DESC:DETAILED DESCRIPTION OF THE INVENTION
Definitions
The term "contacting" as used herein, refers to applying a solution, e.g., a mixture comprising a protein product and a contaminant, as described herein, to a chromatography matrix. In some embodiments, the term "contacting" is synonymous with "loading" a solution onto a chromatography column. A "chromatography support" as used herein refers to the adsorbent solid material contained within a chromatography column.
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 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 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 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.
A "contaminant" or an “impurity”, as used interchangeably herein, is a material that is different from the desired polypeptide product. The contaminant may be a variant or isoform 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 nucleic acid, endotoxin, etc. A contaminant can also be process related, for example - Protein-A-leachates.
The terms “variant” and “isoform” have been used interchangeably throughout the document.
“High molecular weight aggregates” as referred herein encompasses association of at least two molecules of a product of interest, e.g., antibody or any antigen-binding fragment thereof. The association of at least two molecules of a product 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 non-reducible crosslinking. An aggregate can be a dimer, trimer, tetramer, or a multimer greater than a tetramer, etc.
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 limited to, 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 “about” as used herein, would mean and include a variation of upto 20 % from the particular value.
The term “large scale” as used herein, refers to manufacturing/producing/ obtaining/processing of the desired protein in a bioreactor (or equivalent thereof) of capacity 1000–Liter or more.
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).
The term "Hydrophobic Interaction Chromatography" refers to a form of chromatography that uses a chromatographic support with functional groups that separate proteins on the basis of their hydrophobicity.
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 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 total interaction, the target protein and one or more impurities can be separated under various conditions.
The term "Anion Exchange Chromatography'" refers to a form of ion-exchange chromatography that uses a support with functional groups that exchange anions.
The term "load" herein refers to the composition loaded onto the chromatography material, i.e., protein-A support, or 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 target protein substantially binds to the chromatographic support, and is subsequently eluted from the chromatographic support.
The term "flow-through" chromatography, as used herein, refers to a form of chromatography, wherein a chromatography support is used that is not intended to retain or specifically bind with the target protein, e.g., the protein product (e.g, the monoclonal antibody). Under "flow-through" chromatography, a mixture comprising the target protein is applied to the chromatography support, and the effluent comprises the target protein.
Aggregate concentration can be measured in a protein sample using Size Exclusion Chromatography (SEC), a well-known and widely accepted method in 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.
Detailed Description of the Embodiments
The present invention discloses a method to obtain a purified composition comprising an antibody or antigen binding fragments thereof from a composition comprising the antibody and one or more contaminants, for example, high molecular weight aggregates, host cell proteins/nucleic acids, protein-A leachates, the method comprises the use of a combination of one or more chromatography steps, selected from affinity chromatography, cation exchange chromatography and anion exchange chromatography.
In an embodiment, the method is used to obtain a purified composition comprising an antibody or antigen binding fragments thereof from a composition comprising an antibody and one or more contaminants, the method comprises use of affinity chromatography, cation exchange chromatography and anion exchange chromatography, wherein the affinity chromatography is the first chromatography step.
In some embodiments, the method is used to obtain a purified composition comprising an antibody or antigen binding fragments thereof from a composition comprising an antibody and one or more contaminants, the method comprises use of affinity chromatography in bind and elute mode, cation exchange chromatography in bind and elute mode and anion exchange chromatography in flow-through mode, wherein the affinity chromatography is the first chromatography step.
In some embodiments, the method is used to obtain a purified composition of vedolizumab or antigen binding fragments thereof from a composition comprising vedolizumab and one or more contaminants, the method comprises use of affinity chromatography, cation exchange chromatography and anion exchange chromatography, wherein the affinity chromatography is the first chromatography step.
In some embodiments, the method is used to obtain a purified composition comprising an antibody or antigen binding fragments thereof from a composition comprising an antibody and one or more contaminants, the method comprises use of affinity chromatography, cation exchange chromatography and anion exchange chromatography, wherein the affinity chromatography is the first chromatography step and wherein the method is devoid of hydrophobic interaction chromatography.
In some embodiments, the method is used to obtain a purified composition of vedolizumab or antigen binding fragments thereof from a composition comprising vedolizumab and one or more contaminants, the method comprises use of affinity chromatography, cation exchange chromatography and anion exchange chromatography, wherein the affinity chromatography is the first chromatography step and wherein the method is devoid of hydrophobic interaction chromatography.
In some embodiments, the method is used to obtain a purified composition comprising an antibody or antigen binding fragments thereof from a composition comprising an antibody and one or more contaminants, the method comprises use of affinity chromatography, cation exchange chromatography and anion exchange chromatography, wherein the affinity chromatography is the first chromatography step and wherein the method is devoid of hydrophobic interaction chromatography and mixed mode chromatography.
In some embodiments, the method is used to obtain a purified composition comprising an antibody or antigen binding fragments thereof from a composition comprising an antibody and one or more contaminants, the method comprises use of affinity chromatography, cation exchange chromatography and anion exchange chromatography, in the above order, and wherein the method is devoid of hydrophobic interaction chromatography and mixed mode chromatography.
In any of the above mentioned embodiments, the antibody is an anti-a4ß7 antibody or antigen binding fragment thereof.
In some embodiments, the method is used to obtain a purified composition of vedolizumab or antigen binding fragments thereof from a composition comprising vedolizumab and one or more contaminants, the method comprises use of affinity chromatography, cation exchange chromatography and anion exchange chromatography, wherein the affinity chromatography is the first chromatography step and wherein the method is devoid of hydrophobic interaction chromatography and mixed mode chromatography.
In some embodiments, the buffer solutions used at specific pH and conductivity values in AEX and CEX steps leads to the maintenance of the charge isoforms of the antibody in a particular targeted range.
In some embodiments, the method further includes additional purification steps including, but not limited to, viral inactivation, depth filtration, diafiltration, ultrafiltration, tangential flow filtration and other steps well known to a person skilled in the art. These additional purification steps may be interspersed between the chromatography steps.
In some embodiments, anion exchange chromatography is the final chromatography step.
In some embodiments, the purified composition comprises less than 1% HMW aggregates.
In some embodiments, the purified composition comprises less than 0.5% HMW aggregates.
In some embodiments, the purified composition comprises from about 65% to about 75% major isoform. In certain embodiments, the purified composition comprises from about 65% to about 72% major isoform.
In some embodiments, the purified composition comprises less than 16% basic isoforms. In some embodiments, the purified composition comprises 14%-16%, 12%-14%, 10%-12%, 8%-10%, 6%-8% or 4%-6% basic isoforms. In some embodiments, the purified composition comprises 8%-12% basic isoforms. In certain embodiments, the purified composition comprises 9%-11% basic isoforms.
In some embodiments, the purified composition comprises about 15%-25% acidic isoforms. In certain embodiments, the purified composition comprises about 16%-21% acidic isoforms.
In some embodiments, recovery of the antibody from the method disclosed is about 98% or more.
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 protein-A leachates, host cell proteins, host cell DNA, etc.
In any of the above mentioned embodiments, the composition comprising the antibody and one or more contaminants may be partially purified or may be obtained directly from cell culture.
In an embodiment, the invention discloses a large scale purification method to obtain a purified composition comprising an antibody or antigen binding fragments thereof from a composition comprising an antibody and one or more contaminants, the method comprises use of affinity chromatography, cation exchange chromatography and anion exchange chromatography, in the above order.
In an embodiment, the invention discloses a large scale purification method to obtain a purified composition comprising an antibody or antigen binding fragments thereof from a composition comprising an antibody and one or more contaminants, the method comprises use of affinity chromatography, cation exchange chromatography and anion exchange chromatography, in the above order and wherein, the method is devoid of hydrophobic interaction chromatography and mixed mode chromatography.
In the above mentioned embodiments, the antibody is an anti-a4ß7 antibody or antigen binding fragment thereof.
In the above embodiment, recovery of the anti-a4?7 antibody from the large scale purification method is about 98% or more.
In some embodiments, the anti-a4?7 antibody monomer content from the large scale purification method is about 99.5% or more.
In any of the above mentioned embodiments, the anti-a4?7 antibody is vedolizumab.
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.
EXAMPLES
Example 1: Purification using Affinity Chromatography
A therapeutic monoclonal antibody which binds to human a4ß7 integrin, vedolizumab, was cloned and expressed in a Chinese Hamster Ovary cell line and the cell culture broth containing the expressed antibody was harvested and clarified. The clarified cell culture broth was then loaded onto a protein-A chromatography resin (MabSelect™ PrismA) that was pre-equilibrated with a 20 mM phosphate buffer (pH 7.0) containing 150 mM sodium chloride. The column was then washed with the same buffer, followed by a high-salt wash with 200 mM phosphate buffer, 1.5 M NaCl (pH 6.3). The column was then washed with 20 mM phosphate buffer (pH 6.0). The bound protein was eluted using elution buffer containing 124 mM sodium acetate at a pH of 3.4. UV absorbance at 280 nm was measured and the eluate was collected from rising value of absorbance 125 mAU to declining value of absorbace 125 mAU. The eluate from Protein-A affinity chromatography was subjected to low-pH incubation (pH: 3.5 ± 0.2) and depth filtration.
Example 2: Purification using cation exchange chromatography
This example describes further purification steps of the therapeutic antibody described in example 1. 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 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, post-load wash and elution) 4.0
Load Factor (g/L) 40
Table 1: Chromatography conditions used in CEX
Stage Buffer pH Conductivity (mS/cm)
Equilibration 50 mM phosphate buffer 5.9 4±1
Wash 50 mM phosphate buffer 5.9 4±1
Elution Linear gradient, 0%-30% B in 15 CV
A: 50 mM phosphate buffer,
B: 50 mM phosphate buffer, 250 mM NaCl 5.9 A: 4±1
B: 26±2
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 in the eluate obtained from CEX at elution buffer pH of 5.9.
Batch HMW % (load) HMW% (eluate) % Reduction % Recovery
Vmab-1 1.5 0.3 80.0% 91
Vmab-2 1.6 0.3 81.3% 94
Vmab-3 1.4 0.3 78.6% 100
Table 3: HMW aggregate level in CEX load and CEX eluate
Similarly, the levels of HCP and protein-A leachates were determined in CEX load and eluate and are represented in Table 4 along with HCD content in CEX eluate.
Batch number HCP (ng/mg) PAL (ng/mg) HCD (pg/mg)
CEX load CEX eluate CEX load CEX eluate CEX eluate
Vmab-1 170 4.4 14.0 1.6 1.3
Vmab-2 194 4.6 16.0 2.2 0.2
Vmab-3 173 4.1 16.5 1.2 0.8
Table 4: HCP and PAL levels at AEX load and AEX FT stages
It is evident from Tables 3 and 4 that the disclosed method is able to significantly reduce the levels of HMW aggregate in addition to the removal of HCP and protein-A leachates.
Example 3: Purification using anion exchange chromatography
This example describes the further purification of the therapeutic antibody described in example 1. The eluate from protein-A affinity chromatography was subjected to low-pH incubation and depth filtration, and further polishing steps, including cation exchange chromatography (CEX). The eluate obtained from CEX was subjected to tangential flow filtration (TFF) and the permeate obtained from TFF was used as the load for anion exchange chromatography (AEX). At this stage, HMW aggregate level was determined using analytical size exclusion chromatography and was found to be significantly increased as compared to the level of HMW aggregates in the previous step, i.e., CEX eluate (see Table 7). AEX was then carried out in flow-through mode and the specific pH and conductivity of the loading buffer was used to reduce the level of HMW aggregates. Details of AEX chromatography are given in Tables 5 and 6. It is to be noted that samples Vmab-1 to Vmab-7 were processed at the 50-liter scale and samples Vmab-8 to Vmab-10 were processed at the 1000-liter scale.
Resin Q Sepharose FF
Bed Height (cm) 18 – 22
Residence Time (min) (loading and Post-load wash) 6.0
Load Factor (g/L) 150
Table 5: Chromatography conditions used in AEX
Stage Buffer pH Conductivity (mS/cm)
Pre-equilibration 200 mM phosphate buffer, 1.5 M NaCl 6.30 120±5
Equilibration 35 mM phosphate buffer 7.0 4±1
Post load wash 35 mM phosphate buffer 7.0 4±1
Regeneration 0.5 M NaOH NA NA
Table 6: Details of buffer used in AEX

Table 7 shows the HMW aggregate data at the TFF input and output stages.
Batch HMW % (TFF input) HMW% (TFF output) % increase
Vmab-1 0.3 0.6 100%
Vmab-2 0.3 0.6 100%
Vmab-3 0.3 0.6 100%
Table 7: HMW level at TFF stage
The flow-through material obtained from AEX was subjected to analytical SEC to determine the level of HMW aggregate. Table 8 summarizes the HMW aggregate level at the time of loading onto AEX and in the flow-through obtained from AEX.
Batch HMW % (load) HMW% (flow-though) % Reduction
Vmab-1 0.7 0.3 57%
Vmab-2 0.7 0.3 57%
Vmab-3 0.6 0.3 50%
Table 8: HMW aggregate level in AEX load and AEX flow-through stages
Similarly, the levels of HCP and protein-A leachates were determined at the AEX load and flow-through stages and are represented in Table 9 along with HCD content in AEX flow-through.
Batch number HCP (ng/mg) PAL (ng/mg) HCD (pg/mg)
AEX load AEX FT AEX load AEX FT AEX FT
Vmab-1 4.50 0.40 3.10 0.10 0.5
Vmab-2 5.51 0.40 4.30 0.10 0.2
Vmab-3 4.20 0.40 3.00 0.10 0.1
Table 9: HCP and PAL levels at AEX load and AEX FT stages
It is evident from Tables 8 and 9 that the disclosed method is able to significantly reduce the levels of HMW aggregate in addition to the removal of HCP and protein-A leachates.
Further, the pH of the flow-through obtained from AEX chromatography was adjusted to pH 6.0 using 2M acetic acid. TFF was carried out post pH adjustment to concentrate the protein to not less than 70 mg/mL and the TFF retentate was diluted with formulation buffer to prepare the drug substance at a concentration of 65 mg/mL.
The following tables show the level of impurities (charge isoforms, HMW aggregates, HCPs, HCDs, and Protein-A leachates) at the neutralized eluate level, NTEL (post protein-A chromatography stage) and in the drug substance (DS).
Table 10 shows the HMW aggregate level.
Batch # Monomer (%) HMW (%)
NTEL DS NTEL DS
Vmab-1 98.1 99.5 1.8 0.5
Vmab-2 98.0 99.5 1.9 0.4
Vmab-3 98.1 99.6 1.8 0.4
Vmab-4 98.0 99.5 2.0 0.4
Vmab-5 98.0 99.2 2.0 0.6
Vmab-6 98.1 99.5 1.9 0.4
Vmab-7 98.2 99.5 1.7 0.3
Vmab-8 98.7 99.6 1.2 0.2
Vmab-9 98.9 99.5 1.1 0.3
Vmab-10 98.9 99.8 1.2 0.2
Table 10: HMW aggregate level at NTEL and DS stage
Table 11 shows the level of charge isoforms.
Batch # Acidic isoform (%) Major isoform (%) Basic isoform (%)
NTEL DS NTEL DS NTEL DS
Vmab-1 21.6 17.8 67.9 65.1 10.50 10.0
Vmab-2 20.1 17.2 68.9 66.9 11.00 10.2
Vmab-3 19.6 16.7 69.0 67.7 11.40 9.7
Vmab-4 16.1 17.5 69.5 72.1 14.4 10.4
Vmab-5 18.2 19.4 71.5 70.5 10.3 10.1
Vmab-6 18.4 20.5 71.1 70.2 10.5 9.4
Vmab-7 18.8 20.2 71.4 70.7 9.9 9.1
Table 11: Charge variant level at NTEL and DS stage

Table 12 shows the level of HCPs, HCDs and Protein-A leachates.
Batch # HCP (ng/mg) HCD (pg/mg) Protein-A leachate (ng/mg)
NTEL DS NTEL DS NTEL DS
Vmab-1 477 0.1 89 0.1 68 0.1
Vmab-2 516 0.1 34 0.2 54 0.1
Vmab-3 384 0.1 44 0.1 68 0.1
Vmab-4 477 1.3 217 0.2 34 0.1
Vmab-5 677 1.1 181 0.1 66 0.1
Vmab-6 527 0.4 129 0.5 52 0.1
Vmab-7 666 0.3 117 0.1 67 0.1
Vmab -8 563 <0.4 195 <0.07 <31.3 <0.07
Vmab-9 525 <0.4 173 <0.07 <30.58 <0.07
Vmab-10 498 <0.4 120 <0.07 <29.15 <0.07
Table 12: Process related impurities at NTEL and DS stage
Table 13 shows the recovery % of the antibody from various process steps.
Batch Protein-A eluate CEX eluate AEX flow-through DS
Vmab-8 94.4 92.9 97.4 98.0
Vmab-9 104.1 92.9 101.6 99.0
Vmab-10 99.8 94.8 98.9 100.0
,CLAIMS:We claim:
1. A method to obtain a purified composition comprising an anti-a4?7 antibody or antigen binding fragments thereof from a composition comprising anti-a4?7 antibody and one or more contaminants, the method comprises use of affinity chromatography, cation exchange chromatography and anion exchange chromatography, wherein the affinity chromatography is the first chromatography step.
2. The method as claimed in claim 1, wherein the anion exchange chromatography is the final chromatography step.
3. The method as claimed in claim 1, wherein the method is devoid of hydrophobic interaction chromatography.
4. The method as claimed in claim 1, wherein the method is devoid of hydrophobic interaction and mixed mode chromatography.
5. The method as claimed in claim 1, wherein the method is a large scale purification method.
6. The method as claimed in claim 1, wherein the purified anti-a4?7 antibody composition has about 99.5 % or more of the antibody in its monomeric form.
7. The method as claimed in claim 1, wherein the anti-a4?7 antibody has a recovery of about 98% or more.
8. The method as claimed in claim 1, wherein the contaminants are selected from high molecular weight aggregates, host cell protein, host cell DNA, protein-A leachates, endotoxins, acidic variants and basic variants.
9. The method as claimed in claim 8, wherein the acidic variants content in the purified composition is about 16% - 21%.

10. The method as claimed in claim 8, wherein the basic variants content in the purified composition is about 9% -11%.