DESC:FIELD OF THE INVENTION
The present invention relates to protein purification methods. In particular, the
invention relates to methods for purifying a composition comprising
immunoglobulin using a combination of various chromatography steps.
5 BACKGROUND OF THE INVENTION
Monoclonal antibodies (mAbs) are effective targeted therapeutic agents. The high
specificity of the antibodies makes them ideal to reach their intended target and
hence is useful to treat a wide variety of diseases.
The commercial production of recombinant human monoclonal antibody
10 therapeutics demands robust processes, i.e., the purification scheme needs to
reliably and predictably produce an antibody composition intended for use in
humans. A purification process should be designed to remove product related
contaminants such as high molecular weight (HMW) aggregates, variants such as
charge variants (acidic, deamidated/oxidized, basic), sequence variants and other
15 variants, as well as process related contaminants such as leached Protein-A, host
cell protein, DNA, adventitious and endogenous viruses, endotoxin, extractable
from resins and filters, process buffers and agents such as detergents that may have
been employed for virus reduction. In designing a purification scheme and other
conditions for each of the chromatographic steps, along with removal of
20 contaminants, an important consideration is recovery from each step of the
purification scheme and from the overall purification scheme. Hence, for a
commercially viable process, the purification scheme needs to be designed to
ensure adequate removal of contaminants from an antibody composition while
maintaining the yield of the same.
25 Product-related and process-related impurities, including aggregates and charge
variants, have the potential to interfere with the purification process, affect the
protein during storage, and/or can potentially be a cause of adverse reactions upon
administration of an antibody to a subject as a pharmaceutical. Therefore,
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separation of the desired recombinant therapeutic protein from product- and
process-related impurities to a purity sufficient for use as a human therapeutic
poses a formidable challenge. Chromatographic techniques exploit the physical
and chemical differences between the antibodies and the contaminant for the
5 separation. Majority of purification schemes for mAbs involve a Protein-A based
chromatography, which results in a high degree of purity and recovery in a single
step. One or two additional chromatography steps are employed as polishing steps,
generally selected from cation exchange chromatography, anion exchange
chromatography, hydrophobic interaction chromatography or mixed-mode
10 chromatography. The selection of the polishing chromatography steps is dictated
majorly by the target antibody to be purified because every antibody is different in
terms of its physico-chemical properties and may need a different purification
scheme and/or different purification conditions for an efficient separation from
impurities.
15 Removal of HMW aggregates, especially soluble aggregates, presents a challenge
due to the physical and chemical similarity of the aggregates to the drug product
itself, which is usually a monomer. In addition, the presence of unwanted charge
variants such as far-basic variants pose additional challenges, which require
additional efforts to fine-tune the purification scheme.
20 Hence, there is a need for an improved purification scheme to control the productand process related species, including HMW aggregates and certain charge
variants, in the final drug substance of a therapeutic antibody composition.
SUMMARY OF THE INVENTION
The present invention discloses a method for purifying an antibody composition
25 comprising the target antibody and one or more contaminants, the method
comprising the steps of affinity chromatography, cation exchange chromatography,
and mixed-mode chromatography, wherein the cation exchange chromatography
step comprises the steps of contacting the antibody composition with a negatively
charged resin at an antibody concentration of less than about 40 grams per liter of
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the resin, wherein a significant amount of the target antibody binds to the resin,
washing the resin with a wash buffer solution, and eluting the bound antibody with
an elution buffer comprising citrate at pH about 6.0 and conductivity less than 10
mS/cm.
5 The specific elution conditions employed effect up to 95% reduction of HMW
aggregates, further maintaining the recovery of the antibody to be about 85% or
more.
The method disclosed as per the current invention results in a significant reduction
of HMW aggregates and the complete removal of a far-basic variant of the target
10 antibody.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
The phrase "ion exchange material" refers to a solid phase which is negatively
charged (i.e., a cation exchange resin) or positively charged (i.e., an anion
15 exchange resin). The charge may be provided by attaching one or more charged
ligands to the solid phase, e.g. by covalent linking. Alternatively, or in addition,
the charge may be an inherent property of the solid phase (e.g. as is the case for
silica, which has an overall negative charge).
The term "conductivity" refers to the ability of an aqueous solution to conduct an
20 electric current between two electrodes. In solution, the current flows by ion
transport. Therefore, with an increasing amount of ions present in the aqueous
solution, the solution will have a higher conductivity. The unit of measurement
for conductivity is mS/cm, and can be measured using a conductivity meter, e.g.,
by Orion. The conductivity of a solution may be altered by changing the
25 concentration of ions therein. For example, the concentration of a buffering agent
and/or concentration of a salt (e.g. NaCl or KCl) in the solution may be altered in
order to achieve the desired conductivity.
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A "contaminant" is a material that is different from the desired polypeptide
product. The contaminant may be a variant of the desired polypeptide (e.g. a
deamidated variant or an aminoaspartate variant of the desired polypeptide) or
another non-product related polypeptide, for e.g., host cell protein, host cell
5 nucleic acid, endotoxin, etc. A contaminant can also be process related, for
example - Protein-A-leachates.
“High molecular weight aggregates” as referred herein encompasses association
of at least two molecules of a product of interest, e.g., antibody or any antigenbinding fragment thereof. The association of at least two molecules of a product
10 of interest may arise by any means including, but not limited to, non-covalent
interactions such as, e.g., charge-charge, hydrophobic and van der Waals
interactions; and covalent interactions such as, e.g., disulfide interaction or nonreducible crosslinking. An aggregate can be a dimer, trimer, tetramer, or a
multimer greater than a tetramer, etc.
15 The term “process or product related impurities” as used herein refer to the
contaminants which may be derived from the manufacturing process, for
example, but not limited to, cell culture, downstream or cell substrates and may
include host cell proteins, host cell DNA, nucleic acid, protein-A leachates etc.,
or may be molecular variants of the protein of interest, for example, but not
20 limited to, high molecular weight (HMW) aggregates, acidic variants, basic
variants, low molecular weight variants etc., and may be formed during
expression, manufacture or storage of the protein.
The term ‘variants’ as used here in refers to a group of low-pI, mid-pI and high-pI
variants, and are described as “acidic variants”, “mid peak” and “basic variants”
25 respectively, based on their differential elution from an analytical ion-exchange
HPLC.
An ‘acidic variant’ is a variant of a polypeptide of interest which is more acidic
than the polypeptide of interest. An acidic variant species elute earlier than the
main peak when determined by a standard cation exchange chromatography.
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A ‘basic variant’ is a variant of a polypeptide of interest which is more basic than
the polypeptide of interest. A basic variant species elute later than the main peak
when determined by a standard cation exchange chromatography. A ‘far basic
variant’ is a variant that is more basic than the basic variant of a polypeptide of
5 interest. A far basic variant species elute later than the basic variant when
determined by a standard cation exchange chromatography.
The term “about” as used herein, means an acceptable error range for the
particular value as determined by one of ordinary skill in the art. For example,
“about” can mean a range of up to 20%.
10 The "composition" to be purified herein comprises the protein of interest and one
or more contaminants. The composition 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 antibody (e.g., the composition may
comprise harvested cell culture fluid).
15 The term "load" herein refers to the composition loaded onto the chromatography
material, i.e., ion exchange support. Preferably, the chromatography material is
equilibrated with an equilibration buffer prior to loading the composition which is
to be purified.
The term "bind and elute mode" as used herein refers to a process wherein the
20 target protein substantially binds to the chromatographic support, and is
subsequently eluted from the chromatographic support.
The term “Mixed-Mode Chromatography” refers to a form of chromatography
that uses a chromatographic support with at least two unique types of functional
groups, each interacting with the molecule or protein of interest. Mixed-mode
25 chromatography generally uses ligands that have more than one type of
interaction with target proteins and/or impurities. For example, a charge-charge
type of interaction and/or a hydrophobic or hydrophilic type of interaction, or an
electroreceptor-donor type interaction. In general, based on the difference in the
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total interaction, the target protein and one or more impurities can be separated
under various conditions.
Aggregate concentration can be measured in a protein sample using Size
Exclusion Chromatography (SEC), a well-known and widely accepted method in
5 the art. Size exclusion chromatography uses a molecular sieving retention
mechanism, based on differences in the hydrodynamic radii or differences in size
of proteins. Large molecular weight aggregates cannot penetrate or only partially
penetrate the pores of the stationary phase. Hence, the larger aggregates elute first
and smaller molecules elute later, the order of elution being a function of the size.
10 Detailed Description of the Embodiments
The present invention discloses a method to purify an antibody composition
comprising the target antibody and one or more contaminants, for example, high
molecular weight aggregates, host cell proteins/nucleic acids, protein-A leachates,
and charge variants, the method comprises the use of a combination of affinity
15 chromatography, cation exchange chromatography and mixed-mode
chromatography.
In an embodiment, the method disclosed herein is used to reduce the level of
process and product related impurities in an antibody composition comprising an
anti-PD1 antibody and one or more said impurities using a combination of
20 affinity chromatography, cation exchange chromatography and mixed-mode
chromatography.
In another embodiment, the method disclosed herein is used to reduce the level of
process and product related impurities in an antibody composition comprising an
anti-PD1 antibody and one or more said impurities, wherein the method
25 comprises the following steps:
(a) cell culture clarification
(b) affinity chromatography
(c) low-pH viral inactivation and pH neutralization
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(d) depth filtration
(e) cation exchange chromatography
(f) mixed-mode chromatography
(g) nano-filtration
5 (h) ultrafiltration/diafiltration
In another embodiment, the method disclosed herein is used to reduce the level of
process and product related impurities in an antibody composition comprising
nivolumab and one or more said impurities, wherein the method comprises the
following steps:
10 (a) cell culture clarification
(b) affinity chromatography
(c) low-pH viral inactivation and pH neutralization
(d) depth filtration
(e) cation exchange chromatography
15 (f) mixed-mode chromatography
(g) nano-filtration
(h) ultrafiltration/diafiltration
In yet another embodiment, the method disclosed herein is used to reduce the
level of impurities such as HMW aggregates and far-basic variants in an antibody
20 composition comprising an anti-PD1 antibody and one or more said impurities
using cation exchange chromatography, wherein the antibody composition is
loaded onto the cation exchange support in the presence of a loading buffer
solution under such conditions that the target antibody substantially binds to the
cation exchange support, and the bound antibody is eluted from the cation
25 exchange support by a buffer solution comprising citrate, and wherein the cation
exchange support is washed using a wash buffer only once between the loading
and the elution steps.
In yet another embodiment, the method disclosed herein is used to reduce the
level of impurities such as HMW aggregates and far-basic variants in an antibody
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composition comprising an anti-PD1 antibody and one or more said impurities,
the method comprising steps of:
(a) loading the antibody composition onto a cation exchange support in the
presence of a loading buffer solution under conditions such that the anti-PD1
5 antibody substantially binds to the cation exchange support,
(b) washing the cation exchange support with a wash buffer solution,
(c) eluting the bound antibody using an elution buffer, and
(d) collecting the eluate from the cation exchange support,
wherein the elution buffer solution comprises citrate,
10 and wherein the cation exchange support is washed using a wash buffer only once
between the loading and the elution steps.
In a further embodiment, the method disclosed herein is used to reduce the level
of impurities such as HMW aggregates and far-basic variants in an antibody
composition comprising an anti-PD1 antibody and one or more said impurities,
15 the method comprising steps of:
(a) loading the antibody composition onto a cation exchange support in the
presence of a loading buffer solution under conditions such that the anti-PD1
antibody substantially binds to the cation exchange support,
(b) washing the cation exchange support with a wash buffer solution,
20 (c) eluting the bound antibody using an elution buffer, and
(d) collecting the eluate from the cation exchange support,
wherein the elution buffer solution comprises citrate, has a pH of about 6.0 and
conductivity less than 10 mS/cm,
and wherein the cation exchange support is washed using a wash buffer only once
25 between the loading and the elution steps.
In a further embodiment, the method disclosed herein is used to reduce the level
of impurities such as HMW aggregates and far-basic variants in an antibody
composition comprising nivolumab and one or more said impurities, the method
comprising steps of:
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(a) loading the antibody composition onto a cation exchange support in the
presence of a loading buffer solution under conditions such that nivolumab
substantially binds to the cation exchange support,
(b) washing the cation exchange support with a wash buffer solution,
5 (c) eluting the bound antibody using an elution buffer, and
(d) collecting the eluate from the cation exchange support,
wherein the elution buffer solution comprises citrate,
and wherein the cation exchange support is washed using a wash buffer only once
between the loading and the elution steps.
10 In a further embodiment, the method disclosed herein is used to reduce the level
of impurities such as HMW aggregates and far-basic variants in an antibody
composition comprising nivolumab and one or more said impurities, the method
comprising steps of:
(a) loading the antibody composition onto a cation exchange support in the
15 presence of a loading buffer solution under conditions such that nivolumab
substantially binds to the cation exchange support,
(b) washing the cation exchange support with a wash buffer solution,
(c) eluting the bound antibody using an elution buffer, and
(d) collecting the eluate from the cation exchange support,
20 wherein the elution buffer solution comprises citrate, has a pH of about 6.0 and
conductivity less than 10 mS/cm,
and wherein the cation exchange support is washed using a wash buffer only once
between the loading and the elution steps.
In any of the above mentioned embodiments, the high molecular weight
25 aggregates are reduced by up to 95% in the eluate collected from the cation
exchange support as compared to the level of high molecular weight aggregates
in the antibody composition loaded onto the cation exchange support.
In any of the above mentioned embodiments, the method results in complete
removal of the far-basic variant of the antibody.
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In any of the above mentioned embodiments, the method is also used to reduce
the level of other process-related impurities, including but not limited to proteinA leachates, host cell proteins, host cell DNA, etc.
In any of the above mentioned embodiments, the CEX is operated in bind and
5 elute mode.
In any of the above mentioned embodiments, the antibody is an anti-PD-1
antibody or antigen binding fragment thereof.
In any of the above mentioned embodiments, the antibody is nivolumab.
The invention is more fully understood by reference to the following examples.
10 These examples should not, however, be construed as limiting the scope of the
invention.
EXAMPLES
Example 1
A therapeutic monoclonal antibody which binds and blocks programmed death
15 receptor-1 (PD-1) was cloned and expressed in a Chinese Hamster Ovary cell line
and the cell culture broth containing the expressed antibody was harvested,
clarified and subjected to protein-A affinity chromatography. The eluate from
protein-A affinity chromatography was subjected to low-pH incubation and depth
filtration, and the filtered liquid comprising the antibody composition was further
20 purified using cation exchange chromatography (CEX). Level of impurities was
determined in both load and eluate of CEX. Details of CEX chromatography are
given in Tables 1 and 2.
Resin Poros™ XS
Bed Height (cm) 18 – 22
Residence Time (min) (loading, postload wash and elution)
4.0
Load Factor (g/L) <40
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Table 1: Chromatography conditions used in CEX
Stage Buffer pH
Conductivity
(mS/cm)
Equilibration 10 mM sodium citrate buffer 6.0±0.2 4.2±0.5
Wash
10 mM sodium citrate + 20 mM
NaCl
6.0±0.2 4.2±0.5
Elution
10 mM sodium citrate + 70 mM
NaCl
6.0±0.2 9.2±1.0
Regeneration 0.5 M NaOH NA NA
Table 2: Details of buffers used in CEX
Table 3 summarizes the HMW aggregate level at the time of loading onto CEX and
5 in the eluate obtained from CEX at elution buffer pH of 5.9.
Batch HMW % (load) HMW% (eluate) % Reduction % Recovery
Nmab-1 6.7 0.3 95.5 88
Nmab-2 4.6 0.4 91.3 83
Nmab-3 1.4 0.3 78.6 90
Nmab-4 1.4 0.3 78.6 89
Average 86.0 87.5
Table 3: HMW aggregate level in CEX load and CEX eluate
Similarly, the levels of far-basic variants were determined in CEX load and eluate
and are represented in Table 4 along with HCD content in CEX eluate.
Batch Far Basic variant %
(load)
Far Basic variant %
(eluate)
%
Reduction
Nmab-1 6.9 0 100%
Nmab-2 7.9 0 100%
Nmab-3 9.0 0 100%
Nmab-4 4.9 0 100%
10 Table 4: Far-basic variant levels at CEX load and CEX eluate stages
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It is evident from Tables 3 and 4 that the disclosed method is able to significantly
reduce the levels of HMW aggregates and completely removes the far-basic
variants from the antibody composition. ,CLAIMS:We Claim:
1. A method to reduce the level of high molecular weight aggregates and far-basic
variants impurities content in an antibody composition, comprising an anti-PD1
antibody and one or more said impurities, using cation exchange chromatography
wherein the antibody composition is loaded onto the cation exchange support in the
presence of a loading buffer solution under such conditions that the target antibody
substantially binds to the cation exchange support, and the bound antibody is eluted
from the cation exchange support by a buffer solution comprising citrate, and wherein
the cation exchange support is washed using a wash buffer only once between the
loading and the elution steps.
2. A method disclosed to reduce the level of high molecular weight aggregates and
far-basic variants impurities content in an antibody composition, comprising an antiPD1 antibody and one or more said impurities, the method comprising steps of:
(a) loading the antibody composition onto a cation exchange support in the presence
of a loading buffer solution under conditions such that the anti-PD1 antibody
substantially binds to the cation exchange support,
(b) washing the cation exchange support with a wash buffer solution,
(c) eluting the bound antibody using an elution buffer, and
(d) collecting the eluate from the cation exchange support,
wherein the elution buffer solution comprises citrate, has a pH of about 6.0 and
conductivity less than 10 mS/cm,
and wherein the cation exchange support is washed using a wash buffer only once
between the loading and the elution steps.
3.The method according to claim 1 or 2, wherein the high molecular weight aggregates
are reduced by up to 95% in the eluate collected from the cation exchange support as
compared to the level of high molecular weight aggregates in the antibody
composition loaded onto the cation exchange support.
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4. The method according to claim 1 or 2, wherein the far-basic variants of the
antibody are completely removed / 100 % removed in the eluate collected from
the cation exchange support.
5. The method according to claim 1 or 2, wherein the method is preceded by an
affinity chromatography step.
6. The method according to claim 1 or 2, wherein the method comprises a mixed
mode chromatography step.
7. The method according to claim 1 or 2, further comprises one or more filtration
steps.
8. The anti-PD1 antibody according to claim 1 or 2, is nivolumab.