A process for producing FC-fusion protein in mammalian cell culture

Abstract

The present disclosure relates to an improved process to control product quality parameters selected from total sialyation profile and increased main peak percentage of the Fc-Fusion protein specifically CTLA4-IgG (Abatacept) during production and / or at harvest level Furthermore, the present disclosure also reduces and / or decreases low molecular weight species (LMWs) and improves titer of the fusion protein during the mammalian cell culture.

Description

[0001] A PROCESS FOR PRODUCING FC-FUSION PROTEIN IN MAMMALIAN CELL CULTURE

[0002] FIELD OF INVENTION:

[0003] The present disclosure relates to the process for production of a Fc- fusion protein. Particularly the process relates to control product quality parameters selected from total sialyation profile and / or galactosylation profile and increased main peak percentage of the Fc-Fusion protein specifically, CTLA4-IgG during the mammalian cell culture and / or at harvest level. Furthermore, the present process also reduces and / or decreases low molecular weight species (LMWs) and improves titer of the fusion protein during the mammalian cell culture.

[0004] BACKGROUND OF INVENTION:

[0005] Mammalian cell cultures, such as Chinese hamster ovary (CHO) cells, are extensively used in the commercial production of therapeutic biomolecules, including monoclonal antibodies and other proteins. Various process parameters, such as pH, temperature, pO2, CO2, air flow, feeding, and agitation rate, are critical for sustaining cells at high productivity levels. Among these, temperature and pH are particularly crucial and must be optimized to enhance cell growth, viability, and productivity for generating therapeutic biomolecules. Optimizing fed-batch culture conditions for producing Fc-Fusion proteins with higher titer and improved quality is complex and often specific to the biotherapeutic molecule in question.

[0006] Recombinant protein products are increasingly recognized for their medical and clinical significance in therapeutic, treatment, and prophylactic applications. Thus, developing reliable cell culture processes for the production of fusion protein CTLA4 IgGl is challenging and optimization of the cell culture duration, selection and addition of feed, and respective concentration and ratio requires substantial scientific efforts and thought process which are not expected or predicted by the skilled person. The process conditions are important to achieve higher viable cell densities, resulting in increased final protein product concentration, addresses both a key goal and a pressing need in the field.

[0007] The present invention offers an enhanced cell culture process for producing CTLA4-IgGl during the production phase while controlling product quality attributes, such as total sialylation profile and / or galactosylation profile and increased main peak percentage. Additionally, the invention provides a method for reducing impurities like low molecular weight species (LMWs) and increasing the titer of the fusion protein.

[0008] SUMMARY OF INVENTION:

[0009] In an embodiment, a process for the production of Fc fusion protein comprising; a) culturing a mammalian cell in a cell culture medium, and b) harvesting the Fc fusion protein; wherein the cell culture medium comprises: i.a basal medium and ii.a feed medium wherein the feed medium comprises an initial feed regime and a production feed regime; wherein the production feed regime is supplemented in a less amount than the amount of initial feed regime; wherein the harvested Fc-fusion protein comprises one or more improved characteristics selected from the total sialylation of more than about 50%, more than about 30% of mono sialyation, more than about 15% of bi sialyation, more than about 1% of tri sialyation analysed by hydrophobic interaction liquid chromatography (HILIC), more than about 25% of acidic charged species analysed by anion exchange chromatography (AEX), more than about 25% of total galactosylation analysed by Hydrophilic Interaction Ultra-Performance Liquid Chromatography (HILIC -UPLC)), main peak percentage more than about 50% analysed by CE-SDS, and less than about 25% of total low molecular weight percentage analysed by CE-SDS.

[0010] In an embodiment, wherein the initial feed regime and the production feed regime is supplemented in a suitable ratio during the culturing of mammalian cell.

[0011] In an embodiment, the production feed regime is at least 70%, 75%, or 80% lower in amount than the initial feed regime.

[0012] In an embodiment, the production feed regime is at least 75% lower in amount than the initial feed regime.

[0013] In an embodiment, the ratio of the initial feed regime to the production feed regime is about 7:4 or higher. In an embodiment, the initial feed regime comprises of a primary feeding and a tertiary feeding; wherein the primary feeding and the tertiary feeding is supplemented in a suitable ratio; wherein the tertiary feeding is at least about 10% lower than the primary feeding.

[0014] In one embodiment, the ratio of the primary feeding to the tertiary feeding is at least 10: 1 or less.

[0015] In an embodiment, the production feed regime comprises of a secondary feeding and a quaternary feeding; wherein the secondary feeding and the quaternary feeding is supplemented in a suitable ratio; wherein the quaternary feeding is at least about 10% lower than the secondary feeding.

[0016] In one embodiment, the ratio of secondary feeding to the quaternary feeding is at least 10: 1 or less.

[0017] In an embodiment, the primary feeding comprises a media 1; wherein the secondary feeding comprises a media 1; wherein the tertiary feeding comprises a media 2; and wherein the quaternary feeding comprises a media 2.

[0018] In an embodiment, the cell culture process is for at least more than Day 10 up to Day 16.

[0019] In an embodiment, the cell culture process is for at least more than Day 10 up to Day 14.

[0020] In an embodiment, the cell culture process is for at least more than Day 10 up to Day 12.

[0021] In an embodiment, the initial feed regime is supplemented during growth phase when viable cell density of the cell culture is 0.3 * 106cells / mL.

[0022] In an embodiment, the initial feed regime is supplemented on a suitable day selected from day 2, day 3, day 4, day 5, day 6, day 7 and day 8, if the cell culture duration is Day 12.

[0023] In an embodiment, the production feed regime is supplemented on a suitable day selected from day 7, day 8, day 9, day 10, and day 11, if the cell culture duration is Day 12.

[0024] In an embodiment, the production feed regime is supplemented when the viable cell density of the cell culture reaches at least 18* 106cells / ml. In an embodiment, the primary feeding is at least about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, and about 80% higher than the secondary feeding media.

[0025] In an embodiment, the secondary feeding is at least about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, and about 80% lower than the primary feeding.

[0026] In an embodiment, the tertiary feeding media is at least about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, and about 80% higher than the quaternary feeding

[0027] In an embodiment, the quaternary feeding is at least about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, and about 80% lower than the tertiary feeding.

[0028] In an embodiment, the ratio of the primary feeding to the secondary feeding is at least about 7:4, or about 2: 1. In an embodiment, the ratio of the tertiary feeding to the quaternary feeding is at least about 7:4, or about 2: 1.

[0029] In an embodiment, the cell culture is subjected to a feed medium regimen continuously or periodically.

[0030] In an embodiment, the mammalian cell is cultured in basal medium for at least 24 hours to 48 hours.

[0031] In an embodiment, the basal medium is supplemented with glutamine and poloxamer in a suitable amount.

[0032] In an embodiment, the basal medium is supplemented with glutamine in an amount from about 3mM to about 8mM and with poloxamer in amount from about 2g / L to about 7g / L.

[0033] In an embodiment, the initial viable cell density of the cell culture is from about O. l x lO6cells / mL to about 0.7* 106cells / mL.

[0034] In an embodiment, the initial viable cell density of the cell culture is from about 0.3>< 106cells / mL to about 0.5x l06cells / mL.

[0035] In an embodiment, feeding of the cell culture is initiated when the glucose concentration drops below 6.5g / L .

[0036] In an embodiment, the cell culture is performed at a single temperature of about 37°C ±1.

[0037] In an embodiment, the cell culture is performed at one temperature shift; wherein the temperature shift comprises of a first temperature and a second temperature wherein the second temperature is at least 3 degrees lower than the first temperature. In an embodiment, the first temperature is 37°C.

[0038] In an embodiment, the dissolved oxygen (DO) level of the cell culture is maintained from about 50% to about 70% throughout the process till the harvesting.

[0039] In an embodiment, the pH of the cell culture is maintained from about 6.85 to about 7.15 throughout the process till the harvesting.

[0040] In one embodiment, the pH is 7.

[0041] In an embodiment, the harvesting of Fc-Fusion protein is performed on day 11, or on day 12, or on day 13, and on day 14. In an embodiment, the harvesting of Fc-Fusion protein is performed on day 11, and on day 12.

[0042] In an embodiment, the harvesting of Fc-Fusion protein is performed when the cell viability drops below about 50%, about 55%, about 60%, and about 65%.

[0043] In an embodiment, wherein the harvested Fc-fusion protein shows one or more improved characteristics selected from at least about 50% to about 60% of total sialylation, at least about 30% to 40% of mono sialyation, at least about 15% to about 20% of bi sialyation, at least about 1% to about 5% of tri sialyation analysed by hydrophobic interaction liquid chromatography (HILIC), at least about 25% to about 30% of acidic charged species analysed by Anion exchange chromatography (AEX), at least about 30% to about 35% of total galactosylation analysed by Hydrophilic Interaction Ultra-Performance Liquid Chromatography (HILIC- UPLC)), main peak percentage of at least about 50% to 200% analysed by CE-SDS, and less than about 25% to about 30% of total low molecular weight percentage analysed by CE-SDS.

[0044] In an embodiment, the titer of the harvested Fc-Fusion protein is more than about 50%, analyzed by Protein-A chromatography.

[0045] In an embodiment, the production feed regime is supplemented about 75% less in amount than the amount of initial feed regime; wherein the initial feed regime and the production feed regime is supplemented in a suitable ratio of about 7:4; wherein the initial feed regime comprises a primary feeding and a tertiary feeding, where the tertiary feeding is at least about 10% lower than the primary feeding; where the ratio of primary feeding to tertiary feeding is about 10: 1; wherein the production feed regime comprises of a secondary feeding and a quaternary feeding, where the quaternary feeding is at least about 10% lower than the secondary feeding; where the ratio of secondary feeding to quaternary feeding is about 10: 1; wherein the initial feed regime is supplemented on a suitable day selected from day 3, day 4, day 5, day 6, and day 7, if the cell culture duration is Day 12; wherein the production feed regime is supplemented on a suitable day selected from day 8, day 9, day 10 and day 11, if the cell culture duration is Day 12; wherein the cell culture is subjected to a feed medium regimen continuously; wherein the dissolved oxygen (DO) level of the cell culture is maintained at about 60%; wherein the cells are cultured through fed-batch process in a production bioreactor for a total run period of 12 days; wherein the harvested and purified Fc-fusion protein shows one or more improved characteristics selected from at least about 53% of total sialylation, at least about 35% of mono sialyation, at least about 18% of bi sialyation, at least about 3% of tri sialyation analysed by hydrophobic interaction liquid chromatography (HILIC), at least about 28% of acidic charged species analysed by anion exchange chromatography (AEX), at least about 33% of total galactosylation analysed by Hydrophilic Interaction Ultra-Performance Liquid Chromatography (HILIC- UPLC)), main peak percentage of at least more than 70% analysed by CE-SDS, and less 28% of total low molecular weight percentage analysed by CE-SDS; wherein the titre of the harvested Fc-Fusion protein is more than 51%, analysed by Protein- A chromatography.

[0046] In an embodiment, the Fc-fusion protein is CTLA4-IgG fusion protein, or Abatacept, and Belatacept.

[0047] In an embodiment, the Fc-fusion protein is CTLA4-IgGl fusion protein, or Abatacept.

[0048] In an embodiment, the harvested Fc fusion protein has at least about more than 3% of increased total sialyation when performed with varying feed media compared to the total sialyation when performed without varying feed media.

[0049] In an embodiment, the harvested Fc fusion protein has at least about more than 4% of increased total sialyation when performed with varying feed media compared to the total sialyation when performed without varying feed media.

[0050] In an embodiment, the harvested Fc fusion protein has at least about more than 5% of increased total sialyation when performed with varying feed media compared to the total sialyation when performed without varying feed media. In an embodiment, acomposition of CTLA4-IgGl comprising one or more quality parameters selected from a total sialylation of at least about more than 51%, a mono sialylation of at least about more than 31%, a bi sialylation of at least about more than 16%, a tri sialyation of at least about more than 1%, an acidic variant of at least about more than 26%, a total galactosylation of at least about more than 30%, and a LMW impurities less than about 26%; wherein the main peak is more than 60%. In an embodiment, acomposition of CTLA4-IgGl comprising one or more quality parameters selected from a total sialylation of at least about 51%, a mono sialylation of at least about 31%, a bi sialylation of at least about 16%, a tri sialyation of at least about 1%, an acidic variant of at least about 26%, a total galactosylation of at least about 30%, and a LMW impurities less than about 26%; wherein the main peak is at least about 60%.

[0051] In an embodiment, a composition of CTLA4-IgGl comprising one or more quality parameters selected from a total sialylation of at least 53%, a mono sialylation of at least 35%, a bi sialylation of at least 18%, a tri sialyation of at least 3%, an acidic variant of at least 25% and a LMW impurities less than about 28%; at least about 33% of total galactosylation; wherein the main peak is more than 70%.

[0052] In an embodiment, the titer of harvested CTLA4-IgGl at NPEL stage is more than 51% , analyzed by Protein A chromatography.

[0053] In an embodiment, the harvested CTLA4-IgGl fusion protein is Abatacept.

[0054] In an embodiment, the present disclosure is related to a process that can control the sialylation profile of CTLA4-IgG to obtain desired level of sialylated CTLA4-IgG and galactosylation.

[0055] In an embodiment, the total sialyaled profile comprises mono-sialylated species, bi- sialylated species and tri-sialylated species.

[0056] In an embodiment, mono-sialylated species of CTLA4-IgG comprises about 32% to about 35% of total sialylated species.

[0057] In an embodiment, bi-sialylated species of CTLA4-IgG comprises about 15% to about 20% of total sialylated species.

[0058] In an embodiment, tri-sialylated species of CTLA4-IgG comprises about 1% to about 5% of total sialylated species. In an embodiment, the sialylation profile analysed by hydrophobic interaction liquid chromatography (HILIC).

[0059] In an embodiment, total acidic species of CTLA4-IgG comprises about 23% to about 28% analysed by by anion exchange chromatography (AEX).

[0060] In an embodiment, total galactosylation of CTLA4-IgG comprises about 25% to about 35% by Hydrophilic Interaction Ultra-Performance Liquid Chromatography (HILIC- UPLC)).

[0061] In embodiment, total LMW impurities of the harvested CTLA4-IgG is decreased and / or reduced about 5% to about 200% or more analysed by CE-SDS.

[0062] In an embodiment, the LMW impurities obtained from the cell culture can be further be reduced and / or decreased in subsequent purification steps. The present invention results low and / or decreased LMW impurities, which further reduces and / or lowers the burden on the skilled person during the downstream purification steps.

[0063] In an embodiment, total main peak percentage of the harvested CTLA4-IgG is increased about 5% to about 200% or more analysed by CE-SDS.

[0064] In an embodiment, titer of the harvested CTLA4-IgG is increased and / or improved about 5% to about 100% or more analysed by Protein-A chromatography.

[0065] BRIEF DESCRIPTION OF DRAWINGS:

[0066] Figure 1: depicts the total CTLA4-IgGl harvest titer analysed by Protein-A chromatography.

[0067] Figure 2: depicts the per centage of total sialylated species of CTLA4-IgGl harvest analysed by HILIC.

[0068] Figure 3: depicts the percentage of total mono-sialylated species of CTLA4-IgGl harvest analysed by HILIC.

[0069] Figure 4: depicts the percentage of main peak purity of CTLA4-IgGl harvest analysed by CE-SDS.

[0070] Figure 5: depicts the percentage of total low molecular weight species (LMWs) of CTLA4- IgGl harvest analysed by CE-SDS.

[0071] DETAILED DESCRIPTION OF THE INVENTION: As used herein, the following terms shall have the meanings as indicated below unless otherwise specified or required by context:

[0072] The term “comprises” or “comprising” is used in the present description, it does not exclude other elements or steps. For purpose of the present invention, the term “consisting of is considered to be an optional embodiment, of the term “comprising of . If hereinafter a group is defined to comprise at least a certain number of embodiments, this is also to be understood to disclose a group which optionally consists only of these embodiments.

[0073] As used throughout the specification and in the appended claims, the singular forms “a,” “an,” and “the” include the plural reference unless the context clearly dictates otherwise. The term “polypeptide” or “protein” or “antibody of interest” or “glycosylated protein” refers to monoclonal antibody comprised of polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. The polypeptides are produced by mammalian cell line such as Chinese Hamster Ovary (CHO) cell line.

[0074] The term “CTLA4-IgGl” or “CTLA4IgG” or “Abatacepf ’ used herein refers to the soluble fusion protein comprising an extracellular domain of wild type CTLA4, or a portion thereof that binds B7, joined to an immunoglobulin (Ig) molecule, or a portion thereof. The extracellular domain of CTLA4 or portion thereof is joined to an Ig moiety comprising all or a portion of an immunoglobulin molecule, preferably all or a portion of an immunoglobulin constant region such as all or a portion of IgCyl (IgCgammal), IgCy2 (IgCgamma2), IgCy3 (IgCgamma3), IgCy4 (IgCgamma4), IgCp (IgCmu), IgCal (IgCalphal), IgCa2 (IgCalpha2), IgCS (IgCdelta) or IgCs (IgCepsilon), rendering the fusion molecule soluble. The Ig moiety can include the hinge, CH2 and CH3 domains, or the CHI, hinge, CH2 and CH3 domains, of the aforementioned constant regions or other constant regions. The CTLA4-IgGl molecule further also considered as dimer (having two monomers i.e., the CTLA4 portion and the Fc portion)

[0075] The term “purified” used herein refers to a composition comprising a CTLA4-Ig molecule or a selected population of CTLA4-Ig molecules that is removed from its natural environment (e.g., is isolated) and is at least 90% free, 91% free, 92% free, 93% free, 94% free, 95% free, 96% free, 97% free, 98% free, 99% free, 99.5% free, or 99.9% free from other components, such as cellular material or culture medium, with which it is naturally associated. “Purified” can also refer to a composition comprising a CTLA4-Ig molecule or a selected population of CTLA4-Ig molecules that is removed from its natural environment and is at least 60% free, 65% free, 70% free, 75% free, 80% free, or 85% free from other components, such as cellular material or culture medium, with which it is naturally associated.

[0076] The term “about”, as used herein, is intended to refer to ranges of approximately 5% greater than or less than the referenced value. In certain circumstances, one of skill in the art will recognize that, due to the nature of the referenced value, the term “about” can mean more or less than a 5% deviation from that value.

[0077] The term “and / or” used herein is referred as specific disclosure of each of the two specified features or components with or without the other. Thus, the term “and / or” as used in a phrase such as “A and / or B” herein is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Likewise, the term “and / or” as used in a phrase such as “A, B, and / or C” is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0078] The use of the alternative (e.g., “or”) should be understood to mean either one, both, and any combination thereof of the alternatives. As used herein, the indefinite articles “a” or “an” should be understood to refer to “one or more” of any recited or enumerated component.

[0079] The term “harvested Fc- Fusion protein”, “Harvested composition” refers to a CTLA4- IgGl composition which is not a purified composition, and it is obtained directly from cell culture without the involvement of any purification steps.

[0080] The term “main peak percentage (%)” refers to the purity of the desired protein of interest, in the present invention the desired protein of interest is a Fc-Fusion protein, more specifically a CTLA4-IgG, CTLA4-IgGl molecule. A skilled person in the art can easily understand the true meaning of main peak. The main peak percentage is the peak area that majorly presents in a high amount compared to the pre and post peak.

[0081] The term used “low molecular weight” or “LMW” species which is a protein backbone- truncated fragments & considered as product-related impurities that contribute to the size heterogeneity of fusion protein. LMW species often have low or substantially reduced activity relative to the monomeric form of the fusion protein and can lead to immunogenicity or potentially impact pharmacokinetic properties in vivo. As a result, LMW species are considered critical quality attributes that are routinely monitored during drug development and as part of release testing of purified drug product during manufacturing. In the present invention the “LMW” or “Low molecular weight” are fragments of CTLA4-IgGl molecule, for e.g., CTLA4, the Fc portion etc.

[0082] The term “Cell Viability” or “% Viability” refers to percentage of viable cells out of total number of cells including the viable and non-viable cells at a particular time point in cell culture. In an embodiment, the cell viability refers to the harvest cell viability.

[0083] The term “bioreactor” used herein, refers to a vessel for culturing or growing cells in nutritive media that supports towards a biologically active environment for culturing of cells, equipped with impeller, spargers etc. The environmental conditions inside the bioreactor, such as temperature, nutrient concentrations, pH, and dissolved gases affect the growth and productivity of the organisms monitored, adjusted, and controlled. The cultures can be grown in shake flasks, small scale bioreactors, and / or large-scale bioreactors. The temperature of the cell culture is maintained by a cooling jacket, coils, or both. The pH of the cell culture is measured and adjusted with small amounts of acid or base, depending upon the biological reaction.

[0084] The term “culture vessel” is used interchangeably with “bioreactor”, “reactor” and “tank”. A bioreactor used on an industrial scale can be at least 50 L, at least 100 L, at least 200 L, at least 300 L, at least 500 L, at least 1000 L, at least 2,000 L, at least 5,000 L, at least 10,000 L, at least 15,000 L, at least 20,000 L, at least 25,000 L, or any size appropriate for the large production scale necessary to produce an industrial supply.

[0085] The term “liquid culture” used herein refers to cells (for example, bacteria, plant, insect, yeast, or animal cells) grown on supports, or growing suspended in a liquid nutrient medium.

[0086] The term “seed culture” used herein refers to a cell culture grown in order to be used to inoculate larger volumes of culture medium. The seed culture can be used to inoculate larger volumes of media in order to expand the number of cells growing in the culture (for example, cells grown in suspension).

[0087] The terms “culture medium” and “cell culture medium” and “fermentation medium” used herein refer to a nutrient solution used for growing and or maintaining cells, especially mammalian cells. Without limitation, these solutions ordinarily provide at least one component from one or more of the following categories: (1) an energy source, usually in the form of a carbohydrate such as glucose; (2) all essential amino acids, and usually the basic set of twenty amino acids plus cysteine; (3) vitamins and / or other organic compounds required at low concentrations; (4) free fatty acids or lipids, for example linoleic acid; and (5) trace elements, where trace elements are defined as inorganic compounds or naturally occurring elements that are typically required at very low concentrations, usually in the micromolar range. The nutrient solution can be supplemented electively with one or more components from any of the following categories: (1) hormones and other growth factors such as, serum, insulin, transferrin, and epidermal growth factor; (2) salts, for example, magnesium, calcium, and phosphate; (3) buffers, such as HEPES; (4) nucleosides and bases such as, adenosine, thymidine, and hypoxanthine; (5) protein and tissue hydrolysates, for example peptone or peptone mixtures which can be obtained from purified gelatin, plant material, or animal byproducts; (6) antibiotics, such as gentamycin; (7) cell protective agents, for example pluronic polyol; and (8) galactose. Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma)), RPM 1-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), (Sigma)) are suitable for culturing the host cells. In addition, any of the media described in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980) can be used as culture media for the host cells. Any other necessary supplements can also be included at appropriate concentrations.

[0088] The term “basal media” used herein refers to a nutrient-rich solution designed to support the basic metabolic needs of cells in vitro. This type of medium provides essential nutrients, such as amino acids, vitamins, minerals, glucose, and salts, that help cells maintain their normal functions, including growth, proliferation, and survival. Basal media generally do not contain serum or specific growth factors, which can be added separately based on the needs of specific cell types or experiments. Further, the basal media refer to the initial growth media wherein the cells are cultured.

[0089] The term “feed” or “feed medium” or “feed media” or “feeding” or “feed regime” are interchangeable and refers to the cell culture media specifically formulated to supplement and sustain nutrient levels in dense or high-yield cultures, enhancing overall productivity and cell health. Feed media can be added or supplemented to the cell culture in the bioreactor daily, periodically, alternative day or in continuous mode.

[0090] The term “cell culture solution” used herein refers to the cell culture medium containing cells.

[0091] The term “varying amount” used herein refers to the amount of feed media added to the cell culture during the cell culture process. The amount of feed media is calculated based on the initial or current volume and / or weight of the cell culture in v / v percentage and / or w / w percentage. To calculate the percentage volume by volume (% v / v) of a solution, below formula can be used:

[0092] % v / v = mL of feed media / 100 mL of cell culture solution.

[0093] The term “initial feed regime” used herein refers to the feed media that used in the initial days of cell culture process, where the initial feed regime comprises combination of primary feeding and tertiary feeding. For example, the initial feed regime is added or supplemented to the cell culture comprising primary feeding and tertiary feeding from about day 3 to about day 7 when the entire cell culture duration is about 12 days. A skilled person can increase or decrease the duration (number of days) of supplementing initial feed regime with respect to the duration of the entire cell culture process.

[0094] The term “production feed regime” used herein refers to the feed media that used in the later days of cell culture process, where the production feed regime comprises combination of secondary feeding and quaternary feeding. For example, the production feed regime is added or supplemented to the cell culture comprising secondary feeding and quaternary feeding in an amount from day 8 to till the day before harvesting when the entire cell culture duration is about 12 days. A skilled person can increase or decrease the duration (number of days) of supplementing production feed regime with respect to the duration of the entire cell culture process.

[0095] In an embodiment, the primary feed media and tertiary feed media are added in combination and considering the sum of the primary feed media and tertiary feed media herein in the present invention as “Initial media” or “Initial feed media”.

[0096] In an embodiment, the secondary feed media and quaternary feed media are added in combination and considering the sum of the secondary feed media and quaternary feed media herein in the present invention as “Production media” or “Production feed media”.

[0097] One can thus understand the meaning of the terms “primary feeding”, “secondary feeding”, “tertiary feeding” and “quaternary feeding” used herein from the details provided for initial feed regime, production feed regime and composition of feeds.

[0098] The term “primary feeding” or “primary feed” or “primary feed media” or “primary media” used herein are interchangeable.

[0099] The term “secondary feeding” or “secondary feed” or “secondary feed media” or “secondary media” used herein are interchangeable.

[0100] The term “ tertiary feeding” or “ tertiary feed” or “tertiary feed media” or “tertiary media” used herein are interchangeable.

[0101] The term “quaternary feeding” or “quaternary feed” or “quaternary feed media” or “quaternary media” used herein are interchangeable.

[0102] The term “Feed media 1” or “Feed medium 1” comprises primary feeding and secondary feeding. The term “Feed media 2” or “Additional Feed medium 2” are interchangeable and comprises tertiary and quaternary feeding.

[0103] The term “Total Feed Media 1” refers to the sum of Primary and Secondary feeding.

[0104] The term “total additional feed” or “total additional feed media 2” used herein are interchangeable and refers to the total feed of tertiary feed media and quaternary feed media.

[0105] The term “total feed” used herein refers to the total feed media added or supplemented into the cell culture from day 3 to the day before harvest. The total feed is the total of feed media comprising primary, secondary, tertiary and quaternary feed media. For example: i) the primary feed media and the secondary feed media are added into the cell culture from day 3 to day 7 and day 8 to day 11 is 3.5% v / v and 2.0% v / v respectively. ii) the tertiary feed media and the quaternary feed media are added into the cell culture from day 3 to day 7 and day 8 to day 11 is 0.35% v / v and 0.2% v / v respectively,

[0106] Therefore, the total of the feed media defining as “Total feed” is added into the cell culture calculated at the day before harvesting i.e., on day 11 end or dayl2 will be 28.05% v / v. In another word, the term “Total Feed” refer to the Sum of Total Feed Media 1 and Total additional Feed Media 2 calculated at the end of cell culture process.

[0107] The term “total primary and secondary media” or “total primary feed and secondary feed” refers to the total % v / v of total cell culture volume that added to the cell culture calculated on the day before harvest. For example, primary feed and secondary feed added into the cell culture on day 3 to day 7 and day 8 to day 11 are 3.5 % v / v and 0.35 % v / v of total cell culture volume respectively, then the total primary and secondary feed media added in to the cell culture will be 25.5% v / v.

[0108] The term “total tertiary and quaternary feed media” refers to the total % v / v of total cell culture volume that added to the cell culture calculated on the day before harvest. For example, tertiary feed and quaternary feed added into the cell culture on day 3 to day 7 and day 8 to day 11 are 0.35 % v / v and 0.2 % v / v of total cell culture volume respectively, then the total tertiary and quaternary feed media added in to the cell culture will be 2.55% v / v. The term “protein of interest” used herein refers to the Fc-fusion protein molecule, specifically Abatacept (CTLA4-IgGl), Etanercept, Aflibercept, Romiplostim, Belatacept. The term “protein of interest” used herein also refers to the antibodies selected from Omalizumab, Pembrolizumab, Certolizumab, Nemolizumab, Tarlatamab, Crovalimab, Camrelizumab, Serplulimab, Faricimab, Romosozumab, Durvalumab, Emicizumab, Benralizumab, Ocrelizumab, Guselkumab, Dupilumab, Avelumab, Atezolizumab, Daratumumab, Mepolizumab, Evolocumab, Secukinumab, Nivolumab, Blinatumomab, Vedolizumab, Pertuzumab, Belimumab, Ipilimumab, Denosumab, Tocilizumab, Ofatumumab, Golimumab, Ustekinumab, Certolizumab pegol, Eculizumab, Ranibizumab, Natalizumab, Adalimumab, Rituximab.

[0109] The term “host cell” or “cell line” refers to mammalian cells i.e., Chinese hamster ovary (CHO) cells cultured in suitable medium to express protein of interest. In an embodiment, the Chinese hamster ovary cell being utilized to produce polypeptide directly into the cell culture.

[0110] The terms “fed batch cell culture” and “fed batch culture” refers to a cell culture wherein the cells, preferably mammalian (Chinese hamster ovary), and culture media are supplied to the culturing vessel initially and additional culture nutrients are fed, continuously or in discrete increments, to the culture during culturing, with or without periodic cell and / or product harvest before termination of culture. A “fed batch method,” refers to a method by which a fed batch cell culture is supplied with additional nutrients. A fed batch method may comprise adding supplemental media as per determined feeding schedule.

[0111] The term “perfusion batch culture” or “continuous cell culture” as used herein refers to a process that uses a method to keep cells in a bioreactor while continuously exchanging culture medium. Fresh medium replenishes nutrients and carbon sources, while cellular waste and medium depleted of nutrients are removed. This exchange of medium is commonly expressed as the number of operating vessel volumes per day (VVD). The term “continuous large-scale 10 fermenter” or “perfusion-batch cell culture” or “continuous cell culture” as used herein are interchangeable.

[0112] The term “Titer” or “Yield” or “expression of protein” as used herein, refers to the total amount of protein produced by a cell culture in given amount of cell culture volume.

[0113] “Bioreactor” herein is referred as vessel, equipment or a system to support the growth and development of microorganisms or cell lines under aseptic condition. The bioreactor body can be made of materials selected from Stainless steel, glass or polymers like polystyrene, polycarbonate, Polyethylene terephthalate, PETG, LDPE, PVA, PVC, PVA or PP.

[0114] “Product quality” is the identity, purity, potency, stability, and safety of the protein of interest determined by the predefined product quality attributes for example glycan profile, charge variants profile, size variants profile, etc. which impacts the degree to which a drug substance or product meets its intended use and fulfils its inherent properties.

[0115] The term “growth phase”, “initial phase” as used herein of the cell culture as used herein refers to the period of exponential cell growth (for example, the log phase) where cells are primarily dividing rapidly. During this phase, the rate of increase in the density of viable cells is higher than at any other time point.

[0116] The term “production phase” of the cell culture as used herein refers to the period of time during which cell growth is stationary or is maintained at a near constant level. The density of viable cells remains approximately constant over a given period of time. Logarithmic cell growth has terminated, and protein production is the primary activity during the production phase. The medium at this time is generally supplemented to support continued protein production and to achieve the desired glycoprotein product. The term “mammalian host cell” herein refers to a cell which is highly organised, has dynamic structure that compartmentalises its many functions into organelles such as the nucleus, Golgi, and endoplasmic reticulum. The nucleus retains the genetic material for cell maintenance and replication, whereby efficient signal dependent targeting of cellular proteins into or out of the nucleus. Herein mammalian cell refers to the host cells which can grow in culture and expressing a desired recombinant product protein. In an embodiment the mammalian cell is CHO cell.

[0117] The term “neutralized protein A eluate” or NPEL herein refers to is the final product of Protein A affinity chromatography, where the acidic solution used to elute a target protein (like an antibody) has been brought back to a neutral pH (around 5-8) to prevent protein damage.

[0118] The invention is further described hereinafter with reference to its general method steps, that exemplifies but do not limit the scope of the invention.

[0119] An embodiment of the invention discloses a process for production of a Fc- Fusion protein. The process comprises steps: a) culturing a mammalian cell in a cell culture medium, and b) harvesting the Fc fusion protein.

[0120] In an embodiment, the cell culture medium comprises a basal medium and a feed medium.

[0121] In an embodiment, the process to produce an Fc-fusion protein comprises: a) culturing a mammalian cell in a cell culture medium, and b) harvesting the Fc fusion protein wherein the cell culture medium comprises i. a basal medium and ii. a feed medium

[0122] In an embodiment, the basal medium utilized for mammalian cell culture is supplemented with glutamine and poloxamer amongst others.

[0123] In one embodiment the amount of glutamine supplemented in the basal media is about 3mM to about 8mM. In another embodiment the amount of poloxamer supplemented to the basal media is 2g / L to about 7g / L.

[0124] In an embodiment, the feed medium comprises of more than one feeding regime.

[0125] In an embodiment, the feed medium comprises of an initial feed regime and a production feed regime.

[0126] In a certain embodiment, the initial feed regime and the production feed regime is supplemented in a suitable ratio during the culturing of mammalian cell. In one embodiment, the production feed regime is supplemented in less amount than the initial feed regime.

[0127] In another embodiment, the production feed regime is lower in amount than the initial feed regime, where the production feed regime is 70%, 75%, or 80% lower in amount than the initial feed regime. In one embodiment, the production feed regime is at least 75% lower in amount than the initial feed regime.

[0128] In another embodiment, the initial feed regime comprises of a primary feeding and a tertiary feeding. In another embodiment, the production feed regime comprises of a secondary feeding and a quaternary feeding.

[0129] In one embodiment the ratio of initial feed regime to production feed regime is selected from 7:4, or higher.

[0130] In another embodiment, the ratio of the primary feeding to the tertiary feeding supplemented in the initial feed regime is selected from 7: 1, 8:1, 9: 1, 10: 1, 11 : 1, 12: 1, and 13:1. In one embodiment, the ratio of the primary feeding to the tertiary feeding supplemented in the initial feed regime is 10: 1 or less.

[0131] In another embodiment, the ratio of the secondary feeding to the quaternary feeding supplemented in the production feed regime is selected from 7: 1, 8: 1, 9: 1, 10: 1, 11 : 1, 12: 1, and 13: 1. In one embodiment, the ratio of the secondary feeding to the quaternary feeding supplemented in the production feed regime is 10: 1 or less.

[0132] In an embodiment, the primary feeding and secondary feeding are same and are supplemented to the cell culture as a media 1. In another embodiment, the tertiary and quaternary media are same and are supplemented to the cell culture as a media 2. The composition of primary feed media and secondary feed media are same, but they are defined as primary feed media and secondary feed media based on the addition time into the cell culture and volume added. For example, primary and secondary feed media used herein in the present invention is HyClone Cell Boost 7a Supplement and added on the day 3 to till day 7. In similar context, the composition of tertiary feed media and quaternary feed media are same, but they are defined as tertiary feed media and quaternary feed media based on the addition time into the cell culture and volume added. For example, tertiary and quaternary feed media used herein in the present invention is HyClone Cell Boost 7b Supplement and added on the day 8 onwards to till day 11 or to the day before harvest. However, the scope of the present invention should not be considered limiting to the HyClone Cell Boost 7a and HyClone Cell Boost 7b. A skilled person can be utilized the disclosed invention and concept with other feed media as well e.g., 4X Feed A / Feed B- Sartorius, Efficient Feed C+ - Sigma, BCD Feed 4- Irvine, Cellist Feed 2- Ajinomoto, Advance Feed 1- Sigma, Vento Feed 4- Merck, 4X Feed A (w / o Glucose)- Sartorius.

[0133] In an embodiment, the secondary feeding is lower than the primary feeding wherein the secondary feeding is at least about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60% lower than the primary feeding.

[0134] In an embodiment, the secondary feeding is performed till the day before harvesting of the protein of interest.

[0135] In an embodiment, the primary feeding is higher than the secondary feeding, wherein the primary feeding is at least about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80% higher than the secondary feeding. In certain embodiments, the primary feeding is performed predominantly during the initial or growth phase and partially during the initial time period of production phase.

[0136] In an embodiment, the cell culture is performed by providing an additional feed along with either primary or secondary feeding.

[0137] In another embodiment, the additional feed comprising initial feed regime and production feed regime, wherein the initial feed regime comprises tertiary feeding and production feed regime comprises quaternary feeding.

[0138] In an embodiment, the quaternary feeding is lower amount than the tertiary feeding during the cell culture process.

[0139] In an embodiment, the quaternary feeding is lower than the tertiary feeding wherein the quaternary feeding is at least about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60% lower than the tertiary feeding.

[0140] In an embodiment, the quaternary feeding is performed till the day before harvesting of the protein of interest.

[0141] In an embodiment, the tertiary feeding is higher than the quaternary feeding wherein the tertiary feeding is at least about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 55%, about 56%, about

[0142] 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about

[0143] 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about

[0144] 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about

[0145] 78%, about 79%, about 80% higher than the quaternary feeding.

[0146] In another embodiment, the tertiary feeding is performed during the initial or growth phase.

[0147] In certain embodiment, the tertiary feeding is performed predominantly during the initial or growth phase.

[0148] In certain embodiment, the tertiary feeding is performed predominantly during the growth phase and partially during the initial time period of production phase. In certain embodiment, the ratio of the primary feeding to the secondary feeding is at least about 7:4, or about 2: 1.

[0149] In certain embodiment, wherein the ratio of the tertiary feeding to the quaternary feeding is at least about 7 :4, or about 2: 1.

[0150] In an embodiment, the initial feed regime is supplemented during the initial or growth phase of the cell culture. In one embodiment, the production feed regime is supplemented during the production phase of the cell culture.

[0151] In an embodiment, the mammalian cell is cultured for a period of more than 10 days. In one embodiment, the mammalian cell is cultured for a period of more than 10 days until Day 12. In an exemplary embodiment, the mammalian cell is cultured for a period of more than 10 days until Day 14.

[0152] In an embodiment, the mammalian cell is cultured for a period of Day 10, Day 11, Day 12, Day 13, and Dayl4.

[0153] In an embodiment, the feed medium is supplemented continuously or periodically. In one embodiment, the feed medium is supplemented continuously.

[0154] In an embodiment, the initial feed regime is supplemented on as suitable day selected from day 2, day 3, day 4, day 5, day 6, day 7 and day 8, for a 12-day cell culture.

[0155] In an embodiment, the mammalian cell is cultured in basal medium for at least 24 hours to 48 hours.

[0156] In an embodiment , the initial viable cell density of the cell culture is from about 0.1 * 106cells / mL to about 0.7* 106cells / ml.

[0157] In one embodiment, the initial viable cell density of the cell culture is from about 0.3 * 106cells / ml.

[0158] In one embodiment, the initial feed regime is supplemented on a suitable day of initial or growth phase, selected from day 3, day 4, day 5, day 6, and day 7, for a 12-day cell culture.

[0159] In one exemplary embodiment, the primary feed media is supplemented selected from day 2, day 3, day 4, day 5, day 6, day 7, and day 8, for a 12-day cell culture. In another exemplary embodiment, the tertiary feed media is supplemented on a suitable day selected from day 2, day 3, day 4, day 5, day 6, day 7, and day, for a 12-day cell culture.

[0160] In another embodiment, the production feed regime is supplemented on suitable day of the production phase, selected from day 5, day 6, day 7, day 8, day 9, day 10 and day 11, for a 12-day cell culture. The production phase can be determined by a skilled person based on the duration of the cell culture process, viable cell density, production of the protein of interest. For an example, the instant application has a 14-day cell culture process where the production phase is considered from day 8 onwards.

[0161] In an embodiment, the production feed regime is initiated when the viable cell density of the mammalian cell culture is 18 * 106cells / ml.

[0162] In one embodiment, the production feed regime is supplemented on a suitable day selected from day 8, day 9, day 10 and day 11, for a 12-day cell culture.

[0163] In one embodiment, the secondary feed media is supplemented on a suitable day selected from day 5, day 6, day 7, day 8, day 9, day 10 and day 11, for a 12-day cell culture.

[0164] In one exemplary embodiment, the secondary feed media is supplemented on suitable day selected from day 8, day 9, day 10 and day 11, for a 12-day cell culture. In one embodiment, the quaternary feed media is supplemented on a suitable day selected from day 5, day 6, day 7, day 8, day 9, day 10 and day 11, for a 12-day cell culture.

[0165] In one exemplary embodiment, the quaternary feed media is supplemented on suitable day selected from day 8, day 9, day 10 and day 11, for a 12-day cell culture. In an embodiment, the tertiary feeding and the primary feeding is performed on suitable day selected from day 2, day 3, day 4, day 5, day 6, day 7, day 8, for a 12- day cell culture.

[0166] In an embodiment, the tertiary feeding and the primary feeding is performed on suitable day selected from day 3, day 4, day 5, day 6, and day 7, for a 12-day cell culture. In an embodiment, the primary feeding is provided in higher amount than tertiary feeding during the cell culture, for a 12-day cell culture.

[0167] In an embodiment, the secondary feeding and the quaternary feeding is performed till the day before harvesting of the protein of interest.

[0168] In one embodiment, the total feed (primary feed, secondary feed, tertiary feed, and quaternary feed) used in the cell culture till the day before harvesting is selected from about 20% v / v 29% v / v or about 35% v / v 40% v / v of cell culture.

[0169] In one embodiment, the total feed used in the cell culture does not contain the amount from 31% v / v to below 35% v / v.

[0170] In an embodiment, the total additional feed media comprising tertiary feed, and quaternary feed used in the cell culture till the day before harvesting is selected from about 1.5% v / v to about 4.0% v / v of cell culture.

[0171] In an embodiment, the cell culture comprising addition of feed medium 1 and additional feed medium 2 wherein the feed medium 1 is used in total higher than additional feed medium 2 during the cell culture wherein the total feed medium 1 is used in the amount from 20% v / v to about 28% v / v of the total cell culture volume.

[0172] In an embodiment, the total primary and secondary feed media added in the cell culture till the day before harvesting is selected from about 15% v / v to about 40% v / v of cell culture.

[0173] In an embodiment, the total primary and secondary feed media added in the cell culture till the day before harvesting is selected from about 17% v / v, about 18% v / v, about 19% v / v, about 20% v / v, about 21% v / v, about 22% v / v, about 23% v / v, about 24% v / v, about 25% v / v, about 26% v / v, about 27% v / v, about 28% v / v, about 29% v / v, about 30% v / v, about 31% v / v, about 32% v / v, about 33% v / v, about 34% v / v, about 35% v / v, about 36% v / v, about 37% v / v, about 38% v / v, about 39% v / v of cell culture.

[0174] In an embodiment, the total primary and secondary feed media added in the cell culture till the day before harvesting is selected from about 21% v / v, about 21.5% v / v, about 22% v / v, about 22.5% v / v, about 23% v / v, about 23.5% v / v, about 24% v / v, about 24.5% v / v, about 25% v / v, about 25.5% v / v, about 26% v / v, about 26.5% N / N, about 27% v / v, about 27.5% v / v, about 28% v / v, about 28.5% v / v, about 29% v / v, about 29.5% v / v, about 30% v / v, about 30.5% v / v, about 31% v / v, about 31.5% v / v, about 32% v / v, about 32.5% v / v, about 33% v / v, about 33.5% v / v, about 34% v / v, about 34.5% v / v, about 35% v / v, about 35.5% v / v, about 36% v / v, about 36.5% v / v, about 37% v / v, about 37.5% v / v, about 38% v / v, about 38% v / v of cell culture. In an embodiment, the total tertiary and quaternary feed media added in the cell culture till the day before harvesting is selected from about 1.5% v / v to about 4.0% v / v of cell culture.

[0175] In an embodiment, the total tertiary and quaternary feed media added in the cell culture till the day before harvesting is selected from about 1.6% v / v, about 1.7% v / v, about 1.8% v / v, about 1.9% v / v, about 2.0% v / v, about 2.1% v / v, about 2.2% v / v, about 2.3% v / v, about 2.4% v / v, about 2.5% v / v, about 2.6% v / v, about 2.7% v / v, about 2.8% v / v, about 2.9% v / v, about 3.0% v / v, about 3.1% v / v, about 3.2% v / v, about 3.3% v / v, about 3.4% v / v, about 3.5% v / v, about 3.6% v / v, about 3.7% v / v, about 3.8% v / v, about 3.9% v / v of cell culture.

[0176] In an alternative embodiment, the daily feeding of media 1 decreases from day 8 onwards to till the day before harvesting. For example, feeding of media 1 is from day 3 onwards to till day 7 is about 3.5% v / v of culture volume and further decreases from day 8 onwards to till the day before harvesting is about 2% v / v of culture volume.

[0177] In an alternative embodiment, the daily feeding of media 1 may decrease from day 8 onwards to till the day before harvesting.

[0178] In an alternative embodiment, the daily feeding of media 2 maintains constant feeding of about 0.1% v / v to about 0.4% v / v from day 3 onwards to till the day before harvesting.

[0179] In an embodiment, the daily feeding of media 2 is constant feeding of about 0.25% v / v to about 0.45% v / v from day 3 onwards to day 7 and then decreases from day 8 to about 0.1% v / v to about 0.3% v / v till the day before harvesting.

[0180] In an embodiment, the daily feeding of media 2 is constant feeding of about 0.35% v / v from day 3 onwards to day 7 and then decreases the feeding of the media 2 from day 8 to about 0.2% v / v till the day before harvesting. In an embodiment, harvesting is performed on day 11, or on day 12, or on day 13, or on day 14.

[0181] In an embodiment, harvesting is performed when cell viability drops below about 50%, about 55%, about 60%, about 65%.

[0182] In another embodiment, the ratio of total primary feed media and secondary feed media to tertiary and quaternary feed media are selected from about 5: 1, about 6: 1, about 7: 1, about 8: 1, about 9: 1, about 10: 1, about 11 : 1, about 12: 1, about 13: 1, about 14: 1, about 15: 1. For example, primary feed media and secondary feed media is added on day 3 onwards to till the day before harvesting in a total amount of about 16 g / L, wherein the tertiary media and quaternary feed media is added on day 3 onwards to till the day before harvesting in a total amount of about 2 g / L wherein the ratio is 8: 1. For example, primary feed media and secondary feed media is added on day 3 onwards to till the day before harvesting in a total amount of about 30 g / L, wherein the tertiary feed media and quaternary feed media is added on day 3 onwards to till the day before harvesting in a total amount of about 2 g / L wherein the ratio is 15:1.

[0183] In another embodiment, the ratio of total primary feed media and secondary feed media to tertiary and quaternary feed media are selected from about 5: 1, about 6: 1, about 7: 1, about 8: 1, about 9: 1, about 10: 1, about 11 : 1, about 12: 1, about 13: 1, about 14: 1, about 15: 1. For example, primary feed media and secondary feed media is added on day 3 onwards to till the day before harvesting in a total amount of about 16% v / v of culture volume, wherein the tertiary feed media and quaternary feed media is added on day 3 onwards to till the day before harvesting in a total amount of about 2% v / v of culture volume, wherein the ratio is 8: 1. For example, primary feed media and secondary feed media is added on day 3 onwards to till the day before harvesting in a total amount of about 30% v / v of culture volume, wherein the tertiary feed media and quaternary feed media is added on day 3 onwards to till the day before harvesting in a total amount of about 2% v / v of culture volume, wherein the ratio is 15: 1.

[0184] In an embodiment, the ratio of total primary feed media and secondary feed media to tertiary and quaternary feed media are selected from about 5: 1, about 6: 1, about 7:1, about 8: l, about 9: l, about 10: 1, about 11 : 1, about 12: 1, about 13: 1, about 14: 1, about 15: 1. For example, primary feed media and secondary feed media is added on day 3 onwards to till the day before harvesting in a total amount of about 25.5% v / v of culture volume, wherein the tertiary feed media and quaternary feed media is added on day 3 onwards to till the day before harvesting in a total amount of about 2.55% v / v of culture volume, wherein the ratio is 10: 1. For example, primary feed media and secondary feed media is added on day 3 onwards to till the day before harvesting in a total amount of about 37.8% v / v of culture volume, wherein the tertiary feed media and quaternary feed media is added on day 3 onwards to till the day before harvesting in a total amount of about 3.15% v / v of culture volume, wherein the ratio is 12: 1.

[0185] In an alternative embodiment, the daily feeding of primary feed media decreases from day 8 onwards to till the day before harvesting which is considering herein in the present invention as “secondary feed media”. For example, feeding of primary feed media is from day 3 onwards to till day 7 is about 3.5% v / v of culture volume and further decreases from day 8 onwards to till the day before harvesting is about 2% v / v of culture volume.

[0186] In an alternative embodiment, the daily feeding of primary feed media is decreased on day 8. In an embodiment, the decreased feeding of primary feed media then further maintains till the day before harvesting which is considering herein in the present invention as “secondary feed media”. For example, feeding of primary feed media is from day 3 onwards to till day 7 is about 3% v / v of culture volume and further decreases from day 8 onwards to till the day before harvesting is about 1.7% v / v of culture volume i.e., secondary feed media is maintaining from day 8 onwards to till the day before harvesting is about 1.7% v / v of culture volume.

[0187] In an alternative embodiment, the daily feeding of tertiary feed media maintains constant feeding of about 0.1% v / v to about 0.4% v / v from day 3 onwards to till the day before harvesting.

[0188] In an embodiment, the daily feeding of tertiary feed media maintains constant feeding of about 0.25% v / v to about 0.45% v / v from day 3 onwards to day 7 and then decreases from day 8 to about 0.1% v / v to about 0.3% v / v till the day before harvesting which is considering herein in the present invention as “quaternary feed media”.

[0189] In an embodiment, the daily feeding of tertiary feed media maintains constant feeding of about 0.35% v / v from day 3 onwards to day 7 and then decreases the feeding of tertiary feed media from day 8 to about 0.2% v / v till the day before harvesting which is considering herein in the present invention as “quaternary feed media”.

[0190] In another embodiment, the sum total of primary feed and secondary feed is added to the cell culture is about 15% v / v to about 40% v / v calculated from day 3 to the day before harvesting, wherein the sum total of tertiary feed media and quaternary feed media is added to the cell culture is about 1.5% v / v to about 4.0% v / v calculated from day 3 to the day before harvesting.

[0191] In an embodiment, the feeding primary, secondary, third, and quaternary feed media is performed daily basis, periodically basis, alternative day basis.

[0192] In an embodiment, feeding is started from day 3 or day 4 to till one day before harvest, two days before harvest and / or three days before harvest.

[0193] In an embodiment, the feeding of the cell culture is initiated when the glucose concentration drops below 6.5g / L.

[0194] In an embodiment, the cell culture is performed at a single temperature of about 37°C ±1.

[0195] In an embodiment, the cell culture is performed at one temperature shift comprising of a first temperature and a second temperature. In one embodiment, the second temperature is at least 3 degrees lower than the first temperature.

[0196] In one embodiment, the first temperature is about 37°C.

[0197] In an embodiment, the dissolved oxygen (DO) level of the cell culture is maintained from about 50 to about 70% throughout the process till the harvesting. In one embodiment the dissolved oxygen (DO) level of the cell culture is about 60%.

[0198] In an embodiment, the pH of the cell culture is maintained from about 6.85 to about 7.15 throughout the process till the harvesting. In one embodiment, the pH of the cell culture is 7. In an embodiment, the cell culture can be performed at different scales selected from 250ml scale, or 51tr scale, or 501tr scale, or 5001tr scale or 20001tr scale.

[0199] In an embodiment, the air flow rate for the cell culture is maintained from about Oml / min to about 25ml / min.

[0200] In an embodiment, the O2 flow rate is maintained during the cell culture in the bioreactor is about Oml / min to about 25ml / min.

[0201] In an embodiment, the agitation rate for the cell culture is maintained during the cell culture in the bioreactor is about 550-900 RPM.

[0202] In an embodiment, the air overlay flow rate is maintained during the cell culture in the bioreactor is about 2.5 mL / min.

[0203] The process parameters are not limited to those described and may be adapted by one skilled in the art for different process scales.

[0204] In an embodiment, the harvested cell culture obtained from the cell culture process is further subjected for one or more purification steps comprising downstream chromatography purifications, tangential flow filtration, depth filtration, one or more viral inactivation steps and not only limited to the recited purification steps but includes one or more steps.

[0205] In an embodiment, as shown in fig.1 to fig.5, the Fc-fusion protein harvested shows one or more improved characteristics selected from total dialkylated, mono sialylated species, bi sialyated species, tri sialyated species, main peak percentage, total low molecular weight percentage, acidic charged variants, total galactosylation and titer.

[0206] In one embodiment, the Fc-fusion protein is harvested at NPEL (Neutralized Protein A eluate) stage shows one or more improved characteristics selected from at least about 50% to about 55% of total sialylation, at least about 30% to 37% of mono sialyation, at least about 15% to about 20% of bi sialyation, at least about 1% to about 5% of tri sialyation analyzed by hydrophobic interaction liquid chromatography (HILIC), at least about 25% to about 30% of acidic charged species analyzed by Anion exchange chromatography (AEX), at least about 25% to about 35% of total galactosylation analysed by Hydrophilic Interaction UltraPerformance Liquid Chromatography (HILIC -UPLC))main peak percentage of at least about 50% to 200% analyzed by CE-SDS, and less about 25% to about 30% of total low molecular weight percentage analyzed by CE-SDS.

[0207] In an embodiment, the present invention provides the cell culture process for production of Fc- fusion protein comprising: a) culturing a mammalian cell in a cell culture medium, and b) harvesting the Fc fusion protein; wherein the cell culture medium comprises: i. a basal medium and ii. a feed medium wherein the feed medium comprises an initial feed regime and a production feed regime; wherein the initial feed regime and the production feed regime is supplemented in a suitable ratio during the culturing of mammalian cell; wherein the production feed regime is supplemented 75% less amount than the amount of initial feed regime; wherein the Fc- fusion protein harvested shows one or more improved characteristics selected from total sialylated species, mono sialylated species, bi sialylated species, tri dialkylated species, main peak percentage, total low molecular weight percentage, acidic charge variants, total galactosylation, and titre.

[0208] In an embodiment, the present invention provides the cell culture process for production of Fc- fusion protein comprising: a) culturing a mammalian cell in a cell culture medium, and b) harvesting the Fc fusion protein; wherein the cell culture medium comprises: i. a basal medium and ii. a feed medium wherein the initial feed comprising of primary feed and tertiary feed; wherein the production feed is comprising of secondary feed and quaternary feed; wherein the production feed is supplemented 75% less amount than the amount of initial feed regime; wherein the ratio of the initial feed regime to the production feed regime is about 7:4; wherein the Fc- fusion protein harvested shows one or more improved characteristics selected from total sialylated species, mono sialylated species, bi sialylated species, tri sialylated species, acidic charged variants, total galactosylation, main peak percentage, total low molecular weight percentage and titre.

[0209] In an embodiment, the present invention provides the cell culture process for production of Fc- fusion protein comprising: a) culturing a mammalian cell in a cell culture medium, and b) harvesting the Fc fusion protein; wherein the cell culture medium comprises: i. a basal medium and ii. a feed medium wherein the initial feed comprising of primary feed and tertiary feed; wherein the primary feed and tertiary feed is present in suitable ratio selected from 7: 1, 8: 1, 9: 1, 10: 1, 11 : 1, 12: 1, and 13: 1; wherein the production feed is comprising of secondary feed and quaternary feed; wherein the secondary feed and quaternary feed is present in suitable ratio selected from 7: 1, 8: 1, 9: 1, 10: 1, 11 : 1, 12: 1, and 13: 1; wherein the production feed is supplemented 75% less amount than the amount of initial feed regime; wherein the ratio of the initial feed regime to the production feed regime is about 7:4; wherein the production feed is comprising of secondary feed and quaternary feed; wherein the Fc- fusion protein harvested shows one or more improved characteristics selected from total sialylated species, mono sialylated species, bi sialylated species, tri sialylated species, main peak percentage, total low molecular weight percentage, acidic charged variants, total galactosylation, and titre.

[0210] In an embodiment, the present invention provides the cell culture process for production of Fc- fusion protein comprising: a) culturing a mammalian cell in a cell culture medium, and b) harvesting the Fc fusion protein; wherein the cell culture medium comprises: i. a basal medium and ii. a feed medium wherein the initial feed comprising of primary feed and tertiary feed; wherein the primary feed and tertiary feed is present in suitable ratio selected from 7:1, 8: 1, 9: 1, 10: 1, 11 : 1, 12: 1, and 13: 1; where the tertiary feeding is at least about 10% lower than the primary feeding; where the ratio of primary feeding to tertiary feeding is about 10: 1; wherein the initial feed is provided on suitable day selected from day 2, day 3, day 4, day 5, day 6, day 7 and day 8; wherein the production feed is comprising of secondary feed and quaternary feed; wherein the secondary feed and quaternary feed is present in suitable ratio selected from 7:1, 8: 1, 9: 1, 10:1, 11 : 1, 12: 1 and 13: 1; where the quaternary feeding is at least about 10% lower than the secondary feeding; where the ratio of secondary feeding to quaternary feeding is about 10: 1; wherein the production feed is provided on suitable day selected from day 5, day 6, day 7, day 8, day 9, day 10 and day 11; wherein the production feed is lower in the amount than the initial feed; wherein the Fc- fusion protein harvested shows one or more improved characteristics selected from total sialylated species, mono sialylated species, bi sialylated species, tri sialylated species, main peak percentage, total low molecular weight percentage, acidic charged variants, total galactosylation and titre.

[0211] In an embodiment, the present invention provides the cell culture process for production of Fc- fusion protein comprising: a) culturing a mammalian cell in a cell culture medium, and b) harvesting the Fc fusion protein; wherein the cell culture medium comprises: i. a basal medium and ii. a feed medium wherein the initial feed comprising of primary feed and tertiary feed; wherein the primary feed and tertiary feed is present in the ratio of 10: 1; wherein the initial feed is provided on day 3, day 4, day 5, day 6 and day 7; wherein the production feed is comprising of secondary feed and quaternary feed; wherein the production feed is provided on day 8, day 9, day 10 and day 11; wherein the production feed is lower in the amount than the initial feed; wherein the Fc- fusion protein harvested shows one or more improved characteristics selected from total sialylated species, mono sialylated species, bi sialylated species, tri sialylated species, main peak percentage, total low molecular weight percentage, acidic charged variants, total galactosylation, and titre.

[0212] In an exemplary embodiment the process of present invention: a. culturing a mammalian cell in a cell culture medium, and b. harvesting the Fc fusion protein; wherein the cell culture medium comprises: i. a basal medium and ii. a feed medium wherein the feed medium comprises an initial feed regime and a production feed regime; wherein the initial feed regime and the production feed regime is supplemented in a suitable ratio during the culturing of mammalian cell; wherein the production feed regime is supplemented in less amount than the amount of initial feed regime; wherein the production feed regime is supplemented about 75% less amount than the amount of initial feed regime; wherein the ratio of the initial feed regime to the production feed regime is about 7:4; wherein the initial feed regime comprises a primary feeding and a tertiary feeding, where the tertiary feeding is at least about 10% lower than the primary feeding; where the ratio of primary feeding to tertiary feeding is about 10: 1; wherein the production feed regime comprises of a secondary feeding and a quaternary feeding, where the quaternary feeding is at least about 10% lower than the secondary feeding; where the ratio of secondary feeding to quaternary feeding is about 10: 1; wherein the initial feed regime is supplemented on a suitable day selected from day 3, day 4, day 5, day 6, and day 7, if the cell culture duration is Day 12; wherein the production feed regime is supplemented on a suitable day selected from day 8, day 9, day 10 and day 11, if the cell culture duration is Day 12; wherein the cell culture is subjected to a feed medium regimen continuously; wherein the initial viable cell density of the cell culture is about 0.3 * 106cells / mL wherein the temperature of the cell culture throughout the process is maintained at 37°C; wherein the pH of the cell culture throughout the process is 7; wherein the dissolved oxygen (DO) level of the cell culture is maintained at about 60%; wherein the cells are cultured through fed-batch process in a production bioreactor for a total run period of 12 days; wherein the harvested and purified Fc-fusion protein shows one or more improved characteristics selected from at least about 53% of total sialylation, at least about 35% of mono sialyation, at least about 18% of bi sialyation, at least about 3% of tri sialyation analysed by hydrophobic interaction liquid chromatography (HILIC), at least about 28% of acidic charged species analysed by anion exchange chromatography (AEX), at least about 33% of total galactosylation analysed by Hydrophilic Interaction Ultra-Performance Liquid Chromatography (HILIC- UPLC)), main peak percentage of at least more than 70% analysed by CE-SDS, and less 28% of total low molecular weight percentage analysed by CE-SDS; wherein the titer of the harvested Fc-Fusion protein is more than 51%, analysed by Protein- A chromatography.

[0213] In an embodiment, the Fc-fusion protein is CTLA4-IgG or Abatacept, or Belatacept.

[0214] In an embodiment, the Fc-fusion protein is CTLA4-IgGl fusion protein, or Abatacept.

[0215] In an embodiment, the present disclosure is related to a process that can control the sialylation profile of CTLA4-IgG to obtain desired level of sialylated CTLA4-IgG. In an embodiment, the total sialyaled profile comprises mono-sialylated species. In an embodiment, mono-sialylated species of CTLA4-IgG comprises about 32% to about 35% of total sialylated species.

[0216] In an embodiment, the sialylation profile analysed by hydrophobic interaction liquid chromatography (HILIC).

[0217] In embodiment, total LMW impurities of the harvested CTLA4-IgG is decreased and / or reduced about 5% to about 200% or more analysed by CE-SDS.

[0218] In an embodiment, the total galactosylation of the harvested CTLA4-IgG is at least 25% to 35%.

[0219] In an embodiment, the acidic charged variants of the harvested CTLA4-IgG is at least 25% to 40%.

[0220] In an embodiment, the LMW impurities obtained from the cell culture can be further be reduced and / or decreased in subsequent purification steps. The present invention results low and / or decreased LMW impurities, which further reduces and / or lowers the burden on the skilled person during the downstream purification steps.

[0221] In an embodiment, total main peak percentage of the harvested CTLA4-IgG is increased about 5% to about 200% or more analysed by CE-SDS.

[0222] In an embodiment, titer of the harvested CTLA4-IgG is increased and / or improved about 5% to about 100% or more analysed by Protein-A chromatography.

[0223] In an embodiment , the present invention discloses a composition of CTLA4-IgGl . The composition of CTLA4-IgGl comprising one or more quality parameters selected from a total sialylation of at least 53%, a mono sialylation of at least 35%, a bi sialylation of at least 18%, a tri sialyation of at least 3%, an acidic variant of at least 25%, total galactosylation of at least 33%, and a LMW impurities less than about 28%; wherein the main peak is more than 70%.

[0224] In an embodiment, the titer of harvested CTLA4-IgGl at NPEL stage is more than 51% , analysed by Protein A chromatography.

[0225] A harvested CTLA4-IgGl composition comprising total sialylation (%) at least 51% measured and / or analysed by hydrophobic interaction liquid chromatography (HILIC) and main peak at least more than 70% determined by CE-SDS. A harvested CTLA4-IgGl composition comprising total sialylation (%) at least 52% measured and / or analysed by hydrophobic interaction liquid chromatography (HILIC) and main peak at least more than 70% determined by CE-SDS.

[0226] A harvested CTLA4-IgGl composition comprising total sialylation (%) at least 53% measured and / or analysed by hydrophobic interaction liquid chromatography (HILIC) and main peak at least more than 70% determined by CE-SDS.

[0227] A harvested CTLA4-IgGl composition comprising mono sialylation (%) at least 32.4% measured and / or analysed by hydrophobic interaction liquid chromatography (HILIC) and main peak at least more than 70% determined by CE- SDS.

[0228] A harvested CTLA4-IgGl composition comprising mono sialylation (%) at least 33% measured and / or analysed by hydrophobic interaction liquid chromatography (HILIC) and main peak at least more than 70% determined by CE-SDS.

[0229] A harvested CTLA4-IgGl composition comprising mono sialylation (%) at least 34% measured and / or analysed by hydrophobic interaction liquid chromatography (HILIC) and main peak at least more than 70% determined by CE-SDS.

[0230] A harvested CTLA4-IgGl composition comprising mono sialylation (%) at least 35% measured and / or analysed by hydrophobic interaction liquid chromatography (HILIC) and main peak at least more than 70% determined by CE-SDS.

[0231] In an embodiment, the main peak percentage is more than about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% determined by CE-SDS.

[0232] The harvested Fc fusion protein has at least about more than 3% of increased total sialyation when performed with varying feed media compared to the total sialyation when performed without varying feed media. The harvested Fc fusion protein has at least about more than 4% of increased total sialyation when performed with varying feed media compared to the total sialyation when performed without varying feed media.

[0233] The harvested Fc fusion protein has at least about more than 5% of increased total sialyation when performed with varying feed media compared to the total sialyation when performed without varying feed media.

[0234] A composition of CTLA4-IgGl comprising one or more quality parameters selected from a total sialylation of at least about more than 51%, a mono sialylation of at least about more than 31%, a bi sialylation of at least about more than 16%, a tri sialyation of at least about more than 1%, an acidic variant of at least about more than 26% a total galactosylation of at least about more than 30%, and a LMW impurities less than about 26%; wherein the main peak is more than 60%.

[0235] A composition of CTLA4-IgGl comprising one or more quality parameters selected from a total sialylation of at least about 51%, a mono sialylation of at least about 31%, a bi sialylation of at least about 16%, a tri sialyation of at least about 1%, an acidic variant of at least about 26% a total galactosylation of at least about 30%, and a LMW impurities less than about 26%; wherein the main peak is more than 70%.

[0236] A harvested CTLA4-IgGl composition comprising reduced and / or decreased total LMW impurities (%) wherein the total LMW impurities is present less than 28% and main peak percentage at least more than 70% determined and / or measured by CE-SDS.

[0237] A harvested CTLA4-IgGl composition comprising titer at least 51% measured by Protein-A chromatography and main peak percentage at least more than 70% determined by CE-SDS.

[0238] In an embodiment, the present invention provides a cell culture process for producing CTLA4-IgGl . The examples below are for illustrative purposes, and the scope of the invention should not be considered limited to them.

[0239] Example:

[0240] Effect of varying feed media regime / ratio and concentration in CTLA4-IgGl

[0241] 5 production process on titer and product quality

[0242] The CTLA4-IgGl (Abatacept) cell bank vial was thawed in to Shake flask 125 (SF125) with seed media and cell counts were checked. The incubation was continued for 3-5 days during which cells reach the optimum cell density for subculture, cells were subsequently expanded to Shake flask 250(SF250) / 500(SF500) / 1000(SF1000) to generate sufficient Inoculum depending upon the inoculum requirement to inoculate Ambr®250 bioreactors (250 mL). Production Fed batch bioreactor run was executed at Ambr®250 bioreactor using process parameters as mentioned below table 1.

[0243] Table 1: Process parameters for production of CTLA4-IgGl

[0244] The control process was run as per the parameters mentioned in the above table with Total feed media 1 addition of 31.5% and does not involve any feed regime variation during the entire culture run. Product quality of the clarified harvest was checked at the end of the cell culture r6n i.e. on day 12 for various quality attributes. Further experiments were also conducted for process 1 and process 2 with varying concentration and / or amount of 21.8% and 22.5% respectively of feed medium 1 throughout the entire production culture. The additional feed medium 2 was also varied in Process 2. It was observed that, changing the total concentration of Feed media 1 and Additional feed medium 2 triggered significant changes on overall culture productivity and product quality. The concentration of peak ammonia (NH3) for the control process was observed to be 11.05mmHg whereas peak ammonia for the present process was decreased to 8.17mmHg. Overall changes are summarised in the following table 2:

[0245] Table 2: Results of varying feed media regime / ratio and concentration.

[0246] 5

[0247] 10

[0248] 15

Claims

Claims:We claim:

1. A process for the production of Fc fusion protein comprising; a. culturing a mammalian cell in a cell culture medium, and b. harvesting the Fc fusion protein; wherein the cell culture medium comprises: i. a basal medium and ii. a feed medium wherein the feed medium comprises an initial feed regime and a production feed regime; wherein the production feed regime is supplemented in a less amount than the amount of initial feed regime; wherein the harvested Fc-fusion protein comprises one or more improved characteristics selected from the total sialylation of more than about 50%, more than about 30% of mono sialyation, more than about 15% of bi sialyation, more than about 1% of tri sialyation analysed by hydrophobic interaction liquid chromatography (HILIC), more than about 25% of acidic charged species analysed by anion exchange chromatography (AEX), more than about 25% of total galactosylation analysed by Hydrophilic Interaction Ultra-Performance Liquid Chromatography (HILIC -UPLC)), main peak percentage more than about 50% analysed by CE-SDS, and less than about 25% of total low molecular weight percentage analysed by CE-SDS.

2. The process according to claim 1, wherein the initial feed regime and the production feed regime is supplemented in a suitable ratio during the culturing of mammalian cell.

3. The process according to claim 1, wherein the production feed regime is at least 70%, 75%, or 80% lower in amount than the initial feed regime.

4. The process according to claim 1, wherein the production feed regime is at least 75% lower in amount than the initial feed regime.

5. The process according to claim 1 , wherein the ratio of the initial feed regime to the production feed regime is about 7:4 or higher.

6. The process according to claim 1, wherein the initial feed regime comprises of a primary feeding and a tertiary feeding.

7. The process according to claim 6, wherein the primary feeding and the tertiary feeding is supplemented in a suitable ratio; wherein the tertiary feeding is at least about 10% lower than the primary feeding.

8. The process according to claim 6, wherein the ratio of the primary feeding to the tertiary feeding is at least 10: 1 or less.

9. The process according to claim 1, wherein the production feed regime comprises of a secondary feeding and a quaternary feeding.

10. The process according to claim 9, wherein the secondary feeding and the quaternary feeding is supplemented in a suitable ratio; wherein the quaternary feeding is at least about 10% lower than the secondary feeding .

11. The process according to claim 9, wherein the ratio of tertiary feeding to the quaternary feeding is at least 10: 1 or less.

12. The process according to any of the preceding claims, wherein the primary feeding comprises a media 1; wherein the secondary feeding comprises a media 1; wherein the tertiary feeding comprises a media 2; and wherein the quaternary feeding comprises a media 2.

13. The process according to claim 1, wherein the cell culture process is for at least more than Day 10 up to Day 16.

14. The process according to claim 1, wherein the cell culture process is for at least more than Day 10 up to Day 14.

15. The process according to claim 1, wherein the cell culture process is for at least more than Day 10 up to Day 12.

16. The process according to claim 1, wherein the initial feed regime is supplemented during growth phase to achieve the viable cell density of the cell culture 0.3 * 106cells / mL.

17. The process according to claim 1, wherein the initial feed regime is supplemented on a suitable day selected from day 2, day 3, day 4, day 5, day 6, day 7 and day 8, if the cell culture duration is Day 12.

18. The process according to claim 1, wherein the production feed regime is supplemented on a suitable day selected from day 7, day 8, day 9, day 10, and day 11, if the cell culture duration is Day 12.

19. The process according to claim 18, wherein the production feed regime is supplemented when the viable cell density of the cell culture reaches at least 18* 106cells / ml.

20. The process according to any of the preceding claims, wherein the primary feeding is at least about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%,about 75%, about 76%, about 77%, about 78%, about 79%, and about 80% higher than the secondary feeding media.

21. The process according to any of the preceding claims, wherein the secondary feeding is at least about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, and about 80% lower than the primary feeding.

22. The process according to any of the preceding claims, wherein the tertiary feeding media is at least about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 55%, about56%, about 57%, about 58%, about 59%, about 60%, about 61%, about62%, about 63%, about 64%, about 65%, about 66%, about 67%, about68%, about 69%, about 70%, about 71%, about 72%, about 73%, about74%, about 75%, about 76%, about 77%, about 78%, about 79%, and about 80% higher than the quaternary feeding.

23. The process according to any of the preceding claims, wherein the quaternary feeding is at least about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, and about 80% lower than the tertiary feeding.

24. The process according to any of the preceding claims, wherein the ratio of the primary feeding to the secondary feeding is at least about 7:4, or about 2: 1.

25. The process according to any of the preceding claims, wherein the ratio of the tertiary feeding to the quaternary feeding is at least about 7:4, or about 2: 1.

26. The process according to claim 1, wherein the cell culture is subjected to a feed medium regimen continuously or periodically.

27. The process according to claim 1, wherein the mammalian cell is cultured in basal medium for at least 24 hours to 48 hours.

28. The process according to claim 1, wherein the basal medium is supplemented with glutamine and poloxamer in a suitable amount.

29. The process according to claim 28 wherein the basal medium is supplemented with glutamine in an amount from about 3mM to about 8mM and with poloxamer in amount from about 2g / L to about 7g / L.

30. The process according to claim 1, wherein the initial viable cell density of the cell culture is from about 0.1 * 106cells / mL to about 0.7* 106cells / mL.

31. The process according to claim 30, wherein the initial viable cell density of the cell culture is from about 0.3* 106cells / mL to about 0.5* 106cells / mL.

32. The process according to claim 1, wherein feeding of the cell culture is initiated when the glucose concentration drops below 6.5g / L .

33. The process according to claim 1, wherein the cell culture is performed at a single temperature of about 37°C ±1.

34. The process according to claim 1, wherein the cell culture is performed at one temperature shift; wherein the temperature shift comprises of a first temperature and a second temperature wherein the second temperature is at least 3 degrees lower than the first temperature.

35. The process according to claim 1, wherein the first temperature is 37°C.

36. The process according to claim 1, wherein the dissolved oxygen (DO) level of the cell culture is maintained from about 50% to about 70% throughout the process till the harvesting.

37. The process according to claim 1, wherein the pH of the cell culture is maintained from about 6.85 to about 7.15 throughout the process till the harvesting.

38. The process according to claim 1, wherein the harvesting of Fc-Fusion protein is performed on day 11, or on day 12, or on day 13, and on day 14.

39. The process according to claiml, wherein the harvesting of Fc-Fusion protein is performed on day 11, and on day 12.

40. The process according claim 1, wherein the harvesting of Fc-Fusion protein is performed when the cell viability drops below about 50%, about 55%, about 60%, and about 65%.

41. The process according claim 1, wherein the harvested Fc-fusion protein shows one or more improved characteristics selected from at least about 50% to about 60% of total sialylation, at least about 30% to 40% of monosialyation, at least about 15% to about 20% of bi sialyation, at least about 1% to about 5% of tri sialyation analysed by hydrophobic interaction liquid chromatography (HILIC), at least about 25% to about 30% of acidic charged species analysed by Anion exchange chromatography (AEX), at least about 30% to about 35% of total galactosylation analysed by Hydrophilic Interaction Ultra-Performance Liquid Chromatography (HILIC -UPLC)), main peak percentage of at least about 50% to 200% analysed by CE-SDS, and less than about 25% to about 30% of total low molecular weight percentage analysed by CE-SDS.

42. The process according claim 1, wherein the titter of the harvested Fc-Fusion protein is more than about 50%, analysed by Protein-A chromatography.

43. The process according to claim 1, wherein the production feed regime is supplemented about 75% less in amount than the amount of initial feed regime; wherein the initial feed regime and the production feed regime is supplemented in a suitable ratio of about 7:4; wherein the initial feed regime comprises a primary feeding and a tertiary feeding, where the tertiary feeding is at least about 10% lower than the primary feeding; where the ratio of primary feeding to tertiary feeding is about 10: 1; wherein the production feed regime comprises of a secondary feeding and a quaternary feeding, where the quaternary feeding is at least about 10% lower than the secondary feeding; where the ratio of secondary feeding to quaternary feeding is about 10: 1; wherein the initial feed regime is supplemented on a suitable day selected from day 3, day 4, day 5, day 6, and day 7, if the cell culture duration is Day 12;wherein the production feed regime is supplemented on a suitable day selected from day 8, day 9, day 10 and day 11, if the cell culture duration is Day 12; wherein the cell culture is subjected to a feed medium regimen continuously; wherein the dissolved oxygen (DO) level of the cell culture is maintained at about 60%; wherein the cells are cultured through fed-batch process in a production bioreactor for a total run period of 12 days; wherein the harvested and purified Fc-fusion protein shows one or more improved characteristics selected from at least about 53% of total sialylation, at least about 35% of mono sialyation, at least about 18% of bi sialyation, at least about 3% of tri sialyation analysed by hydrophobic interaction liquid chromatography (HILIC), at least about 28% of acidic charged species analysed by anion exchange chromatography (AEX), at least about 33% of total galactosylation analysed by Hydrophilic Interaction Ultra-Performance Liquid Chromatography (HILIC -UPLC)), main peak percentage of at least more than 70% analysed by CE-SDS, and less 28% of total low molecular weight percentage analysed by CE-SDS; wherein the titre of the harvested Fc-Fusion protein is more than 51%, analysed by Protein-A chromatography.

44. The process according to claiml, wherein the Fc-fusion protein is CTLA4- IgG fusion protein, or Abatacept, and Belatacept.

45. The process according to claim 1, wherein the Fc-fusion protein is CTLA4- IgGl fusion protein, or Abatacept.

46. The process according to claim 1, wherein the harvested Fc fusion protein has at least about more than 3% of increased total sialyation when performed with varying feed media compared to the total sialyation when performed without varying feed media.

47. The process according to claim 1, wherein the harvested Fc fusion protein has at least about more than 4% of increased total sialyation when performed with varying feed media compared to the total sialyation when performed without varying feed media.

48. The process according to claim 1, wherein the harvested Fc fusion protein has at least about more than 5% of increased total sialyation when performed with varying feed media compared to the total sialyation when performed without varying feed media.

49. A composition of CTLA4-IgGl comprising one or more quality parameters selected from a total sialylation of at least about more than 51%, a mono sialylation of at least about more than 31%, a bi sialylation of at least about more than 16%, a tri sialyation of at least about more than 1%, an acidic variant of at least about more than 26% a total galactosylation of at least about more than 30%, and a LMW impurities less than about 26%; wherein the main peak is more than 60%.

50. A composition of CTLA4-IgGl comprising one or more quality parameters selected from a total sialylation of at least about 51%, a mono sialylation of at least about 31%, a bi sialylation of at least about 16%, a tri sialyation of at least about 1%, an acidic variant of at least about 26% a total galactosylation of at least about 30%, and a LMW impurities less than about 26%;wherein the main peak is more than 70%.

51. A composition of CTLA4-IgGl comprising one or more quality parameters selected from a total sialylation of at least 53%, a mono sialylation of at least 35%, a bi sialylation of at least 18%, a tri sialyation of at least 3%, an acidic variant of at least 25% a total galactosylation of at least 33%, and a LMW impurities less than about 28%; wherein the main peak is more than 70%.

52. The composition according to claim 49, wherein the titre of harvested CTLA4-IgGl at NPEL stage is more than 51% , analysed by Protein A chromatography.

53. The composition according to claim 49, wherein the CTLA4-IgGl fusion protein is Abatacept.

Images