DESC:A CELL CULTURE PROCESS TO PRODUCE AN ANTIBODY COMPOSITION
FIELD OF INVENTION
The Biological material used in the invention was not obtained from India.
The present invention relates to a cell culture process for culturing mammalian cells producing an antibody composition. The present invention discloses a method of reducing the % of acidic variants in an antibody composition, the method comprising culturing the mammalian cells in cell culture medium having low cystine content, wherein the % of acidic variant is reduced as compared to a composition produced using culture medium having high cystine content. Further, the present invention provides a method comprises reducing the cysteinylation and/or glutathionylation in a cell culture process producing an antibody composition by using cell culture medium having low cystine content as compared to a cell culture process using culture medium having high cystine content.
BACKGROUND OF THE INVENTION
Recombinant monoclonal antibodies (mAbs) have emerged as an important class of biopharmaceutical drugs. Recombinant mAbs are inherently complex and heterogenous molecules which is due to various factors like post-translational modifications that occur within the cells they are expressed and chemical modifications that may occur during different steps of manufacturing. The resultant mAb variants often lead unwanted effect on stability and activity of these drugs. Therefore, regulatory agencies warrant that these recombinant mAb variants are well managed to ensure their safety and intended efficacy.
Most of the recombinant mAbs are of IgG isotype and they share the evolutionary conserved disulfide bonds between the cysteine residues described for naturally human IgG. While number of intra-chain disulfide bonds in the various IgG subclasses is same, the number of inter-chain disulfide bonds varies amongst the various IgG subclasses. It is presumed that sulfhydryl group of all cysteines in` IgG are in disulfide bonded state. However, presence of free sulfhydryls has been reported both in human IgGs as well as recombinant mAbs which are attributable reduction of intra-chain as well as intra-chain disulfide bonds (Liu and May, MAbs. 2012 Jan-Feb;4(1):17-23). Further still, monoclonal antibodies have been reported wherein cysteine residues other than canonical disulfide linkage associated cysteines have been described, which have free sulfhydrl group (Gadgil et al. Anal. Biochem. 355 (2006) 165–174; McSherry et. al. MABS (2016), VOL. 8, NO. 4, 718–725).
The free cysteines are susceptible to chemical modification such as cysteinylation and glutathionylation. Such chemical modification results to acidic variants of the antibodies. The increased level of acidic variants can affect the biophysical and biochemical properties in some cases the bioactivity of the protein (Prade et.al. Cysteine in cell culture media induces acidic IgG1 species by disrupting the disulfide bond network. Biotech and Bioeng. 2021;118:1091–1104).
Secukinumab is a recombinant fully humanized monoclonal immunoglobulin G1 (IgG1)/? antibody that selectively targets IL-17A and blocks its interaction with the IL-17 receptor. It has a non-canonical cysteine present at 97th position in the light chain (CysL97) of secukinumab (WO2016103146). This cysteine is in the CDR3 region of the light chain of the antibody. Any chemical modification of these non-canonical cysteines resulting to high levels of acidic variants that may negatively affect the activity and stability of the antibody.
The present invention discloses a method to modulate these chemical modifications of the non-canonical free cysteine. The method comprises reduction of cysteinylation and/or glutathionylation in a cell culture process expressing an antibody composition using cell culture medium having low cystine content.
SUMMARY OF THE INVENTION
The present invention relates to an antibody composition comprising antibody variants resulted due to chemical modifications, which are the critical quality attributes (CQA) of the antibody. The invention also discloses a method to reduce the % of acidic variants in an antibody composition by culturing mammalian cells in cell culture medium having low cystine content. The present invention provides a method to reduce cysteinylated and/or glutathionylated variants in an antibody composition by using a cell culture process comprising culturing mammalian cells producing the said antibody in cell culture medium having low cystine content.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
The term “about” refers to a range of values that are similar to the stated reference value to a range of values that fall within 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 percent or less of the stated reference value.
The term “antibody” as used herein encompasses whole antibodies and any antigen binding fragment (i.e., “antigen-binding portion”) or single chains or fusion protein thereof. An “antibody” refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, or an antigen binding portion thereof.
The term "biosimilar" refers to a recombinant pharmaceutical protein, commonly with identical amino acid sequence to a reference product that contains, similar, very similar to or same post-translational modifications as the reference product yielding no clinically meaningful difference in terms of safety, purity and potency.
The term “canonical cysteine” refers to a cysteine residue in an antibody sequence which are involved in evolutionary conserved disulfide linkages described in immunoglobulins.
The term “cell culture process” as used herein refers to a process of culturing a population of cells that are capable of producing recombinant protein of interest or antibody.
The term “charge variants” i.e. variant species having acidic or basic charge due to various post translational modification can be detected by Ion exchange chromatography (IEX) or Isoelectric focusing (IEF). The term ‘main peak’ refers to the peak that elutes in abundance (major peak) during ion exchange chromatography. In particular, during cation exchange chromatography (CEX), the peak that elutes earlier than the main peak has charge that is acidic relative to main peak and is termed as acidic variant peak (ACV). The peak that elutes later than the main peak has charge that is basic relative to the main peak and is termed as basic variant peak (BCV).
The term “cysteinylation” refers to a protein modification that effectively converts an L-cysteine residue to S-(L-cysteinyl)-L-cysteine, forming a disulfide bond with free cysteine. The “cysteinylated variant” refers to the thiol variant comprising such bonded cysteine in their amino acid chain.
The term “free cysteine” refers to a cysteine that is not involved in disulfide bonding. These include free cysteine residues resulting due to reduction of the cysteines involved in conserved disulfide linkages in immunoglobulins and native free cysteine residues that are not involved in conserved disulfide linkages described in immunoglobulins.
The term “glutathionylation” refers to modification of protein wherein formation of disulfide bond between protein cysteines and glutathione (GSH) cysteine takes place. The term “glutathionylated variant” refers to the variant comprising such bonded cysteine with glutathione.
The term “target/predetermined value” refers to the quantifiable value for antibody characteristic that is indicative of its biosimilarity of a biosimilar antibody to the ‘reference product’.
The term “thiol variant” refers an antibody variant that may result due to chemical modification of sulfhydryl group present in free cysteine in the antibody. The exemplary chemical modifications include cysteinylation, cystinylation and glutathionylation.
DETAILED DESCRIPTION OF THE EMBODIMENTS
The present invention discloses a method of reducing the % of acidic variants in an antibody composition. Further, the present invention discloses a method of reducing cysteinylation and/or glutathionylation in a cell culture process producing the antibody composition by using cell culture medium having low cystine content.
Any mammalian cell or cell type which is suitable for expression of recombinant proteins in a cell culture medium may be used for the present invention. Non-limiting examples of mammalian cells that may be used with the present invention include Chinese hamster ovary (CHO) cells, baby hamster kidney (BHK21) cells and murine myeloma cells (NS0 and Sp2/0) human retinoblasts (PER.C6 cell line), human embryonic kidney cell line (HEK-293 cell line) (Dumont, J., et al., Human cell lines for biopharmaceutical manufacturing: history, status, and future perspectives. Crit Rev Biotechnol, 2016. 36(6): p. 1110-1122). In a preferred embodiment, CHO cell lines expressing recombinant proteins may be used in accordance with the present invention.
Cell culture medium is understood by those skilled in the art to refer to a nutrient solution in which cells, such as animal or mammalian cells, are grown. A cell culture medium generally includes one or more of the following components: an energy source (e.g., a carbohydrate such as glucose); amino acids; vitamins; lipids or free fatty acids; and trace elements, e.g., inorganic compounds or naturally occurring elements in the micromolar range. Cell culture medium can also contain additional components, such as hormones and other growth factors (e.g., insulin, transferrin, epidermal growth factor, serum, and the like); salts (e.g., calcium, magnesium and phosphate); sugars (e.g. mannose, galactose, fucose); amino acids (glutamine); buffers (e.g., HEPES); nucleosides and bases (e.g., adenosine, thymidine, hypoxanthine); antibiotics ( e.g., gentamycin); and cell protective agents ( e.g., a Pluronic polyol (Pluronic F68). Commercially available media can be utilized in accordance with the present invention, for example, Dulbecco's Modified Eagles Medium (DMEM, Sigma-Aldrich); RPMI-1640 Medium (Sigma-Aldrich); EX-CELL® Advanced CHO Fed-batch Medium (Sigma-Aldrich); Cell Boost™ 7a and 7b (GE Healthcare Bio-Sciences AB). One skilled in the art would appreciate that some cell culture media are suited to support cells through their initial growth phase (basal medium) while some sustain cells through the later growth phase and production phase of cell culture (feed medium), and would be able to choose appropriate culture medium.
The methods described in the present invention are in recognition of the fact that various parameters of the cell culture process may be used to obtain antibody composition of desired antibody composition. In an embodiment, the method of the present invention envisages a cell culture process to reduce cysteinylation and/or glutathionylation, the method comprising culturing mammalian cells in a cell culture medium having low cystine content.
A person of ordinary skill in the art would be able to characterize and analyse the various antibody variants present in the antibody composition produced by the cell culture process described herein using the state of the art techniques.
In an embodiment the present invention provides a method of reducing acidic variant content in an antibody composition, the method comprising culturing the cells producing the said antibody in a lower cystine containing cell culture medium and the said reduction in acidic variant is in comparison with an antibody composition produced in cell culture medium having higher cystine content. In an embodiment, the acidic variants in the said antibody composition comprises of thiol variants.
In an embodiment, the present invention discloses a method of producing an antibody composition, the method comprises culturing the mammalian cells expressing the antibody in cell culture medium having low cystine content, wherein the % of acidic variant of the antibody is about 18.0% and/or the % of main species of the antibody is about 74%.
In an embodiment the invention of the present application provides a method of producing a homogenous antibody composition comprising of a thiol variant, wherein the said method comprises culturing of cells producing the said antibody in a low cystine containing cell culture medium.
In an embodiment, the present invention discloses a method to reduce cysteinylation and/or glutathionylation in an antibody composition, the method comprising culturing mammalian cells in a cell culture medium having low cystine content, wherein the obtained antibody composition comprises reduced cysteinylated and/or glutathionylated variants of the antibody as compared to the antibody composition obtained using cell culture medium having high cystine content.
In an embodiment, the present invention discloses a method to improve an anti-IL-17A antibody composition, the method comprises culturing the mammalian cell in cell culture medium having low cystine content, wherein the improvement in the antibody composition is in terms of increased % of free cysteine variant and decreased % of cysteinylated variant, and decreased % of glutathionylated variant, when compared with the antibody composition produced using cell culture medium having high cystine content.
A method of reducing thiol variant heterogeneity in an antibody composition, wherein the said method comprises culturing the cells producing the said antibody in a low cystine containing cell culture medium and the said reduction in thiol variant heterogeneity is in comparison with an antibody composition produced in cell culture medium having higher cystine content.
In any of the above embodiment, the antibody produced using the present invention is a biosimilar monoclonal antibody.
In an embodiment, the present invention discloses a method to produce a biosimilar antibody composition, wherein the method comprises culturing the mammalian cells expressing the biosimilar antibody in a cell culture medium having low cystine content, thereby the obtained biosimilar antibody composition comprises acidic variant, wherein the % of acidic variant is in a target/predetermined value.
Further, in an embodiment, the present invention discloses a method to produce a biosimilar antibody composition, wherein the method comprises culturing the mammalian cells expressing the biosimilar antibody in a cell culture medium having low cystine content, thereby the obtained biosimilar antibody composition comprises cysteinylated and/or glutathionylated variants of the antibody, wherein the % of cysteinylated and/or glutathionylated variants are in target/predetermined value.
In an embodiment, the method of the present invention is applicable to antibody composition comprising thiol variants wherein the thiol variants arise from presence of free cysteine or chemical modification of free sulfhydryl present on free cysteines derived from other wise disulfide bonded cysteines and/or non-canonical free cysteines. In another embodiment, the method of the present invention is applicable to antibody composition of antibodies having a non-canonical cysteine residue in the light chain. In yet another embodiment, the said non-canonical residue is in the CDR region of the light chain, preferably in the CDR3 region of the light chain. In preferred embodiment, the method of the present invention is applicable to antibody composition of antibodies having a non-canonical cysteine at 97th position of light chain, the position being designated according to Kabat numbering scheme.
In yet another embodiment, the method of present invention is applicable to antibody molecules which have more than one cysteine residues that can give rise to thiol variants, and wherein these multiple cysteine residues are homogenously modified or have mix of possible chemical modification. For example, an antibody molecule having a free cysteine in each of its light chain, each of the cysteine residue has same modification e.g. cysteinylation, or has different modification on each light chain e.g. cysteinylation in one light chain while glutathionylation in other light chain.
In an embodiment, the thiol variants in said antibody composition comprises from the variant/variants selected from the group comprising free cysteine variant, cysteinylated variant, glutathionylated variant, having intramolecular or intermolecular disulfide bond scrambling, antibody dimer variants.
In a preferred embodiment, the method of the present invention results in a homogenous antibody composition wherein the free cysteine variant is the most abundant species present in the composition. In a preferred embodiment, the method of the present invention reduces the thiol variants heterogeneity arising due to modification of the free cysteine in the said antibody composition and the resultant composition is essentially free of any modification of the free cysteines.
In an embodiment, the cell culture process disclosed in the present invention further comprises of a temperature shift from 370C to 320C. Further, the temperature shift in the present invention is performed on day 5.
In an embodiment, the cell culture process disclosed in the present invention further comprises of supplementation of glucose.
In an embodiment, the cell culture disclosed further comprises of addition of feed medium on day 3, 5, 7, 9, 11 and 13.
In an embodiment, the mammalian cell used to produce the antibody composition of the present invention is Chinese hamster ovary (CHO) cells.
In an embodiment, the antibody produced using the cell culture process of the present invention is an anti-IL-17A antibody. In a preferred embodiment, the antibody produced using the cell culture method of the present invention has the following light and heavy chain amino acid sequences:
> Light Chain
EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPCTFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
> Heavy Chain
EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYWMNWVRQAPGKGLEWVAAINQDGSEKYYVGSVKGRFTISRDNAKNSLYLQMNSLRVEDTAVYYCVRDYYDILTDYYIHYWYFDLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
EXAMPLES:
Example I
An anti-IL-17A antibody having light chain and heavy chain as described above was cloned and expressed in a recombinant CHO (rCHO) cell line using techniques described in detail in “Molecular Cloning: A Laboratory Manual (Fourth Edition)”. rCHO cells expressing the antibody were seeded at a density of about 0.5 million cells/mL in basal cell culture medium I and cultured at a temperature of about 37°C. The cell culture medium was supplemented with glucose. A temperature shift was applied on day 5, thereby reducing the culture temperature to about 32°C. The feed medium I was added on day 3, 5, 7, 9, 11 and 13. The culture was harvested on day 13. The cystine content in basal cell culture medium and feed medium is given in table 1. The titer of the antibody produced is given in table 2. The charge variant content and the thiol variant content in the antibody composition produced is given in table 2 and 3 respectively.
Example II
The cell culture process described in Example I was carried with following modifications. Basal cell culture medium II and the feed medium II was used for culturing cells. The culture was harvested on day 14. The titer of the antibody produced is given in table 2. The charge variant content and the thiol variant content in the antibody composition produced is given in table 2 and 3 respectively.
Table 1. Cystine content in the cell culture medium used.
Examples Media & Feed Cystine content Total Cystine content
I Basal Medium I 0.25 mM 3.15 mM
Feed I 2.9 mM
II Basal Medium II 0.46 mM 1.08mM
Feed II 0.62 mM
Table 2. Titer of the antibody
Examples Titer (g/L)
I 4.2
II 4.5
Table 3. Charge variants present in antibody composition
Examples Acidic Variants Main species Basic Variants
Cosentyx 12.7 % 77.2 % 10.1 %
I 39.5 % 57.6 % 2.9 %
II 18.0% 74.3 % 7.7 %

Table 4. Thiol variants present in antibody composition.
Examples Free Cys 1 Cys 2 Cys 1 GSH 2GSH 1 Cys +
1 GSH
Cosentyx 98.9 % 0.0 % 0.0 % 0.0 % 0.7 % 0.4 %
I 68.9 % 30.8 % 11.3 % 8.9 % 0.6 % 4.1 %
II 100 % 0.0 % 0.0 % 0.0 % 0.0 % 0.0 %

,CLAIMS:We claim:
1. A method for reducing acidic variant content in an antibody composition, the said method comprising
culturing mammalian cells producing the said antibody composition in a low cystine containing cell culture medium,
to obtain an antibody composition comprising reduced % of acidic variant in the antibody composition as compared to the composition of the said antibody obtained using a cell culture medium having high cystine content.
2. An antibody composition obtained from the method in claim 1 which is comprised of about 18.0% acidic variants and/or about 74% main species of the antibody.
3. A method for reducing cysteinylation and/or glutathionylation in an antibody composition, wherein the method comprising
culturing mammalian cells producing the said antibody composition in a low cystine containing cell culture medium,
wherein the reduction in cysteinylated variants and/or glutathionylated variants of the antibody is in comparison to the composition of the said antibody obtained using a cell culture medium having high cystine content.
4. A method for reducing thiol variant heterogeneity in an antibody composition produced in a cell culture, the method comprising
culturing mammalian cells producing the said antibody composition in a low cystine containing cell culture medium,
wherein the reduction is in comparison to composition of the said antibody obtained using cell culture medium having high cystine content.
5. A method for reducing thiol variant heterogeneity in an antibody composition as claimed in claim 4, wherein the thiol variant heterogeneity is imparted by free cysteine variants, cysteinylated variants and/or glutathionylated variants.
6. A method for reducing thiol variant heterogeneity in an antibody composition as claimed in claim 5, wherein the reduced heterogeneity of thiol variants in the antibody composition is in terms of increased % of free cysteine variants and/or decreased % of cysteinylated variants and/or decreased % of glutathionylated variants.
7. A method for reducing thiol variant heterogeneity in an antibody composition as claimed in claim 4-6, wherein the free cysteine variant is the most abundant species of thiol variants in the antibody composition.
8. A method for producing an antibody composition as claimed in the preceding claims, wherein the antibody produced has a non-canonical cysteine residue at 97th position of light chain, as designated according to kabat numbering scheme.
9. A method for producing a biosimilar antibody composition in a cell culture, wherein the method comprising
culturing mammalian cells producing the said biosimilar antibody composition in a low cystine containing cell culture medium,
wherein the said biosimilar antibody has a non-canonical cysteine residue at 97th position of light chain, as designated according to kabat numbering scheme, and is comprised of acidic variants, cysteinylated variants and/or glutathionylated variants in a target/predetermined value.

10. A method for producing an antibody composition in a cell culture as claimed in the preceding claims, wherein the antibody produced is an anti-IL-17A antibody.
11. A method for producing an antibody composition in a cell culture as claimed in the preceding claims, wherein the method comprises a temperature shift from 370C to 320C.
12. A method for producing an antibody composition in a cell culture as claimed in the preceding claims, wherein the method comprises addition of feed medium on day 3, 5, 7, 9, 11 and 13 of the cell culture.