DESC:FIELD OF THE INVENTION
The present invention relates to a method of reducing impurities and heterogeneity of an antibody in a cell culture media, wherein the method comprises a step of acidification of cell culture media, before the harvest of the cell culture broth.
BACKGROUND OF THE INVENTION
Large-scale purification of proteins remains a significant challenge in the biopharmaceutical industry. Efficient and cost-effective methods are constantly in demand to achieve desired yields and purity levels. Therapeutic proteins are primarily products of recombinant DNA technology, i.e., cloning and expression of a heterologous gene in prokaryotic or eukaryotic systems. However, proteins expressed by recombinant DNA methods are typically associated with impurities and heterogeneity. Heterogeneity in the expression of the desired protein can be in several forms, e.g. in the form of charged variants (typically acidic charge variants, 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 and/or heterogeneity of an antibody, 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 (deamidation, aggregates or degradation products) and process related (media components, HCP, DNA, chromatographic media used in purification, endotoxins, viruses, etc). See, Office of Biologics 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.
The prior art discloses various methods for purification of crude or partially purified antibodies. WO 89/05157 teaches purification of immunoglobulins by cation-exchange 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 1999/057134 describes the use of ion exchange chromatography for purification of polypeptides by varying conductivity and/or pH. US 5110913 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 deals with high amount of impurities and heterogeneity and hence require frequent use of low pH wash conditions, and/or accompanied by frequent and significant change in pH conditions during a chromatography step. However, frequent alterations of pH conditions during a 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 during 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 present invention aims at reducing the impurities (such as aggregates, host cell DNA and host cell proteins) and heterogeneity of the antibody viz., charge variants, at the harvest stage itself. The principle object of the present invention is to reduce the above said impurities and charge variants in the antibody sample that is being loaded onto the downstream chromatographic step(s), resulting in effective separation of charge variants, increased purity and recovery of the desired antibody.
SUMMARY OF THE INVENTION
The present invention describes a method of reducing impurities and heterogeneity in an antibody composition obtained from a cell culture process, wherein the method comprises a step of addition of an acid to the cell culture process (or culture media), before harvesting the antibody. The method of acidification, results in reduction of impurities (such as aggregates, host cell DNA and host cell proteins) and heterogeneity of the antibody (viz., charge variants) at the harvest stage itself, and thus reduce the impurity and heterogeneity in the antibody containing cell culture sample, that is being loaded onto downstream chromatographic steps.
The harvested antibody, obtained after acidification of cell culture media, can be held at room temperature, up to 72 hours, without an increase in turbidity. Further, the harvested antibody, obtained from the above said cell culture method, when subjected to chromatographic purification, was found to have reduced acidic variant content in the eluate, as compared to the antibody obtained from a cell culture method that does not involve acidification.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is an illustration of comparison of turbidity of acidified and non-acidified clarified cell culture broth (CCCB) at room temperature over a period of time. The clarified sample is stable at room temperature (RT) beyond the 48 hour hold time as can be inferred by the near constant turbidity even beyond 72 hour hold time.
Figure 2 is an illustration of comparison of turbidity of samples from neutralized protein-A eluate of acidified and non-acidified cell culture broth when the samples are kept at room temperature over a specified period of time. Turbidity for protein-A eluate generated from non-acidified CCCB was higher than the one generated from acidified CCCB and increased over time at room temperature.
Figure 3 is an illustration of comparison of acidic variants (AcV) in samples taken from protein-A eluate of acidified and non-acidified cell culture broth when the samples are kept at room temperature over a specified period of time. Percentage of acidic variants increased over time for both the samples with higher increase for non-acidified CCCB.
DETAILED DESCRIPTION OF THE INVENTION
The present invention describes a process for increasing the purity of an antibody composition by reducing the impurities and/or heterogeneity in the antibody, at harvest stage during a cell culture process. The process described in the present invention includes a step of acidification of the cell culture process/media, before harvest. The conditions described in the present invention results in effective reduction of acidic variants, as well as removal of impurities 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.
“Turbidity” of a solution has been used here to give a quantitative measurement to the impurity in the solution. Higher the turbidity, higher the impurity level in the antibody solution.
The term “acidic species/variants” refer to a characteristic of a population of proteins wherein the population includes a distribution of product-related impurities identifiable by the presence of charge heterogeneities. For example, in monoclonal antibody (mAb) preparations, such acidic species heterogeneities can be detected by various methods, such as, for example, WCX-10 HPLC (a weak cation exchange chromatography), or IEF (isoelectric focusing). In other terms, acidic variants is a variant of protein of interest which is more acidic (as determined by cation exchange chromatography) than the protein of interest. An example of acidic variant includes deamidated variant.
The term “cell culture” refers to a method/process by which cells are grown under controlled conditions, generally outside their natural environment. To grow cells in controlled conditions involves use of cell culture media.
The term “acidified cell culture” in the invention refers to a cell culture process in which to which an acid is added to the culture media which is used for culturing the cells.
The term “non-acidified cell culture” in the invention refers to a cell culture process in which to which an acid is not added to the culture media which is used for culturing the cells.
In an embodiment, the present invention discloses a method of reducing impurities and/or heterogeneity in an antibody composition, comprising;
a) Culturing cells expressing the antibody of interest in a cell culture growth media at a suitable pH,
b) Reducing the pH of the cell culture media in step a) by addition of an acid,
c) Harvesting the cell culture media containing the antibody of interest,
d) Clarifying the culture obtained in step c) to prepare Cell Culture Clarified Broth (CCCB),
e) Subjecting the CCCB containing the antibody to one or more chromatographic steps and
f) Recovering the antibody from the chromatographic step.
In above mentioned embodiment of the invention, the recovered antibody from step f) of acidified cell culture contains reduced impurities as compared to the recovered antibody obtained from non-acidified cell culture containing the same antibody.
In above mentioned embodiment of the invention, the antibody obtained from the step f) of acidified cell culture sample contains at least 10% reduced acidic species as compared to non-acidified culture sample containing the same antibody.
In any of the above mentioned embodiment of the invention, the chromatographic steps include affinity chromatography and/or an ion exchange chromatography, wherein the affinity chromatography include protein-A chromatography, and may be followed by one or more ion-exchange chromatography such as anion/cation exchange chromatography.
In any of the above embodiment, the antibody is an anti-TNF-a antibody.
In the above embodiment, the antibody may be a chimeric, humanized or a human antibody.
In any of the above mentioned embodiments of the invention, the pH of the cell culture in step b) is reduced at least one unit compared to the pH of the cell culture in step a).
In any of the above mentioned embodiments, the pH of the cell culture in step b) is reduced by addition of a weak acid to the cell culture broth.
In the above mentioned embodiment of the invention, the weak acid is acetic acid.
In any of the above embodiment, the pH of the acidified pre-harvest cell culture media in step b) is about 6.0.
The CCCB obtained in step d) from any of the above mentioned embodiments is stable at least 72 hours at room temperature without much increase in turbidity as compared to non-acidified cell culture.
In any of the above mentioned embodiments, the CCCB obtained in step d) is subjected to protein-A chromatography and the eluate obtained from the protein-A chromatography is stable at least 72 hours at room temperature as compared to non-acidified cell culture.
Holding the CCCB or protein-A chromatography eluate at room temperature for at least 72 hours is one of the advantages of the invention, as it enables storage of the sample during non-availability of downstream process facility or manufacturing facility.
The embodiments mentioned herein may optionally include one or more tangential flow filtration, concentration, diafiltration or ultrafiltration steps.
The embodiments mentioned herein may 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 MabselectTM (GE-Healthcare Life sciences), an affinity matrix with recombinant Protein-A ligand. The resin is made of highly cross-linked 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-CH2CHOHCH2OCH2CHOHCH2N+(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 example. The example should not, however, be construed as limiting the scope of the invention.
EXAMPLES
Example-1
TNFa antibody was cloned and expressed in a recombinant CHO (rCHO) cell line as described in detail in Molecular Cloning: A laboratory Manual by Green and 15 Sambrook, which is incorporated herein by reference. rCHO cells expressing antibody at a seeding density of about 0.3 million cells/mL were seeded in culture medium at 37°C and pH 7.2. Subsequently, on day 4, temperature was shifted to at least two units and the pH was shifted to at least 0.2 units (temperature and the pH of the cell culture were shifted to 34 °C and to 6.9 respectively). Feed was added to the cell culture medium on day 3, 5 and 7. Two batches of the cell cultures were cultured under above mentioned conditions. Further, 2 M acetic acid is added to one batch of the cell culture on day 12, just before harvesting and the pH of the cell culture is adjusted to pH 6.0. Another culture, to which acetic acid is not added, is maintained as control and referred as non-acidified cell culture in further description of the invention.
Example -2
After harvesting, both acidified and non-acidified cell culture samples were clarified using depth filtration. The clarified cell culture broth (CCCB’) obtained from both acidified and non-acidified cell cultures were held at room temperature. The turbidity of the samples were measured using turbidometry and the results are represented in Figure 1.
Example-3
The clarified cell culture broth obtained from acidified cell culture was loaded onto a protein-A chromatography column (Mabselect, VL44x250, 205 mL) that was pre-equilibrated with Tris buffer solution (pH 7.0) (equilibration buffer). The column was then washed with equilibration buffer. This was followed by a wash step wherein the column was washed with Tris buffer (pH 7.0) with higher conductivity, and a further wash with citrate buffer at pH 5. The bound antibody was eluted using citrate buffer, pH 2.5 – 3.5. The eluate was neutralized using 1M Tris-acetate buffer containing pH of 9.0.
Alternatively, procedure exemplified in Example-3 was repeated for the cell culture broth obtained from the non-acidified cell culture.
The neutralized eluates (NTEL) from acidified and non-acidified cell cultures were held at room temperature up to 72 hours. The turbidity and acidic variants content were measured using turbidometry and weak cation exchange chromatography respectively. Results of the experiment are represented as Figure 2 and 3.
,CLAIMS:CLAIMS
We claim:
1) A method of reducing impurities and/or heterogeneity in an antibody composition, the method comprising:
a) culturing cells expressing the antibody of interest in a cell culture media at a suitable pH,
b) reducing the pH of the cell culture media in step a) by addition of an acid (acidified cell culture),
c) harvesting the cell culture media containing the antibody of interest,
d) clarifying the culture obtained in step c) to prepare Cell Culture Clarified Broth (CCCB),
e) subjecting the CCCB containing the antibody to one or more chromatographic steps and
f) recovering the antibody from the chromatographic step
wherein, the recovered antibody composition comprises reduced impurities and/or heterogeneity as compared to the antibody recovered from non-acidified cell culture.
2) The method of claim 1, wherein the pH of the cell culture media in step b) is reduced by at least one unit compared to the pH of the cell culture media.
3) The method of claim 1 and 6, wherein the pH of the cell culture media in step b) is reduced by addition of a weak acid such as acetic acid to the cell culture media.
4) The method of claim 1, wherein the pH of the acidified cell culture media in step b) is about 6.0.
5) The method of claim 1, wherein one or more chromatographic steps include an affinity chromatography and /or an ion exchange chromatography.
6) The method of claim 1, wherein one or more chromatographic steps include a protein-A chromatography.
7) The method of claim 1, wherein the antibody is an anti-TNF-a antibody.
8) The method of claim 1, wherein the CCCB obtained in step d) is stable for at least 72 hours at room temperature without an increase in turbidity as compared to non-acidified cell culture.
9) The method of claim 1, wherein in step e), the chromatographic step is a protein A chromatography, and the eluate so obtained from protein A chromatography is stable for at least 72 hours at room temperature without an increase in turbidity as compared to non-acidified cell culture.
10) The method of claim 1, wherein antibody from step (f) contains at least 10% reduced acidic species as compared to the recovered antibody obtained from a non-acidified cell culture.