Title of Invention

"METHOD FOR ALBUMIN PURIFICATION"

Abstract A method of purifying recombinant human serum albumin (rHSA) from a solution, which method comprises subjecting a cell culture supernatant (CCS) comprising rHSA to the following chromatographic steps: (a) cation exchange on a bimodal high salt tolerant matrix, wherein each ligand comprises two groups capable of interacting with the substance to be isolated. (b) hydrophobic interaction chromatography (HIC); and (c) anion exchange.
Full Text Technical field
The present invention relates to the field of protein purification, and especially to the purification of human serum albumin. The method utilises a series of chromatography steps, which results in an efficient purification suitable for use in large-scale operations.
Background
Human serum albumin (HSA) is the most abundant protein present in blood plasma, where its role is to contribute to the maintenance of osmotic pressure and to bind nutrients and metabolites, to thereby enable transport thereof. There is a large pharmaceutical and scientific interest in HSA, e.g. as a drug for treating hypoalbuminemia caused by a loss of albumin or a reduction in albumin synthesis, in hemorrhagic shock etc. In the earliest methods available, the HSA was purified from blood. However, such methods brought about problems, for example a sporadic supply of blood, economical disadvantages and contamination with undesirable substances such as hepatitis virus and not least AIDS virus. To avoid these problems, alternative methods based on recombinant DNA techniques have more recently been developed to produce recombinant HSA (rHSA). Even though a number of recombinant methods have been suggested, it has been shown that the purification of the rHSA from the fermentation broth is a crucial step and there is an ongoing need of improvements to this end.
EP 0 612 761 discloses a method of producing recombinant human serum albumin of high purity, which does not contain free non-antigenic contaminants. The method utilises hydrophobic interaction chromatography (HIC) under specified conditions combined with other steps such as ion exchange chromatography, treatment with boric acid or a salt thereof followed by ultrafiltration, and heat treatment. However, a series of that many steps will still be too complex and accordingly too expensive for satisfactory use in large-scale operation in industry. 0 570 916 also discloses a process (or producing recombinant human scrum albumin
by gene manipulation Icchniques, wherein purification is by a combination of steps in which a culture supernatant is subjected lo ultrafiltration, heal treatment, acid treatment and another ullrallllralioii, followed by subsequent treatments with a cation exchanger, a hydrophobic chromatography carrier and an anion exchanger and by sailing out. I low-ever, similar lo the above-mentioned patent, this purification scheme is too complex, time-consuming and accordingly loo expensive to provide an efficient procedure for use in large-scale operation.
\,V 0 699 6S7 discloses a method of purification of rl ISA wherein culture medium is heal treated to inactivate proteases and then contacted with a fluidised bed of cation exchange parlicles. The eluent can subsequently be subject to ultrafiltration, UK! and anion exchange chromatography. However, use of a fluidised bed will require equipment different lo the conventional packed bed chromatographic step. Accordingly, there is still a need of more efficient and economically attractive procedures tor purification of rl ISA from a culture brolh.
Summary of (lie invention
One object of (be present invention is lo provide a method (or purification of recombinant [ISA that is easily adapted lo large scale operation. This is achieved by a method, which comprises subjecting a cell culture supernatant (COS) comprising rl ISA to the following slops:
(a) cation exchange chromatography on a bimodal high salt tolerant matrix;
(b) hydrophobic interaction chromatography (MIC);
(c) anion exchange chromatography.
'flic method according, lo the invention comprises fewer process steps than (he above-discussed methods. In addition, the bimodal high salt toleranl cation exchange matrix used in step (a) allows use of a cell culture supernatant without any further dilution, which is an advantageous feature since it greatly reduces the total volume and hence the costs as compared lo the prior ail methods.
Another object of the invention is to improve the adsorption capacity of the chromatographic steps used in purification ofrl ISA. (his can he achieved by the method de scribed above wherein a cation exchanger, comprising ligands known as high salt brands (I ISI,), is used in step (a). Said liquids arc bimodal in the sense that they comprise at least two sites that interact with the substance to he isolated, one providing a charged interaction and one providing an interaction based on hydrogen bonds and/or a hydrophobic interaction.
Another object of the invention is to further decrease the colour content, and more specifically to further increase the purity, of the final product. This can be achieved by use of the method described above, wherein a weak anion exchanger is used.
jJrief description pfdrawings
bigurc I A-l) illustrates possible matrix materials suitable for use in the different steps of cation exchange, hydrophobic interaction and anion exchange chromatography according, to the invention.
figure 2 shows the results of cation exchange chromatography according to step (a) of the method according to (he invention of an undiluted cell culture supernatant (CCS). Fraction 2H contains the rlISA.
Figure 3 shows the results of UK! of fraction 2H from big 2. The rllSA is eluled in fraction 3A.
Figure 4 shows the results of anion exchange chromatography of fraction 3 A from big '.\. The purified rl ISA is eluled in fraction 4B.
I'igure S shows electrophoresis analyses of the main fractions obtained using the three step purification protocol according to the present invention.
l).etai.!ed description of the invention
One aspect of (lie present invention is a method of purifying recombinant human serum albumin (if ISA) from a solution, which method comprises subjecting a cell culture supernatant (CCS) comprising rl ISA to the following chromatographic steps (a) cation exchange on a bimodal high sail tolerant matrix;
(b) hydrophobic interaction chromatography (H1C); and (e) anion exchange.
In a specific embodiment of the prcscnl method, (In: conduclivily of the CCS is above about. 10 mS/cin, such as above about 15 and preferably above about 20, e.g. about 2.S-50, or more specifically 23 30 m.S/cni, when applied lo step (a), which is possible due lo the kind of cation exchanger used, which comprises liquids of the type known as high sail liquids (I LSI,). Accordingly, an important advantage with the present method is that it avoids the need lo dilute the CCS, and thereby allows use of much reduced volumes of sample during the cation exchange step as compared to the previously described methods for purification of rllSA.
The (X!S can be any fermentation broth from which cells preferably have been removed e.g. by cenliifugalion. Accordingly, the origin of the rllSA isolated according to the present invention can be any suitable host, such as microbial cells, such as Ksc/uri.shia coli, iiucillus suhtilis etc, yeast cells, such as Stwcharomyccs cvrevisiav, I'ichia pastoris etc, or animal cell lines. In an advantageous embodiment, the host cell is IHchia pastoris. Methods of producing a recombinant host cell and conditions for expression of a protein such as 11 ISA therefrom are well known, see e.g. the above discussed I'.V 0 612 761 for a reference lo various patent applications within this Held.
The main purpose of step (a) is lo eliminate low molecular weight coloured substances such as pigments that are negatively charged. Thus, step (a) utilises a cation exchanger, which comprises ligands of the type known as high salt ligaiuls (llttL). In this context, "high salt" refers to the above-mentioned properly of high salt concentration tolerance that is characteristic for this class of ion exchangers. This property is provided by the nature of the ligands, which is bimodal in the sense (hat each ligand comprises two groups capable of interacting with the substance to be isolated, in the present case rl ISA. The primary binding mode is provided by a charged binding group, i.e. an ion exchanging group, hence the classification of the I INI-type of matrices as ion exchangers. A second binding mode is provided by a secondary binding group, which provides an ad-
dilional interaction willi the substance to be isolated. Usually, the secondary binding
group provides a hydrogen bonding or a hydrophobic inlcraction, but other interactions
can be envisaged, a.s will be discussed in more detail below. In this context, it is to be
noted that the term "bimodal" is used herein to define thai two or more binding modes
are involved, and it is therefore not limited to only two binding modes. In the present
application, cation exchangers of I LSI,-type are utilised, and such cation exchangers
have; been disclosed in detail, see e.g. PCT/EP'01/08203 (Aincrsham Pharmacia Biotech
AH). However, a general description thereof will follow below.
The charged binding group present on a calionie high salt ligaiul (MSI.) can be selected from the group comprised ofsulphonatc ( SO5 /-NO,l I), sulphate (-OSO3 )S()(I I), eai-boxylate ( (COO/-COOHI), phosphate (-OPO /-0PO),V-OPOH2 and phosphouatc ( PO2/-PO, I l/-Pt)|ll;). In one advantageous embodiment, the IISL-lypc of cation exchanger is a weak cat ion-exchanger, i.e. cation-exchangers that have a pKa above .\. In an alternative embodiment, they are strong cation exchangers that have a pKa below 3. Typical examples of such weak cation exchangers are carboxylate (COO/-COOOH2), phosphate (-()POOl»OPOH1 /-OPO l2 and phosphonale (-PO32-PO3H).,III /-PO3H2;).
The secondary binding group comprises at least one hydrogen-bonding atom, which is located at a distance of 1-7 atoms from the cation-exchanging group. A hydrogen-bonding atom is an atom that is capable of participating in hydrogen bonding (except hydrogen), see Karger el al., An Introduction into Separation Science, John Wiley & Sous (1973) page 42. The hydrogen-bonding atom can be selected from the group that consists of hctcroatoms, .such as oxygens (caibonyl oxygen, ether oxygen, ester oxygen, hydroxy oxygen, sulphone oxygen, sulphonc amide oxygen, sulfoxide oxygen, oxygen in aromatic tings etc), nitrogens (amide nitrogen, nitrogen in aromatic rings etc), .sulphurs (thioethcr sulphur, sulphur in aromatic rings etc); and sp- and spz-hybridised carbons; and halo groups, such as fliioro, chloro, bromo or iodo, preferably fluoro. The second binding group typically contains no charged atom or atom that is chargeable by a pH change.
The stability ol' the cation exchange ligands used in step (a) can in {general lenn.s he de-lined as (he capacity to resist 0.1 or I M NaOl I in water Coral least 40 hours, i'or illus dative examples of suitable chemical ligand structures of the cation exchangers useful in step (a) of the present method, see above-mentioned I'CT/lil'O1/087.03. In a specific embodiment of the present method, step (a) utilises the I LSI. cation exchange ligand illustrated in I'igl A of the present specification.
Defined in functional terms, the cation exchanger used in step (a) of the present method is capable of
(a) binding rllSA by cation-exchange in an aqueous reference liquid at an
ionic strength corresponding to 0.3 M NuC'l and,
(b) permitting a break through capacity lor the substance > 200 %, such as > 300% or
> 500% or > 1000 %, of the break through capacity of the substance lor a reference
cation-exchanger containing sulphopropyl groups -CI l/CH/MI^SO," at the ionic
strengths shown above.
I'd'/1'TO 1/08203 describes such a reference ion exchanger in more detail. The level o( cation exchange ligunds in the cation exchangers used in the inventive method is usually selected in the interval of I-4000 pmol/ml matrix, such as 2-MK) pmol/ml matrix, with preference for .S-300 pmol/ml matrix. Possible and preferred ranges are, among others, determined by the nature of the matrix, ligand etc. Thus, the level of cation-exchange ligands is usually within (he range of 10-300 for agarose based matrices, f'ordextran-based matrices, the interval is typically 10-600 pmol/ml matrix.
The above mentioned IX "171'IPO 1/08203 also comprises an extensive discussion of ma Irix materials useful with cation exchangers of I LSI,-type. In brief, such matrices can be based on organic or inorganic material. It is preferably hydropliilic and in the form of a polymer, which is insoluble and more or less swellable in water. Hydrophobic polymers that have been dcrivali/.cd to become hydropliilic are included in (bis definition. Suitable polymers are polyhydroxy polymers, e.g. based on polysaccharides, such as agarose,
extran, cellulose, starch, pullulau, clc. and completely synthetic polymers, such as poly-aery lie amide, polymelhacrylic amide, poly (hydroxyalkylvinyl ethers), poly(hydroxyalkylacrylales) and polymelhacrylates (e.g. polyglyoidylmelliacrylale), polyvinylalcohols and polymers based on slyrenes and divinylhen/enes, and co-polymers in which Iwo or more of the monomers corresponding to the above-mentioned polymers are included. I'olymers, which arc soluble in water, may he deiivati/ed to hecome in soluble, e.g. by cross linnking and by coupling lo an insoluble body via adsorption or co-valenl binding. Mydrophilie groups can be introduced on hydrophobic polymers (e.g. on co-polymcrs of monovinyl and divinylben/enes) by polymerisation of monomers exhibiting groups which can be converted lo Ol I, or by hydrophilisalion of the final polymer, e.g. by adsorption of suitable compounds, such as hydrophilic polymers. An illustrative example ofa suitable matrix is the commercially available beaded Scpharosc, which is agarose-bascd and available from Amcrshani llioscicnccs, Uppsala, Sweden. Suitable inorganic materials to be used as support matrices arc silica, zirconium oxide, graphite, tantalum oxide etc.
The above mentioned IHT/EP01/08203 comprises an extensive disclosure of the preparation of cation exchangers of IISI,-typc. Also, a review of methods of immobilising li-gand-forming compounds to surfaces is given in llcrmansou, (i. T., Mallia, A. K. & Smith, I*. K,, (Ivds.), Immobilisation Affinity Ligantl Techniques, Academic I'ress, INC, 1992.
As mentioned above, one of the benefits of the I LSI,-type of ion exchangers is that it is possible to run adsorption lo the column, i.e. binding of rl ISA to the ligand, at elevated ionic strengths compared to what lias normally been done for conventional cation-exchangers, for instance the reference sulphopropyl cation-exchanger discussed above. The exact ionic slienglh to be used dining binding depends on the nature of the protein and Hie type and concentration of the ligand on the matrix. Useful ionic strengths often correspond to Na(!l concentrations (pure water) > 0.1 M, such as 1> 0.3 M or even > 0..S M. Desorplion can be performed e.g. by increasing the ionic strength and/or by change of the pi I. Typical sails to be used for changing the ionic strength are selected among
soluble ammonium or inclal salts of phosphates, sulphates, etc, in particular alkali metal
and/or alkaline earth metal salts. The same salts can also be used in the adsorption steps,
but then often in lower concentrations.
In one embodiment of the present method, the amount of cation exchange matrix used in step (a) is about half the amount of UK.' matrix used in step (b). Accordingly, one advantage of the present invention is the outstanding binding capacity of the bimodal ca lion exchange matrix that allows a reduction in volume and therefore operational costs as compared to conventional cation exchangers. In the presence of a high salt concentration, such as a conductivity of about 23-30 mS/cm, the adsorption of commercially available SI' Scpharo.se for rl ISA is about 2 4 nig/ml, packed gel, while that of the high salt ligand prototype (big I A) has been shown to be at least 50 rng/ml,. Accordingly, the use ofthe bimodal high salt ligand (IISL) clearly simplifies and improves the purification process significantly and reduces the cost of operation on large scale.
One embodiment ofthe present method comprises heal treatment of the CCS before step (a), The heating can be performed directly i.e. while the host cells arc still present or alia icmovnl thereof, such as by cenlrifugation, ultrafiltration or any other suitable method. The healing can IK; performed at SO-I00°C during a period of time of from I minute up to several hours, preferably at (>0-75°C for 20 minutes lo 3 hours and inosl preferably at about 68°C for about 30 minutes. The healing is conveniently performed in a water bath equipped with thermostat. In one embodiment, a stabiliser is added before (he heating, such as sodium caprylale at a pH of about 6.0. Other stabilisers can be used, such as acctyltiyplophan, organic cai boxy lie acids etc. After the heating, the pll ofthe CCS is preferably adjusted lo a lower value suitable for the subsequent adsorption on a cation exchanger, such as pi I 4.S.
In another embodiment of the present method (he product from step (a), i.e. (he fraction bound to the column comprising the IISL type matrix, is heal-lreated before step (b). Preferably, a reducing agent, such as cysteine, is added. Other examples of useful reducing agents are mercaptoclhauol, reduced glutathione etc. The purpose of (his is to fa-
cilitatc the removel of coloured subnstances during step (b). 't'his heat treatment is in gen
eral pet formed as described above, even though in the preferred embodiment, a slightly
lower temperature such as about 60°C for a slightly longer period of lime such as about
60 minutes is used.
As mentioned above, step (b) of the present method utilises hydrophobic interaction chromatography (I IIC). The main purpose of .step (b) is to remove proteolytic degradation products ol'rllSA, which products arc: usually ofa si/,c of about 10-50 kl)a. UK' is a well known principle in the ail of'chromatography, and provides a versatile tool for separation based on differences in surface hydrophobieity. Many biomoleculcs that are considered to be hydrophilic have been shown to still expose sufficient hydrophobic groups to allow interaction with the hydrophobic ligands attached to the chromatographic matrix. IIIC,' has already been suggested for the purification of rlISA, see e.g. EP' 0 6°9 6X7. Compared with another well known separation principle, namely reverse phase chromatography, MIC utilises a much lower density of ligand on (he matrix. This feature promotes a higher degree of selectivity, while allowing mild elation conditions to help preserve the biological activity of the protein of interest. In the present conlexl, the IIIC step is used to adsorb (he above-mentioned proteolytic degradation producls of 11 ISA, while the lull length il ISA is elided in (lie unbound fraction. The hydrophobic interaction between (he rl ISA and the immobilised ligand on the matrix is enhanced by a small increase in the ionic .strength of the buffers used. There are many separation male rials commercially available for IMC today, and the present invention is not limited to any specific matrix and/or ligand. Thus, in general terms, the matrix used in step (b) can be based on an organic or inorganic material. In the case of organic materials, it can e.g. be a native polymer, such as agarose, dexlran, cellulose, starch etc, or a synthetic polymer, such as divinylben/.ene, styrene etc. In the case of inorganic matrix materials, silica is a well-known and commonly used material. In an advantageous embodiment, the ma Irix is cross-linked agarose, which is commercially available from a number of compa nies, such as Scpharose™ from Amersham Bioscienees (Uppsala, Sweden). In one embodiment, the I IIC matrix used in step (b) exhibits one or more hydrophobic ligands ca pable of interaction with rl ISA, selected from the group that consists of phenyl, butyl,
such as n-bulyl, octyl, such as n octyl, etc, preferably on an agarose matrix. Alternatively, hydrophobic ligands such as others, isopropyl or phenyl arc presenl on a divinyl-benzene matrix, such as Source™ from Aniershani Biosciences (Uppsala, Sweden). In the most preferred embodiment, phenyl liquids on a crosslinked agarose matrix are used Cor step ([)). The matrix is preferably comprised of porous heads, which can have a water content of above about 90%, preferably about 94%. The average particle size can e.g. be between 10 and 150 pin as measured on a wet bead, preferably below 100, such as about 90 pin. The liquid density on the matrix can for example be between 20 and 60, such as about 40 µmol/ml gel. As a specific example, the matrix used is I'henyl Sephnrose™ 6 last Flow from Amersham Bioseiences (Uppsala, Sweden). In this specific case, the denotation last blow is used for a mtrix the cross-linking of which has been optimised to give process adapted How characteristics with typical ilow rates of 300-400 cm/h through a 15 cm bed height at a pressure of I bar. However, the skilled person in this field can easily adapt such process parameters depending on the scale of the operation. This slop can be performed at a pH of about 4-8, such as 6.5-7, and at a salt concentration of about 0.01 to 0.5 M, such as 0.05 to 0.2 M.
As is also mentioned above, step (c) of the presenl method utilises an anion exchanger, preferably a weak anion exchanger, for removing minor impurities from step (b), and especially to remove undesired compounds, such as low molecular weight pigments. The invention is not limited to use of any specific anion exchanger material, as long as it exhibits a suffieient amunt of ligands capable of binding compounds of negative charge thai are undesirable in the linul rl ISA product. The anion exchange chromatographic step can e.g. be performed at a pi I of about 5.0-8.0 and a salt concentration of 0.01 to 0.2 M for removal of the impurities. As regards the matrix material, it may be ol any organic oi' inorganic material, as discussed above. In one preferred embodiment, the matrix is comprised of porous beads of cross-linked agarose, such as Sephnrose™ from Aiucr-sham Biosciences (Uppsala, Sweden). The ligands attached thereto are weak anion exchangers, the binding group of which is for example a primary or secondary amine. Such a binding group can be attached to (he matrix e.g. via an alky] chain with an ether group closest to the matrix. The literature describes many ways of attaching a binding group to

ii matrix via spacers, arms etc, and as mentioned above the present invention is not limited to any specific structure. In an illustrative embodiment, an agarose bead having lig ands according to (lie follow in/4 formula O-CH2CH2-N-(C2H5)2H,DEAE Sephn-rose™ from Amersham Hioscienecs, is used. This embodiment will result in rlISA in both the bound and the unbound fraction elated from the column which, for some applications, is satisfactory.
However, an alternative embodiment which is more advantageous as far as purity of the final product is concerned, is using an agarose bead having ligands that comprise two ester groups, and preferably also two hydroxy I groups. The binding group is then preferably primary amine. The advantage with this embodiment is that (he 11 ISA will be present only in the fraction that is moderately bound to the matrix, resulting in a much-improved purity and operational convenience. An illustrative general formula for this last mentioned embodiment is presented in Fig 1, however it is to be understood that the present invention also comprises a method wherein similar structures are used in step
flowever, it is to be understood that (he present invention encompasses use also of ma trices similar to the above, which are based on the same general ligand structure. Also, the denotation "Gel in the formula of Fig IA is understood to include any matrix, as discussed above in relation to step (b). One commercially available example of a matrix comprising, the above-described ligand is ButylSephaiose™ (Amersham Mioscienees, I Ippsala, Sweden)- In the experiments presented below, a ligand density of 160 jrmoles/ml was used. Accordingly, it appears that especially advantageous results can be achieved by optimising the ligand density of the matrix. It also appears that when the first mentioned kind of anion exchangers are used, i.e. DEAE kind of matrices, a larger volume is required, such as about three times (he volume, as compared to the last mentioned Butyl-Sepharose media. Accordingly, the preferred embodiment of the present method utilises a secondary amine as the anion-exchangiug group during step (c) and a ligand density of at least about 100 µmoles/ml.
In n specific embodiment, the ligand density of the union exchanger is in the range of
50-300, such as 100-200 and preferably about 160 µmoles/ml. One advantage is that the
purified rIISA can then be recovered only from the bound fraction from step (c), as
compared to e.g. DEAE when it may be present in both bound and unbound fractions.
Detailed description of drawings
figure I A-l) illustrates possible ligand structures suitable for use in the present method. More specificaly, Fig IA shows a cation exchanger of high salt gaud (I LSI.) type, fig, IB shows a hydrophobic interaction chromatography matrix namely 1'hcnyl Sepharose, and fig, 1 figure 2 shows (he results from step (a), i.e. cation exchange chromatography of 147 ml -undiluted cell culture supernatant (CCS) on a 20 ml, column comprising a cation exchange matrix with the ligand structure illustrated in fig, I A. fraction II) contains the rl ISA, which is clearly separated from the impurities represented by fraction 2A. figure .\ shows (he results from step (h), i.e. hydrophobic interaction chromatography (IIIC) of fraction 21J from fig 7. on a 40 ml column comprising Phenyl Sepharose as il lust rated in lig 1 I), fraction A represents the 11 ISA.
figure 4 shows (he results from step (c), i.e. union chromatography of fraction .1 A from fig 3 on a 40 ml column comprising the modified butyl-Sepliarose as described in the experimental part below. The purified 11 ISA is in fraction 4B.
figure 5 shows native PAGE (8-25%) and SI.)S-PAGE (10-15%) analyses of the main fractions obtained using the three step method according to the invention, for native PACK visualised by silver staining (5A), about 3.3 µg of protein per spot was applied, for SDS-PAGE visualised by silver staining (51)), about 2 pg of protein per spot was applied, for SI)S-I'A( if visualised by Coomassic staining (5C), about 10 µg of protein per spot was applied. I: CCS; 2: IISL Cat.Ex. (unbound); 3: I LSI.. Cut. Ex. (bound); 4:
Phenyl (unbound); 5. Phenyl (bound); 6: I liSub. Butyl; 7: IliSuh. Hulyl (hound); 8: USA (control). The arrows show the positions of rf ISA.
EXPERIMENTAL PART Materials and methods
The cell culture supernatant (CCS) containing rI ISA was prepared by 1'ornicnling genetically modified lljmstoris cells for 2 weeks or nunc, followed by separation of the cells by filtration. The CCS, which was green in colour, was divided into aliquols of about 200 ml and stored at 2()°C until use. The quality of the CCS was determined by gel nitration on an analytical column of Superdex™ 200 UK 10/30 (Amcrsham Rioscienecs, Uppsala, Sweden). This analysis gave the relative amounts of high molecular weight (I IMW) and low molecular weight (MViW) impurities in the CCS as well as the approximate content ofllie monomeric form of 11 ISA.
Sodium caprylate (oclanoic acid, Na salt) and I, cysteine were bought from SIGMA Chemical Co. Chromalographically purified MSA from human plasma was kindly provided by I. Audersson at the plasma processing unit of Amcrsham liiosciences, Uppsala, Sweden. The concentration of protein in various samples was determined using the liio Kad Protein Assay kit (known as the Bradford method). Hovine serum albumin (MSA) was used to construct the standard curve. UV/Vis absorption measurements were made using a tthimad/u UV I60A recording spectrophotometer (Shimad/.u Corporation, Japan). All other chemicals used were of analytical or reagent grade.
Analytical electrophoresis was performed using a Phastgel electrophoresis system and appropriate PhasKJel media and buffer Strips (all from Amcrsham liiosciences, Uppsala, Sweden). The elect!ophoretic analyses were performed using iialivc-l'ACilC (8-25%) or Sl.XS-I'ACili (IIOII reduced, 10-15%) gels according, to the Manufacturer's recommendations. The amount of sample applied per spot was as follows: about 3.3 |ig for native samples and 2|ig for the SDS-trealed samples, both of which were stained with Silver
Staining Kit (Amersham Bioseienoes, Uppsala, Sweden); 10 p.g for the SPS-treated
samples that were stained with Cooiuassio. Brilliant Blue (CBB).
Mass spcclromelric analysis (aimed at determining the mass of purified rl ISA and plasma-derived MSA) were done by \h: J. Flonsburg at Amersliam Bioseiences, Uppsala, Sweden, using a MALDI TOF instrument. I'cptidc mapping of the tryplic digest of the native and recombinant I ISA was also done using this instrument. The results obtained from the latter analysis serve to establish the most probable primary sequence of the illSA with reference to the known sequences of the tryptie peptides generated from purified MSA.
Mali ices and ehiomalqgraphy system
The chromatographic experiments were performed ill room temperature (about 23°C) using an AKTA™ Explorer 100 system controlled by UNICORN™ (Version 3.1) soil-ware (Amersham Bioseicnces, Uppsala, Sweden). The separation matrix used for step (I)) is Phenyl Scpharoso™ 6 his! Flow (high sub), a regular product of Amersham Bio-seieuees, Uppsala, Sweden. For step (o), cilhoi commercially available DIvAU Scpharoso™ Fast Flow (Amersham liioscicnccs, Uppsala, Sweden) or a modified matrix was used: Butyl Sopharose™ 6 Fast Flow (Amersham liioscicnccs, Uppsala, Sweden) was produced with an increased ligand density (batch 1)238025:160 µmol/ml) as compared to the commercial product (70-40 µmol/ml gel). This modified matrix will herein be denoted "modified Butyl Sepharose". Furthermore, step (a) utilised a prototype matrix of MSL-typo, cation-exchanger, sec Fig 1 A. This medium was packed in a XK26/20 glass column as a thick suspension in 20% cthanol to obtain a bed volume of 40 ml. A linear flow rate of 300 cm/h was used, flic packed column was washed with about 7. bed vol umes of dc-iouised water to eiute most of the clhanol and then equilibrated with the appropriate buffer solution prior to sample application. The amount of buffer required for each of the chromatographic steps using the various media is shown in Table I below.
Buffers
Buffer_A: 25 mM sodium acetate, pH 4.5
Mix 25 nil, of I M sodium acetate and 40 ml, of 1 M acetic acid and dilute to I I, with
de-ionised water. Conductivity: about 2mS/cm at room temperature (RT).
Buffer B 50 mM sodium phosphate, 0.1 M NaCl, 10 mM .sodium caprylalc, pH 7.0 Mix 155 ml, 0.2 M Na?lll'()4 I 95 ml, of 0.2 M N«II2I»04 I 5.8 g of NaCI I 1.60 g sodium caprylalc and dilute lo 1 1, with de-ionised water. Conductivity: about l(> mS/cm at ItT.
Buffer C: 50 mM sodium phosphate, 0.1 M NaCI, pHI 6.0
Mix 212 ml, 0.2 M Nnll2PO4 I 38 ml. of 0.2 M Na,IIP04 I 5.8 g of NaCI ami dilute to
I I, with dc ionised water. Conductivity: 14 mS/cm at UT.
liultcr I): 50 mM sodium phosphate, 0.2 M NaCI, pi I 6.0
Mix2l2ml,0.2M Null2l>(>4 I 38 ml. ol(>.2 M Na;lll>(.)4 I 11.7 g of NaCI and dilute to
I I, with de-ionised water. Conductivity: 22 inS/eui at KT.
IJ.ulTei If,: Cleaning in-place(ClI') solution 30% isopropanol dissolve in I M NaOl I solution.
Example Purification rllSA Heat treaement of cell culture supernatant (CCS)
Before cation exchange chromatography, the CCS was heat-treated primarily lo inactivate proteolytic enzymes produced during fer mentation of/', pastorix. This was per formed as follows:
The frozen sample of CCS was thawed and 10 niM Na-caprylate was dissolved. The pi I was adjusted to 6.0 and it was healed for 30 minutes in a water bath (maintained at 68°C by thermostat). The sample was cooled to room temperature and its pi I adjusted to 4,5. If a conventional cation exchange medium, such as SI' Sepharose IJI3, was lo be used for .step (a), it would have been required to dilute the CCS 2-8 times, depending on the original conductivity of the solution, with do ionised water lo reach a conductivity of about 5-10 mS/cm (approximately 0.1 M salt concentration).
However,the HSI,-type matrix used according to the present invention is much more
tolerant to increased salt concentrations, and therefore the heat-treated CCS can normally he applied to step (a) without any further dilution, as long as the conductivity I hereof is less than ahout 30 mS/cm.
The partially purified rl ISA obtained alter the cation exchange according to step (a) (i.e. the fraction hound to (he IISI .-type matrix) was also heal- treated prior to slop (b) as follows: The pH of the sample was adjusted to 6.0 with I M NaOII and cysteine was dis solved (herein to a concentration of 5mM lo serve as a reducing agent. This solution was then heated for 60 minutes in a water bath maintained at 60°C. The main purpose of this operation is to facilitate the removal of coloured substances by the IIIC matrix.
Step (a)Captuyre using cation exchange chromatography,
The cation-exchange medium was packed in an XK 16/20 column (packed bed volume 20 ml.,) and washed with 2 column volumes (CV) of Uuffer A for equilibration. The heat-treated CCS was applied lo the column via a 150 ml, Superloop™ (Amersbam Itio-scienees, Uppsala, Sweden) at a flow rate of 300 mL/h (I50cm/h). The amount of r) ISA applied was about I g (i.e. 50 mg rlISA/ml of packed gel). After sample application, the unbound material waseluted with 3 CV of Uuffer A followed by elution of the bound rllSA with 5 CV of Uuffer I!. The two fractions were pooled separately and the pi I of the bound fraction was adjusted lo 6.0 with a I M NaOII solution. The solution was then heated as described above, cooled lo room temperature and further purified on a NIC column as described below. An I ml, aliquot from each pooled fraction was saved for analytical purposes (i.e. lo determine protein content, the A350/A280 ratio and electrophoresis analysis).
Regeneration: The column was washed with 2 CV of Uuffer Iv to elute very strongly bound substances and restore the function of (he gel. The column was allowed to stand overnight in the same solution and then washed with 4 CV of de-ionised water lo elute most of the NaOII and iso-propanol. The regenerated column was rc-equilibrated with A CV of Uuffer A prior to the next cycle of adsorplion/desorption process.
Step(b):Purification step using 1-IIC
The rl ISA containing fraction from the previous step was transferred to a 150 ml, Su-
perloop™ and applied to an XK 26/20 column (lacked with Phenyl Sepharose™ Past
Flow (high sub), packed hed volume 40 mL. The column was prc-cquilil>ratcd with .CV of Buffer C. After sample application, the column was washed with 2 CV of Buffer
C to elulo the unbound material that contains the rllSA. The hound material (containing
mainly the 45 kDa degraded form ol'rIISA) was elutcd with 2 CV of de-ionised water.
Regeneration: The same procedure as above.
Step (e); Polishing step using weak anion exchange
The two fractions obtained from the previous IIIC step were pooled and I ml - aliquots from each were saved for analytical determinations (see above). A column (XK26/20) was packed with DEAE Sepharose™ F'ast flow or the above described modified Bulyl-Sepharose to obtain a (lacked bed volume of 40 ml,. Kadi of the packed media was washed with 2 C'V of de-ionised water and (hen with about 5 CV of Buffer C to equilibrate them. The unbound fraction obtained from the 1IIC step was transferred to a 150 inL Supcrloop and applied to one or the other of the above two columns. The unbound fraction was elutcd with 6 CV of Buffer C (from the DKAH Sepharose hast Mow column) or with 2 CV from the modified Butyl-Sepharosc column. The bound fraction was elutcd with 2 CV of a 2. M solution of NaCI (for the DEAE column) or with 5 CV of Duffer I) for the modified Butyl-sepharose column. The flow rate was maintained at 90 cm/h throughout.
Regeneration: the same procedure as above.
The elulion protocols optimised according to the invention for each of the media used are summarised in Table I. The skilled person in this field can easily scale up the above-described process to pilot or production-scale operations.

Tabic I:
The number of column volumes ((IV) of' equilibration, clution and
regeneration solution required for each chromatography step
(Table Removed)
Results
Since the three step purification process is bused on step-wise edition, it is easily adaptable to large-scale operation. Use of a high ligand ButySepharose for step (c) results in efficient removal of L M W impurities (hat chile as a group in the unbound fraction. Use of the high ligand-BulylSepharose also results in a better A350/A580 ratio than use of the DHAE type of matrix for step (c).













We Claim:
1. A method of purifying recombinant human serum albumin (rHS A) from a solution, which
method comprises subjecting a cell culture supernatant (CCS) comprising rHSA to the
following chromatographic steps:
(a) cation exchange oh a bimodal high salt tolerant matrix, wherein each ligand comprises two groups capable of interacting with the substance to be isolated.
(b) hydrophobic interaction chromatography (HIC); and
(c) anion exchange.

2. A method as claimed in claim 1, wherein the conductivity of the CCS is above 10, such as above 15 and preferably above 20, e.g. 25-50 mS/cm, when applied to step (a).
3. A method as claimed in claim 1 or 2, wherein the bimodal cation exchange matrix is capable of interacting with rHSA by charge interaction and by hydrogen bonding and/or hydrophobic interaction.
4. A method as claimed in any one of the preceding claims, which also comprises heat treatment of the CCS before step (a).
5. A method as claimed in any one of the preceding claims, which also comprises heat treatment of the CCS before step (b) in the presence of a reducing agent.
6. A method as claimed in any one of the preceding claims, wherein step (b) utilises a HIC matrix comprising phenyl, aliphatic and/or heterocyclic ligands.
7. A method as claimed in any one of the preceding claims, wherein the amount of cation exchange matrix used in step (a) is half the amount of HIC matrix used in step (b).
8. A method as claimed in any one of the preceding claims, wherein step (c) is a weak anion exchange step.

9. A method as claimed in any one of the preceding claims, wherein the ligand density of
the weak anion exchanger is >50 µmol/ml gel/matrix, preferably >100 µmol/ml
gel/matrix and most preferably 160 µmol/ml gel/matrix.
10. A method as claimed in claim 9, wherein the purified rHSA is recovered only from the bound fraction from step (c).

Documents:

3350-DELNP-2004-Abstract-(19-01-2011).pdf

3350-delnp-2004-abstract.pdf

3350-DELNP-2004-Claims-(19-01-2011).pdf

3350-delnp-2004-claims.pdf

3350-delnp-2004-Correspondence Others-(07-07-2011).pdf

3350-DELNP-2004-Correspondence-Others-(02-08-2010).pdf

3350-delnp-2004-Correspondence-Others-(06-01-2011).pdf

3350-DELNP-2004-Correspondence-Others-(19-01-2011).pdf

3350-delnp-2004-correspondence-others.pdf

3350-DELNP-2004-Description (Complete)-(19-01-2011).pdf

3350-delnp-2004-description (complete).pdf

3350-DELNP-2004-Drawings-(19-01-2011).pdf

3350-delnp-2004-drawings.pdf

3350-delnp-2004-form-1.pdf

3350-delnp-2004-form-13.pdf

3350-delnp-2004-form-18.pdf

3350-delnp-2004-form-2.pdf

3350-DELNP-2004-Form-3-(02-08-2010).pdf

3350-delnp-2004-Form-3-(06-01-2011).pdf

3350-delnp-2004-form-3.pdf

3350-delnp-2004-form-5.pdf

3350-DELNP-2004-GPA-(19-01-2011).pdf

3350-delnp-2004-gpa.pdf

3350-delnp-2004-pct-101.pdf

3350-delnp-2004-pct-201.pdf

3350-delnp-2004-pct-304.pdf

3350-delnp-2004-pct-345.pdf

3350-delnp-2004-pct-409.pdf

3350-delnp-2004-pct-416.pdf

3350-DELNP-2004-Petition 137-(02-08-2010).pdf


Patent Number 249705
Indian Patent Application Number 3350/DELNP/2004
PG Journal Number 45/2011
Publication Date 11-Nov-2011
Grant Date 04-Nov-2011
Date of Filing 28-Oct-2004
Name of Patentee NORTH CHINA PHARMACEUTICAL GROUP CORPORATION
Applicant Address R&D CENTER, 388 HEPING EAST ROAD, HEBEI PROVINCE, SHIJIAZHUANG 050015, CHINA
Inventors:
# Inventor's Name Inventor's Address
1 MAKONNEN BELEW AMERSHAM BIOSCIENCES AB, BJORKGATAN 30, S-751 84 UPPSALA, SWEDEN
2 MEI YAN LI NORTH CHINA PHARMACEUTICAL GROUP CORP., 388 HEPING EAST ROAD, HEBEI PROVINCE, SHIJIAZHUANG 050015, CHINA
3 WEI ZHANG NORTH CHINA PHARMACEUTICAL GROUP CORP., 388 HEPING EAST ROAD, HEBEI PROVINCE, SHIJIAZHUANG 050015, CHINA
PCT International Classification Number C07K 14/765
PCT International Application Number PCT/SE2003/00766
PCT International Filing date 2003-05-09
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 0201518-8 2002-05-15 Sweden