WO2012160530A1 - Purification of antibodies - Google Patents
Purification of antibodies Download PDFInfo
- Publication number
- WO2012160530A1 WO2012160530A1 PCT/IB2012/052594 IB2012052594W WO2012160530A1 WO 2012160530 A1 WO2012160530 A1 WO 2012160530A1 IB 2012052594 W IB2012052594 W IB 2012052594W WO 2012160530 A1 WO2012160530 A1 WO 2012160530A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- buffer
- exchange chromatography
- process according
- antibody
- wash
- Prior art date
Links
Classifications
-
- 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
- C07K1/165—Extraction; Separation; Purification by chromatography mixed-mode chromatography
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
- A61K39/39591—Stabilisation, fragmentation
-
- 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
- C07K1/20—Partition-, reverse-phase or hydrophobic interaction 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
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2887—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD20
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Medicinal Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Molecular Biology (AREA)
- Genetics & Genomics (AREA)
- Analytical Chemistry (AREA)
- Immunology (AREA)
- Animal Behavior & Ethology (AREA)
- Veterinary Medicine (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Public Health (AREA)
- Microbiology (AREA)
- Epidemiology (AREA)
- Pharmacology & Pharmacy (AREA)
- Engineering & Computer Science (AREA)
- Mycology (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention provides a method of purification of antibodies using chromatographic technique. The method involves the use of cation -exchange chromatography for the purification of the antibody. The purified antibody can be used as a therapeutic composition.
Description
PURIFICATION OF ANTIBODIES
FIELD OF THE INVENTION
The present invention relates to a method of purification of antibodies comprising a cation exchange chromatography step.
BACKGROUND OF THE INVENTION
Large-scale purification of proteins remains a significant challenge in the biopharmaceutical industry as efficient and cost-effective methods are required to achieve desired yields and purity levels. Therapeutic proteins are primarily products of recombinant DNA technology, i.e., cloning and expression of a heterologus gene in prokaryotic or eukaryotic systems. However, proteins expressed by recombinant DNA methods are typically associated with impurities such as host cell proteins (HCP), host cell DNA (HCD), viruses, etc. Also, there is significant heterogeneity in the expression of the desired protein, in the form of charged variants (typically acidic, lower pi variants and basic, higher pi variants). Further, multimeric proteins, such as antibodies, have a higher tendency to aggregate, contributing to significantly increased impurity levels.
The presence of these impurities, including aggregates and undesirable charged variants, is a potential health risk, and hence their removal from a final product is a regulatory requirement. Thus drug regulatory agencies such as United States Food and Drug administration (FDA) require that biopharmaceuticals be free from impurities, both product related (aggregates or degradation products) and process related (media components, HCP, DNA, chromatographic media used in purification, endotoxins, viruses, etc). See, Office of Biologies Research and Review, Food and Drug Administration, Points to consider in the production and testing of new drugs and biologicals produced by recombinant DNA technology (Draft), 1985. Thus, elimination of impurities from a final product is mandatory and poses a significant challenge in the development of methods for the purification of therapeutic proteins.
Antibodies constitute one of the most important classes of therapeutic proteins, especially in the areas of oncology, arthritis and other chronic diseases. The prior art discloses various methods for purification of crude or partially purified antibodies. WO 89/05157 teaches purification of immunoglobulins by cation-
exchange chromatography using varying pH and salt concentrations in the wash and elution steps. 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.
However the prior art typically describe the use of low pH wash conditions that may be accompanied by frequent and significant change in pH conditions during load/wash/elution steps. Such frequent alterations of pH conditions during
chromatography may result in considerable reduction of antibody yield and, further, may decrease the stability of the antibody. Moreover frequent pH changes during chromatography pose difficulties in scale up.
Given the cumbersome nature of the conditions described in the prior art, alternative methods for purification of antibodies that alleviate some of the difficulties described above is desirable. The principle object of the present invention is to provide an improved method for obtaining antibody preparations that avoids use of low pH conditions and significantly reduces alterations in pH during a
chromatographic step resulting in effective separation of charge variants, increased purity and recovery of the desired antibody.
SUMMARY OF THE INVENTION
The present invention describes a process for the purification of antibodies from a mixture of impurities using cation exchange chromatography wherein the pH of a first wash buffer is similar to the pH of the load buffer, and pH of a second wash buffer is similar to the pH of the elution buffer.
BRIEF DESCRIPTION OF THE DRAWINGS
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 is an illustration of a chromatogram from the procedure as described in Example 2. The consistency of elution in multiple runs is shown.
Fig. 4 is an illustration of a chromatogram from the procedure as described in Example 3. Figure represents the flow- through fraction of the anion exchange chromatography. DETAILED DESCRIPTION OF THE INVENTION
The present invention describes a process for purification of antibodies by cation exchange chromatography. The chromatographic conditions require minimal pH adjustments during load/wash/elution steps. The process described in the present invention also avoids use of low pH buffers. The conditions described in the present invention results in effective separation of charged variants, as well as removal of impurities such as HCP, aggregates and Protein A leachates and an optimum yield of the desired antibody.
The term "antibody" as used herein 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.
The term "impurities" as used herein 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, endotoxin etc.
The term "low pH" as used herein refers to a pH of less than 6.0
The term "load buffer" as used herein refers to the buffer that is used to load the composition comprising the antibody of interest and one or more impurities onto the ion exchange support.
The term "wash buffer" as used herein refers to a buffer that is used to wash or re-equilibrate the ion exchange support, or to elute one or more impurities from the ion exchange support, prior to elution of the antibody of interest.
The "elution buffer" is used to elute the antibody of interest from the ion exchange support.
In an embodiment, the invention provides a method of the purification of antibodies comprising,
a) Loading the antibody containing solution onto a cation exchange support at a particular pH
b) Washing the support with a first wash buffer at a pH similar to the pH of the load buffer
c) Washing the support with a second wash buffer at a second pH
d) Eluting the antibody from the support using an elution buffer at a pH similar to the second wash buffer,
wherein the pH of the two wash buffers are greater than 6.
In an embodiment, the pH of the second wash buffer is less than the pH of the first wash buffer.
In another embodiment, the invention provides a method for the purification of antibodies comprising,
a) Loading the antibody containing solution onto a cation exchange support with a buffer of pH value from about pH 6.0 to about pH 8.0
b) Washing the support with a wash buffer of pH value from about pH 6.0 to about pH 8.0
c) Washing the support with a second wash buffer of pH about 6.5
d) Eluting the antibody from the support using an elution buffer of pH about 6.5 In an embodiment, a protein-A chromatography, may precede the cation exchange chromatography.
In an embodiment, another ion-exchange chromatography or a hydrophobic interaction chromatography may precede or follow the cation exchange
chromatography.
The embodiments mentioned herein may optionally include one or more tangential flow filtration, concentration, diafiltration or ultra filtration steps.
The embodiments 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. In
embodiments, the protein A resin is Mabselect™ (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.
Alternatively, the support could be a monolithic column, disk or tubular, that performs the function of a cation exchanger. In embodiments of the invention, 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. In embodiments of the invention, a strong anion exchange resin, such as Q- Sepharose Fast Flow® (GE Healthcare Life Sciences) is used. This resin is made of highly cross-linked, 6 % agarose matrix attached to -O-CH2CHOHCH2OCH2CHOHCH2N+(CH3)3 functional group.
Examples of 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.
The invention is more fully understood by reference to the following examples. These examples should not, however, be construed as limiting the scope of the invention.
EXAMPLE 1
Protein A chromatography
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
Cation exchange chromatography
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) at a conductivity of 3.0 to 6.0 mS/cm. This was followed by washing the resin with a wash buffer of Tris buffer (pH 7.5) at a conductivity of 3.0 to 6.0 mS/cm. A second wash step was performed with wash buffer consisting of citrate buffer, pH 6.5, at a conductivity of 3.0 to 6.0. The bound antibody was eluted using a buffer of citrate buffer, pH 6.5 at conductivity between 9-12 mS/cm.
EXAMPLE 3
Anion exchange chromatography
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 a Tris buffer (pH 7.5) equilibration buffer.
This was followed by a post load wash with equilibration buffer and the load and wash flow-through was collected.
Table 1 :
BDL: Below Detection Limit
Table 2:
K0: Species devoid of the C-terminal lysine residue
BDL: Below Detection Limit
Claims
1. A process for purification of an antibody, comprising, a) loading the antibody containing solution onto a cation exchange chromatography support at a particular pH
b) washing the support with a first wash buffer at a pH similar to the pH of the load buffer
c) washing the support with a second wash buffer at a second pH and
d) eluting the antibody from the support with an elution buffer at a pH similar to the second wash buffer,
wherein the pH of the two wash buffers are greater than 6.
2. A process according to claim 1 , wherein the pH value of the first wash buffer is from about pH 6.0 to about pH 8.0
3. A process according to claim 2, wherein the pH value is from about pH 6.5 to about pH 7.5.
4. A process according to claim 3, wherein the pH value is about pH 7.5.
5. A process according to claim 1 , wherein the pH value of the second wash buffer is about 6.5.
6. A process according to claim 1 , wherein the pH value of the elution buffer is about 6.5.
7. A process according to claim 1 , wherein the cation-exchange
chromatography is preceded by a protein-A affinity chromatography step.
8. A process according to claim 1 , wherein the cation-exchange
chromatography is preceded by an anion exchange chromatography step or a hydrophobic interaction chromatography step.
9. A process according to claim 1 , wherein the cation- exchange
chromatography step is followed by an anion exchange chromatography step or a hydrophobic interaction chromatography step.
10. A process according to claim 1 , wherein the cation- exchange chromatography step is preceded and/or followed by one or more ion exchange chromatography steps.
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US14/118,813 US20140107321A1 (en) | 2011-05-26 | 2012-05-24 | Purification of antibodies |
EP12790188.2A EP2714713B1 (en) | 2011-05-26 | 2012-05-24 | Purification of anti-cd20 antibodies |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
IN1783CH2011 | 2011-05-26 | ||
IN1783/CHE/2011 | 2011-05-26 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2012160530A1 true WO2012160530A1 (en) | 2012-11-29 |
Family
ID=47216681
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/IB2012/052594 WO2012160530A1 (en) | 2011-05-26 | 2012-05-24 | Purification of antibodies |
Country Status (3)
Country | Link |
---|---|
US (1) | US20140107321A1 (en) |
EP (1) | EP2714713B1 (en) |
WO (1) | WO2012160530A1 (en) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2014118685A3 (en) * | 2013-01-29 | 2014-12-31 | Dr. Reddy's Laboratories Limited | Method of altering the acidic variant content of antibody |
EP3643322A1 (en) * | 2018-10-26 | 2020-04-29 | Mabion SA | Low aggregate anti cd20 ligand formulation |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN111902720A (en) | 2018-03-21 | 2020-11-06 | 沃特世科技公司 | Non-antibody high affinity based sample preparation, adsorbents, devices and methods |
Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2001032878A2 (en) * | 1999-11-04 | 2001-05-10 | University College London | A novel polypeptide hormone phosphatonin |
WO2002012455A1 (en) * | 2000-08-07 | 2002-02-14 | Avigen, Inc. | LARGE-SCALE RECOMBINANT ADENO-ASSOCIATED VIRUS (rAAV) PRODUCTION AND PURIFICATION |
WO2004024866A2 (en) * | 2002-09-11 | 2004-03-25 | Genentech, Inc. | Protein purification |
US7381560B2 (en) | 1992-11-13 | 2008-06-03 | Biogen Idec Inc. | Expression and use of anti-CD20 antibodies |
WO2009058812A1 (en) * | 2007-10-30 | 2009-05-07 | Genentech, Inc. | Antibody purification by cation exchange chromatography |
WO2011009623A1 (en) * | 2009-07-24 | 2011-01-27 | F. Hoffmann-La Roche Ag | Optimizing the production of antibodies |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR20090005315A (en) * | 2006-04-05 | 2009-01-13 | 애보트 바이오테크놀로지 리미티드 | Antibody purification |
WO2011037983A1 (en) * | 2009-09-23 | 2011-03-31 | Medarex, Inc. | Cation exchange chromatography |
-
2012
- 2012-05-24 EP EP12790188.2A patent/EP2714713B1/en active Active
- 2012-05-24 US US14/118,813 patent/US20140107321A1/en not_active Abandoned
- 2012-05-24 WO PCT/IB2012/052594 patent/WO2012160530A1/en active Application Filing
Patent Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7381560B2 (en) | 1992-11-13 | 2008-06-03 | Biogen Idec Inc. | Expression and use of anti-CD20 antibodies |
WO2001032878A2 (en) * | 1999-11-04 | 2001-05-10 | University College London | A novel polypeptide hormone phosphatonin |
WO2002012455A1 (en) * | 2000-08-07 | 2002-02-14 | Avigen, Inc. | LARGE-SCALE RECOMBINANT ADENO-ASSOCIATED VIRUS (rAAV) PRODUCTION AND PURIFICATION |
WO2004024866A2 (en) * | 2002-09-11 | 2004-03-25 | Genentech, Inc. | Protein purification |
WO2009058812A1 (en) * | 2007-10-30 | 2009-05-07 | Genentech, Inc. | Antibody purification by cation exchange chromatography |
WO2011009623A1 (en) * | 2009-07-24 | 2011-01-27 | F. Hoffmann-La Roche Ag | Optimizing the production of antibodies |
Non-Patent Citations (1)
Title |
---|
See also references of EP2714713A4 * |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2014118685A3 (en) * | 2013-01-29 | 2014-12-31 | Dr. Reddy's Laboratories Limited | Method of altering the acidic variant content of antibody |
EP3643322A1 (en) * | 2018-10-26 | 2020-04-29 | Mabion SA | Low aggregate anti cd20 ligand formulation |
Also Published As
Publication number | Publication date |
---|---|
US20140107321A1 (en) | 2014-04-17 |
EP2714713A4 (en) | 2014-10-29 |
EP2714713B1 (en) | 2018-05-09 |
EP2714713A1 (en) | 2014-04-09 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US20130178608A1 (en) | Protein purification by ion exchange | |
US20200282332A1 (en) | Use of alkaline washes during chromatography to remove impurities | |
WO2012160536A1 (en) | Antibody purification | |
AU2017204893A1 (en) | Method for purifying Fc-fusion protein | |
EP2480561B1 (en) | Cation exchange chromatography | |
US20140128577A1 (en) | Purification of chimeric protein | |
US20200283472A1 (en) | A process for purification of fc-fusion proteins | |
WO2013158279A1 (en) | Protein purification methods to reduce acidic species | |
US20180094023A1 (en) | Method for separation of monomeric polypeptides from aggregated polypeptides | |
MX2009002014A (en) | Process for the purification of fc-containing proteins. | |
US20130116413A1 (en) | Purification of proteins | |
US20210054024A1 (en) | Use of caprylic acid precipitation for protein purification | |
CN112313248A (en) | Method for purifying monomeric monoclonal antibodies | |
EP2714713B1 (en) | Purification of anti-cd20 antibodies | |
WO2014102814A1 (en) | Process for the purification of fc fusion proteins | |
WO2013054250A1 (en) | Purification method | |
Zhao et al. | Applications of ion-exchange chromatography for the purification of antibodies | |
KR20230132470A (en) | Protein compositions and methods of producing and using them |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 12790188 Country of ref document: EP Kind code of ref document: A1 |
|
WWE | Wipo information: entry into national phase |
Ref document number: 14118813 Country of ref document: US |
|
NENP | Non-entry into the national phase |
Ref country code: DE |