Cell culture additive and uses thereof

Abstract

Provided is a cell culture additive or supplement that contains histidine, methionine, glycine, a zinc-based inorganic compound, and an optional antioxidant, as well as its use in fed-batch culture of a mammalian cell for recombinant protein production.

Description

Cell culture additive and uses thereofINCORPORATION BY REFERENCE

[0001] All documents cited or referenced herein (including without limitation all literature documents, patents, published patent applications cited herein) (“herein cited documents”), and all documents cited or referenced in herein cited documents, together with any manufacturer’s instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated herein by reference, and may be employed in the practice of the invention. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference. Any Genbank sequences mentioned in this disclosure are incorporated by reference with the Genbank sequence to be that of the earliest effective filing date of this disclosure.FIELD OF THE INVENTION

[0002] The present application generally relates to a cell culture additive or supplement that may comprise histidine, methionine, glycine, a zinc-based inorganic compound, and an optional antioxidant. The present application also relates to the use of the cell culture additive or supplement in culturing a mammalian cell, for enhancing the cell’s viability in the late culture stage, or for improving recombinant protein production in the mammalian cell.BACKGROUND OF THE INVENTION

[0003] By the end of 2023, the National Medical Products Administration (NMPA) has approved more than 70 antibody drugs for treatment of various diseases such as cancers and inflammatory diseases, and it is expected that the number of antibody drugs approved in China will exceed 100 in 2025. Monoclonal antibodies (mAbs) have become the fastest commercialized therapeutic molecules due to their high antigen specificity' and slight side effects. However, the administered dose of monoclonal antibodies is usually much higher than that of other t pes of biologies. In order to reduce the unit production cost of mAb drugs and ease the patients’ cost burden, increasing mAb production titer is an eternal topic in the industry.

[0004] Among the therapeutic protein expression systems, mammalian cells such as Chinese hamster ovary (CHO) cells are often used, due to the advantages of e.g., easy scale-up and culture, high yield, and protein post-translational modification similar to those in human cells. The mAh production in CHO cells is mainly performed with fed- batch processes where the initial cellular growth is supported by the basal medium and concentrated feed medium is added to replenish nutrients. The mAb titer may be improved by manipulating either the host cell or the expression vector. Alternatively, a high-performance cell culture medium may be used to improve the titer.

[0005] The mainstream CHO cell culture media on the market are all chemically defined, including Thermo's DynamisTM / FeedB+fed-batch combination, and Cytiva's ActiproTM / 7a / 7b fed-batch combination. Many companies also have their in-house prepared media. These media contain nutrients that support cell growth, cell proliferation, and protein production, including amino acids (both essential and non- essential), carbon sources, and inorganic salts. The higher nutrient levels in a basal or feed medium may not always result in a high protein production level. For example, ActiCHO Feed™-A and -B feed media, high in amino acids and glucose levels, rendered the BC-G CHO cells produce IgGs with lower titer than Efficient Feed™ A and B feed media having much lower nutrient levels, when used in combination w ith CD FortiCHO™ or ActiCHO-P basal medium. Cell size increase was observed when ActiCHO Feed™-A and -B media were used, and it was assumed that such cell size change may be caused by overfed nutrients-mediated cell cycle arrest (Pan X, et al., (2017) Selection of chemically defined media for CHO cell fed-batch culture processes. Cytotechnology. 69( 1 ) : 39-56).

[0006] Some compounds are introduced to the cell culture as additives or supplements to enhance mAb production. For example, Kishishita et al. found that serine, cysteine, and tyrosine increased the titer of mAbs (Kishishita S, et al., (2015) Optimization of chemically defined feed media for monoclonal antibody production in Chinese hamster ovary cells. Journal of Bioscience and Bioengineering. 120(l):78-84. Takagi et al. found that the addition of pyrimidine nucleosides, such as deoxyuridine, thymidine and deoxy cytidine, could further improve protein titer by promoting cell growth (Takagi Y, et al. , (2017) The enhancement of antibody concentration and achievement of high cell density CHO cell cultivation by adding nucleoside. Cytotechnology. 69(3): 511-521). Gangwar et al. found that pyridoxine could increase protein productivity (Gangwar N,et al., (2021) Effect of vitamins and metal ions on productivity and charge heterogeneity of IgGl expressed in CHO cells. Biotechnol. J. 16(8):e2000464). Kruger et al. found that the mixture of polyamine and iron could increase the cell density, viability and protein titer of CHO cells (US 8,637,312). Professor Tan Wensong of East China University7of Science and Technology and others found that a mixture of glucose and amino acids can achieve high-density growth and high-titer expression of CHO cells (CN 102229908B). It has also been reported that Zinc and histidine can improve protein production (RocaBC, etal., (2019) Zinc supplementation increases protein titer of recombinant CHO cells. Cytotechnology. 71 (5): 915-924). In addition, antioxidants can potentially increase antibody production by reducing the damage of free radicals to cells (Chevallier V, et al. , (2019) Oxidative stress-alleviating strategies to improve recombinant protein production in CHO cells. Biotechnology bioengineering. 117(4):1172-1186).

[0007] To avoid or minimize the adverse effects on cell growth and proliferation and in the meanwhile to improve protein production by the cultured cells, the compounds for enhancing mAb production may need be introduced to the cell culture in certain cell culture stages at relatively low levels.

[0008] Citation or identification of any document in this application is not an admission that such document is available as prior art to the present invention.SUMMARY OF THE INVENTION

[0009] The inventors of the present application, have designed a cell culture additive or supplement, that simply contains three amino acids, a zinc-based inorganic compound and an optional antioxidant. When added to CHO fed-batch culture in the early exponential grow th phase (e.g., Day 3 of cultivation), the cell culture additive or supplement surprisingly alleviated the cell viability decline in the later stage, and enhanced the recombinant protein production by the CHO cells, as measured by e.g., the increased protein level, no matter what basal and feed media were used in the cell culture.

[0010] Methionine was reported to be a limiting essential amino acid (Zizhuo Xing et al., (2011) Optimizing amino acid composition of CHO cell culture media for a fusion protein production. Process Biochemistry. 46: 1423-1429), and is provided in most commercially7available cell culture media at a level exceeding the cell consumptionrate (Pan X, et al., (2017) supra). The effect of the excess of methionine on the cell culture, especially the protein production titer in the cell culture, was actually unpredictable before the fding date of the present application, and has now been approved by the inventors of the disclosure to be beneficial.

[0011] The glycine in the cell culture medium was said to increase ammonium production and thus should be decreased in amount (Zizhuo Xing et al.. (2011) supra). The introduction of additional glycine to the cell culture, in addition to that in the basal and feed media, in the early exponential growth phase (e.g.. Day 3 of the fed-batch culture), surprisingly improved recombinant protein production by the cultured cells.

[0012] Studies have shown the zinc consumption occurs primarily during the lag phase and becomes minimal thereafter, and the impact of the zinc supplement on betaglycuronidase (GUS) production was marginal (< 6%) (Ryan J. Graham, et al., (2001) Zinc supplementation modulates intracellular metal uptake and oxidative stress defense mechanisms in CHO cell cultures. Biochemical Engineering Journal 169: 107928). Surprisingly, the addition of the zinc-based inorganic compound to the cell culture in the early exponential growth phase (e.g.. Day 3 of the fed-batch culture) has been found to benefit the protein production.

[0013] The addition of the antioxidants in the disclosure surprisingly decreased viable cell density from early log phase. However, more surprisingly, the evidently less cells produced absolutely more antibodies.

[0014] The cell culture additive or supplement of the disclosure may be also used in the culture of other mammalian cells, e.g., CHO-K1, CHO-S and CHO-DG44 cells, for enhancing cell growth and / or protein production.

[0015] Therefore, in a first aspect, the disclosure provides a cell culture additive or supplement composition, which may comprise histidine, methionine, glycine, and a zinc-based inorganic compound.

[0016] The histidine may be L-histidine or a salt thereof such as L-histidine hydrochloride. In certain embodiments, the histidine may be L-histidine hydrochloride. In certain embodiments, the histidine may be L-histidine hydrochloride monohydrate.

[0017] The zinc-based inorganic compound may be an inorganic salt of zinc, e.g., zinc sulfate, or zinc chloride. In certain embodiments, the zinc-based inorganic compound is zinc sulfate. In certain embodiments, the zinc-based inorganic compound is zinc sulfate heptahydrate.

[0018] The cell culture additive or supplement composition of the disclosure may comprise histidine, methionine, glycine, and the zinc-based inorganic compound in a (2.39-4.77): (0.47-4.69): (0.67-6.66): (0.01-0.1) molar ratio, e.g., in a 2.39:0.47:0.67:0.01; 4.77:4.69:6.66:0.1; 4.77:0.47:3.33:0.01; 4.77:4.69:6.66:0.01; or 4.77:0.47:6.66:0.1 molar ratio. In certain embodiments, the cell culture additive or supplement composition of the disclosure may comprise 28-89 wt% L-histidine hydrochloride monohydrate, 4-56 wt% methionine, 3-47 wt% glycine and 0.1-5 wt% zinc sulfate heptahydrate. In certain embodiments, the cell culture additive or supplement composition of the disclosure may comprise 80.3 wt% L-histidine hydrochloride monohydrate, 11.2 wt% methionine, 8.0 wt% glycine and 0.5 wt% zinc sulfate heptahydrate.

[0019] The cell culture additive or supplement composition of the disclosure may further comprise an antioxidant. The antioxidant may be a selenium-based compound, glutathione, or the combination thereof. The selenium-based compound may be a selenite, e.g.. sodium selenite. The glutathione may be glutathione reduced, or a L- glutathione. In certain embodiments, the glutathione may be L-glutathione reduced.

[0020] The cell culture additive or supplement composition of the disclosure may comprise histidine, methionine, glycine, the zinc-based inorganic compound and the antioxidant in a (2.39-4.77): (0.47-4.69): (0.67-6.66): (0.01-0.1): (0.81-1.63) molar ratio, e.g., a (2.39-4.77): (0.47-4.69): (0.67-3.33): (0.01-0.1): (0.81-1.63) molar ratio, or a (2.39-4.77): (0.47-4.69): (3.33-6.66): (0.01-0.1): (0.81-1.63) molar ratio. In certain embodiments, the cell culture additive or supplement composition of the disclosure may comprise histidine, methionine, glycine, the zinc-based inorganic compound and the antioxidant in e.g.. a 2.39:0.47:0.67:0.01 : 1.63; 4.77:4.69:6.66:0.1: 1.63; 2.39:0.47:0.67:0.01 :0.81; 4.77:0.47:3.33:0.01: 1.63; 4.77:4.69:6.66:0.01 : 1.63; or 4.77:0.47:6.66:0.1:0.81 molar ratio. In certain embodiments, the cell culture additive or supplement composition of the disclosure may comprise histidine, methionine, glycine, the zinc-based inorganic compound, the selenite and glutathione in a (2.39- 4.77): (0.47-4.69): (0.67-6.66): (0.01-0.1): (0.00014-0.00145): (0.81-1.63) molar ratio, e.g., a (2.39-4.77): (0.47-4.69): (0.67-3.33): (0.01-0.1): (0.00014-0.00145): (0.81-1.63) molar ratio, or a (2.39-4.77): (0.47-4.69): (3.33-6.66): (0.01-0.1): (0.00014-0.00145): (0.81-1.63) molar ratio. In certain embodiments, the cell culture additive or supplement composition of the disclosure may comprise histidine, methionine, glycine, the zinc-based inorganic compound, the selenite and glutathione in e.g., a 2.39:0.47:0.67:0.01:0.00145: 1.63; 4.77:4.69:6.66:0.1:0.00145:1.63;2.39:0.47:0.67:0.01:0.00014:0.81; 4.77:0.47:3.33:0.01:0.00145:1.63;4.77:4.69:6.66:0.01:0.00014:1.63; or 4.77:0.47:6.66:0.1:0.00014:0.81 molar ratio. In certain embodiments, the cell culture additive or supplement composition of the disclosure may comprise 22-73 wt% L-histidine hydrochloride monohydrate, 3-47 wt% methionine, 2-38 wt% glycine, 0.1-3 wt% zinc sulfate heptahydrate, 0.001-0.03 wt% sodium selenite, and 10-45 wt% L-glutathione reduced. In certain embodiments, the cell culture additive or supplement composition of the disclosure may comprise 44.5 wt% L-histidine hydrochloride monohydrate, 6.2 wt% methionine, 4.5 wt% glycine, 0.27 wt% zinc sulfate heptahydrate, 0.022 wt% sodium selenite, and 44.5 wt% L- glutathione reduced; 36.6 wt% L-histidine hydrochloride monohydrate, 25.6 wt% methionine, 18.3 wt% glycine, 1.10 wt% zinc sulfate heptahydrate, 0.009 wt% sodium selenite, and 18.3 wt% L-glutathione reduced; 57.3 wt% L-histidine hydrochloride monohydrate, 8.0 wt% methionine, 5.7 wt% glycine. 0.34 wt% zinc sulfate heptahydrate, 0.003 wt% sodium selenite, and 28.6 wt% L-glutathione reduced; 54.8 wt% L-histidine hydrochloride monohydrate, 3.8 wt% methionine, 13.7 \\t% glycine, 0.16 wt% zinc sulfate heptahydrate, 0.014 wt% sodium selenite, and 27.4 wt% L- glutathione reduced; 37.0 wt% L-histidine hydrochloride monohydrate, 25.9 wt% methionine, 18.5 wt% glycine. 0.11 wt% zinc sulfate heptahydrate, 0.001 wt% sodium selenite, and 18.5 wt% L-glutathione reduced; or 54.1 wt% L-histidine hydrochloride monohydrate, 3.8 wt% methionine, 27.0 wt% glycine, 1.62 wt% zinc sulfate heptahydrate, 0.001 wt% sodium selenite, and 13.5 wt% L-glutathione reduced.

[0021] In certain embodiments, the cell culture additive or supplement composition of the disclosure may consist of histidine, methionine, glycine, and the zinc-based inorganic compound in a (2.39-4.77): (0.47-4.69): (0.67-6.66): (0.01-0.1) molar ratio, e.g., in a 2.39:0.47:0.67:0.01; 4.77:4.69:6.66:0.1; 4.77:0.47:3.33:0.01;4.77:4.69:6.66:0.01; or 4.77:0.47:6.66:0.1 molar ratio. In certain embodiments, the cell culture additive or supplement composition of the disclosure may consist of 28-89 wt% L-histidine hydrochloride monohydrate, 4-56 wt% methionine, 3-47 wt% glycine and 0.1-5 wt% zinc sulfate heptahydrate. In certain embodiments, the cell culture additive or supplement composition of the disclosure may consist of 80.3 wt% L-histidine hydrochloride monohydrate. 11.2 wt% methionine, 8.0 wt% glycine and 0.5 wt% zincsulfate heptahydrate.

[0022] In certain embodiments, the cell culture additive or supplement composition of the disclosure may consist of histidine, methionine, glycine, the zinc-based inorganic compound and the antioxidant in a (2.39-4.77): (0.47-4.69): (0.67-6.66): (0.01-0.1): (0.81-1.63) molar ratio, e.g., a (2.39-4.77): (0.47-4.69): (0.67-3.33): (0.01-0.1): (0.81- 1.63) molar ratio, or a (2.39-4.77): (0.47-4.69): (3.33-6.66): (0.01-0.1): (0.81-1.63) molar ratio, particularly in e.g., a 2.39:0.47:0.67:0.01 : 1.63; 4.77:4.69:6.66:0.1 : 1.63; 2.39:0.47:0.67:0.01 :0.81; 4.77:0.47:3.33:0.01 : 1.63; 4.77:4.69:6.66:0.01 : 1.63; or 4.77:0.47:6.66:0.1 :0.81 molar ratio. In certain embodiments, the cell culture additive or supplement composition of the disclosure may consist of histidine, methionine, glycine, the zinc-based inorganic compound, the selenite and glutathione in a (2.39- 4.77): (0.47-4.69): (0.67-6.66): (0.01-0.1): (0.00014-0.00145): (0.81-1.63) molar ratio, e.g., a (2.39-4.77): (0.47-4.69): (0.67-3.33): (0.01-0.1): (0.00014-0.00145): (0.81-1.63) molar ratio, or a (2.39-4.77): (0.47-4.69): (3.33-6.66): (0.01-0.1): (0.00014-0.00145): (0.81-1.63) molar ratio, particularly in e.g., a 2.39:0.47:0.67:0.01 :0.00145: 1.63; 4.77:4.69:6.66:0.1:0.00145: 1.63; 2.39:0.47:0.67:0.01:0.00014:0.81;4.77:0.47:3.33:0.01 :0.00145: 1.63; 4.77:4.69:6.66:0.01:0.00014: 1.63; or4.77:0.47:6.66:0.1:0.00014:0.81 molar ratio. In certain embodiments, the cell culture additive or supplement composition of the disclosure may consist of 22-73 wt% L- histidine hydrochloride monohydrate, 3-47 wt% methionine, 2-38 wt% glycine, 0.1-3 wt% zinc sulfate heptahydrate, 0.001 -0.03 wt% sodium selenite, and 10-45 wt% L- glutathione reduced. In certain embodiments, the cell culture additive or supplement composition of the disclosure may consist of 44.5 wt% L-histidine hydrochloride monohydrate, 6.2 wt% methionine, 4.5 wt% glycine. 0.27 wt% zinc sulfate heptahydrate, 0.022 wt% sodium selenite, and 44.5 wt% L-glutathione reduced; 36.6 wt% L-histidine hydrochloride monohydrate, 25.6 wt% methionine, 18.3 wt% glycine, 1.10 wt% zinc sulfate heptahydrate, 0.009 wt% sodium selenite, and 18.3 wt% L- glutathione reduced; 57.3 wt% L-histidine hydrochloride monohydrate, 8.0 wt% methionine, 5.7 wt% glycine, 0.34 wt% zinc sulfate heptahydrate, 0.003 wt% sodium selenite, and 28.6 wt% L-glutathione reduced; 54.8 wt% L-histidine hydrochloride monohydrate, 3.8 wt% methionine, 13.7 wt% glycine, 0.16 wt% zinc sulfate heptahydrate, 0.014 wt% sodium selenite, and 27.4 \\t% L-glutathione reduced; 37.0 wt% L-histidine hydrochloride monohydrate, 25.9 wt% methionine, 18.5 wt% glycine.0.11 wt% zinc sulfate heptahydrate, 0.001 wt% sodium selenite, and 18.5 wt% L- glutathione reduced; or 54.1 wt% L-histidine hydrochloride monohydrate, 3.8 wt% methionine, 27.0 wt% glycine, 1.62 wt% zinc sulfate heptahydrate, 0.001 wt% sodium selenite, and 13.5 wt% L-glutathione reduced.

[0023] The disclosure also provides a feed medium, which may comprise the cell culture additive or supplement composition of the disclosure.

[0024] The cell culture additive or supplement composition, or the feed medium, of the disclosure may be added to a fed-batch culture of a mammalian cell, such as a Chinese ovary (CHO) cell, e.g., a CHO-K1 cell, especially those adapted for suspension culture, in the early exponential phase (e g., Day 3 of the fed-batch culture), for enhancing cell growth and / or protein production.

[0025] In a second aspect, the disclosure provides the use of the cell culture additive or supplement composition, or the feed medium, of the disclosure, in culturing a mammalian cell.

[0026] The mammalian cell may be a Chinese ovary (CHO) cell, e.g., a CHO-K1, CHO-S or CHO-DG44 cell. The mammalian cell may have been adapted for suspension culture. The mammalian cell may be a CHO cell, e g., a CHO-K1 cell, for suspension culture.

[0027] The mammalian cell may be a recombinant cell that over-expresses a recombinant protein. The mammalian cell may comprise a nucleic acid molecule for over-expressing the recombinant protein, integrated into its genome, or in an expression vector. The expression vector may be a DNA molecule (e.g., a plasmid vector), a RNA molecule (e.g., a circular RNA molecule), or a DNA-RNA molecule. The mammalian cell may be introduced by e.g., transfection, with the nucleic acid molecule for overexpressing the recombinant protein in e.g., a vector. The vector may be a DNA molecule, e.g., a plasmid vector such as an adeno-associated virus (AAV) vector. The recombinant protein may be an antibody. The nucleic acid molecule may comprise the sequences for encoding the heavy chain variable region, light chain variable region, heavy chain constant region and / or light chain constant region, as required, with a sequence coding for e.g., a 2A peptide positioned between each two of these sequences.

[0028] The cell culture additive or supplement composition, or the feed medium, of the disclosure may be added to a fed-batch culture of the mammalian cell in the early exponential phase (e.g., Day 3 of the fed-batch culture).

[0029] Particularly, the disclosure provides a method for culturing a mammalian cell, which may comprise culturing the mammalian cell in a fed-batch culture, and adding to the cell in the early exponential phase (e.g., Day 3 of the fed-batch culture) the cell culture additive or supplement composition, or the feed medium, of the disclosure.

[0030] The step of adding the cell culture additive or supplement composition, or the feed medium, of the disclosure may comprise adding histidine, methionine, glycine, and a zinc-based inorganic compound at final concentrations of 2.39-4.77 mmol / L, 0.47-4.69 mmol / L, 0.67-6.66 mmol / L, and 0.01-0.1 mmol / L, respectively, e g., at final concentrations of 2.39-4.77 mmol / L, 0.47-4.69 mmol / L, 0.67-3.33 mmol / L, and 0.01- 0.1 mmol / L, respectively, or at final concentrations of 2.39-4.77 mmol / L, 0.47-4.69 mmol / L, 3.33-6.66 mmol / L. and 0.01-0.1 mmol / L, respectively. In certain embodiments, the step of adding the cell culture additive or supplement composition, or the feed medium, of the disclosure may comprise adding histidine, methionine, glycine, and a zinc-based inorganic compound at final concentrations of 2.39 mmol / L, 0.47 mmol / L, 0.67 mmol / L. and 0.01 mmol / L, respectively: 4.77 mmol / L. 4.69 mmol / L, 6.66 mmol / L, and 0.1 mmol / L, respectively; 4.77 mmol / L, 0.47 mmol / L, 3.33 mmol / L, and 0.01 mmol / L, respectively; 4.77 mmol / L, 4.69 mmol / L, 6.66 mmol / L, and 0.01 mmol / L, respectively; or 4.77 mmol / L, 0.47 mmol / L, 6.66 mmol / L, and 0.1 mmol / L, respectively. The step of adding the cell culture additive or supplement composition, or the feed medium, of the disclosure may comprise adding L-histidine hydrochloride monohydrate, methionine, glycine, and zinc sulfate heptahydrate at final concentrations of 500-1000 mg / L, 70-700 mg / L, 50-500 mg / L and 3-30 mg / L, respectively. In certain embodiments, the step of adding the cell culture additive or supplement composition, or the feed medium, of the disclosure may comprise adding L-histidine hydrochloride monohydrate, methionine, glycine, and zinc sulfate heptahydrate at final concentrations of 500 mg / L, 70 mg / L, 50 mg / L and 3 mg / L, respectively.

[0031] The step of adding the cell culture additive or supplement composition, or the feed medium, of the disclosure may comprise adding histidine, methionine, glycine, a zinc-based inorganic compound and an antioxidant at final concentrations of 2.39-4.77 mmol / L, 0.47-4.69 mmol / L, 0.67-6.66 mmol / L, 0.01-0.1 mmol / L, and 0.81-1.63 mmol / L, respectively, e.g., at the final concentrations of 2.39-4.77 mmol / L, 0.47-4.69 mmol / L, 0.67-3.33 mmol / L, 0.01-0.1 mmol / L, and 0.81-1.63 mmol / L, respectively, orat the final concentrations of 2.39-4.77 mmol / L, 0.47-4.69 mmol / L, 3.33-6.66 mmol / L, 0.01-0.1 mmol / L, and 0.81-1.63 mmol / L. respectively. In certain embodiments, the step of adding the cell culture additive or supplement composition, or the feed medium, of the disclosure may comprise adding histidine, methionine, glycine, a zinc-based inorganic compound and an antioxidant at final concentrations of 2.39 mmol / L, 0.47 mmol / L, 0.67 mmol / L, 0.01 mmol / L. and 1.63 mmol / L. respectively; 4.77 mmol / L, 4.69 mmol / L, 6.66 mmol / L, 0.1 mmol / L and 1.63 mmol / L, respectively; 2.39 mmol / L, 0.47 mmol / L, 0.67 mmol / L, 0.01 mmol / L, and 0.81 mmol / L, respectively; 4.77 mmol / L, 0.47 mmol / L, 3.33 mmol / L, 0.01 mmol / L and 1.63 mmol / L, respectively; 4.77 mmol / L, 4.69 mmol / L, 6.66 mmol / L, 0.01 mmol / L and 1.63 mmol / L, respectively;4.77 mmol / L, 0.47 mmol / L, 6.66 mmol / L, 0.1 mmol / L and 0.81 mmol / L, respectively. The step of adding the cell culture additive or supplement composition, or the feed medium, of the disclosure may comprise adding L-histidine hydrochloride monohydrate, methionine, glycine, zinc sulfate heptahydrate, sodium selenite, and L-glutathione reduced at final concentrations of 500-1000 mg / L, 70-700 mg / L, 50-500 mg / L, 3-30 mg / L, 0.025-0.25 mg / L and 250-500 mg / L, respectively, e.g., at final concentrations of 500-1000 mg / L, 70-700 mg / L, 50-250 mg / L, 3-30 mg / L, 0.025-0.25 mg / L and 250-500 mg / L, respectively, or at final concentrations of 500-1000 mg / L, 70-700 mg / L, 250-500 mg / L, 3-30 mg / L, 0.025-0.25 mg / L and 250-500 mg / L, respectively. In certain embodiments, the step of adding the cell culture additive or supplement composition, or the feed medium, of the disclosure may comprise adding L-histidine hydrochloride monohydrate, methionine, glycine, zinc sulfate heptahydrate, sodium selenite, and L- glutathione reduced at final concentrations of 500 mg / L, 70 mg / L, 50 mg / L, 3 mg / L, 0.25 mg / L and 500 mg / L, respectively; 1000 mg / L, 700 mg / L, 500 mg / L, 30 mg / L, 0.25 mg / L and 500 mg / L, respectively; 500 mg / L, 70 mg / L, 50 mg / L, 3 mg / L, 0.025 mg / L and 250 mg / L, respectively; 1000 mg / L, 70 mg / L, 250 mg / L, 3 mg / L, 0.25 mg / L and 500 mg / L, respectively; 1000 mg / L, 700 mg / L, 500 mg / L, 3 mg / L, 0.025 mg / L and 500 mg / L, respectively; or 1000 mg / L, 70 mg / L, 500 mg / L, 30 mg / L, 0.025 mg / L and 50 mg / L, respectively.

[0032] The method may further comprise maintaining the glucose concentration at about 7 g / L during the fed-batch culture. In certain embodiment, the method may comprise supplementing glucose even' day, from e.g., Day 3 of the fed-batch culture, to bring glucose concentration to about 7 g / L.

[0033] The method may comprise inoculating the mammalian cell at a suitable seeding density. In certain embodiments, the method may comprise inoculating the mammalian cell at a cell density of 0.4 to 0.6xl06 / mL.

[0034] The method may comprise culturing the mammalian cell under a suitable condition. In certain embodiments, the method may comprise culturing the mammalian cell at a shaking speed of 280 rpm under 5%CCh and 80% humidity.

[0035] The method may comprise culturing the mammalian cell for 11 days or more. In certain embodiments, the method may comprise culturing the mammalian cell for 11 days.

[0036] The method may comprise culturing the mammalian cell in chemically defined basal and feed media. In certain embodiment, the method may comprise culturing the mammalian cell in Dynamis™ AGT™ basal medium and 3x concentrated EfficientFeedB+ AGTfeed medium. In certain embodiment, the method may comprise culturing the mammalian cell in UproCHO M01 basal medium and UproCHO FOIA and UproCHO F01B feed media. The method may comprise adding the basal medium on Day 1 of the batch-fed culture. The method may comprise adding the feed medium every other day, beginning on Day 3 of the batch-fed culture.

[0037] The mammalian cell may be a Chinese ovary (CHO) cell, e.g., a CHO-K1 cell. The mammalian cell may have been adapted for suspension culture. The mammalian cell may be a CHO cell, e.g.. a CHO-K1 cell, for suspension culture.

[0038] The mammalian cell may be a recombinant cell that over-expresses a recombinant protein. The mammalian cell may comprise a nucleic acid molecule for over-expressing the recombinant protein, integrated into its genome, or in an expression vector. The expression vector may be a DNA molecule (e.g., a plasmid vector), a RNA molecule (e.g., a circular RNA molecule), or a DNA-RNA molecule. The mammalian cell may be introduced by e.g., transfection, with the nucleic acid molecule for overexpressing the recombinant protein in e.g., a vector. The vector may be a DNA molecule, e.g., a plasmid vector such as an adeno-associated virus (AAV) vector. The recombinant protein may be an antibody. The nucleic acid molecule may comprise the sequences for coding the heavy chain variable region, light chain variable region, heavy chain constant region and / or light chain constant region, as required, with a sequence coding for e.g., a 2A peptide positioned between each two of these sequences.

[0039] In a third aspect, the disclosure provides the use of the cell culture additive orsupplement composition, or the feed medium, of the disclosure, in producing a recombinant protein in a mammalian cell.

[0040] The mammalian cell may be a Chinese ovary (CHO) cell, e.g., a CHO-K1 cell. The mammalian cell may have been adapted for suspension culture. The mammalian cell may be a CHO cell, e.g., a CHO-K1 cell, for suspension culture.

[0041] The mammalian cell may be a recombinant cell that over-expresses the recombinant protein. The mammalian cell may comprise a nucleic acid molecule for over-expressing the recombinant protein, integrated into its genome, or in an expression vector. The expression vector may be a DNA molecule (e.g., a plasmid vector), a RNA molecule (e.g., a circular RNA molecule), or a DNA-RNA molecule. The mammalian cell may be introduced by e.g., transfection, with the nucleic acid molecule for overexpressing the recombinant protein in e.g., a vector. The vector may be a DNA molecule, e.g., a plasmid vector such as an adeno-associated virus (AAV) vector. The recombinant protein may be an antibody . The nucleic acid molecule may comprise the sequences for coding the heavy chain variable region, light chain variable region, heavy chain constant region and / or light chain constant region, as required, with a sequence coding for e.g., a 2A peptide positioned between each two of these sequences . The cell culture additive or supplement composition, or the feed medium, of the disclosure may be added to a fed-batch culture of the mammalian cell in the early exponential phase (e.g.. Day 3 of the fed-batch culture).

[0042] Particularly, the disclosure provides a method for producing a recombinant protein in a mammalian cell, wherein the mammalian cell over-expresses a recombinant protein, which may comprise performing the method according to the second aspect of the disclosure.

[0043] The method may further comprise collecting the recombinant protein from the cell culture.

[0044] The mammalian cell may be a Chinese ovar7(CHO) cell, e.g., a CHO-K1 cell. The mammalian cell may have been adapted for suspension culture. The mammalian cell may be a CHO cell, e.g., a CHO-K1 cell, for suspension culture.

[0045] The mammalian cell may be a recombinant cell that over-expresses a recombinant protein. The mammalian cell may comprise a nucleic acid molecule for over-expressing the recombinant protein, integrated into its genome, or in an expression vector. The expression vector may be a DNA molecule (e.g., a plasmid vector), a RNAmolecule (e.g., a circular RNA molecule), or a DNA-RNA molecule. The mammalian cell may be introduced by e.g., transfection, with the nucleic acid molecule for overexpressing the recombinant protein in e.g., a vector. The vector may be a DNA molecule, e.g., a plasmid vector such as an adeno-associated virus (AAV) vector. The recombinant protein may be an antibody . The nucleic acid molecule may comprise the sequences for coding the heavy chain variable region, light chain variable region, heavy chain constant region and / or light chain constant region, as required, with a sequence coding for e g., a 2A peptide positioned between each two of these sequences.

[0046] In a fourth aspect, the disclosure provides the use of the cell culture additive or supplement composition, or the feed medium, of the disclosure, in enhancing cell growth. The enhancement of cell growth includes prevention of cell viability decrease or drop in the late culture stage (e.g., the late log phase and / or early stationary phase).

[0047] The mammalian cell may be a Chinese ovary (CHO) cell, e.g., a CHO-K1 cell. The mammalian cell may have been adapted for suspension culture. The mammalian cell may be a CHO cell, e.g.. a CHO-K1 cell, for suspension culture.

[0048] The cell culture additive or supplement composition, or the feed medium, of the disclosure may be added to a fed-batch culture of the mammalian cell in the early exponential phase (e.g., Day 3 of the fed-batch culture).

[0049] Particularly, the disclosure provides a method for enhancing cell culture of a mammalian cell, which may comprise performing the method according to the second aspect of the disclosure. Particularly, the method may comprise culturing the mammalian cell in a fed-batch culture, and adding to the cell in the early exponential phase (e.g., Day 3 of the fed-batch culture) the cell culture additive or supplement composition, or the feed medium, of the disclosure.

[0050] Other features and advantages of the instant disclosure will be apparent from the following detailed description and examples, which should not be construed as limiting. The contents of all references, Genbank entries, patents and published patent applications cited throughout this application are expressly incorporated herein by reference.

[0051] Accordingly, it is an object of the invention not to encompass within the invention any previously known product, process of making the product, or method of using the product such that Applicants reserve the right and hereby disclose a disclaimer of any previously known product, process, or method. It is further noted that theinvention does not intend to encompass within the scope of the invention any product, process, or making of the product or method of using the product, which does not meet the written description and enablement requirements of the USPTO (35 U.S.C. §112, first paragraph) or the EPO (Article 83 of the EPC), such that Applicants reserve the right and hereby disclose a disclaimer of any previously described product, process of making the product, or method of using the product. It may be advantageous in the practice of the invention to be in compliance with Art. 53(c) EPC and Rule 28(b) and (c) EPC. All rights to explicitly disclaim any embodiments that are the subject of any granted patent(s) of applicant in the lineage of this application or in any other lineage or in any prior filed application of any third part}' is explicitly reserved. Nothing herein is to be construed as a promise.

[0052] It is noted that in this disclosure and particularly in the claims and / or paragraphs, terms such as "comprises", "comprised", "comprising" and the like can have the meaning attributed to it in U.S. Patent law; e.g., they can mean "includes", "included", "including", and the like; and that terms such as "consisting essentially of' and "consists essentially of' have the meaning ascribed to them in U.S. Patent law, e.g., they allow for elements not explicitly recited, but exclude elements that are found in the prior art or that affect a basic or novel characteristic of the invention.DESCRIPTION OF THE DRAWINGS

[0053] The following detailed description, given by way of example, but not intended to limit the invention solely to the specific embodiments described, may best be understood in conjunction with the accompanying drawings.

[0054] FIG. 1A and IB show the viable cell density (A) and cell viability (B) of CHO cells during fed-batch culture to which a cell culture additive composition 1 or a cell culture additive composition 2 was added on Day 3.

[0055] FIG. 2A and 2B show the viable cell density (A) and cell viability' (B) of CHO cells during fed-batch culture to which the cell culture additive composition 1 was added on Day 3 with varying component concentrations.

[0056] FIG. 3A and 3B show the viable cell density (A) and cell viability (B) of CHO cells during fed-batch culture using a different basal medium and a different feed medium to which the cell culture additive composition 1 was added on Day 3 with varying component concentrations.DETAILED DESCRIPTION OF THE INVENTION

[0057] The present disclosure is now further described with the non-limiting examples below.

[0058] As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0059] The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within one or more than one standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, up to 15%, up to 10%, up to 5%, or up to 1% of a given value.

[0060] The term “comprising”, “comprise”, “containing”, or “contain” is inclusive and does not exclude additional, unrecited elements or features, as long as they do not render the invention unworkable. The term “consist” or “consisting” excludes any element, step, or ingredient not specifically mentioned after said wording. Furthermore, to the extent that the terms “including”, “includes”, “having”, “has”, “with”, or variants thereof are used in either the detailed description and / or the claims, such terms are intended to be inclusive in a manner similar to the term “comprising”.

[0061] A cell culture “additive” or “supplement” refers to a substance or substances added to the cell culture medium in e.g., small quantities, to maintain and / or promote the growth and / or differentiation of cells, to extend or strengthen an attribute of the culture or cells as a whole, or to make up for a deficiency. The cell culture additive or supplement of the disclosure is mainly used to prevent cell viabi li ty drop in the late culture stage, and / or to improve recombinant production by the cultured cells.

[0062] A “fed-batch” culture is a modified batch culture method that adds compounds needed for cell growth, cell proliferation and / or recombinant protein production periodically. In a fed-batch process, a basal medium supports initial growth and production, and a feed medium prevents depletion of nutrients and sustains the production phase.

[0063] The “over-expression” of a gene or a protein refers to an unnatural or abnormal increase in the production or expression of a gene in an organism, e.g.. a recombinant host cell, resulting in increased RNA or protein levels.

[0064] The term “host cell” refers to a living cell invaded by or capable of being invaded by a foreign nucleic acid-carrying vector, e g., a plasmid vector.

[0065] A “recombinant” cell is a cell that receives DNA from an outside source and incorporates it into its own. The process of creating recombinant cells involves combining genetic material from two different sources to alter their characteristics.

[0066] The term “transfecting” or “transfection” refers to a technique or process that introduces a foreign nucleic acid (DNA or RNA) into a cell using non-viral methods. The foreign nucleic acid as introduced into the cell may integrate into the cell genome, or exist as an independent molecule but interferes with the cell’s function.

[0067] An “antibody”, also known as an immunoglobulin, is a glycoprotein produced by e.g., plasma cells, or in vitro recombinantly produced, that recognizes and specifically binds a target, through at least one antigen-binding site. An antibody can be any of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes) thereof (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2), based on the identity of their heavy-chain constant domains referred to as alpha, delta, epsilon, gamma, and mu, respectively. The different classes of immunoglobulins have different and well-known subunit structures and three-dimensional configurations. A full-length IgG antibody may comprise two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain may be comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (CH). The heavy chain constant region may be comprised of three domains, CHI, CH2 and C . Each light chain may be comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region (CL). The light chain constant region may be comprised of one domain. The heavy chain constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system, while the light chain constant region may function to stabilize the antibody structure.

[0068] The term “antibody” herein also includes the “antigen-binding portion” of an antibody, which refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term “antigen-binding portion” of anantibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab')2 fragment, a bivalent fragment which may comprise two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, and (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546), which consists of a VH domain.

[0069] The term "monoclonal antibody7’ or "m Ab” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations and / or post-translation modifications (e.g., isomerizations, amidations) that may be present in minor amounts.

[0070] The term “vector” refers to a substance that is used to carry or include a nucleic acid sequence, including for example, in order to introduce a nucleic acid sequence into a host cell and optionally express a nucleic acid sequence in the host cell. Vectors applicable for use include, for example, expression vectors, plasmids, phage vectors, viral vectors, episomes and artificial chromosomes, which can include selection sequences or markers operable for replication in a host cell or for stable integration into a host cell’s chromosome. Additionally, the vectors can include one or more selectable marker genes and appropriate expression control sequences. Selectable marker genes that can be included, for example, to provide resistance to antibiotics or toxins, complement auxotrophic deficiencies, or supply critical nutrients not in the culture media. Expression control sequences can include constitutive and inducible promoters, transcription enhancers, transcription terminators, and the like which are well known in the art. The term “promoter” as used herein, generally refers to the regulatory DNA region which controls transcription or expression of a gene and which may be located adjacent to or overlapping a nucleotide or region of nucleotides at which RNA transcription is initiated. A promoter may contain specific DNA sequences which bind protein factors, often referred to as transcription factors, which facilitate binding of RNA polymerase to the DNA leading to gene transcription. When two or more nucleic acid molecules are to be co-expressed, both nucleic acid molecules can be inserted, for example, into a single expression vector or in separate expression vectors. For single vector expression, the encoding nucleic acids can be operationally linked to one common expression control sequence or linked to different expression controlsequences, such as one inducible promoter and one constitutive promoter. The introduction of nucleic acid molecules into a host cell can be confirmed using methods well known in the art. It is understood by those skilled in the art that the nucleic acid molecules are expressed in a sufficient amount to produce a desired product, and it is further understood that expression levels can be optimized to obtain sufficient expression using methods well known in the art. Particularly, an ‘“expression vector” is associated with actual expression of a coding DNA fragment into an mRNA and a protein in the target cell or organism, and usually contains all the regulatory sequences, such as promoter, ribosomal binding site, transcription initiation site, translation initiation site, which are essential for getting maximum expression. The expression vector in the disclosure may be a DNA vector or an RNA vector, e.g., an mRNA or a circular RNA.

[0071] The term “integrated into” or “integrating into” the genome of a cell means the insertion or addition of a correct DNA sequence into a cell genome at a proper site to produce a functional protein or functional proteins.

[0072] In the current biologic drug market, the monoclonal antibody occupies a large share due to its simple structure, easy production and high antigen specificity, but it is usually administered in a higher dose than other types of biologies. Thus, a relatively high production titer is required for mAbs, to reduce the unit production cost of mAb drugs and ease the patients’ cost burden.

[0073] The mAb production is often done in mammalian cells such as CHO cells in a fed-batch culture. A fed-batch culture is a semi-batch operation in which the nutrients necessary for cell growth and target product production, including the carbon source, nitrogen source, amino acids, and some kinds of inducers, are fed either intermittently or continuously during the course of an otherwise batch operation. The culture broth is harvested only at the end of the operational period. The over-supply or under-supply of the carbon source or any other compounds during the cell culture may adversely affect the cell growth and product production. For example, during the penicillin fermentation, the over-supply of a carbon source resulted in more mycelial growth and low- penicillin formation, while under-supply caused slower mycelial growth and eventually slower penicillin formation (Henry C. Lim, Hwa Sung Shin, (2013) Fed- Batch Cultures: Principles and Applications of Semi-Batch Bioreactors, Cambridge University Press, ISBN: 9780521513364). Therefore, it is the goal in the fed-batchculture to manipulate the feed rates, such that the concentrations of the nutrients in the culture can either remain at a constant level or follow a predetermined optimal profde, which is however time and labor consuming.

[0074] The most effective and straightforward way to improve the product production (e.g., mAh production) may be to select a basal medium and a feed medium with good performance. Some components in the media may be not at the amounts optimal for mAb production, as these components in high levels may be not easy to dissolve, or even have inhibitory effect on cell growth or proliferation. However, these components may be added as a cell culture additive or supplement when needed.

[0075] The inventors of the disclosure, have designed a cell culture additive or supplement, that simply contains three amino acids, a zinc-based inorganic compound and an optional antioxidant. When added to CHO cells in a fed-batch culture in the early exponential phase (e.g., Day 3 of the fed-batch culture), the cell culture additive or supplement surprisingly prevented cell viability' decrease or drop in the late culture stage (e.g., the late log phase and / or early stationary phase), and enhanced recombinant protein production by the CHO cells as measured by e.g., the increased protein level, no matter what basal and feed media were used in the cell culture.

[0076] The three amino acids contained in the cell culture additive or supplement of the disclosure are histidine, methionine, glycine, among which histidine and methionine are essential amino acids, and glycine is the non-essential amino acid. Amino acids are the basic building blocks of proteins and logically constitute all proteinaceous material of the cell. A large proportion of amino acids in the cell culture medium are diverted to the non-proteogenic pathways, including the TCA cycle, that in turn may affect the fate of the cells (Salazar A, et al., (2016) Amino acids in the cultivation of mammalian cells. Amino Acids. 48(5): 1161-71). The essential amino acids are those cannot be synthesized by the cells and thus must be included in the culture medium. They may be required for cell proliferation and their concentrations may determine the maximum achievable cell density. The nonessential amino acids are those can be made by the cells themselves, but can still be added to the cell culture to stimulate cell growth and prolong cell viability7(Arora M. (2013) Cell culture media: a review. Mater Methods . 3: 175). The consumption rates of histidine and methionine in the cells during the fed- batch culture are relatively low as compared to other essential amino acids, either in the log phase or the stationary phase (Pan X, et al.. (2017) supra). The concentrations ofthese two amino acids in the cell culture medium range from about 0.015 g / L to about 0. 150 g / L, and the concentration of glycine in the cell culture medium is about 0.008 to 0.330 g / L (Salazar A, et al., (2016) supra). It is reported that methionine is a limiting amino acid, while glycine may increase ammonium production and should be decreased (Zizhuo Xing et al., (2011) supra). The methionine is provided in most commercially available cell culture media at a higher concentration that exceeds the consumption rate (Pan X. et al., (2017) supra).

[0077] The further addition of histidine, methionine, glycine to the cell culture respectively at final concentrations of 500-1000 mg / L, 70-700 mg / L and 50-500 mg / L, as described in the present application, at Day 3 of the cell culture, surprisingly prevented the cell viability drop in the late culture stages, and more importantly, improved recombinant protein production by the cultured cells.

[0078] The zinc-based inorganic compound, e.g., zinc sulfate, as contained in the cell culture additive or supplement, is a trace element that is needed in minute amount for proper cell growth. Ryan J. Graham et al., found that most zinc consumption occurs primarily during the lag phase and becomes minimal thereafter, and the impact of the zinc supplement on beta-glycuronidase (GUS) production was marginal (< 6%) (Ryan J. Graham, et al., (2001) supra).

[0079] Surprisingly, the addition of a zinc-based inorganic compound, with the three amino acids, to the cell culture at the final concentration of about 3-30 mg / L, prevented the cell viability’ drop in the late culture stages, and more importantly, improved recombinant protein production by the cultured cells.

[0080] Most cells in animals are exposed to low O2 concentrations, in the range of 1- 10 mm Hg, while the in vitro cell culture is commonly performed under 95% air / 5% CO2, generating more reactive oxygen species (ROS) and imposing an oxidative stress on the cells. The cell culture media are frequently deficient in the antioxidants that are normally obtained in the animal diet and delivered to cells by bloodstream. Neither vitamin E nor vitamin C is added to the cell culture medium as being insoluble or unstable. The lack of selenium in the culture medium may decrease the activity of thioredoxin reductase, and thus impair cell metabolism (Halliwell B. (2014) Cell culture, oxidative stress, and antioxidants: avoiding pitfalls. Biomed J. 37(3):99-105).

[0081] The addition of two antioxidants, namely sodium selenite, and L-glutathione reduced, to the cell culture surprisingly decreased viable cell density from early logphase. However, more surprisingly, the evidently less cells produced more antibodies, as shown in Example 1 below.

[0082] Therefore, the disclosure provides a cell culture additive or supplement composition, which may comprise histidine, methionine, glycine, and a zinc-based inorganic compound.

[0083] The cell culture additive or supplement composition of the disclosure may comprise or consist of histidine, methionine, glycine, and the zinc-based inorganic compound in a (2.39-4.77): (0.47-4.69): (0.67-6.66): (0.01-0.1) molar ratio. In certain embodiments, the cell culture additive or supplement composition of the disclosure may comprise or consist of 28-89 wt% L-histidine hydrochloride monohydrate, 4-56 wt% methionine, 3-47 wt% glycine and 0. 1-5 wt% zinc sulfate heptahydrate.

[0084] Alternatively, the cell culture additive or supplement composition of the disclosure may comprise or consist of histidine, methionine, glycine, the zinc-based inorganic compound and the antioxidant in a (2.39-4.77): (0.47-4.69): (0.67-6.66): (0.01-0.1): (0.81-1.63) molar ratio. In certain embodiments, the cell culture additive or supplement composition of the disclosure may comprise or consist of histidine, methionine, glycine, the zinc-based inorganic compound, the selenite and glutathione in a (2.39-4.77): (0.47-4.69): (0.67-6.66) :(0.01-0.1): (0.00014-0.00145): (0.81-1.63) molar ratio. In certain embodiments, the cell culture additive or supplement composition of the disclosure may comprise or consist of 22-73 wt% L-histidine hydrochloride monohydrate, 3-47 wt% methionine, 2-38 wt% glycine, 0.1 -3 wt% zinc sulfate heptahydrate, 0.001-0.03 wt% sodium selenite, and 10-45 wt% L-glutathione reduced.

[0085] The cell culture additive or supplement composition of the disclosure may comprise 22 wt% or more, 36.6 wt% or more, 37.0 wt% or more, 44.5 wt% or more, 54.1 \\1% or more, 54.8 wt% or more, 57.3 wt% or more, or 73 wt% or more of L- histidine hydrochloride monohydrate. The cell culture additive or supplement composition of the disclosure may comprise 73 wt% or less, 57.3 wt% or less, 54.8 wt% or less, 54.1 wt% or less, 44.5 wt% or less, 37.0 wt% or less, 36.6 wt% or less, or 22 wt% or less of L-histidine hydrochloride monohydrate.

[0086] The cell culture additive or supplement composition of the disclosure may comprise 3 wt% or more, 3.8 wt% or more, 6.2 wt% or more, 8.0 wt% or more, 25.6 wt% or more, 25.9 wt% or more, or 47 wt% or more of methionine The cell cultureadditive or supplement composition of the disclosure may comprise 47 wt% or less, 25.9 wt% or less, 25.6 wt% or less, 8.0 wt% or less, 6.2 wt% or less. 3.8 wt% or less, or 3 wt% or less of methionine.

[0087] The cell culture additive or supplement composition of the disclosure may comprise 2 wt% or more, 4.5 wt% or more, 5.7 wt% or more, 13.7 wt% or more, 18.3 wt% or more, 18.5 wt% or more, 27.0 wt% or more, or 38.0 wt% or more of glycine. The cell culture additive or supplement composition of the disclosure may comprise 38.0 wt% or less, 27.0 wt% or less, 18.5 wt% or less, 18.3 wt% or less, 13.7 \\t% or less, 5.7 wt% or less, 4.5 wt% or less, or 2 wt% or less of glycine.

[0088] The cell culture additive or supplement composition of the disclosure may comprise 0.1 wt% or more, 0.11 wt% or more, 0.16 wt% or more, 0.27 wt% or more, 0.34 wt% or more, 1.10 wt% or more, 1.62 wt% or more, or 3.0 wt% or more of zinc sulfate heptahydrate. The cell culture additive or supplement composition of the disclosure may comprise 3.0 wt% or less, 1.62 wt% or less, 1.10 wt% or less, 0.34 wt% or less, 0.27 wt% or less. 0. 16 wt% or less, 0. 11 wt% or less, or 0. 1 wt% or less of zinc sulfate heptahydrate.

[0089] The cell culture additive or supplement composition of the disclosure may comprise 0.001 wt% or more, 0.003 wt% or more, 0.009 wt% or more, 0.014 wt% or more, 0.022 wt% or more, or 0.03 wt% or more of sodium selenite. The cell culture additive or supplement composition of the disclosure may comprise 0.03 wt% or less. 0.022 wt% or less, 0.014 wt% or less, 0.009 wt% or less, 0.003 wt% or less, or 0.001 wt% or less of sodium selenite.

[0090] The cell culture additive or supplement composition of the disclosure may comprise 10.0 wt% or more, 13.5 wt% or more, 18.3 wt% or more, 18.5 wt% or more, 27.4 wt% or more, 28.6 wt% or more, 44.5 wt% or more, or 45.0 wt% or more of L- glutathione reduced. The cell culture additive or supplement composition of the disclosure may comprise 45.0 wt% or less, 44.5 wt% or less, 28.6 wt% or less, 27.4 wt% or less, 18.5 wt% or less. 18.3 wt% or less, 13.5 t% or less, or 10.0 wt% or less of L- glutathione reduced.

[0091] The cell culture additive or supplement composition, or the feed medium, of the disclosure may be added to a fed-batch culture of a mammalian cell, such as a Chinese ovary (CHO) cell, e.g., a CHO-K1 cell, especially those adapted for suspension culture, in the early exponential phase (e.g., Day 3 of the fed-batch culture), forenhancing cell growth and / or protein production.

[0092] In another respect, the disclosure provides a method for culturing a mammalian cell, which may comprise culturing the mammalian cell in a fed-batch culture, and adding to the cell in the early exponential phase (e.g., Day 3 of the fed-batch culture) the cell culture additive or supplement composition, or the feed medium, of the disclosure.

[0093] This method may be used to produce a recombinant protein, e.g., an antibody, especially a monoclonal antibody, resulting in improved protein titer.

[0094] This method may be used to culture a mammalian cell, resulting in high viability in the late culture stages.

[0095] The step of adding the cell culture additive or supplement composition, or the feed medium, of the disclosure may comprise adding histidine, methionine, glycine, and a zinc-based inorganic compound at final concentrations of 2.39-4.77 mmol / L, 0.47-4.69 mmol / L, 0.67-6.66 mmol / L, and 0.01-0.1 mmol / L, respectively. The step of adding the cell culture additive or supplement composition, or the feed medium, of the disclosure may comprise adding L-histidine hydrochloride monohydrate, methionine, glycine, and zinc sulfate heptahydrate at final concentrations of 500-1000 mg / L, 70- 700 mg / L, 50-500 mg / L and 3-30 mg / L, respectively.

[0096] The step of adding the cell culture additive or supplement composition, or the feed medium, of the disclosure may comprise adding histidine, methionine, glycine, a zinc-based inorganic compound and an antioxidant at final concentrations of 2.39-4.77 mmol / L, 0.47-4.69 mmol / L, 0.67-6.66 mmol / L, 0.01-0.1 mmol / L, and 0.81-1.63 mmol / L, respectively. The step of adding the cell culture additive or supplement composition, or the feed medium, of the disclosure may comprise adding L-histidine hydrochloride monohydrate, methionine, glycine, zinc sulfate heptahydrate, sodium selenite, and L-glutathione reduced at final concentrations of 500-1000 mg / L, 70-700 mg / L, 50-500 mg / L, 3-30 mg / L, 0.025-0.25 mg / L and 250-500 mg / L„ respectively.

[0097] The method may further comprise supplementing glucose every day, from e.g., Day 3 of the fed-batch culture, to bring glucose concentration to a suitable level, e.g., about 7 g / L. The method may comprise inoculating the mammalian cell at a suitable seeding density, e.g., 0.4 to 0.6><106 / mL. The method may comprise culturing the mammalian cell under a suitable condition.

[0098] The method may comprise culturing the mammalian cell for 11 days or more.The data showed that the viable cell density or cell viability was still quite high on Day 11, meaning the cell culture may last for more than 11 days, if required.

[0099] The additive or supplement of the disclosure may be used in cell culture using any suitable cell culture media. In the Examples of the disclosure, two basal and feed media combinations were used in the tests. It turned out that the addition of the additive or supplement at the early culture phase, e.g., Day 3 of the cell culture, resulted in enhanced cell viability in the late culture stages and more importantly higher antibody titer, no matter what cell culture media were used. In certain embodiments, the method may comprise culturing the mammalian cell in chemically defined basal and feed media.

[0100] The mammalian cell may be a Chinese ovary (CHO) cell, e g., a CHO-K1 cell. The mammalian cell may have been adapted for suspension culture. The mammalian cell may be a CHO cell, e.g., a CHO-K1 cell, for suspension culture.

[0101] The mammalian cell may be a recombinant cell that over-expresses a recombinant protein. The mammalian cell may comprise a nucleic acid molecule for over-expressing the recombinant protein, integrated into its genome, or in an expression vector. The recombinant protein may be an antibody. The nucleic acid molecule for expressing the desired protein, e.g., an antibody, may be in a vector and introduced into the cell with the vector. The nucleic acid molecule may express the protein in the vector. Alternatively, the nucleic acid molecule may be integrated or incorporated into the genome of the host cell to obtain stable cell lines. The skilled in the art know the technique for integrating foreign DNAs into the genome of a host cell. For example, a vector carrying a foreign DNA may incorporate the foreign DNA into the host cell genome by random integration, where the DNA inserts into random locations within the genome. Alternatively, the integration can occur through homologous recombination, where the foreign DNA is precisely inserted at a specific site guided by homologous sequences. Another method involves the use of transposon elements, e.g., the transposase and transposon-derived ITRs, which facilitate the movement and integration of the DNA into various genomic locations. In the Examples below, the mammalian cell used for over-expressing the antibody was constructed by introducing the antibody-encoding sequences in a vector (e g., a plasmid) into the cell by transfection, and integrating the sequences into the cell’s genome by random integration.

[0102] The present invention will be further illustrated in the following Examples which are given for illustration purposes only and are not intended to limit the inventionin any way.EXAMPLESExample 1. Addition of cell culture additives in fed-batch culture of CHO cells

[0103] CHO-K1 cells, obtained by adapting CHO-K1 cells (ECACC, Cat. No.: 85051005) to suspension culture, were transfected, by electroporation, with a plasmid vector (pGenHT 1.0-UP, GenScript Probio) containing a DNA fragment encoding a full-length human IgG antibody, and screened for monoclonal stable cell lines capable of producing the antibody.

[0104] One of the monoclonal stable cell lines as obtained was seeded into a 50 mb spin tube in 10 mL Dynamis™ AGT™ basal medium (Thermo Fischer Technologies, A26175-02) at the cell density of 0.5 ( ± 0. l)x lO6 / mL, and cultured in a shaking incubator at the shaking speed of 280 rpm under 5%CO2 and 80% humidity.

[0105] On Day 3 of the cell culturing, a cell culture additive composition 1, containing L-histidine hydrochloride monohydrate (PanReac Applichem, A1591,1000), methionine (PanReac Applichem, A1340,1000), glycine (PanReac Applichem, 141340), zinc sulfate heptahydrate (Sigma Aldrich, Z0251-100g), sodium selenite (Sigma Aldrich, S5261-10g), and L-glutathione reduced (Sigma Aldrich, G6013-5g), was added to a group of cells, and a cell culture additive composition 2, containing L- histidine hydrochloride monohydrate, methionine, glycine, and zinc sulfate heptahydrate, was added to another group of cells, with the final concentration of each additive component in the cell culture set forth in Table 1. No cell culture additive was added to the control cells.

[0106] Beginning on Day 3, 4% v / v 3x concentrated Effi ci entFeedB+ AGT(Thermo Fischer Technologies, A2503001) as the feed medium was supplemented every other day according to the initial cell culture medium volume. The glucose concentration in the cell culture was measured every day, and a 400 g / kg of glucose stock solution was added to the culture medium every day as needed to maintain the glucose concentration at about 7 g / L.

[0107] The viable cell density (VCD), cell viability (VIA), cell diameter (DIA), and the glucose, lactate and ammonium levels were monitored regularly throughout the 11- day cell culture, and the antibody titer or level in the cell culture supernatant on Day 11 was measured.Table 1. Final concentration of each additive component in cell culture

[0108] FIG. 1A and IB showed the viable cell density (VCD, *106cells / mL, A) anc viability (%, B) of the cells from the 3 groups during the 11-day cell culture. It can be seen that the additive composition 1 reduced the VCD a bit, but both additive composition 1 and additive composition 2 prevented cell viability (%) decrease at the late culture stage.

[0109] The antibody titers as measured on Day 11 were set forth in Table 2. The addition of both additive compositions promoted the antibody production as compared to the control group, with the antibody titer from the cells added with the additive composition 1 being the highest.Table 2. Effect of cell culture additive composition on antibody productionExample 2, Addition of cell culture additives at different concentrations

[0110] Following the protocol of Example 1. the same stable CHO-K1 cells were subjected to 11-day fed-batch culture, except that the additive composition 1 was added to the cell culture on Day 3 with different final concentrations of the additive components as set forth in Table 3.Table 3. Final concentration of each additive component in cell culture medium

[0111] FIG. 2A and 2B showed the viable cell density (VCD, xlO6cells / mL, A) and viability' (%, B) of the cells from the 5 groups during the 11-day cell culture. It can be seen that the additive composition 1, when added at different final component concentrations, reduced the VCD, but prevented cell viability (%) decrease to some extent at the late culture stage.

[0112] More importantly, as shown in Table 4, the addition of the additive composition 1, in spite of the different final component concentrations, promoted the antibody production as compared to the control group.Table 4. Effect of cell culture additive composition 1 on antibody productionExample 3, Addition of cell culture additives in fed-batch culture of CHO cells with other basal and feed media

[0113] Following the protocol of Example 1, the same stable CHO-K1 cells were subjected to 11-day fed-batch culture, wherein the additive composition 1 was added to the cell culture on Day 3 with different final concentrations of the additive components as set forth in Table 5, and the cells used UproCHO MOI (ProBio, PBM001) as the basel medium and added with 4% (v / v) of UproCHO FOIA (ProBio, PBFA001) and 0.4% (v / v) of UproCHO F01B (ProBio, PBFB001) as the feed media.Table 5. Final concentration of each additive component in cell culture medium

[0114] FIG. 3A and 3B showed the viable cell density (VCD, *106cells / mL, A) and viability (%, B) of the cells from the 3 groups during the 11-day cell culture. It can be seen that the additive composition 1, when added to the cell culture, maintained the comparable VCD as compared to the control group, and prevented cell viability (%) decrease to some extent at the late culture stage.

[0115] Similarly, as shown in Table 6, the addition of the additive composition 1 promoted the antibody production, although different basal and feed media were used in the cell culture.Table 6. Effect of cell culture additive composition 1 on antibody production

[0116] Having thus described in detail preferred embodiments of the present invention, it is to be understood that the invention is not to be limited to particular details set forth in the above description as many apparent variations thereof are possible without departing from the spirit or scope of the present invention.

Claims

We claim:

1. A cell culture additive composition, comprising histidine, methionine, glycine, and a zinc-based inorganic compound.

2. The cell culture additive composition of claim 1, further comprising an antioxidant.

3. The cell culture additive composition of claim 1, wherein the zinc-based inorganic compound is zinc sulfate.

4. The cell culture additive composition of claim 2, wherein the antioxidant is selenite and / or glutathione.

5. The cell culture additive composition of claim 3, comprising L-histidine, methionine, glycine, and zinc sulfate in a (2.39-4.77): (0.47-4.69): (0.67-6.66): (0.01- 0.1) molar ratio.

6. The cell culture additive composition of claim 5, comprising 28-89 wt% L- histidine hydrochloride monohydrate, 4-56 wt% methionine, 3-47 wt% glycine and 0.1- 5 wt% zinc sulfate heptahydrate.

7. The cell culture additive composition of claim 6, comprising 80.3 wt% L- histidine hydrochloride monohydrate, 11.2 wt% methionine, 8.0 wt% glycine and 0.5 wt% zinc sulfate heptahydrate.

8. The cell culture additive composition of claim 4, comprising L-histidine hydrochloride, methionine, glycine, zinc sulfate heptahydrate, sodium selenite, and L- glutathione in a (2.39-4.77): (0.47-4.69): (0.67-6.66): (0.01-0.1): (0.81-1.63) molar ratio.

9. The cell culture additive composition of claim 8, comprising 22-73 wt% L- histidine hydrochloride monohydrate, 3-47 wt% methionine, 2-38 wt% glycine, 0.1-3 wt% zinc sulfate heptahydrate, 0.001-0.03 wt% sodium selenite, and 10-45 wt% L-glutathione reduced.

10. The cell culture additive composition of claim 9, comprising 44.5 wt% L- histidine hydrochloride monohydrate, 6.2 wt% methionine, 4.5 wt% glycine, 0.27 wt% zinc sulfate heptahydrate, 0.022 wt% sodium selenite, and 44.5 wt% L-glutathione reduced;36.6 wt% L-histidine hydrochloride monohydrate, 25.6 wt% methionine, 18.3 wt% glycine, 1.10 wt% zinc sulfate heptahydrate, 0.009 wt% sodium selenite, and 18.3 wt% L-glutathione reduced;57.3 wt% L-histidine hydrochloride monohydrate, 8.0 wt% methionine. 5.7 wt% glycine, 0.34 wt% zinc sulfate heptahydrate. 0.003 wt% sodium selenite, and 28.6 wt% L-glutathione reduced;54.8 wt% L-histidine hydrochloride monohydrate, 3.8 wt% methionine, 13.7 wt% glycine, 0.16 wt% zinc sulfate heptahydrate, 0.014 wt% sodium selenite, and 27.4 wt% L-glutathione reduced;37.0 wt% L-histidine hydrochloride monohydrate, 25.9 wt% methionine, 18.5 wt% glycine, 0.11 wt% zinc sulfate heptahydrate, 0.001 wt% sodium selenite, and 18.5 wt% L-glutathione reduced; or54.1 wt% L-histidine hydrochloride monohydrate, 3.8 wt% methionine, 27.0 wt% glycine, 1.62 wt% zinc sulfate heptahydrate. 0.001 wt% sodium selenite, and 13.5 wt% L-glutathione reduced.

11. A method for culturing a mammalian cell, comprising culturing the mammalian cell in a fed-batch culture, and adding to the cell on 3rdday of the fed-batch culture histidine, methionine, glycine, and a zinc-based inorganic compound at final concentrations of 2.39-4.77 mmol / L, 0.47-4.69 mmol / L, 0.67-6.66 mmol / L, and 0.01- 0.1 mmol / L, respectively.

12. The method of claim 11, further comprising adding to the cell on 3rdday of the fed-batch culture a selenite and glutathione at final concentrations of 0.00014- 0.00145 mmol / L and 0.81-1.63 mmol / L, respectively.

13. The method of claim 11. comprising adding to the cell on 3rdday of the fed-batch culture L-histidine hydrochloride monohydrate, methionine, glycine, and zinc sulfate heptahydrate at final concentrations of 500-1000 mg / L, 70-700 mg / L, 50-500 mg / L and 3-30 mg / L, respectively.

14. The method of claim 13, comprising adding to the cell on 3rdday of the fed- batch culture L-histidine hydrochloride monohydrate, methionine, glycine, and zinc sulfate heptahydrate at final concentrations of 500 mg / L, 70 mg / L, 50 mg / L and 3 mg / L, respectively.

15. The method of claim 12, comprising adding to the cell on 3rdday of the fed- batch culture L-histidine hydrochloride monohydrate, methionine, glycine, zinc sulfate heptahydrate, sodium selenite, and L-glutathione reduced at final concentrations of 500-1000 mg / L, 70-700 mg / L, 50-500 mg / L, 3-30 mg / L, 0.025-0.25 mg / L and 250-500 mg / L, respectively.

16. The method of claim 15, comprising adding to the cell on 3rdday of the fed- batch culture L-histidine hydrochloride monohydrate, methionine, glycine, zinc sulfate heptahydrate, sodium selenite, and L-glutathione reduced at final concentrations of 500 mg / L, 70 mg / L, 50 mg / L, 3 mg / L, 0.25 mg / L and 500 mg / L, respectively; 1000 mg / L, 700 mg / L, 500 mg / L, 30 mg / L, 0.25 mg / L and 500 mg / L, respectively; 500 mg / L, 70 mg / L, 50 mg / L, 3 mg / L, 0.025 mg / L and 250 mg / L, respectively; 1000 mg / L, 70 mg / L, 250 mg / L, 3 mg / L, 0.25 mg / L and 500 mg / L, respectively; 1000 mg / L, 700 mg / L, 500 mg / L, 3 mg / L, 0.025 mg / L and 500 mg / L, respectively; or 1000 mg / L, 70 mg / L, 500 mg / L, 30 mg / L, 0.025 mg / L and 50 mg / L. respectively.

17. The method of claim 11 , comprising maintaining glucose concentration at about 7 g / L during the fed-batch culture.

18. The method of claim 11, comprising adding to the cell a basal medium on 1stday of the batch-fed culture, and adding a feed medium beginning on 3rdday of the batch-fed culture.

19. The method of claim 11, wherein the mammalian cell comprises a nucleic acidfragment for over-expressing a recombinant protein, integrated into its genome, or in an expression vector.

20. The method of claim 11, wherein the mammalian cell is a CHO cell, e.g., a CHO-K1 cell.

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