WO2012160536A1 - Purification d'anticorps - Google Patents

Purification d'anticorps Download PDF

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Publication number
WO2012160536A1
WO2012160536A1 PCT/IB2012/052610 IB2012052610W WO2012160536A1 WO 2012160536 A1 WO2012160536 A1 WO 2012160536A1 IB 2012052610 W IB2012052610 W IB 2012052610W WO 2012160536 A1 WO2012160536 A1 WO 2012160536A1
Authority
WO
WIPO (PCT)
Prior art keywords
exchange chromatography
buffer
chromatography
anion exchange
anion
Prior art date
Application number
PCT/IB2012/052610
Other languages
English (en)
Inventor
Kishore Jahagirdar
Neeru Gupta
Original Assignee
Dr Reddy's Laboratories Limited
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Dr Reddy's Laboratories Limited filed Critical Dr Reddy's Laboratories Limited
Publication of WO2012160536A1 publication Critical patent/WO2012160536A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/18—Ion-exchange chromatography
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/36—Extraction; Separation; Purification by a combination of two or more processes of different types

Definitions

  • the present invention relates to a method of purification of antibodies comprising an anion exchange chromatography.
  • Therapeutic proteins are primarily products of recombinant DNA technology, i.e., cloning and expression of a heterologus gene in prokaryotic or eukaryotic systems.
  • proteins expressed by recombinant DNA methods are typically associated with impurities such as host cell proteins (HCP), host cell DNA (HCD), viruses, etc.
  • HCP host cell proteins
  • HCD host cell DNA
  • viruses etc.
  • HCP host cell proteins
  • HCD host cell DNA
  • heterogeneity in the expression of the desired protein in the form of charged variants (typically acidic, lower pi variants and basic, higher pi variants).
  • multimeric proteins, such as antibodies have a higher tendency to aggregate, contributing to significantly increased impurity levels.
  • Antibodies constitute one of the most important classes of therapeutic proteins, especially in the areas of oncology, arthritis and other chronic diseases.
  • Purification of antibodies often involves a combination of different chromatographic steps, that generally begins with “a capture step” by protein A affinity chromatography, followed by one or more additional separation steps.
  • a final “polishing step” is often necessary for the removal of trace amounts of HCP, HCD, viruses or endotoxins.
  • the polishing step is typically carried out by anion exchange chromatography performed in a flow-through mode.
  • WO 2004/024866 describes a method of purifying a polypeptide by ion exchange chromatography in which a gradient wash with differing salt concentrations is used to resolve the polypeptide.
  • WO 1999/057134 describes the use of ion exchange chromatography for purification of polypeptides by varying conductivity and/or pH .
  • US 51 10913 claims purification of murine antibodies using low pH and at least three different pH conditions in the ion-exchange chromatographic step.
  • US 7847071 describes purification of antibodies using series of chromatographic techniques wherein the anion-exchange chromatography is performed in the flow-through mode using a buffer of pH 8.0 and a displacer salt.
  • WO 2010072381 reports purification of immunoglobulin in the flow-through from the anion exchange chromatography wherein the buffer has pH value of from 8.0 to 8.5.
  • the principle object of the present invention is to provide an improved method for obtaining antibody preparations that avoid use of buffers at very low or high pH during anion exchange chromatography.
  • the present invention provides an anion exchange chromatography with buffer conditions in the neutral pH range, for the purification of antibodies.
  • the invention results in effective clearance of viruses, separation of charge variants, increased purity and recovery of the desired antibody.
  • the present invention describes a process for the purification of antibodies by anion exchange chromatography performed in flow-through mode in the neutral pH range.
  • Fig. 1 is an illustration of a chromatogram from the procedure as described in Example 1 .
  • the line marked “Cond” represents the increase in conductivity in mS/cm.
  • Peak A represents the eluate obtained from protein A chromatography resin.
  • Fig. 2 is an illustration of a chromatogram from the procedure as described in Example 2.
  • Peak A and B represent the eluate obtained from cation exchange chromatography.
  • Peak A and B are charge variants of the anti-CD20 antibody.
  • FIG. 3 and 4 are illustrations of a chromatogram from the procedure as described in Example 3. Figures represent the flow-through fraction of the anion exchange chromatography.
  • the present invention describes a process for purification of antibodies by anion-exchange chromatography.
  • the process avoids use of very low or high pH buffers.
  • the neutral pH buffer conditions described in the invention also facilitates easy "switch over" between alternate chromatographic steps without need for significant buffer exchange or neutralization steps.
  • the conditions described in the present invention results in effective separation of charge variants, removal of impurities such as HCP, HCD and viruses and results in optimum yield of the desired antibody.
  • antibody refers to immunoglobulins and can be isolated from various sources, such as murine, human, recombinant etc. In its broadest sense it includes monoclonal antibodies, polyclonal antibodies, multispecific antibodies and antibody fragments. It also includes truncated antibodies, chimeric, humanized or pegylated antibodies, isotypes, allotypes and alleles of immunoglobulin genes and fusion proteins, which contain an immunoglobulin moiety.
  • impurities refers to a material that is different from the desired polypeptide. They may be nucleic acids such as host cell DNA, host cell proteins, variants of the desired polypeptide, another polypeptide, viruses, endotoxin etc.
  • flow-through mode refers to a process wherein the desired protein is not bound to a chromatographic resin but is instead obtained in the unbound or "flow-through" fraction during loading or post loading washes of a chromatography support.
  • the desired protein in the flow-through can be collected as various fractions and pooled together or can be collected as a single fraction.
  • neutral pH range refers to the pH range of about 7.0 to about 7.5.
  • the invention provides a method of the purification of an antibody comprising an anion exchange chromatography operated in flow-through mode, wherein the buffer solution used is in the pH range of about 7.0 to about 7.5.
  • the pH of the buffer is about 7.0.
  • the pH of the buffer is about 7.5.
  • the invention provides a method for the purification of an antibody wherein a cation exchange chromatography precedes or follows the said anion exchange chromatography.
  • a protein-A chromatography precedes the cation exchange and anion exchange chromatography.
  • inventions mentioned herein may optionally include one or more tangential flow filtration, concentration, diafiltration or ultra filtration steps.
  • inventions mentioned herein optionally include one or more viral inactivation steps or sterile filtration or nano filtration steps.
  • the embodiments mentioned herein may include one or more neutralization steps.
  • the protein A chromatographic resin used may be any protein A or variant or a functional fragment thereof coupled to any chromatographic support.
  • the protein A resin is MabselectTM (GE-Healthcare Life sciences), an affinity matrix with recombinant Protein-A ligand.
  • the resin is made of highly cross- linked agarose matrix.
  • Cation exchange chromatographic step mentioned in the embodiments may be carried out using any weak or strong cation exchange chromatographic resin or a membrane, which could function as a weak or a strong cation exchanger.
  • Commercially available cation exchange support include a resin, but are not limited to, those having a sulfonate based group e.g., MonoS, MiniS, Source 15S and 30S, SP Sepharose Fast Flow, SP Sepharose High Performance from GE Healthcare, Toyopearl SP-650S and SP-650M from Tosoh, S-Ceramic Hyper D, from Pall Corporation or a carboxymethyl based group e.g., CM Sepharose Fast Flow from GE Healthcare, Macro-Prep CM from BioRad, CM-Ceramic Hyper D, from Pall Corporation, Toyopearl CM-650S, CM-650M and CM-650C from Tosoh.
  • a resin but are not limited to, those having
  • a strong cation exchange resin such as SP- Sepharose ® (GE Healthcare Life Sciences) is used. This resin is made using a highly cross-linked, 6 % agarose matrix attached to a sulfopropyl functional group.
  • Anion exchange chromatography mentioned in the embodiments may be carried out using any weak or strong anion exchange chromatographic resin or a membrane, which could function as a weak or a strong anion exchanger.
  • Commercially available anion exchange resins include, but are not limited to, DEAE cellulose, Poros PI 20, PI 50, HQ 10, HQ 20, HQ 50, D 50 from Applied Biosystems, MonoQ, MiniQ, Source 15Q and 3OQ, Q, DEAE and ANX Sepharose Fast Flow, Q Sepharose high Performance, QAE SEPHADEX and FAST Q SEPHAROSE from GE Healthcare, Macro-Prep DEAE and Macro-Prep High Q from Biorad, Q-Ceramic Hyper D, DEAE-Ceramic Hyper D, from Pall Corporation.
  • a strong anion exchange resin such as Q- Sepharose Fast Flow ® (GE Healthcare Life Sciences) is used.
  • This resin is made using a highly cross-linked, 6 % agarose matrix attached to -O-CH 2 CHOHCH2OCH2CHOHCH 2 N + (CH3)3 functional group.
  • the anion exchange chromatography could be carried out using a monolithic column, disk or tubular, that functions as an anion exchanger.
  • buffering agents used in the buffer solutions include, but are not limited to, TRIS, phosphate, citrate, acetate, succinate, MES, MOPS, or ammonium and their salts or derivatives thereof.
  • An anti-CD20 antibody was cloned and expressed in a CHO cell line as described in U.S. Patent No. 7,381 ,560, which is incorporated herein by reference.
  • the cell culture broth containing the expressed antibody was harvested, clarified and subjected to protein A affinity chromatography as described below.
  • the clarified cell culture broth was loaded onto a protein A chromatography column (Mabselect, VL44x250, 205 mL) that was pre-equilibrated with Tris buffer solution (pH 7.0). The column was then washed with equilibration buffer. This was followed by a wash with Tris buffer (pH 7.0) with higher conductivity and a final wash with citrate buffer at pH 5. The bound antibody was eluted using citrate buffer, pH 2.5 - 3.5.
  • Example 2 The eluate obtained from the protein A chromatography procedure described in Example 1 was loaded onto a cation exchange resin (SP Sepharose, VL44x250, 304 mL) pre-equilibrated with Tris buffer (pH 7.5). This was followed by washing the resin with a wash buffer of Tris buffer (pH 7.5). A second wash step was performed with wash buffer consisting of citrate buffer, pH 6.5. The bound antibody was eluted using a buffer of citrate buffer, pH 6.5 at conductivity between 9-12 mS/cm.
  • a cation exchange resin SP Sepharose, VL44x250, 304 mL
  • the eluate obtained from the cation exchange chromatography procedure described in Example 2 was loaded onto an anion exchange resin (Q-Sepharose FF, VL32x250, 80 mL) pre-equilibrated with 5 column volume of an equilibration buffer (40 mM Tris buffer, pH 7.5, at a conductivity value of 3.0 to 6.0 mS/cm). This was followed by a post load wash with the equilibration buffer and the load and wash flow-through was collected.
  • an anion exchange resin Q-Sepharose FF, VL32x250, 80 mL
  • Nanofiltered filtrate composition may then be collected and used.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Analytical Chemistry (AREA)
  • Immunology (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention concerne un procédé permettant de purifier des anticorps par chromatographie. Le procédé consiste à utiliser la chromatographie à échange d'anions pour purifier l'anticorps. l'anticorps purifié peut être utilisée en tant que compositions thérapeutiques.
PCT/IB2012/052610 2011-05-26 2012-05-24 Purification d'anticorps WO2012160536A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
IN1782CH2011 2011-05-26
IN1782/CHE/2011 2011-05-26

Publications (1)

Publication Number Publication Date
WO2012160536A1 true WO2012160536A1 (fr) 2012-11-29

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015001504A3 (fr) * 2013-07-04 2015-08-06 Prothena Biosciences Limited Formulations d'anticorps et procédés correspondants
US9217030B2 (en) 2012-01-27 2015-12-22 Prothena Biosciences Limited Humanized antibodies that recognize alpha-synuclein
US9556259B2 (en) 2011-10-28 2017-01-31 Prothena Biosciences Limited Humanized antibodies that recognize alpha-synuclein
US9605056B2 (en) 2012-10-08 2017-03-28 Prothena Biosciences Limited Antibodies recognizing alpha-synuclein
US10329323B2 (en) * 2014-07-25 2019-06-25 The United States Of America, As Represented By The Secretary, Department Of Health & Human Services Method for purifying antibodies using PBS
US10562973B2 (en) 2014-04-08 2020-02-18 Prothena Bioscience Limited Blood-brain barrier shuttles containing antibodies recognizing alpha-synuclein
US11155575B2 (en) 2018-03-21 2021-10-26 Waters Technologies Corporation Non-antibody high-affinity-based sample preparation, sorbent, devices and methods

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1771260A (zh) * 2003-02-28 2006-05-10 英国龙沙生物医药股份有限公司 用蛋白质a和离子交换层析来纯化抗体
CN101454025A (zh) * 2006-04-05 2009-06-10 艾博特生物技术有限公司 抗体纯化
WO2010141039A1 (fr) * 2008-10-20 2010-12-09 Abbott Laboratories Isolement et purification d'anticorps par chromatographie d'affinité de la protéine a

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1771260A (zh) * 2003-02-28 2006-05-10 英国龙沙生物医药股份有限公司 用蛋白质a和离子交换层析来纯化抗体
CN101454025A (zh) * 2006-04-05 2009-06-10 艾博特生物技术有限公司 抗体纯化
WO2010141039A1 (fr) * 2008-10-20 2010-12-09 Abbott Laboratories Isolement et purification d'anticorps par chromatographie d'affinité de la protéine a

Cited By (24)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10723792B2 (en) 2011-10-28 2020-07-28 Prothena Biosciences Limited Humanized antibodies that recognize alpha-synuclein
US9884906B2 (en) 2011-10-28 2018-02-06 Prothena Biosciences Limited Humanized antibodies that recognize alpha-synuclein
US10450369B2 (en) 2011-10-28 2019-10-22 Prothena Biosciences Limited Humanized antibodies that recognize alpha-synuclein
US11345749B2 (en) 2011-10-28 2022-05-31 Prothena Biosciences Limited Humanized antibodies that recognize alpha-synuclein
US9556259B2 (en) 2011-10-28 2017-01-31 Prothena Biosciences Limited Humanized antibodies that recognize alpha-synuclein
US10597441B2 (en) 2012-01-27 2020-03-24 Prothena Biosciences Limited Humanized antibodies that recognize alpha-synuclein
US9217030B2 (en) 2012-01-27 2015-12-22 Prothena Biosciences Limited Humanized antibodies that recognize alpha-synuclein
US9670273B2 (en) 2012-01-27 2017-06-06 Prothena Biosciences Limited Humanized antibodies that recognize alpha-synuclein
US10875909B2 (en) 2012-01-27 2020-12-29 Prothena Biosciences Limited Humanized antibodies that recognize alpha-synuclein
US9234031B2 (en) 2012-01-27 2016-01-12 Prothena Biosciences Limited Humanized antibodies that recognize alpha-synuclein
US9605056B2 (en) 2012-10-08 2017-03-28 Prothena Biosciences Limited Antibodies recognizing alpha-synuclein
US10081674B2 (en) 2012-10-08 2018-09-25 Prothena Biosciences Limited Antibodies recognizing α-synuclein
US10301382B2 (en) 2012-10-08 2019-05-28 Prothena Biosciences Limited Antibodies recognizing alpha-synuclein
US10875910B2 (en) 2012-10-08 2020-12-29 Prothena Biosciences Limited Antibodies recognizing alpha-synuclein
US10669331B2 (en) 2012-10-08 2020-06-02 Prothena Biosciences Limited Antibodies recognizing α-synuclein
CN105492019A (zh) * 2013-07-04 2016-04-13 普罗塞纳生物科学有限公司 抗体制剂和方法
US10513555B2 (en) 2013-07-04 2019-12-24 Prothena Biosciences Limited Antibody formulations and methods
WO2015001504A3 (fr) * 2013-07-04 2015-08-06 Prothena Biosciences Limited Formulations d'anticorps et procédés correspondants
CN105492019B (zh) * 2013-07-04 2020-02-11 普罗塞纳生物科学有限公司 抗体制剂和方法
EP3524264A1 (fr) * 2013-07-04 2019-08-14 Prothena Biosciences Limited Procédés pour le purification d'anticorps
US10562973B2 (en) 2014-04-08 2020-02-18 Prothena Bioscience Limited Blood-brain barrier shuttles containing antibodies recognizing alpha-synuclein
US10906935B2 (en) 2014-07-25 2021-02-02 United Therapeutics Corporation Method for purifying antibodies
US10329323B2 (en) * 2014-07-25 2019-06-25 The United States Of America, As Represented By The Secretary, Department Of Health & Human Services Method for purifying antibodies using PBS
US11155575B2 (en) 2018-03-21 2021-10-26 Waters Technologies Corporation Non-antibody high-affinity-based sample preparation, sorbent, devices and methods

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