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 a pharmaceutical composition. The invention provides a mammalian cell culture process comprising culturing the mammalian cells at a pH range of 6.7 to 7.2 and performing a temperature shift, to obtain an antibody composition comprising charge variant.

BACKGROUND OF THE INVENTION

Monoclonal antibodies (mAbs) are the fastest growing pharmaceutical products used for diseases like cancer, autoimmune, cardiovascular and inflammatory disorders. They are produced in mammalian cells like Chinese hamster ovary (CHO) cells since they have post-translational modifications (PTMs) which are integral to the effector function of the therapeutic antibody molecules. PTMs refer to enzymatic modification of proteins following its translation which generally result in mature protein/antibody. It may also lead to various variants viz. glycoforms, charge variants and size variants. As a result of these modifications, the structure, stability and function of the proteins are altered. Hence, it is critical to monitor the levels of these variants for the antibody to maintain its biological activity and stability.

Vedolizumab, a recombinant humanized lgG1 monoclonal antibody binding to human a4[37 integrin, is approved for the treatment of moderate to severely active ulcerative colitis and Crohn’s disease in adults who have failed at least one conventional therapy. It consists of an asparagine-linked glycosylation site on each of the heavy chain. (Wyant et. al. An Overview of the Mechanism of Action of the Monoclonal Antibody Vedolizumab. Journal of Crohn's and Colitis, 2016, 1437-1444). Biosimilars , sometimes called similar biological medicinal product or ‘follow-on biologic’ or ‘subsequent entry biologic’, include therapeutic mAbs which are similar to already licensed therapeutic product (the ‘reference product’). Regulatory agencies mandate that biosimilars must demonstrate high similarity to the reference product, in terms of quality characteristics, biological activity, safety and efficacy, in order to have marketing approval (Sullivan, P.M. and L.M. DiGrazia, Analytic characterization of biosimilars. Am J Health SystPharm, 2017. 74(8): p. 568-579; Guttman et. al. Assessing Glycosimilarity of Biotherapeutics. 2018 https://sciex.com/content/dam/SCIEX/pdf/tech- notes/all/Glycosimilarity. pdf) .

Therefore, the present invention relates to a cell culture process for producing an antibody composition, the process comprising culturing mammalian cells at a pH range of 6.7 to 7.2, performing a temperature shift from a first culture temperature to a second culture temperature, thereby obtaining an antibody composition comprising charge variants, wherein the percentage of basic charge variants is not less than 16%.

SUMMARY OF THE INVENTION

The present invention relates to a cell culture process for producing a pharmaceutical monoclonal antibody composition, the process comprising culturing mammalian cells expressing the monoclonal antibody at a pH range of about 6.7 to about 7.2, performing a temperature shift from a first culture temperature to a second culture temperature, thereby obtaining an antibody composition comprising charge variant. Further, the present invention discloses that the obtained antibody composition comprises of basic charge variants, wherein the percentage of basic charge variants is not less than 16%.

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” or “monoclonal antibody” refers to an intact antibody or an antigen binding fragment thereof.

The term “antibody composition” refers to a population of antibody molecules or fragments thereof that is produced by mammalian cell culture. The population of antibody molecules may have one or several post translational modifications (PTM), imparting the antibody molecules a different molecular weight, charge, solubility or combinations 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 “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 reference product refers to a currently or previously marketed recombinant protein, also described as the "originator product" or "branded product" serving as a comparator in the studies.

The term “temperature shift” refers to the change in temperature during the cell culture process.

DETAILED DESCRIPTION OF THE EMBODIMENTS

The present invention provides a cell culture process for culturing mammalian cells expressing an antibody, the process comprising culturing the cells at a pH range of 6.7 to 7.2 and lowering of temperature of the cell culture from a first temperature to a second temperature, to obtain antibody composition comprising charge variants.

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 (NSO 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 Biotech not, 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 comprising various variants. In an embodiment, the cell culture process envisages culturing the cells at a pH range of about 6.7 to about 7.2 and performing a temperature shift.

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 discloses a cell culture process for producing an antibody composition, wherein the said process comprises a) providing mammalian cells expressing the said antibody b) culturing the cells at a pH range of about 6.7 to about 7.2 c) performing a temperature shift during the cell culture from a first culture temperature to a second culture temperature, wherein the second culture temperature is lower than the first culture temperature d) recovering the said recombinant antibody composition wherein the antibody composition produced comprises of not less than 16.45% basic charge variants.

In an embodiment, the present invention discloses a cell culture process for producing an antibody composition, wherein the said process comprises a) providing mammalian cells expressing the said antibody b) culturing the cells at a pH range of about 6.7 to about 7.2 c) performing a temperature shift during the cell culture from a first culture temperature to a second culture temperature, wherein the second culture temperature is lower than the first culture temperature d) recovering the said recombinant antibody composition wherein the antibody composition produced comprises of about 19.1 % acidic variants, about 16.45% or more basic charge variants.

In an embodiment, the present invention discloses a cell culture process for producing an antibody composition, wherein the said process comprises a) providing mammalian cells expressing the said antibody b) culturing the cells at a pH range of about 6.7 to about 7.2 c) performing a temperature shift during the cell culture from a first culture temperature to a second culture temperature, wherein the second culture temperature is lower than the first culture temperature d) recovering the said recombinant antibody composition wherein the antibody composition produced comprises of about 19.1 % acidic variant, about 64.5% main species of the antibody and about 16.45% of basic variant.

In any of the embodiments mentioned above, the temperature shift is performed on day 5 of the cell culture, day 6 of the cell culture, or day 7 of the cell culture. In yet another embodiment, the temperature shift is performed on day 6 of the cell culture.

In any of the embodiments mentioned above, the difference between the first culture temperature and the second culture temperature is about 6.5. In another embodiment, the culture temperature before the temperature shift is about 37°C and the culture temperature after the temperature shift is about 30°C.

In a further embodiment, the antibody produced in any of the above embodiment is a biosimilar antibody.

In yet another embodiment, the antibody produced using the present invention is an anti a4[37 integrin antibody. In a preferred embodiment, the antibody produced using the present invention is vedolizumab.

Those skilled in the art will recognize that several embodiments are possible within the scope and spirit of this invention. The invention will now be described in greater detail by reference to the following non-limiting example. The following example further illustrates the invention but, of course, should not be construed as in any way limiting its scope.

EXAMPLE

An anti-a4[37 integrin antibody having a heavy chain and light chain as described in W02007061679A1 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 and cultured at an initial temperature of 36.5°C. The pH range of the cell culture is maintained between pH 6.7 to 7.2. On day 6, a temperature shift was performed, thereby reducing the culture temperature to 30°C. The cell culture was added with 25% feed medium. The culture was harvested on day 13. The antibody composition and titer obtained is depicted in Table 1 .

Table 1. The composition and titer of the anti-a4[37 integrin antibody.