Title of Invention

A METHOD FOR PURIFYING POLYPEPTIDE USING TWO-STEP ION EXCHANGE CHROMATOGRAPHY

Abstract A method for purifying a target polypeptide from a solution using two - step ion exchange chromatography wherein separating the target polypeptide by binding the impurities in one step, reloading and binding the target polypeptide in another step using a single ion exchange column by adjusting ionic strength of the carrier solvent.
Full Text FORM 2
THE PATENTS ACT, 1970 (39 of 1970)
COMPLETE SPECIFICATION [See section 10]
A METHOD FOR PURIFYING
POLYPEPTIDE USING TWO-STEP ION EXCHANGE CHROMATOGRAPHY;
SERUM INSTITUTE OF INDIA LIMITED, A COMPANY INCORPORATED UNDER THE COMPANIES ACT, 1956, WHOSE ADDRESS IS "SAROSH BHAVAN" 16-B/1, DR. AMBEDKAR ROAD, PUNE - 411 001, MAHARASHTRA, INDIA.
THE FOLLOWING SPECIFICATION PARTICULARLY DESCRIBES THE NATURE OF THIS INVENTION AND THE MANNER IN WHICH IT IS TO BE PERFORMED.
5 MAR 2004 GRANTED

25-3-2004

BACKGROUND OF INVENTION
1. Field of Invention:
The preferred embodiments of the invention relates to a process for purifying a polypeptide from a mixture containing said polypeptide and related or un-related impurities in a two-step ion exchange chromatography process using a conventional step and a negative step in any preferred order.
2. Related (Prior) art:
The biotechnology industry is based on purification of physiologically active proteins from natural sources or genetically engineered proteins from culture or fermentation medium for the manufacture of various commercially useful vaccines, anti-cancer drugs, hormones, blood proteins, therapeutic agents, etc.
For such usage, it is necessary to have the polypeptide in a pure form. Protein purification requires separation of target protein from all other proteins and other impurities taking advantage of the physico-chemical differences between them. One such property is that of charge. Different proteins have different combinations of amino acids giving each protein a unique spread of ionic charges.
Ion exchange chromatography (IEC) is one of the widely practiced purification methods to purify proteins or peptides based on the net charge on the proteins. The IEC can be used in two different methods: anion exchange and cation exchange based on the type of charged ligands of the chromatography matrix.
A conventional IEC method usually consists of one or more stages of: cleaning, regeneration, equilibration, loading, washing, elution, and regeneration. (Remington's Pharmaceutical Sciences, Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990).
For industrial application of IEC, the elution is achieved by increasing the concentration of salt in an aqueous buffer solution, either as step(s) or linear gradient, optionally controlled by modulating the pH (S. Bj. O slashed.rn and L. Thim, Activation of Coagulation Factor VII to VIIa. Res. Disl. No. 269, 564-565, 1986).
2-

In a conventional application of IEC on an industrial level the loading conditions favor the binding of the target protein to chromatography matrix, followed by eluting the said target polypeptide preferentially by altering the ionic strength of the carrier solvent.
In a conventional IEC, on application of the sample the conditions are created to favor the binding of the protein of interest on the chromatography matrix. Alternatively for research purpose, the conventional IEC can be modified so as to favor the binding of the impurities without letting the target protein to bind the matrix (isocratic elution), referred hitherto as negative purification (Crane, A. BIOL 405, Cell Biology, Dr. Keller, Dec. 6, 1999). However, commercial application of negative purification even though possible is not practiced. In the present invention, a novel IEC method based on both principles (the negative and the conventional) have been applied in tandem in any order, for the purification of recombinant Hepatitis B surface antigen.
BRIEF SUMMARY OF THE INVENTION
In contrast to the conventional single-stage IEC method of purifying target polypeptide by preferential binding followed by changing solvent conditions in step(s) or linear gradient that favor preferential elution, the present invention relates to a novel two-stage ion exchange chromatography process and relates to the following aspects:
Aspect 1:
One of the embodiments of the present invention relates to a two-stage anion exchange chromatography process, comprising of:
(a) Wherein, in the first stage, the carrier solution is adjusted by increasing the ionic strength of the carrier solution, at pH values optionally maintained with buffer, so that the related or unrelated impurities, having a localized or net negative charge higher than that of the target polypeptide and the carrier solution are preferentially bound to an anion exchange matrix leaving the target polypeptide in the un-bound fraction. Normally, the lowest ionic strength or optionally highest pH that does not allow the binding of the target polypeptide to the matrix during loading is used.
3

(b) In the second stage re-loading of the un-bound material containing the target polypeptide on to a cleaned, regenerated and equilibrated anion-exchange matrix; after diluting it with water or buffer such that the overall ionic strength of the loading solution preferentially favors the binding of the target polypeptide having localized or net negative charge, washing the matrix and eluting the bound target polypeptide preferentially from the matrix with a step or linear gradient of higher counter-ionic aqueous solution, optionally having a salt component and / or pH maintained optionally with buffer. Normally the highest ionic strength that permits binding of the target polypeptide during loading and the lowest counter-ionic strength that effects elution is used.
Aspect 2:
In another embodiment of the present invention relates to a two-stage cation exchange chromatography process, comprising of:
(a) Wherein, in the first stage, the carrier solution is adjusted by increasing the ionic strength, of the carrier solution, at pH values optionally maintained with buffer, so that the related or unrelated impurities having a localized or net positive charge higher than that of the target polypeptide and the concentration of charge ions in the carrier solution are preferentially bound to a cation exchange matrix leaving the target polypeptide in the un-bound fraction. Normally, the lowest ionic strength or optionally lowest pH that does not allow the binding of the target polypeptide to the matrix during loading is used.
(b) In the second stage, re-loading of the un-bound material containing the target polypeptide on to a cleaned, regenerated and equilibrated cation-exchange matrix; after diluting it with water or buffer such that the overall ionic strength of the loading solution preferentially favors the binding of the target polypeptide having localized or net positive charge, washing of the matrix and elution the bound target polypeptide preferentially from the matrix with a step or linear gradient of higher counter-ionic aqueous solution, optionally having a salt component and / or pH optionally maintained with buffer. Normally the highest ionic strength that permits binding of the target polypeptide during loading and the lowest counter-ionic strength that effects elution is used.
4

Aspect 3:
Another embodiment of the present invention relates to a two-stage ion exchange chromatography process, comprising of:
(a) Wherein, in the first stage, the carrier solution is adjusted by increasing the ionic strength of the carrier solution, at pH values optionally maintained with buffer, so that the related or unrelated impurities, having a localized or net negative charge higher than that of the target polypeptide and the concentration of charge ions in the carrier solution are preferentially bound to an anion exchange matrix leaving the target polypeptide in the un-bound fraction. Normally, the lowest ionic strength or optionally highest pH that does not allow the binding of the target polypeptide to the matrix during loading is used.
(b) In the second stage, re-loading of the un-bound material containing the target polypeptide on to a cleaned, regenerated and equilibrated cation-exchange matrix; after diluting it with water or buffer such that the overall ionic strength of the loading solution preferentially favors the binding of the target polypeptide having localized or net positive charge, washing of the matrix and elution the bound target polypeptide preferentially from the matrix with a step or linear gradient of higher counter-ionic aqueous solution, optionally having a salt component and / or pH optionally maintained with buffer. Normally the highest ionic strength that permits binding of the target polypeptide during loading and the lowest counter-ionic strength that effects elution is used.
Aspect 4:
A further embodiment of the present invention relates to a two-stage ion exchange chromatography process, comprising of:
(a) Wlierein, in the first stage, the carrier solution is adjusted by increasing the ionic strength of the carrier solution, at pH values optionally maintained with buffer, so that the related or unrelated impurities having a localized or net positive charge higher than that of the said polypeptide and the concentration of charge ions in the carrier solution are preferentially bound to a cation exchange matrix leaving the target polypeptide in the un-bound fraction. Normally, the lowest ionic strength or optionally highest pH that does not allow the binding of the target polypeptide to the matrix during loading is used.
5

(b) In the second stage re-loading of the un-bound material containing the target polypeptide on to a cleaned, regenerated and equilibrated anion-exchange matrix; after diluting it with water or buffer such that the overall ionic strength of the loading solution preferentially favors the binding of the target polypeptide having localized or net negative charge, washing the matrix and eluting the bound target polypeptide preferentially from the matrix with a step or linear gradient of higher counter-ionic aqueous solution, optionally having a salt component and / or pH maintained optionally with buffer. Normally the highest ionic strength that permits binding of the target polypeptide during loading and the lowest counter-ionic strength that effects elution is used.
Aspect 5:
The process according to any one of the aspects 1 to 4, wherein the type of ion exchange chromatography matrix is chosen depending on the quantity and charge characteristic of the target polypeptide and that of the impurities, and includes anion exchanger in both, that is stage (a) and stage (b) or cation exchanger in both, stage (a) and stage (b) or anion exchanger in stage (a) and cation exchanger in stage (b).or cation exchanger in stage (a) and anion exchanger in stage (b).
Aspect 6:
The process according to any one of the aspects 1 to 5, wherein depending on the quantity and charge characteristic of the target polypeptide and that of the impurities, the sequence of the said two stages, stage (a) and stage (b) is optionally reversed by modifying the ionic strength, and optionally pH, of the loading solution containing the said polypeptide. That is, modifying the ionic strength of the loading solution, and / or pH, to preferentially bind the impurities to the matrix in stage (a) and to preferentially bind the target polypeptide to the matrix in stage (b). Optionally, modifying the ionic strength of the loading solution, and / or pH, to preferentially bind the said polypeptide to the matrix in stage (a) and to preferentially bind impurities to the matrix in stage (b). Use of either option constitutes an alternative embodiment of the present invention. In a further broad embodiment of the present invention, include modifying the ionic strength of the loading solution, and /or pH, to preferentially exclude the binding of the target polypeptide to the chromatography matrix in both, stage (a) and stage (b).
6

Aspect 7:
In a further embodiment of the present invention one or more of the salt component used in either stage (a) or stage (b) is selected from any organic or inorganic salt or mixtures thereof, preferably sodium chloride, potassium chloride, calcium chloride, sodium acetate, potassium acetate, ammonium acetate, sodium citrate, potassium citrate, ammonium citrate, sodium sulphate, potassium sulph?.t Aspect 8:
The process according to any one of the aspects 1 - 4 wherein, optionally no salt component is present in stage (a).
Aspect 9:
The process according to any one of the aspects 1 - 4 wherein, optionally no salt component is present in stage (b).
Aspect 10:
The process according to any one of the aspects 1 - 4 wherein, optionally no buffer is present in stage (a).
Aspect 1 1:
The process according to any one of the aspects 1 - 4 wherein, optionally no buffer is present in stage (b),
AsDect 12:
In a further embodiment of the present invention also relates to the choice of buffer used in the two stages can be independently selected, and would be known to the person skilled in the art. It can be determined according to the well-known techniques such as conventional test-tube methods as described (Handbook from Amersham-Pharmacia Biotech), the buffer component used in either stage (a) and / or stage (b) is independently selected from phosphate buffers, tris (Trizma) buffers, citrate buffers, borate buffers, lactate buffers, glycyl-glycine buffers, tricine buffers, arginine buffers, carbonate buffers, acetate buffers, glutamate
7

buffers, ammonium buffers, glycine buffers, alkylamine buffers, aminoethyl alcohol buffers, ethylenediamine buffers, tri-ethanol amine, imidazole buffers, pyridine buffers and barbiturate buffers and mixtures thereof, preferably citric acid, sodium citrate, sodium phosphate, phosphoric acid, glutamic acid, sodium glutamate, glycine, sodium carbonate, potassium citrate, potassium phosphate, potassium glutamate, potassium carbonate, tris-hydroxymethylamino methane and boric acid and mixtures thereof (Remington's Pharmaceutical Sciences, Gennaro, Ed. Mack Publishing Co., Easton, Pa., 1990, or Remington: The Science and Practice of Pharmacy, 19th Edition, 1995). The choice of pH, buffer and ionic strength required in the two stages is determined according to the well-known techniques such as conventional test-tube methods as described (Handbook from Amersham-Pharmacia Biotech.), and could be known to the person skilled in the art (e.g. the buffer used for the purpose depends on the polypeptide and the anionic or cationic nature of the matrix; for anion exchange, preferably the buffer is Tris or phosphate buffer and for cation exchange the buffer is preferably acetate or phosphate buffer). Each of these buffers constitutes an alternative embodiment of the present invention.
Aspect 13:
The functional group used for anion exchange resin is chosen depending on the affinity of the target polypeptide vis-a-vis impurities and includes diethylaminoethyl (DEAE), quaternary aminoethyl (QAE), quaternary ammonium (Q), and the like. Suitable anion exchange resins having such functional groups include, for example, DE92 (Whatman); DEAE ^CELLULOSE (Sigma), BAKERBOND ABX 40 mu (J. T. Baker, Inc.); FRACTOGEL EMD DEAE-650 (EM Separtations), FRACTOGEL EMD TMAE-650 (S) TM (EM Science), TOYOPEARL DEAE 650 M (Tosohass), DEAE-SEPHAROSE CL-6BT", RESOURCE QTm and Q SEPHAROSE (QSFF) (Amersham Pharmacia Biotech AB), DEAE MERE SEP. 1000 TM (Millipore), and DEAE SPHERODEX (Sepracor); MACRO-PEP QTM (Bio-Rad Laboratories); Q-HYPERD (BioSepra, Inc.); and the like. Other suitable anion exchange chromatography matrices know to those skilled in the art, also find use herein.
Aspect 14:
The functional group used for cation exchange resin is chosen depending on the charge of the target polypeptide vis-a-vis impurities and which are capable of binding polypeptides over a wide pH range. These include a wide variety of
8

materials known in the art, for example carboxymethyl (CM), sulphopropyl (SP) and Methyl sulphonate (S) and the like. Cation exchange resins having such functional groups include, for example, CM52 CELLULOSETM (Whatman, Inc.); S-HYPERDTM and CM SPHERODEX (Secpracor); SP SEPHAROSE FF, DEAE SEPHAROSE FF, CM-SEPHAROSE, and RESOURCE STM (Amersham Pharmacia Biotech AB); and JT BAKER CSxTS' (J. T. Baker, Inc.), TOYOPEARL SP550CTM, TOYOPEARL DEAE 650M (Tosohass) and FRACTOGEL EMDTM S03—650(m) (MERCK). Although other suitable materials for use in cation exchange chromatography are know to those skilled in the art, also find use herein.
Aspect 15:
The ion exchange chromatography matrix is a solid phase chosen depending on the type of flow properties desired. The matrices can be different in the characteristics such as fibrous, microgranular, and beaded matrices. The solid phase may be a purification column, a discontinuous phase of discrete particles in a mixing vessel, or a membrane or filter made of matrix material. The solid phase include, but not limited to, polysaccharide (cellulose, agarose, dextran) or other rigid matrices (e.g. controlled pore-glass, polyacylamide, methylacrylate, polyether, ceramic, polystyrenedivinyl benzene or composites. For example matrix based on dextran (e.g. Sephadex) or on cross-linked agarose (e.g. Sepharose, reverse phase STREAMLINE) or cross-linked cellulose (Sephacel) or methylacrylate (e.g. Toyo Pearl) or polystyrene/divinyl benzene beads (SOURCE) are some examples.
The preferred matrices include, but not limited to, Sepharose matrix, Sephadex matrix, Streamline matrix, and Source matrix from Amersham-Pharmacia Biotech, HyperD matrix, Trisacryl matrix, and Spherosil matrix from BioSepra, TSK gel matrix and Toyopearl matrix from TosoHaas, Fractogel EMD matrix from Merck, Poros resins from Perspective Biosystems, Macro-Prep resins from BioRAD, Expression resins from Whatman etc. These matrices include, either cation exchange matrix or anion exchange matrix.
Aspect 16:
The choice of pH, in the two stages is determined according to the well-known techniques such as conventional test-tube methods as described (Handbook from Amersham-Pharmacia Biotech). The present invention also relates to the choice
R

of pH, which would be known to the person skilled in the art (e.g. for cation exchange resins preferably pH 4-7, the exact pH usually below the isoelectric point (pi) of the polypeptide and for anion exchange resins preferably pH 6-9, the exact pH usually above the pi of the target polypeptide).
Aspect 17:
The present invention also relates to the choice of ionic strength required for the two stages, which can be independently selected depending on the nature and content of impurities, range of selectivity desired (level of purity), and can be determined according to the well-known techniques such as conventional test-tube methods as described (Handbook from Amersham-Pharmacia Biotech). Appropriate salt component is used to achieve the desired ionic strength of the elution solution. Changing the pH and thereby altering the charge of the molecule is another way to achieve binding and elution of the molecule (target protein/ impurities). In the preferred embodiment of the invention, a single parameter (that is either conductivity or pH) is changed to achieve elution of both the polypeptide and contaminant, while the other parameter (that is pH or conductivity, respectively) remaining constant. For example, while the conductivity of the various solutions (such as loading solution, washing solution, elution solution, regeneration solution and equilibration solution) may differ, the pH of these solutions may be essentially the same.
Aspect 18:
In a further embodiment of the present invention, the buffer concentration used in stage (a) and / or (b) is selected from the range of 0.1 mM to 3000 mM, preferably 1.0 mM to 1000 mM, most preferably 20 mM to 800 mM. Each of these ranges constitutes an alternative embodiment of the present invention. The quantify of buffer used can vary widely and will generally be in the range of about 1 to 40 column volumes, preferably 2 to 20 column volumes.
Aspect 19:
In a further embodiment of the present invention, the salt concentration used in stage (a) is selected from the range of 0.1 mM to 3000 mM, preferably 1.0 mM to 1000 mM, most preferably 20 mM to 800 mM. Each of these ranges constitutes an alternative embodiment of the present invention.
10

Aspect 20:
In a further embodiment of the present invention, the step increase in salt concentration in stage (b) is selected from the range of 0.1 mM to 3000 mM. preferably 1.0 mM to 1000 mM, most preferably 20 mM to 800 mM. Each of these ranges constitutes an alternative embodiment of the present invention.
Aspect 21:
In a further embodiment of the present invention, the concentration of salt component in stage (b) is a increased preferably in a linear gradient from 0.1 mM to 3000 mM, preferably 1.0 mM to 1000 mM, most preferably 20 mM to 800 mM. Each of these ranges constitutes an alternative embodiment of the present invention.
Aspect 22:
In a further embodiment of the present invention, the target proteins, polypeptides, oligopeptides, to be purified is Hepatitis B surface antigen (S, L, M, mixed particles). Each of these proteins, polypeptides, and their combinations as well as their homologues, analogs and derivatives thereof, constitutes an alternative embodiment of the present invention.
Aspect 23:
Another aspect of the invention relates to a mixture comprising of the target polypeptide, optionally modified chemically by conventional techniques such as alkylation, acylatation, esterification or amide formation or the like, so as to aid in the separation of impurities from the target substance and is one aspect of the present invention to any derivatization of proteins or polypeptides such as active site blockage using cholomethyl ketone (S. Higashi, H. Nishimura, S. Fuiji, K. Takada, S, Iwanaga, 1992. J. Biol. Chem. 267, 17990; J. H. Lavvson, S. Butenas, K. Mann, 1992. J. Biol. Chem. 267, 4834; J. Contrino, G. A. Hair, M. A. Schimeizl, F. R. Rickles, D. L. Kreutzer, 1994. Am. J. Pathol. 145, 1315); so as to block the active sites or protect the target protein from proteases.
11

Aspect 24:
Peptides can be produced on an industrial scale according to conventional organic peptide-synthesis chemistry' either as native peptide or a mixture of their homologues, analogs or derivatives.
Industrial-scale polypeptide production can also be achieved using Continuous Exchange Cell-free Rapid Translation System (ProteoMaster from Roche Applied Systems, Penzberg Germany).
Polypeptides can also be produced by a method which comprises of cultiiring or fermenting a host cell containing the DNA sequence(s) introduced in to the host cell by suitable standard techniques (e.g. Sambrook, J, Fritseh, E. F. and Maniatis, T. Molecular Cloning; A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, 1989) encoding, usually one, or at times more than one, polypeptide or protein under suitable nutrient medium conditions permitting the expression of the protein(s) or polypeptide(s), after which the resulting polypeptide is recovered from the culture or fermentation broth or transgenic animals expressing the target protein in mammary glands.
The DNA sequence encoding the parent peptide encoding a part of the target polypeptide may be of genomic or cDNA origin prepared either by polymerase chain reaction using suitable primers (Burden and Whitney, Biotechnology: Proteins to PCR: A course in Strategies and Lab Techniques, 1995) or cloned in a suitable vector (e.g. as described in Sambrook, J., Fritseh, E.F. and Maniatis, T. Molecular Cloning: A Laboratory' Manual, Cold Spring Harbor Laboratory Press, New York, 1989) or synthesized by established chemical methods (e.g. Beaucage and Caruthers, Tetrahedron Letters 22 (1981), 1859-1869; Matthes et al. EMBO Journal 3 (1984), 801-805) The nucleic acid encoding the target polypeptide is isolated by any of these techniques is inserted into a replicable vector for expression. The vector components generally include, but not limited to, one or more of the following: a signal sequence, a promoter sequence, autonomous replication sequence, one or more marker genes, a termination sequence (e.g. as described in U.S. Pat. No. 5389525)
The medium used to culture the cells may be any conventional medium suitable for growing the host cells, such as minimal or complex media containing appropriate supplements as available from commercial suppliers or as published recipes (e.g. Difco Manual). The polypeptide produced by the cell may be
12

intracellular or secreted by the cells and may be recovered by disrupting the cells in presence of protease inhibitor(s) or by purifying the extra-cellular polypeptide from the cell-free culture medium, respectively.
Aspect 25:
The conventional down-stream processing methods are then employed for the purification of polypeptides arising out of any one of the industrial production methods described in aspect-24. The down-stream processing includes one or more steps, for example, centrifugation or filtration to recover either the culture supernatant (if the target polypeptide is secreted by the cells) or the cells (if the target polypeptide is intracellular). For the latter proteins, the first step of a purification process involves disruption of cells by one or more passages through any of the known mechanical disruption devices such as the glass ball mill (e.g. DynoMill and Fryma Coball Mill), high pressure homogenizer (e.g. Nanojet), by enzymatic treatment or osmotic shock method to release the intracellular protein, optionally in presence of a protease inhibitor, optionally precipitating the proteinaceous components present in the homogenate or culture supernatant by means of one or more precipitating agents such as ammonium sulphate, polyethylene glycol, urea so as to precipitate out preferentially the target protein or the impurities excluding the target protein, The resulting precipitated solution is clarified by means of differential centrifugation or by filtration and subsequently subjected the supernatant or filtrate to one or more chromatographic techniques such as hydrophobic interaction chromatography (HIC), size-exclusion or gel-filtration chromatography (GFC), reverse-phase chromatography, affinity chromatography, ion exchange chromatography (IEC) according to the present invention, and optionally subjecting to further purification steps, if necessary, by any of the known techniques. Exemplary further purification steps include but not restricted to, filtration, iso-pycnic or equilibrium density gradient centrifugation, sterilization and others. The sequential order of the purification steps, based on the characteristics of the target polypeptide and on the characteristics of the related or unrelated impurities accompanying the polypeptide of interest, may vary. Thus, in a broad aspect, any industrial process comprising one or more steps described herein, for production of a pure target polypeptide, which also includes an IEC process according to the present invention, is within the embodiment of the present application.
Optionally, the target polypeptide is conjugated or mixed (but not conjugated) with one or more heterologous molecules as desired. The heterologous molecule
13

may for example be one which is used as a - stabilizer (e.g. polyethylene glycol, glycine, glutamic acid, asparagines, histidine, arginine or lysine, monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, dextrins, trehalose), a carrier (e.g. sorbitol, gelatin), a excipient (e.g. buffers such as Tris, phosphate, citrate), an adjuvant (e.g. aluminum hydroxide, aluminum phosphate), an antioxidant (e.g. ascorbic acid, methionine), a preservative (e.g. phenol, benzalkonium chloride, thiomersol), a bulking agent (manirol, sorbitol, dextrose), a non-ionic surfactanats (tween-20), a metal complexes (e.g. Zn-protein complexes), a cytokine (e.g. interleukin), additional (one or more) active compounds (e.g. additional cnemotherapeutic agents, antigens, enzymes, fluroscent label, radionuclide, toxins, lectins, targeting antibodies, cnemotherapeutic drugs), a hydrophilic polymers (e.g. polyvinylpyrrolidone), a cryoprotectant (e.g. glycine, DMSO, ethyleneglycol) a chelating agent (e.g. EDTA), a sustained release preparation (e.g. copolymer).
The target polypeptide may also be entrapped in appropriate semipermeable matrices, films, microcapsules for sustained release (e.g. polyester hydrogels, poly(vinyl alcohol), polylactides (U.S. Pat. No. 3,773,919), copolymers (e.g. lactic acid-glycolic acid), synthetic or natural lipid-based earner particles (e.g. liposomes (WO Pat. No 0154666), HBsAg particles (WO Pat. No 9939736)
14

Definitions:
The term "polypeptide" as used herein refers to peptide and protein having more than about 10 amino acids.
The term "target polypeptide" as used herein refers to peptide and protein having more than about 10 amino acids and which is the polypeptide of interest, purified using ion exchange chromatography process according to the present invention. The target polypeptide in the present invention is Hepatitis B surface antigen.
The term "related impurities" as used herein, is intended to mean one or more impurities with a different local or overall net charge from the polypeptide of interest, for example truncated forms, with extra amino acids, various derivatives, deaminated forms, incorrectly folded forms, varying glycosylation and sialyation, lack of y-carboxy glutamic acid, and others.
The term "unrelated impurities" as used herein, is intended to mean one or more impurities with a different local or overall net charge from the polypeptide of interest, and may be either non-proteinaceous for example nucleotides, DNA, lipids, carbohydrates, and others; or proteinaceous for example polypeptide with different amino acid sequence from the target polypeptide.
The term "low ionic strength aqueous solution" as used herein, in connection with washing solution having lower negative (or positive) ionic concentration than the net charge of the target polypeptide and is intended to mean that the numeric value of the local concentration of ions surrounding the polypeptide is lower than the numeric value of the local or overall net charge on the polypeptide.
The term "higher counter-ionic aqueous solution" as used herein, in connection with elution solution having higher negative (or positive) ionic concentration than the net charge surrounding the target polypeptide bound to the matrix and is intended to mean that the numeric value of the local concentration of ions surrounding the polypeptide is higher than the numeric value of the overall net charge on the polypeptide.
The term "conductivity" as used herein, refers to the ability of an aqueous solution to conduct an electric current between two electrodes. In solution, the current flows by ion transport. Therefore, with an increasing amount of ions present in the aqueous solution, the solution will have a higher conductivity. The unit of measurement for conductivity is mmhos (mS/cm), and can be measured using a
15

conductivity meter used on-line or off-line. Changing the concentration of ions therein may alter the conductivity of the solution. Thus by changing the concentration of the buffering agent and / or salt (e.g. NaCl or KC1) in the solution may be altered in order to achieve the desired conductivity.
The term "water" as used herein, is intended to mean either pure water, demineralized water or water for injection.
The term "loading solution" as used herein, is an aqueous mobile phase used for loading of the target polypeptide and one or more impurities onto the ion exchange matrix. Optionally the loading solution contains one or more buffering agent(s) and/or salt component(s). The loading solution has a conductivity and/or pH such that the polypeptide of interest (and generally one or more contaminants) is/are bound to the ion exchange matrix.
The term "carrier solution" as used herein, is an aqueous mobile phase used as carrier of the target polypeptide and one or more impurities onto the ion exchange matrix. Optionally the carrier solution contains one or more buffering agent(s) and/or salt component(s). The carrier solution has a conductivity and/or pH such that the polypeptide of interest (and generally one or more contaminants) does not bind to the ion exchange matrix.
The term "negative ion exchange chromatography" as used herein, is intended to mean that the concentration of ions in the carrier solution is higher than the net charge of the target polypeptide so that the said polypeptide is not preferentially bound to the ion exchange chromatography matrix. The concentration of ions in the carrier solution is modified by addition of appropriate concentration of one or more salt component(s) and / or optionally with one or more buffering agent(s).
The term "binding" as used herein is meant to immobilize by virtue of ionic interactions between a charged molecule (target protein/impurities) and oppositely charged ion exchange matrix under appropriate conditions (pH/conductivity).
The term "unbound fraction" as used herein is meant the flow-through solution containing material that did not bind the matrix.
The term "washing" as used herein is meant passing aqueous solution of low ionic concentration, optionally buffered to maintain pH and optionally containing a salt component, through or over the ion exchange matrix to remove unbound material.
16

The tern "elution" as used herein is meant displacing (releasing) the bound molecule (target polypeptide/impurities) from an ion exchange chromatography matrix by increasing the ionic strength of the solution surrounding the bound molecule such that the free ions in the solution competes with the localized or net charge on the molecule for the oppositely charged sites on the ion exchange matrix. Appropriate salt component is used to achieve the desired ionic strength of the elution solution. Changing the pH and thereby altering the charge of the molecule is another way to achieve binding and elution of the molecule.
The term "cleaned" as used herein is meant passing aqueous solution containing appropriate cleaning and sanitizing agent such as 0.05M to 2.0M NaOH, with or without the presence of up to 2 M salt component, preferably NaCl, over the ion exchange matrix.
The tenn '"regenerated" as used herein is meant passing solution having conductivity and/or pH as required to re-generate the ion exchange resins such that it can be re-used.
The tenn "equilibrated" as used herein is meant passing solution having conductivity and/or pH as required to equilibrate the ion exchange resins such that the ionic strength, and optionally pH, of the mobile phase is similar to that of the carrier solution or that of the loading solution.
The term "isoelectric point" or "pi" as used herein refers to the pH at which the polypeptide's positive net charge balances its negative net charge, pi can be determined from calculating the net charge of the amino acid residues of the polypeptide or can be determined from isoelectric focusing.
The tenn "localized or net charge" as used herein is intended to mean a numeric value of either positive or negative charge, localized on the surface of the molecule (i.e. patches of residues with similar charge) or sum of all the charges on the surface of the molecule, which interacts with the charged ligands of the chromatography matrix.
The term "ligand" as used herein is meant either positively or negatively charged functional groups immobilized on a solid phase (chromatography matrix), and which can participate in a charged interaction with oppositely charged molecule in the mobile phase.
17

BRIEF DESCRIPTION OF THE EXAMPLES:
The present invention is illustrated by the following examples, which, are not to be construed as limiting the scope of protection.
Hepatitis B surface antigen (HBsAg), a particulate lipoprotein, was expressed in a methylotrophic yeast Hamemda polymorpha K3 / 8-1 (EP 173378) by conventional recombinant DNA technology. The recombinant yeast strain was fennented in synthetic media using limiting feeds of glycerol or methanol or mixture of glycerol and methanol to achieve an optimum expression of HBsAg under the control of formate dehydrogenase promoter. The fermentation was carried out in three phases: an initial growth phase with higher glycerol concentration (2.0 to 2.5%v-glycerol/v-broth), was followed by derepression phase with dissolved-oxygen-dependent glycerol feeding as the sole carbon source and the final methanol induction phase with time-based feeding of glycerol and methanol (0.1-1.0%v-glycerol/v-broth and 0.5-8.0%v- methanol/v-broth). Upon completion of the fennentation process the cells are separated from the medium components by either tangential-flow micro-filtration system or centrifugation followed by disruption in a glass-bead mill (e.g. Dyno Mill KD20) at a cell density of 50-150 g. dry cell weight / litre in presence of a protease inhibitor (PMSF). The disrupted cells are precipitated using polyethylene glycol to remove cell debris and impurities by removing the precipitate by centrifugation followed by trapping and desorption of the antigen from fused silica (Aerosil 380 V) hitherto referred as the desorbate. The desorbate is adjusted for conductivity prior to ion exchange chromatography as described herein:
EXAMPLE-1: Anion exchange chromatography - Stage (a) followed by Stage (b):
Stage (a)- Negative TEC:
The conductivity of the desorbate, containing the target protein (HBsAg) at a concentration of 0.1 - 1.5 mg/ml, preferably at 0.3 to 1.0 mg/ml and having total protein content of 0.5 - 2.0 mg/ml, preferably 0.8 to 1.4 mg/ml, was adjusted to 13-16 mS/cm by addition of 5 M NaCl before loading on the chromatography column containing Toyopearl DEAE 650M (TosoHass) anion exchanger at a flow rate of 76 cm per hour. The resulting flow through (that is unbound fraction) was collected, followed by passing 2 column volumes of washing buffer, 2 column volumes of elution solution-I containing 0.2 M NaCl in 50 mM Tris buffer, pH
IS

8.5 and conductivity of 18-22 mS/cm, 2 column volumes of elution solution-II containing 0.2 M NaCl in 50mM Tris buffer, pH 8.5 and conductivity of 50 mS/cm, cleaned with 2 column volumes of 0.5 M NaOH in water, regenerated with 2 column volumes of elution solution-II and equilibrated with 2 column volumes of 50mM Tris at pH 8.5 and conductivity of 2.0 mS/cm.
Stage (b) - Conventional IEC.
The flow-through (unbound fraction) obtained in stage (a) was diluted with loading buffer to give a final conductivity of 10 (+/-2 mS/cm) before loading on a cleaned, regenerated and equilibrated chromatography column containing Toyopearl DEAE 650M (TosoHass) anion exchanger at a flow rate of 76 cm per hour, followed by passing sequentially: 2 column volumes of 50mM Tris at pH 8.5 and conductivity of 8 mS/cm, 2 column volumes of elution buffer-I and 2 column volumes of elution buffer-II. The individual fractions collected for each buffer passed through the column was analyzed separately for protein, HBsAg, DNA, lipid and protein impurities using SDA-PAGE. For each solution passed, absorbance (at 280 nm) and conductivity using on-line detectors was recorded.
Total protein and HBsAg concentrations of each chromatography fraction (flow through, wash fraction, elution buffer-I fraction, elution buffer-II fraction) was detennined on the basis of UV spectrophotometer scans of each sample and from the A28o value, the concentration of protein and HBsAg was detennined from the fonnula:
Protein concentration in the Desorbate = A280 X Dilution factor X 3.5*
HBsAg concentration in the Desorbate = A2so X Dilution factor X 6.0*
Protein concentration in IEC eluate buffer-I = A2so X Dilution factor X 3.5*
HBsAg concentration in IEC eluate buffer-I = A28oX Dilution factor X 5.0*
^Extinction coefficient
19

EXAMPLE-2: Anion exchange chromatography -Stage (b) followed by Stage (a):
The same desorbate used in Example-1 was purified by reversing the two stages, that is stage (b) prior to stage (a).
Stage (b) - Conventional IEC.
The desorbate conductivity was adjusted to 8 mS/cm using 1 M NaCl and buffered with 50 raM Tris at pH 8.5, prior to loading on a pre-equilibrated reduced-scale column containing at a flow rate of 76 cm per hour, followed by passing sequentially: 2 column volumes of 50mM Tris at pH 8.5 and conductivity of 8 mS/cm, 2 column volumes of elution buffer-I containing 0.2 M NaCl in 50mM Tris buffer, pH 8.5 and conductivity of 18-22 mS/cm, and 2 column volumes of elution buffer-II containing 0.2 M NaCl in 50mM Tris buffer, pH 8.5 and conductivity of 50 mS/cm. The individual fractions collected for each buffer passed through the column was analyzed separately for protein, HBsAg, DNA, lipid and protein impurities using SDA-PAGE. For each solution passed, absorbance (at 280 nm) and conductivity using on-line detectors was recorded.
The resulting elution buffer-1 fraction from the column was collected and purified further by concentrating on a tangential-flow ultra-filtration system using a 300kDa nominal cut-off membrane and subjecting the concentrate to cesium chloride equilibrium density gradient centrifugation (at 45000 rpm for 42 hours), followed by gel-filtration (or size exclusion) chromatography.
Stage (a)- Negative IEC:
In a separate study, the resulting elution buffer-I fraction from the anion exchange column [Example 2, Stage (b)] was collected and diluted with 50 mM Tris (pH 8.5) to give a conductivity of 13-16 mS/cm prior to loading on a cleaned, regenerated and equilibrated chromatography column containing Toyopearl DEAE 650M (TosoHass) anion exchanger at a flow rate of 76 cm per hour. The loading was followed by passing sequentially through the matrix: 2 column volumes of 50mM Tris at pH 8.5 and conductivity of 8 mS/cm, 2 column volumes of elution buffer-I, followed by 2 column volumes of elution buffer-II. Out flowing material from the column was separately collected and analyzed for each of the buffers passed.
20

RESULTS AND DISCUSSION:
The target protein was eluted and collected as a single peak and analyzed for protein impurities by SDS-PAGE, Ratio of A280/A260, Size-exclusion chromatography, Ratio of HBsAg to protein content and DNA content.
The above-described two-stage method gave higher level of purity (e.g. 95% as compared to 75%) with similar yield of the target polypeptide in comparison with the single-stage conventional anion exchange chromatography as described above in Stage (b) as shown in TABLE-1.
There was a considerable improvement in the step yield of HBsAg (up to 200%) when the eluate from the two-stage anion exchange chromatography was concentrated on a cross-flow ultra-filtration membrane (of 300kDa nominal cut off) and subjected to cesium chloride equilibrium density gradient centrifligation (at 45000 rpm for 40 hours) when compared with the recovery of HBsAg purified using a conventional anion exchange chromatography [EXAMPLE-2, Stage (b)] and further purified using an identical purification scheme of ultra-filtration and density gradient centrifugation (TABLE-1).
21

Table 1
Comparative analysis of batches processed using the modified lEC protocol with the
conventional protocol
Batch Sample Average Volume
(D HBsAg Purity
(Based on SDS-
PAGE scans)
(Average %) Average DNA
Content (Microgram) Step Recovery
of HBsAg (Average %) Cummulative recovery of
HBsAg (Average %)
084043 to 084045 lEC Pool 52.6 72 7 34
084047 to 084049
45.8 95 8 31

084043 to 084045 CsCI Pool 2.27 95.4 2 35 10
084047 to 084049
2.68 99.1 4 69 21
Batches 084043 to 084045 are performed with the conventional single step lEC method
Batches 084047 to 084049 are performed with the modified dual step lEC method


WE CLAIM
1. A method for purifying a target polypeptide from a solution using two - step ion exchange chromatography wherein separating the target polypeptide by binding the impurities in one step, reloading and binding the target polypeptide in another step using a single ion exchange column by adjusting ionic strength of the carrier solvent.
2. The method according to claim 1, wherein the sequence of the two steps can optionally be reversed depending on the quantity and charge characteristics of impurities and that of the target polypeptide to be purified.
3. The method according to claim 1, wherein the target polypeptide is preferentially bound or remains un-bound to ion exchange matrix depending on the net charges of said polypeptide and ionic concentration of the carrier solvent.

4 The method according to claim 3, wherein the said carrier solvent comprises water optionally with salt component(s), buffer component(s)
5 The method according to claim 3, wherein the ion exchange matrix is cationic, anionic or both.
6 The method as claimed in claim 1, wherein the said target polypeptide is Hepatitis B surface antigen (S, L, M mixed particles), mixed particles, each of these proteins or polypeptides or their homologous, analogs and derivatives thereof.
23

7. The method according to claim 1 is an industrial method for purifying
a target polypeptide from a mixture of related or non -related
impurities.
8. The method according to claim 7, wherein the impurities are related
impurities including impurities with a different local or overall net charge
from the polypeptide of interest, truncated forms with extra amino acids,
various derivatives, deaminated forms, incorrectly folded forms, varying
glycosylation and sialyation, lack of gamma carboxy glutamic acid.
9. The method according to claim 7, wherein the impurities are un-related
impurities including impurities with a different local or overall net charge
from the polypeptide of interest, and may be either non-proteinaceous for
example nucleotides, DNA, lipids, carbohydrates; or proteinaceous for
example polypeptide with different amino acid sequence from the target
polypeptide.
10. The method as claimed in claim 1, wherein the polypeptide is optionally
subjected to further purification.
Dated this 29th Day of August, 2003
FOR SERUM INSTITUTE OF INIDA LIMITED their Agent

(MANISH SAURASTRI) KRISHNA & SAURASTRI

Documents:

860-mum-2003-cancelled pages(25-03-2004).pdf

860-mum-2003-claims(granted)-(25-03-2004).doc

860-mum-2003-claims(granted)-(25-03-2004).pdf

860-mum-2003-correspondence(02-12-2004).pdf

860-mum-2003-correspondence(ipo)-(21-10-2004).pdf

860-mum-2003-form 1(29-08-2003).pdf

860-mum-2003-form 19(29-08-2003).pdf

860-mum-2003-form 2(granted)-(25-03-2004).doc

860-mum-2003-form 2(granted)-(25-03-2004).pdf

860-mum-2003-form 3(29-08-2003).pdf

860-mum-2003-form 5(28-09-2003).pdf

860-mum-2003-power of attorney(01-12-2003).pdf


Patent Number 207060
Indian Patent Application Number 860/MUM/2003
PG Journal Number 30/2007
Publication Date 27-Jul-2007
Grant Date 21-May-2007
Date of Filing 29-Aug-2003
Name of Patentee SERUM INSTITUTE OF INDIA LIMITED
Applicant Address SAROSH BHAVAN 16-B/1, DR. AMBEDKAR ROAD, PUNE,
Inventors:
# Inventor's Name Inventor's Address
1 RUSTOM MODY FLAT NO 902, BUILDIING "W", SACRED HEART CO-OP HOUSING SOCIETY NO. 75/2/2B, 9TH FLOOR, WANAWADI, PUNE 411 040,
PCT International Classification Number C07K 1/16
PCT International Application Number N/A
PCT International Filing date
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 NA