient already suffering from a disease, condition or

disorder, in an amount sufficient to cure or at least partially arrest the symptoms of the disease,
disorder or condition. Such an amount is defined to be a "therapeutically effective amount," and
will depend on the severity and course of the disease, disorder or condition, previous therapy, the
patient's health status and response to the drugs, and the judgment of the treating physician. It is
considered well within the skill of the art for one to determine such therapeutically effective
amounts by routine experimentation (e.g.9 a dose escalation clinical trial).
[149] The term "treating" is used to refer to either prophylactic and/or therapeutic
treatments.
[150] Non-naturally encoded amino acid polypeptides may be metabolized upon
administration to an organism in need to produce a metabolite that is then used to produce a desired
effect, including a desired therapeutic effect
[151] Unless otherwise indicated, conventional methods of mass spectroscopy, NMR,
HPLC, protein chemistry, biochemistry, recombinant DNA techniques and pharmacology, within ,
the skill of the art are employed.
DETAILED DESCRIPTION
J. Introduction
[152]- hGH molecules comprising at least one unnatural amino acid are provided in the;
invention. In certain embodiments of the invention, the hGH polypeptide with at least oneμlunnatural amino acid includes at least one post-translational modification. In one embodiment, theμlat least one post-translational modification comprises attachment of a molecule including but notμllimited to, a label, a dye, a polymer, a water-soluble polymer, a derivative of polyethylene glycol, aμlphotocrosslinker, a radionuclide, a cytotoxic compound, a drug, an affinity label, a photoaffinityμllabel, a reactive compound, a resin, a second protein or polypeptide or polypeptide analog, anμlantibody or antibody fragment, a metal chelator, a cofactor, a fatty acid, a carbohydrate, aμlpolynucleotide, a DNA, a RNA, an antisense polynucleotide, a saccharide, a water-solubleμldendrimer, a cyclodextrin, an inhibitory ribonucleic acid, a biomaterial, a nanoparticle, a spin label,μla fluorophore, a metal-containing moiety, a radioactive moiety, a novel functional group, a groupμlthat covalently or noncovalently interacts with other molecules, a photocaged moiety, an actinicμlradiation excitable moiety, a photoisomerizable moiety, biotin, a derivative of biotin, a biotin

analogue, a moiety incorporating a heavy atom, a chemically cleavable group, a photocleavableμlgroup, an elongated side chain, a carbon-linked sugar, a redox-active agent, an amino thioacid, aμltoxic moiety, an isotopically labeled moiety, a biophysical probe, a phosphorescent group, aμlchemiluminescent group, an electron dense group, a magnetic group, an intercalating group, aμlchromophore, an energy transfer agent, a biologically active agent, a detectable label, a smallμlmolecule, a quantum dot, a nanotransmitter, a radionucleotide, a radiotransmitter, a neutron-captureμlagent, or any combination of the above or any other desirable compound or substance, comprising aμlsecond reactive group to at least one unnatural amino acid comprising a first reactive group utilizingμlchemistry methodology that is known to one of ordinary skill in the art to be suitable for theμlparticular reactive groups.
[153] The protein or polypeptide of interest can contain at least one, at least two, at least
three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or ten or moreμlunnatural amino acids. The unnatural amino acids can be the same or different, for example, there ".μlcan be 1,2, 3,4, 5, 6, 7, 8, 9, 10 or more different sites in the protein that comprise 1, 2, 3, 4, 5, 6, 7,μl8, 9, 10 or more different unnatural amino acids. In certain embodiments, at least one, but fewer 'μlthan all, of a particular amino acid present in a naturally occurring version of the protein aTeμlsubstituted with an unnatural amino acid.
[154] The present invention provides methods and compositions based on members of the
GH supergene family, in particular hGH, comprising at least one non-naturally encoded amino acid.μlIntroduction of at least one non-naturally encoded amino acid into a GH supergene family memberμlsuch as hGH can allow for the application of conjugation chemistries that involve specific chemicalμlreactions, including, but not limited to, with one or more non-naturally encoded amino acids whileμlnot reacting with the commonly occurring 20 amino acids. In some embodiments, the GHμlsupergene family member comprising the non-naturally encoded amino acid is linked to a waterμlsoluble polymer, such as polyethylene glycol (PEG), via the side chain of the non-naturally encodedμlamino acid. This invention provides a highly efficient method for the selective modification ofμlproteins with PEG derivatives, which involves the selective incorporation of non-geneticallyμlencoded amino acids, including but not limited to, those amino acids containing functional groupsμlor substituents not found in the 20 naturally incorporated amino acids, including but not limited to aμlketone, an azide or acetylene moiety, into proteins in response to a selector codon and the

subsequent modification of those amino acids with a suitably reactive PEG derivative. Onceμlincorporated, the amino acid side chains can then be modified by utilizing chemistry methodologiesμlknown to those of ordinary skill in the art to be suitable for the particular functional groups orμlsubstituents present in the non-naturally encoded amino acid. Known chemistry methodologies of aμlwide variety are suitable for use in the present invention to incorporate a water soluble polymer intoμlthe protein.
[155] It is well established in the art that PEG can be used to modify the surfaces of
biomaterials (see, e.g., U.S. Patent 6,610,281; Mehvar, R., J. Pharaiaceut. Sci., 3(1):125-136 (2000)μlwhich are incorporated by reference herein).
[156] A discussion of recombinant nucleic acid methods, selector codons, orthogonal
tRNAs, orthogonal aminoacyl tRNA synthetases, and non-naturally encoded amino acids withμlvarious reactive groups, including but not limited to, carbonyl groups, hydrazine, hydrazide,μlaminooxy, azide, and alkyne groups, is provided in U.S. Patent Application Serial No. 11/046,432μlentitled "Modified Human Growth Hormone Polypeptides and Their Uses," which is incorporatedμlby reference in its entirety herein. Cellular uptake and biosynthesis of non-naturally encoded amino 'μlacids are also discussed in this application. This application also details sites for incorporation ofμlone or more non-naturally encoded amino acids into hGH and expression of hGH polypeptides. Theμlsynthesis of non-natural amino acids containing carbonyl groups such as p-acetyl-(-7-)- 'μlphenylalanine and m-acetyl-(+A)-phenylalanine is described in Zhang, Z., et al., Biochemistry 42:μl6735-6746 (2003), which is incorporated by reference herein.
II. Polypeptides with Unnatural Amino Acids
[157] The incorporation of an unnatural amino acid can be done for a variety of purposes,
including but not limited to, tailoring changes in protein structure and/or function, changing size,μlacidity, nucleophilicity, hydrogen bonding, hydrophobicity, accessibility of protease target sites,μltargeting to a moiety (including but not limited to, for a protein array), adding a biologically activeμlmolecule, attaching a polymer, attaching a radionuclide, modulating serum half-life, modulatingμltissue penetration (e.g., tumors), modulating active transport, modulating tissue, cell or organμlspecificity or distribution, modulating immunogenicity, modulating protease resistance, etc.μlProteins that include an unnatural amino acid can have enhanced or even entirely new catalytic orμlbiophysical properties. For example, the following properties are optionally modified by inclusion

of an unnatural amino acid into a protein: toxicity, biodistribution, structural properties,μlspectroscopic properties, chemical and/or photochemical properties, catalytic ability, half-lifeμl(including but not limited to, serum half-life), ability to react with other molecules, including butμlnot limited to, covalently or noncovalently, and the like. The compositions including proteins thatμlinclude at least one unnatural amino acid are useful for, including but not limited to, novelμltherapeutics, diagnostics, catalytic enzymes, industrial enzymes, binding proteins (including but notμllimited to, antibodies), and including but not limited to, the study of protein structure and function.μlSee, e.g., Dougherty, (2000) Unnatural Amino Acids as Probes of Protein Structure and Function,μlCurrent Opinion in Chemical Biology. 4:645-652.
[158] In one aspect of the invention, a composition includes at least one protein with at least
one, including but not limited to, at least two, at least three, at least four, at least five, at least six, atμlleast seven, at least eight, at least nine, or at least ten or more unnatural amino acids. The unnaturalμlamino acids can be the same or different, including but not limited to, there can be 1,2, 3,4, 5, 6, 7, 'μl8, 9, or 10 or more different sites in the protein that comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or moreμldifferent unnatural amino acids, in another aspect, a composition includes a protein with at least μlone, but fewer than all, of a particular amino acid present in the protein is substituted with theμlunnatural amino acid. For a given protein with more than one unnatural amino acids, the unnatural .μlamino acids can be identical or different (including but not limited to, the protein can include two or '.μlmore different types of unnatural amino acids, or can include two of the same unnatural aminoμlacid). For a given protein with more than two unnatural amino acids, the unnatural amino acids canμlbe the same, different or a combination of a multiple unnatural amino acid of the same kind with atμlleast one different unnatural amino acid.
[159] Proteins or polypeptides of interest with at least one unnatural amino acid are a
feature of the invention. The invention also includes polypeptides or proteins wife at least oneμlunnatural amino acid produced using the compositions and methods of the invention. An excipientμl(including but not limited to, a pharmaceutically acceptable excipient) can also be present with theμlprotein.
[160] By producing proteins or polypeptides of interest with at least one unnatural amino
acid in eukaryotic cells, proteins or polypeptides will typically include eukaryotic post-translationalμlmodifications. In certain embodiments, a protein includes at least one unnatural amino acid and at

least one post-translational modification that is made in vivo by a eukaryotic cell, where the post-μltranslational modification is not made by a prokaryotic cell. For example, the post-translationμlmodification includes, including but not limited to, acetylation, acylation, lipid-modification,μlpalmitoylation, palmitate addition, phosphorylation, glycolipid-linkage modification, glycosylation,μland the like. In one aspect, the post-translational modification includes attachment of anμloligosaccharide (including but not limited to, (GlcNAc-Man)2-Man-GlcNAc-GlcNAc)) to anμlasparagine by a GlcNAc-asparagine linkage. See Table 1 of U.S. Patent Application No.μl11/046,432 entitled "Modifed Human Growth Hormone Polypeptides and Their Uses," which isμlincorporated by reference herein, which lists some examples of N-linked oligosaccharides ofμleukaryotic proteins (additional residues can also be present, which are not shown). In anotherμlaspect, the post-translational modification includes attachment of an oligosaccharide (including butμlnot limited to, Gal-GalNAc, Gal-GlcNAc, etc.) to a serine or threonine by a GalNAc-serine orμlGalNAc-threonine linkage, or a GlcNAc-serine or a GlcNAc-threonine linkage.
[161] In yet another aspect, the post-translation modification includes proteolytic
processing of precursors (including but not limited to, calcitonin precursor, calcitonin gene-relatedμlpeptide precursor, preproparathyroid hormone, preproinsulin, proinsulin, prepro-opiomelanocortin,μlpro-opiomelanocortin and the like), assembly into a multisubunit protein or macromolecular ;μlassembly, translation to another site in the cell (including but not limited to, to organelles, such asμlthe endoplasmic reticulum, the Golgi apparatus, the nucleus, lysosomes, peroxisomes,μlmitochondria, chloroplasts, vacuoles, etc., or through the secretory pathway). In certainμlembodiments, the protein comprises a secretion or localization sequence, an epitope tag, a FLAGμltag, a polyhistidine tag, a GST fusion, or the like. U.S. Patent Nos. 4,963,495 and 6,436,674, whichμlare incorporated herein by reference, detail constructs designed to improve secretion of hGHμlpolypeptides.
[162] One advantage of an unnatural amino acid is that it presents additional chemical
moieties that can be used to add additional molecules. These modifications can be made in vivo inμla eukaryotic or non-eukaryotic cell, or in vitro. Thus, in certain embodiments, the post-translationalμlmodification is througji the unnatural amino acid. For example, the post-translational modificationμlcan be through a nucleophilic-electrophilic reaction. Most reactions currently used for the selectiveμlmodification of proteins involve covalent bond formation between nucleophilic and electrophilic

reaction partners, including but not limited to the reaction of a-haloketones with histidine orμlcysteine side chains. Selectivity in these cases is determined by the number and accessibility of theμlnucleophilic residues in the protein. In proteins of the invention, other more selective reactions canμlbe used such as the reaction of an unnatural keto-amino acid with hydrazides or aminooxyμlcompounds, in vitro and in vivo. See, e.g., Cornish, et al., (1996) J. Am. Chem. Soc 118:8150-μl8151; Mahal, et al., (1997) Science. 276:1125-1128; Wang, et al., (2001) Science 292:498-500;μlChin, et al, (2002) J. Am. Chem. Soc. 124:9026-9027; Chin, et al., (2002) Proc. Natl. Acad. ScLμl99:11020-11024; Wang, et al., (2003) Proc. Natl. Acad. ScL 100:56-61; Zhang, et al, (2003)μlBiochemistry, 42:6735-6746; and, Chin, et al., (2003) Science, 301:964-7, all of which areμlincorporated by reference herein. This allows the selective labeling of virtually any protein with aμlhost of reagents including fluorophores, crosslinking agents, saccharide derivatives and cytotoxicμlmolecules. See also, U.S. Patent No. 6,927,042 entitled "Glycoprotein synthesis," which isμlincorporated by reference herein. Molecules that may be attached to hGH include, but are not .μllimited to, dyes, fluorophores, crosslinking agents, saccharide derivatives, polymers (including but tμlnot limited to, derivatives of polyethylene glycol), photocrosslinkers, cytotoxic compounds, affinity -μllabels, derivatives of biotin, resins, beads, a second protein or polypeptide (or more),μlpolynucleotide(s) (including but not limited to, DNA, RNA, etc.), metal chelators, cofactors, fatty .μlacids, carbohydrates, and the like.
[163] This invention provides formulations of non-natural amino acid polypeptides
generated via selective modification of proteins, which involves the genetic incorporation ofμlunnatural amino acids.
III. In vivo generation of hGH polypeptides comprising non-genetically-encoded
amino acids
[164] The hGH polypeptides of the invention can be generated in vivo using modified
tRNA and tRNA synthetases to add to or substitute amino acids that are not encoded in naturally-μloccurring systems.
[165] Methods for generating tRNAs and tRNA synthetases which use amino acids that are
not encoded in naturally-occurring systems are described in, e.g., U.S. Patent ApplicationμlPublications 2003/0082575 (Serial No. 10/126,927) and 2003/0108885 (Serial No. 10/126,931)μlwhich are incorporated by reference herein. These methods involve generating a translationalμlmachinery that functions independently of the synthetases and tRNAs endogenous to the translation

system (and are therefore sometimes referred to as "orthogonal"). Typically, the translation systemμlcomprises an orthogonal tRNA (O-tRNA) and an orthogonal aminoacyl tRNA synthetase (O-RS).μlTypically, the O-RS preferentially aminoacylates the O-tRNA with at least one non-naturallyμloccurring amino acid in the translation system and the O-tRNA recognizes at least one selectorμlcodon that is not recognized by other tRNAs in the system. The translation system thus inserts theμlnon-naturally-encoded amino acid into a protein produced in the system, in response to an encodedμlselector codon, thereby "substituting" an amino acid into a position in the encoded polypeptide.μl[166] A wide variety of orthogonal tRNAs and aminoacyl tRNA synthetases have been
described in the art for inserting particular synthetic amino acids into polypeptides, and areμlgenerally suitable for use in the present invention. For example, keto-specific O-tRNA/aminoacyl-μltRNA synthetases are described in Wang, L., et al, Proc. Natl Acad. Sci. USA 100:56-61 (2003)μland Zhang, Z. et al., Biochem. 42(22):6735-6746 (2003). Exemplary O-RS, or portions thereof, areμlencoded by polynucleotide sequences and include, amino acid sequences disclosed in U.S. Patent.(μlApplication Publications 2003/0082575 and 2003/0108885, each incorporated herein by reference.μlCorresponding O-tRNA molecules for use with the O-RSs are also described in U.S. Patent ,;μlApplication Publications 2003/0082575 (Serial No. 10/126,927) and 2003/0108885 (Serial No.μl10/126,931) which are incorporated by reference herein.
[167] An example of an azide-specific O-tRNA/aminoacyl-tRNA synthetase system is ,,
described in Chin, J. W., et al, J. Am. Chem. Soc. 124:9026-9027 (2002). Exemplary O-RS . [168] Several other orthogonal pairs have been reported. Glutaminyl (see, e.g., Liu, D. R.,
and Schultz, P. G. (1999) Proc. Natl. Acad. Sci. U. S. A. 96:4780-4785), aspartyl (see, e.g.,

Pastrnak, M., et al., (2000) Helv. Chim. Acta 83:2277-2286), and tyrosyl (see, e.g., Ohno, S., et al.,μl(1998) J. Biochem. (Tokyo, Jpn.) 124:1065-1068; and, Kowal, A. K., et al., (2001) Proc. Natl.μlAcad. Sci. U. S. A. 98:2268-2273) systems derived from S. cerevisiae tRNA's and synthetases haveμlbeen described for the potential incorporation of unnatural amino acids in E. coli. Systems derivedμlfrom the E. coli glutaminyl {see, e.g., Kowal, A. K., et al., (2001) Proc. Natl. Acad. Sci. U. S. A.μl98:2268-2273) and tyrosyl (see, e.g., Edwards, H., and Schimmel, P. (1990) Mol. Cell. Biol.μl10:1633-1641) synthetases have been described for use in S. cerevisiae. The E. coli tyrosyl systemμlhas been used for the incorporation of 3-iodo-L-tyrosine in vivo, in mammalian cells. See,μlSakamoto, K., et al., (2002) Nucleic Acids Res. 30:4692-4699.
[169J Use of O-tRNA/aminoacyl-tRNA synthetases involves selection of a specific codon
which encodes the non-naturally encoded amino acid. While any codon can be used, it is generallyμldesirable to select a codon that is rarely or never used in the cell in which the O-tRNA/aminoacyl-μltRNA synthetase is expressed. For example, exemplary codons include nonsense codon such as.μlstop codons (amber, ochre, and opal), four or more base codons and other natural three-base codons „μlthat are rarely or unused.
[170] Specific selector codon(s) can be introduced into appropriate positions in the hGH
polynucleotide coding sequence using mutagenesis methods known in the art (including but notμllimited to, site-specific mutagenesis, cassette mutagenesis, restriction selection mutagenesis, etc.).μl[171] Methods for generating components of the protein biosynthetic machinery, such as
O-RSs, O-tRNAs, and orthogonal O-tRNA/O-RS pairs that can be used to incorporate a non-μlnaturally encoded amino acid are described in Wang, L., et al, Science 292: 498-500 (2001); Chin,μlJ. W., et al, J. Am. Chem. Soc. 124:9026-9027 (2002); Zhang, Z. et al, Biochemistry 42: 6735-μl6746 (2003). Methods and compositions for the in vivo incorporation of non-naturally encodedμlamino acids are described in U.S. Patent Application Publication 2003/0082575 (Serial No.μl10/126,927) which is incorporated by reference herein. Methods for selecting an orthogonal tRNA-μltRNA synthetase pair for use in in vivo translation system of an organism are also described in U.S.μlPatent Application Publications 2003/0082575 (Serial No. 10/126,927) and 2003/0108885 (SerialμlNo. 10/126,931) which are incorporated by reference herein. PCT Publication No. W0 04/035743μlentitled "Site Specific Incorporation of Keto Amino Acids into Proteins," which is incorporated byμlreference herein in its entirety, describes orthogonal RS and tRNA pairs for the incorporation ofμlketo amino acids. PCT Publication No. WO 04/094593 entitled "Expanding the Eukaryotic Genetic

Code," which is incorporated by reference herein in its entirety, describes orthogonal RS and tRNAμlpairs for the incorporation of non-naturally encoded amino acids in eukaryotic host cells. Suchμlmethods are also detailed in U.S. Patent Application No. 11/046,432 entitled "Modifed HumanμlGrowth Hormone Polypeptides and Their Uses," which is incorporated by reference herein.μl[172] The organisms used in methods generating orthogonal tRNA and RS pairs comprise
a variety of organisms and a variety of combinations. For example, the first and the secondμlorganisms of the methods can be the same or different In one embodiment, the organisms areμloptionally a prokaryotic organism, including but not limited to, Methanococcus jannaschii,μlMethanobacterium thermoautotrophicum, Halobacterium, Escherichia colμAfulgidus, P.furiosus,μlP. horikoshiμA. pernix, T. thermophilic, or the like. Alternatively, the organisms optionallyμlcomprise a eukaryotic organism, including but not limited "to, plants (including but not limited to,μlcomplex plants such as monocots, or dicots), algae, protists, fungi (including but not limited to,μlyeast, etc), animals (including but not limited to, mammals, insects, arthropods, etc.), or the like. Inμlanother embodiment, the second organism is a prokaryotic organism, including but not limited to,μlMethanococcus jannaschii9 Methanobacterium thermoautotrophicwn9 Halobacterium, Escherichia )μlcolμA.fulgidus, Halobacterium, P.furiosus, P. horikoshiμA. pernix, T. thermophilus, or the like. .μlAlternatively, the second organism can be a eukaryotic organism, including but not limited to, a .. «μlyeast, a animal cell, a plant cell, a fungus, a mammalian cell, or the like. In various embodiments ."„μlthe first and second organisms are different
K Location ofnon-naturally-occurring amino acids in hGHpolypeptides
[173] The present invention contemplates incorporation of one or more non-naturally-
occurring amino acids into hGH polypeptides. One or more non-naturally-occurring amino acidsμlmay be incorporated at a particular position which does not disrupt activity of the polypeptide. Thisμlcan be achieved by making "conservative" substitutions, including but not limited to, substitutingμlhydrophobic amino acids with hydrophobic amino acids, bulky amino acids for bulky amino acids,μlhydrophilic amino acids for hydrophilic amino acids, and/or inserting the non-naturally-occurringμlamino acid in a location that is not required for activity.
[174] Regions of hGH can be illustrated as follows, wherein the amino acid positions in
hGH are indicated in the middle row (SEQ ID NO: 2 of U.S. Patent Application No. 11/046,432,μlwhich is incorporated by reference herein):


desired sites for substitution with a non-naturally encoded amino acid within the hGH polypeptide.μlIt is readily apparent to those of ordinary skill in the art that any position of the polypeptide chain isμlsuitable for selection to incorporate a non-naturally encoded amino acid, and selection may be basedμlon rational design or by random selection for any or no particular desired purpose. Selection ofμldesired sites may be for producing a hGH molecule having any desired property or activity,μlincluding but not limited to, agonists, super-agonists, inverse agonists, antagonists, receptor bindingμlmodulators, receptor activity modulators, dimer or multimer formation, no change to activity orμlproperty compared to the native molecule, or manipulating any physical or chemical property of theμlpolypeptide such as solubility, aggregation, or stability. For example, locations in the polypeptideμlrequired for biological activity of hGH polypeptides can be identified using point mutation analysis,μlalanine scanning or homolog scanning methods known in the art. See, e.g., Cunningham, B. and 'μlWells, J., Science, 244:1081-1085 (1989) (identifying 14 residues that are critical for hGHμlbioactivity) and Cunningham, B.5 et ah Science 243: 1330-1336 (1989) (identifying antibody andμlreceptor epitopes using homolog scanning mutagenesis). U.S. Patent No. 5,580,723; 5,834,250;μl6,013,478; 6,428,954; and 6,451,561, which are incorporated by reference herein, describe methodsμlfor the systematic analysis of the structure and function of polypeptides such as hGH by identifyingμlactive domains which influence the activity of the polypeptide with a target substance. Residuesμlother than those identified as critical to biological activity by alanine or homolog scanningμlmutagenesis may be good candidates for substitution with a non-naturally encoded amino acidμldepending on the desired activity sought for the polypeptide. Alternatively, the sites identified asμlcritical to biological activity may also be good candidates for substitution with a non-naturallyμlencoded amino acid, again depending on the desired activity sought for the polypeptide. Anotherμlalternative would be to simply make serial substitutions in each position on the polypeptide chainμlwith a non-naturally encoded amino acid and observe the effect on the activities of the polypeptide.μlIt is readily apparent to those of ordinary skill in the art that any means, technique, or method forμlselecting a position for substitution with a non-natural amino acid into any polypeptide is suitableμlfor use in the present invention.

[176] The structure and activity of naturally-occurring mutants of hGH polypeptides that
contain deletions can also be examined to determine regions of the protein that are likely to beμltolerant of substitution with a non-naturally encoded amino acid. See, e.g., Kostyo et al, Biochem.μlBiophys. Acta, 925: 314 (1987); Lewis, U., et al, 1 Biol Chem., 253:2679-2687 (1978) for hGH.μlIn a similar manner, protease digestion and monoclonal antibodies can be used to identify regions ofμlhGH that are responsible for binding the hGH receptor. See, e.g., Cunningham, B., et al Scienceμl243: 1330-1336 (1989); Mills, J., et al, Endocrinology, 107:391-399 (1980); LμC, Mol CellμlBiochem., 46:31-41 (1982) (indicating that amino acids between residues 134-149 can be deletedμlwithout a loss of activity). Once residues that are likely to be intolerant to substitution with non-μlnaturally encoded amino acids have been eliminated, the impact of proposed substitutions at each ofμlthe remaining positions can be examined from the three-dimensional crystal structure of the hGHμland its binding proteins. See de Vos, A., et al, Science, 255:306-312 (1992) for hGH; all crystalμlstructures of hGH are available in the Protein Data Bank (including 3HHR, 1AXμand 1HWG)μl(PDB, available on the World Wide Web at rcsb.org), a centralized database containing three- .μldimensional structural data of large molecules of proteins and nucleic acids. Thus, those of skill in ;iμlthe art can readily identify amino acid positions that can be substituted with non-naturally encoded μlamino acids.
[177J In some embodiments, the hGH polypeptides of the invention comprise one or more '
non-naturally occurring amino acids positioned in a region of the protein that does not disrupt the vμlhelices or beta sheet secondary structure of the polypeptide.
[178] Exemplary residues of incorporation of a non-naturally encoded amino acid may be
those that are excluded from potential receptor binding regions (including but not limited to, Site Iμland Site II), may be fully or partially solvent exposed, have minimal or no hydrogen-bondingμlinteractions with nearby residues, may be minimally exposed to nearby reactive residues, and mayμlbe in regions that are highly flexible (including but not limited to, C-D loop) or structurally rigidμl(including but not limited to, B helix) as predicted by the three-dimensional crystal structure,μlsecondary, tertiary or quaternary structure of the hGH polypeptide bound or unbound to its receptor.μl[179J In some embodiments, one or more non-naturally encoded amino acids are
incorporated at any position in one or more of the following regions corresponding to secondaryμlstructures in hGH as follows: positions corresponding to 1-5 (N-terminus), 6-33 (A helix), 34-74μl(region between A helix and B helix, the A-B loop), 75-96 (B helix), 97-105 (region between B

helix and C helix, the B-C loop), 106-129 (C helix), 130-153 (region between C helix and D helix,μlthe C-D loop), 154-183 (D helix), 184-191 (C-terminus) from SEQ ID NO: 2. In otherμlembodiments, GH polypeptides, e.g., hGH polypeptides of the invention comprise at least one non-μlnaturally-occurring amino acid substituted for at least one amino acid located in at least one regionμlof GH, e.g., hGH selected from the group consisting regions corresponding to the N-terminus (1-5),μlthe N-terminal end of the A-B loop (32-46); the B-C loop (97-105), the C-D loop (132-149), and theμlC-terminus (184-191) of SEQ ID NO: 2. In some embodiments, one or more non-naturally encodedμlamino acids are incorporated at one or more of the following positions of GH, e.g., hGHμlcorresponding to: before position 1 (i.e. at the N-terminus), 1, 2, 3, 4, 5, 8, 9, 11, 12, 15, 16, 19, 22,μl29, 30, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 52, 55, 57, 59, 65, 66,μl69, 70, 71, 74, 88, 91, 92, 94, 95, 97, 98, 99,100, 101, 102, 103,104, 105, 106, 107, 108, 109, 111,μl112, 113, 115, 116, 119, 120, 122, 123, 126, 127, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138,
139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 158, 159, /.μl161, 168, 172, 183, 184, 185, 186,187, 188, 189,190,191, 192 (i.e., at the carboxyl terminus of theμlprotein) of SEQ ID NO: 2 or the corresponding amino acids of SEQ ED NO: 1 or 3. Vμl[180] Exemplary sites of incorporation of one or more non-naturally encoded amino acidsμlinclude sites corresponding to 29, 30, 33, 34, 35, 37, 39, 40, 49, 57, 59, 66, 69, 70, 71, 74, 88, 91,μl92, 94, 95, 98, 99, 101, 103, 107, 108, 111, 122, 126, 129, 130, 131, 133, 134, 135, 136, 137, 139, .
140, 141, 142, 143, 145, 147; 154, 155, 156, 159, 183, 186, and 187, or any combination thereof .μlfrom SEQ ED NO: 2 or the corresponding amino acids of SEQ ID NO: 1 or 3.
[181] A subset of exemplary sites for incorporation of one or more non-naturally encoded
amino acid include sites corresponding to 29, 33, 35, 37, 39, 49, 57, 69, 70, 71, 74, 88, 91, 92, 94,μl95, 98, 99, 101, 103, 107, 108, 111, 129, 130, 131, 133, 134, 135, 136, 137, 139, 140, 141, 142,μl143, 145, 147, 154, 155, 156, 186, and 187, or any combination thereof from SEQ ID NO: 2 or theμlcorresponding amino acids of SEQ ID NO: 1 or 3. An examination of the crystal structure of GH,μle.g., hGH and its interactions with the GH, e.g., hGH receptor indicates that the side chains of theseμlamino acid residues are fully or partially accessible to solvent and the side chain of a non-naturallyμlencoded amino acid may point away from the protein surface and out into the solvent.μl[182] Exemplary positions for incorporation of one or more non-naturally encoded amino
acids include sites corresponding to 35, 88, 91, 92, 94, 95, 99, 101, 103, 111, 131, 133, 134, 135,μl136, 139, 140, 143, 145, and 155, or any combination thereof from SEQ ID NO: 2 or the

corresponding amino acids of SEQ ID NO: 1 or 3. An examination of the crystal structure of GH,μle.g., hGH and its interactions with the GH, e.g., hGH receptor indicates that the side chains of theseμlamino acid residues are fully exposed to the solvent and the side chain of the native residue pointsμlout into the solvent.
[183] A subset of exemplary sites for incorporation of one or more non-naturally encoded
amino acids include sites corresponding to 30, 74, 103, or any combination thereof, from SEQ IDμlNO: 2 or the corresponding amino acids of SEQ ID NO: 1 or 3. Another subset of exemplary sitesμlfor incorporation of one or more non-naturally encoded amino acids include sites corresponding toμl35,92,143,145, or any combination thereof, from SEQ ID NO: 2 or the corresponding amino acidsμlof SEQ ID NO: 1 or 3. A further subset of exemplary sites for incorporation of one or more non-μlnaturally encoded amino acids include sites corresponding to 35, 92, 131, 134, 143, 145, or anyμlcombination thereof, from SEQ ID NO: 2 or the corresponding amino acids of SEQ ID NO: 1 or 3.μlStill a further subset of exemplary sites for incorporation of one or more non-naturally encoded .μlamino acids include sites corresponding to 30, 35, 74, 92, 103, 145, or any combination thereof, ..μlfrom SEQ ID NO: 2 or the corresponding amino acids of SEQ ID NO: 1 or 3. Yet a further subset -.μlof exemplary sites for incorporation of one or more non-naturally encoded amino acids include sitesμlcorresponding to 35, 92, 143, 145, or any combination thereof from SEQ ID NO: 2 or theμlcorresponding amino acids of SEQ ID NO: 1 or 3. In certain embodiments, sites for incorporationμlof one or more non-naturally encoded amino acids include a site corresponding to 35 from SEQ ID ;»'μlNO: 2 or the corresponding amino acids of SEQ ID NO: 1 or 3.
[184] In some embodiments, at least one of the non-naturally encoded amino acids
incorporated into the GH, e.g., hGH, contains a carbonyl group, e.g., a ketone group. In certainμlembodiments, at least one of the non-naturally encoded amino acids incorporated into the GH, e.g.,μlhGH is para-acetylphenylalanine. In some embodiments in which the GH, e.g., hGH contains aμlplurality of non-naturally-encoded amino acids, more than one of the non-naturally-encoded aminoμlacids incorporated into the GH, e.g., hGH is para-acetylphenylalanine. In some embodiments inμlwhich the GH, e.g., hGH contains a plurality of non-naturally-encoded amino acids, substantially allμlof the non-naturally-encoded amino acids incorporated into the GH, e.g., hGH are para-μlacetylphenylalanine.
[185] In some embodiments, the non-naturally occurring amino acid is linked to a water
soluble polymer at one or more positions, including but not limited to, positions corresponding to:

before position 1 (i.e. at the N-terminus), 1, 2, 3, 4, 5, 8, 9, 11, 12, 15, 16, 19, 22, 29, 30, 32, 33, 34,μl35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 52, 55, 57, 59, 65, 66, 69, 70, 71, 74, 88,μl91, 92, 94, 95, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 111, 112, 113, 115,μl116, 119, 120, 122, 123, 126, 127, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141,μl142,143, 144, 145,146, 147, 148, 149, 150, 151, 152,153, 154,155, 156, 158,159,161, 168, 172,μl183,184,185, 186,187, 188, 189,190,191, 192 (i.e., at the carboxyl terminus of the protein) (SEQμlID NO: 2 or the corresponding amino acids of SEQ ID NO: 1 or 3). In some embodiments, the non-μlnaturally occurring amino acid is linked to a water soluble polymer at positions including but notμllimited to, positions corresponding to one or more of these positions: 30, 35, 74, 92, 103, 143, 145μl(SEQ ED NO: 2 or the corresponding amino acids of SEQ ID NO: 1 or 3). In some embodiments,μlthe non-naturally occurring amino acid is linked to a water soluble polymer at positions includingμlbut not limited to, positions corresponding to one or more of these positions: 35, 92, 143, 145 (SEQμlID NO: 2 or the corresponding amino acids of SEQ ID NO: 1 or 3). In some embodiments, the non- vμlnaturally occurring amino acid is linked to a water soluble polymer at positions including but not ;μllimited to, positions corresponding to one or more of these positions: 35, 92, 131, 134, 143, 145, or >μlany combination thereof, from SEQ ID NO: 2 or the corresponding amino acids of JSEQ ID NO: 1 .μlor 3. In some embodiments, the non-naturally occurring amino acid is linked to a water soluble ,μlpolymer at positions including but not limited to, positions corresponding to one or more of these,'μlpositions: 30, 35, 74, 92, 103, 145, or any combination thereof, from SEQ ID NO: 2 or the ,μl'corresponding amino acids of SEQ ID NO: 1 or 3. In some embodiments, the non-naturallyμloccurring amino acid is linked to a water soluble polymer at positions including but not limited to,μlpositions corresponding to one or more of these positions: 35, 92, 143, 145, or any combinationμlthereof, from SEQ ID NO: 2 or the corresponding amino acids of SEQ ID NO: 1 or 3. In someμlembodiments, the non-naturally occurring amino acid is linked to a water-soluble polymer at aμlposition corresponding to, but not limited to, position 35 from SEQ ID NO: 2 or the correspondingμlamino acids of SEQ ID NO: 1 or 3 is linked to a water-soluble polymer.
[186] In some embodiments the water-soluble polymer(s) linked to the GH, e.g., hGH,
include one or more polyethylene glycol molecules (PEGs). The polymer, e.g., PEG, may be linearμlor branched. Typically, linear polymers, e.g., PEGs, used in the invention can have a MW of aboutμl0.1 to about 100 kDa, or about 1 to about 60 kDa, or about 20 to about 40 kDa, or about 30 kDa.μlTypically, branched polymers, e.g., PEGs, used in the invention can have a MW of about 1 to about

100 kDa, or about 30 to about 50 kDa, or about 40 kDa. Polymers such as PEGs are describedμlfurther herein. In certain embodiments, the linkage between the GH, e.g., hGH and the water-μlsoluble polymer, e.g., PEG, is an oxime bond.
[187] Certain embodiments of the invention encompass compositions that include a GH,
e.g., hGH, linked to at least one water-soluble polymer by a covalent bond, where the covalent bondμlis an oxime bond. In some embodiments, the water-soluble polymer is a PEG, e.g., a linear PEG.μlIn some embodiments encompassing at least one linear PEG linked by an oxime bond to a GH, e.g.,μlhGH, the PEG can have a MW of about 0.1 to about 100 kDa, or about 1 to about 60 kDa, or aboutμl20 to about 40 kDa, or about 30 kDa. In certain embodiments encompassing a linear PEG linked byμlan oxime bond to a GH, e.g., hGH, the PEG has a MW of about 30 kDa. In some embodimentsμlencompassing at least one branched PEG linked by an oxime bond to a GH, e.g., hGH, the PEG canμlhave a MW of about 1 to about 100 kDa or about 30 to about 50 kDa, or about 40 kDa. In certainμlembodiments encompassing a branched PEG linked by an oxime bond to a GH, e.g., hGH, the PEG .μlhas a MW of about 40 kDa. In some embodiments, the GH is a GH, e.g., hGH and in certain of ;>μlthese embodiments, the GH, e.g., hGH has a sequence that is at least about 80% identical to SEQ ID μlNO: 2; in some embodiments the GH, e.g., hGH has a sequence that is the sequence of SEQ ID NO:μl2. In some embodiments, the GH, e.g., hGH, contains at least one non-naturally-encoded amino .μlacid; in some of these embodiments, at least one oxime bond is between the non-naturally-encoded .μlamino acid and at least one water-soluble polymer. In some embodiments, the non-naturally- ,μlencoded amino acid contains a carbonyl group, such as a ketone group; in some embodiments, the.μlnon-naturally-encoded amino acid is para-acetylphenylalanine. In some embodiments, the para-μlacetylphenylalanine is substituted at a position corresponding to position 35 of SEQ ID NO: 2.μl[188] Thus, in some embodiments, the invention provides a GH, e.g., hGH, linked to at
least one water-soluble polymer, e.g., a PEG, by a covalent bond, where the covalent bond is anμloxime bond. In certain embodiments, the water-soluble polymer is a PEG and the PEG is a linearμlPEG. In these embodiments, the linear PEG has a MW of about 0,1 to about 100 kDa, or about 1 toμlabout 60 kDa, or about 20 to about 40 kDa, or about 30 kDa. In certain embodiments encompassingμla linear PEG linked by an oxime bond to a GH, e.g., hGH, the PEG has a MW of about 30 kDa. Inμlcertain embodiments, the water-soluble polymer is a PEG that is a branched PEG. In theseμlembodiments, the branched PEG has a MW of about 1 to about 100 kDa, or about 30 to about 50

kDa, or about 40 kDa. In certain embodiments encompassing a branched PEG linked by an oximeμlbond to a GH, e.g., hGH3 the PEG has a MW of about 40 kDa.
[189] In some embodiments, the invention provides a GH, e.g., hGH, where the GH, e.g.,
hGH contains a non-naturally encoded amino acid, where the GH is linked to at least one water-μlsoluble polymer, e.g., a PEG, by a covalent bond, and where the covalent bond is an oxime bondμlbetween the non-naturally encoded amino acid and the water-soluble polymer, e.g., PEG. In someμlembodiments, the non-naturally-encoded amino acid is incorporated into the GH, e.g., hGH, at aμlposition corresponding to position 35 of SEQ ID NO: 2. In certain embodiments where the water-μlsoluble polymer is a PEG, the PEG is a linear PEG. In these embodiments, the linear PEG has aμlMW of about 0.1 to about 100 kDa, or about 1 to about 60 kDa, or about 20 to about 40 kDa, orμlabout 30 kDa. In certain embodiments encompassing a linear PEG linked by an oxime bond to aμlGH, e.g., hGH, the PEG has a MW of about 30 kDa. In certain embodiments where the water-μlsoluble polymer is a PEG, the PEG is a branched PEG. In these embodiments, the branched PEGμlhas a MW of about 1 to about 100 kDa, or about 30 to about 50 kDa, or about 40 kDa. In certain μlembodiments encompassing a branched PEG linked by an oxime bond to a GH, e.g., hGH, the PEG rμlhas a MW of about 40 kDa.
[190] In some embodiments, the invention provides a GH, e.g., hGH, where the GH, e.g.,,
hGH contains a non-naturally encoded amino acid that is a carbonyl-containing non-naturally:.μlencoded amino acid, where the GH is linked to at least one water-soluble polymer, e.g., a PEG, by a μlcovalent bond, and where the covalent bond is an oxime bond between the non-naturally encodedμlcarbonyl-containing amino acid and the water-soluble polymer, e.g., PEG. In some embodiments,μlthe non-naturally-encoded carbonyl-containing amino acid is incorporated into the GH, e.g., hGH,μlat a position corresponding to position 35 of SEQ ID NO: 2. In certain embodiments where theμlwater-soluble polymer is a PEG, the PEG is a linear PEG. In these embodiments, the linear PEGμlhas a MW of about 0.1 to about 100 kDa, or about 1 to about 60 kDa, or about 20 to about 40 kDa,μlor about 30 kDa. In certain embodiments encompassing a linear PEG linked by an oxime bond to aμlGH, e.g., hGH, the PEG has a MW of about 30 kDa. In certain embodiments where the water-μlsoluble polymer is a PEG, the PEG is a branched PEG. hi these embodiments, the branched PEGμlhas a MW of about 1 to about 100 kDa, or about 30 to about 50 kDa, or about 40 kDa. In certainμlembodiments encompassing a branched PEG linked by an oxime bond to a GH, e.g., hGH, the PEGμlhas a MW of about 40 kDa.

[191] In some embodiments, the invention provides a GH, e.g., hGH, that contains a non-
naturally encoded amino acid that includes a ketone group, where the GH is linked to at least oneμlwater-soluble polymer, e.g., a PEG, by a covalent bond, and where the covalent bond is an oximeμlbond between the non-naturally encoded amino acid containing a ketone group and the water-μlsoluble polymer, e.g., PEG. In some embodiments, the non-naturally-encoded amino acidμlcontaining a ketone group is incorporated into the GH, e.g., hGH, at a position corresponding toμlposition 35 of SEQ ID NO: 2. In certain embodiments where the water-soluble polymer is a PEG,μlthe PEG is a linear PEG. In these embodiments, the linear PEG has a MW of about 0.1 to aboutμl100 kDa, or about 1 to about 60 kDa, or about 20 to about 40 kDa, or about 30 kDa. In certainμlembodiments encompassing a linear PEG linked by an oxime bond to a GH, e.g., hGH, the PEG hasμla MW of about 30 kDa. In certain embodiments where the water-soluble polymer is a PEG, theμlPEG is a branched PEG. In these embodiments, the branched PEG has a MW of about 1 to aboutμl100 kDa, or about 30 to about 50 kDa, or about 40 kDa. In certain embodiments encompassing a 'μlbranched PEG linked by an oxime bond to a GH, e.g., hGH, the PEG has a MW of about 40 kDa..».μl[192] In some embodiments, the invention provides a GH, e.g., hGH, that contains a non- }
naturally encoded amino acid that is a para-acetylphenylalanine, where the GH linked to at least oneμlwater-soluble polymer, e.g., a PEG, by a covalent bond, and where the covalent bond is an oxime μlbond between the para-acetylphenylalanine and the water-soluble polymer, e.g., PEG. In some μlembodiments, the para-acetylphenylalanine is incorporated into the GH, e.g., hGH, at a position .:;μlcorresponding to position 35 of SEQ ID NO: 2. In certain embodiments where the water-solubleμlpolymer is a PEG, the PEG is a linear PEG. In these embodiments, the linear PEG has a MW ofμlabout 0.1 to about 100 kDa, or about 1 to about 60 kDa, or about 20 to about 40 kDa, or about 30μlkDa. In certain embodiments encompassing a linear PEG linked by an oxime bond to a GH, e.g.,μlhGH, the PEG has a MW of about 30 kDa. In certain embodiments where the water-solubleμlpolymer is a PEG, the PEG is a branched PEG. In these embodiments, the branched PEG has aμlMW of about 1 to about 100 kDa, or about 30 to about 50 kDa, or about 40 kDa. In certainμlembodiments encompassing a branched PEG linked by an oxime bond to a GH, e.g., hGH, the PEGμlhas a MW of about 40 kDa.
[193] In certain embodiments the invention provides a GH, e.g., hGH that includes SEQ ID
NO: 2, and where the GH, e.g., hGH is substituted at a position corresponding to position 35 of

SEQ ID NO: 2 with a para-acetylphenylalanine that is linked by an oxime linkage to a linear PEGμlof MW of about 30 kDa.
[194] In some embodiments, the invention provides a hormone composition that includes a
GH, e.g., hGH, linked via an oxime bond to at least one PEG, e.g., a linear PEG, where the GH,μle.g., hGH comprises the amino acid sequence of SEQ ID NO: 2, and where the GH, e.g., hGHμlcontains at least one non-naturally-encoded amino acid substituted at one or more positionsμlincluding, but not limited to, positions corresponding to: before position 1 (i.e. at the N-terminus),μl1, 2, 3, 4, 5, 8, 9,11, 12, 15, 16, 19, 22, 29, 30, 32,33, 34, 35, 36, 37, 38, 39,40,41,42,43, 44, 45,μl465 47, 48, 49, 52, 55, 57, 59, 65, 66, 69, 70, 71, 74, 88, 91, 92, 94, 95, 97, 98, 99, 100, 101, 102,μl103, 104, 105, 106, 107, 108, 109, 111, 112, 113, 115, 116, 119, 120, 122, 123, 126,127, 129, 130,μl131, 132, 133, 134, 135, 136,137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147,148, 149, 150,μl151, 152, 153, 154, 155, 156, 158, 159, 161, 168, 172, 183, 184, 185, 186, 187,188, 189, 190, 191,μl192 (i.e., at the caiboxyl terminus of the protein) (SEQ ID NO: 2 or the corresponding amino acids ?μlof SEQ ID NO: 1 or 3). In some embodiments, the invention provides a hormone composition that iμlincludes a GH, e.g., hGH, linked via an oxime bond to at least one PEG, e.g., a linear PEG, where iμlthe GH, e.g., hGH comprises the amino acid sequence of SEQ ID NO: 2, and where the GH, e.g.,μlhGH contains at least one non-naturally-encoded amino acid substituted at one or more positionsμlincluding, but not limited to, positions corresponding to: 30, 35, 74, 92, 103, 143, 145 (SEQ ID NO: μl2 or die corresponding amino acids of SEQ ID NO: 1 or 3). In some embodiments, the inventionμlprovides a hormone composition that includes a GH, e.g., hGH, linked via an oxime bond to at leastμlone PEG, e.g., a linear PEG, where the GH, e.g., hGH comprises the amino acid sequence of SEQμlID NO: 2, and where the GH, e.g., hGH contains at least one non-naturally-encoded amino acidμlsubstituted at one or more positions including, but not limited to, positions corresponding to: 35, 92,μl143, 145 (SEQ ID NO: 2 or the corresponding amino acids of SEQ ID NO: 1 or 3). In someμlembodiments, the invention provides a hormone composition that includes a GH, e.g., hGH, linkedμlvia an oxime bond to at least one PEG, e.g., a linear PEG, where the GH, e.g., hGH comprises theμlamino acid sequence of SEQ ID NO: 2, and where the GH, e.g., hGH contains at least one non-μlnaturally-encoded amino acid substituted at one or more positions including, but not limited to,μlpositions corresponding to: 35, 92, 131, 134, 143, 145, or any combination thereof, from SEQ IDμlNO: 2 or the corresponding amino acids of SEQ ID NO: 1 or 3. In some embodiments, theμlinvention provides a hormone composition that includes a GH, e.g., hGH, linked via an oxime bond

to at least one PEG, e.g., a linear PEG, where the GH, e.g., hGH comprises the amino acid sequenceμlof SEQ ID NO: 2, and where the GH, e.g., hGH contains at least one non-naturally-encoded aminoμlacid substituted at one or more positions including, but not limited to, positions corresponding to;μl30, 35, 74, 92, 103, 145, or any combination thereof, from SEQ ED NO: 2 or the correspondingμlamino acids of SEQ ED NO: 1 or 3. In some embodiments, the invention provides a hormoneμlcomposition that includes a GH, e.g., hGH, linked via an oxime bond to at least one PEG, e.g., aμllinear PEG, where the GH, e.g., hGH comprises the amino acid sequence of SEQ ED NO: 2, andμlwhere the GH, e.g., hGH contains at least one non-naturally-encoded amino acid substituted at oneμlor more positions including, but not limited to, positions corresponding to: 35, 92, 143, 145, or anyμlcombination thereof, from SEQ ED NO: 2 or the corresponding amino acids of SEQ ED NO: 1 or 3.μlIn some embodiments, the invention provides a hormone composition that includes a GH, e.g.,μlhGH, linked via an oxime bond to at least one PEG, e.g., a linear PEG, where the GH, e.g., hGHμlcomprises the amino acid sequence of SEQ ID NO: 2, and where the GH, e.g., hGH contains at least -μlone non-naturally-encoded amino acid substituted at one or more positions including, but not ;:μllimited to, positions corresponding to position 35 from SEQ ID NO: 2 or the corresponding amino .'-.μlacids of SEQ ID NO: 1 or 3. In embodiments in which the PEG is a linear PEG, the PEG can haveμla MW of about 0.1 to about 100 kDa, or about 1 to about 60 kDa, or about 20 to about 40 kDa, orμlabout 30 kDa.
[195] hi some embodiments, the invention provides a hormone composition that includes a
GH, e.g., hGH, linked via an oxime bond to at least one PEG, e.g., a linear PEG, where the GH,μle.g., hGH includes the amino acid sequence of SEQ ID NO: 2, and where the GH, e.g., hGHμlcontains at least one non-naturally-encoded amino acid that is a para-acetylphenylalanineμlsubstituted at one or more positions including, but not limited to, positions corresponding to: beforeμlposition 1 (i.e. at the N-terminus), 1,2,3,4,5,8,9,11,12, 15, 16, 19,22,29,30,32,33,34,35,36,μl37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 52, 55, 57, 59, 65, 66, 69, 70, 71, 74, 88, 91, 92,μl94, 95, 97, 98, 99, 100,101, 102, 103,104, 105, 106, 107, 108, 109, 111, 112, 113, 115, 116, 119,μl120, 122, 123,126, 127,129,130, 131,132, 133, 134,135, 136, 137, 138, 139, 140, 141, 142, 143,μl144, 145, 146,147, 148,149,150, 151,152, 153, 154,155, 156, 158, 159, 161, 168, 172, 183, 184,μl185, 186, 187,188,189,190,191,192 (i.e., at the carboxyl terminus of the protein) (SEQ ID NO: 2μlor the corresponding amino acids of SEQ ID NO: 1 or 3). In some embodiments, the inventionμlprovides a hormone composition that includes a GH, e.g., hGH, linked via an oxime bond to at least

one PEG, e.g., a linear PEG, where the GH, e.g., hGH comprises the amino acid sequence of SEQμlID NO: 2, and where the GH, e.g., hGH contains at least one non-naturally-encoded amino acid thatμlis a para-acetylphenylalanine substituted at one or more positions including, but not limited to,μlpositions corresponding to: 30, 35, 74, 92, 103, 143, 145 (SEQ ID NO: 2 or the correspondingμlamino acids of SEQ ID NO: 1 or 3). In some embodiments, the invention provides a hormoneμlcomposition that includes a GH, e.g., hGH, linked via an oxime bond to at least one PEG, e.g., aμllinear PEG, where the GH, e.g., hGH comprises the amino acid sequence of SEQ ID NO: 2, andμlwhere the GH, e.g., hGH contains at least one non-naturally-encoded amino acid that is a para-μlacetylphenylalanine substituted at one or more positions including, but not limited to, positionsμlcorresponding to: 35, 92, 143, 145 (SEQ ID NO: 2 or the corresponding amino acids of SEQ IDμlNO: 1 or 3). In some embodiments, the invention provides a hormone composition that includes aμlGH, e.g., hGH, linked via an oxime bond to at least one PEG, e.g., a linear PEG, where the GH,μle.g., hGH comprises the amino acid sequence of SEQ ID NO: 2, and where the GH, e.g., hGHvμlcontains at least one non-naturally-encoded amino acid that is a para-acetylphenylalanine μlsubstituted at one or more positions including, but not limited to, positions corresponding to: 35, 92, rf. .μl131, 134, 143, 145, or any combination thereof from SEQ ID NO: 2 or the corresponding amino....μlacids of SEQ ID NO: 1 or 3. In some embodiments, the invention provides a hormone composition ,μlthat includes a GH, e.g., hGH, linked via an oxime bond to at least one PEG, e.g., a linear PEG, ,μlwhere the GH, e.g., hGH comprises the amino acid sequence of SEQ ID NO: 2, and where the GH,μle.g., hGH contains at least one non-naturally-encoded amino acid that is a para-acetylphenylalanineμlsubstituted at one or more positions including, but not limited to, positions corresponding to: 30, 35,μl74, 92, 103, 145, or any combination thereof, from SEQ ID NO: 2 or the corresponding amino acidsμlof SEQ ED NO: 1 or 3. In some embodiments, the invention provides a hormone composition thatμlincludes a GH, e.g., hGH, linked via an oxime bond to at least one PEG, e.g., a linear PEG, whereμlthe GH, e.g., hGH comprises the amino acid sequence of SEQ ID NO: 2, and where the GH, e.g.,μlhGH contains at least one non-naturally-encoded amino acid that is a para-acetylphenylalanineμlsubstituted at one or more positions including, but not limited to, positions corresponding to: 35, 92,μl143, 145, or any combination thereof, from SEQ ID NO: 2 or the corresponding amino acids ofμlSEQ ED NO: 1 or 3. In some embodiments, the invention provides a hormone composition thatμlincludes a GH, e.g., hGH, linked via an oxime bond to at least one PEG, e.g., a linear PEG, whereμlthe GH, e.g., hGH comprises the amino acid sequence of SEQ ID NO: 2, and where the GH, e.g.,

hGH contains at least one non-naturally-encoded amino acid that is a para-acetylphenylalanineμlsubstituted at one or more positions including, but not limited to, positions corresponding toμlposition 35 from SEQ ID NO: 2 or the corresponding amino acids of SEQ ID NO: 1 or 3. Inμlembodiments in which the PEG is a linear PEG, the PEG can have a MW of about 0.1 to about 100μlkDa, or about 1 to about 60 kDa, or about 20 to about 40 kDa, or about 30 kDa.μl[196] In some embodiments, the invention provides a GH, e.g., hGH, where the GH, e.g.,
hGH contains at least one non-naturally encoded amino acid, where the GH is linked to a pluralityμlof water-soluble polymers, e.g., a plurality of PEGs, by covalent bonds, where one or more of theμlcovalent bond is an oxime bond between at least one of the non-naturally encoded amino acid andμlthe water-soluble polymer, e.g., PEG. The GH, e.g., hGH, may be linked to about 2-100 water-μlsoluble polymers, e.g., PEGs, or about 2-50 water-soluble polymers, e.g., PEGs, or about 2-25μlwater-soluble polymers, e.g., PEGs, or about 2-10 water-soluble polymers, e.g., PEGs, or about 2-5μlwater-soluble polymers, e.g., PEGs, or about 5-100 water-soluble polymers, e.g., PEGs, or about 5- '.μl50 water-soluble polymers, e.g., PEGs, or about 5-25 water-soluble polymers, e.g., PEGs, or about ...»μl5-10 water-soluble polymers, e.g., PEGs, or about 10-100 water-soluble polymers, e.g., PEGs, or μlabout 10-50 water-soluble polymers, e.g., PEGs, or about 10-20 water-soluble polymers, e.g., μlPEGs, or about 20-100 water-soluble polymers, e.g., PEGs, or about 20-50 water-soluble polymers, -μle.g., PEGs, or about 50-100 water-soluble polymers, e.g., PEGs. The one or more non-naturally- .μlencoded amino acids may be incorporated into the GH, e.g., hGH, at any position described herein. μlIn some embodiments, at least one non-naturally-encoded amino acid is incorporated into the GH, -μle.g., hGH, at a position corresponding to position 35 of SEQ ID NO: 2. In some embodiments, theμlnon-naturally encoded amino acids include at least one non-naturally encoded amino acid that is aμlcarbonyl-containing non-naturally encoded amino acid, e.g., a ketone-containing non-naturallyμlencoded amino acid such as a para-acetylphenylalanine. In some embodiments, the GH, e.g., hGH,μlincludes a para-acetylphenylalanine. In some embodiments, the para-acetylphenylalanine isμlincorporated into the GH, e.g., hGH, at a position corresponding to position 35 of SEQ ED NO: 2,μlwhere the para-acetylphenylalanine is linked to one of the polymers, e.g., one of the PEGs, by anμloxime bond. In some embodiments, at least one of the water-soluble polymers, e.g., PEGs, is linkedμlto the GH, e.g., hGH, by a covalent bond to at least one of the non-naturally-encoded amino acids.μlIn some embodiments, the covalent bond is an oxime bond, hi some embodiments, a plurality ofμlthe water-soluble polymers, e.g., PEGs, are linked to the GH, e.g., hGH, by covalent bonds to a

plurality of the non-naturally-encoded amino acids. In some embodiments, at least one the covalentμlbonds is an oxime bond; in some embodiments, a plurality of the covalent bonds are oxime bonds;μlin some embodiments, substantially all of the bonds are oxime bonds. The plurality of water-μlsoluble polymers, e.g., PEG, may be linear, branched, or any combination thereof. In embodimentsμlthat incorporate one or more linear PEGs, the linear PEGs have a MW of about 0.1 to about 100μlkDa, or about 1 to about 60 kDa, or about 20 to about 40 fcDa, or about 30 fcDa. In embodimentsμlthat incorporate one or more branched PEGs, the branched PEGs have a MW of about 1 to aboutμl100 kDa, or about 30 to about 50 kDa, or about 40 kDa. It will be appreciated that embodimentsμlemploying a plurality of water-soluble polymers, e.g., PEGs, will, in general, employ such polymersμlat lower MWs than embodiments in which a single PEG is used. Thus, in some embodiments, theμloverall MW of the plurality of PEGs is about 0.1-500 kDa, or about 0.1-200 kDa, or about 0.1-100μlkDa, or about 1-1000 kDa, or about 1-500 kDa, or about 1-200 kDa, or about 1-100 kDa, or aboutμl10-1000 kDa, or about 10-500 kDa, or about 10-200 kDa, or about 10-100 kDa, or about 10-50 .μlkDa, or about 20-1000 kDa, or about 20-500 kDa, or about 20-200 kDa, or about 20-100 kDa, orμlabout 20-80 kDa, about 20-60 kDa, about 5-100kDa, about 5-50 kDa, or about 5-20 kDa.μl[197] Human GH antagonists include, but are not limited to, those with substitutions at: 1,
2, 3, 4, 5, 8, 9, 11, 12, 15, 16, 19, 22, 103, 109, 112, 113, 115, 116, 119, 120, 123, and 127 or anμladdition at position 1 (i.e., at the N-terminus), or any combination thereof (SEQ ID NO:2, or theμlcorresponding amino acid in SEQ ID NO: 1,3, or any other GH sequence).
[198] A wide variety of non-naturally encoded amino acids can be substituted for, or
incorporated into, a given position in a hGH polypeptide. In general, a particular non-naturallyμlencoded amino acid is selected for incorporation based on an examination of the three dimensionalμlcrystal structure of a hGH polypeptide with its receptor, a preference for conservative substitutionsμl(i.e., aryl-based non-naturally encoded amino acids, such as p-acetylphenylalanine or O-μlpropargyltyrosine substituting for Phe, Tyr or Trp), and the specific conjugation chemistry that oneμldesires to introduce into the hGH polypeptide (e.g., the introduction of 4-azidophenylalanme if oneμlwants to effect a Huisgen [3+2] cycloaddition with a water soluble polymer bearing an alkyneμlmoiety or a amide bond formation with a water soluble polymer that bears an aryl ester that, in turn,μlincorporates a phosphine moiety).
[199] In one embodiment, the method further includes incorporating into the protein the
unnatural amino acid, where the unnatural amino acid comprises a first reactive group; and

contacting the protein with a molecule (including but not limited to, a label, a dye, a polymer, aμlwater-soluble polymer, a derivative of polyethylene glycol, a photocrosslinker, a radionuclide, aμlcytotoxic compound, a drug, an affinity label, a photoaffinity label, a reactive compound, a resin, aμlsecond protein or polypeptide or polypeptide analog, an antibody or antibody fragment, a metalμlchelator, a cofactor, a fatty acid, a carbohydrate, a polynucleotide, a DNA, a RNA, an antisenseμlpolynucleotide, a saccharide, a water-soluble dendrimer, a cyclodextrin, an inhibitory ribonucleicμlacid, a biomaterial, a nanoparticle, a spin label, a fluorophore, a metal-containing moiety, aμlradioactive moiety, a novel functional group, a group that covalently or noncovalently interacts withμlother molecules, a photocaged moiety, an actinic radiation excitable moiety, a photoisomerizableμlmoiety, biotin, a derivative of biotin, a biotin analogue, a moiety incorporating a heavy atom, aμlchemically cleavable group, a photocleavable group, an elongated side chain, a carbon-linked sugar,μla redox-active agent, an amino thioacid, a toxic moiety, an isotopically labeled moiety, aμlbiophysical probe, a phosphorescent group, a chemiluminescent group, an electron dense group, a 'tμlmagnetic group, an intercalating group, a chromophore, an energy transfer agent, a biologicallyμlactive agent, a detectable label, a small molecule, a quantum dot, a nanotransmitter, a )μlradionucleotide, a radiotransmitter, a neutron-capture agent, or any combination of the above, or l-μlany other desirable compound or substance that comprises a second reactive group.μl[200] In some cases, the non-naturally encoded amino acid substitution(s) will be '
combined with other additions, substitutions or deletions within the hGH polypeptide to affect other ';μlbiological traits of the hGH polypeptide. In some cases, the other additions, substitutions orμldeletions may increase the stability (including but not limited to, resistance to proteolyticμldegradation) of the hGH polypeptide or increase affinity of the hGH polypeptide for its receptor. Inμlsome cases, the other additions, substitutions or deletions may increase the solubility (including butμlnot limited to, when expressed in E. coli or other host cells) of the hGH polypeptide. In someμlembodiments additions, substitutions or deletions may increase the polypeptide solubility followingμlexpression in E. coli or other recombinant host cells. In some embodiments sites are selected forμlsubstitution with a naturally encoded or non-natural amino acid in addition to another site forμlincorporation of a non-natural amino acid that results in increasing the polypeptide solubilityμlfollowing expression in E. coli or other recombinant host cells, hi some embodiments, the hGHμlpolypeptides comprise another addition, substitution or deletion that modulates affinity for the hGHμlpolypeptide receptor, modulates (including but not limited to, increases or decreases) receptor

dimerization, stabilizes receptor dimers, modulates circulating half-life, modulates release or bio-μlavailabilty, facilitates purification, or improves or alters a particular route of administration. Aμlnumber of such alterations are described in U.S. Patent Application No. 11/046,432 entitledμl"Modifed Human Growth Hormone Polypeptides and Their Uses," which is incorporated byμlreference herein in its entirety. Similarly, hGH polypeptides can comprise protease cleavageμlsequences, reactive groups, antibody-binding domains (including but not limited to, FLAG or poly-μlHis) or other affinity based sequences (including, but not limited to, FLAG, poly-His, GST, etc.) orμllinked molecules (including, but not limited to, biotin) that improve detection (including, but notμllimited to, GFP), purification or other traits of the polypeptide.
[201] In some embodiments, the substitution of a non-naturally encoded amino acid
generates an GH, e.g., hGH antagonist. A subset of exemplary sites for incorporation of one orμlmore non-naturally encoded amino acid include: 1, 2, 3, 4, 5, 8, 9, 11, 12, 15, 16, 19, 22, 103, 109,μl112, 113, 115, 116, 119, 120, 123, 127, or an addition before position 1 (SEQ ID NO: 2, or theμlcorresponding amino acid in SEQ ID NO: 1, 3, or any other GH sequence). In some embodiments, ..μlGH, e.g., hGH antagonists comprise at least one substitution in the regions 1-5 (N-terminus), 6-33 .:μl(A helix), 34-74 (region between A helix and B helix, the A-B loop), 75-96 (B helix), 97-105 ':μl(region between B helix and C helix, the B-C loop), 106-129 (C helix), 130-153 (region between Cμlhelix and D helix, the C-D loop), 154-183 (D helix), 184-191 (C-terminus) that cause GH to act as .μlan antagonist. In other embodiments, the exemplary sites of incorporation of a non-naturallyμlencoded amino acid include residues within the amino terminal region of helix A and a portion ofμlhelix C. In another embodiment, substitution of G120 with a non-naturally encoded amino acidμlsuch as p-azido-L-phenyalanine or O-propargyl-L-tyrosine. In other embodiments, the above-listedμlsubstitutions are combined with additional substitutions that cause the GH, e.g., hGH polypeptide toμlbe an GH, e.g., hGH antagonist. For instance, a non-naturally encoded amino acid is substituted atμlone of the positions identified herein and a simultaneous substitution is introduced at G120 (e.g.,μlG120R, G120K, G120W, G120Y, G120F, or G120E). In some embodiments, the GH, e.g., hGHμlantagonist comprises a non-naturally encoded amino acid linked to a water soluble polymer that isμlpresent in a receptor binding region of the GH, e.g., hGH molecule.
[202] In some cases, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids are substituted with
one or more non-naturally-encoded amino acids. In some cases, the GH, e.g., hGH polypeptideμlfurther includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more substitutions of one or more non-naturally

encoaea ammo acms ror naturaily-occurrmg amino acids. For example, in some embodiments, oneμlor more residues in the following regions of GH, e.g., hGH are substituted with one or more non-μlnaturally encoded amino acids: 1-5 (N-terminus); 32-46 (N-terminal end of the A-B loop); 97-105μl(B-C loop); and 132-149 (C-D loop); and 184-191 (C-terminus). In some embodiments, one orμlmore residues in the following regions of GH, e.g., hGH are substituted with one or more non-μlnaturally encoded amino acids: 1-5 (N-terminus), 6-33 (A helix), 34-74 (region between A helixμland B helix, the A-B loop), 75-96 (B helix), 97-105 (region between B helix and C helix, the B-Cμlloop), 106-129 (C helix), 130-153 (region between C helix and D helix, the C-D loop), 154-183 (Dμlhelix), 184-191 (C-terminus). In some cases, the one or more non-naturally encoded residues areμllinked to one or more lower molecular weight linear or branched PEGs (approximately - 5-20 kDaμlin mass or less), thereby enhancing binding affinity and comparable serum half-life relative to theμlspecies attached to a single, higher molecular weight PEG.
[203] In some embodiments, up to two of the following residues of GH, e.g., hGH are ;
substituted with one or more non-naturally-encoded amino acids at position: 29, 30, 33, 34, 35, 37,μl39, 40, 49, 57, 59, 66, 69, 70, 71, 74, 88, 91, 92, 94, 95, 98, 99, 101, 103, 107, 108, 111, 122,126, ]μl129,130, 131, 133, 134, 135, 136, 137, 139, 140, 141, 142, 143, 145, 147, 154, 155, 156, 159, 183, ;μl186, and 187. In some cases, any of the following pairs of substitutions are made: K38X and ..μlK140X; K41X and K145X; Y35X and E88X; Y35X and F92X; Y35X and Y143X; μlF92X and Y143X wherein X represents a non-naturally encoded amino acid. Preferred sites for μlincorporation of two or more non-naturally encoded amino acids include combinations of theμlfollowing residues: 29, 33, 35, 37, 39, 49, 57, 69, 70, 71, 74, 88, 91, 92, 94, 95, 98, 99, 101, 103,μl107, 108, 111, 129, 130, 131, 133, 134, 135, 136, 137, 139, 140, 141, 142, 143, 145, 147, 154, 155,μl156, 186, and 187. Particularly preferred sites for incorporation of two or more non-naturallyμlencoded amino acids include combinations of the following residues: 35, 88, 91, 92, 94, 95, 99,μl101,103, 111, 131,133, 134,135,136,139,140,143,145, and 155.
[204] Preferred sites for incorporation in GH, e.g., hGH of two or more non-naturally
encoded amino acids include combinations of the following residues: before position 1 (i.e. at theμlN-terminus), 1, 2, 3, 4, 5, 8, 9, 11, 12, 15, 16, 19, 22, 29, 30, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,μl42, 43, 44, 45, 46, 47, 48, 49, 52, 55, 57, 59, 65, 66, 69, 70, 71, 74, 88, 91, 92, 94, 95, 97, 98, 99,μl100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 111, 112, 113, 115, 116, 119, 120, 122, 123, 126,μl127, 129,130, 131,132, 133, 134, 135, 136, 137,138,139, 140, 141, 142, 143, 144, 145, 146, 147,

148, 149, 150, 151, 152, 153, 154, 155, 156, 158, 159, 161, 168, 172, 183, 184, 185, 186, 187, 188,μl189, 190, 191, 192 (i.e. at the carboxyl terminus of the protein) or any combination thereof fromμlSEQ ID NO: 2.
V. Expression in Non-eukaryotes andEukaryotes
[205] To obtain high level expression of a cloned hGH polynucleotide, one typically
subclones polynucleotides encoding a hGH polypeptide of the invention into an expression vectorμlthat contains a strong promoter to direct transcription, a transcription/translation terminator, and ifμlfor a nucleic acid encoding a protein, a ribosome binding site for translational initiation. Suitableμlbacterial promoters are known to those of ordinary skill in the art and described, e.g., in Sambrookμlet al. Molecular Cloning, A Laboratory Manual (2001) and Ausubel et ah Current Protocols inμlMolecular Biology (1999).
[206] Bacterial expression systems for expressing hGH polypeptides of the invention are
available in, including but not limited to, E. colμ Bacillus sp., Pseudomonas fluorescens,.μlPseudomonas aeruginosa, Pseudomonas putida, and Salmonella (Palva et al., Gene 22:229-235 ?μl(1983); Mosbach et al.9 Nature 302:543-545 (1983)). Kits for such expression systems are"μlcommercially available. Eukaryotic expression systems for mammalian cells, yeast, and insect cellsμlare known to those of ordinary skill in the art and are also commercially available. In cases whereμlorthogonal tRNAs and aminoacyl tRNA synthetases (described above) are used to express the hGH 'μlpolypeptides of the invention, host cells for expression are selected based on their ability to use the μlorthogonal components. Exemplary host cells include Gram-positive bacteria (including but notμllimited to B. brevis, B. subtilis, or Streptomyces) and Gram-negative bacteria (E. colμPseudomonasμlfluorescens, Pseudomonas aeruginosa, Pseudomonas putida)9 as well as yeast and other eukaryoticμlcells. Cells comprising O-tRNA/O-RS pairs can be used as described herein.μl[207] A eukaryotic host cell or non-eukaryotic host cell of the present invention provides
the ability to synthesize proteins that comprise unnatural amino acids in large useful quantities. Inμlone aspect, the composition optionally includes, including but not limited to, at least 10μlmicrograms, at least 50 micrograms, at least 75 micrograms, at least 100 micrograms, at least 200μlmicrograms, at least 250 micrograms, at least 500 micrograms, at least 1 milligram, at least 10μlmilligrams, at least 100 milligrams, at least one gram, or more of the protein that comprises anμlunnatural amino acid, or an amount that can be achieved with in vivo protein production methodsμl(details on recombinant protein production and purification are provided herein). In another aspect,

the protein is optionally present in the composition at a concentration of, including but not limitedμlto, at least 10 micrograms of protein per liter, at least 50 micrograms of protein per liter, at least 75μlmicrograms of protein per liter, at least 100 micrograms of protein per liter, at least 200 microgramsμlof protein per liter, at least 250 micrograms of protein per liter, at least 500 micrograms of proteinμlper liter, at least 1 milligram of protein per liter, or at least 10 milligrams of protein per liter orμlmore, in, including but not limited to, a cell lysate, a buffer, a pharmaceutical buffer, or other liquidμlsuspension (including but not limited to, in a volume of, including but not limited to, anywhereμlfrom about 1 nl to about 100 L or more). The production of large quantities (including but notμllimited to, greater that that typically possible with other methods, including but not limited to, inμlvitro translation) of a protein in a eukaryotic cell including at least one unnatural amino acid is aμlfeature of the invention.
[208] A eukaryotic host cell or non-eukaryotic host cell of the present invention provides
the ability to biosynthesize proteins that comprise unnatural amino acids in large useful quantities.μlFor example, proteins comprising an unnatural amino acid can be produced at a concentration of, .μlincluding but not limited to, at least 10 pg/liter, at least 50 |xg/liter, at least 75 jig/liter, at least 100μljig/liter, at least 200 ng/liter, at least 250 fig/liter, or at least 500 jig/liter, at least lmg/liter, at leastμl2mg/liter, at least 3 mg/liter, at least 4 mg/liter, at least 5 mg/liter, at least 6 mg/liter, at least 7μlmg/liter, at least 8 mg/liter, at least 9 mg/liter, at least 10 mg/liter, at least 20, 30, 40, 50, 60, 70, 80, 'μl90, 100, 200, 300, 400, 500, 600, 700, 800, 900 mg/liter, 1 g/liter, 5 g/liter, 10 g/liter or more ofμlprotein in a cell extract, cell lysate, culture medium, a buffer, and/or the like.μl[209] Expression systems, vectors, host cells, culturing conditions and medium, and
isolation from host cells of hGH polypeptides are further described in U.S. Patent Application No.μl11/046,432 entitled "Modifed Human Growth Hormone Polypeptides and Their Uses," which isμlincorporated by reference herein.μlK General Purification Methods
[210] The hGH polypeptides of the present invention are normally purified after expression
in recombinant systems. The hGH polypeptide may be purified from host cells by a variety ofμlmethods known to the art. hGH polypeptides produced in bacterial host cells may be poorly solubleμlor insoluble (in the form of inclusion bodies). Amino acid substitutions may readily be made in theμlhGH polypeptide that are selected for the purpose of increasing the solubility of the recombinantlyμlproduced protein utilizing the methods disclosed herein as well as those known in the art. In the

case of insoluble protein, the protein may be collected from host cell lysates by centrifugation andμlmay further be followed by homogenization of the cells. In the case of poorly soluble protein,μlcompounds including, but not limited to, polyethylene imine (PEI) may be added to induce theμlprecipitation of partially soluble protein. The precipitated protein may then be convenientlyμlcollected by centrifugation. Recombinant host cells may be disrupted or homogenized to release theμlinclusion bodies from within the cells using a variety of methods known to those of ordinary skill inμlthe art. Host cell disruption or homogenization may be performed using techniques known to thoseμlof ordinary skill in the art, including, but not limited to, enzymatic cell disruption, sonication,μldounce homogenization, or high pressure release disruption. In one embodiment of the method ofμlthe present invention, the high pressure release technique is used to disrupt the E. coli host cells toμlrelease the inclusion bodies of the hGH polypeptides. When handling inclusion bodies of hGHμlpolypeptide, it is advantageous to minimize the homogenization time on repetitions in order toμlmaximize the yield of inclusion bodies without loss due to factors such as solubilization, ;:μlmechanical shearing or proteolysis.
[211] Insoluble or precipitated hGH polypeptide may then be solubilized using any of a
number of suitable solubilization agents known to the art. The hGH polyeptide maybe solubilized μlwith urea or guanidine hydrochloride. The volume of the solubilized hGH polypeptide should be .μlminimized so that large batches may be produced using conveniently manageable batch sizes. Thisμlfactor may be significant in a large-scale commercial setting where the recombinant host may beμlgrown in batches that are thousands of liters in volume. In addition, when manufacturing hGHμlpolypeptide in a large-scale commercial setting, in particular for human pharmaceutical uses, theμlavoidance of harsh chemicals that can damage the machinery and container, or the protein productμlitself, should be avoided, if possible. It has been shown in the method of the present invention thatμlthe milder denaturing agent urea can be used to solubilize the hGH polypeptide inclusion bodies inμlplace of the harsher denaturing agent guanidine hydrochloride. The use of urea significantlyμlreduces the risk of damage to stainless steel equipment utilized in the manufacturing andμlpurification process of hGH polypeptide while efficiently solubilizing the hGH polypeptideμlinclusion bodies.
{212] In the case of soluble hGH protein, the hGH may be secreted into the periplasmic
space or into the culture medium. In addition^ soluble hGH may be present in the cytoplasm of theμlhost cells. It may be desired to concentrate soluble hGH prior to performing purification steps.

Standard techniques known to those of ordinary skill in the art may be used to concentrate solubleμlhGH from, for example, cell lysates or culture medium. In addition, standard techniques known toμlthose of ordinary skill in the art may be used to disrupt host cells and release soluble hGH from theμlcytoplasm or periplasmic space of the host cells.
[213] When hGH polypeptide is produced as a fusion protein, the fusion sequence may be
removed. Removal of a fusion sequence may be accomplished by enzymatic or chemical cleavage.μlEnzymatic removal of fusion sequences may be accomplished using methods known to those ofμlordinary skill in the art. The choice of enzyme for removal of the fusion sequence will beμldetermined by the identity of the fusion, and the reaction conditions will be specified by the choiceμlof enzyme as will be apparent to one of ordinary skill in the art Chemical cleavage may beμlaccomplished using reagents known to those of ordinary skill in the art, including but not limited to,μlcyanogen bromide, TEV protease, and other reagents. The cleaved hGH polypeptide may beμlpurified from the cleaved fusion sequence by methods known to those of ordinary skill in the art. 'μlSuch methods will be determined by the identity and properties of the fusion sequence and the hGH 'μlpolypeptide, as will be apparent to one of ordinary skill in the art. Methods for purification may μg' .μlinclude, but are not limited to, size-exclusion chromatography, hydrophobic interactionμlchromatography, ion-exchange chromatography or dialysis or any combination thereof.μl[214] The hGH polypeptide may also be purified to remove DNA from the protein .
solution. DNA may be removed by any suitable method known to the art, such as precipitation orμlion exchange chromatography, but may be removed by precipitation with a nucleic acidμlprecipitating agent, such as, but not limited to, protamine sulfate. The hGH polypeptide may beμlseparated from the precipitated DNA using standard methods known to those of ordinary skill in theμlart, including, but not limited to, centrifugation or filtration. Removal of host nucleic acid moleculesμlis an important factor in a setting where the hGH polypeptide is to be used to treat humans and theμlmethods of the present invention reduce host cell DNA to pharmaceutically acceptable levels.μl[215] Methods for small-scale or large-scale fermentation can also be used in protein
expression, including but not limited to, fermentors, shake flasks, fluidized bed bioreactors, hollowμlfiber bioreactors, roller bottle culture systems, and stirred tank bioreactor systems. Each of theseμlmethods can be performed in a batch, fed-batch, or continuous mode process.μl[216] Human hGH polypeptides of the invention can generally be recovered using methods
standard in the art. For example, culture medium or cell lysate can be centrifuged or filtered to

remove cellular debris. The supernatant may be concentrated or diluted to a desired volume orμldiafiltered into a suitable buffer to condition the preparation for further purification. Furtherμlpurification of the hGH polypeptide of the present invention includes separating deamidated andμlclipped forms of the hGH polypeptide variant from the intact form.
[217J Any of the following exemplary procedures can be employed for purification of hGH
polypeptides of the invention: affinity chromatography; anion- or cation-exchange chromatographyμl(using, including but not limited to, DEAE SEPHAROSE); chromatography on silica; highμlperformance liquid chromatography (HPLC), reverse phase HPLC (RP-HPLC); gel filtrationμlchromatography (using, including but not limited to, SEPHADEX G-75); hydrophobic interactionμlchromatography; size-exclusion chromatography, metal-chelate chromatography;μlultrafiltration/diafiltration; ethanol precipitation; ammonium sulfate precipitation;μlchromatofocusing; displacement chromatography; electrophoretic procedures (including but notμllimited to preparative isoelectric focusing), differential solubility (including but not limited to μlammonium sulfate precipitation), SDS-PAGE, or extraction.
[218] Proteins of the present invention, including but not limited to, proteins comprising i
unnatural amino acids, antibodies to proteins comprising unnatural amino acids, binding partnersμlfor proteins comprising unnatural amino acids, etc., can be purified, either partially or substantially μlto homogeneity, according to standard procedures known to and used by those of skill in the art. 'μlAccordingly, polypeptides of the invention can be recovered and purified by any of a number ofμlmethods known to those of ordinary skill in the art, including but not limited to, ammonium sulfateμlor ethanol precipitation, acid or base extraction, column chromatography, affinity columnμlchromatography, anion or cation, exchange chromatography, phosphocellulose chromatography,μlhydrophobic interaction chromatography, hydroxylapatite chromatography, lectin chromatography,μlgel electrophoresis and the like. Protein refolding steps can be used, as desired, in making correctlyμlfolded mature proteins. High performance liquid chromatography (HPLC), affinityμlchromatography or other suitable methods can be employed in final purification steps where highμlpurity is desired. In one embodiment, antibodies made against unnatural amino acids (or proteinsμlcomprising unnatural amino acids) are used as purification reagents, including but not limited to, forμlaffinity-based purification of proteins comprising one or more unnatural amino acid(s). Onceμlpurified, partially or to homogeneity, as desired, the polypeptides are optionally used for a wide

variety of utilities, including but not limited to, as assay components, therapeutics, prophylaxis,μldiagnostics, research reagents, and/or as immunogens for antibody production.
[219] In addition to other references noted herein, a variety of purification/protein folding
methods are known to those of ordinary skill in the art, including, but not limited to, those set forthμlin R. Scopes, Protein Purification. Springer-Verlag, N.Y. (1982); Deutscher, Methods inμlEnzymologv Vol. 182: Guide to Protein Purification. Academic Press, Inc. N.Y. (1990); Sandana,μl(1997) Bioseparation of Proteins. Academic Press, hie; Bollag et al. (1996) Protein Methods. 2ndμlEdition Wiley-Liss, NY; Walker, (1996) The Protein Protocols Handbook Humana Press, NJ,μlHarris and Angal, (1990) Protein Purification Applications: A Practical Approach IRL Press atμlOxford, Oxford, England; Harris and Angal, Protein Purification Methods: A Practical ApproachμlIRL Press at Oxford, Oxford, England; Scopes, (1993) Protein Purification: Principles and Practiceμl3rd Edition Springer Verlag, NY; Janson and Ryden, (1998) Protein Purification: Principles. HighμlResolution Methods and Applications. Second Edition Wiley-VCH, NY; and Walker (1998),μlProtein Protocols on CD-ROM Humana Press, NJ; and the references cited therein.
[220] One advantage of producing a protein or polypeptide of interest with an unnatural
amino acid in a eukaryotic host cell or non-eukaryotic host cell is that typically the proteins orμlpolypeptides will be folded in their native conformations. However, in certain embodiments of theμlinvention, those of skill in the art will recognize that, after synthesis, expression and/or purification,μlproteins can possess a conformation different from the desired conformations of the relevant,μlpolypeptides. In one aspect of the invention, the expressed protein is optionally denatured and thenμlrenatured. This is accomplished utilizing methods known in the art, including but not limited to, byμladding a chaperonin to the protein or polypeptide of interest, by solubilizing the proteins in aμlchaotropic agent such as guanidine HC1, utilizing protein disulfide isomerase, etc.
[221] In general, it is occasionally desirable to denature and reduce expressed polypeptides
and then to cause the polypeptides to re-fold into the preferred conformation. For example,μlguanidine, urea, DTT, DTE, and/or a chaperonin can be added to a translation product of interest.μlMethods of reducing, denaturing and renaturing proteins are known to those of ordinary skill in theμlart (see, the references above, and Debinskμet al. (1993) J. Biol. Chem., 268: 14065-14070;μlKreitman and Pastan (1993) Bioconiug. Chem.. 4: 581-585; and Buchner, et al., (1992) Anal.μlBiochenu 205: 263-270). Debinskμet al., for example, describe the denaturation and reduction of

inclusion body proteins in guanidine-DTE. The proteins can be refolded in a redox bufferμlcontaining, including but not limited to, oxidized glutathione and L-arginine. Refolding reagentsμlcan be flowed or otherwise moved into contact with the one or more polypeptide or otherμlexpression product, or vice-versa.
[222] In the case of prokaryotic production of hGH polypeptide, the hGH polypeptide thus
produced may be misfolded and thus lacks or has reduced biological activity. The bioactivity of theμlprotein may be restored by "refolding". In general, misfolded hGH polypeptide is refolded byμlsolubilizing (where the hGH polypeptide is also insoluble), unfolding and reducing the polypeptideμlchain using, for example, one or more chaotropic agents (e.g. urea and/or guanidine) and a reducingμlagent capable of reducing disulfide bonds (e.g. dithiothreitol, DTT or 2-mercaptoethanol, 2-ME).μlAt a moderate concentration of chaotrope, an oxidizing agent is then added (e.g., oxygen, cystine orμlcystamine), which allows the reformation of disulfide bonds. hGH polypeptide may be refoldedμlusing standard methods known in the art, such as those described in U.S. Pat. Nos. 4,511,502,μl4,511,503, and 4,512,922, which are incorporated by reference herein. The hGH polypeptide mayμlalso be cofolded with other proteins to form heterodimers or heteromultimers.μl[223] After refolding or cofolding, the hGH polypeptide may be further purified.
Purification of hGH may be accomplished using a variety of techniques known to those of ordinaryμlskill in the art, including hydrophobic interaction chromatography, size exclusion chromatography, ;μlion exchange chromatography, reverse-phase high performance liquid chromatography, affinity 'μlchromatography, and the like or any combination thereof. Additional purification may also includeμla step of drying or precipitation of the purified protein.
[224] After purification, hGH may be exchanged into different buffers and/or concentrated
by any of a variety of methods known to those of ordinary skill in the art, including, but not limitedμlto, diafiltration and dialysis. hGH that is provided as a single purified protein may be subject toμlaggregation and precipitation.
[225] The purified hGH may be at least 90% pure (as measured by reverse phase high
performance liquid chromatography, RP-HPLC, or sodium dodecyl sulfate-polyacrylamide gelμlelectrophoresis, SDS-PAGE) or at least 95% pure, or at least 98% pure, or at least 99% or greater^μlpure. Regardless of the exact numerical value of the purity of the hGH, the hGH is sufficientlyμlpure for use as a pharmaceutical product or for further processing, such as conjugation with a waterμlsoluble polymer such as PEG.

1-226] Certain hGH molecules may be used as therapeutic agents in the absence of other
active ingredients or proteins (other than excipients, carriers, and stabilizers, serum albumin and theμllike), or they may be complexed with another protein or a polymer.
[227] Any one of a variety of isolation steps may be performed on the cell lysate, extract,
culture medium, inclusion bodies, periplasmic space of the host cells, cytoplasm of the host cells, orμlother material, comprising hGH polypeptide or on any hGH polypeptide mixtures resulting fromμlany isolation steps including, but not limited to, affinity chromatography, ion exchangeμlchromatography, hydrophobic interaction chromatography, gel filtration chromatography, highμlperformance liquid chromatography ("HPLC"), reversed phase-HPLC ("RP-HPLC"), expanded bedμladsorption, or any combination and/or repetition thereof and in any appropriate order.μl[228] Equipment and other necessary materials used in performing the techniques
described herein are commercially available. Pumps, fraction collectors, monitors, recorders, andμlentire systems are available from, for example, Applied Biosystems (Foster City, CA), Bio-Rad.μlLaboratories, Inc. (Hercules, CA), and Amersham Biosciences, Inc. (Piscataway, NJ). .^μlChromatographic materials including, but not limited to, exchange matrix materials, media, and vμlbuffers are also available from such companies.
[229J Equilibration, and other steps in the column chromatography processes described ' f
herein such as washing and elution, may be more rapidly accomplished using specialized equipment }μlsuch as a pump. Commercially available pumps include, but are not limited to, HILOAD® Pump P-μl50, Peristaltic Pump P-l, Pump P-901, and Pump P-903 (Amersham Biosciences, Piscataway, NJ).μl[230] Examples of fraction collectors include RediFrac Fraction Collector, FRAC-100 and
FRAC-2dO Fraction Collectors, and SUPERFRAC® Fraction Collector (Amersham Biosciences,μlPiscataway, NJ). Mixers are also available to form pH and linear concentration gradients.μlCommercially available mixers include Gradient Mixer GM-1 and In-Line Mixers (AmershamμlBiosciencfes, Piscataway, NJ).
[231] The chromatographic process may be monitored using any commercially available
monitor. Such monitors may be used to gather information like UV, pH, and conductivity.μlExamples of detectors include Monitor UV-1, UVICORD® S n, Monitor UV-M n, Monitor UV-μl900, Monitor UPC-900, Monitor pH/C-900, and Conductivity Monitor (Amersham Biosciences,μlPiscataway, NJ). Indeed, entire systems are commercially available including the various AKTA®μlsystems from Amersham Biosciences (Piscataway, NJ).

[232] In one embodiment of the present invention, for example, the hGH polypeptide may
be reduced and denatured by first denaturing the resultant purified hGH polypeptide in urea,μlfollowed by dilution into TRIS buffer containing a reducing agent (such as DTT) at a suitable pH.μlIn another embodiment, the hGH polypeptide is denatured in urea in a concentration range ofμlbetween about 2 M to about 9 M, followed by dilution in TRIS buffer at a pH in the range of aboutμl5.0 to about 8.0. The refolding mixture of this embodiment may then be incubated. In oneμlembodiment, the refolding mixture is incubated at room temperature for four to twenty-four hours.μlThe reduced and denatured hGH polypeptide mixture may then be further isolated or purified.μl[233] As stated herein, the pH of the first hGH polypeptide mixture may be adjusted prior
to performing any subsequent isolation steps. In addition, the first hGH polypeptide mixture or anyμlsubsequent mixture thereof may be concentrated using techniques known in the art. Moreover, theμlelution buffer comprising the first hGH polypeptide mixture or any subsequent mixture thereof mayμlbe exchanged for a buffer suitable for the next isolation step using techniques known to those of ;,μlordinary skill in the art.
[234] Ion Exchange Chromatography Ion exchange chromatography may be performed. /.
See generally ION EXCHANGE CHROMATOGRAPHY: PRINCIPLES AND METHODS (Cat. No. 18-1114-21,μlAmersham Biosciences (Piscataway, NJ)).. Commercially available ion exchange columns includeμlHITRAP®, HIPREP®, and HILOAD® Columns (Amersham Biosciences, Piscataway, NJ). Such ,μlcolumns utilize strong anion exchangers such as Q SEPHAROSE® Fast Flow, Q SEPHAROSE® μlHigh Performance, and Q SEPHAROSE® XL; strong cation exchangers such as SP SEPHAROSE®μlHigh Performance, SP SEPHAROSE® Fast Flow, and SP SEPHAROSE® XL; weak anionμlexchangers such as DEAE SEPHAROSE® Fast How; and weak cation exchangers such as CMμlSEPHAROSE® Fast Flow (Amersham Biosciences, Piscataway, NJ). Anion or cation exchangeμlcolumn chromatography may be performed on the hGH polypeptide at any stage of the purificationμlprocess to isolate substantially purified hGH polypeptide.
[235J The cation exchange chromatography step may be performed using any suitable
cation exchange matrix. Useful cation exchange matrices include, but are not limited to, fibrous,μlporous, non-porous, microgranular, beaded, or cross-linked cation exchange matrix materials. Suchμlcation exchange matrix materials include, but are not limited to, cellulose, agarose, dextran,μlpolyacrylate, polyvinyl, polystyrene, silica, polyether, or composites of any of the foregoing.

[236] The cation exchange matrix may be any suitable cation exchanger including strong
and weak cation exchangers. Strong cation exchangers may remain ionized over a wide pH rangeμland thus, may be capable of binding hGH over a wide pH range. Weak cation exchangers,μlhowever, may lose ionization as a function of pH. For example, a weak cation exchanger may loseμlcharge when the pH drops below about pH 4 or pH 5. Suitable strong cation exchangers include,μlbut are not limited to, charged functional groups such as sulfopropyl (SP), methyl sulfonate (S), orμlsulfoethyl (SE). The cation exchange matrix may be a strong cation exchanger, having an hGHμlbinding pH range of about 2.5 to about 6.0. Alternatively, the strong cation exchanger may have anμlhGH binding pH range of about pH 2.5 to about pH 5.5. The cation exchange matrix may be aμlstrong cation exchanger having an hGH binding pH of about 3.0. Alternatively, the cationμlexchange matrix may be a strong cation exchanger, having an hGH binding pH range of about 6.0μlto about 8.0. The cation exchange matrix may be a strong cation exchanger having an hGHμlbinding pH range of about 8.0 to about 12.5. Alternatively, the strong cation exchanger may have .μlan hGH binding pH range of about pH 8.0 to about pH 12.0.
[237] Prior to loading the hGH, the cation exchange matrix may be equilibrated, for ;:
example, using several column volumes of a dilute, weak acid, e.g., four column volumes of 20 mMμlacetic acid, pH 3. Following equilibration, the hGH may be added and the column may be washed ,.μlone to several times, prior to elution of substantially purified hGH, also using a weak acid solution .:.μlsuch as a weak acetic acid or phosphoric acid solution. For example, approximately 2-4 columnμlvolumes of 20 mM acetic acid, pH 3, may be used to wash the column. Additional washes using,μle.g., 2-4 column volumes of 0.05 M sodium acetate, pH 5.5, or 0.05 M sodium acetate mixed withμl0.1 M sodium chloride, pH 5.5, may also be used. Alternatively, using methods known in the art,μlthe cation exchange matrix may be equilibrated using several column volumes of a dilute, weakμlbase.
[238] Alternatively, substantially purified hGH may be eluted by contacting the cation
exchanger matrix with a buffer having a sufficiently low pH or ionic strength to displace the hGHμlfrom the matrix. The pH of the elution buffer may range from about pH 2.5 to about pH 6.0. Moreμlspecifically, the pH of the elution buffer may range from about pH 2.5 to about pH 5.5, about pHμl2.5 to about pH 5.0. The elution buffer may have a pH of about 3.0. In addition, the quantity ofμlelution buffer may vary widely and will generally be in the range of about 2 to about 10 columnμlvolumes. Moreover, suitable buffers known to those of skill in the art may include, but not limited

to, citrate, phosphate, formate, HEPES, and MES buffers ranging in concentration from at leastμlabout 5 raM to at least about 100 mM.
[239] Following adsorption of the hGH to the cation exchanger matrix, substantially
purified hGH may be eluted by contacting the matrix with a buffer having a sufficiently high pH orμlionic strength to displace the hGH from the matrix. The pH of the elution buffer may range fromμlabout pH 8.0 to about pH 12.5. More specifically, the elution buffer may range from about pH 8.0μlto about pH 12.0. Suitable buffers for use in high pH elution of substantially purified hGH include,μlbut are not limited to, citrate, phosphate, formate, acetate, HEPES, and MES buffers ranging inμlconcentration from at least about 5 mM to at least about 100 mM. In addition, a buffer having 0.1μlM potassium borate, 0.6 M potassium chloride, 0.1 mM EDTA, pH 8.7 may be used. Substantiallyμlpurified hGH may also be eluted using standard buffers, such as a bicine buffer which includesμlabout 50 to 100 mM bicine, about 75 mM bicine; 25 to about 100 mM sodium chloride, about 50μlmM sodium chloride, and about 0.05 to about 0.5 EDTA, about 0.1 mM EDTA, pH 7.5.μl[240] Reverse-Phase Chromatography RP-HPLC may be performed to purify proteins
following suitable protocols that are known to those of ordinary skill in the art. See, e.g., Pearson et. ;μlal., ANAL BIOCHEM. (1982) 124:217-230 (1982); Rivier et al., J. CHROM. (1983) 268:112-119;μlKunitani et al., J. CHROM. (1986) 359:391-402. RP-HPLC may be performed on the hGHμlpolypeptide to isolate substantially purified hGH polypeptide. In this regard, silica derivatized,.μlresins with alkyl functionalities with a wide variety of lengths, including, but not limited to, at leastμlabout C3 to at least about C30, at least about C3 to at least about C2C or at least about C3 to at leastμlabout Ci8, resins may be used. Alternatively, a polymeric resin may be used. For example,μlTosoHaas Amberchrome CGlOOOsd resin may be used, which is a styrene polymer resin. Cyano orμlpolymeric resins with a wide variety of alkyl chain lengths may also be used. Furthermore, the RP-μlHPLC column may be washed with a solvent such as ethanol. The Source RP column is anotherμlexample of a RP-HPLC column.
[241] A suitable elution buffer containing an ion pairing agent and an organic modifier
such as methanol, isopropanol, tetrahydrofiiran, acetonitrile or ethanol, may be used to elute theμlhGH polypeptide from the RP-HPLC column. The most commonly used ion pairing agentsμlinclude, but are not limited to, acetic acid, formic acid, perchloric acid, phosphoric acid,μltrifluoroacetic acid, heptafluorobutyric acid, triethylamine, tetramethylammonium,μltetrabutylammonium, and triethylammonium acetate. Elution may be performed using one or more

gradients or isocratic conditions, with gradient conditions preferred to reduce the separation timeμland to decrease peak width. Another method involves the use of two gradients with differentμlsolvent concentration ranges. Examples of suitable elution buffers for use herein may include, butμlare not limited to, ammonium acetate and acetonitrile solutions.
[242] hGH may be isolated or purified, for example, using a SOURCE RP column, with an
acetonitrile gradient
[243] Hydrophobic Interaction Chromatography Purification Techniques Hydrophobic
interaction chromatography (HIC) may be performed on the hGH polypeptide. See generallyμlHYDROPHOBIC INTERACTION CHROMATOGRAPHY HANDBOOK: PRINCIPLES AND METHODS (Cat. No.μl18-1020-90, Amersham Biosciences (Piscataway, NJ) which is incorporated by reference herein.μlSuitable HIC matrices may include, but are not limited to, alkyl- or aryl-substituted matrices, suchμlas butyl-, hexyl-, octyl- or phenyl-substituted matrices including agarose, cross-linked agarose,μlsepharose, cellulose, silica, dextran, polystyrene, poly(methacrylate) matrices, and mixed modeμlresins, including but not limited to, a polyethyleneamine resin or a butyl- or phenyl-substituted ,μlpoly(methacrylate) matrix. Commercially available sources for hydrophobic interaction column .;μlchromatography include, but are not limited to, HITRAP®, HIPREP® and HILOAD® columns .μl(Amersham Biosciences, Piscataway, NJ).
[244] Briefly, prior to loading, the HIC column may be equilibrated using standard buffers ;
known to those of ordinary skill in the art, such as an acetic acid/sodium chloride solution orμlHEPES containing ammonium sulfate. Ammonium sulfate may be used as the buffer for loadingμlthe HIC column. After loading the hGH polypeptide, the column may then washed using standardμlbuffers and conditions to remove unwanted materials but retaining the hGH polypeptide on the HICμlcolumn. The hGH polypeptide may be eluted with about 3 to about 10 column volumes of aμlstandard buffer, such as a HEPES buffer containing EDTA and lower ammonium sulfateμlconcentration than the equilibrating buffer, or an acetic acid/sodium chloride buffer, among others.μlA decreasing linear salt gradient using, for example, a gradient of potassium phosphate, may also beμlused to elute the hGH molecules. The eluant may then be concentrated, for example, by filtrationμlsuch as diafiltration or ultrafiltration. Diafiltration may be utilized to remove the salt used to eluteμlthe hGH polypeptide.
[245] Isolation steps using, for example, gel filtration (GEL FILTRATION: PRINCIPLES AND
METHODS (Cat. No. 18-1022-18, Amersham Biosciences, Piscataway, NJ) which is incorporated by

reference herein, hydroxyapatite chromatography (suitable matrices include, but are not limited to,μlHA-Ultrogel, High Resolution (Calbiochem), CHT Ceramic Hydroxyapatite (BioRad), Bio - GelμlHTP Hydroxyapatite (BioRad)), HPLC, expanded bed adsorption, ultrafiltration, diafiltration,μllyophilization, and the like, may be performed on the first hGH polypeptide mixture or anyμlsubsequent mixture thereof, to remove any excess salts and to replace the buffer with a suitableμlbuffer for the next isolation step or even formulation of the final drug product.μl[246] The yield of hGH polypeptide, including substantially purified hGH polypeptide,
may be monitored at each step described herein using techniques known to those of ordinary skill inμlthe art. Such techniques may also be used to assess the yield of substantially purified hGHμlpolypeptide following the last isolation step. For example, the yield of hGH polypeptide may beμlmonitored using any of several reverse phase high pressure liquid chromatography columns, havingμla variety of alkyl chain lengths such as cyano RP-HPLC, CigRP-HPLC; as well as cation exchangeμlHPLC and gel filtration HPLC.
[247] Purity may be determined using standard techniques, such as SDS-PAGE, or by
measuring hGH polypeptide using Western blot and ELISA assays. For example, polyclonal μlantibodies may be generated against proteins isolated from negative control yeast fermentation and μlthe cation exchange recovery. The antibodies may also be used to probe for the presence of μlcontaminating host cell proteins.
[248] RP-HPLC material Vydac C4 (Vydac) consists of silica gel particles, the surfaces of
which carry C4-alkyl chains. The separation of hGH polypeptide from the proteinaceous impuritiesμlis based on differences in the strength of hydrophobic interactions. Elution is performed with anμlacetonitrile gradient in diluted trifluoroacetic acid. Preparative HPLC is performed using a stainlessμlsteel column (filled with 2.8 to 3.2 liter of Vydac C4 silicagel). The Hydroxyapatite Ultrogel eluateμlis acidified by adding trifluoroacetic acid and loaded onto the Vydac C4 column. For washing andμlelution an acetonitrile gradient in diluted trifluoroacetic acid is used. Fractions are collected andμlimmediately neutralized with phosphate buffer. The hGH polypeptide fractions which are within theμlIPC limits are pooled.
[249] DEAE Sepharose (GE Healthcare) material consists of diethylaminoethyl (DEAE)-
groups which are covalently bound to the surface of Sepharose beads. The:binding of hGHμlpolypeptide to the DEAE groups is mediated by ionic interactions. Acetonitrile and trifluoroaceticμlacid pass through the column without being retained. After these substances have been washed off,

trace impurities are removed by washing the column with acetate butter at a low pH. Then theμlcolumn is washed with neutral phosphate buffer and hGH polypeptide is eluted with a buffer withμlincreased ionic strength. The column is packed with DEAE Sepharose fast flow. The columnμlvolume is adjusted to assure a hGH polypeptide load in the range of 3-10 mg hGH polypeptide/mlμlgel. The column is washed with water and equilibration buffer (sodium/potassium phosphate). Theμlpooled fractions of the HPLC eluate are loaded and the column is washed with equilibration buffer.μlThen the column is washed with washing buffer (sodium acetate buffer) followed by washing withμlequilibration buffer. Subsequently, hGH polypeptide is eluted from the column with elution bufferμl(sodium chloride, sodium/potassium phosphate) and collected in a single fraction in accordanceμlwith the master elution profile. The eluate of the DEAE Sepharose column is adjusted to theμlspecified conductivity. The resulting drug substance is sterile filtered into Teflon bottles and storedμlat-70°C.
[250] Additional methods that may be employed include, but are not limited to, steps to -
remove endotoxins. Endotoxins are lipopoly-saccharides (LPSs) which are located on the outer μlmembrane of Gram-negative host cells, such as, for example, Escherichia coli. Methods for ..μlreducing endotoxin levels are known to one of ordinary skill in the art and include, but are notμllimited to, purification techniques using silica supports, glass powder or hydroxyapatite, reverse-μlphase, affinity, size-exclusion, anion-exchange chromatography, hydrophobic interactionμlchromatography, a combination of these methods, and the like. Modifications or additional μlmethods may be required to remove contaminants such as co-migrating proteins from theμlpolypeptide of interest. Methods for measuring endotoxin levels are known to one of ordinary skillμlin the art and include, but are not limited to, Limulus Amebocyte Lysate (LAL) assays.μl[251] A wide variety of methods and procedures can be used to assess the yield and purity
of a hGH protein one or more non-naturally encoded amino acids, including but not limited to, theμlBradford assay, SDS-PAGE, silver stained SDS-PAGE, coomassie stained SDS-PAGE, massμlspectrometry (including but not limited to, MALDI-TOF) and other methods for characterizingμlproteins known to one of ordinary skill in the art. Additional methods include, but are not limitedμlto: SDS-PAGE coupled with protein staining methods, immunoblotting, matrix assisted laserμldesorption/ionization-mass spectrometry (MALDI-MS), liquid chromatography/mass spectrometry,μlisoelectric focusing, analytical anion exchange, chromatofocusing, and circular dichroism.

[252] Procedures including, but not limited to, those listed herein tor isolation and
purification may also be used in formulation studies to assess the stability of hGH polypeptides ofμlthe invention, the stability of PEGylated forms of hGH polypeptides, and progress of theμlPEGylation reaction.
VII. Expression in Alternate Systems
[253] Several strategies have been employed to introduce unnatural amino acids into
proteins in non-recombinant host cells, mutagenized host cells, or in cell-free systems. Theseμlsystems are also suitable for use in making the hGH polypeptides comprising a non-naturallyμlencoded amino acid. Derivatization of amino acids with reactive side-chains such as Lys, Cys andμlTyr resulted in the conversion of lysine to N -acetyl-lysine. Chemical synthesis also provides aμlstraightforward method to incorporate unnatural amino acids. With the recent development ofμlen2ymatic ligation and native chemical ligation of peptide fragments, it is possible to make largerμlproteins. See, e.g., P. E. Dawson and S. B. H. Kent, Annu. Rev. Biochem. 69:923 (2000).μlChemical peptide ligation and native chemical ligation are described in U.S. Patent No. 6,184,344, /μlU.S. Patent Publication No. 2004/0138412, U.S. Patent Publication No. 2003/0208046, WO ;μl02/098902, and WO 03/042235, which are incorporated by reference herein. A general in vitro μlbiosynthetic method in which a suppressor tRNA chemically acylated with the desired unnatural ,-μlamino acid is added to an in vitro extract capable of supporting protein biosynthesis, has been used ..-μlto site-specifically incorporate over 100 unnatural amino acids into a variety of proteins of virtually'μlany size. See, e.g.y V. W. Cornish, D. Mendel and P. G. Schultz, Angew. Chem. Int. Ed. Engl.,μl1995, 34:621 (1995); CJ. Noren, SJ. Anfhony-Cahill, M.C. Griffith, P.G. Schultz, A generalμlmethod for site-specific incorporation of unnatural amino acids into proteins, Science 244:182-188μl(1989); and, J.D. Bain, C.G. Glabe, T.A. Dix, A.R. Chamberlin, E.S. Diala, Biosynthetic site-μlspecific incorporation of a non-natural amino acid into a polypeptide, J. Am. Chem. Soc.μl111:8013-8014 (1989). A broad range of functional groups has been introduced into proteins forμlstudies of protein stability, protein folding, enzyme mechanism, and signal transduction.μl[254] An in vivo method, termed selective pressure incorporation, was developed to
exploit the promiscuity of wild-type synthetases. See, e.g.t N. Budisa, C. Minks, S. Alefelder, W.μlWenger, R M. Dong, L. Moroder and R. Huber, FASEBJ., 13:41 (1999). An auxotrophic strain, inμlwhich the relevant metabolic pathway supplying the cell with a particular natural amino acid isμlswitched off, is grown in minimal media containing limited concentrations of the natural amino

acid, while transcription of the target gene is repressed. At the onset of a stationary growth phase,μlthe natural amino acid is depleted and replaced with the unnatural amino acid analog. Induction ofμlexpression of the recombinant protein results in the accumulation of a protein containing theμlunnatural analog. For example, using this strategy, o, m and p-fluorophenylalanines have beenμlincorporated into proteins, and exhibit two characteristic shoulders in the UV spectrum which canμlbe easily identified, see, e.g, C. Minks, R. Huber, L. Moroder and N. Budisa, Anal. Biochem..μl284:29 (2000); trifluoromethionine has been used to replace methionine in bacteriophage T4μllysozyme to study its interaction with chitooligosaccharide ligands by 19F NMR, see, e.g., H.μlDuewel, E. Daub, V. Robinson and J. F. Honek, Biochemistry, 36:3404 (1997); and trifluoroleucineμlhas been incorporated in place of leucine, resulting in increased thermal and chemical stability of aμlleucine-zipper protein. See, e.g.9 Y. Tang, G. Ghirlanda, W. A. Petka, T. Nakajima, W. F. DeGradoμland D. A. Tirrell, Angew. Chem. frit Ed. EngL, 40:1494 (2001). Moreover, selenomethionine andμltelluromethionine are incorporated into various recombinant proteins to facilitate the solution ofμlphases in X-ray crystallography. See, e.g, W. A. Hendrickson, J. R. Horton and D. M. Lemaster, .μlEMBO J.. 9:1665 (1990); J. O. Boles, K. LewinskμM. Kunkle, J. D. Odom, B. Dunlap, L. Lebioda μland M. Hatada, Nat. Struct. Biol., 1:283 (1994); N. Budisa, B. Steipe, P. Demange, C. Eckerskorn, ,;μlJ. Kellermann and R. Huber, Eur. J. Biochenu 230:788 (1995); and, N. Budisa, W. Karnbrock, S. μlSteinbacher, A. Hunan, L. Prade, T. Neuefeind, L. Moroder and R. Huber, J. Mol. Biol.» 270:616 ..μl(1997). Methionine analogs with alkene or alkyne functionalities have also been incorporated,μlefficiently, allowing for additional modification of proteins by chemical means. See, e.g., J. C. vanμlHest andD. A. Tirrell, FEBS Lett.. 428:68 (1998); J. C. van Hest, K. L. Kiick and D. A. Tirrell, LμlAm. Chem. Soc.. 122:1282 (2000); and, K. L. Kiick and D. A. Tirrell, Tetrahedron, 56:9487 (2000);μlU.S. Patent No. 6,586,207; U.S. Patent Publication 2002/0042097, which are incorporated byμlreference herein.
[255] The success of this method depends on the recognition of the unnatural amino acid
analogs by aminoacyl-tRNA synthetases, which, in general, require high selectivity to insure theμlfidelity of protein translation. One way to expand the scope of this method is to relax the substrateμlspecificity of aminoacyl-tRNA synthetases, which has been achieved in a limited number of cases.μlFor example, replacement of Ala294 by Gly in Escherichia coli phenylalanyl-tRNA synthetaseμl(PheRS) increases the size of substrate binding pocket, and results in the acylation of tRNAPhe byμlp-Cl-phenylalanine (p-Cl-Phe). See, M. Ibba, P. Kast and H. Hennecke, Biochemistry. 33:7107

(1994). An Escherichia coli strain harboring this mutant PheRS allows the incorporation of p-CJi-μlphenylalanine or p-Br-phenylalanine in place of phenylalanine. See, e.g., M. Ibba and H.μlHennecke, FEBS Lett,, 364:272 (1995); and, N. Sharma, R. Furter, P. Kast and D. A. Tirrell, FEBSμlLett., 467:37 (2000). Similarly, a point mutation Phel30Ser near the amino acid binding site ofμlEscherichia coli tyrosyl-tRNA synthetase was shown to allow azatyrosine to be incorporated moreμlefficiently than tyrosine. See, F. Hamano-Takaku, T. Iwama, S. Saito-Yano, K. Takaku, Y.μlMonden, M. Kitabatake, D. Soil and S. Nishimura, J. Biol. Chem., 275:40324 (2000).μl[256] Another strategy to incorporate unnatural amino acids into proteins in vivo is to
modify synthetases that have proofreading mechanisms. These synthetases cannot discriminate andμltherefore activate amino acids that are structurally similar to the cognate natural amino acids. Thisμlerror is corrected at a separate site, which deacylates the mischarged amino acid from the tRNA toμlmaintain the fidelity of protein translation. If the proofreading activity of the synthetase is disabled,μlstructural analogs that are misactivated may escape the editing function and be incorporated. This1μlapproach has been demonstrated recently with the valyl-tRNA synthetase (ValRS). See, V. Doring, μlH. D. Mootz, L. A. Nangle, T. L. Hendrickson, V. de Crecy-Lagard, P. Schimmel and P. Marliere;?μlScience, 292:501 (2001). ValRS can misaminoacylate tRNAVal with Cys, Thr, or aminobutyrate/1μl(Abu); these noncognate amino acids are subsequently hydrolyzed by the editing domain. After;μlrandom mutagenesis of the Escherichia coli chromosome, a mutant Escherichia coli strain was',μlselected that has a mutation in the editing site of ValRS. This edit-defective ValRS incorrectly^μlcharges tRNAVal with Cys. Because Abu sterically resembles Cys (—SH group of Cys is replacedμlwith -CH3 in Abu), the mutant ValRS also incorporates Abu into proteins when this mutantμlEscherichia coli strain is grown in the presence of Abu. Mass spectrometric analysis shows thatμlabout 24% of valines are replaced by Abu at each valine position in the native protein.μl[257] Solid-phase synthesis and semisynthetic methods have also allowed for the synthesis
of a number of proteins containing novel amino acids. For example, see the following publicationsμland references cited within, which are as follows: Crick, F.H.C., Barrett, L. Brenner, S. Watts-μlTobin, R. General nature of the genetic code for proteins. Nature. 192:1227-1232 (1961);μlHofmann, K., Bohn, H. Studies on polypeptides. XXXVI. The effect of pyrazole-imidazoleμlreplacements on the S-protein activating potency of an S-peptide fragment; J. Am Chem,μl88(24):5914-5919 (1966); Kaiser, E.T. Synthetic approaches to biologically active peptides andμlproteins including enyzmes, Ace Chem Res. 22:47-54 (1989); Nakatsuka, T., SasakμT., Kaiser,

E.T. Peptide segment coupling catalyzed by the se?nisynthetic enzyme thiosubtilisin, J Am ChemμlSoc, 109:3808-3810 (1987); Schnolzer, M., Kent, SBE Constructing proteins by dovetailingμlunprotected synthetic peptides: backbone-engineered HIV protease, Science, 256(5054):221-225μl(1992); Chaiken, I.M. Semisynthetic peptides and proteins, CRC Crit Rev Biochem, 11(3):255-301μl(1981); Offord, R.E. Protein engineering by chemical means? Protein Bng.. 1(3):15M57 (1987);μland, Jackson, D.Y., Burnier, J., Quan, C, Stanley, M., Tom, J., Wells, J.A. A Desigfied PeptideμlLigase for Total Synthesis of Ribonuclease A with Unnatural Catalytic Residues, Science,μl266(5183):243(1994).
[258] Chemical modification has been used to introduce a variety of unnatural side chains,
including cofactors, spin labels and oligonucleotides into proteins in vitro. See, e.g., Corey, D.R.,μlSchultz, P.G. Generation of a hybrid sequence-specific single-stranded deoxyribonuclease, Science.μl238(4832):1401-1403 (1987); Kaiser, E.T., Lawrence D.S., Rokita, S.E. The chemical modificationμlof enzymatic specificity, Annu Rev Biochem. 54:565-595 (1985); Kaiser, E.T., Lawrence, D.S. μlChemical mutation ofenyzme active sites, Science, 226(4674):505-511 (1984); Neet, K.E., Nanci A, ,;μlKoshland, D.E. Properties ofthiol-subtilisin, J Biol. Chem. 243(24):6392-6401 (1968); Polgar, L. et ;μlM.L. Bender. A new enzyme containing a synthetically formed active site. Thiol-subtilisin. J. Am ,μlChem Soc. 88:3153-3154 (1966); and, Pollack, SJ., Nakayama, G. Schultz, P.G. Introduction ofμlnucleophiles and spectroscopic probes into antibody combining sites, Science, 242(4881): 1038- ,μl1040(1988).
[259] Alternatively, biosynthetic methods that employ chemically modified aminoacyl-
tRNAs have been used to incorporate several biophysical probes into proteins synthesized in vitro.μlSee the following publications and references cited within: Brunner, J. New Photolabeling andμlcrosslinking methods, Annu. Rev Biochem, 62:483-514 (1993); and, Krieg, U.C., Walter, P.,μlHohnson, A.E. PhotocrosslinMng of the signal sequence of nascent preprolactin of the 54-μlkilodalton polypeptide of the signal recognition particle, Proc. Natl. Acad. Sci. 83(22):8604-8608μl(1986).
[260] Previously, it has been shown that unnatural amino acids can be site-specifically
incorporated into proteins in vitro by the addition of chemically aminoacylated suppressor tRNAs toμlprotein synthesis reactions programmed with a gene containing a desired amber nonsense mutation.μlUsing these approaches, one can substitute a number of the common twenty amino acids with closeμlstructural homologues, e.g., fluorophenylalanine for phenylalanine, using strains auxotropic for a

particular amino acid. See, e.g., Noren, C.J., Anthony-Cahill, Griffith, M.C., Schultz, P.G. Aμlgeneral method for site-specific incorporation of unnatural amino acids into proteins, Science, 244:μl182-188 (1989); M.W. Nowak, et al., Science 268:439-42 (1995); Bain, ID., Glabe, C.G., Dix,μlT.A., Chamberlin, A.R., Diala, E.S. Biosynthetic site-specific Incorporation of a non-natural aminoμlacid into a polypeptide, J. Am Chem Soc, 111:8013-8014 (1989); N. Budisa et al., FASEB J. 13:41-μl51 (1999); Ellman, J.A., Mendel, D., Anthony-Cahill, S., Noren, CJ., Schultz, P.G. Biosyntheticμlmethod for introducing unnatural amino acids site-specifically into proteins Methods in Enz.. vol.μl202, 301-336 (1992); and, Mendel, D., Cornish, V.W. & Schultz, P.G. Site-Directed Mutagenesisμlwith an Expanded Genetic Code, Annu Rev Biophvs. Biomol Struct. 24,435-62 (1995).μl[261] For example, a suppressor tRNA was prepared that recognized the stop codon UAG
and was chemically aminoacylated with an unnatural amino acid. Conventional site-directedμlmutagenesis was used to introduce the stop codon TAG, at the site of interest in the protein gene.μlSee, e.g., Sayers, J.R., Schmidt, W. Eckstein, F. 5'-3? Exonucleases in phosphorothioate-basedμlolignoucleotide-directed mutagensis. Nucleic Acids Res. 16(3):791-802 (1988). When the acylated ,;μlsuppressor tRNA and the mutant gene were combined in an in vitro transcriptibn/translation system, TΜLthe unnatural amino acid was incorporated in response to the UAG codon which gave a protein ,μlcontaining that amino acid at the specified position. Experiments using [3HJ-Phe and experimentsμlwith a-hydroxy acids demonstrated that only the desired amino acid is incorporated at the position .μlspecified by the UAG codon and that this amino acid is not incorporated at any other site in theμlprotein. See, e.g., Noren, et al, supra; Kobayashi et al., (2003) Nature Structural Biologyμl10(6):425r432; and, EUman, J.A., Mendel, D., Schultz, P.G. Site-specific incorporation of novelμlbackbone structures into proteins, Science, 255(5041):197-200 (1992).
[262] A tRNA may be aminoacylated with a desired amino acid by any method or
technique, including but not limited to, chemical or enzymatic aminoacylation.
[263] Aminoacylation may be accomplished by aminoacyl tRNA synthetases or by other
enzymatic molecules, including but not limited to, ribozymes. The term "ribozyme" isμlinterchangeable with "catalytic RNA." Cech and coworkers (Cech, 1987, Science, 236:1532-1539;μlMcCorkle et al., 1987, Concepts Biochem. 64:221-226) demonstrated the presence of naturallyμloccurring RNAs that can act as catalysts (ribozymes). However, although these natural RNAμlcatalysts have only been shown to act on ribonucleic acid substrates for cleavage and splicing, theμlrecent development of artificial evolution of ribozymes has expanded the repertoire of catalysis to

vanous chemical reactions. Studies nave laenttnea KINA molecules mat can catalyze ammoacyl-μlRNA bonds on their own (2f)3'-termini (Dlangakekare et al., 1995 Science 267:643-647), and anμlRNA molecule which can transfer an amino acid from one RNA molecule to another (Lohse et al.,μl1996, Nature 381:442-444).
[264] U.S. Patent Application Publication 2003/0228593, which is incorporated by
reference herein, describes methods to construct ribozymes and their use in aminoacylation ofμltRNAs with naturally encoded and non-naturally encoded amino acids. Substrate-immobilizedμlforms of enzymatic molecules that can aminoacylate tRNAs, including but not limited to,μlribozymes, may enable efficient affinity purification of the aminoacylated products. Examples ofμlsuitable substrates include agarose, sepharose, and magnetic beads. The production and use of aμlsubstrate-immobilized form of ribozyme for aminoacylation is described in Chemistry and Biologyμl2003,10:1077-1084 and U.S. Patent Application Publication 2003/0228593, which are incorporatedμlby reference herein.
[265] Chemical aminoacylation methods include, but are not limited to, those introduced
by Hecht and coworkers (Hecht, S. M. Ace. Chem. Res. 1992, 25, 545; Heckler, T. G.; Roesser, J.μlR.; Xu, C; Chang, P.; Hecht, S. M. Biochemistry 1988, 27, 7254; Efecht, s> M. Alford, B. L.;μlKuroda, Y.; Kitano, S. J. Biol. Chem. 1978, 253, 4517) and by Schultz, ChamberliiμDougherty andμlothers (Cornish, V. W.; Mendel, D.; Schultz, P. G. Angew. Chem. Int. Ed. Engl. 1995, 34, 621;μlRobertson, S. A.; Ellman, J. A.; Schultz, P. G. J. Am. Chem. Soc. 1991, 113, 2722; Noren, C. J.;μlAnthony-Cahill, S. J.; Griffith, M. C; Schultz, P. G. Science 1989, 244, 182; Bain, J. D.; Glabe, C.μlG.; Dix, T. A.; Chamberlin, A. R. J. Am. Chem. Soc. 1989, 111, 8013; Bain, J. D. et al. Natureμl1992, 356, 537; Gallivan, J. P.; Lester, H. A.; Dougherty, D. A. Chem. Biol. 1997, 4, 740; Turcatti,μlet al. J. Biol. Chem. 1996, 271, 19991; Nowak, M. W. et al. Science, 1995, 268, 439; Saks, M. E. etμlal. J. Biol. Chem. 1996,271,23169; Hohsaka, T. et al. J. Am. Chem. Soc. 1999,121, 34), which areμlincorporated by reference herein, to avoid the use of synthetases in aminoacylation. Such methodsμlor other chemical aminoacylation methods may be used to aminoacylate tRNA molecules.
[266] Methods for generating catalytic RNA may involve generating separate pools of
randomized ribozyme sequences, performing directed evolution on the pools, screening the poolsμlfor desirable aminoacylation activity, and selecting sequences of those ribozymes exhibiting desiredμlaminoacylation activity.

[267] Ribozymes can comprise motifs and/or regions that facilitate acylation activity, such
as a GGU motif and a U-rich region. For example, it has been reported that U-rich regions canμlfacilitate recognition of an amino acid substrate, and a GGU-motif can form base pairs with the 3?μltermini of a tRNA. In combination, the GGU and motif and U-rich region facilitate simultaneousμlrecognition of both the amino acid and tRNA simultaneously, and thereby facilitate aminoacylationμlof the 3r terminus of the tRNA.
[268] Ribozymes can be generated by in vitro selection using a partially randomized
»j.μlr24mini conjugated with tRNA CCCG» followed by systematic engineering of a consensus sequence
found in the active clones. An exemplary ribozyme obtained by this method is termed "Fx3
ribozyme" and is described in U.S. Pub. App. No. 2003/0228593, the contents of which is
incorporated by reference herein, acts as a versatile catalyst for the synthesis of various aminoacyl-
tRNAs charged with cognate non-natural amino acids.
[269] Immobilization on a substrate may be used to enable efficient affinity purification of ,
the aminoacylated tRNAs. Examples of suitable substrates include, but are not limited to, agarose, ,μlsepharose, and magnetic beads. Ribozymes can be immobilized on resins by taking advantage of .μlthe chemical structure of RNA, such as the 3f-cis-diol on the ribose of RNA can be oxidized withμlperiodate to yield the corresponding dialdehyde to facilitate immobilization of the RNA on the .μlresin. Various types of resins can be used including inexpensive hydrazide resins wherein reductive -μlanimation makes the interaction between the resin and the ribozyme an irreversible linkage.μlSynthesis of aminoacyl-tRNAs can be significantly facilitated by this on-column aminoacylationμltechnique. Kourouklis et al. Methods 2005; 36:239-4 describe a column-based aminoacylationμlsystem.
[270] Isolation of the aminoacylated tRNAs can be accomplished in a variety of ways. One
suitable method is to elute the aminoacylated tRNAs from a column with a buffer such as a sodiumμlacetate solution with 10 mM EDTA, a buffer containing 50 mM N-(2-hydroxyethyl)piperazine-N'-μl(3-propanesulfonic acid), 12.5 mM KC1, pH 7.0,10 mM EDTA, or simply an EDTA buffered waterμl(pH 7.0).
[271] The aminoacylated tRNAs can be added to translation reactions in order to
incorporate the amino acid with which the tRNA was aminoacylated in a position of choice in aμlpolypeptide made by the translation reaction. Examples of translation systems in which the

aminoacylated tRNAs of the present invention may be used include, but are not limited to cellμllysates. Cell lysates provide reaction components necessary for in vitro translation of a polypeptideμlfrom an input mRNA. Examples of such reaction components include but are not limited toμlribosomal proteins, rRNA, amino acids, tRNAs, GIT, ATP, translation initiation and elongationμlfactors and additional factors associated with translation. Additionally, translation systems may beμlbatch translations or compartmentalized translation. Batch translation systems combine reactionμlcomponents in a single compartment while compartmentalized translation systems separate theμltranslation reaction components from reaction products that can inhibit the translation efficiency.μlSuch translation systems are available commercially.
[272] Further, a coupled transcription/translation system may be used. Coupled
transcription/translation systems allow for both transcription of an input DNA into a correspondingμlmRNA, which is in turn translated by the reaction components. An example of a commerciallyμlavailable coupled transcription/translation is the Rapid Translation System (RTS, Roche.Inc.). Theμlsystem includes a mixture containing E. coli lysate for providing translational components such asμlribosomes and translation factors. Additionally, an RNA polymerase is included for the 'μltranscription of the input DNA into an mRNA template for use in translation. RTS can use μlcompartmentalization of the reaction components by way of a membrane interposed betweenμlreaction compartments, including a supply/waste compartment and a transcription/translationμlcompartment.
[273] Aminoacylation of tRNA may be performed by other agents, including but not
limited to, transferases, polymerases, catalytic antibodies, multi-functional proteins, and the like.μl[274] Lu et al. in Mol Cell. 2001 Oct;8(4):759-69 describe a method in which a protein is
chemically ligated to a synthetic peptide containing unnatural amino acids (expressed proteinμlligation).
[275] Microinjection techniques have also been use incorporate unnatural amino acids into
proteins. See, e.g.t M. W. Nowak, P. C. Kearney, J. R. Sampson, M. E. Saks, C. G. Labarca, S. K.μlSilverman, W. G. Zhong, J. Thorson, J. N. Abelson, N. Davidson, P. G. Schultz, D. A. Doughertyμland H. A. Lester, Science, 268:439 (1995); and, D. A. Dougherty, Curr. Opin. Chem. Biol.. 4:645μl(2000). A Xenopus oocyte was coinjected with two RNA species made in vitro: an mRNAμlencoding the target protein with a UAG stop codon at the amino acid position of interest and anμlamber suppressor tRNA aminoacylated with the desired unnatural amino acid. The translational

machinery of the oocyte then inserts the unnatural amino acid at the position specified by UAG.μlThis method has allowed in vivo structure-function studies of integral membrane proteins, whichμlare generally not amenable to in vitro expression systems. Examples include the incorporation of aμlfluorescent amino acid into tachykinin neurokinin-2 receptor to measure distances by fluorescenceμlresonance energy transfer, see, e.g., G. TurcattμK. Nemeth, M. D. Edgerton, U. Meseth, F. Talabot,μlM. Peitsch, J. Knowles, H. Vogel and A. Chollet, J. Biol. Chem., 271:19991 (1996); theμlincorporation of biotinylated amino acids to identify surface-exposed residues in ion channels, see,μle.g., J. P. Gallivan, H. A. Lester and D. A. Dougherty, Chem. Biol.. 4:739 (1997); the use of cagedμltyrosine analogs to monitor conformational changes in an ion channel in real time, see, e.g., J. C.μlMiller, S. K. Silverman, P. M. England, D. A. Dougherty and H. A. Lester, Neuron, 20:619 (1998);μland, the use of alpha hydroxy amino acids to change ion channel backbones for probing their gatingμlmechanisms. See, e.g., P. M. England, Y. Zhang, D. A. Dougherty and H. A. Lester, Cell, 96:89μl(1999); and, T. Lu, A. Y. Ting, J. Mainland, L. Y. Jan, P. G. Schultz and J. Yang, Nat Neurosci., .μl4:239(2001).
[276] The ability to incorporate unnatural amino acids directly into proteins in vivo offers a -'
wide variety of advantages including but not limited to, high yields of mutant proteins, technical μlease, the potential to study the mutant proteins in cells or possibly in living organisms and the use of .μlthese mutant proteins in therapeutic treatments and diagnostic uses. The ability to include unnaturalμlamino acids with various sizes, acidities, nucleophilicities, hydrophobicities, and other propertiesμlinto proteins can greatly expand our ability to rationally and systematically manipulate theμlstructures of proteins, both to probe protein function and create new proteins or organisms withμlnovel properties.
[277] In one attempt to site-specifically incorporate para-F-Phe, a yeast amber suppressor
tRNAPheCUA /phenylalanyl-tRNA synthetase pair was used in a p-F-Phe resistant, Pheμlauxotrophic Escherichia colt strain. See, e.g., R. Furter, Protein Sci.. 7:419 (1998).μl[278] It may also be possible to obtain expression of a hGH polynucleotide of the present
invention using a cell-free (in-vitro) translational system. Translation systems may be cellular orμlcell-free, and may be prokaryotic or eukaryotic. Cellular translation systems include, but are notμllimited to, whole cell preparations such as permeabilized cells or cell cultures wherein a desiredμlnucleic acid sequence can be transcribed to mRNA and the mRNA translated. Cell-free translationμlsystems are commercially available and many different types and systems are well-known.

Examples of cell-free systems include, but are not limited to, prokaryotic lysates such asμlEscherichia coli lysates, and eukaryotic lysates such as wheat germ extracts, insect cell lysates,μlrabbit reticulocyte lysates, rabbit oocyte lysates and human cell lysates. Eukaryotic extracts orμllysates may be preferred when the resulting protein is glycosylated, phosphorylated or otherwiseμlmodified because many such modifications are only possible in eukaryotic systems. Some of theseμlextracts and lysates are available commercially (Promega; Madison, Wis.; Stratagene; La Jolla,μlCalif.; Amersham; Arlington Heights, El.; GEBCO/BRL; Grand Island, N.Y.). Membranousμlextracts, such as the canine pancreatic extracts containing microsomal membranes, are alsoμlavailable which are useful for translating secretory proteins. In these systems, which can includeμleither mRNA as a template (in-vitro translation) or DNA as a template (combined in-vitroμltranscription and translation), the in vitro synthesis is directed by the ribosomes. Considerableμleffort has been applied to the development of cell-free protein expression systems. See, e.g., Kim,μlD.M. and J.R. Swartz, Biotechnology andBioengineering, 74309-316 (2001); Kim, D.M. and J.R.μlSwartz, Biotechnology Letters, 22, 1537-1542, (2000); Kim, D.M., and J.R. Swartz, BiotechnologyμlProgress, 16, 385-390, (2000); Kim, D.M., and J.R. Swartz, Biotechnology and Bioengineering, 66,μl180-188, (1999); and Patnaik, R. and J.R. Swartz, Biotechniques 24, 862-868, (1998); U.S. PatentμlNo. 6,337,191; U.S. Patent Publication No. 2002/0081660; WO 00/55353; WO 90/05785, which areμlincorporated by reference herein. Another approach that may be applied to the expression of hGHμlpolypeptides comprising a non-naturally encoded amino acid includes the mRNA-peptide fusionμltechnique. See, e.g., R. Roberts and J. Szostak, Proa Natl Acad. Sci. (USA) 94:12297-12302μl(1997); A. Frankel, et al, Chemistry & Biology 10:1043-1050 (2003). In this approach, an mRNAμltemplate linked to puromycin is translated into peptide on the ribosome. If one or more tRNAμlmolecules has been modified, non-natural amino acids can be incorporated into the peptide as well.μlAfter the last mRNA codon has been read, puromycin captures the C-terminus of the peptide. If theμlresulting mRNA-peptide conjugate is found to have interesting properties in an in vitro assay, itsμlidentity can be easily revealed from the mRNA sequence. In this way, one may screen libraries ofμlhGH polypeptides comprising one or more non-naturally encoded amino acids to identifyμlpolypeptides having desired properties. More recently, in vitro ribosome translations with purifiedμlcomponents have been reported that permit the synthesis of peptides substituted with non-naturallyμlencoded amino acids. See, e.g., A. Forster et aL9 Proc. Natl Acad. Sci. (USA) 100:6353 (2003).

[279] Reconstituted translation systems may also be used. Mixtures of puniied translation
factors have also been used successfully to translate mRNA into protein as well as combinations ofμllysates or lysates supplemented with purified translation factors such as initiation factor-1 (IF-1),μlIF-2, IF-3 (a or )3)5 elongation factor T (EF-Tu), or termination factors. Cell-free systems may alsoμlbe coupled transcription/translation systems wherein DNA is introduced to the system, transcribedμlinto mRNA and the mRNA translated as described in Current Protocols in Molecular Biology (F.μlM. Ausubel et al. editors, Wiley Interscience, 1993), which is hereby specifically incorporated byμlreference. RNA transcribed in eukaryotic transcription system may be in the form of heteronuclearμlRNA (hnRNA) or 5'-end caps (7-methyl guanosine) and 3'-end poly A tailed mature mRNA, whichμlcan be an advantage in certain translation systems. For example, capped mRNAs are translated withμlhigh efficiency in the reticulocyte lysate system.μlVIIL Macromoleadar Polymers Coupled to hGHPolypeptides
[280] Various modifications to the non-natural amino acid polypeptides described herein .;
can be effected using the compositions, methods, techniques and strategies described herein. These ,μlmodifications include the incorporation of further functionality onto the non-natural amino acidμlcomponent of the polypeptide, including but not limited to, a label; a dye; a polymer; a water-μlsoluble polymer, a derivative of polyethylene glycol; a photocrosslinker; a radionuclide; a cytotoxic ;μlcompound; a drug; an affinity label; a photoaffinity label; a reactive compound; a resin; a second μlprotein or polypeptide or polypeptide analog; an antibody or antibody fragment; a metal chelator; a .μlcofactor; a fatty acid; a carbohydrate; a polynucleotide; a DNA; a RNA; an antisenseμlpolynucleotide; a saccharide; a water-soluble dendrimer, a cyclodextrin; an inhibitory ribonucleicμlacid; a biomaterial; a nanoparticle; a spin label; a fluorophore, a metal-containing moiety; aμlradioactive moiety; a novel functional group; a group that covalently or noncovalently interacts withμlother molecules; a photocaged moiety; an actinic radiation excitable moiety; a photoisomerizableμlmoiety; biotin; a derivative of biotin; a biotin analogue; a moiety incorporating a heavy atom; aμlchemically cleavable group; a photocleavable group; an elongated side chain; a carbon-linkedμlsugar; a redox-active agent; an amino thioacid; a toxic moiety; an isotopically labeled moiety; aμlbiophysical probe; a phosphorescent group; a chemiluminescent group; an electron dense group; aμlmagnetic group; an intercalating group; a chromophore; an energy transfer agent; a biologicallyμlactive agent; a detectable label; a small molecule; a quantum dot; a nanotransmitter, aμlradionucleotide; a radiotransmitter; a neutron-capture agent; or any combination of the above, or

any other desirable compound or substance. As an illustrative, non-limiting example of theμlcompositions, methods, techniques and strategies described herein, the following description willμlfocus on adding macromolecular polymers to the non-natural amino acid polypeptide with theμlunderstanding that the compositions, methods, techniques and strategies described thereto are alsoμlapplicable (with appropriate modifications, if necessary and for which one of skill in the art couldμlmake with the disclosures herein) to adding other functionalities, including but not limited to thoseμllisted above.
[281] A wide variety of macromolecular polymers and other molecules can be linked to
hGH polypeptides of the present invention to modulate biological properties of the hGHμlpolypeptide, and/or provide new biological properties to the hGH molecule. These macromolecularμlpolymers can be linked to the hGH polypeptide via a naturally encoded amino acid, via a non-μlnaturally encoded amino acid, or any functional substituent of a natural or non-natural amino acid,μlor any substituent or functional group added to a natural or non-natural amino acid. The molecular μlweight of the polymer may be of a wide range, including but not limited to, between about 100 Da -,μland about 100,000 Da or more. The molecular weight of the polymer may be between about 100.;μlDa and about 100,000 Da, including but notlimited to, 100,000 Da, 95,000 Da, 90,000 Da, 85,000 ...μlDa, 80,000 Da, 75,000 Da, 70,000 Da, 65,000 Da, 60,000 Da, 55,000 Da, 50,000 Da, 45,000 Da,μl40,000 Da, 35,000 Da, 30,000 Da, 25,000 Da, 20,000 Da, 15,000 Da, 10,000 Da, 9,000 Da, 8,000μlDa, 7,000 Da, 6,000 Da, 5,000 Da, 4,000 Da, 3,000 Da, 2,000 Da, 1,000 Da, 900 Da, 800 Da, 700.rμlDa, 600 Da, 500 Da, 400 Da, 300 Da, 200 Da, and 100 Da. In some embodiments, the molecularμlweight of the polymer is between about 100 Da and 50,000 Da. In some embodiments, theμlmolecular weight of the polymer is between about 100 Da and 40,000 Da. In some embodiments,μlthe molecular weight of the polymer is between about 1,000 Da and 40,000 Da. In someμlembodiments, the molecular weight of the polymer is between about 5,000 Da and 40,000 Da. Inμlsome embodiments, the molecular weight of the polymer is between about 10,000 Da and 40,000μlDa.
[282] The present invention provides substantially homogenous preparations of
polymerprotein conjugates. "Substantially homogenous" as used herein means that polymerrproteinμlconjugate molecules are observed to be greater than half of the total protein. The polymerrproteinμlconjugate has biological activity and the present "substantially homogenous" PEGylated hGHμlpolypeptide preparations provided herein are those which are homogenous enough to display the

advantages of a homogenous preparation, e.g., ease in clinical application in predictability of lot toμllot pharmacokinetics.
[283] One may also choose to prepare a mixture of polymerrprotein conjugate molecules,
and the advantage provided herein is that one may select the proportion of mono-polymer:proteinμlconjugate to include in the mixture. Thus, if desired, one may prepare a mixture of various proteinsμlwith various numbers of polymer moieties attached (i.e., di-, tri-, tetra-, etc.) and combine saidμlconjugates with the mono-polymenprotein conjugate prepared using the methods of the presentμlinvention, and have a mixture with a predetermined proportion of mono-polymer:proteinμlconjugates.
[284] The polymer selected may be water soluble so that the protein to which it is attached
does not precipitate in an aqueous environment, such as a physiological environment. The polymerμlmay be branched or unbranched. For therapeutic use of the end-product preparation, the polymerμlwill be pharmaceutically acceptable.
[285] The proportion of polyethylene glycol molecules to protein molecules will vary, as
will their concentrations in the reaction mixture. In general, the optimum ratio (in terms of rμlefficiency of reaction in that there is minimal excess unreacted protein or polymer) may be -μldetermined by the molecular weight of the polyethylene glycol selected and on fee number of ;μlavailable reactive groups available. As relates to molecular weight, typically the higher theμlmolecular weight of the polymer, the fewer number of polymer molecules which may be attached to μlthe protein. Similarly, branching of the polymer should be taken into account when optimizing theseμlparameters. Generally, the higher the molecular weight (or the more branches) the higher theμlpolymerrprotein ratio.
[286] Examples of polymers include but are not limited to polyalkyl ethers and alkoxy-
capped analogs thereof (e.g., polyoxyethylene glycol, polyoxyethylene/propylene glycol, andμlmethoxy or ethoxy-capped analogs thereof, especially polyoxyethylene glycol, the latter is alsoμlknown as polyethyleneglycol or PEG); polyvinylpyrrolidones; polyvinylalkyl ethers;μlpolyoxazolines, polyalkyl oxazolines and polyhydroxyalkyl oxazolines; polyacrylamides, polyalkylμlacrylamides, and polyhydroxyalkyl acrylamides (e.g., polyhydroxypropylmethacrylamide andμlderivatives thereof); polyhydroxyalkyl acrylates; polysialic acids and analogs thereof; hydrophilicμlpeptide sequences; polysaccharides and their derivatives, including dextran and dextran derivatives,μle.g., carboxymethyldextran, dextran sulfates, aminodextran; cellulose and its derivatives, e.g.,

carboxymethyl cellulose, hydroxyalkyl celluloses; chitin and its derivatives, e.g., chitosan, succinylμlchitosan, carboxymethylchitin, carboxymethylchitosan; hyaluronic acid and its derivatives;μlstarches; alginates; chondroitin sulfate; albumin; pullulan and carboxymethyl pullulan;μlpolyaminoacids and derivatives thereof, e.g., polyglutamic acids, polylysines, polyaspartic acids,μlpolyaspartamides; maleic anhydride copolymers such as: styrene maleic anhydride copolymer,μldivinylethyl ether maleic anhydride copolymer; polyvinyl alcohols; copolymers thereof;μlterpolymers thereof; mixtures thereof; and derivatives of the foregoing.
[287] The proportion of polyethylene glycol molecules to protein molecules will vary, as
will their concentrations in the reaction mixture. In general, the optimum ratio (in terms ofμlefficiency of reaction in that there is minimal excess unreacted protein or polymer) may beμldetermined by the molecular weight of the polyethylene glycol selected and on the number ofμlavailable reactive groups available. As relates to molecular weight, typically the higher theμlmolecular weight of the polymer, the fewer number of polymer molecules which may be attached to ;μlthe protein. Similarly, branching of the polymer should be taken into account when optimizing these .;μlparameters. Generally, the higher the molecular weight (or the more branches) the higher the ;:.μlpolymer:protein ratio. ,.!
[288] As used herein, and when contemplating PEGrhGH polypeptide conjugates, the term v
"therapeutically effective amount" refers to an amount which gives the desired benefit to a patient. ,μlThe amount will vary from one individual to another and will depend upon a number of factors,μlincluding the overall physical condition of the patient and the underlying cause of the condition toμlbe treated. The amount of hGH polypeptide used for therapy gives an acceptable rate of change andμlmaintains the desired change at a beneficial level. A therapeutically effective amount of the presentμlcompositions may be readily ascertained by one of ordinary skill in the art using publicly availableμlmaterials and procedures.
[289] The water soluble polymer may be any structural form including but not limited to
linear, forked or branched. Typically, the water soluble polymer is a poly(alkylene glycol), such asμlpoly(ethylene glycol) (PEG), but other water soluble polymers can also be employed. By way ofμlexample, PEG is used to describe certain embodiments of this invention.
[290] PEG is a well-known, water soluble polymer that is commercially available or can be
prepared by ring-opening polymerization of ethylene glycol according to methods known to thoseμlof ordinary skill in the art (Sandler and Karo, Polymer Synthesis, Academic Press, New York, Vol.

3, pages 138-161). The term "PEG" is used broadly to encompass any polyethylene glycolμlmolecule, without regard to size or to modification at an end of the PEG, and can be represented asμllinked to the hGH polypeptide by the formula:μlXO~(CH2CH2OVCH2CH2-Y
where n is 2 to 10,000 and X is H or a terminal modification, including but not limited to, a CMΜLalkyl, a protecting group, or a terminal functional group.
[291] In some cases, a PEG used in the invention terminates on one end with hydroxy or
methoxy, i.e., X is H or CH3 ("methoxy PEG"). Alternatively, the PEG can terminate with aμlreactive group, thereby forming a bifunctional polymer. Typical reactive groups can include thoseμlreactive groups that are commonly used to react with the functional groups found in the 20 commonμlamino acids (including but not limited to, maleimide groups, activated carbonates (including but notμllimited to, p-nitrophenyl ester), activated esters (including but not limited to, N-μlhydroxysuccinimide, p-nitrophenyl ester) and aldehydes) as well as functional groups that are inertμlto the 20 common amino acids but that react specifically with complementary functional groups .'μlpresent in non-naturally encoded amino acids (including but not limited to, azide groups, alkyneμlgroups). It is noted that the other end of the PEG, which is shown in the above formula by Y, will ,vμlattach either directly or indirectly to a hGH polypeptide via a naturally-occurring or non-naturally iμlencoded amino acid. For instance, Y may be an amide, carbamate or urea linkage to an amine μlgroup (including but not limited to, the epsilon amine of lysine or the TV-terminus) of theμlpolypeptide. Alternatively, Y may be a maleimide linkage to a thiol group (including but notμllimited to, the thiol group of cysteine). Alternatively, Y may be a linkage to a residue notμlcommonly accessible via the 20 common amino acids. For example, an azide group on the PEGμlcan be reacted with an alkyne group on the hGH polypeptide to form a Huisgen [3+2] cycloadditionμlproduct. Alternatively, an alkyne group on the PEG can be reacted with an azide group present in aμlnon-naturally encoded amino acid to form a similar product. In some embodiments, a strongμlnucleophile (including but not limited to, hydrazine, hydrazide, hydroxylamine, semicarbazide) canμlbe reacted with an aldehyde or ketone group present in a non-naturally encoded amino acid to formμla hydrazone, oxime or semicarbazone, as applicable, which in some cases can be further reduced byμltreatment with an appropriate reducing agent. Alternatively, the strong nucleophile can beμlincorporated into the hGH polypeptide via a non-naturally encoded amino acid and used to reactμlpreferentially with a ketone or aldehyde group present in the water soluble polymer.

[292] Any molecular mass for a PEG can be used as practically desired, including but not
limited to, from about 100 Daltons (Da) to 100,000 Da or more as desired (including but not limitedμlto, sometimes 0.1-50 kDa or 10-40 kDa). The molecular weight of the PEG may be of a wide range,μlincluding but not limited to, between about 100 Da and about 100,000 Da or more. The molecularμlweight of the PEG may be between about 100 Da and about 100,000 Da, including but not limitedμlto, 100,000 Da, 95,000 Da, 90,000 Da, 85,000 Da, 80,000 Da, 75,000 Da, 70,000 Da, 65,000 Da,μl60,000 Da, 55,000 Da, 50,000 Da, 45,000 Da, 40,000 Da, 35,000 Da, 30,000 Da, 25,000 Da, 20,000μlDa, 15,000 Da, 10,000 Da, 9,000 Da, 8,000 Da, 7,000 Da, 6,000 Da, 5,000 Da, 4,000 Da, 3,000 Da,μl2,000 Da, 1,000 Da, 900 Da, 800 Da, 700 Da, 600 Da, 500 Da, 400 Da, 300 Da, 200 Da, and 100μlDa. In some embodiments, the molecular weight of the PEG is between about 100 Da and 50,000μlDa. In some embodiments, the molecular weight of the PEG is between about 100 Da and 40,000μlDa. In some embodiments, the molecular weight of the PEG is between about 1,000 Da and 40,000μlDa. In some embodiments, the molecular weight of the PEG is between about 5,000 Da and 40,000 .μlDa. In some embodiments, the molecular weight of the PEG is between about 10,000 Da and rμl40,000 Da. Branched chain PEGs, including but not limited to, PEG molecules with each chain ;μlhaving a MW ranging from 1-100 kDa (including but not limited to, 1-50 kDa or 5-20 kDa) can also ;μlbe used. The molecular weight of the branched chain PEG may be, including but not limited to,μlbetween about 1,000 Da and about 100,000 Da or more. The molecular weight of the branched vμlchain PEG may be between about 1,000 Da and about 100,000 Da, including but not limited to, μl100,000 Da, 95,000 Da, 90,000 Da, 85,000 Da, 80,000 Da, 75,000 Da, 70,000 Da, 65,000 Da,μl60,000 Da,55,000 Da, 50,000 Da, 45,000 Da, 40,000 Da, 35,000 Da, 30,000 Da, 25,000 Da, 20,000μlDa, 15,000 Da, 10,000 Da, 9,000 Da, 8,000 Da, 7,000 Da, 6,000 Da, 5,000 Da, 4,000 Da, 3,000 Da,μl2,000 Da, and 1,000 Da. In some embodiments, the molecular weight of the branched chain PEG isμlbetween about 1,000 Da and 50,000 Da. In some embodiments, the molecular weight of theμlbranched chain PEG is between about 1,000 Da and 40,000 Da. In some embodiments, theμlmolecular weight of the branched chain PEG is between about 5,000 Da and 40,000 Da. In someμlembodiments, the molecular weight of the branched chain PEG is between about 5,000 Da andμl20,000 Da. A wide range of PEG molecules are described in, including but not limited to, theμlShearwater Polymers, Inc. catalog, Nektar Therapeutics catalog, incorporated herein by reference.μl[293] Generally, at least one terminus of the PEG molecule is available for reaction with the
non-naturally-encoded amino acid. For example, PEG derivatives bearing alkyne and azide

moieties for reaction with amino acid side chains can be used to attach PEG to non-naturallyμlencoded amino acid. If the non-naturally encoded amino acid comprises an azide, then the PEGμlwill typically contain either an alkyne moiety to effect formation of the [3+2] cycloaddition productμlor an activated PEG species (i.e., ester, carbonate) containing a phosphine group to effect formationμlof the amide linkage. Alternatively, if the non-naturally encoded amino acid comprises an alkyne,μlthen the PEG will typically contain an azide moiety to effect formation of the [3+2] Huisgenμlcycloaddition product. If the non-naturally encoded amino acid comprises a carbonyl group, theμlPEG will typically comprise a potent nucleophile (including but not limited to, a hydrazide,μlhydrazine, hydroxylamine, or semicarbazide functionality) in order to effect formation ofμlcorresponding hydrazone, oxime, and semicarbazone linkages, respectively. In other alternatives, aμlreverse of the orientation of the reactive groups described above can be used, i.e., an azide moiety inμlthe non-naturally encoded amino acid can be reacted with a PEG derivative containing an alkyne.μl[294] In some embodiments, the hGH polypeptide variant with a PEG derivative contains a
chemical functionality that is reactive with the chemical functionality present on the side chain ofμlthe non-naturally encoded amino acid.
[295] The invention provides in some embodiments azide- and acetylene-containing 'r
polymer derivatives comprising a water soluble polymer backbone having an average molecular 'μlweight from about 800 Da to about 100,000 Da. The polymer backbone of the water-solubleμlpolymer can be poly(ethylene glycol). However, it should be understood that a wide variety ofμlwater soluble polymers including but not limited to poly(ethylene)gIycol and other relatedμlpolymers, including poly(dextran) and poly(propylene glycol), are also suitable for use in theμlpractice of this invention and that the use of the term PEG or poly(ethylene glycol) is intended toμlencompass and include all such molecules. The term PEG includes, but is not limited to,μlpoly(ethylene glycol) in any of its forms, including Afunctional PEG, multiaimed PEG, derivatizedμlPEG, forked PEG, branched PEG, pendent PEG (i.e. PEG or related polymers having one or moreμlfunctional groups pendent to the polymer backbone), or PEG with degradable linkages therein.μl[296] PEG is typically clear, colorless, odorless, soluble in water, stable to heat, inert to
many chemical agents, does not hydrolyze or deteriorate, and is generally non-toxic. Polyethyleneμlglycol) is considered to be biocompatible, which is to say that PEG is capable of coexistence withμlliving tissues or organisms without causing harm. More specifically, PEG is substantially non-

immunogenic, which is to say that PEG does not tend to produce an immune response in the body.μlWhen attached to a molecule having some desirable function in the body, such as a biologicallyμlactive agent, the PEG tends to mask the agent and can reduce or eliminate any immune response soμlthat an organism can tolerate the presence of the agent. PEG conjugates tend not to produce aμlsubstantial immune response or cause clotting or other undesirable effects. PEG having the formulaμl- CH2CH20-(CH2CH20)n - CH2CH2~, where n is from about 3 to about 4000, typically fromμlabout 20 to about 2000, is suitable for use in the present invention. PEG having a molecular weightμlof from about 800 Da to about 100,000 Da are in some embodiments of the present inventionμlparticularly useful as the polymer backbone. The molecular weight of PEG may be of a wide range,μlincluding but not limited to, between about 100 Da and about 100,000 Da or more. The molecularμlweight of PEG may be between about 100 Da and about 100,000 Da, including but not limited to,μl100,000 Da, 95,000 Da, 90,000 Da, 85,000 Da, 80,000 Da, 75,000 Da, 70,000 Da, 65,000 Da, „μl60,000 Da, 55,000 Da, 50,000 Da, 45,000 Da, 40,000 Da, 35,000 Da, 30,000 Da, 25,000 Da, 20,000μlDa, 15,000 Da, 10,000 Da, 9,000 Da, 8,000 Da, 7,000 Da, 6,000 Da, 5,000 Da, 4,000 Da, 3,000 Da, .μl2,000 Da, 1,000 Da, 900 Da, 800 Da, 700 Da, 600 Da, 500 Da, 400 Da, 300 Da, 200 Da, and 100..μlDa. In some embodiments, the molecular weight of PEG is between about 100 Da and 50,000 Da.μlIn some embodiments, the molecular weight of PEG is between about 100 Da and 40,000 Da. Inμlsome embodiments, the molecular weight of PEG is between about 1,000 Da and 40,000 Da. In.μlsome embodiments, the molecular weight of PEG is between about 5,000 Da and 40,000 Da. Inμlsome embodiments, the molecular weight of PEG is between about 10,000 Da and 40,000 Da.μl[297] The polymer backbone can be linear or branched. Branched polymer backbones are
generally known in the art. Typically, a branched polymer has a central branch core moiety and aμlplurality of linear polymer chains linked to the central branch core. PEG is commonly used inμlbranched forms that can be prepared by addition of ethylene oxide to various polyols, such asμlglycerol, glycerol oligomers, pentaerythritol and sorbitol. The central branch moiety can also beμlderived from several amino acids, such as lysine. The branched poly(ethylene glycol) can beμlrepresented in general form as R(-PEG-OH)m in which R is derived from a core moiety, such asμlglycerol, glycerol oligomers, or pentaerythritol, and m represents the number of arms. Multi-armedμlPEG molecules, such as those described in U.S. Pat. Nos. 5,932,462 5,643,575; 5,229,490;

4,289,872; U.S. Pat. Appl. 2003/0143596; WO 96/21469; and WO 93/21259, each of which isμlincorporated by reference herein in its entirety, can also be used as the polymer backbone.μl[298] Branched PEG can also be in the form of a forked PEG represented by PEG(~
YCHZ2)n, where Y is a linking group and Z is an activated terminal group linked to CH by a chainμlof atoms of defined length.
[299] Yet another branched form, the pendant PEG, has reactive groups, such as carboxyl,
along the PEG backbone rather than at the end of PEG chains.
[300] In addition to these forms of PEG, the polymer can also be prepared with weak or
degradable linkages in the backbone. For example, PEG can be prepared with ester linkages in theμlpolymer backbone that are subject to hydrolysis. As shown below, this hydrolysis results inμlcleavage of the polymer into fragments of lower molecular weight:μl-PEG-CO2-PEG-+H2O -> PEG-C02H+HO-PEG~
It is understood by those of ordinary skill in the art that the term poly(ethylene glycol) or PEG .P-ΜLrepresents or includes all the forms known in the art including but not limited to those disclosed...μlherein. .',
[301] Many other polymers are also suitable for use in the present invention. In some (s
embodiments, polymer backbones that are water-soluble, with from 2 to about 300 terminμare μlparticularly useful in the invention. Examples of suitable polymers include, but are not limited to, μlother poly(alkylene glycols), such as poly(propylene glycol) ("PPG")> copolymers thereofμl(including but not limited to copolymers of ethylene glycol and propylene glycol), terpolymersμlthereof, mixtures thereof, and the like. Although the molecular weight of each chain of the polymerμlbackbone can vary, it is typically in the range of from about 800 Da to about 100,000 Da, oftenμlfrom about 6,000 Da to about 80,000 Da. The molecular weight of each chain of the polymerμlbackbone may be between about 100 Da and about 100,000 Da, including but not limited to,μl100,000 Da, 95,000 Da, 90,000 Da, 85,000 Da, 80,000 Da, 75,000 Da, 70,000 Da, 65,000 Da,μl60,000 Da, 55,000 Da, 50,000 Da, 45,000 Da, 40,000 Da, 35,000 Da, 30,000 Da, 25,000 Da, 20,000μlDa, 15,000 Da, 10,000 Da, 9,000 Da, 8,000 Da, 7,000 Da, 6,000 Da, 5,000 Da, 4,000 Da, 3,000 Da,μl2,000 Da, 1,000 Da, 900 Da, 800 Da, 700 Da, 600 Da, 500 Da, 400 Da, 300 Da, 200 Da, and 100μlDa. In some embodiments, the molecular weight of each chain of the polymer backbone is betweenμlabout 100 Da and 50,000 Da. In some embodiments, the molecular weight of each chain of the

polymer backbone is between about 100 Da and 40,000 Da. In some embodiments, the molecularμlweight of each chain of the polymer backbone is between about 1,000 Da and 40,000 Da. In someμlembodiments, the molecular weight of each chain of the polymer backbone is between about 5,000μlDa and 40,000 Da. In some embodiments, the molecular weight of each chain of the polymerμlbackbone is between about 10,000 Da and 40,000 Da.
[302] Those of ordinary skill in the art will recognize that the foregoing list for
substantially water soluble backbones is by no means exhaustive and is merely illustrative, and thatμlall polymeric materials having the qualities described above are contemplated as being suitable forμluse in the present invention.
[303] In some embodiments of the present invention the polymer derivatives are "multi-
functional", meaning that the polymer backbone has at least two terminμand possibly as many asμlabout 300 terminμfunctionalized or activated with a functional group. Multifunctional polymerμlderivatives include, but are not limited to, linear polymers having two terminμeach terminus beingμl. bonded to a functional group which may be the same or different

X is a second functional group.
Examples of a linking moiety for A and B include, but are not limited to, a multiply-functionalizedμlalkyl group containing up to 18, including but not limited to, between 1-10 carbon atoms. Aμlheteroatom such as nitrogen, oxygen or sulfur may be included with the alkyl chain. The alkylμlchain may also be branched at a heteroatom. Other examples of a linking moiety for A and Bμlinclude, but are not limited to, a multiply functionalized aryl group, containing up to 10, includingμlbut not limited to, 5-6 carbon atoms. The aryl group may be substituted with one more carbon

atoms, nitrogen, oxygen or sulfur atoms. Other examples of suitable linking groups include thoseμllinking groups described in U.S. Pat. Nos. 5,932,462; 5,643,575; and U.S. Pat Appl. Publicationμl2003/0143596, each of which is incorporated by reference herein. Those of ordinary skill in the artμlwill recognize that the foregoing list for linking moieties is by no means exhaustive and is merelyμlillustrative, and that all linking moieties having the qualities described above are contemplated to beμlsuitable for use in the present invention.
[305] Examples of suitable functional groups for use as X include, but are not limited to,
hydroxy!, protected hydroxyl, alkoxyl, active ester, such as N-hydroxysuccinimidyl esters and 1-μlbenzotriazolyl esters, active carbonate, such as N-hydroxysuccinimidyl carbonates and 1-μlbenzotriazolyl carbonates, acetal, aldehyde, aldehyde hydrates, alkenyl, acrylate, methacrylate,μlacrylamide, active sulfone, amine, aminooxy, protected amine, hydrazide, protected hydrazide,μlprotected thiol, carboxylic acid, protected carboxylic acid, isocyanate, isothiocyanate, maleimide, .μlvinylsulfone, dithiopyridine, vinylpyridine, iodoacetamide, epoxide, glyoxals, diones, mesylates, »μltosylates, tresylate, alkene, ketone, and azide. As is understood by those of ordinary skill in the art, ;μlthe selected X moiety should be compatible with the azide group so that reaction with the azide μlgroup does not occur. The azide-containing polymer derivatives may be homobifunctional, ,μlmeaning that the second functional group (i.e., X) is also an azide moiety, or heterobifunctional, μlmeaning that the second functional group is a different functional group.
[306] The term "protected" refers to the presence of a protecting group or moiety that
prevents reaction of the chemically reactive functional group under certain reaction conditions. Theμlprotecting group will vary depending on the type of chemically reactive group being protected. Forμlexample, if the chemically reactive group is an amine or a hydrazide, the protecting group can beμlselected from the group of tert-butyloxycarbonyl (t-Boc) and 9-fluorenylmethoxycarbonyl (Fmoc).μlIf the chemically reactive group is a thiol, the protecting group can be orthopyridyldisulfide. If theμlchemically reactive group is a carboxylic acid, such as butanoic or propionic acid, or a hydroxylμlgroup, the protecting group can be benzyl or an alkyl group such as methyl, ethyl, or tert-butyl.μlOther protecting groups known in the art may also be used in the present invention.μl[307] Purification of the crude product can usually be accomplished by methods known in
the art including, but are not limited to, precipitation of the product followed by chromatography, ifμlnecessary.

[308] Water soluble polymers can be linked to the hGH polypeptides of the invention. The
water soluble polymers may be linked via a non-naturally encoded amino acid incorporated in theμlhGH polypeptide or any functional group or substituent of a non-naturally encoded or naturallyμlencoded amino acid, or any functional group or substituent added to a non-naturally encoded orμlnaturally encoded amino acid. Alternatively, the water soluble polymers are linked to a hGHμlpolypeptide incorporating a non-naturally encoded amino acid via a naturally-occurring amino acidμl(including but not limited to, cysteine, lysine or the amine group of the N-terminal residue). Inμlsome cases, the hGH polypeptides of the invention comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 non-naturalμlamino acids, wherein one or more non-naturally-encoded amino acid(s) are linked to water solubleμlpolymer(s) (including but not limited to, PEG and/or oligosaccharides). In some cases, the hGHμlpolypeptides of the invention further comprise 1, 2, 3,4, 5, 6, 7, 8, 9, 10, or more naturally-encodedμlamino acid(s) linked to water soluble polymers. In some cases, the hGH polypeptides of theμlinvention comprise one or more non-naturally encoded amino acid(s) linked to water solubleμlpolymers and one or more naturally-occurring amino acids linked to water soluble polymers. Inμlsome embodiments, the water soluble polymers used in the present invention enhance the serumμlhalf-life of the hGH polypeptide relative to the unconjugated form.
[309] The number of water soluble polymers linked to a hGH polypeptide (i.e., the extent
of PEGylation or glycosylation) of the present invention can be adjusted to provide an alteredμl(including but not limited to, increased or decreased) pharmacologic, pharmacokinetic orμlpharmacodynamic characteristic such as in vivo half-life. In some embodiments, the half-life ofμlhGH is increased at least about 10, 20, 30, 40, 50, 60, 70, 80, 90 percent, 2- fold, 5-fold, 6-fold, 7-μlfold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold,μl19-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 50-fold, or at least about 100-fold over anμlunmodified polypeptide.


[311] In some embodiments, the hydroxylamine-terminal PEG derivative will have the
structure:
RO-(CH2CH20)n-0-(CH2)m-0-NH2
where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10 and n is 100-1,000 (i.e., average
molecular weight is between 5-40 kDa).
[312] In some embodiments, the hydrazine- or hydrazide-containing PEG derivative will
have the structure:
RO-(CH2CH2OVO-(CH2)m-X-NH-NH2
where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10 and n is 100-1,000 and X is
optionally a carbonyl group (C=0) that can be present or absent.
[313] In some embodiments, the semicarbazide-containing PEG derivative will have the
structure:
RO-(CH2CH20)n -0-(CH2)m-NH-C(0)-NH-NH2
where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10 and n is 100-1,000.
... '»
[314] In another embodiment of the invention, a hGH polypeptide comprising a carbonyl-
containing amino acid is modified with a PEG derivative that contains a terminal hydroxylamine, .
hydrazide, hydrazine, or semicarbazide moiety that is linked to the PEG backbone by means of an '
amide linkage.
[315] In some embodiments, the hydroxylamine-terminal PEG derivatives have the
structure:
RO-(CH2CH20)n-0-(CH2)2.NH-C(0)(CH2)m-0-NH2
where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10 and n is 100-1,000 (i.e., average
molecular weight is between 5-40 kDa).
[316] In some embodiments, the hydrazine- or hydrazide-containing PEG derivatives have
the structure:
RO-(CH2CH20)n-0-(CH2)2-NH-C(0)(CH2)m-X-NH-NH2
where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10, n is 100-1,000 and X is optionally
a carbonyl group (C=0) that can be present or absent.
[317] In some embodiments, the semicarbazide-containing PEG derivatives have the
structure:
RO-(CH2CH20)n-0-(CH2)2-NH-C(0)(CH2)m-NH-C(0)-NH-NH2

where K is a simple alkyl (methyl, ethyl, propyl, etc.;, m is 2-10 and n is 100-1,000.
[318] In another embodiment of the invention, a hGH polypeptide comprising a carbonyl-
containing amino acid is modified with a branched PEG derivative that contains a terminal
hydrazine, hydroxylamine, hydrazide or semicarbazide moiety, with each chain of the branched
PEG having a MW ranging from 10-40 kDa. Each chain of the branched PEG may have a MW
ranging from 5-20 kDa.
[319] In another embodiment of the invention, a hGH polypeptide comprising a non-
naturally encoded amino acid is modified with a PEG derivative having a branched structure. For
instance, in some embodiments, the hydrazine- or hydrazide-terminal PEG derivative will have the
following structure:
[RO-(CH2CH20)n-O-(CH2)2-NH-C(O)]2CM(CH2)m"X-NH-NH2
where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10 and n is 100-1,000, and X is
optionally a carbonyl group (C=0) that can be present or absent.
[320] In some embodiments, the PEG derivatives containing a semicarbazide group will
have the structure:
[RO-(CH2CH20)n-0-(CH2^
where R is a simple alkyl (methyl, ethyl, propyl, etc.), X is optionally NH, O, S, C(O) or not j,
present, m is 2-10 and n is 100-1,000.
[321] In some embodiments, the PEG derivatives containing a hydroxylamine group will ;
have the structure:

where R is a simple alkyl (methyl, ethyl, propyl, etc.), X is optionally NH, O, S, C(0) or not
present m is 2-10 and n is 100-1,000.
[322] The degree and sites at which the water soluble polymer(s) are linked to the hGH
polypeptide can modulate the binding of the hGH polypeptide to the hGH polypeptide receptor at
Site 1. In some embodiments, the linkages are arranged such that the hGH polypeptide binds the
hGH polypeptide receptor at Site 1 with a Kd of about 400 nM or lower, with a Kd of 150 nM or
lower, and in some cases with a Kd of 100 nM or lower, as measured by an equilibrium binding
assay, such as that described in Spencer et al9 J. Biol. Chem., 263:7862-7867 (1988) for hGH.
[323] Methods and chemistry for activation of polymers as well as for conjugation of
peptides are described in the literature and are known in the art. Commonly used methods for

activation of polymers include, but are not limited to, activation of functional groups with cyanogenμlbromide, periodate, glutaraldehyde, biepoxides, epichlorohydrin, divinylsulfone, carbodiimide,μlsulfonyl halides, trichlorotriazine, eta (see, R. F. Taylor, (1991), PROTEIN IMMOBILISATION.μlFUNDAMENTAL AND APPLICATIONS, Marcel Dekker, N.Y.; S. S. Wong, (1992), CHEMISTRY OFΜLPROTEIN CONJUGATION AND CROSSLINKING, CRC Press, Boca Raton; G. T. Hermanson et aL,μl(1993), IMMOBILIZED AFFINITY LIGAND TECHNIQUES, Academic Press, N.Y.; Dunn, RX., et al,μlEds. POLYMERIC DRUGS AND DRUG DELIVERY SYSTEMS, ACS Symposium Series Vol.μl469, American Chemical Society, Washington, D.C. 1991).
[324] Several reviews and monographs on the functionalization and conjugation of PEG are
available. See, for example, Harris, MacromoL Chem. Phys. C25: 325-373 (1985); Scouten,μlMethods in Enzymology 135: 30-65 (1987); Wong et al, Enzyme Microb. Technol 14: 866-874μl(1992); Delgado et al, Critical Reviews in Therapeutic Drug Carrier Systems 9: 249-304 (1992);μlZalipsky, Bioconjugate Chem. 6: 150-165 (1995).
[325] Methods for activation of polymers can also be found in WO 94/17039, U.S. Pat. No. .
5,324,844, WO 94/18247, WO 94/04193, U.S. Pat. No. 5,219,564, U.S. Pat. No. 5,122,614, WO 'μl90/13540, U.S. Pat No. 5,281,698, and WO 93/15189, and for conjugation between activatedμlpolymers and enzymes including but not limited to Coagulation Factor VIII (WO 94/15625), μlhemoglobin (WO 94/09027), oxygen carrying molecule (U.S. Pat. No. 4,412,989), ribonuclease andμlsuperoxide dismutase (Veronese at aL, App. Biochem. Biotech. 11: 141-52 (1985)). All references μland patents cited are incorporated by reference herein.
[326] PEGylation (i.e., addition of any water soluble polymer) of hGH polypeptides
containing a non-naturally encoded amino acid, such as p-azido-L-phenylalanine, is carried out byμlany convenient method. For example, hGH polypeptide is PEGylated with an alkyne-terminatedμlmPEG derivative. Briefly, an excess of solid mPEG(5000)-0-CH2-OCH is added, with stirring, toμlan aqueous solution ofp-azido-L-Phe-containing hGH polypeptide at room temperature. Typically,μlthe aqueous solution is buffered with a buffer having a pKa near the pH at which the reaction is toμlbe carried out (generally about pH 4-10). Examples of suitable buffers for PEGylation at pH 7.5,μlfor instance, include, but are not limited to, HEPES, phosphate, borate, TRIS-HC1, EPPS, and TES.μlThe pH is continuously monitored and adjusted if necessary. The reaction is typically allowed toμlcontinue for between about 1-48 hours.

[327] The reaction products are subsequently subjected to hydrophobic interaction
chromatography to separate the PEGylated hGH polypeptide variants from free mPEG(5000)-O-μlCH2-OCH and any high-molecular weight complexes of the PEGylated hGH polypeptide whichμlmay form when unblocked PEG is activated at both ends of the molecule, thereby crosslinking hGHμlpolypeptide variant molecules. The conditions during hydrophobic interaction chromatography areμlsuch that free mPEG(5000)-0-CH2-OCH flows through the column, while any crosslinkedμlPEGylated hGH polypeptide variant complexes elute after the desired forms, which contain oneμlhGH polypeptide variant molecule conjugated to one or more PEG groups. Suitable conditions varyμldepending on the relative sizes of the cross-linked complexes versus the desired conjugates and areμlreadily determined by those of ordinary skill in the art. The eluent containing the desiredμlconjugates is concentrated by ultrafiltration and desalted by diafiltration.
[328] If necessary, the PEGylated hGH polypeptide obtained from the hydrophobic
chromatography can be purified further by one or more procedures known to those of ordinary skillμlin the art including, but are not limited to, affinity chromatography, anion- or cation-exchangeμlchromatography (using, including but not limited to, DEAE SEPHAROSE); chromatography on'μlsilica; reverse phase HPLC; gel filtration (using, including but not limited to, SEPHADEX G-75); -μlhydrophobic interaction chromatography; size-exclusion chromatography, metal-chelate μlchromatography; ultrafiltration/diafiltration; ethanol precipitation; ammonium sulfate precipitation;μlchromatofocusing; displacement chromatography; electrophoretic procedures (including but not μllimited to preparative isoelectric focusing), differential solubility (including but not limited toμlammonium sulfate precipitation), or extraction. Apparent molecular weight may be estimated byμlGPC by comparison to globular protein standards (Preneta, AZ in PROTEIN PURIFICATION METHODS,μlA PRACTICAL APPROACH (Harris & Angal, Eds.) IRL Press 1989,293-306). The purity of the hGH-μlPEG conjugate can be assessed by proteolytic degradation (including but not limited to, trypsinμlcleavage) followed by mass spectrometry analysis. Pepinsky RB, et al.B J. PharrncoL & Exp. Ther.μl297(3):1059-66(2001).
[329] A water soluble polymer linked to an amino acid of a hGH polypeptide of the
invention can be further derivatized or substituted without limitation.

Other PEG derivatives and General PEGylation techniques
[330] Azide-containing, alkyne-containing, and phosphine-containing PEG derivatives are
described in U.S. Patent Application 11/046,432, entitled "Modifed Human Growth HormoneμlPolypeptides and Their Uses".
[331] Other exemplary PEG molecules that may be linked to hGH polypeptides, as well as
PEGylation methods include those described in, e.g., U.S. Patent Publication No. 2004/0001838;μl2002/0052009; 2003/0162949; 2004/0013637; 2003/0228274; 2003/0220447; 2003/0158333;μl2003/0143596; 2003/0114647; 2003/0105275; 2003/0105224; 2003/0023023; 2002/0156047;μl2002/0099133; 2002/0086939; 2002/0082345; 2002/0072573; 2002/0052430; 2002/0040076;μl2002/0037949; 2002/0002250; 2001/0056171; 2001/0044526; 2001/0021763; U.S. Patent No.μl6,646,110; 5,824,778; 5,476,653; 5,219,564; 5,629,384; 5,736,625; 4,902,502; 5,281,698;μl5,122,614; 5,473,034; 5,516,673; 5,382,657; 6,552,167; 6,610,281; 6,515,100; 6,461,603;μl6,436,386; 6,214,966; 5,990,237; 5,900,461; 5,739,208; 5,672,662; 5,446,090; 5,808,096;μl5,612,460; 5,324,844; 5,252,714; 6,420,339; 6,201,072; 6,451,346; 6,306,821; 5,559,213; μl5,747,646; 5,834,594; 5,849,860; 5,980,948; 6,004,573; 6,129,912; WO 97/32607, EP 229,108, EP μl402,378, WO 92/16555, WO 94/04193, WO 94/14758, WO 94/17039, WO 94/18247, WO μl94/28024, WO 95/00162, WO 95/11924, WO95/13090, WO 95/33490, WO 96/00080, WO μl97/18832, WO 98/41562, WO 98/48837, WO 99/32134, WO 99/32139, WO 99/32140, WO μl96/40791, WO 98/32466, WO 95/06058, EP 439 508, WO 97/03106, WO 96/21469, WO 95/13312, μlEP 921 131, WO 98/05363, EP 809 996, WO 96/41813, WO 96/07670, EP 605 963, EP 510 356,μlEP 400 472, EP 183 503 and EP 154 316, which are incorporated by reference herein. Any of theμlPEG molecules described herein may be used in any form, including but not limited to, single chain,μlbranched chain, multiarm chain, single functional, bi-functional, multi-functional, or anyμlcombination thereof. U.S. Patent Application Serial No. 11/046,432 entitled 'Modified HumanμlGrowth Hormone Polypeptides and Their Uses," which is incorporated by reference, providesμlfurther discussion of PEG and forms thereof.
IX. Measurement of Potency, Functional In Vivo Half-Life, and Pharmacokinetic
Parameters
[332] An important aspect of the invention is the prolonged biological half-life that is
obtained by construction of the hGH polypeptide with or without conjugation of the polypeptide toμla water soluble polymer moiety. The rapid decrease of hGH polypeptide serum concentrations has

made it important to evaluate biological responses to treatment with conjugated and non-conjugated
hGH polypeptide and variants thereof. The conjugated and non-conjugated hGH polypeptide and
variants thereof of the present invention may have prolonged serum half-lives also after
subcutaneous or i.v. administration, making it possible to measure by, e.g. ELISA method or by a
primary screening assay. ELISA or RIA kits from either BioSource International (Camarillo, CA)
or Diagnostic Systems Laboratories (Webster, TX) may be used. Measurement of in vivo biological
half-life is carried out as described herein.
[333] The potency and functional in vivo half-life of an hGH polypeptide comprising a non-
naturally encoded amino acid can be determined according to the protocol described in Clark, R., et
aμJ. Biol Chem. 271(36):21969-21977 (1996).
[334] Pharmacokinetic parameters for a hGH polypeptide comprising a non-naturally
encoded amino acid can be evaluated in normal Sprague-Dawley male rats (N=5 animals per .
treatment group). Animals will receive either a single dose of 25 ug/rat iv or 50 ug/rat sc, and
approximately 5-7 blood samples will be taken according to a pre-defined time course, generally
covering about 6 hours for a hGH polypeptide comprising a nbn-naturally encoded amino acid not
conjugated to a water soluble polymer and about 4 days for a hGH polypeptide comprising a non-
naturally encoded amino acid and conjugated to a water soluble polymer. Pharmacokinetic data for
hGH polypeptides is well-studied in several species and can be compared directly to the data .
obtained for hGH polypeptides comprising a non-naturally encoded amino acid. See Mordenti J., et
al, Pharm. Res. 8(11):1351-59 (1991), which is incorporated by reference herein, for studies related
to hGH.
[335] Pharmacokinetic parameters can also be evaluated in a primate, e.g., cynomolgus
monkeys. A single injection may be administered either subcutaneously or intravenously, and
serum hGH levels are monitored over time.
[336] The specific activity of hGH polypeptides in accordance with this invention can be
determined by various assays known in the art. The biological activity of the hGH polypeptide
muteins, or fragments thereof, obtained and purified in accordance with this invention can be tested
by methods described or referenced herein or known to those of ordinary skill in the art.
X. Administration and Pharmaceutical Compositions
[337] The polypeptides or proteins of the invention (including but not limited to, hGH,
synthetases, proteins comprising one or more unnatural amino acid, etc.) are optionally employed

for therapeutic uses, including but not limited to, in combination with a suitable pharmaceuticalμlcarrier. Such compositions, for example, comprise a therapeutically effective amount of theμlcompound, and a pharmaceutically acceptable carrier or excipient. Such a carrier or excipientμlincludes, but is not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and/orμlcombinations thereof. The formulation is made to suit the mode of administration. In general,μlmethods of administering proteins are known to those of ordinary skill in the art and can be appliedμlto administration of the polypeptides of the invention.
[338] Therapeutic compositions comprising one or more polypeptide of the invention are
optionally tested in one or more appropriate in vitro and/or in vivo animal models of disease, toμlconfirm efficacy, tissue metabolism, and to estimate dosages, according to methods known to thoseμlof ordinary skill in the art. In particular, dosages can be initially determined by activity, stability orμlother suitable measures of unnatural herein to natural amino acid homologues (including but notμllimited to, comparison of a hGH polypeptide modified to include one or more unnatural amino.μlacids to a natural amino acid hGH polypeptide), i.e., in a relevant assay.
[339] Administration is by any of the routes normally used for introducing a molecule into
ultimate contact with blood or tissue cells. The unnatural amino acid polypeptides of the inventionμlare administered in any suitable manner, optionally with one or more pharmaceutically acceptableμlcarriers. Suitable methods of administering such polypeptides in the context of the presentμlinvention to a patient are available, and, although more than one route can be used to administer aμlparticular composition, a particular route can often provide a more immediate and more effectiveμlaction or reaction than another route.
[340] Pharmaceutically acceptable carriers are determined in part by the particular
composition being administered, as well as by the particular method used to administer theμlcomposition. Accordingly, there is a wide variety of suitable formulations of pharmaceuticalμlcompositions of the present invention.
[341] hGH polypeptides of the invention, including but not limited to PEGylated hGH,
may be administered by any conventional route suitable for proteins or peptides, including, but notμllimited to parenterally, e.g. injections including, but not limited to, subcutaneously or intravenouslyμlor any other form of injections or infusions. Polypeptide compositions can be administered by aμlnumber of routes including, but not limited to oral, intravenous, intraperitoneal, intramuscular,

transdermal, subcutaneous, topical, sublingual, or rectal means, uompositions composing non-μlnatural amino acid polypeptides, modified or unmodified, can also be administered via liposomes.μlSuch administration routes and appropriate formulations are generally known to those of skill in theμlart. The hGH polypeptide, including but not limited to PEGylated hGH, comprising a non-naturallyμlencoded amino acid may be used alone or in combination with other suitable components such as aμlpharmaceutical carrier.
[342] The hGH polypeptide comprising a non-natural amino acid, alone or in combination
with other suitable components, can also be made into aerosol formulations (i.e., they can beμl"nebulized') to be administered via inhalation. Aerosol formulations can be placed into pressurizedμlacceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like.
[343J Formulations suitable for parenteral administration, such as, for example, by
intraarticular (in the joints), intravenous, intramuscular, intradermal, intraperitoneal, andμlsubcutaneous routes, include aqueous and non-aqueous, isotonic sterile injection solutions, whichμlcan contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic withμlthe blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that canμlinclude suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. Theμlformulations of hGH can be presented in unit-dose or multi-dose sealed containers, such as ampulesμland vials.
[344] Parenteral administration and intravenous administration are preferred methods of
administration. In particular, the routes of administration already in use for natural amino acidμlhomologue therapeutics (including but not limited to, those typically used for EPO, GH, G-CSF,μlGM-CSF, IFNs, interleukins, antibodies, and/or any other pharmaceutically delivered protein),μlalong with formulations in current use, provide preferred routes of administration and formulationμlfor the polypeptides of the invention.
[345] The viscosity and formulation profiles of the polypeptide composition may allow
administration with a small gauge needle by using smaller plunger pressures compared toμlconventional formulations. Smaller gauge needles including, but not limited to, 27, 28, 29, 30, orμl31 gauge needles may be used to administer the polypeptide composition of the present invention toμlsubjects. U.S. Patent No. 6,875,432, which is incorporated by reference herein, discusses theμlviscosity of protein formulations and problems associated with viscosity of protein formulations for

subcutaneous administration. Small gauge needles are an advantage due to reduced pain ofμlinjection which improves patient compliance with dosing regimens. Smaller plunger pressuresμlneeded for injecting render the hGH polypeptide, including but not limited to PEGylated hGH,μleasier to administer with short injection duration. Types of needles include, but are not limited to,μlpen needles, thin walled, normal walled, and luer needles.
[346] Viscosity may be measured by standard techniques known to those of ordinary skill
in the art. 'Viscosity" may be "kinematic viscosity" or "absolute viscosity." "Kinematic viscosity"μlis a measure of the resistive flow of a fluid under the influence of gravity. When two fluids of equalμlvolume are placed in identical capillary viscometers and allowed to flow by gravity, a viscous fluidμltakes longer than a less viscous fluid to flow through the capillary. If one fluid takes 200 seconds toμlcomplete its flow and another fluid takes 400 seconds, the second fluid is twice as viscous as theμlfirst on a kinematic viscosity scale. "Absolute viscosity", sometimes called dynamic or simple ,μlviscosity, is the product of kinematic viscosity and fluid density:μlAbsolute Viscosity==Kinematic Viscosity x Density
The dimension of kinematic viscosity is L2 /T where L is a length and T is a time. Commonly,μlkinematic viscosity is expressed in centistokes (cSt). The SI unit of kinematic viscosity is mm /s,μl. which is 1 cSt. Absolute viscosity is expressed in units of centipoise (cP). The SI unit of absoluteμlviscosity is the milliPascal-second (mPa-s), where 1 cP=l mPa-s.
[347] The dose administered to a patient, in the context of the present invention, is
sufficient to have a beneficial therapeutic response in the patient over time, or other appropriateμlactivity, depending on the application. The dose is determined by the efficacy of the particularμlvector, or formulation, and the activity, stability or serum half-life of the unnatural amino acidμlpolypeptide employed and the condition of the patient, as well as the body weight or surface area ofμlthe patient to be treated. The size of the dose is also determined by the existence, nature, and extentμlof any adverse side-effects that accompany the administration of a particular vector, formulation, orμlthe like in a particular patient
[348] In determining the effective amount of the vector or formulation to be administered
in the treatment or prophylaxis of disease (including but not limited to, cancers, inherited diseases,μldiabetes, ADDS, or the like), the physician evaluates circulating plasma levels^ formulation

toxicities, progression of the disease, and/or where relevant, the production of anti- unnatural aminoμlacid polypeptide antibodies.
[349] The dose administered, for example, to a 70 kilogram patient, is typically in the
range equivalent to dosages of currently-used therapeutic proteins, adjusted for the altered activityμlor serum half-life of the relevant composition. The vectors or pharmaceutical formulations of thisμlinvention can supplement treatment conditions by any known conventional therapy, includingμlantibody administration, vaccine administration, administration of cytotoxic agents, natural aminoμlacid polypeptides, nucleic acids, nucleotide analogues, biologic response modifiers, and the like.
[350] For administration, formulations of the present invention are administered at a rate
determined by the LD-50 or ED-50 of the relevant formulation, and/or observation of any side-μleffects of the unnatural amino acid polypeptides at various concentrations, including but not limitedμlto, as applied to the mass and overall health of the patient. Administration can be accomplished viaμlsingle or divided doses.
[351J If a patient undergoing infusion of a formulation develops fevers, chills, or muscle
aches, he/she receives the appropriate dose of aspirin, ibuprofeh, acetaminophen or other pain/feverμlcontrolling drug. Patients who experience reactions to the infusion such as fever, muscle aches, andμlchills are premedicated 30 minutes prior to the future infusions with either aspirin, acetaminophen,μlor, including but not limited to, diphenhydramine. Meperidine is used for more severe chills andμlmuscle aches that do not quickly respond to antipyretics and antihistamines. Cell infusion is slowedμlor discontinued depending upon the severity of the reaction.
[352] Human hGH polypeptides of the invention can be administered directly to a
mammalian subject. Administration is by any of the routes normally used for introducing hGHμlpolypeptide to a subject The hGH polypeptide compositions according to embodiments of theμlpresent invention include those suitable for oral, rectal, topical, inhalation (including but not limitedμlto, via an aerosol), buccal (including but not limited to, sub-lingual), vaginal, parenteral (includingμlbut not limited to, subcutaneous, intramuscular, intradermal, intraarticular, intrapleural,μlintraperitoneal, inracerebral, intraarterial, or intravenous), topical (i.e., both skin and mucosalμlsurfaces, including airway surfaces) and transdermal administration, although the most suitableμlroute in any given case will depend on the nature and severity of the condition being treated.μlAdministration can be either local or systemic. The formulations of compounds can be presented in

unit-dose or multi-dose sealed containers, such as ampules and vials. hUM polypeptides ot theμlinvention can be prepared in a mixture in a unit dosage injectable form (including but not limited to,μlsolution, suspension, or emulsion) with a pharmaceutically acceptable carrier or excipient. hGHμlpolypeptides of the invention can also be administered by continuous infusion (using, including butμlnot limited to, minipumps such as osmotic pumps), single bolus or slow-release depot formulations.μl[353] Formulations suitable for administration include aqueous and non-aqueous solutions,
isotonic sterile solutions, which can contain antioxidants, buffers, bacteriostats, and solutes thatμlrender the formulation isotonic, and aqueous and non-aqueous sterile suspensions that can includeμlsuspending agents, solubilizers, thickening agents, stabilizers, and preservatives. Solutions andμlsuspensions can be prepared from sterile powders, granules, and tablets of the kind previouslyμldescribed.
[354] Freeze-drying is a commonly employed technique for presenting proteins which
serves to remove water from the protein preparation of interest Freeze-drying, or lyophilization, is aμlprocess by which the material to be dried is first frozen and then the ice or frozen solvent isμlremoved by sublimation in a vacuum environment. An excipient may be included in pre-lyophilizedμlformulations to enhance stability during the freeze-drying process and/or to improve stability of theμllyophilized product upon storage. Pikal, M. Biopharm. 3(9)26-30 (1990) and Arakawa et al. Pharm.μlRes. 8(3):285-291 (1991).
[355] The spray drying of pharmaceuticals is also known to those of ordinary skill in the art.
For example, see Broadhead, J. et al., "The Spray Drying of Pharmaceuticals," in Drug Dev. Ind.μlPharm, 18 (11 & 12), 1169-1206(1992). In addition to small molecule pharmaceuticals, a variety ofμlbiological materials have been spray dried and these include: enzymes, sera, plasma, microμlorganisms and yeasts. Spray drying is a useful technique because it can convert a liquidμlpharmaceutical preparation into a fine, dustless or agglomerated powder in a one-step process. Theμlbasic technique comprises the following four steps: a) atomization of the feed solution into a spray,μlb) spray-air contact; c) drying of the spray; and d) separation of the dried product from the dryingμlair. U.S. Patent Nos. 6,235,710 and 6,001,800, which are incorporated by reference herein, describeμlthe preparation of recombinant erythropoietin by spray drying.
[3561 The pharmaceutical compositions and formulations of the present invention may
comprise a pharmaceutically acceptable carrier, excipient, or stabilizer. Pharmaceuticallyμlacceptable carriers are determined in part by the particular composition being administered, as well

as by the particular method used to administer the composition. Accordingly, there is a wideμlvariety of suitable formulations of pharmaceutical compositions (including optionalμlpharmaceutically acceptable carriers, excipients, or stabilizers) of the present invention {see, e.g.,μlRemington's Pharmaceutical Sciences', 17 ed. 1985)).
[357] Suitable carriers include, but are not limited to, buffers containing succinate,
phosphate, borate, HEPES, citrate, histidine or histidine derivatives, imidazole, acetate, bicarbonate,μland other organic acids; antioxidants including but not limited to, ascorbic acid; low molecularμlweight polypeptides including but not limited to those less than about 10 residues; proteins,μlincluding but not limited to, serum albumin, gelatin, or immunoglobulins; hydrophilic polymersμlincluding but not limited to, polyvinylpyrrolidone; amino acids including but not limited to, glycine,μlglutamine, histidine or histidine derivatives, methionine, asparagine, arginine, glutamate, or lysine;μlmonosaccharides, disaccharides, and other carbohydrates, including but not limited to, trehalose,μlsucrose, glucose, mannose, or dextrins; chelating agents including but not limited to, EDTA;μldivalent metal ions including but not limited to, zinc, cobalt, or copper; sugar alcohols including butμlnot limited to, mannitol or sorbitol; salt-forming counter ions including but not limited to, sodium;μland/or nonionic surfactants including but not limited to Tween™ (including but not limited to,μlTween 80 (polysorbate 80) and Tween 20 (polysorbate 20; PS20)), Pluronics™ and other pluronicμlacids, including but not limited to, pluronic acid F68 (poloxamer 188), or PEG. Suitableμlsurfactants include for example but are not limited to polyethers based upon poly(ethylene oxide)-μlpoly(propylene oxide)-poly(ethylene oxide), i.e., (PEO-PPO-PEO), or poly(propylene oxide)-μlpoly(ethylene oxide)-poly(propylene oxide), i.e., (PPO-PEO-PPO), or a combination thereof. PEO-μlPPO-PEO and PPO-PEO-PPO are commercially available under the trade names Pluronics™, R-μlPluronics™, Tetronics™ and R-Tetronics™ (BASF Wyandotte Corp., Wyandotte, Mich.) and areμlfurther described in U.S. Pat. No. 4,820,352 incorporated herein in its entirety by reference. Otherμlethylene/polypropylene block polymers may be suitable surfactants. A surfactant or a combinationμlof surfactants may be used to stabilize PEGylated hGH against one or more stresses including butμlnot limited to stress that results from agitation. Some of the above may be referred to as "bulkingμlagents." Some may also be referred to as "tonicity modifiers." Additional carriers include, but areμlnot limited to, ammonium sulfate ((NH^SO^. Ammonium sulfate ((NKL^SGO may be used in aμlformulation of the present invention at a range of about 0.1 mM to about 200 mM, including but notμllimited to, 200 mM, 190 mM, 180 mM, 170 mM, 160 mM, 150 mM, 140 mM, 130 mM, 120 mM,

110 mM, 100 mM, 95 mM, 90 raM, 85 mM, 80 mM, 75 mM, 70 mM, 65 mM, 60 mM, 55 mM, 50μlmM, 45 mM, 40 mM, 35 mM, 30 mM, 25 mM, 20 mM, 15 mM, 10 mM, 5 mM, 1 mM, 0.9 mM,μl0.8 mM, 0.7 mM, 0.6 mM, 0.5 mM, 0.4 mM, 0.3 mM, 0.2 mM, and 0.1 mM. Histidine may be usedμlin a formulation of the present invention at a range of about 0.1 mM to about 200 mM, includingμlbut not limited to, 200 mM, 190 mM, 180 mM, 170 mM, 160 mM, 150 mM, 140 mM, 130 mM,μl120 mM, 110 mM, 100 mM, 95 mM, 90 mM, 85 mM, 80 mM, 75 mM, 70 mM, 65 mM, 60 mM, 55μlmM, 50 mM, 45 mM, 40 mM, 35 mM, 30 mM, 25 mM, 20 mM, 15 mM, 10 mM, 5 mM, 1 mM, 0.9μlmM, 0.8 mM, 0.7 mM, 0.6 mM, 0.5 mM, 0.4 mM, 0.3 mM, 0.2 mM, and 0.1 mM. In someμlembodiments, Histidine is between about 5 mM and about 30 mM in the formulation.μl[358] A buffer of a formulation of the present invention may be in a range of about 0.1 mM
to about 200 mM, including but not limited to, 200 mM, 190 mM, 180 mM, 170 mM, 160 mM, 150μlmM, 140 mM, 130 mM, 120 mM, 110 mM, 100 mM, 95 mM, 90 mM, 85 mM, 80 mM, 75 mM, 70μlmM, 65 mM, 60 mM, 55 mM, 50 mM, 45 mM, 40 mM, 35 mM, 30 mM, 25 mM, 20 mM, 19 mM,μl18 mM, 17 mM, 16 mM, 15 mM, 14 mM, 13 mM, 12 mM, 11 mM, 10 mM, 9 mM, 8 mM, 7 mM, 6μlmM, 5 mM, 4 mM, 3 mM, 2 mM, 1 mM, 0.9 mM, 0.8 mM, 0.7 mM, 0.6 mM, 0.5 mM, 0.4 mM, 0.3μlmM, 0.2 mM, and 0.1 mM. In some embodiments, the buffer is at a concentration of about 0.1 mMμlto about 200 mM. In some embodiments, the buffer is at a concentration of about 1 mM to about 75μlmM. m some embodiments, the buffer is at a concentration of about 1 mM to about 20 mM. Inμlsome embodiments, the buffer is at a concentration of about 5 mM to about 30 mM. The buffer of aμlformulation of the present invention may provide a pH range from about pH 4.0 to about pH 8.5,μlincluding but not limited to, pH 8.5, pH 8.0, pH 7.5, pH 7.0, pH 6.5, pH 6.0, pH 5.5, pH 5.0, pH 4.5,μland pH 4.0. The pH is any tenth pH value within those enumerated above; for example, pH 8.5, pHμl8.4, pH 8.3, pH 8.2, pH 8.1, pH 8.0, pH 7.9, pH 7.8, pH 7.7, pH 7.6, pH 7.5, pH 7.4, pH 7.3, pH 7.2,μlpH 7.1, pH 7.0, pH 6.9, pH 6.8, pH 6.7, pH 6.6, pH 6.5, pH 6.4, pH 6.3, pH 6.2, pH 6.1, pH 6.0, pHμl5.9, pH 5.8, pH 5.7, pH 5.6, pH 5.5, pH 5.4, pH 5.3, pH 5.2, pH 5.1, pH 5.0, pH 4.9, pH 4.8, pH 4.7,μlpH 4.6, pH 4.5, pH 4.4, pH 4.3, pH 4.2, pH 4.1, and pH 4.0. In some embodiments, the pH isμlbetween about pH 6.0 and about pH 7.3. In some embodiments, the pH is between about pH 5.5μland about 8.0. In some embodiments, the pH is between about 4.0 and about 8.5. In someμlembodiments, the pH is between about 4.0 and about 7.5. In some embodiments, the pH is betweenμlabout 6.0 and about 7.5.

I35yj An amino acid ot a tormuJation oi tne present invention may be m a range ot about
0.1 g/L to 100 g/L, including but not limited to, 100 g/L, 95 g/L, 90 g/L, 85 g/L, 80 g/L, 75 g/L, 70μlg/L, 65 g/L, 60 g/L, 55 g/L, 50 g/L, 45 g/L, 40 g/L, 35 g/L, 30 g/L, 25 g/L, 20 g/L, 19 g/L, 18 g/L,μl17 g/L, 16 g/L, 15 g/L, 14 g/L, 13 g/L, 12 g/L, 11 g/L, 10 g/L, 9 g/L, 8 g/L, 7 g/L, 6 g/L, 5 g/L, 4μlg/L, 3 g/L, 2 g/L, 1 g/L, 0.9 g/L, 0.8 g/L, 0.7 g/L, 0.6 g/L, 0.5 g/L, 0.4 g/L, 0.3 g/L, 0.2 g/L, and 0.1μlg/L. In some embodiments, the amino acid is between about 0.1 g/L and 60 g/L. In someμlembodiments, the amino acid is between about 0.1 g/L and 100 g/L. In some embodiments, theμlamino acid is between about 1 g/L and 50 g/L. In some embodiments, the amino acid is betweenμlabout 5 g/L and 25 g/L.
[360] A sugar alcohol of a formulation of the present invention may be in a range of about
0.1 g/L to 100 g/L, including but not limited to, 100 g/L, 95 g/L, 90 g/L, 85 g/L, 80 g/L, 75 g/L, 70μlg/L, 65 g/L, 60 g/L, 55 g/L, 50 g/L, 45 g/L, 40 g/L, 35 g/L, 30 g/L, 25 g/L, 20 g/L, 19 g/L, 18 g/L,μl17 g/L, 16 g/L, 15 g/L, 14 g/L, 13 g/L, 12 g/L, 11 g/L, 10 g/L, 9 g/L, 8 g/L, 7 g/L, 6 g/L, 5 g/L, 4μlg/L, 3 g/L, 2 g/L, 1 g/L, 0.9 g/L, 0.8 g/L, 0.7 g/L, 0.6 g/L, 0.5 g/L, 0.4 g/L, 0.3 g/L, 0.2 g/L, and 0.1μlg/L. In some embodiments, the sugar alcohol is between about 0.1 g/L and about 60 g/L. In someμlembodiments, the sugar alcohol is between about 0.1 g/L and about 100 g/L. In someμlembodiments, the sugar alcohol is between about 1 g/L and about 50 g/L. In some embodiments,μlthe sugar alcohol is between about 2 g/L and about 25 g/L.
[361] A carbohydrate of a formulation of the present invention may be in a range of about
0.1 g/L to 100 g/L, including but not limited to, 100 g/L, 95 g/L, 90 g/L, 85 g/L, 80 g/L, 75 g/L, 70μlg/L, 65 g/L, 60 g/L, 55 g/L, 50 g/L, 45 g/L, 40 g/L, 35 g/L, 30 g/L, 25 g/L, 20 g/L, 19 g/L, 18 g/L,μl17 g/L, 16 g/L, 15 g/L, 14 g/L, 13 g/L, 12 g/L, 11 g/L, 10 g/L, 9 g/L, 8 g/L, 7 g/L, 6 g/L, 5 g/L, 4μlg/L, 3 g/L, 2 g/L, 1 g/L, 0.9 g/L, 0.8 g/L, 0.7 g/L, 0.6 g/L, 0.5 g/L, 0.4 g/L, 0.3 g/L, 0.2 g/L, and 0.1μlg/L. In some embodiments, the carbohydrate is at about 0.1 g/L to about 100 g/L. In someμlembodiments, the carbohydrate is at about 1 g/L to about 50 g/L. In some embodiments, theμlcarbohydrate is at about 2 g/L to about 25 g/L. In some embodiments, the carbohydrate is at aboutμl0.1 g/L to about 50 g/L.
[362] A disaccharide of a formulation of the present invention may be in a range of about
0.1 g/L to 100 g/L, including but not limited to, 100 g/L, 95 g/L, 90 g/L, 85 g/L, 80 g/L, 75 g/L, 70μlg/L, 65 g/L, 60 g/L, 55 g/L, 50 g/L, 45 g/L, 40 g/L, 35 g/L, 30 g/L, 25 g/L, 20 g/L, 19 g/L, 18 g/L,μl17 g/L, 16 g/L, 15 g/L, 14 g/L, 13 g/L, 12 g/L, 11 g/L, 10 g/L, 9 g/L, 8 g/L, 7 g/L, 6 g/L, 5 g/L, 4

g/L, 3 g/L, 2 g/L, 1 g/L, 0.9 g/L, 0.8 g/L, 0.7 g/L, 0.6 g/L, 0.5 g/L, 0.4 g/L, 0.3 g/L, 0.2 g/L, and 0.1μlg/L. In some embodiments, the disaccharide is between about 0.1 g/L and about 50 g/L.μl[363] A monosaccharide of a formulation of the present invention may be in a range of
about 0.1 g/L to 100 g/L, including but not limited to, 100 g/L, 95 g/L, 90 g/L, 85 g/L, 80 g/L, 75μlg/L, 70 g/L, 65 g/L, 60 g/L, 55 g/L, 50 g/L, 45 g/L, 40 g/L, 35 g/L, 30 g/L, 25 g/L, 20 g/L, 19 g/L,μl18 g/L, 17 g/L, 16 g/L, 15 g/L, 14 g/L, 13 g/L, 12 g/L, 11 g/L, 10 g/L, 9 g/L, 8 g/L, 7 g/L, 6 g/L, 5μlg/L, 4 g/L, 3 g/L, 2 g/L, 1 g/L, 0.9 g/L, 0.8 g/L, 0.7 g/L, 0.6 g/L, 0.5 g/L, 0.4 g/L, 0.3 g/L, 0.2 g/L,μland 0.1 g/L.
[364] A non-ionic surfactant of a formulation of the present invention may be in a range of
about 0.01% to about 10%, including but not limited to, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%,μl1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%,μl0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.0085%, 0.008%, 0.0075%, 0.007%, 0.0065%,μl0.006%, 0.0055%, 0.005%, 0.0045%, 0.004%, 0.0035%, 0.003%, 0.0025%, 0.002%, 0.0015%,μl0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002%, andμl0.0001%. In some embodiments, the non-ionic surfactant is at about 0.0001% to about 10%. Inμlsome embodiments, the non-ionic surfactant is at about 0.01% to about 10%. In someμlembodiments, the non-ionic surfactant is at about 0.1% to about 5%. In some embodiments, theμlnon-ionic surfactant is at about 0.1% to about 1%. In some embodiments, the non-ionic surfactantμlis at about 0.0001% to about 1%.
[365] The pharmaceutical quantity of hGH in a formulation of the present invention may be
in a range of about 0.1 mg to about 50 mg, including but not limited to, 50 mg, 49 mg, 48 mg, 47μlmg, 46 mg, 45 mg, 44 mg, 43 mg, 42 mg, 41 mg, 40 mg, 39 mg, 38 mg, 37 mg, 36 mg, 35 mg, 34μlmg, 33 mg, 32 mg, 31 mg, 30 mg, 29.5 mg, 29 mg, 28.5 mg, 28 mg, 27.5 mg, 27 mg, 26.5 mg, 26μlmg, 25.5 mg, 25 mg, 24.5 mg, 24 mg, 23.5 mg, 23 mg, 22.5 mg, 22 mg, 21.5 mg, 21 mg, 20.5 mg,μl20 mg, 19.5 mg, 19 mg, 18.5 mg, 18 mg, 17.5 mg, 17 mg, 16.5 mg, 16 mg, 15.5 mg, 15 mg, 14.5μlmg, 14 mg, 13.5 mg, 13 mg, 12.5 mg, 12 mg, 11.5 mg, 11 mg, 10.5 mg, 10 mg, 9.5 mg, 9 mg, 8.5μlmg, 8 mg, 7.5 mg, 7.0 mg, 6.5 mg, 6.0 mg, 5.5 mg, 5.0 mg, 4.5 mg, 4.0 mg, 3.5 mg, 3.0 mg, 2.5 mg,μl2.0 mg, 1.5 mg, 1.0 mg, 0.9 mg, 0.8 mg, 0.7 mg, 0.6 mg, 0.5 mg, 0.4 mg, 0.3 mg, 0.2 mg, and 0.1μlmg. In some embodiments, the pharmaceutical quantity of hGH in a formulation is between about 2μlmg and about 30 mg. In some embodiments, the pharmaceutical quantity of hGH in a formulationμlis between about 2 mg and about 25 mg. In some embodiments, the pharmaceutical quantity of hGH

in a formulation is between about 0.1 mg and about 30 mg. In some embodiments, theμlpharmaceutical quantity of hGH in a formulation, is between about 0.1 mg and about 8 mg. In someμlembodiments, the pharmaceutical quantity of hGH in a formulation is between about 0.5 mg andμlabout 8 mg. In some embodiments, the pharmaceutical quantity of hGH in a formulation is betweenμlabout 0.5 mg and about 6 mg. hi some embodiments, the pharmaceutical quantity of hGH in aμlformulation is between about 1 mg and about 5 mg.
[366] hGH polypeptides of the invention, including those linked to water soluble polymers
such as PEG can also be administered by or as part of sustained-release systems. Sustained-releaseμlcompositions include, including but not limited to, semi-permeable polymer matrices in the form ofμlshaped articles, including but not limited to, films, or microcapsules. Sustained-release matricesμlinclude from biocompatible materials such as poly(2-hydroxyethyl methacrylate) (Langer et aLy J.μlBiomed. Mater Res., 15: 267-277 (1981); Langer, Chem. Tech., 12: 98-105 (1982), ethylene vinylμlacetate (Langer et al9 supra) or poly-D-(-)-3-hydroxybutyric acid (EP 133,988), polylactide;μl(polylactic acid) (U.S. Patent No. 3,773,919; EP 58,481), polyglycolide (polymer of glycolic acid)μlpolylactide co-glycolide (copolymers of lactic acid and glycolic acid) polyanhydrides, copolymer;μlof L-glutamic acid and gamma-ethyl-L-glutamate (Sidman et aL9 Biopolymers, 22, 547-556 (1983)μlpoly(ortho)esters, polypeptides, hyaluronic acid, collagen, chondroitin sulfate, carboxylic acids,μlfatty acids, phospholipids, polysaccharides, nucleic acids, polyamino acids, amino acids such as.μlphenylalanine, tyrosine, isoleucine, polynucleotides, polyvinyl propylene, polyvinylpyrrolidone andμlsilicone. Sustained-release compositions also include a liposomally entrapped compound.μlLiposomes containing the compound are prepared by methods known per se: DE 3,218,121;μlEppstein et aL, Proc. Natl Acad. ScL U.S.A., 82: 3688-3692 (1985); Hwang et als Proc. Natl Acad.μlSet U.S.A., IT. 4030-4034 (1980); EP 52,322; EP 36,676; U.S. Patent No. 4,619,794; EP 143,949;μlU.S. Patent No. 5,021,234; Japanese Pat. Appln. 83-118008; U.S. Pat. Nos. 4,485,045 andμl4,544,545; and EP 102,324. All references and patents cited are incorporated by reference herein.μl[367] Liposomally entrapped hGH polypeptides can be prepared by methods described in,
e.g., DE 3,218,121; Eppstein et al, Proc. Natl Acad. ScL U.S.A., 82: 3688-3692 (1985); Hwang etμlal, Proc. Natl Acad. Set U.S.A., 77: 4030-4034 (1980); EP 52,322; EP 36,676; U.S. Patent No.μl4,619,794; EP 143,949; U.S. Patent No. 5,021,234; Japanese Pat. Appln. 83-118008; U.S. PatentμlNos. 4,485,045 and 4,544,545; and EP 102,324. Composition and size of liposomes are well knownμlor able to be readily determined empirically by one of ordinary skill in the art. Some examples of

liposomes as described in, e.g., Park JW, et aL, Proc. Natl. Acad. Sci. USA 92:1327-1331 (1995);μlLasic D and Papahadjopoulos D (eds): MEDICAL APPLICATIONS OF LIPOSOMES (1998); DrammondμlDC, et aL, Liposomal drug delivery systems for cancer therapy, in Teicher B (ed): CANCER DRUGμlDISCOVERY AND DEVELOPMENT (2002); Park JW, et aL, Clin. Cancer Res. 8:1172-1181 (2002);μlNielsen UB, etaL,Biochim. Biophys. Acta 1591(1-3):109-118 (2002); MamotC, et aL, Cancer Res.μl63: 3154-3161 (2003). All references and patents cited are incorporated by reference herein.μl[368] The dose administered to a patient in the context of the present invention should be
sufficient to cause a beneficial response in the subject over time. Generally, the totalμlpharmaceutical^ effective amount of the hGH polypeptide of the present invention administeredμlparenterally per dose is in the range of about 0.01 /ig/kg/day to about 100 /xg/kg, or about 0.05μlmg/kg to about 1 mg/kg, of patient body weight, although this is subject to therapeutic discretion.μlThe frequency of dosing is also subject to therapeutic discretion, and may be more frequent or lessμlfrequent than the commercially available hGH polypeptide products approved for use in humans.μlGenerally, a PEGylated hGH polypeptide of the invention can be administered by any of the routesμlof administration described above.
XI. Therapeutic Uses ofh GH Polypeptides of the Invention
[369] The hGH polypeptides of the invention are useful for treating a wide range oi
disorders.
[370] The hGH agonist polypeptides of the invention may be useful, for example, foi
treating growth deficiency, immune disorders, and for stimulating heart function. Individuals withμlgrowth deficiencies include, e.g., individuals with Tinner's Syndrome, GH-deficient individualsμl(including children), children who experience a slowing or retardation in their normal growth curveμlabout 2-3 years before their growth plate closes (sometimes known as "short normal children"), andμlindividuals where the insulin-like growth factor-I (IGF-I) response to GH has been blockedμlchemically (i.e., by glucocorticoid treatment) or by a natural condition such as in adult patientsμlwhere the IGF-I response to GH is naturally reduced. The hGH polypeptides of the invention mayμlbe useful for treating individuals with the following conditions: pediatric growth hormoneμldeficiency, idiopathic short stature, adult growth hormone deficiency of childhood onset, adultμlgrowth hormone deficiency of adult onset, or secondary growth hormone deficiency. Adultsμldiagnosed with growth hormone deficiency in adulthood may have had a pituitary tumor or

radiation. Conditions including but not limited to, metabolic syndrome, head injury, obesity,μlosteoporosis, or depression may result in growth hormone deficiency-like symptoms in adults.μl[371] An agonist hGH variant may act to stimulate the immune system of a mammal by
increasing its immune function, whether the increase is due to antibody mediation or cell mediation,μland whether the immune system is endogenous to the host treated with the hGH polypeptide or isμltransplanted from a donor to the host recipient given the hGH polypeptide (as in bone marrowμltransplants). "Immune disorders" include any condition in which the immune system of anμlindividual has a reduced antibody or cellular response to antigens than normal, including thoseμlindividuals with small spleens with reduced immunity due to drug (e.g., chemotherapeutic)μltreatments. Examples individuals with immune disorders include, e.g., elderly patients, individualsμlundergoing chemotherapy or radiation therapy, individuals recovering from a major illness, or aboutμlto undergo surgery, individuals with AIDS, Patients with congenital and acquired B-cellμl.deficiencies such as hypogammaglobulinemia, common varied agammaglobulinemia, and selectiveμlimmunoglobulin deficiencies (e.g., IgA deficiency, patients infected with a virus such as rabies withμlan incubation time shorter than the immune response of the patient; and individuals with hereditaryμldisorders such as diGeorge syndrome.
[372] hGH antagonist polypeptides of the invention may be useful for the treatment of
gigantism and acromegaly, diabetes and complications (diabetic retinopathy, diabetic neuropathy)μlarising from diabetes, vascular eye diseases (e.g., involving proliferative neovascularization),μlnephropathy, and GH-responsive malignancies.
[373] Vascular eye diseases include, e.g., retinopathy (caused by, e.g., pre-maturity or sickle
cell anemia) and macular degeneration.
[374] GH-responsive malignancies include, e.g., Wilm's tumor, sarcomas (e.g., osteogenic
sarcoma), breast, colon, prostate, and thyroid cancer, and cancers of tissues that express GHμlreceptor mRNA (i.e., placenta, thymus, brain, salivary gland, prostate, bone marrow, skeletalμlmuscle, trachea, spinal cord, retina, lymph node and from Burkitt's lymphoma, colorectalμlcarcinoma, lung carcinoma, lymphoblastic leukemia, and melanoma).
[375] The GH, e.g., hGH agonist polypeptides of the invention may be useful, for example,
for treating chronic renal failure, growth failure associated with chronic renal insufficiency (CRI),μlshort stature associated with Turner Syndrome, pediatric Prader-Willi Syndrome (PWS), HIV

patients with wasting or cachexia, children born small for gestational age (SGA), obesity, andμlosteoporosis.
[376] Average quantities of the hGH may vary and in particular should be based upon the
recommendations and prescription of a qualified physician. The exact amount of hGH is a matter ofμlpreference, subject to such factors as the exact type of condition being treated, the condition of theμlpatient being treated, as well as the other ingredients in the composition. The amount to be givenμlmay be readily determined by one of ordinary skill in the art based upon therapy with hGH.μl[377] Pharmaceutical compositions of the invention may be manufactured in conventional
manner.
XIL - Articles of Manufacture
[378] In another embodiment of the invention, an article of manufacture is provided which
contains the formulation of the present invention and provides instructions for its use. The article ofμlmanufacture comprises a container. The article of manufacture may contain the formulation of theμlpresent invention, including but not limited to, lyophilized, liquid, spray dried, frozen or other formsμlthereof, and instructions for its preparation or reconstitution, if required. Suitable containersμlinclude, but are not limited to, for example, bottles, vials, syringes, auto-injection devices, and testμltubes. The container may be formed from a variety of materials such as glass or plastic. Theμlcontainer holds the formulation and the label on, or associated with, the container may indicateμldirections for reconstitution of the lyophilized formulation, if required, and/or use. For example, theμllabel may indicate that the formulation is reconstituted to specific protein concentrations. The labelμlmay further indicate that the formulation is useful or intended for subcutaneous administration. Theμlcontainer holding the formulation may be a single-use or a multi-use vial. In one embodiment, theμlcontainer holding the formulation is a single-use vial. The article of manufacture may furtherμlcomprise a second container comprising a suitable diluent. In one embodiment, the suitable diluentμlis sterile water or bacteriostatic water for injection, USP. Upon mixing of a diluent and theμllyophilized formulation, if required, the final protein concentration in the formulation may generallyμlbe between about 2 mg/ml and about 50 mg/ml. Upon mixing of a diluent and the lyophilizedμlformulation, if required, the final protein concentration in the formulation may generally be betweenμlabout 2 mg/ml and about 25 mg/ml. The final protein concentration in the formulation mayμlgenerally be between about 2 mg/ml and about 25 mg/ml. In one embodiment, the final proteinμlconcentration is about 8 mg/ml. The article of manufacture may further include other materials

desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles,μlsyringes, and package inserts with instructions for use.
EXAMPLES
[379] The following examples are offered to illustrate, but not to limit the claimed
invention.
Example 1 Materials and Methods for Formulations for Met-Y35pAF hGHμl[380] This example describes a formulations study that identified and evaluated suitable
conditions and excipients that preserve the protein structure and activity of Met-Y35pAF hGHμlduring storage before conjugation with PEG. Liquid formulations of hGH are frozen formulationsμlwhich contain (1) 2.5 g/L sodium bicarbonate, 20 g/L glycine, 2 g/L mannitol, 2 g/L lactose, at pHμl7.3; or 2) sodium citrate, 20 g/L glycine, 5g/L mannitol, at pH 6.0. An estimated dose of 0.5-4μlmg/ml of PEG-hGH is added as an active ingredient. These formulations are currently stored at -μl20°C. Development of a single-dose lyophilized formulation of PEGylated hGH comprising a non-μlnaturally encoded amino acid or liquid formulation of PEGylated hGH comprising a non-naturallyμlencoded amino acid is desired.
Methods
[381] Several methods were implemented to characterize the physical and chemical
stability of Met-Y35pAF hGH. These methods may be used in the formulations study with
PEGylated hGH.
[382] SDS-PAGE with a Reducing Gel (SDS-PAGE-R) provided separations of hGH
based exclusively on molecular weight allowing any potential degradation products to be visualized,
since this technique results in all disulfide bonds being completely cleaved and causes the
polypeptides to be completely unfolded. SDS-PAGE with a Non-reducing Gel (SDS-PAGE-NR)
provided separation of molecules based on molecular weight, allowing the identification of potential
dimers of hGH since the protein is unfolded without a reducing agent, thus leaving disulfide bonds
intact.

[383] Differential Scanning Calorimetry (DSC) was used to measure the AH (enthalpy) of
unfolding due to heat denaturation. This technique evaluated the effects of different solutionμlconditions and excipients on protein stability which was reflected in either an increase or decreaseμlin the overall melting temperature (Tm) of the protein. Another technique used was Reverse PhaseμlHPLC (RP-HPLC) to separate molecules on the basis of relative hydrophobicities. Specifically, thisμlmethod was used to separate hGH based on subtle differences in hydrophobicity and retentionμlbehavior associated with structural modifications such as deamidation and oxidation.


Sample Preparation
[387] The sample of methionyl-hGH with the non-natural amino acid p-acetyl-
phenylalanine substituted at position 35 (MetY35pAF cBl) in "WHO buffer was allocated into 6
different vials at 4.425 mg/ml and frozen at -80°C until the day of the study. On the day of
experiment, MetY35pAF was buffer exchanged into the specific buffer at 4°C by dialysis in 0.1 -
0.5 mL Slide-a-lyzers. Approximately 800μl of 425 mg/ml Met-Y35pAF hGH was dialyzed in 250
mL buffer for at least four hours, and the dialysis buffer was changed (250 mL) for overnight
dialysis. Using 500 mL provided a 10,000,000-fold dilution of WHO buffer excipients.
[388] Samples were removed from dialysis and concentrations were measured. Each
sample was diluted to the appropriate concentration. For DSC, the desired final concentration was
0.75 mg/ml (450 ul volume). For RP-HPLC, the final concentration was 2 mg/ ml (30 ul volume).
Ten samples were analyzed on RP-HPLC for each individual group of the matrix. Enough sample
to allow for four time points (3 days, 1 week, 2 weeks, and 1 months) was stored in a tube at 4°C.
Enough sample for 5 freeze/thaw cycles was stored in a vial at -80°C. There may have been one oi
two additional freeze/thaw cycles prior to the number of freeze/thaw cycles (1, 2, 3, 4 and 5 cycles]
completed as part of this study. The sample for the 0 time point at 4°C was immediately placed in
an HPLC vial.
[389] For SDS-PAGE-R, 30 ug (0.015 mL @ 2 mg/mL) of material was used in the
analysis. Samples were aliquoted into three vials as indicated for RP-HPLC.
[390] For SDS-PAGE-NR, 30 ug (0.015 mL @ 2 mg/mL) of material was used in the
analysis. Samples were aliquoted into three vials as indicated for RP-HPLC. In all cases, any
remaining sample was immediately frozen at -80°C.
Example 2 Results of Formulation Study for Met-Y35pAF hGH
Buffer pH
[391] The pH of all formulation buffers was analyzed after six weeks at 4°C. It was found
that the pH of all buffers chosen was stable over a six week period, with the exception of the
sodium bicarbonate buffer (Group E) as shown in Figure 1. The pH of Group E increased over
time.

SDS-PAGE-R and SDS-PAGE-NR
[392] Analysis of samples with SDS-PAGE reducing and non-reducing gels showed no
change either after four weeks (1 month) at 4°C or during five freeze/thaw cycles. No dimerμlformation or degradation products were found by this method. Reducing gels for group A (at t=0,μlt= 4 weeks at 4°C) are shown as Fig. 2C and 2D, and non-reducing gels for group B (at t=0, t= 4μlweeks at 4°C) are shown as Fig. 2A and 2B.μlDifferential Scanning Calorimetry (DSC)
[393] The effects of buffer and excipient on thermal stability of MetY35pAF hGH are
shown as Figures 3-8. Figure 3A-F shows overlaid thermoprofiles of formulation groups A-F.μlFigure 5 provides a table summarizing the DSC melting temperatures and changes to Tm for the fullμlmatrix. Thermoprofiles from B7 and F2 of the matrix are shown in Figure 4A-B.μl[394] Buffer A demonstrated the lowest Tm, as shown in Figures 5 and 6. Arginine raised
the Tm in Groups B and C, as shown in B7 and C7 of the matrix on Figures 5 and 6. At higher pHsμl(i.e. pH 7.0), MetY35pAF hGH exhibited greater thermostability; however additionalμlsubpopulations were seen that have much higjier melting temperatures and broader curves as shownμlin Figures 3 and 4. These subpopulations may exhibit greater stability or aggregation tendencies.μlAmino acids, in particular Arg and Glu, as well as (NILt)2S04 decreased the Tm at higher pHs.μlSugars did not have an effect on the overall Tm-
[395] The ratio of AHV/AH was analyzed for all groups as shown in Figure 7. Recombinant
hGH is known to exhibit a AHv/AH 4. Variations in this ratio were observed; these valuesμldemonstrate that the unfolding of MetY35pAF is irreversible. As the pH increased, additionalμlsubpopulations were identified (the ratio for a 2nd subpopulation of Group F was also plotted at pHμl7.5 on Figure 7). The change in enthalpy (AUC) of each sample was similar as shown in Figure 8,μlexcept at higher pHs. Differences in AH at higher pHs may be due to missed broad transitions,μlaccounting for a lower AUC. AH (AUC of the fit) was plotted for every group to ensure similarμlvalues were obtained within each group.
Reverse Phase HPLC (RP-HPLC)
[396] RP-HPLC was used to analyze the purity and chemical degradation of MetY35pAF
hGH during storage at 4°C for four weeks. Datasets for Groups B, C, E and F each comparing

MetY35pAF hGH with the WHO hGH standard are shown in Figures 9A-D. Figure 10A illustratesμlthe use of Agilent Chemstation software in analyzing the different peaks present in group E5 (theμlmain peak, primary deamidation/oxidation peak, and the secondary deamidation peak). The primaryμldeamidation/oxidation peak increased over time with Group E as shown in Figures 10B and 11;μlalso, higher pHs exhibited greater amounts of deamidation/oxidation than lower pHs. Figure 12μlshows that the secondary deamidation/oxidation peak demonstrated less variability over time withμlall formulations groups. The main GH peak decreased over time for all formulations groups asμlshown in in Figure 13, though groups B and C exhibited less change compared to the other groups.μlGroup A exhibited very little deamidation, but the main GH peak did decrease over time andμladditional peaks, other than known deamidation/oxidation peaks, did develop over time. The dataμlof Group A did, however, demonstrate that MetY35pAF is fairly stable from 0 to 3 days at this pH,μlwhich is critical since the PEGylation reaction takes place at pH 4.0 over one to two days.μl[397] RP-HPLC was also used to analyze the samples that had undergone freeze-thaw
cycles. Individual groups within B and C appeared similar as shown in Figure 14 (primaryμldeamidation/oxidation peak) and Figure 15 (secondary deamidation peak). After two freeze/thawμlcycles, the main GH peak decreased as shown in Figure 16, indicating freeze/thaw effects.μlHowever, one or two freeze/thaw cycles did occur with this sample prior to the initiation of thisμlformulations study.μlSummary of Findings
[398] It is well established that hGH exhibits a highly flexible structure at lower pH, and
hGH forms a very rigid structure at higher pH (Kasimova et al. J. Mol. Biol. 2002 318:679-695).μl[399] Of the buffers tested, it was found that Buffer B (20 mM sodium citrate) at pH 6.0
performed best for chemical and thermodynamic stability of MetY35pAF hGH. The addition ofμlArginine at pH 6.0 increased the thermostability of MetY35pAF hGH. At this time, sugars did notμlhave any positive or negative effect on MetY35pAF hGH; however, sugars may be important forμlpH shifts pre -and post-PEGylation events. The oxime linkage over time is also likely to be moreμlstable at pH 6.0 than at a higher pH such as pH 7.5.μlExample 3
[400] The stability profile of PEG-hGH is examined with key formulation parameters
under accelerated stability conditions, major degradation products are identified, and stabilityμlindicating assays are confirmed. The main objective of single-dose formulation development is to

optimize the formulation for sufficient storage stability minimally at refrigerated temperature andμlmay have sufficient storage stability when stored at ambient temperature. In addition to theμlIyophilized formulation development, additional studies are investigating the sensitivity to lightμlexposure and agitation, development of the RP-HPLC method distinguishing the PEGylated hGHμlfrom the non-PEGylated form, structural analysis, and other studies. Successful Iyophilizedμlformulations normally show the following attributes: stability in solution for handling duringμlformulation and fill-finish, good stability during freeze-drying, good storage stability, nativeμlstructure in the dried state (if relevant), no obvious sample collapse or melt back, optimum moistureμlcontent level, high glass transition temperature, rugged cake structure, efficient drying cycle,μlminimum injection associated pain, and dissolves in water-filled injectable within one minute whenμlreconstituted. In some embodiments, the optimum moisture content level is3% water for aμlIyophilized formulation. In preferred embodiments, the optimum moisture content level is1%μlwater for a Iyophilized formulation.
[401] The formulation variables are determined. The stability of Iyophilized formulation
candidates are examined at 40°C: ambient temperature, and refrigerated temperature with weekl>μltime points for up to 4 weeks, 6 weeks, and 2 months. Also, the stability of Iyophilized formulationμlcandidates are examined at ambient temperature and refrigerated temperature for up to 3, 4, and €μlmonth increments. The stability of the reconstituted form of each formulation during storage at 2-μl8°C for a week is also be tested. Analytical methods include the SDS-PAGE method describedμlabove, SEC-HPLC, Ion-exchange HPLC, RP-HPLC, and other structural analyses.μl[402] Basic parameters useful for optimizing lyophilization cycle for most stable
formulations are determined. For example, collapse temperature (or glass transition temperature),μlannealing temperature, and other important physical properties of frozen formulations areμldetermined. A test lyophilization process is run to confirm that ideal cake can be obtained with theμloptimized cycle.μlExample 4
Reverse Phase High Performance Liquid Chromatography (RP-HPLC)
[403] Reverse Phase High Performance Liquid Chromatography (RP-HPLC) is a technique
that separates molecules on the basis of relative hydrophobicities. Samples are passed over aμlstationary phase of silica covalently bonded to hydrocarbon chains. The molecules of interest areμlretarded by the stationary phase and eluted with an isocratic solvent The chromatographic elution

time is characteristic for a particular molecule. This method separates hGH based on subtleμldifferences in hydrophobicity and retention behavior associated with structural modifications suchμlas deamidation
[404] C4 RP-HPLC was used to assess relative purity and potential chemical degradation
(deamidation and oxidation) of recombinant human growth hormone (hGH). This method was usedμlto support identification and purity assessment of hGH. Some partial degradation products of hGHμlwere observed using this technique. References for this technique include, EuropeanμlPharmacopoeia 2002, p. 193; British Pharmacopoeia 2001, p.1938 - 1939; and "A Reversed-PhaseμlHigh Performance Liquid Chromatographic Method for Characterization of Biosynthetic HumanμlGrowth Hormone" by R.M. Riggin et al. Analytical Biochemistry 167,199-209 (1987).μl[405] Equipment for this procedure included, the following or equivalents thereof: UV/Vis
Spectrophotometer (Agilent 8453 or equivalent); 50 ul quartz cuvette; PD-10, Nap-10, or Nap 5μldesalting columns (depending on sample volume; Amersham Biosciences Nap5 column 17-0853-02μlor equivalent); 0.5 mL Vivaspin concentrators (if needed; Vivascience 10,000 MWCO, PES,μlVS0102 or equivalent); HPLC vials and gaps (Alltech 100 ul screw cap polypropylene vialsμl#12962, TFE liner caps #73048, open hole screw caps #73044, or equivalent); clean 1 and 2 L glassμlbottles; a column such as Vydac C4 214TP54, a C4-silica reversed phase HPLC column with aμldimension of 4.6 x 250mm, particle size of 5 p. and pore size of 300A; and High-pressure liquidμlchromatography instrument capable of performing linear gradients (such as Agilent 1100 HPLCμlequipped with a vacuum degasser, quaternary pump, thermostatted autosampler, thermostattedμlcolumn compartment, diode array detector (DAD), and Chemstation chromatography software).μl[406] Reagents for this procedure included solid chemicals that were analytical grade or
better and solvents that were HPLC grade or better, unless otherwise noted. Examples of suchμlchemicals include TRIS - Tromethamine, U.S.P. grade, Spectrum TR149, or equivalent; N-μlpropanol, HPLC grade, 99.9%, Sigma Aldrich 34871, or equivalent; and Ammonium Bicarbonate,μlUltra 99.5%, Fluka # 09830, or equivalent. Additional solutions include: Buffer for DeamidationμlControl (30 mM Ammonium Bicarbonate, pH 9.0) and mobile phase solution. For the buffer forμldeamidation control, 2.37g of Ammonium bicarbonate was dissolved in 0.95 L Milli-Q H2O, pH toμl9.0 with NaOH, and the volume was brought up to 1L with Milli-Q H2O. The resulting buffer wasμlsterile filtered using 0.22 xm PES filters (Corning #431098, or equivalent). For the Mobile phaseμlsolution (50 mM Tris-HCl, pH 7.5 and 29% n-propanol)), 6.05g Tromethamine (USP grade,

Spectrum Kl4y, or equivalent) was dissolved m 0.95 L Milli-Q H2O. The solution was brought to pH 7.5 with HC1, and the volume brought up to 1L with Milli-Q H20. The two solvents (TRIS and propanol) were mixed, and the mixture was sterile filtered using 0.22μmPES filters (Corning #431098, or equivalent).
[407] Samples for use as standards in RP-HPLC include World Health, Organization
(WHO) hGH (Cat. # 98/574) reconstituted to 1.9 - 2.1 mg/ml with 1.0 ml of water. Other hGH reference standards may be used at 1.9 - 2.1 mg/ml concentration. For the resolution solution, the hGH standard is buffer exchanged into 30 mM Ammonium Bicarbonate, pH 9.0 buffer using a PD-10, Nap-10, or Nap-5 desalting column (depending on sample volume). The standard was concentrated using a 0.5 mL Vivaspin concentrator to 1.9-2,1 mg/ml, incubated at 37°C for 24 hours, and then stored at -80°C until use. Test material was diluted to 2.0 mg/ml protein concentration for analysis. Sample concentrations were measured using standard techniques. Procedure
[408] The instrument was set-up with the following conditions: 1) Column: Vydac C4
214TP54 column; 2) Pump Setup—gradient: isocratic; flow rate: 0.5 ml/min; duration: 90min; Max Pressure: 200 bar; 3) Autosampler Temperature: 4°C; 4) Injector Setup— Injection: Standard Injection; Injection Volume: 20μl; Draw Speed: 100 μl/min; Needle Wash: 100 ul with water; Injection Speed: 100μl l/min; Stop Time: As pump; 5) DAD Signals-Table 2; Peak Width0.1 min; Slit: 4 ran; Stop Time: 75 min;

6) Column Thermostat—Temperature: 45°C; Store: Temperature; 7) Preliminary Integration Events-
-Slope Sensitivity: 0.1; Peak Width: 0.5; Area Reject: 1.0; Height Reject: 1.0; Integration ON: 10
min.
[409] The column was equilibrated with 10 column volumes (41.5 ml = 83 min at 0.5
ml/min) of the mobile phase. 20μl of the standard was injected using the autosampler, and the
HPLC program was run. If the retention time of the WHO standard was not between 33 - 35
minutes, or the other standard was not between 37-40 minutes, the mobile phase composition was

adjusted, the column was re-equilibrated, and the standard was re-run. Suggested adjustments
included adding less than 5 ml of 50 mM Tris-HCl pH 7.5 per liter of mobile phase if retention time
was less than 33 minutes, and less than 2 ml of n-propanol if retention time greater than 35. Since
evaporation of the propanol may occur, a standard was run on each day that samples were tested,
and buffers were adjusted accordingly.
[410] 20nl of the resolution solution was injected, and the HPLC program was run.
Desamido-hGH appeared as a small peak at a retention time of about 0.85 relative to the principal
peak. The test was not valid unless the resolution between the peaks corresponding to hGH and
desamido-hGH was at least 1.0 and the symmetry factor of the hGH peak was 0.8 to 1.8. 20jil of
the test article was injected, and the HPLC program was run. Samples were run in triplicate.
Average retention times were reported.
[411] Modifications to various conditions and/or parameters may be required to analyze
PEGylated hGH and other forms. Modifications to RP-HPLC are known to those of ordinary skill
in the art.
Data Analysis
[412] The average retention time of the test article was compared with the standard. The
average purity of the test article was calculated: Integration area of the main peak / integration
areas of all peaks) x 100%. Any peak(s) due to the solvent were disregarded.
[413] Results from RP-HPLC for MetY35pAF hGH with the WHO hGH standard are
provided in Table 3 and Figure 17. Specifications such as the retention time difference between the
test article and the reference standard and purity may vary with different molecules or forms of a
molecule (i.e. Y35pAF hGH compared to metY35pAF hGH).



Example 5
Size Exclusion Chromatography
[414] Size-exclusion high performance liquid chromatography (SEC-HPLC) is a technique
using the stationary phase as a porous matrix which is permeated by mobile phase molecules.
Sample molecules small enough to enter the pore structure are retarded, while larger molecules are
excluded and therefore rapidly carried through the column. Thus, size exclusion chromatography
provides separation of molecules by size and the chromatographic elution time is characteristic for a
particular molecule.
[415J SEC-HPLC was used to assess recombinant human growth hormone (hGH) potency.
SEC-HPLC was a method to determine the percentage of monomer (PEGylated and non-
PEGylated) hGH. Dimer and other high molecular weight proteins were observable using this
technique. Thus, this technique separates monomer from dimer and other higher molecular weight
substances in the sample, as well as PEGylated and non-PEGylated forms. References for this
technique, include, but are not limited to, European Pharmacopoeia 2002, p. 193; British
Pharmacopoeia 2001, p. 1941; and "High-Performance Size-Exclusion Chromatographic
Determination of the Potency of Biosynthetic Human Growth Hormone Products" by R.M. Riggin
et al. Journal of Chromatography 435(1988), p. 307-318.
[416] Equipment for this procedure included, the following or equivalents thereof: a
UV/Vis Spectrophotometer (Agilent 8453 or equivalent); 50 ul quartz cuvette; 0.5 mL Vivaspin
concentrators (if needed; Vivascience 10,000 MWCO, PES, VS0102 or equivalent); HPLC vials
and caps (Alltech 100 ul screw cap polypropylene vials #12962, TFE liner caps #73048, open hole
screw caps #73044, or equivalent); clean 1 and 2 L glass bottles; a column such as Tosohaas TSK
Super SW3000 18675 and Super SW Guard Column 18762, a silica-based size exclusion HPLC

column with a dimension of 4.6 x 300 mm, particle size of 4μmand pore size of 250A along with aμlguard column having a dimension of 4.6 x 35 mm and 4 x particle size); and High-pressure liquidμlchromatography instrument capable of performing linear gradients (such as Agilent 1100 HPLCμlequipped with a vacuum degasser, quaternary pump, thermostatted autosampler, thermostattedμlcolumn compartment, diode array detector (DAD), Refractive Index detector (RID) andμlChemstation chromatography software).
[417] Reagents for this procedure included solid chemicals that were analytical grade or
better and solvents that were HPLC grade or better, unless otherwise noted. Examples of suchμlchemicals include, Monobasic Sodium Phosphate, Spectrum U.S.P. grade S0130, or equivalent;μlDibasic Sodium Phosphate, Spectrum U.S.P. grade S0140, or equivalent; 2-propanol, Fisher HPLCμlgrade A451-4, or equivalent. Additional solutions include: mobile phase solution (97% of 63 mMμlsodium phosphate pH 7.0; 3% of 2-propanol) and solution A. To make mobile phase solution,μl26.8g of dibasic sodium phosphate was dissolved in 1 L Milli-Q H2O, and 13.79 g monobasicμlsodium phosphate was dissolved in 1L Mill-Q H2O. The two solutions were mixed to give aμlsodium phosphate buffer of pH 7.0. The 100 mM sodium phosphate, pH 7.0 buffer was diluted toμl63 mM with Mill-Q H2O. 970 mL of 63 mM sodium phosphate pH 7.0 was mixed with 30 mL ofμl2-propanol (or other appropriate volumes to obtain 97% of 63 mM sodium phosphate pH 7.0, 3% ofμl2-propanol). The resulting buffer was sterile filtered using 0.22 xm PES filters (Corning #431098,μlor equivalent). Solution A is 25 mM Sodium Phosphate, pH 7.0. 250 mL of lOOmM sodiumμlphosphate, pH 7.0 buffer was diluted with 750 mL Milli-Q H2O. The resulting buffer was sterileμlfiltered using 0.22 im PES filters (Corning #431098, or equivalent).
[418] Samples for use as standards in SEC-HPLC include World Health, Organization
(WHO) hGH (Cat. # 98/574) reconstituted with 1.0 ml of water and diluted to 0.9 - 1.1 mg/ml.μlOther hGH reference standards may be used at 0.9 - 1.1 mg/ml concentration. For the resolutionμlsolution, the hGH standard was incubated at 50°C for 12-24 hours, dissolved in solution A, thenμldiluted to 1 mg/ml with solution A, and stored at -80°C until use. The test material was diluted toμl1.0 mg/ml with Solution A. Sample concentrations were measured using standard techniques.μlProcedure
[419] The instrument was set-up with the following conditions: 1) Column: TSK Super
SW3000 18675 and Guard Column 18762 Auto sampler; 2) Temperature: room temperature; 3)μlPump Setup—gradient: isocratic; flow rate: 0.3 ml/min: duration: 25 min; Max Pressure: 120 bar; 4)

Injector setup—Injection: Standard Injection; Injection Volume: 20μl; Draw Speed: 100 iVmin;μlInjection Speed: 100 pJ/min; Needle wash: 100 ul H20; Stop Time: As pump; 4) DAD Signals-μlTable 4; Peak Width0.05 ruin; Slit: 2 ran; Stop Time: As pump;

5) RID Signal—Temperature: 35°C; Response Time:0.2 min 4s, standard; 6) Column Thermostat:μlTemperature: 23 °C; Store: Temperature.
[420] The column was equilibrated with 10 column volumes (50 ml = 166 minutes at 0.3
ml/minutes) of the mobile phase, and the RID was purged for 20 minutes before injecting theμlsamples. 20(4,1 of the resolution solution was injected. In the chromatogram obtained, 1) the mainμlnon-PEGyl&ted peak eluted at a retention time of approximately 13 - 13.5 minutes, 2) the peaksμlcorresponding to the hGH dimer eluted at a retention time of approximately 12.2 - 12.6 minutes,μland 3) the higher molecular weight proteins (non-PEGylated) eluted at relative retention time of 7.4μl- 8.0 minutes. The main PEGylated peak eluted at a retention time of approximately 8.3 - 8.8μlminutes. 20 jil of the standard was injected, and the HPLC program was run. 20jal of the testμlarticle was injected, and the HPLC program was run. Samples were run in triplicate. Averageμlretention times were recorded.
[421] Modifications to various conditions and/or parameters may be required to further
characterize PEGylated hGH or other forms. Modifications to SEC-HPLC are known to those ofμlordinary skill in the art.μlData Analysis
[422] The Retention Time of the hGH test article is compared with the standard. For
purity determinations of non-PEGylated hGH, the integrated main peak areas of the hGH test articleμland the standard is compared, and the percentage of monomer in the hGH test article is calculated:μl(peak area of hGH sample / peak area of standard) x 100%. The percentage of dimer and/or higherμlaggregates in the hGH test article is also calculated. For purity determinations of PEGylated hGH,μlthe integrated main peak areas of the PEGylated hGH sample and the standard is compared, and the

percentage of PEGylated monomer in PEGylated-hGH sample is calculated by: (peak area of
PEGylated hGH sample / peak area of standard) x 100%. The percentage of PEGylated dimer,
higher aggregates, and non-PEGylated monomer in the hGH test article are calculated also.
Alternatively, purity is determined for non-PEGylated hGH or PEGylated hGH by: (Integration area
of the main peak of hGH sample / integration areas of all peaks of hGH sample) x 100%. Any
peak(s) due to the solvent were disregarded. Purity determinations are also calculated using
absolute mass or direct peak area rather than by percent of a reference standard.
[423] Specifications: The retention time difference between the hGH test article and the
standard is approximately ±30 seconds. For dimer and higher molecular weight proteins, in the
chromatogram obtained with the test article, the sum of the areas of any peak with a retention time
less than that of the principal peak is not greater than 4.0% or 6.0% of the total area of the peaks,
respectively. Any peak(s) due to the solvent are disregarded.
[424] Figure 18 shows SEC-HPLC data from 30K PEG~metY35pAF and metY35pAF.
Example 6
Cation-Exchange High Performance Liquid Chromatography (cIEX-HPLC)
[425] Cation-exchange high performance liquid chromatography (cIEX-HPLC) is a
technique that relies on charge-charge interactions between a protein and the charges immobilized
on the resin. Cation exchange chromatography takes advantage of the positively charged ions of a
protein that bind to the negatively charged resin. A common structural modification of hGH is
deamidation of asparagine (Asn) residues, and this cIEX-HPLC method permits the separation of
deamidated and deamidation intermediates of PEGylated and non-PEGylated hGH.
[426] The following method was used to assess relative purity and potential chemical
degradation (i.e. deamidation) of recombinant human growth hormone (hGH). This method was
used to support identification and purity assessment of PEGylated and non-PEGylated hGH. Some
partial degradation products of hGH were observable using this technique.
[427] Equipment for this procedure included, the following or equivalents thereof: UV/Vis
Spectrophotometer (Agilent 8453 or equivalent); 50 ul quartz cuvette; 0.5 mL Vivaspin
concentrators (if needed; Vivascience 10,000 MWCO, PES, VS0102 or equivalent); HPLC vials
and caps (Alltech 100 ul screw cap polypropylene vials #12962, TEE liner caps #73048, open hole
screw caps #73044, or equivalent); clean 1 and 2 L glass bottles; a column such as PolyCAT A 4.6
x 200 mm, 5μ, 1000 A (204CT0510) and PolyCAT A guard column, 4.6 x 10 mm, 5μ1000 A

(JGCCT0510); and High-pressure liquid chromatography instrument capable of performing linearμlgradients (such as Agilent 1100 HPLC equipped with a vacuum degasser, quaternary pump,μlthermostatted autosampler, thermostatted column compartment, diode array detector (DAD),μlRefractive Index detector (RED) and Chemstation chromatography software).μl[428] Reagents for this procedure included solid chemicals that were analytical grade or
better and solvents that were HPLC grade or better, unless otherwise noted. Examples of suchμlchemicals include, Ammonium Acetate, Spectrum HPLC grade A2149, or equivalent; Acetonitrile,μlFisher HPLC grade A998, or equivalent, Ammonium Bicarbonate. Additional solutions include:μlMobile phase A (40% Acetonitrile, H2O); Mobile phase B (40% Acetonitrile, 500 mM AmmoniumμlAcetate, pH 4.5) and Buffer for Deamidation Control (30 mM Ammonium Bicarbonate, pH 9.0).μlFor Mobile phase A buffer, 400 mL HPLC grade Acetonitrile was mixed with 600 ml sterile filteredμlMilli-Q H2O, and the resulting mixture was sterile filtered using 0.22 pm PES filters (Corningμl#431098), or equivalent). For Mobile phase B buffer, 38.54 g ammonium acetate was dissolved inμl970 mL Milli-Q H2O. The solution was brought to pH 4.5 with glacial acetic acid, and then theμlvolume brought up to 1000 mL volume with Milli-Q H2O. The buffer was sterile filtered using 0.22μlpm PES filters (Corning #431098). 100 mL Milli-Q H20, 400 mL HPLC grade Acetonitrile, andμl500 mL 500 mM Ammonium Acetate, pH 4.5 were mixed. For the Buffer for Deamidation Control,μl2.37g of Ammonium bicarbonate was dissolved in 0.95 L Milli-Q H2O, and the pH was brought toμl9.0 with NaOH. The volume was then brought up to 1L with Milli-Q H2O. The resulting bufferμlwas sterile filtered using 0.22 pm PES filters (Corning #431098, or equivalent).μl[429] Samples for use as standards in cIEX-HPLC included World Health, Organization
(WHO) hGH (Cat. # 98/574) reconstituted with 1.0 ml of water and diluted to 0.9 - 1.1 mg/ml.μlOther hGH reference standards may be used at 0.9 - 1.1 mg/ml concentration. For the resolutionμlsolution, hGH standard(s) were buffer exchanged into 30 mM Ammonium Bicarbonate, pH 9.0μlbuffer using a PD-10, Nap-10, or Nap-5 desalting column (depending on sample volume). Theμlstandard was concentrated using a 0.5 mL Vivaspin concentrator to 0.9 - 1.1 mg/ml, incubated atμl37°C for 24 hours, and then stored at -80°C until use. The test article was diluted to 1.0 mg/ml. Aμldeamidated resolution standard was included. Alternatively, the test article and standards areμldiluted over a range of concentrations. Sample concentrations were measured using standardμltechniques.μlProcedure

[430J The instrument was set-up with the following conditions: I) Uolumn: jfolyUAl" A
204CT0510 and JGCCT0510; 2) Auto sampler Temperature: 4°C; 3) Pump Setup-gradient: 50 50-μl250 mM Ammonium Acetate pH 4.5;

6) Column Thermostat: Temperature: 20°C; Store: Temperature.
[431] The column was equilibrated with 10 column volumes of 90% mobile phase A and
10% mobile phase B. 15pi of the resolution solution was injected. In the chromatogram obtained,μlthe main non-PEGylated peak elutes at a retention time of approximately 64 - 67 minutes, the non-μlPEGylated deamidated peak elutes at a retention time of approximately 62 - 65 minutes, the mainμlPEGylated peak elutes at a retention time of approximately 43 - 46 minutes, and the PEGylatedμldeamidated peak elutes at a retention time of approximately 41-44 minutes. 15 fxl of the standardμlwas injected, and the HPLC program was run. 15|xl of the test article was injected, and the HPLCμlprogram was run. Samples were run in triplicate. Average retention times were reported.

[432] Modifications to various conditions and/or parameters may be required to analyze
PEGylated hGH and other forms. Modifications to cIEX-HPLC are known to those of ordinaryμlskill in the art. Additional reference standards include, but are not limited to, in-process pY35pAFμlfor in-process release of bulk pY35pAF, purified pY35pAF for quantifying residual free pY35pAFμlin PEGylated-pY35pAF, and purified PEG-pY35pAF.μlData Analysis
[433] The retention times of the hGH test article with the standard were compared and
purity determinations of non-PEGylated hGH and PEGylated hGH calculated. Purity may beμldetermined by calculations such as: (Integration area of the main peak of hGH sample / integrationμlareas of all peaks of hGH sample) x 100%. Any peak(s) due to the solvent were disregarded.μl[434] ' Table 6 and Figure 19 show cIEX-HPLC analysis of 30K PEGmY35pAF hGH and
mY35pAF hGH.


stirred at room temperature (RT) for 1 hour, methoxy-polyethylene glycol amine (m-PEG-NEk, 7.5μlg, 0.25 mmol, Mt. 30 K, from BioVectra) and Diisopropylethylamine (0.1 mL, 0.5 mmol) wereμladded. The reaction was stirred at RT for 48 hours, and then was concentrated to about 100 mL. Theμlmixture was added dropwise to cold ether (800 mL). The t-Boc-protected product precipitated outμland was collected by filtering, washed by ether 3xl00mL. It was further purified by re-dissolving inμlDCM (100 mL) and precipitating in ether (800 mL) twice. The product was dried in vacuumμlyielding 7.2 g (96%), confirmed by NMR and Nihydrin test
[438] The deBoc of the protected product (7.0 g) obtained above was carried out in 50%
TFA/DCM (40 mL) at 0 °C for 1 hour and then at RT for 1.5 hour. After removing most of TFA inμlvacuum, the TFA salt of the hydroxylamine derivative was converted to the HC1 salt by adding 4NμlHC1 in dioxane (ImL) to the residue. The precipitate was dissolved in DCM (50 mL) and re-μlprecipitated in ether (800 mL). The final product (6.8 g, 97%) was collected by filtering, washedμlwith ether 3x lOOrnL, dried in vacuum, stored under nitrogen. Other PEG (5K, 20K) hydroxylamineμlderivatives were synthesized using the same procedure.μlExample 8
[439] This example describes expression and purification methods used for hGH,
polypeptides comprising a non-natural amino acid. Host cells have been transformed with,μlorthogonal tRNA, orthogonal aminoacyl tRNA synthetase, and hGH constructs.μl[440] A small stab from a frozen glycerol stock of the transformed DH10B(fis3) cells were
first grown in 2 ml defined medium (glucose minimal medium supplemented with leucine,μlisoleucine, trace metals, and vitamins) with 100 |ig/ml ampicillin at 37 °C. When the ODsooμlreached 2-5, 60 JJ! was transferred to 60 ml fresh defined medium with 100 jig/ml ampicillin andμlagain grown at 37 °C to an ODeoo of 2-5. 50 ml of the culture was transferred to 2 liters of definedμlmedium with 100 |ag/ml ampicillin in a 5 liter fermenter (Sartorius BBI). The fermenter pH wasμlcontrolled at pH 6.9 with potassium carbonate, the temperature at 37 °C, the air flow rate at 5 1pm,μland foam with the polyalkylene defoamer KFO Fl 19 (Lubrizol). Stirrer speeds were automaticallyμladjusted to maintain dissolved oxygen levels30% and pure oxygen was used to supplement theμlair sparging if stirrer speeds reached their maximum value. After 8 hours at 37 °C, the culture wasμlfed a 5 OX concentrate of the defined medium at an exponentially increasing rate to maintain aμlspecific growth rate of 0.15 hour"1. When the OD«v» reached approximately 100, a racemic mixture

of para-acetyl-phenylalanine was added to a final concentration of 3.3 mM, and the temperature wasμllowered to 28°C. After 0.75 hour, isopropyl-b-D-thiogalactopyranoside was added to a finalμlconcentration of 0.25 mM. Cells were grown an additional 8 hour at 28 °C, pelleted, and frozen at -μl80 °C until further processing,
[441] The His-tagged mutant hGH proteins were purified using the ProBond Nickel-
Chelating Resin (Invitrogen, Carlsbad, CA) via the standard His-tagged protein purificationμlprocedures provided by Invitrogen's instruction manual, followed by an anion exchange column.μl[442] The purified hGH was concentrated to 8 mg/ml and buffer exchanged to the reaction
buffer (20 mM sodium acetate, 150 mM NaCl, 1 mM EDTA, pH 4.0). MPEG-Oxyamine powderμlwas added to the hGH solution at a 20:1 molar ratio of PEG:hGH. The reaction was carried out atμl28°C for 2 days with gentle shaking. The PEG-hGH was purified from un-reacted PEG and hGH viaμlan anion exchange column.μlExample 9
Purity Analysis by SDS-PAGE
[443] The following method was used to evaluate the purity of PEG-recombinant hGH
conjugates by SDS-PAGE, followed by total protein staining. Any charged molecule such as aμlprotein will migrate when placed in an electric field. The velocity of migration of a protein in anμlelectric field depends on the strength of the electric field, the net electric charge on the protein, andμlthe frictional resistance. The frictional resistance is the function of the size and shape of the protein.μlWhen denatured in the presence of excess SDS, most proteins bind SDS in a constant weight ratioμlsuch that they have essentially identical charge densities and migrate in polyacrylamide gelsμlaccording to protein size. Proteins separated by gel electrophoresis can be detected by CoomassieμlBrilliant Blue staining.
[444] Equipment for this procedure included, the following or equivalents thereof: XCell
Surelock Mini-Cell (Invitrogen), heat block set to +70-80°C, power supply (up to 200V),μlmicrocentrifuge (such as Beckman Coulter Microfuge 18 or 22R), and reciprocal shaker. Reagentsμlincluded NuPAGE MOPS SDS Running Buffer (20X, Invitrogen PN NP0001); NuPAGE MESμlSDS Running Buffer (20X, Invitrogen PN NP0002); NuPAGE LDS Sample Buffer (4X, InvitrogenμlPN NP0007); NuPAGE Sample Reducing Agent (10X, Invitrogen PN NP0009); 12% Bis-TrisμlNuPAGE precast gel, 1.0mm x 10-well (Invitrogen PN NP0341BOX); 4-12% Bis-Tris NuPAGE

precast gel, 1.0mm x 10-well (Invitrogen PN NP0321BOX); Pre-Stained Molecular Weight Markerμl(SeeBlue PIus2, Invitrogen PN LC5925); MilliQ-quality H20 or equivalent; SimplyBlue SafeStainμl(Invitrogen PN LC6065) or equivalent; reference standard (WHO rhGH standard; calibrationμlsolutions for rhGH (Y35pAF-pB2/pB3, 2 mg/ml); calibration solutions for the pEG-rhGH conjugateμl(PEG30-pY35pAF-01, 2 mg/mL). Protein concentrations of the standards and the test article wereμlmeasured using standard techniques known in the art.μlAnalysis of PEGylated rhGH product
[445] 10 jig of the reference standard (RS, e.g. calibration solution PEG30-pY35pAF-01)
was prepared under non-reducing and reducing conditions. 10 ug of PEG30-pY35pAF-01 (2μlmg/mL) was added to 4X LDS and MilliQ H20 to obtain a final 28μl sample in IX LDS. Forμlreduced conditions, 10 |xg of reference standard was added to 4X LDS, 10X Reducing Agent, andμlMilliQ H2O to obtain a 28 (xl sample in IX LDS and IX Reducing Agent. Similarly, lOjig ofμlPEGylated rhGH test articles were also prepared under non-reduced and reduced conditions. TheμlPEG-rhGH test articles and PEG-rhGH reference standards were not heated, but were snapμlcentrifuged prior to loading on 4-12% Bis-Tris NuPAGE precast gels prepared with IX MES SDSμlRunning Buffer according to manufacturer's instructions. The gels were loaded in the order of Pre-μlStained Molecular Weight Marker, 10 fig reference standard, blank lane (recommended to minimizeμlpotential carryover effects), followed by the test articles with a maximum setting of 200V for 35μlminutes. The gels were incubated in di-HaO, stained with shaking using SimplyBlue or anμlequivalent, and destained with water.
[446] The electropherogram of the PEG-rhGH test article should conform to the
electropherogram obtained with the PEG-rhGH reference standard. Any bands that do not matchμlthe reference standard may be degradation products or aggregates. Higher molecular weight bandsμlmay represent aggregates, and lower molecular weight bands may represent polypeptide that is noμllonger conjugated to PEG.μlExample 10
Purity and Chemical Degradation Analysis of rhGH by CEX-HPLC/EEX-HPLCμl[447] The following method was used to assess relative purity and potential chemical
degradation (i.e. deamidation) of PEGylated recombinant human growth hormone (rhGH) byμlcation-exchange high performance liquid chromatography (CEX-HPLC). CEX-HPLC is a

technique that relies on charge-charge interactions between a protein and the charges immobilizedμlon the resin. Cation exchange chromatography takes advantage of the positively charged ions of aμlprotein that bind to the negatively charged resin. A common structural modification of rhGHμldeamidation of asparagine (Asn) residues, and this CEX-HPLC method permits the separation ofμldeamidated and deamidation intermediates of PEGylated and nonPEGylated rhGH. This methodμlwas used to support identification and purity assessment of PEGylated rhGH. Some partialμldegradation products of rhGH are observable using this technique.
[448] Equipment for this procedure included, the following or equivalents thereof: UVYVis
Spectrophotometer (Agilent 8453 or equivalent); 50 il quartz cuvette; 0.5 mL Vivaspinμlconcentrators (if needed; Vivascience 10,000 MWCO, PES, VS0102 or equivalent); PD-10, NAP-μl10, or NAP-5 column (GE Healthcare, Cat #17-0851-01, 17-0853-01, 17-0854-01); HPLC vialsμland caps (Alltech 100 x screw cap polypropylene vials #12962, TFE liner caps #73048, open holeμlscrew caps #73044, or equivalent); clean 1 and 2 L glass bottles; column - PolyCAT A 4.6 x 200μlmm, 5x , 1000 A (PolyLC, 204CT0510) and PolyCAT A guard column, 4.6 x 10 mm, 5μ1000 Aμl(PolyLC, JGCCT0510); high-pressure liquid chromatography instrument capable of performingμllinear gradients (such as Agilent 1100 HPLC equipped with a vacuum degasser, quaternary pump,μlthermostatted autosampler, thermostatted column compartment, diode array detector (DAD), andμlChemstation chromatography software).
(449] Reagents for this procedure included water (Milli-Q quality or equivalent) and solid
chemicals are analytical grade or better and solvents are HPLC grade or better, unless otherwiseμlnoted. Storage of reagents and procedural steps occurred at room temperature, unless otherwiseμlindicated. Examples of such chemicals include Ammonium Acetate, Spectrum A2149, HPLCμlgrade, or equivalent; Acetonitrile, Fisher A998, HPLC grade, or equivalent; AmmoniumμlBicarbonate, Fluka # 09830, Ultra 99.5%, or equivalent; Glacial Acetic Acid, Fisher # 64-19-7,μlHPLC grade, or equivalent; Sodium Citrate Dihydrate, Spectrum S0165, USP grade, or equivalent;μlGlycine, Spectrum AM125 or equivalent; Mannitol, Spectrum MA165, or equivalent; 6N HO,μlMallinckrodt 2662-46, or equivalent.
[450] Mobile phase A buffer was 50 mM Ammonium Acetate, pH 4.25, 40% Acetonitrile
(AcCN), and Mobile Phase B buffer was 500 mM Ammonium Acetate, pH 4.25, 40% AcCN.μlAdditional reagents prepared were 10% acetic acid; Buffer for Deamidation: 30 mM Ammonium

Bicarbonate, pH 9.0; and Sample Dilution Buffer: 20 mM Sodium Citrate, 20g/L Glycine, 5 g/LμlMannitol, pH 6.0, each sterile filtered using 0.22 |j.m PES filters (Coming #431098, or equivalent).μl[451] World Health Organization (WHO) rhGH (Cat # 98/574) was used as a non-
PEGylated hGH standard. It was reconstituted in 1.0 ml of water and diluted to 1.1 mg/ml usingμldilution buffer. 10% (v/v) of 10% acetic acid was added to bring the pH between pH 3.8 - 4.3 withμla final concentration of 1.0 mg/ml (acceptable range 0.9 - 1.1 mg/ml). Another non-PEGylatedμlhGH standard, the calibration solution Y35pAF-pB2/pB3, was prepared in a similar fashion. AμlPEGylated hGH standard, calibration solution PEG30-pY35pAF-01, was also prepared in a similarμlfashion.
[452] For the PEGylated Resolution Solution, the PEG30-pY35pAF-01 calibration solution
was buffer exchanged into 30 mM Ammonium Bicarbonate, pH 9.0 buffer using a PD-10, Nap-10,μlor Nap-5 desalting column. The standard was concentrated using a 0.5 mL Vivaspin concentrator toμlapproximately 2 mg/ml (acceptable range 1.9-2.1 mg/ml), and the sample was incubated at 37°Cμlfor 24 hours. The sample or portion of the sample needed was diluted to 1.1 mg/ml using dilutionμlbuffer, and 10% (v/v) of 10% acetic acid was added to bring pH between pH 3.8 - 4.3 with a finalμlconcentration of 1.0 mg/ml (acceptable range 0.9 - 1.1 mg/ml).
[453] The test article was diluted to 1.1 mg/ml using dilution buffer and 10% (v/v) of 10%
acetic acid was added to bring pH between pH 3.8 - 4.3 with a final concentration of 1.0 mg/mlμl(acceptable range 0.9 — 1.0 mg/ml). Protein concentrations of the standards and the test article wereμlmeasured using standard techniques known in the art.μlProcedure
[454] The instrument was set-up with the following conditions: 1) Column: PolyCAT A
204CT0510 and JGCCT0510; 2) Auto sampler Temperature: room temperature; 3) Pump Setup:μlstep gradient: 81.5 - 108.5 mM Ammonium Acetate pH 4.25 (7 - 13% B), followed by 108.5 - 500μlmM Ammonium Acetate pH 4.25 (13 - 100% B); 4) Table 7;



Peak Width:0.1 min; Slit: 4 nm; Stop Time: as pump; 7) Column Thermostat: Temperature: 30°C;μlrecord the temperature.
[455] The column was equilibrated with 10-15 column volumes of 100% mobile phase A.
25-50 jal of the PEGylated calibration solution PEG30-pY35pAF-01 was injected. The mainμlPEGylated peak eluted at a retention time of 56.97 min (± 0.5 min). Next, 25-50μl of the WHO orμlcalibration solution Y35pAF-pB2/pB3 was injected and the HPLC program was run. The mainμlnon-PEGylated peak eluted at a retention time of 98.54 min (± 0.5 min), a relative retention time ofμl1.73 ±0.01 to the main PEGylated peak.
[456] 25-50 jLtl of the PEGylated resolution solution was then injected. hi the
chromatogram obtained, the main PEGylated peak eluted at a retention time of 56.97 min (± 0.5μlmin), and the PEGylated deamidated peak eluted at a retention time of 0.79 ± 0.02 relative to theμlmain peak (45.23 ± 0.3 min; (current conditions result in a resolution of 2.3 ± 0.02).μl[457] 25-50 x of the PEGylated test article was then injected, and the HPLC program was
run. The samples were run in triplicate, and average retention times were noted. Chromatogramsμlwere generated with absorbance (280 nm).μlData Analysis

[458] The retention time of the PEGylated rhGH test article was compared with the
calibration solution PEG30-pY35pAF-01. The average purity of the test article was calculatedμlusing: (Integration area of the main peak / integration areas of all peaks) x 100%. Any peak(s) dueμlto the solvent were disregarded.μlExample 11
Purity Determination of rhGH by SEC-HPLC
[459] This procedure was used to assess the purity of recombinant human growth hormone
(rhGH) and PEGylated rhGH by size-exclusion high performance liquid chromatography (SEC-μlHPLC). This test separates monomer from dimer and other related substances of higher molecularμlweight in the sample, as well as PEGylated and nonPEGylated samples. SEC-HPLC is a techniqueμlusing the stationary phase as a porous matrix which is permeated by mobile phase molecules.μlSample molecules small enough to enter the pore structure are retarded, while larger molecules areμlexcluded and therefore rapidly carried through the column. Thus, size exclusion chromatograph)μlmeans separation of molecules by size and the chromatographic elution time is characteristic for zμlparticular molecule. This procedure is used to determine the percentage of monomer (PEGylatedμland unPEGylated) rhGH. Dimer and other high molecular weight proteins are observable using thisμltechnique. An example of SEC-HPLC integration is shown as Figure 20 with the y-axis asμlabsorbance (214 nm) and the x-axis as time (minutes).
[460] References for this technique include European Pharmacopoeia 2002, p. 193; British
Pharmacopoeia 2001, p.1941; "High-Performance Size-Exclusion Chromatographic Determinationμlof the Potency of Biosynthetic Human Growth Hormone Products" by R.M. Riggin et al. Journal ofμlChromatography 435(1988), p. 307-318.
[461] Equipment for this procedure included, the following or equivalents thereof: UV/Vis
Spectrophotometer (Agilent 8453 or equivalent); 50 ul quartz cuvette; 0.5 mL Vivaspinμlconcentrators (if needed; Vivascience 10,000 MWCO, PES, VS0102 or equivalent); HPLC vialsμland caps (Alltech 100 ul screw cap polypropylene vials #12962, TFE liner caps #73048, open holeμlscrew caps #73044, or equivalent); clean 1 and 2 L glass bottles; Column — Tosohaas TSK SuperμlSW3000 18675 and Super SW Guard Column 18762, a silica-based size exclusion HPLC columnμlwith a dimension of 4.6 x 300 mm, particle size of 4 xm and pore size of 250A along with a guardμlcolumn having a dimension of 4.6 x 35 mm and 4 x particle size; High-pressure liquid

chromatography instrument capable of performing linear gradients (such as Agilent 1100 HPLCμlequipped with a vacuum degasser, quaternary pump, thermostatted autosampler, thermostattedμlcolumn compartment, diode array detector (DAD), Refractive Index detector (RID) andμlChemstation chromatography software).
[462] Reagents for this procedure included water (Milli-Q quality or equivalent) and solid
chemicals are analytical grade or better and solvents are HPLC grade or better, unless otherwiseμlnoted. The storage of reagents and procedural steps occurred at room temperarture, unlessμlotherwise indicated. Examples of such chemicals included Monobasic Monohydrate SodiumμlPhosphate, Spectrum U.S.P. grade SOI30, or equivalent; Dibasic Heptahydrate Sodium Phosphate,μlSpectrum U.S.P. grade S0140, or equivalent; 2-propanol, Fisher HPLC grade A451-4, or equivalent.μl[463] Mobile phase buffer was 97% of 63mM sodium phosphate pH 7.0, 3% of 2-
propanol. Solution A was 25 mM Sodium Phosphate, pH 7.0. Both were sterile filtered using 0.22μl|im PES filters (Corning #431098, or equivalent).
[464] World Health Organization (WHO) rhGH (Cat. # 98/574) was used as a non-
PEGylated hGH standard. It was reconstituted with 1.0 ml of water and diluted to 1 mg/mlμlconcentration (acceptable range 0.9 - 1.1 mg/ml) in WHO buffer. Another non-PEGylated hGHμlstandard, calibration solution Y35pAF-pB2/pB3, was prepared in a similar fashion and diluted withμl20 mM sodium citrate, 2% glycine, 0.5% mannitol, pH 6. A PEGylated hGH standard, calibrationμlsolution PEG30-pY35pAF-01, was also prepared in a similar fashion and diluted with 20 mMμlsodium citrate, 2% glycine, 0.5% mannitol, pH 6. For the Resolution Solution, the PEG30-μlpY35pAF-02 higher molecular weight standard was brought to 1 mg/ml concentration (acceptableμlrange 0.9 - 1.1 mg/ml). This solution contains approximately 33% PEG-PEG-GH, 66.5% PEG-μlGH). Test material was diluted to approximately 1.0 mg/ml with Solution A (acceptable range 0.9 -μl1.1 mg/ml). All sample concentrations were measured using standard techniques known in the art.μlThe dilution of samples may be performed with any suitable buffer.μlProcedure
[465] The instrument was set-up with the following conditions: 1) Column: TSK Super
SW3000 18675 and Guard Column 18762; 2) Pump Setup - gradient: isocratic; flow rate: 0.3μlml/tnin; duration: 25min; Max Pressure: 120 bar, 3) Injector Setup — Injection: Standard Injection;


Injection Volume: 10 μl; Draw Speed: 100μl/min; Injection Speed: 100μl/min; Needle wash: 100μlul H20; Stop Time: As pump; 4) DAD Signals: Table 9;

Peak Width:0.05 min; Slit: 2 ran; Stop Time: as pump; 5) RID Signal - Temperature: 35°C;
Response Time:0.2 mm 4s, standard; 6) Column Thermostat: Temperature: 23 °C; record the
temperature.
[466] The column was equilibrated with 10 column volumes (50 ml = 166 min at 0.3
ml/min) of the mobile phase, and the RID was purged for at least 20 minutes before injecting
samples. DAD and RI detectors were autobalanced before sample runs.
[467] 20μl of the calibration solution Y35pAF-pB2/pB3 (or WHO standard) was injected,
and the HPLC program was run. In the chromatogram obtained, the main unPEGylated peak eluted
at a retention time of approximately 12.96 (± 0.05) min. The higher molecular weight
unPEGylated rhGH dimer eluted at a retention time of 0.94 ± 0.02 relative to the main peak.
Higher molecular weight aggregates eluted at retention times of 7.3 - 8.0 min.
[468] 20|il of the calibration solution PEG30-pY35pAF-01 was injected. The main
pegylated peak eluted at a retention time of approximately 8.33 (±0.08) min (relative retention time
of 0.64 to the unPEGylated rhGH). Higher molecular weight PEGylated rhGH aggregates eluted at
times greater than 8.0 min.
[469] 20μl of the resolution solution was injected, and the HPLC program was run. The
main PEGylated peak elutes at a retention time of 8.28 min, and the higher molecular weight
species eluted at 7.54 min, a relative retention time of 0.9 (± 0.05) relative to the main PEGylated
peak.
[470] 20μlof the test article was injected, and the HPLC program was run. Samples were
run in triplicate and average retention times were noted. The retention time of the rhGH test article
was compared with the rhGH standard(s).

[471] The SEC-HPLC data from the test article was compared to data obtained from the
reference standards. To determine the purity of PEGylated rhGH, the integrated main peak areas ofμlthe PEGylated rhGH sample was compared with the total peak area, and the percentage ofμlPEGylated monomer in PEG-rhGH sample was calculated by: (main peak area of PEG- rhGHμlsample / total peak area ) x 100%. The percentage of PEGylated dimer, higher aggregates, andμlnonPEGylated monomer were calculated in the PEGylated hGH test article. Any peak(s) due to theμlsolvent were disregarded. Peaks eluting in the chromatogram prior to the main PEGylated hGHμlpeak represent higher molecular weight species. Such higher molecular weight species may includeμlbut are not limited to dimers (such as PEG-PEG-hGH and other possible dimers) or solubleμlaggregates. Peaks eluting after the main PEGylated hGH peak represent lower molecular weightμlspecies. Such lower molecular weight species may include but are not limited to non-PEGylatedμlmonomer and clipped forms of PEGylated hGH.μlExample 12
Purity and Chemical Degradation Analysis of PEG-hGH by RP-HPLC
[472] The following method was used to assess relative purity and potential chemical
degradation (i.e. oxidation) of PEGylated recombinant human growth hormone (PEG-hGH) byμlreverse phase high performance liquid chromatography (RP-HPLC). RP-HPLC is a technique thatμlseparates molecules on the basis of relative hydrophobicities. Samples are passed over a stationaryμlphase of silica covalently bonded to hydrocarbon chains. The molecules of interest are retarded byμlthe stationary phase and eluted with an isocratic solvent. The chromatographic elution time isμlcharacteristic for a particular molecule. This method separates PEG-hGH from nonPEGylatedμlhGH, as well as isoforms based on subtle differences in hydrophobicity and retention behaviorμlassociated with structural modifications such as oxidation. This method was used to supportμlidentification and purity assessment of PEG-hGH. Some partial degradation products of rhGH areμlobservable using this technique. An example of RP-HPLC integration is shown as Figure 21 withμlthe y-axis as absorbance (214 nm) and the x-axis as time (minutes).
[473] Equipment for this procedure included, the following or equivalents thereof: UVYVis
Spectrophotometer (Agilent 8453 or equivalent); 50quartz cuvette; 0.5 mL Vivaspinμlconcentrators (if needed; Vivascience 10,000 MWCO, PES, VS0102 or equivalent); HPLC vialsμland caps (Ailtech 100 p.1 screw cap polypropylene vials #12962, TFE liner caps #73048, open hole

screw caps #73044, or equivalent); clean 1 and 2 L glass bottles; Column - J.T. Baker Wide PoreμlButyl, 250 x 4.6 mm (cat# 7116-00); High-pressure liquid chromatography instrument capable ofμlperforming linear gradients (such as Agilent 1100 HPLC equipped with a vacuum degasser,μlquaternary pump, thermostatted autosampler, thennostatted column compartment, diode arrayμldetector (DAD), and Chemstation chromatography software).
[474] Reagents for this procedure included water (Milli-Q quality or equivalent) and solid
chemicals are analytical grade or better and solvents are HPLC grade or better, unless otherwiseμlnoted. Storage of reagents and procedural steps occurred at room temperature, unless otherwiseμlindicated. Examples of such chemicals include, Acetonitrile, Fisher A998, HPLC grade, orμlequivalent; Trifluoroacetic Acid, Halocarbon UN2699, Biograde, or equivalent; Sodium Phosphate,μlSpectrum MA 1654, USP grade, or equivalent; Glycine, Spectrum AMI 25 or equivalent; Mannitol,μlSpectrum MA165, or equivalent; Trehalose, Fluka 90208, or equivalent; ION Sodium Hydroxide, orμlequivalent. Mobile Phase A was 0.1% TFA in H2O, and Mobile phase B was 0.1% TFA inμlAcetonitrile.
[475] One of the nonPEGylated standards was World Health Organization (WHO) rhGH
(Cat. # 98/574). It was reconstituted with 1.0 ml of water and diluted to 1.0 mg/ml (acceptableμlrange 0.9 - 1.1 mg/ml) with WHO buffer. Another nonPEGylated standard, calibration solutionμlY35pAF-pB2/pB3, was prepared in a similar fashion and diluted with 20 mM sodium citrate, 2%μlglycine, 0.5% mannitol, pH 6. Calibration solution PEG30-pY35pAF-01, a PEGylated standardμlwas also prepared in a similar fashion and diluted with 20 mM sodium citrate, 2% glycine, 0.5%μlmannitol, pH 6. For the PEGylated Resolution Solution, PEG30-pY35pAF-01 was incubated witibμl0.1% H202 for 24 hours at 4°C to oxidize PEG30-pY35pAF~01. To obtain between 5% and 20%μloxidized PEG30-pY35pAF-01, this incubation time with H2O2 may vary between 4-24 hours.μlCatalase (20 mg/ml stock) can be added to stop the oxidation reaction if needed.μl[476] The test article was diluted to 1.0 mg/ml using dilution buffer (acceptable range 0.9 —
1.0 mg/ml). The sample dilution buffer may be any suitable buffer. Standards and test articleμlprotein concentrations were measured using standard techniques known in the art.μlProcedure


Peak Width:0.1 min; Slit: 4 ran; Stop Time: as pump; 6) Column Thermostat: Temperature: RT;μlrecord the temperature.
[478] New columns were flushed with 10CV of distilled HPLC-grade water to remove the
shipping solvent. The column was cleaned with 10-15 column volumes (CV) of 0.1% TFA in
60% isopropanol in MillQ H20, and 10 - 15 CV H20. This can be followed by 5 CV of 50:50
DMSO:H20 if necessary; otherwise the column was equilibrated with mobile phase A.
[479] The column was equilibrated with 10 - 15 column volumes of 100% mobile phase
A. 10μl of the PEGylated calibration solution PEG30-pY35pAF~01 was injected. The main
pegylated peak eluted at a retention time of 17.05 min (± 0.5 min).
[480] 10 ml of the WHO or calibration solution Y35pAF-pB2/pB3 was injected, and the
HPLC program run. The main nonPEGylated peak elutes at a retention time of 19.04 min (+
0.5min), a relative retention time of 1.1 ± 0.05 to the main PEGylated peak.

[481] 10μl of the PEGylated resolution solution was injected. In the chromatogram
obtained, the main PEGylated peak eluted at a retention time of 17.12 min (± 0.5min), and the
PEGylated oxidized peak eluted at a retention time of 0.93 ± 0.02 relative to the main peak (15.94 ±
0.3 min and a resolution of 2.97 ± 0.05).
[482] 10 i of the PEGylated test article was injected, and the HPLC program was run.
Samples were run in triplicate and average retention times noted.
[483] The retention time (using A214) of the PEGylated rhGH test article was compared
with the calibration solution PEG30-pY35pAF-01. The average purity of the test article (using
A214) was calculated using: (Integration area of the main peak / integration areas of all peaks) x
100%. Any peak(s) due to the solvent were disregarded.
Example 13
[484] A summary of the methods used in formulations development is shown as Table 12.
Long-term storage, basic, or acidic conditions can result in the following product-related impurities:
Deamidated hGH - Asnl49, Asnl52; Oxidized hGH - Metl4, Metl25; Isomerized hGH - Aspl30;
Dehydrated hGH — Asp 130; desPhe/des-Phe-Pro — Phel, Pro2 (non-enzymatic cleavage); Dimer
(covalent and non-covalent species); Clipped hGH (cleavage between Thrl42 and Tyrl43).
Additional methods such as moisture analysis, pH and bioassays including, but not limited to,
proliferation assays are used to evaluate formulations for PEGylated hGH comprising a non-



Example 14
Lyophilized Formulation Study
[485J Purified PEGylated hGH samples comprising a non-naturally encoded amino acid in
20 mM sodium citrate, 2% glycine, 0.5% mannitol, pH 6 were dialyzed into a buffer from theμlformulations matrix (Table 13) prior to stability analysis. A reaction between the non-naturallyμlencoded amino acid (para-acetylphenylalanine) substituted for a tyrosine at position 35 of hGH andμlmPEG-oxyamine formed an oxime bond between hGH and PEG. The samples were thenμllyophilized and stored at 4, 25, and 40°C. After storage of the lyophilized samples for variousμllengths of time (0, 1 week, 2 weeks, 4 weeks, 6 weeks, 2 months, and 3 months), the samples wereμlreconstituted in water to 2 mg/ml and characteristics such as moisture content (Karl Fisher), pH andμlconcentration were observed post reconstitution using standard techniques known to one of ordinaryμlskill in the art. The methods described in Examples 9-13 were used to analyze the PEGylated hGHμlfor degradation products. Sodium phosphate and sodium succinate were included in theμlformulations matrix. Additional analyses include time points at 4 months and 6 months.















































[491] Lower pH formulations were studied for 4 weeks (pH 4) and 6 weeks (pH 5). For the
time-frame studied, the following trends were found. SEC-HPLC and RP-HPLC analysis showedμlthat lower pH formulations (pH 4 and 5) generated increased levels of unPEGylated material afterμlstorage of lyophilized material at 4°C than higher pH formulations (pH 6 and 7). In addition,μlhigher aggregate levels were observed at lower pH formulations at 25 and 40°C than with the pH 6μland 7 formulations. Also, pH 6 and 7 formulations exhibited a greater % of main peak area than theμllower pH formulations as analyzed by cIEX-HPLC.
[492] Throughout the 3 month study timeframe with storage of lyophilized hGH at 25 and
40°C, formulations with Histidine showed the least amount of high molecular weight aggregates byμlSDS-PAGE. Formulations containing mannitol in combination with glycine were found to beμlstabilized hGH against agitation induced aggregation, showing that the combination is a good

bulking agent. However, histidine in combination with mannitol also demonstrated stabilization of
hGH against agitation induced aggregation. Trehalose was an effective stabilizer.
Example 15
Injection Feasibility Study
[493] Injection feasibility studies were performed using a formulation of PEGylated hGH
at four different concentrations. PEG-hGH in 20 mM sodium citrate, 2% glycine, 0.5% mannitol,
pH 6 was buffer exchanged into 10 mM NaBfePCU, 4% mannitol, 2% trehalose, 0.01% polysorbate
20 (PS20), pH 6.0 (Formulation ID = P6MT) via centrifugal concentration. The PEG-hGH in
P6MT buffer was concentrated to 8, 10,12, and 14 mg/mL. 1 mL fill was placed into vials, and the
samples were lyophilized. Lyophilized vials were reconstituted with 1 mL of water. Injection
feasibility was tested by pushing 1 mL of each concentration through 27 gauge needle with 4 lbs of
force. Instant reconstitution was found for all concentrations of PEGylated hGH tested. 8 mg/mL
PEGylated hGH injected in 3.5 s (seconds); 10 mg/mL injected in 3.6 s; 12 mg/mL injected in 3.9 s;
and 14 mg/mL injected in 4.5 s. j
[494J Additional injection feasibility studies were performed with 30 gauge needles. PEG-'
hGH, in 10 mM NaH2P04, 4% mannitol, 2% trehalose, 0.01% polysorbate 20, pH 6.0 (Formulation
ID = P6MT buffer), at 8, 10, 12, and 14 mg/mL was tested for injection feasibility with a 30 gauge:
needle. The samples were pushed through a 30 gauge needle under 8 lbs. of force and timed for
duration. When 1 mL of each concentration was pushed through a 30 gauge needle under 8 lbs. of
force, the 8 mg/mL, 10 mg/mL, 12 mg/mL and 14 mg/mL concentrations injected in 7.4 s
(seconds), 9.9 s, 9.9 s, and 10.7 s, respectively.
[495] Injection of 1 cc within 10 seconds under 8 lbs. of force is generally accepted as the
standard for injection since the average person can apply up to 8 lbs. offeree on a syringe.
Example 16
One Week Reconstitution Study
[496] For this study, reconstituted samples were stored for one week at 4°C. The
formulations matix used for this study was the same as Table 13. After storage for a week, the
concentration and pH were measured. The samples were also analyzed by SEC-HPLC, RP-HPLC,
and SDS-PAGE as shown in Tables 72-74 and Figures 59-60.




[497] One week storage of reconstituted samples at 4°C resulted in slight increase (-1%)
of prepeak 1 of P6MT, P6MTMet, P7GT, and P6GT-P. Lower pH samples showed more
dePEGylation than samples at pH 6 and 7.
Example 17
Agitation/UV studies
t498] Samples were subjected to stresses that may mimic long term storage conditions and
the induction of degradation or aggregation products was observed. Degradation upon exposure to }
UV light may occur in the molecule, or specifically at the oxime or PEG. Two agitation studies
were performed. In the 4 hour agitation study, reconstituted samples from Table 13 were vortexed
vigorously at ambient room temperature for 4 hours prior to measuring concentration and pH, and .
analysis was performed by SEC-HPLC, RP-HPLC, and SDS-PAGE. A subset of these samples
were analyzed one week later after storage at 4°C by SEC-HPLC to investigate if aggregates might
dissociate back to monomer. Control samples were reconstituted samples that were stored at
ambient room temperature for 4 hours.
[499] In the 2 hour agitation study, polysorbate-free samples, their polysorbate-containing
counterparts, and P6MA were vortexed gently for 2 hours at ambient temperature to confirm the
effect of Polysorbate on stability of PEGylated hGH after agitation. See Table 85.
[500] For the UV Exposure study, controls were reconstituted samples stored at ambient
room temperature for 4 hours. Lyophilized samples were exposed to UV light for 4 hours at
ambient temperature and were reconstituted in water prior to analysis by SDS-PAGE, RP-HPLC,
and SEC-HPLC.
[501] See Tables 75-85. SDS-PAGE analysis of these samples is shown in Figures 61-66.
SEC-HPLC data showed that all formulations containing Polysorbate 20 at 0.01% induced

aggregation during agitation (30-80%). Bam, NB et al. describe the use of Tween 20 (Polysorbateμl20) in the inhibition of insoluble aggregates during agitation of recombinant hGH (rhGH) (J PharmμlSci. 1998 Dec; 87(12): 1554-9). Moreover, Maa, YF et al. indicated that polysorbate 20 resulted in aμlreduction of insoluble protein aggregates in the production of a spray-dried rhGH powder (J PharmμlSci 1998 Feb;87(2): 152-9). Agitation-induced aggregates were found to be irreversible non-μlcovalent aggregates. Major light-induced degradation pathways were depegylation and formationμlof covalent prepeak 2.









Example 18μlAgitation Study
[502] Another agitation study was performed with the formulations matrix shown in Table
86. Agitation was performed on 2 mg/mL samples at 250 iL fill in glass vials. Samples wereμlagitated for 4 hours at room temperature. Duplicate set of samples, incubated at room temperatureμlundisturbed, was used as positive controls ("Controls"). The pH and concentration of the samplesμlwas measured, and SEC-HPLC analysis was performed on t=0, control, and agitated samples asμlshown in Tables 87-90.





Example 19
Accelerated Aggregation Studies
[504] Accelerated stress studies were performed to mimic long term storage conditions and
identify potential degradation or aggregation products. Accelerated aggregation studies wereμlperformed using 2 formulations (Formulation ID H7MT-P and H7MGT-P) at approximately 8 andμl14 mg/ml. The components of the formulations are described in Table 91. The "-P" designation'μlindicates no polysorbate 20. Stresses studied were freeze-lhaw conditions, agitation, UV exposure, μland temperature. The methods described above were used to evaluate PEGylated hGH. Samplesμlwere evaluated by SDS-PAGE and SEC-HPLC methods described in Examples 9 and 11,μlrespectively. Additional time points for analysis include 1 month, 2 month, etc. Additional μltechniques for analysis of samples include Dynamic Light Scattering (DLS) and AnalyticalμlUltracentrifugation.


[505] 63 mL of 5.7 mg/mL PEGylated hGH (in 20 mM sodium citrate, 2% glycine, 0.5%
mannitol, pH 6) was concentrated to 14.2 mg/mL. Half of the concentrate was dialyzed againstμlH7MT-P, and the other half against H7MGT-P. Since the dialysis caused the protein to be diluted,μlthe sample in H7MT-P was concentrated to 13.6 mg/mL and the H7MGT-P was concentrated toμl13.5 mg/mL. A portion of the the PEGylated hGH (approximately 4.5 mL) in H7MT-P at 13.6μlmg/mL was diluted to 7.8 mg/mL with H7MT-P buffer. Similarly approximately 4.5 mL ofμlPEGylated hGH in H7MGT-P at 13.5 mg/mL was diluted to 8.0 mg/mL with H7MGT-P buffer.μlFreeze/thaw studies
[506] The formulation was frozen at -70°C for 15 minutes. It was then thawed at 25°C.
The vial was then uncapped to remove a portion of the sample for SEC-HPLC and SDS-PAGEμlanalysis, and the vial re-capped. This procedure was repeated five times. The results of the SEC-μlHPLC analysis are shown in Tables 92-95. SDS-PAGE analysis is shown in Figures 67-70.


- . .
[507] The freeze/thawing of the samples caused a 0.2 — 0.3% increase in higher MW
aggregates, with possible dissociation of higher MW aggregates after the first freeze-thaw cycle in 8μlmg/mL samples.μlVortex/UV Exposure
[508] For this study, the formulations matrix shown in Table 91 was used. For the control
samples in the agitation (vortex) and UV exposure studies, formulations were held at rooirμltemperature for 6 hours. In the agitation study, liquid samples were vortexed at room temperatureμlfor 6 hours at high speed. In the UV exposure study, lyophilized samples were exposed to UV lighlμlat room temperature for 4 hours and reconstituted prior to analysis. Samples were removed foiμlSEC-HPLC and SDS-PAGE analysis. Tables 96-98 show the SEC-HPLC data for the controlμlsamples, the vortexed samples, and the UV-exposed samples. SDS-PAGE analysis of theseμlsamples is shown as Figures 71 and 72.



[509] Histidine formulations without polysorbate 20 were confirmed to reduce the amount
of aggregates formed with vigorous agitation.
[510] DePEGylation was the main degradation event and was more pronounced in samples
with the lower concentration of hGH, UV exposure resulted in an increase in % Pre-peak 2
(dimerization) compared to the control.
[511] In another agitation study, lyophilized samples were stored at 4°C or 40°C for 1 week
or 2 weeks. The samples were then reconstituted prior to analysis by SDS-PAGE and SEC-HPLC.
Tables 99-102 show the SEC-HPLC results. Figures 73-74 show SDS-PAGE analysis for 1 week
samples. Figures 75-76 show SDS-PAGE analysis for 2 week samples.



[512] After 1 week, H7MT-P produced less higher molecular weight aggregate than
H7MGT-P at 4°C. H7MT-P resulted in slightly more dimer formation, but less dePEGylated hGHμlthan H7MGT-P at 40°C. After two weeks, an increased amount of dimerization was observed atμl40°C. Significant de-PEGylation was observed at 40°C. H7MT-P formed less de-PEGylated hGHμlthan H7MGT-P at 40°C. Additional time points include measurements at 4 weeks.μl[513] In addition, samples of PEGylated-hGH were exposed to thermal-unfolding
conditions. The thermal-unfolding samples were incubated for 10 minutes at 85°C, which is aboveμlthe melting temperature (Tm) of 82°C. Thermal-unfolding induced aggregation. Table 103 showsμlthe SEC-HPLC analysis of the samples exposed to thermal-unfolding. SDS-PAGE analysis isμlshown in Figures 73-74.

Example 20
FT/IR (Fourier Transform Infrared Spectroscopy) Scans
[514] Analysis of PEGylated hGH was performed with FT/IR. FT/IR scans were
performed on a Jasco model FT/IR 660 Plus. Each sample was scanned 320 times at a resolution of
4 cm"1 and analyzed with Jasco Spectra Manager vl .53.00. Air backgrounds and water blanks were
taken before each day's sample runs. The elimination of signals derived from water was confirmed

by the absence of absorbance at 1700 cm" After the appropriate background and blank controls
were subtracted from each sample spectrum, second derivative analysis of the remaining protein
and excipient peaks was performed in the Amide I region (1600 -1700 cm"1). As a liquid control,
PEG-hGH bulk at 6 mg/mL was used.
[515] The Amide I signal of PEG-hGH was relatively weaker than other proteins even after
lyophilization. However, most samples showed a-helix signal at 1651 cm"1, so qualitative
comparison among formulation candidates could be accomplished. Glycine shows a strong signal at
Amide I region, so the signal could have been distorted during the course of subtracting glycine
signal from original data.
[516] Second-derivative FTIR spectra [signal intensity (y axis) vs. wave number in cm-1 (x-
axis)] of the P6MT, S4MT, S5MT, H6MT, P6GT, P6MS, P6MTmet, P7MT, and P6MT-P
formulations compared to the liquid control showed: 1) Most mannitol formulations preserved good
a-helix signal regardless of buffer or pH. 2) One formulation, P6GT containing glycine, showed
significant shift in the band signal. 3) Other peaks were observed at 1620, 1630, 1725, and 1750.
Second derivative FTIR spectra of the S5GT, P6MA, P7GT, P6MGT, P6MGT-P, and P6GT-P
formulations had poor a-helix signal. Moreover, all glycine formulations showed significant
deviation from the native signal of liquid sample. The arginine-containing formulation exhibited
major structural changes during lyophilization.
[517] After two months of storage of the lyophilized material at 4°C, FT/IR was
performed. The Amide I signal of PEG-hGH was relatively weaker than other proteins even after
lyophilization. However, most samples showed a-helix signal at 1651 cm"1, so qualitative
comparison among formulation candidates could be accomplished. Glycine showed a strong signal
at Amide I region. At two months, all glycine formulations showed significant deviation from the
native signal of liquid sample. Most mannitol formulations preserved good a-helix signal
regardless of buffer or pH.
Example 21
Agitation (Surfactant testing)
[518] PEG-hGH in 20 mM sodium citrate, 2% glycine, 0.5% mannitol, pH 6 was diluted to
2 mg/mL with H7MT-P. 0.01% polysorbate 20 (PS) was added to one sample, 0.01% Pluronic F68
(F68) to another, and the third had no surfactant added. Agitation was performed with 500 ^L fill

in glass vials. Samples were agitated for 1 hour at room temperature. SEC-HPLC analysis of theμlsamples is shown in Table 104; samples that were agitated are noted with "Vtx/

[519] Relative to the other samples tested, Pluronic F68 was the most effective in
preventing agitation induced aggregation as shown in Table 104.
[520] Additional experiments may include, dialyzing the samples instead of diluting them
to remove residual excipients and/or testing other surfactants, including but not limited to,-μlpolysorbate 80. A surfactant may be used alone or in combination with one or more other'μlsurfactants. Additional studies include testing the effect of the following surfactants on the stabilityμlof PEG-hGH in H7MT formulation against agitation-induced aggregation: 1) no surfactant'μl(negative control); 2) polysorbate 20 at various amounts including, but not limited to, 0.01%; 3)μlPluronic F68 (or Poloxamer 188) at various amounts including, but not limited to, 0.005%, 0.01%,μl0.05%, and 0.1%; 4) polysorbate 80 at various amounts including, but not limited to, 0.01%; and 5)μla combination of pluronic F68 and polysorbate 20 or pluronic F68 and other surfactants. Additionalμllyophilized formulations (H7MT) containing the best surfactant or combination of surfactants willμlbe prepared and their stability at 40°C will be compared to the H7MT formulation. Theμlconcentration of PEG-hGH will be 2 mg/ml, and time points for analysis will be at 0, 1,2, 3, and 4μlweeks, or longer. The techniques described herein will be used for analysis.μlExample 22μlLong term studies
[521] Additional long term stability studies include evaluation of formulations after storage
at various temperatures, including but not limited to, 4°C and 29°C, for 0, 3, 6, 9, 12, 18, and 24

months. The stability of samples reconstituted after lyophilization may be investigated duringμlstorage at 2-S°C for various lengths of time, such as 0, 1 day, 3 days, and 1 week.μlExample 23
Lyophilized Formulation Study
[522] Additional data was generated from the lyophilized formulation study discussed in
Example 14. Data generated for the four month time point include: SDS-gels (samples stored atμl4°C and 25°C for 4 months; Figures 77-80); concentration and pH post reconstitution (Tables 105-μl106); SEC-HPLC analysis of samples stored at 4°C and 25°C for 4 months (Tables 107-108); andμlRP-HPLC analysis of samples stored at 4°C and 25°C for 4 months (Tables 109-110). After fourμlmonths, the formulation containing histidine continued to show the least amount of covalentμlaggregates via SDS-PAGE of the formulations tested. Over the last two months, H6MT and P7MTμlhave showed minimal change at 25°C.






Example 24
Histidine Interaction Study with PEG-HGH
[523] The objective of this study is to investigate the pH drop that has been observed in 10
mM histidine buffer, 4% mannitol, 2% trehalose, pH 7.0 when PEG-hGH is added at concentrations
of at least 8 mg/mL. This study investigated whether the concentration dependent pH change in
histidine formulations is due to the binding of histidine with PEG-hGH.
[524] RP-HPLC and/or IEX-HPLC methods for determining free/bound histidine may be
used in such studies.
Dialysis of 5 mg/mL and 25 mg/mL PEG-hGHμl[525] One or two concentrations of PEG-hGH were dialyzed against one of the following
buffers: 10 mM Na2HP04, pH 7.1; 10 mM histidine (pH 7.0); 10 mM histidine (free base), pH 7.7;μl30 mM histidine (free base), pH 7.7. The pH of the protein was measured after dialysis. Theμlamount of histidine was determined by running SEC-HPLC and measuring the histidine peak whichμlelutes after protein peak. The 214 nm:280 nm was compared to determine if histidine bound to the tμlPEG-hGH.
[526] For 10 mM Histidine (free base), pH 7.7, the 4.5 mg/ml protein sample had a pH of
7.18, and the 14.0 mg/ml protein sample had a pH of 6.89. The 214 nm:280nm ratios were theμlsame (19.2) for both protein concentrations. For 30 mM Histidine (free base), pH 7.7, the 13.3μlmg/ml protein sample had a pH of 7.19. The 214 nm:280 nm ratio was 19.2. For 10 mM His, pHμl7.0, the 4.6 mg/ml protein sample had a pH of 6.94, and the 13.2 mg/ml protein sample had a pH ofμl6.76. The 214 nm:280 nm ratios were the same (19.1) for both protein concentrations. For 10 mMμlNa2HPC>4, pH 7.1, the 4.6 mg/ml protein sample had a pH of 7.10, and the 13.2 mg/ml proteinμlsample had a pH of 7.09. The 214 nm:280 nm ratios were 19.2 for the lower protein concentration,μland 19.1 for the higher protein concentration,μl[527] pH change during concentration
[528] pH changes were measured of the protein during concentration. 10 mM histidine
(free base) and 30 mM His (free base) were tested; the protein concentration started at 1 mg/ml.μlWith 10 mM His (free base), pH 7.7, the following results were found: 1.0 mg/ml protein had pHμl7.41; 11.7mg/mlproteinhadpH6.82;andl5.0mg/mlproteinhadpH6.71. With 30 mM His (free

base), pH 7.7, the following results were found: 1,0 mg/ml protein had pH 7.58; 11.4 mg/ml proteinμlhad pH 7.22; and 17.1 mg/ml protein had pH 7.05.
Addition of histidineμl[529] PEG-hGH was concentrated with 14 mg/ml and dialyzed against water. The
histidine concentration was increased by adding small volumes of concentrated histidine to theμlprotein. The pH was measured at each histidine increase. The following pH measurements wereμldetermined with the histidine concentration (mM): pH 5.57 at 0 mM Histidine, pH 5.68 at 0.25 mMμlHistidine, pH 5.74 at 0.5 mM Histidine, pH 5.86 at 1 mM Histidine, pH 6.13 at 2.5 mM Histidine,μlpH 6.36 at 5 mM Histidine, pH 6.63 at 10 mM Histidine, and pH 7.05 at 30 mM Histidine.μl[530] In these studies, it was found that histidine does not bind to the PEG-hGH. The
buffering capacity of histidine was unable to overcome the buffering capacity of the protein.μlMonobasic phosphate is tested to determine if it can provide enough buffering capacity to maintainμlpH. Any buffer that has a buffering capacity between about pH 5.5 and about pH 8.0, including butμlnot limited to, monobasic phosphate may be suitable. Formulations with histidine had lowerμlamounts of covalent aggregates and aggregates from agitation..
[531] Monobasic phosphate is tested in the following assay. PEG-hGH is concentrated to
14 mg/ml and dialyzed against water. The phosphate concentration is increased by adding smallμlvolumes of concentrated phosphate to the protein. The pH is measured at each phosphate increase,μland a plot is generated with pH vs. phosphate concentration (mM)μlExample 25
[532] Additional data was generated from the accelerated aggregation study discussed in
Example 19. Data generated for the four week time point include: SDS-gels (samples stored at 4°Cμland 40°C for 4 weeks; Figures 81-82); pH (Table 111); and SEC-HPLC analysis of samples storedμlat 4°C and 40°C for 4 weeks (Tables 112-113).



[533] After the four week time period, H7MT-P formed less de-PEGylated hGH than
H7MGT-P at both 4°C and 40°C, and a small amount of covalent aggregates at 40°C. Overall, at
40°C, the main degradation product was de-PEGylated hGH, and there was an increased amount of
dimerization and more covalent aggregates than at 4°C.
Example 26
Agitation study (Surfactants)
[534] A follow-up study was performed to those described in Example 21. PEG-hGH was
dialyzed with H7MT, and the dialyzed protein was diluted to 2 mg/ml with H7MT. Various

amounts of the surfactant Pluronic F68 were added to the samples: no Pluronic F68, 0.01% PluronicμlF68, 0.05% Pluronic F68, 0.1% Pluronic F68, 0.25% Pluronic F68, or 0.5% Pluronic F68. Twoμlcontrol samples contained H7MT buffer with 0.01% Pluronic Acid without protein. One samplμlwas vortexed, whereas the other one was not (t=0). Agitation was performed with 500 uL fill hμlglass vials, and samples were agitated for 2 hours at room temperature. SEC-HPLC analysis waiμlperformed on the various samples, and the results are shown in Table 114.

[535] In a study investigating aggregate reversibility, 0.1% Pluronic F68 was added to the
vortexed control (no surfactant) sample to examine whether agitation-induced aggregates could beμldissociated by Pluronic F68. The samples were analyzed by SEC-HPLC. The results of this studyμlshowed that Pluronic F68 can reduce agitation-induced aggregation, but does not dissociate itμlH7MT with 0.1 % Pluronic F68 had a smaller amount of aggregate than the formulations with lowerμlamounts of Pluronic F68.μlExample 27
Sedimentation Velocity Analysis
[536] This analysis was performed to measure the aggregation of the following three
samples: PEG-hGH polypeptide at 39.9 mg/ml, 24.3 mg/ml, and 1.0 mg/ml. These stocks wereμldiluted to 0.6 mg/ml immediately prior to measurement with Dulbecco's phosphate buffered salineμl(Gibco part no. 144190-144). The high-resolution sedimentation coefficient distributions for theseμlsamples were generated with the vertical axis giving the concentration and the horizontal axisμlshowing the separation on the basis of sedimentation coefficient. Each distribution was normalized


Example 28
AUC/SEC Study
[537] Samples with different concentrations of PEGylated hGH polypeptide (39.9 mg/ml,
24.3 mg/ml, and 1.1 mg/ml) and the formulations shown in Table 115 were found to show similarμlamounts of aggregation by SEC-HPLC, SDS-PAGE, and AUC. Table 116 shows SEC-HPLCμlresults from 20 ug of material loaded onto the column. SDS-PAGE analysis (10 ug loaded in eachμllane) is shown as Figure 83.

Example 29
Intermediate Stability Study
[538] Table 117 details the intermediate stability study. Formulation ID H7.3MT is 30
mM Histidine, 4% Mannitol, 2% Trehalose, pH 7.3. Formulation ID H7.3MT+F is 30 mMμlHistidine, 4% Mannitol, 2% Trehalose, pH 7.3 with 0.1% Pluronic F68. Formulation ID HP7MT is

10 mM Histidine, 10 mM Phosphate, 4% Mannitol, 2% Trehalose, pH 7.0. Formulation IDμlHP7MT+F is 10 mM Histidine, 10 mM Phosphate, 4% Mannitol, 2% Trehalose, pH 7.0 with 0.1%μlPluronic F68. Three different concentrations of PEGylated hlFN with para-acetylphenylalanineμlsubstituted at position 35 are used: 8 mg/ml, 12 mg/ml and 16 mg/mL The study involves analysesμlat t=0, 1 week, 8 weeks, and 24 weeks, and temperatures of 4°C, 25°C, and 40°C. Methodsμlinvolved in this study are as previously described: SDS-PAGE (reduced and non-reduced); SEC-μlHPLC, RP-HPLC, IEX, FTIR, moisture content, etc. Bioactivity is measured using a proliferationμlassay involving BrdU labeling. Briefly, this assay is performed with serum starved rat GHR (L43R)μlexpressing BAF3 cell line, 2E2-2B12-F4. Cells are plated at specified densities of cells/well in aμl96-well plate. The cells are activated with a multi-point dose range of PEGylated hGH polypeptideμland are labeled at the same time with 50 uM BrdU (Sigma, St. Louis, MO). After 48 hours inμlculture, cells are fixed/permeabilized with lOOul of BD cytofix/cytoperm solution (BD Biosciences)μlfor 30 min at room temperature. To expose BrdU epitopes, fixed/permeablilized cells are treatedμlwith 30 ug/well of DNase (Sigma) for 1 hour at 37°C. Immunofluorescent staining with APC-μlconjugated anti-BrdU antibody (BD Biosciences) enables sample analysis on the FACS ArrayμlVariations to this method are known to those of ordinary skill in the art








Pluronic F68. Formulation ID HP7MT is 10 mM Histidine, 10 mM Phosphate, 4% Mannitol, 2%μlTrehalose, pH 7.0. Formulation ID HP7MT+F is 10 mM Histidine, 10 mM Phosphate, 4%μlMannitol, 2% Trehalose, pH 7.0 with 0.1% Pluronic F68. After lyophilized samples are held at 4°Cμlfor 8 weeks, samples are reconstituted and held at room temperature for t=0, 4, 8, and 24 hours andμlanalyzed with methods described previously. See Table 118.



Injection Feasibility Study
[540] Injection feasibility is tested with 8, 12, 16, and 25 mg/ml of PEGylated hGH
polypeptide (Formulation ID H7.3MT). Unconjugated PEG (Formulation ED H7.3MT) is alsoμlanalyzed at approximately 16 mg/ml and 25 mg/ml, as well as a buffer control without polypeptideμlor PEG. An instron machine is used, and 4 lbs offeree is used with 27 and 29 gauge needles.μl[541] Modifications to various conditions and/or parameters to the techniques described
herein are known to those of ordinary skill in the art. Methods such as those described above orμlother methods known to those of ordinary skill in the art may be used in formulation studies.μlPotency studies on samples may be performed with assays known to those of ordinary skill in theμlart.
[542] Suitable formulations include, but are not limited to, H7MT-P with Pluronic Acid
H7MGT-P with Pluronic Acid; H7MT-P; H7MGT-P; H6MT-P with Pluronic Acid; H6MGT-P witμlPluronic Acid; H6MT-P; H6MGT-P; HP7MT; HP7MT with Pluronic Acid; H7.3MT; H7.3MT witμlPluronic Acid. Suitable formulations may have a pH range of about 6 to about 7.3. Suitablμlformulations may have a pH range of about 5.5 to about 8. Suitable formulations may includμlhistidine at about 5 to about 30 mM. Suitable formulations may optionally include mannitol at upμlto about 60 g/L. Suitable formulations may optionally include trehalose at up to about 50 g/L.μlSuitable formulations may optionally include glycine at up to about 60 g/L.
[543] It is understood that the examples and embodiments described herein are for
illustrative purposes only and that various modifications or changes in light thereof will beμlsuggested to persons of ordinary skill in the art and are to be included within the spirit and purviewμlof this application and scope of the appended claims. All publications, patents, and patentμlapplications cited herein are hereby incorporated by reference herein in their entirety for allμlpurposes.





WHAT IS CLAIMED IS:
11. A pharmaceutical formulation of hGH polypeptide comprising one or more non-
2 naturally encoded amino acids wherein said formulation comprises:
3 a) a buffer,
4
5 b) at least one carrier, excipient, or stabilizer;
6
7 c) and a pharmaceutical^ effective quantify of human growth hormone (hGH).
1 2. The hGH polypeptide of claim 1, wherein the polypeptide is linked to a linker,
2 polymer, or biologically active molecule.

1 3. The hGH polypeptide of claim 2, wherein the polypeptide is linked to a water soluble
2 polymer.

1 4. The hGH polypeptide of claim 3, wherein the water soluble polymer comprises a
2 poly(ethylene glycol) moiety.
i
1 5. The formulation of claim 1, wherein the buffer comprises a component selected from
2 the group consisting of phosphate, borate, HEPES, citrate, histidine, a histidine derivative,
3 imidazole, acetate, and bicarbonate.

1 6. The formulation of claim 1, wherein the pH of the buffer is between about pH 4.0
2 and about pH 8.5.

1 7. The formulation of claim 6 wherein the pH of the buffer is between about pH 4.0 and
2 about pH 7.5.

1 8. The formulation of claim 7, wherein the pH of the buffer is between about pH 6.0
2 and about pH 7.5.

1 9. The formulation of claim 8, wherein the pH of the buffer is between about pH 6.0
2 and about pH 7.3.

3 10. The formulation of claim 6, wherein the pH of the buffer is between about pH 5.5
4 and about 8.0.

1 11. The formulation of claim 1, wherein the pharmaceutically effective quantity of hGH
2 is between about 0.1 mg and about 30 mg.

1 12. The formulation of claim 11, wherein the pharmaceutically effective quantity of
2 hGH is between about 2 mg and about 30 mg.

1 13. The formulation of claim 11, wherein the pharmaceutically effective quantity hGH is
2 between about 0.1 mg and about 8 mg.

1 14. The formulation of claim 13, wherein the pharmaceutically effective quantity of
2 hGH is between about 0.5 mg and about 8 mg.

1 15. The formulation of claim 14, wherein the pharmaceutically effective quantity of
2 hGH is between about 0.5 mg and about 6 mg.

1 16. The formulation of claim 15, wherein the pharmaceutically effective quantity of
2 hGH is between about 1 mg and about 5 mg.

1 17. The formulation of claim 12, wherein the pharmaceutically effective quantity of
2 hGH is between about 2 mg and about 25 mg.

1 18. The formulation of claim 1, wherein the buffer is at a concentration of about 0.1 mM
2 to about 200 mM.

1 19. The formulation of claim 18, wherein the buffer is at a concentration of about 1 mM
2 to about 75 mM.

1 20. The formulation of claim 19, wherein the buffer is at a concentration of about 1 mM
2 to about 20 mM.

1 21. The formulation of claim 19, wherein the buffer is at a concentration of about 5 mM
2 to about 30 mM.

3 22. The formulation of claim 1, wherein the at least one carrier, excipient, or stabilizer is
4 selected from the group consisting of an antioxidant, an amino acid, a carbohydrate, a chelating
5 agent, a sugar alcohol, a salt-forming counter ion, and a non-ionic surfactant.

1 23. The formulation of claim 22, wherein the carrier, excipient, or stabilizer is an
2 antioxidant.
1 24. The formulation of claim 23, wherein the antioxidant is ascorbic acid.
1 25. The formulation of claim 22, wherein the carrier, excipient, or stabilizer is an amino
2 acid.

1 26. The formulation of claim 25, wherein the amino acid is selected from the group
2 consisting of glycine, glutamine, histidine, a histidine derivative, methionine, asparagine, glutamate
3 lysine and arginine.

1 27. The formulation of claim 25, wherein the amino acid is at about 0.1 g/L to about 100
2 g/L.

1 28. The formulation of claim 27, wherein the amino acid is at about 1 g/L to about 50
2 g/L.

1 29. The formulation of claim 28, wherein the amino acid is at about 5 g/L to about 25
2 g/L.

1 30. The formulation of claim 27, wherein the amino acid is at about 0.1 g/L to about 60
2 g/L.

1 31. The formulation of claim 22, wherein the carrier, excipient, or stabilizer is a
2 carbohydrate.

1 32. The forumulation of claim 31, wherein the carbohydrate is selected from the group
2 consisting of a monosaccharide, a disaccharide, and other carbohydrate.

3 33. The formulation of claim 32, wherein the carbohydrate is selected from the group
4 consisting of trehalose, sucrose, mannose, glucose, and dextrin.

1 34. The formulation of claim 31, wherein the carbohydrate is at about 0.1 g/L to about
2 100 g/L.
1 35. The formulation of claim 34, wherein the carbohydrate is at about 1 g/L to 50 g/L.
1 36. The formulation of claim 35, wherein the carbohydrate is at about 2 g/L to about 25
2 g/L.

1 37. The formulation of claim 34, wherein the carbohydrate is at about 0.1 g/L to about
2 50 g/L.

1 38. The formulation of claim 22, wherein the carrier, excipient, or stabilizer is a sugar
2 alcohol.
1 39. The formulation of claim 38, wherein the sugar alcohol is mannitol.
1 40. The formulation of claim 38, wherein the sugar alcohol is at about 0.1 g/L to about
2 100 g/L.

1 41. The formulation of claim 40, wherein the sugar alcohol is at about 1 g/L to about 50
2 g/L.
1 42. The formulation of claim 41, wherein the sugar alcohol is at about 2 g/L to about 25
I g/L.
I 43. The formulation of claim 40, wherein the sugar alcohol is at about 0.1 g/L to about
1 60 g/L.
L 44. The formulation of claim 22, wherein the carrier, excipient, or stabilizer is a non-
2 ionic surfactant.

1 45. The formulation of claim 44, wherein the non-ionic surfactant is selected from the
2 group consisting of polysorbate 80 and poloxamer 188 (Pluronic F68).

1 46. The formulation of claim 44, wherein the non-ionic surfactant is at about 0.0001%' to
2 about 10%.

1 47. The formulation of claim 46, wherein the non-ionic surfactant is at about 0.01% to
2 about 10%.

1 48. The formulation of claim 47, wherein the non-ionic surfactant is at about 0.1% to
2 about 5%.

1 49. The formulation of claim 48, wherein the non-ionic surfactant is at about 0.1% to
2 about 1%.

1 50. The formulation of claim 46, wherein the non-ionic surfactant is at about 0.0001% to
2 about 1%.

1 51. The formulation of claim 44, wherein the formulation comprises two non-ionic
2 surfactants.

1 52. The formulation of claim 22, wherein the carrier, excipient, or stabilizer is a
2 chelating agent.
1 53. The formulation of claim 52, wherein the chelating agent is EDTA.
1 54. A method of treating a patient having a disorder modulated by hGH with an effective
2 amount of the formulation of claim 1.

1 55. The formulation of claim 1, wherein the buffer comprises a component that has a
2 buffering capacity of between about pH 5.5 and about 8.0.


Documents:

2690-chenp-2007 -other patent documnet 12-01-2011.pdf

2690-CHENP-2007 AMENDED CLAIMS 12-01-2011.pdf

2690-CHENP-2007 AMENDED PAGES OF SPECIFICATION 12-01-2011.pdf

2690-chenp-2007 form-3 07-03-2011.pdf

2690-chenp-2007 form-3 12-01-2011.pdf

2690-chenp-2007 form-5 12-01-2011.pdf

2690-CHENP-2007 OTHER PATENT DOCUMNET 12-01-2011.pdf

2690-CHENP-2007 CORRESPONDENCE OTHERS 22-07-2010.pdf

2690-CHENP-2007 CORRESPONDENCE OTHERS 01-03-2011.pdf

2690-chenp-2007 correspondence others 07-03-2011.pdf

2690-CHENP-2007 EXAMINATION REPORT REPLY RECEIVED 12-01-2011.pdf

2690-chenp-2007 other patent document 07-03-2011.pdf

2690-chenp-2007-abstract.pdf

2690-chenp-2007-assignement.pdf

2690-chenp-2007-claims.pdf

2690-chenp-2007-correspondnece-others.pdf

2690-chenp-2007-description(complete).pdf

2690-chenp-2007-drawings.pdf

2690-chenp-2007-form 1.pdf

2690-chenp-2007-form 3.pdf

2690-chenp-2007-form 5.pdf

2690-chenp-2007-pct.pdf


Patent Number 247143
Indian Patent Application Number 2690/CHENP/2007
PG Journal Number 13/2011
Publication Date 01-Apr-2011
Grant Date 29-Mar-2011
Date of Filing 21-Jun-2007
Name of Patentee AMBRX ,INC
Applicant Address 10975 NORTH TORREY PINES ROAD, SUITE 100, LA JOLLA,CA 92037,USA
Inventors:
# Inventor's Name Inventor's Address
1 LITZINGER, DAVID 13976 ARBOLITOS DRIVE , POWAY, CA 92064,USA
2 PUTNAM, ANNA-MARIA , A., HAYS 3187 VIA ALICANTE ,#251, LA JOLLA ,CA 92037,USA
3 BUECHLER,YING 1343 CASSINS STREET, CARLSBAD ,CA 92011,USA
PCT International Classification Number C12Q 1/68
PCT International Application Number PCT/US05/47001
PCT International Filing date 2005-12-21
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 60/638,616 2004-12-22 U.S.A.
2 60/680,617 2005-05-13 U.S.A.
3 60/728,035 2005-10-17 U.S.A.