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 89/051 57 teaches purification of immunoglobulins by cationexchange
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 1999/0571 34 describes the use of ion
exchange chromatography for purification of polypeptides by varying conductivity
and/or pH. US 5 11091 3 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 crosslinked
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-CH 2CHOHCH2OCH2CHOHCH 2N+(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 9-1 2 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 :
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.