ANTIBODY PURIFICATION
FIELD OF THE INVENTION
The present invention relates to a method of purification of antibodies
comprising an anion exchange chromatography.
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 a requirement 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.
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.
The prior art discloses various methods for purification of crude or partially
purified antibodies using ion/anion exchange chromatography. 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.
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 201 0072381 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.
However the prior art's use of very low or high pH, in a chromatographic step
may result in considerable reduction in antibody yield and stability. Further, as
antibody purification generally involves multiple chromatographic steps, use of buffer
of either low or high pH in a chromatography necessitates substantial and frequent
pH adjustments, or buffer exchanges, between other chromatographic steps, that in
turn affect efficient and effective scale up of downstream operations.
Hence, 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. Interestingly, and contrary to what
is taught in the prior-art, 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.
SUMMARY OF THE INVENTION
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.
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 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.
DETAILED DESCRIPTION OF THE INVENTION
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.
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, viruses, endotoxin
etc.
The term "flow-through mode" as used herein 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.
The term neutral pH range refers to the pH range of about 7.0 to about 7.5.
In an embodiment, 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.
In one embodiment of the invention, the pH of the buffer is about 7.0.
In another embodiment of the invention, the pH of the buffer is about 7.5.
In yet another embodiment, the invention provides a method for the
purification of an antibody wherein a cation exchange chromatography precedes or
follows the said anion exchange chromatography.
In a further embodiment of the invention, a protein-A chromatography
precedes the cation exchange and anion 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. In
embodiments of the invention, a strong cation exchange resin, such as SPSepharose
® (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 using a highly cross-linked, 6
% agarose matrix attached to -O-CH 2CHOHCH2OCH2CHOHCH 2N+(CH3)3 functional
group. Alternatively, the anion exchange chromatography could be carried out using
a monolithic column, disk or tubular, that functions as an anion exchanger.
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). 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 9-1 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 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.
Table 1
Cation Exchange 0.1 7 0.3 0.81 8 1.1
chromatography
eluate
Anion Exchange 0.08 BDL 0.67 95.6
chromatography
flow through
BDL: Below Detection Limit
Table 2 :
Basic Basic
Acidic
Sample K0 (%) Variant-1 Variant-2
variants (%)
(%) (%)
Protein A eluate 10.64 18.03 64.09 7.24
Cation Exchange 13.73 23.30 62.97 BDL
chromatography
eluate
Anion Exchange 14.03 23.60 62.37 BDL
chromatography flow
through
K0: Species devoid of the C-terminal lysine residue
BDL: Below Detection Limit
Example 4
Nano filtration
The flow-through obtained from the anion exchange chromatography
procedure described in Example 3, was applied onto a nano filtration unit (Ultipor ®
VF grade DV 20, Pall Corporation) using a buffer of 40 mM Tris, pH 7.5, at a
conductivity of 3.0 mS/cm. Nanofiltered filtrate composition may then be collected
and used.
CLAIMS:
1. A process for purification of an antibody, comprising an anion
exchange chromatography operated in flow-through mode, wherein the buffer used
in the said chromatography step is in the pH range of from about 7.0 to about 7.5.
2 . A process according to claim 1, wherein the said pH value is
maintained in the equilibration, load and wash steps of the anion exchange
chromatography step.
3 . A process according to claim 1, wherein the anion-exchange
chromatography step is preceded by a cation-exchange chromatography step.
4 . A process according to claim 1, wherein the anion-exchange
chromatography step is followed by a nanofiltration step.
5 . A process according to either of claims 3 or 4, further comprising a
protein-A affinity chromatography step.
6 . A process for purifying an antibody comprising steps of,
a) protein-A chromatography
b) cation exchange chromatography
c) anion exchange chromatography operated in flow-through mode, wherein
the buffer solution used in the said chromatography step is in the pH range of from
about 7.0 to about 7.5, and a
d) nanofiltration step, wherein said nanofiltration is performed using a buffer
of identical pH and conductivity values as that of the buffer of anion exchange
chromatography.
7 . A process according to claim 8, wherein the buffer used in the
nanofiltration step is at a pH value of from about 7.0 to about 7.5 and at a
conductivity value of about 3.0 to about 6.0 mS/cm.
8 . A process according to either of claims 1 or 6, further comprising a
tangential flow filtration, concentration, diafiltration, ultra filtration, or buffer exchange
step.