DESC:The following specification particularly describes the invention and the manner in which it is to be performed:
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CELL CULTURE PROCESS
INTRODUCTION
The present invention relates to the field of cell culture and in particular to a cell 5 culture process for controlling post translational modifications (PTM’s) in an antibody composition.
BACKGROUND OF INVENTION
Post-translational modifications (PTM’s) are covalent modification that occur in most eukaryotic proteins during or after their biosynthesis and they enrich the structural and 10 biophysical diversity of proteins. PTM’s may be classified according to the modification involved: the addition of functional groups (e.g., phosphorylation and glycosylation); attachment of other polypeptides (e.g., ubiquitination and SUMOylation); changing of the chemical nature of amino acids (e.g., acetylation, deamidation and oxidation); and cleavage of the backbone by proteolysis. 15 A common subset of PTM associated with biopharmaceuticals are charge variants. Modifications such as deamidation, sialylation, formation of various types of covalent adducts, e.g., glycation, and C-terminal lysine cleavage result in an increase in the net negative charge on the mAbs causing a decrease in pI value of the protein, and thereby leading to formation of acidic variants. On the other hand, basic variants arise due to no 20 cleavage/ presence of C-terminal lysine or glycine amidation, succinimide formation, amino
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acid oxidation or removal of sialic acid, which introduce additional positive charges or removal of negative charges. In particular, the acidic species has been demonstrated to have low binding response and binding potency as compared to the main or basic peak. It may thus be desirable to control the acidic species to ensure adequate efficacy in the therapeutic antibody composition. Also, 5 the same has been mandated by the regulatory agencies for biopharmaceutical products. SUMMARY OF THE INVENTION
The present invention describes a cell culture process for controlling the % of acidic variants in an antibody composition, the process comprising modulating cumulative concentration of vitamins and/or organic acids in the culture media used for culturing the cells producing the 10 said antibody composition. In particular, the invention discloses a cell culture process for controlling the % of acidic variants in an antibody composition, the process comprising modulating cumulative concentration of vitamins and/or organic acids in the culture media used for culturing the cells producing the said antibody composition and the culture process is maintained at the same temperature for the entire duration of the cell culture. The disclosed 15 cell culture process does not comprise a step of temperature shift for achieving the said reduction in acidic variant content.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
The term “cell culture process” as used herein refers to a process of culturing a population 20 of cells that are capable of producing recombinant protein of interest or antibody.
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The term “culture media” refers to a solid, liquid or semi-solid containing nutrients to support the growth of cells. Culture media may be chemically defined or alternatively may contain undefined components such as hydrolysates. Typically, a cell culture media contains amino acids, energy sources, lipids, vitamins, growth factors, metal ions and other trace elements etc. The culture medium is usually formulated to a particular osmolality and pH 5 values that are optimal for a particular cell line.
“Antibody composition” refers to a population of antibody molecules or fragments thereof. 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. 10
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 15 (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.
DETAILED DESCRIPTION OF THE EMBODIMENTS
The present invention discloses a cell culture process for controlling the % of acidic variants 20 in an antibody composition, the process comprising modulating cumulative concentration of
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vitamins and/or organic acids in the culture media used for culturing the cells producing the said antibody composition.
An embodiment of the invention discloses a cell culture process for controlling the % of acidic variants in an antibody composition, the process comprising modulating cumulative concentration of vitamins and/or organic acids in the culture media used for culturing the 5 cells producing the said antibody composition and the culture process is maintained at the same temperature for the entire duration of the cell culture.
Another embodiment of the invention discloses a cell culture process for controlling the % of acidic variants in an antibody composition, the process comprising modulating cumulative concentration of vitamins and/or organic acids in the culture media used for culturing the 10 cells producing the said antibody composition and the cell culture process does not comprise a temperature shift.
In any of the above mentioned embodiments, the cell culture is maintained at a temperature of 35oC for the entire culture duration till the harvest of the cell culture.
In any of the above mentioned embodiments, the cells used are mammalian cells, more 15 particularly Chinese Hamster Ovary (CHO) cells.
In any of the above mentioned embodiments, the antibody so obtained is TNF-a antibody, more particularly adalimumab.
In any of the above mentioned embodiments, % of acidic variants is controlled to a value less than or equal to 28%. 20
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In any of the above mentioned embodiments, the modulation of cumulative concentration of vitamins comprises an increase in cumulative concentration of vitamins.
In any of the above mentioned embodiments, the modulation of cumulative concentration of organic acid comprises a decrease in cumulative concentration of organic acids.
In any of the above mentioned embodiments, the modulation of cumulative concentration of 5 vitamins and organic acid comprises an increase in cumulative concentration of vitamins and decrease in cumulative concentration of organic acids.
In any of the above mentioned embodiments, the cumulative concentration of vitamins is greater than about 1000 µM.
In any of the above mentioned embodiments, the cumulative concentration of organic acid 10 is less than about 4 µM.
In any of the above mentioned embodiments, the culture media comprises of one or more vitamins selected from a group consisting of biotin, choline, cyano-cobalamin, folic acid, niacin, nicotinamide, p-aminobenzoic acid, panthotenic acid, pyridoxal, pyridoxamine, pyridoxine, riboflavin, thiamine. 15
In any of the above mentioned embodiments, the culture media comprises of one or more organic acids selected from a group consisting of acetic acid, butyric/2-hydroxy-butyric acids, 3-hydroxybutyric acid, citric acid, formic acid, fumaric acid, isovaleric acid, lactic acid, maleic acid, propionic acid, pyruvic acid, succinic acid, acetone, ethanol, pyroglutamic acid. 20
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The cumulative concentration of vitamins or organic acids in the culture media could be achieved by selection of appropriate basal media and feed.
The cumulative concentration of vitamins in the culture media could be achieved by supplementation of vitamins as individual components.
The cell culture media that are useful in the application include but are not limited to, the 5 commercially available products PF CHO (HyClone®), PowerCHO® 2 (Lo1nza), Zap-CHO (Invitria), CD CHO, CDOptiCHOTM and CHO-S-SFMII (Invitrogen), ProCHOTM (Lonza), CDM4CHOTM (Hyclone), ActiPro (HyClone), Ex-CELL CDCHO Fusion (Merck, Sigma), Ex-CELL Advanced CHO fed batch medium (Merck, Sigma), Ex-CELL Cell Vento 4 CHO medium (Merck Sigma), Dynamis™ AGT™ Medium (Thermo Fisher Scientific) 10 DMEM (Invitrogen), DMEM/F12 (Invitrogen), Ham’s F10 (Sigma), Minimal Essential Media (Sigma), and RPMI -1640 (Sigma) and IS CHO-CD G10.3 (Irvine scientific).
The feed or feed medium in the present invention may be added in a continuous, profile or a bolus mode. One or more feeds may be added in one manner (e.g. profile mode), and other feeds in second manner (e.g. bolus or continuous mode). Further, the feed may be composed 15 of nutrients or other medium components that have been depleted or metabolized by the cells. The feed may be concentrated form of initial cell culture media itself or may be a different culture media. The components may include hormones, growth factors, ions vitamins, nucleoside, nucleotides, trace elements, amino acids, lipids or glucose. Supplementary components may be added at one time or in series of additions to replenish 20 the depleted components. Thus the feed can be a solution of depleted nutrient(s), mixture of nutrient(s) or a mixture of cell culture medium/feed providing such nutrient(s).
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The cell culture feed that are useful in the invention include but are not limited to, the commercially available products EfficientFeed A+ AGT (Product Code A2502301), EfficientFeed B+ AGT (Product Code A2503004) and EfficientFeed C+ AGT (Product Code A2503101) (Thermo Fisher Scientific) Cell Boost 2 (CB-2, GE Healthcare Bio-Sciences AB, Hyclone, Product Code, SH 30596.03), Cell Boost 4 (CB-4, GE Healthcare 5 Bio-Sciences AB , HyClone, Product Code. SH30928), Cell Boost 7a (CB7a, GE Healthcare Bio-Sciences AB, Hyclone, Product Code, SH31026) Cell Boost 7b (CB7b, E Healthcare Bio-Sciences AB, Hyclone, Product Code, SH31027) PF CHO (Thermo Scientific Hyclone, Product Code, SH30333.3).
Certain aspects and embodiments of the invention are more fully defined by reference to the 10 following examples. These examples should not, however, be construed as limiting the scope of the invention.
EXAMPLES
Example – I
An anti-TNF-a antibody was cloned and expressed in a culture media using a CHO cell line 15 as detailed in Molecular Cloning: A laboratory Manual by Green and Sambrook. rCHO cells expressing antibody were seeded at a density of ~0.3 million cells/ml in culture media-I. The cumulative concentration of the vitamins and the organic acids in culture media is given in Table-I. The temperature was initiated at a temperature of 37oC and subsequently on day 4 the temperature was lowered to either one of the temperature values; 31oC on day 4 (Example 20 IA), or 34oC on Day 4 (Example IB). The subsequent lowered temperature value was maintained till the harvest of the cell culture.
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In an alternate, the temperature was initiated at temperature of 35oC (Example IC) and the same temperature was maintained till the harvest of the cell culture.
The culture was harvested on day 12 or at viability less than or equal to 50%, whichever was earlier.
The percentage of acidic variants in the antibody composition produced in each example 5 were determined and are given in Table II.
Example – II
An anti-TNF-a antibody was cloned and expressed in a culture media using a CHO cell line as detailed in Molecular Cloning: A laboratory Manual by Green and Sambrook. rCHO cells expressing antibody were seeded at a density of ~0.3 million cells/ml in culture media-II. 10 The cumulative concentration of the vitamins and the organic acids in culture media is given in Table-I. The temperature was initiated at a temperature of 37oC subsequently on day 4 the temperature was lowered to either one of the temperature values; 31oC on day 4 (Example IIA), or 34oC on day 4 (Example IIB). The subsequent lowered temperature value was maintained till the harvest of the cell culture. 15
In an alternate the temperature was initiated at temperature of 35oC (Example IIC) and the same temperature was maintained till the harvest of the cell culture.
The culture was harvested on day 12 or at viability less than or equal to 60%, whichever was earlier.
The percentage of acidic variants in the antibody composition produced in each example 20 were determined and are given in Table II.
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Media
Vitamins
(µM)
Organic acids
(µM)
Media-I
1101
3.5
Media-II
358
12.5
Table - I: Cumulative concentration of vitamins and organic acid in Media-I and Media-II
Example
% Acidic variants
Example IA
33.5
Example IB
32.2
Example IC
28.8
Example IIA
31.9
Example IIB
31.7
Example IIC
42.8
Table - II: % of acidic variants in the antibody composition as obtained in Example IA-C and Example IIA-C ,CLAIMS:1. A method of producing an antibody composition comprising acidic variants of the antibody, the said method comprising:
culturing a host cell expressing the said antibody in a cell culture media comprising vitamins and organic acids, thereby producing the said composition with lower % of acidic variants as compared to a control composition,
wherein the said control is produced by culturing a host cell expressing the said antibody in control cell culture media comprising vitamins at lower cumulative concentration, and organic acids at higher cumulative concentration than the cell culture media used to obtain the aforesaid antibody composition.
2. The method of the claim 1, wherein the % of acidic variants in the antibody composition produced is less than 30%.
3. The method of the preceding claims, wherein the culture media comprise of one or more vitamins selected from a group consisting of biotin, choline, cyano-cobalamin, folic acid, niacin, nicotinamide, p-aminobenzoic acid, panthotenic acid, pyridoxal, pyridoxamine, pyridoxine, riboflavin, thiamine.
4. The method of claim 3, wherein the cell culture media used comprise a cumulative concentration of vitamins greater than about 1000 µM.
5. The method of the preceding claims, wherein the culture media comprise of one or more organic acids selected from a group consisting of acetic acid, butyric/2-hydroxy-butyric acids, 3-hydroxybutyric acid, citric acid, formic acid, fumaric acid, isovaleric acid,
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lactic acid, maleic acid, propionic acid, pyruvic acid, succinic acid, acetone, ethanol, pyroglutamic acid.
6. The method of claim 5, wherein the cumulative concentration of organic acid is less than about 4 µM.
7. The method of the preceding claims, wherein the cell culture is maintained at a same temperature for its entire duration.
8. The method of claim 7, wherein the temperature maintained is 35oC.
9. The method of the preceding claims, wherein the antibody produced is anti-TNF-a antibody.
10. The method of the preceding claims, wherein the cells used are Chinese Hamster Ovary (CHO) cells.