WO2013102822A1 - Filtration method - Google Patents
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- WO2013102822A1 WO2013102822A1 PCT/IB2012/057547 IB2012057547W WO2013102822A1 WO 2013102822 A1 WO2013102822 A1 WO 2013102822A1 IB 2012057547 W IB2012057547 W IB 2012057547W WO 2013102822 A1 WO2013102822 A1 WO 2013102822A1
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- filtration
- protein
- purification
- chromatography
- antibody
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Classifications
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- 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
- C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/06—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from serum
- C07K16/065—Purification, fragmentation
-
- 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
- C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
-
- 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
Definitions
- the present invention relates to a method of purification of antibody from a composition comprising one or more impurities using depth filtration.
- WO 2008036899 describes a process of removing parvovirus from a therapeutic protein solution by using a depth filter where the protein containing solution is passed through at a pH within 1 pH unit of the isoelectric point (pi) of the virus.
- W01996000237 teaches a method of virus-filtering a macromolecule containing solution by maintaining total salt content in the range of from about 0.2 M to saturation.
- EP 1348445 explains a process for removing viruses from a protein solution by nanofiltration by adding a chaotropic substance to the solution before passing it through the filter.
- WO 2010059232 describes a process of inactivating virus and reducing the infectious titer by adding arginine.
- WO 2008051448 describes a method for reducing protein A contamination by contacting the protein containing solution with a charge-modified depth filter, wherein the depth filter has been pre treated to obtain a pre-treated charge-modified depth filter.
- WO 2000048703 teaches a process for purifying a target substance bound chromatography resin by using a cross-flow filtering technique wherein the chromatography resin(s) is circulated in the cross-flow filter.
- the methods described in the prior art involve selective filtering of virus or modifying a filter system for selective separation of impurities and/ or addition of chaotropic agents to remove contaminants.
- modification, pre-treating of filter systems and/or the use of chaotropes or aggregation inhibitors adds to the complexity of the downstream process.
- the objective of the current invention is to provide an improved method of purification of antibodies with increased clearance of HCD, HCP, Protein A leachates and other impurities using a filtration step.
- Another objective of this invention is to perform the said filtration step before an ion exchange step to remove aggregates such as dimers from the antibody containing sample. This in turn would facilitate loading of the sample onto a subsequent ion-exchange resin and the purification process thereby.
- the present invention discloses a method of purification of antibody containing sample from one or more impurities such as host cell contaminants and aggregates by using a filtration step.
- this invention employs the use of a filtration step prior to an ion exchange chromatography.
- Fig. 1 is an illustration of a chromatogram from the procedure as described example 3.
- the line marked “Cond” represents the increase in conductivity in mS/cm.
- Peak “A” represents the antibody of interest.
- the present invention discloses a method of purification of antibody containing sample from one or more impurities such as host cell contaminants and aggregates by using a filtration step performed prior to an ion exchange step.
- the filtration step mentioned in the embodiment is depth filtration.
- the invention provides a method for the purification of antibodies comprising a filtration step performed before an ion exchange
- the filtration step is performed at a pH of about
- the filtration step may be preceded by Protein-A chromatography.
- the invention provides a method for the purification of antibodies comprising steps of;
- the invention provides a method for the purification of antibodies comprising steps of;
- the invention provides a method for the purification of antibodies comprising steps of; a) Protein-A chromatography
- the invention provides a method for the purification of antibodies comprising steps of;
- the invention provides a method for the purification of antibodies comprising steps of;
- the cation exchange chromatography may be followed by an anion exchange chromatography.
- the protein-A chromatographic resin used may be any protein A or variant or a functional fragment thereof coupled to any chromatographic support.
- Protein A chromatography mentioned in the embodiments may be carried out on columns that are available commercially including ProSep® controlled-pore glass resins produced by Millipore and MabSelectTM, cross-linked agarose resin products produced by Amersham Biosciences, and other types of protein-A affinity chromatography resins, including gel-based resins and silica-based resins.
- protein A resin used is Prosep® VA ultra column (Millipore).
- fresh (i.e. not used before) protein A chromatographic resin may be used to obtain a feed stream for the second chromatographic step.
- sample as used herein comprises the target protein (or antibody of interest) and one or more impurities.
- the composition or sample may be "partially purified” (i.e., having been subjected to one or more purification steps) or may be obtained directly from a host cell or organism producing the polypeptide (e.g., the composition may comprise harvested cell culture fluid).
- impurity refers to material(s) that is different from the desired protein or antibody product. They include, without limitation: host cell materials, such as Chinese Hamster Ovary Proteins (CHOP); leached protein A; nucleic acid(s); a variant, fragment, aggregates or derivative of the desired antibody; another polypeptide; endotoxin; viral contaminant; cell culture media component, etc.
- host cell materials such as Chinese Hamster Ovary Proteins (CHOP); leached protein A; nucleic acid(s); a variant, fragment, aggregates or derivative of the desired antibody; another polypeptide; endotoxin; viral contaminant; cell culture media component, etc.
- a “cation exchange resin” mentioned in the embodiments refers to a solid phase which has a negatively charged ligand such as a carboxylate or sulfonate attached thereto.
- the cation exchange resin can be of any weak or strong cation exchange resin or a membrane which could function as a weak or a strong cation exchanger.
- cation exchange resins include, 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 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
- a strong cation exchange resin such as POROS HS® (Applied Biosystems) is used; the resin is made up of cross-linked poly(styrene- divinylbenzene) flow-through particles surface coated with a polyhydroxylated polymer functionalized with sulfopropyl.
- Depth filter (also referred to as “prefilter” or “media filter”) as used herein, refers to a filter comprising a porous medium that is capable of retaining particles from the mobile liquid phase throughout the filter rather than just on the surface of the filter.
- Depth filters are often composed of a fibrous bed of, for example, cellulose or polypropylene fibers, along with a filter aid or "matrix”, e.g., paper, glass fibers, nylon, polyolefin, carbon, ceramics, diatomaceous earth, or cellulose, which provides a high surface area to the filter.
- Depth filtration mentioned in the embodiments may be carried out by any commercially available depth filters for example Celite (World Minerals, Lompoc, Calif.), Millipore filters (Millipore Corporation, Bedford, Mass.), and Cuno filters (Cuno Corporation, Meriden, Conn.)
- the filter used in the instant invention is Millistak® (COHC or A1 HCseries) Pod Disposable Depth Filter System from Millipore, though the invention may be carried out with filters/membranes that work with similar principle.
- Anti-VEGF antibody was cloned and expressed in a CHO cell line as described in U.S. Patent No. 7,060,269, 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 (Prosep® Ultra) that was pre-equilibrated with Tris buffer solution (pH 7.0).
- the column was then washed with equilibration buffer, followed by a 2nd wash with Tris buffer (pH 7.0) of high conductivity which includes EDTA (pH 5.5), followed by a 3rd wash with Tris acetate buffer (pH 7.0).
- the bound antibody was eluted using 300 mM Tris acetate buffer at a pH of 3.3.
- the protein A eluatefrom example 1 comprising anti-VEGF antibody, was neutralized with 1 M Tris to attain a pH of 5.5 - 6.5 and then passed through a depth Millistak® (C0HC series) at a conductivity of about 6mS/cm to about 8mS/cm.
- a depth Millistak® C0HC series
- HCP Host Cell Protein
- HCD Host Cell DNA
- Protein A Leachates and Dimer content in the eluate before and after depth filtration were then estimated.
- Table 1 Impurity clearance from anti-VEGF antibody comprising sample by Depth Filtration
- the eluate obtained after filtration in example 2 was loaded onto a cation exchange resin (POROS HS 50, VL32X250, 1 60 ml) pre-equilibrated with 50 mM phosphate buffer, pH 6.2, followed by washing the resin with a 60 mM phosphate buffer and eluting the antibody with 90mM phosphate buffer at pH 6.2.
- a cation exchange resin POROS HS 50, VL32X250, 1 60 ml
- Anti-CD20 antibody was cloned and expressed in 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 in Example 1 .
- the protein A eluate comprising anti-CD20 antibody was neutralized with 1 M Tris to pH about 7.0 and then passed through a depth filter Millistak® (COHC series) with a 40 mM Tris buffer at a pH of about 7.0 and at a conductivity of about 3 mS/cm to about 9 mS/cm.
- COHC series depth filter Millistak®
- HCD Host Cell DNA
Abstract
Provided is a method of purification of antibodies from a sample comprising one or more impurities such as host cell contaminants and aggregates, using a filtration step. In particular, the method comprises the step of using of a depth filtration prior to an ion-exchange chromatography.
Description
FILTRATION METHOD
RELATED APPLICATION
This application is related to and takes priority from Indian Provisional application 30/CHE/2012 filed 3 January 2012 and is herein incorporated in its entirety.
FIELD OF THE INVENTION
The present invention relates to a method of purification of antibody from a composition comprising one or more impurities using depth filtration.
BACKGROUND OF THE INVENTION
The design of efficient and economic purification methods for polypeptides produced by recombinant DNA technology is a major challenge in bringing new biopharmaceuticals to the market. Proteins expressed by recombinant DNA methods are typically associated with impurities such as host cell proteins (HCP), host cell DNA (HCD), viruses and aggregate formation. The presence of these impurities is a potential health risk, and hence their removal from the final product is a regulatory requirement and creates a significant challenge in the development of methods for the purification of therapeutic proteins
The challenges of purification vary depending upon the source and location of the product. Regardless of the source (such as expression system type), a purification process normally follows the capture-intermediate purification-polishing approach to attain a purified product. However each chromatographic step requires optimization of several process conditions such as, pH, buffer type, salt type, concentrations etc, Further, optimizing purification protocols can be time consuming, expensive and may result in significant product losses. Considering the cumbersome nature of the steps involved, alternatives that alleviate the complexity of the process are desirable. One such alternative is to incorporate a simple and inexpensive method of filtration in the purification process.
The prior art discloses various methods for purification of proteins by using filtration. WO 2008036899 describes a process of removing parvovirus from a therapeutic protein solution by using a depth filter where the protein containing
solution is passed through at a pH within 1 pH unit of the isoelectric point (pi) of the virus.
W01996000237 teaches a method of virus-filtering a macromolecule containing solution by maintaining total salt content in the range of from about 0.2 M to saturation.
EP 1348445 explains a process for removing viruses from a protein solution by nanofiltration by adding a chaotropic substance to the solution before passing it through the filter. WO 2010059232 describes a process of inactivating virus and reducing the infectious titer by adding arginine.
WO 2008051448 describes a method for reducing protein A contamination by contacting the protein containing solution with a charge-modified depth filter, wherein the depth filter has been pre treated to obtain a pre-treated charge-modified depth filter.
WO 2000048703 teaches a process for purifying a target substance bound chromatography resin by using a cross-flow filtering technique wherein the chromatography resin(s) is circulated in the cross-flow filter.
The methods described in the prior art involve selective filtering of virus or modifying a filter system for selective separation of impurities and/ or addition of chaotropic agents to remove contaminants. However modification, pre-treating of filter systems and/or the use of chaotropes or aggregation inhibitors adds to the complexity of the downstream process.
Hence, the objective of the current invention is to provide an improved method of purification of antibodies with increased clearance of HCD, HCP, Protein A leachates and other impurities using a filtration step. Another objective of this invention is to perform the said filtration step before an ion exchange step to remove aggregates such as dimers from the antibody containing sample. This in turn would facilitate loading of the sample onto a subsequent ion-exchange resin and the purification process thereby. SUMMARY OF THE INVENTION
The present invention discloses a method of purification of antibody containing sample from one or more impurities such as host cell contaminants and aggregates by using a filtration step. In particular this invention employs the use of a filtration step prior to an ion exchange chromatography.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 is an illustration of a chromatogram from the procedure as described example 3. The line marked "Cond" represents the increase in conductivity in mS/cm. Peak "A" represents the antibody of interest.
DETAILED DESCRIPTION OF THE INVENTION
The present invention discloses a method of purification of antibody containing sample from one or more impurities such as host cell contaminants and aggregates by using a filtration step performed prior to an ion exchange step.
The filtration step mentioned in the embodiment is depth filtration.
In an embodiment, the invention provides a method for the purification of antibodies comprising a filtration step performed before an ion exchange
chromatography.
In yet another embodiment, the filtration step is performed at a pH of about
5.0 to about 7.0.
In yet another embodiment the filtration step may be preceded by Protein-A chromatography.
In an embodiment, the invention provides a method for the purification of antibodies comprising steps of;
a) Protein-A chromatography
b) Depth Filtration
c) Ion-exchange chromatography
wherein the said filtration is performed at a pH of about 5.5 to 6.5 and at a
conductivity in the range of 6-8 mS/cm.
In another embodiment, the invention provides a method for the purification of antibodies comprising steps of;
a) Protein-A chromatography
b) Depth Filtration
c) Cation-exchange chromatography
wherein the said filtration is performed at a pH of about 5.5 to 6.5 and at a
conductivity in the range of 6-8 mS/cm.
In an embodiment, the invention provides a method for the purification of antibodies comprising steps of;
a) Protein-A chromatography
b) Depth Filtration
c) Ion-exchange chromatography
wherein the said filtration is performed at a pH of about 5.5 to about 6.5 and at a conductivity of about 3mS/cm to about 9mS/cm.
In another embodiment, the invention provides a method for the purification of antibodies comprising steps of;
a) Protein-A chromatography
b) Depth Filtration
c) Ion-exchange chromatography
wherein the said filtration is performed at a pH of about 5.5 to about 6.5 and at a conductivity of about 3mS/cm.
In another embodiment, the invention provides a method for the purification of antibodies comprising steps of;
a) Protein-A chromatography
b) Depth Filtration
c) Cation-exchange chromatography
wherein the said filtration is performed at a pH of about 5.5 to about 6.5 and at a conductivity of about 3 mS/cm.
In yet another embodiment the cation exchange chromatography may be followed by an anion exchange chromatography.
The protein-A chromatographic resin used may be any protein A or variant or a functional fragment thereof coupled to any chromatographic support. Protein A chromatography mentioned in the embodiments may be carried out on columns that are available commercially including ProSep® controlled-pore glass resins produced by Millipore and MabSelect™, cross-linked agarose resin products produced by Amersham Biosciences, and other types of protein-A affinity chromatography resins, including gel-based resins and silica-based resins. In the embodiments above protein A resin used is Prosep® VA ultra column (Millipore). For the purpose of the invention, fresh (i.e. not used before) protein A chromatographic resin may be used to obtain a feed stream for the second chromatographic step.
The term "sample" as used herein comprises the target protein (or antibody of interest) and one or more impurities. The composition or sample may be "partially purified" (i.e., having been subjected to one or more purification steps) or may be
obtained directly from a host cell or organism producing the polypeptide (e.g., the composition may comprise harvested cell culture fluid).
The term "impurity" or "impurities" as used herein refers to material(s) that is different from the desired protein or antibody product. They include, without limitation: host cell materials, such as Chinese Hamster Ovary Proteins (CHOP); leached protein A; nucleic acid(s); a variant, fragment, aggregates or derivative of the desired antibody; another polypeptide; endotoxin; viral contaminant; cell culture media component, etc.
A "cation exchange resin" mentioned in the embodiments refers to a solid phase which has a negatively charged ligand such as a carboxylate or sulfonate attached thereto. The cation exchange resin can be of any weak or strong cation exchange resin or a membrane which could function as a weak or a strong cation exchanger. Commercially available cation exchange resins include, 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. In embodiments of the invention, a strong cation exchange resin, such as POROS HS® (Applied Biosystems) is used; the resin is made up of cross-linked poly(styrene- divinylbenzene) flow-through particles surface coated with a polyhydroxylated polymer functionalized with sulfopropyl.
"Depth filter" (also referred to as "prefilter" or "media filter") as used herein, refers to a filter comprising a porous medium that is capable of retaining particles from the mobile liquid phase throughout the filter rather than just on the surface of the filter. Depth filters are often composed of a fibrous bed of, for example, cellulose or polypropylene fibers, along with a filter aid or "matrix", e.g., paper, glass fibers, nylon, polyolefin, carbon, ceramics, diatomaceous earth, or cellulose, which provides a high surface area to the filter. Depth filtration mentioned in the embodiments may be carried out by any commercially available depth filters for example Celite (World Minerals, Lompoc, Calif.), Millipore filters (Millipore Corporation, Bedford, Mass.), and Cuno filters (Cuno Corporation, Meriden, Conn.) The filter used in the instant invention is Millistak® (COHC or A1 HCseries) Pod Disposable Depth Filter System
from Millipore, though the invention may be carried out with filters/membranes that work with similar principle.
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
Anti-VEGF antibody was cloned and expressed in a CHO cell line as described in U.S. Patent No. 7,060,269, 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 (Prosep® Ultra) that was pre-equilibrated with Tris buffer solution (pH 7.0). The column was then washed with equilibration buffer, followed by a 2nd wash with Tris buffer (pH 7.0) of high conductivity which includes EDTA (pH 5.5), followed by a 3rd wash with Tris acetate buffer (pH 7.0). The bound antibody was eluted using 300 mM Tris acetate buffer at a pH of 3.3. EXAMPLE 2
Depth Filtration
The protein A eluatefrom example 1 , comprising anti-VEGF antibody,was neutralized with 1 M Tris to attain a pH of 5.5 - 6.5 and then passed through a depth Millistak® (C0HC series) at a conductivity of about 6mS/cm to about 8mS/cm.
Alternatively, filtration experiments performed with phosphate buffer at a pH of about 5.5 to about 6.5 and at a conductivity of about 3 mS/cm yielded similar results.
Host Cell Protein (HCP), Host Cell DNA (HCD), Protein A Leachates and Dimer content in the eluate before and after depth filtration were then estimated. Table 1 Impurity clearance from anti-VEGF antibody comprising sample by Depth Filtration
Impurity Clearance
HCP (ng/mg)
Average Batch 1 Batch 2 Batch 3 Batch 4
Protein A eluate 4.34 5.29 5.75 2.54 3.78
Cation exchange load 1 .18 1 .47 1 .19 0.89 1 .19 (depth filtration eluate)
HCD (ng/mg)
Average Batch 1 Batch 2 Batch 3 Batch 4
Protein A eluate 0.29 0.33 0.21 0.15 0.46
Cation exchange load 0.01 0.02 0 0 0.01 (depth filtration eluate)
Protein A Leachates (ng/mg)
Average Batch 1 Batch 2 Batch 3 Batch 4
Protein A eluate 15.91 23.94 15.38 13.31 1 1 .01
Cation exchange load 4.86 8.53 2.03 5.09 3.79 (depth filtration eluate)
Dimer (%)
Average Batch 1 Batch 2 Batch 3 Batch 4
Protein A eluate 3.14 3.34 3.45 2.97 2.79
Cation exchange load 2.61 2.66 2.76 2.52 2.49 (depth filtration eluate)
Table 2 Monomer recovery from anti-VEGF antibody comprising sample by Depth Filtration
EXAMPLE 3
Cation exchange chromatography
The eluate obtained after filtration in example 2was loaded onto a cation exchange resin (POROS HS 50, VL32X250, 1 60 ml) pre-equilibrated with 50 mM
phosphate buffer, pH 6.2, followed by washing the resin with a 60 mM phosphate buffer and eluting the antibody with 90mM phosphate buffer at pH 6.2.
EXAMPLE 4
The steps of protein-A, depth filtration and cation exchange chromatography were performed with anti-CD20 antibody, using similar conditions.
Anti-CD20 antibody was cloned and expressed in 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 in Example 1 .
The protein A eluate comprising anti-CD20 antibody, was neutralized with 1 M Tris to pH about 7.0 and then passed through a depth filter Millistak® (COHC series) with a 40 mM Tris buffer at a pH of about 7.0 and at a conductivity of about 3 mS/cm to about 9 mS/cm.
Host Cell DNA (HCD) content in the eluate before and after depth filtration was then estimated.
Table 3 HCD clearance from anti-CD20 antibody comprising sample by Depth Filtration
The eluate obtained after depth filtration was subjected to cation exchange chromatography as described in Example 3.
Claims
1. A method of purification of antibody from a sample comprising one or more impurities, comprising a filtration step performed before an ion exchange chromatography, wherein the said filtration step is depth filtration and is performed using a buffer at a pH of about 5.0 to about 7.0.
2. A method of purification of antibody from a sample comprising one or more impurities, comprising steps of:
a) Protein-A chromatography
b) Depth Filtration
c) Ion-exchange chromatography
wherein the said filtration is performed using a buffer at a pH of about 5.0 to about 7.0
3. A method of purification of antibody from a sample comprising one or more impurities, comprising steps of;
a) Protein-A chromatography
b) Depth Filtration
c) Ion-exchange chromatography
wherein the said filtration is performed using a buffer at a pH of about 5.5 to about 6.5 and at a conductivity of about 3 mS/cm to about 9 mS/cm.
4. A method of purification of antibody from a sample comprising one or more impurities, comprising steps of;
a) Protein-A chromatography
b) Depth Filtration
c) Ion-exchange chromatography
wherein the said filtration is performed using a buffer at a pH of about 5.5 to about 6.5 and at a conductivity of about 6 mS/cm to about 8 mS/cm.
5. A method of purification of antibody from a sample comprising one or more impurities comprising steps of; a) Protein-A chromatography
b) Depth Filtration
c) Ion-exchange chromatography
wherein the said filtration is performed using a buffer at a pH of about 5.5 to about 6.5 and at a conductivity of about 3 mS/cm.
6. A method according to claims 1 , 2, 3, 4 or 5 wherein the ion exchange chromatography is a cation exchange chromatography.
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IN30CH2012 | 2012-01-03 | ||
IN30/CHE/2012 | 2012-01-03 |
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WO2016103139A1 (en) * | 2014-12-22 | 2016-06-30 | Dr. Reddy's Laboratories | Method for reduction of aggregates |
CN108017713A (en) * | 2016-11-04 | 2018-05-11 | 郑州伊美诺生物技术有限公司 | One step membrane filter method separates the device and method of milk antibodies |
EP3236772A4 (en) * | 2014-12-22 | 2018-10-17 | Alexion Pharmaceuticals, Inc. | Methods of purifying recombinant proteins |
EP4108674A4 (en) * | 2020-02-21 | 2024-03-06 | Prestige Biopharma Pte Ltd | Optimized method for bevacizumab purification |
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Cited By (4)
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WO2016103139A1 (en) * | 2014-12-22 | 2016-06-30 | Dr. Reddy's Laboratories | Method for reduction of aggregates |
EP3236772A4 (en) * | 2014-12-22 | 2018-10-17 | Alexion Pharmaceuticals, Inc. | Methods of purifying recombinant proteins |
CN108017713A (en) * | 2016-11-04 | 2018-05-11 | 郑州伊美诺生物技术有限公司 | One step membrane filter method separates the device and method of milk antibodies |
EP4108674A4 (en) * | 2020-02-21 | 2024-03-06 | Prestige Biopharma Pte Ltd | Optimized method for bevacizumab purification |
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