Cell culture medium containing small peptides

Abstract

To provide: a cell culture medium, a concentrated culture medium, and a concentrated feed material; a method for manufacturing a cell culture medium and a cell culture feed material; and a method for culturing cells.SOLUTION: The cell culture medium comprises at least two amino acids. One of the amino acids is cysteine or tyrosine. One or more small peptides including a dipeptide is added to the cell culture medium to provide improved conditions and enhanced stability for culturing cells.SELECTED DRAWING: Figure 1

Description

[Background technology]

[0001] background Cell culture media provide the nutrients necessary to maintain and grow cells in a controlled, artificial in vitro environment. The nutrient composition, pH, and osmotic pressure of cell culture media vary according to parameters such as cell type, cell density, and the culture system used.

[0002] Culture medium formulations have been used to culture many cell types, including animal, plant, and bacterial cells. Cultured cells have many applications, including the study of physiological processes and the production of useful biological substances. Examples of such useful products include polypeptides such as monoclonal antibodies, hormones, growth factors, enzymes, and other polypeptides of interest. Such products have many commercial and therapeutic applications, and with the advent of recombinant DNA technology, cells can be manipulated to produce these products in large quantities. Cultured cells are also routinely used for the isolation, identification, and propagation of viruses that can be used as vectors and / or vaccines. Therefore, the ability to culture cells in vitro is not only important for cell physiology studies but also necessary for the production of useful substances that cannot otherwise be obtained by cost-effective means.

[0003] Cell culture medium formulations are well-established in the literature, and many media are commercially available. In early cell culture research, medium formulations were based on the chemical composition and physicochemical properties of blood (e.g., osmotic pressure, pH, etc.) and were referred to as "physiological solutions" (Ringer, S., J. Physiol. 3:380-393 (1980) (Non-patent Literature 1); Waymouth, C., Cells and Tissues in Culture, Vol. 1, Academic Press, London, pp. 99-142 (1965) (Non-patent Literature 2); Waymouth, C., In Vitro 6:109-127 (1970) (Non-patent Literature 3). However, cells in different tissues of mammals are exposed to different microenvironments in terms of oxygen / carbon dioxide partial pressure, as well as concentrations of nutrients, vitamins, and trace elements, and therefore the use of different medium formulations is often necessary for successful in vitro culture of different cell types. Typical components of cell culture media include amino acids, organic and inorganic salts, vitamins, minerals, trace metals, sugars, lipids, and nucleic acids, the types and amounts of which may vary depending on the specific requirements of a given cell or tissue type.

[0004] Glutamine is routinely used in cell culture media because it has been shown to be the primary energy source for cultured cells. In 1959, Eagle showed that the amount of glutamine required for optimal growth of mammalian cell cultures is 3 to 10 times greater than the amount of other amino acids. Eagle et al., Science 130:432-37 (1959) (Non-patent Literature 4). However, glutamine is unstable in aqueous solutions and at high temperatures, forming pyroglutamic acid and ammonia, which can be toxic to certain cells. Roth et al., In Vitro Cellular & Developmental Biology 24(7):696-98 (1988) (Non-patent Literature 5). Therefore, glutamine is typically added to cell culture media immediately before use.

[0005] Alternatively, to avoid the formation of toxic substances such as pyroglutamic acid and ammonia, glutamine-containing dipeptides such as alanylglutamine or glycylglutamine may be used in cell culture media instead of glutamine (Roth et al., In Vitro Cellular & Developmental Biology 24(7):696-98 (1988) (Non-Patent Literature 5)). Glutamic acid is also used instead of glutamine to reduce ammonia accumulation in cell culture media (see Cell Culture Technology for Pharmaceutical and Cell-Based Therapies, 52, Sadettin Ozturk and Wei-Shou Hu eds., Taylor and Francis Group (2006) (Non-Patent Literature 6)).

[0006] Some have proposed acylation of dipeptides such as alanylglutamine to make them more stable under heat sterilization conditions. For example, U.S. Patent No. 5,534,538 (Patent Document 1) describes an N-acyl dipeptide for use in enteral or parenteral nutrition, in which case the N-acyl dipeptide is more stable under heat sterilization conditions than the corresponding non-acylated dipeptide, and the N-acyl group favorably delays the division of the dipeptide until it reaches the kidney. U.S. Patent No. 5,534,538 (Patent Document 1) also found that N-acylaranylglutamine is a suitable glutamine source for cell culture media, as is the corresponding non-acylated dipeptide (alanylglutamine), which is a known component of cell culture media (see Roth et al. above). U.S. Patent No. 5,534,538 (Patent Document 1) further states that N-acyl dipeptides are advantageous over free dipeptides for at least one reason: increased stability of N-acyl dipeptides under heat sterilization conditions. Thus, U.S. Patent No. 5,534,538 (Patent Document 1) actually teaches the avoidance of using dipeptides whose N-terminal amino acid is a free amino group.

[0007] The applicants have found that certain amino acids, such as tyrosine, have limited solubility at the desired concentrations for maximum cell proliferation or protein production. They have also found that other amino acids, such as cysteine, are unstable in aqueous cell culture media, particularly in concentrated cell culture media, and tend to precipitate over time. Specifically, because cysteine ​​has a thiol group, it is susceptible to oxidation reactions in which two cysteine ​​residues are linked by a disulfide bond to form cystine (SCH2CH(NH2)CO2H)2. Cysteine ​​has low solubility in water and readily precipitates from solution. As a result, it remains impossible to create a liquid cell culture medium that can be stored at room temperature and contains the concentrations of tyrosine and cysteine ​​necessary for maximum cell proliferation and / or protein production.

[0008] The applicants have also attempted to address this problem by using lower concentrations of tyrosine or cysteine ​​in aqueous cell culture media than the desired concentration. Doing so makes it possible to achieve an acceptable aqueous storage period. However, this is achieved at the expense of optimal cell growth, protein production, or virus production. In other words, cell culture media containing low concentrations of tyrosine and cysteine ​​support reduced cell growth and / or protein production compared to cell culture media containing optimal concentrations of tyrosine and cysteine.

[0009] Therefore, there is a need for a culture medium, concentrated medium, or concentrated feed supplement containing sufficient amounts of tyrosine and cysteine ​​to support maximum cell growth and / or protein or virus production, while avoiding problems arising from the limited solubility of tyrosine or the limited stability of cysteine, including the tendency of cysteine ​​to precipitate from solution over time. In addition, there is a need for a concentrated feed supplement in a form that does not significantly increase the volume of the final cell culture system upon addition. It is also necessary that the pH and osmotic pressure of the resulting system automatically equilibrate upon addition of such a nutrient supplement, and that the medium or concentrated feed supplement be available in liquid or dry form. Finally, there is a need for a single-registration concentrated feed supplement containing sufficient amounts of cysteine ​​and tyrosine to support maximum cell growth and / or protein production, and / or high-quality expressed proteins. [Prior art documents] [Patent Documents]

[0010] [Patent Document 1] U.S. Patent No. 5,534,538 [Non-patent literature]

[0011] [Non-Patent Document 1] Ringer, S., J. Physiol. 3:380-393 (1980) [Non-Patent Document 2] Waymouth, C., Cells and Tissues in Culture, Vol. 1, Academic Press, London, pp. 99-142 (1965) [Non-Patent Document 3] Waymouth, C., In Vitro 6:109-127 (1970) [Non-Patent Document 4] Eagle et al., Science 130:432-37 (1959)

Non-Patent Document 5

Non-Patent Document 6

Summary of the Invention

[0012] Summary The present invention relates, in part, to a cell culture medium, concentrated medium, or concentrated feed supplement containing concentrations of cysteine ​​and tyrosine that support maximum cell proliferation and / or protein or virus production while avoiding problems arising from the limited solubility and stability of cysteine ​​and tyrosine. In one aspect, the medium, concentrated feed supplement, or concentrated medium may be a serum-free composition. In one aspect, the composition may contain human serum components such as human serum albumin. In a further aspect, the human serum albumin may be a recombinant (r-human serum albumin) derived from a recombinant source, in some preferred cases a plant source such as rice, corn, wheat, or potato, or a fungal source, or yeast or other equivalent microorganisms known to be used in the art (foreign body-free culture). In another aspect, the medium, concentrated feed supplement, or concentrated medium may be a protein-free composition. In yet another aspect, the culture medium, concentrated feed supplement, or concentrated medium may be a protein hydrolysate-free composition, and may not contain any protein hydrolysates. In a particular aspect, the culture medium, concentrated feed supplement, or concentrated medium may be a serum-free, protein-free, and protein hydrolysate-free composition. In a preferred aspect, these compositions may contain components of known composition, be serum-free, protein-free, and free of any protein hydrolysates or any part thereof. In a particular embodiment, a cell culture medium, concentrated medium, or cell culture feed supplement having one or more small peptides or one or more dipeptides may further not contain one or more of the following: lipids, hydrolysates or parts thereof, or growth factors.

[0013] The present invention also relates, in part, to a method for analyzing the above composition for the presence or absence of short peptides containing cysteine ​​or tyrosine. The analysis of the culture medium is performed by any known method in the art, for example, by mass spectrometry (LCMS), capillary electrophoresis, or HPLC.

[0014] In particular, the present disclosure relates to cell culture media, concentrated feeds, or cell culture supplements comprising one or more small peptides having 2 to 6 amino acids as defined later in this application. Accordingly, the present disclosure relates to X 1-5 -Tyrosine, X 1-5 -Cysteine, Tyrosine-X 1-5 and Cysteine-X 1-5 or any small peptide in which cysteine or tyrosine is present anywhere within a short peptide of 1 to 6 amino acids (e.g., X-Cysteine-X 1-4 , X-Tyrosine-X 1-4 -Tyrosine, etc.), or salts thereof, and provides a cell culture medium, concentrated feed, or cell culture supplement comprising one or more small peptides selected therefrom, where X is any amino acid and the N-terminal amino acid of the short peptide has a free amino group. In one embodiment, X is alanine or glycine. In another embodiment, X is serine, valine, proline, aspartic acid, or glutamic acid. In a particular embodiment, the present disclosure provides a cell culture medium, concentrated feed, or cell culture supplement comprising one or more dipeptides selected from X-Tyrosine, X-Cysteine, Tyrosine-X, and Cysteine-X, or salts thereof, where X is any amino acid and the N-terminal amino acid of the dipeptide has a free amino group. In one embodiment, X is alanine or glycine. In another embodiment, X is serine, valine, proline, aspartic acid, or glutamic acid. In yet another embodiment, the one or more dipeptides are alanyl tyrosine and / or alanyl cysteine. The cell culture medium, concentrated feed, or cell culture supplement may be a liquid or a dry powder such as a dry powder medium (DPM) or an agglomerated powder (AGT (trademark)). In one embodiment, the liquid is stored at 2 to 8°C and remains free of precipitates for over 12 months.

[0015] In one embodiment of the present invention, the concentration of tyrosine (contained within the small peptide) in a solution in a culture medium, feed, or supplement is higher than the concentration of tyrosine that would remain soluble in the same solution if it were present as a monomer (i.e., the tyrosine in the small peptide can make the solution "supersaturated with respect to tyrosine"). In one embodiment, the solution has a concentration of about 1 to at least about 100 or about 1 to at least about 25 times the concentration of soluble tyrosine that would be possible if it were present as a monomer. For example, the concentration of small peptides or dipeptides in some cell culture media, concentrated feeds, or concentrated supplements may be about 1 to at least about 5 times, about 1 to at least about 10 times, about 1 to at least about 15 times, about 1 to at least about 20 times, about 1 to at least about 30 times, about 1 to at least about 40 times, about 1 to at least about 50 times, about 1 to at least about 60 times, about 1 to at least about 70 times, about 1 to at least about 80 times, about 1 to at least about 90 times, about 1 to at least about 100 times, about 10 to at least about 20 times, about 10 to at least about 30 times, about 10 to at least about 40 times, about 10 to at least about 50 times, about 10 to at least about 60 times, about 10 to at least about 70 times, etc.

[0016] In one embodiment of the present invention, the concentration of cysteine ​​(contained within a small peptide) in a solution in a culture medium, feed, or supplement is higher in the solution than the concentration of cysteine ​​that would remain soluble in the same solution if it were present as a monomer (i.e., the cysteine ​​in the small peptide can make the solution "supersaturated with respect to cysteine"). In one embodiment, the solubility (or lack thereof) of cysteine ​​includes, for example, the loss of solubility resulting from the conversion of cysteine ​​to cystine. In one embodiment, the solution contains at least about 1 to about 25 times the concentration of soluble cysteine ​​possible as a monomer. For example, the concentrations of small peptides or dipeptides in some cell culture media, concentrated feeds, or concentrated supplements are approximately 1 to at least 5 times, approximately 1 to at least 10 times, approximately 1 to at least 15 times, approximately 1 to at least 20 times, approximately 1 to at least 30 times, approximately 1 to at least 40 times, approximately 1 to at least 50 times, approximately 1 to at least 60 times, approximately 1 to at least 70 times, and approximately 1 to at least 8 times the soluble cysteine ​​concentration where possible in monomer form. The dilution ratio may be 0x, approximately 1x to at least approximately 90x, approximately 1x to at least approximately 100x, approximately 1x to at least approximately 110x, approximately 10x to at least approximately 20x, approximately 10x to at least approximately 30x, approximately 10x to at least approximately 40x, approximately 10x to at least approximately 50x, approximately 10x to at least approximately 60x, approximately 10x to at least approximately 70x, approximately 10x to at least approximately 80x, approximately 10x to at least approximately 90x, approximately 10x to at least approximately 100x, approximately 10x to at least approximately 110x, and so on. In one embodiment, the solution is supersaturated with respect to both tyrosine and cysteine.

[0017] In one embodiment, the solution is supersaturated with respect to both tyrosine and cysteine, for example, within the ranges described above for each of tyrosine or cysteine ​​alone.

[0018] In one embodiment, the addition of the above-mentioned concentrated feed, concentrated medium, or concentrated supplement can reduce the amount of supplement to the culture system, for example, for a fed-batch culture that may already be underway. The culture may be underway for several hours to several days. In another embodiment, increasing the solubility of cysteine ​​and tyrosine contained within the small peptides allows for the design of a single partial concentrated feed, which may also be pH neutral, and this is part of the present invention. The present invention relates, in part, to the preparation of single regimens of concentrated feeds, concentrated mediums, and concentrated supplements containing cysteine ​​and tyrosine contained within small peptides in order to increase the solubility and stability of cysteine ​​and tyrosine in the composition.

[0019] A cell culture medium, concentrate, or cell culture supplement containing one or more small peptides or one or more dipeptides optionally includes one or more of the following: carbohydrates, vitamins, salts, inorganic elements, buffers, and amino acids or salts thereof. In one embodiment, the carbohydrate is a hexose sugar; or pentoses, hexoses, or derivatives thereof, or other equivalents may be used; in some cases, oligosaccharides or derivatives thereof may be used; or the hexose may be selected from the group consisting of glucose, galactose, fructose, and maltose. In a particular embodiment, the carbohydrate is glucose.

[0020] In one embodiment, the amino acid or its salt is one or more of arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

[0021] In one embodiment, a cell culture medium, concentrate, or cell culture supplement comprises (1) a first dipeptide X-tyrosine or a salt thereof, and a second dipeptide X-cysteine ​​or a salt thereof, where X is alanine or glycine, and alanine or glycine has a free amino group; (2) a carbohydrate such as glucose; and (3) an amino acid or a salt thereof. The amino acid or salt thereof may comprise one or more of arginine, asparagine, aspartic acid, cysteine, glutamic acid, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

[0022] In one embodiment, a cell culture medium, concentrate, or cell culture supplement contains one or more small peptides or one or more dipeptides suitable for culturing bacterial cells, yeast cells, plant cells, or animal cells, such as insect cells (e.g., Drosophila cells, Spodoptera cells, or Tricoplusia cells), nematode cells (e.g., C. elegans cells), or mammalian cells (e.g., including CHO cells, COS cells, VERO cells, BHK cells, AE-1 cells, SP2 / 0 cells, L5.1 cells, PerC6, hybridoma cells, HEK 293, and human cells).

[0023] In certain embodiments, a cell culture medium having one or more small peptides or one or more dipeptides is a 1× formulation. In other embodiments, the cell culture medium is concentrated as a 2× or greater than 2× formulation, as discussed elsewhere in this application. In certain embodiments, one or more small peptides or dipeptides, such as X-tyrosine, X-cysteine, tyrosine-X, or cysteine-X, are present in the cell culture medium at concentrations of about 1 g / L to about 16 g / L, about 1 g / L to about 10 g / L, about 1 g / L to about 5 g / L, about 2.5 g / L to about 16 g / L, about 2.5 g / L to about 10 g / L, about 2.5 g / L to about 5 g / L, or about 2.5 g / L to about 8.5 g / L.

[0024] In another aspect, the present disclosure provides a method for culturing cells, comprising the step of contacting the cells with a cell culture medium containing a small peptide or dipeptide as described herein, under conditions that support the culture of the cells. Any cells, in particular bacterial cells, yeast cells, plant cells, or animal cells, can be cultured according to the present method. In one embodiment, animal cells for culture according to the present method are insect cells (e.g., Drosophila cells, Spodoptera cells, or Trichoprusia cells), nematode cells (e.g., C. elegans cells), or mammalian cells (e.g., CHO cells, COS cells, VERO cells, BHK cells, AE-1 cells, SP2 / 0 cells, L5.1 cells, PerC6, hybridoma cells, HEK 293, or other human cells).

[0025] In another embodiment, a method for culturing cells comprises the steps of bringing cells into contact with a cell culture basin medium under conditions that support cell culture, and supplementing the cell culture basin medium with a cell culture medium, cell culture supplement, or cell culture feed having one or more small peptides or one or more dipeptides as described herein. In one embodiment, one or more dipeptides are selected from X-tyrosine, X-cysteine, tyrosine-X, or cysteine-X, or salts thereof, where X is selected from alanine, glycine, serine, valine, proline, aspartic acid, or glutamic acid, and the N-terminal amino acid of one or more dipeptides has a free amino group. In another embodiment, one or more dipeptides are alanyltyrosine and / or alanylcysteine.

[0026] In one embodiment, the cell culture basal medium is supplemented from day two onward with a solution containing cysteine ​​and tyrosine in a small peptide, which may be a concentrated medium or concentrated feed supplement. In certain embodiments, the applicants may refer to the use of a medium, feed, supplement, or other solution containing a small peptide (e.g., a solution containing a dipeptide containing at least one tyrosine or cysteine ​​residue). Indeed, one embodiment of the applicants' invention is the use of such a solution in cell culture. In this regard, those skilled in the art will understand that in certain descriptions herein, the applicants may refer to a particular supplement, medium, feed, or solution merely as an example of an embodiment that may be equally applicable to any of the types of solutions described herein. For example, those skilled in the art will understand that a description of a “supplement” having a particular amino acid composition may be equivalent to a “feed” having a similar composition, and vice versa.

[0027] Cell culture basal media, concentrated media, concentrated feeds, or supplements may contain small peptide or dipeptide-containing media or feeds on days 0, 1, 2, 3, 4, or 5 after the start of cell culture, and may be replenished daily thereafter until day 13 or 14, or until the viability of the culture falls below a predetermined level (e.g., 50%). Optionally, the cell culture basal media may be supplemented with small peptide or dipeptide-containing cell culture media at a rate of approximately 2% of the total volume of the cell culture basal media. In one embodiment, cells produce proteins, peptides, and small RNAs (e.g., miRNA, siRNA, etc.). Protein means recombinant or native protein. Protein also means full-length protein or a portion thereof (such as a domain, motif, polypeptide chain, or polypeptide fragment), whether recombinant or native. Protein also means intracellular protein, extracellular protein, secretory protein, hormone, cytokine, receptor, extracellular matrix protein, immunoglobulin or a portion of immunoglobulin, or fragments thereof, whether recombinant or native. Preferably, protein yields can be higher using cell culture media, cell culture feeds, or cell culture supplements containing the short peptides or dipeptides of the present invention. In certain aspects, for example, the immunoglobulin protein yield may be greater than 3000 mg / L of immunoglobulin after at least 14 days of culture. In another embodiment, cells may produce viruses or VLPs (virus-like particles). In yet another embodiment, cells may produce desired cell products such as vitamins, metabolites, glycoproteins, carbohydrates, lipids, or lipoproteins. In yet another embodiment, the cells themselves are grown and harvested. The culture medium and feed supplement compositions of the present invention contain concentrations of cysteine ​​and tyrosine that support maximum cell growth for virus or VLP production, or maximum vitamin production or glycoprotein or vaccine production, etc., while avoiding problems arising from the limited solubility of cysteine ​​and tyrosine.

[0028] For example, any cells, including bacterial cells, yeast cells, plant cells, insect cells, or mammalian cells, can be cultured according to this method. In one embodiment, the animal cells to be cultured according to this method are insect cells (e.g., Drosophila cells, Spodoptera cells, or Trichoprusia cells), nematode cells (e.g., C. elegans cells), or mammalian cells (e.g., CHO cells, COS cells, VERO cells, BHK cells, AE-1 cells, SP2 / 0 cells, L5.1 cells, PerC6, hybridoma cells, or other human cells). In one embodiment, the cells are CHO cells.

[0029] Another aspect provides a method for preparing a cell culture medium, comprising the step of mixing a carbohydrate such as glucose, and at least one amino acid or a salt thereof, such as arginine, asparagine, aspartic acid, cysteine, glutamic acid, histidine, isoleucine, lysine, methionine, phenylalanine, proline, hydroxyproline, serine, threonine, tryptophan, tyrosine, glutamine, and valine, with one or more small peptides or dipeptides described herein. In one embodiment, one or more dipeptides are selected from X-tyrosine, X-cysteine, tyrosine-X, or cysteine-X, or salts thereof, where X is selected from alanine, glycine, serine, valine, proline, aspartic acid, or glutamic acid, and the N-terminal amino acid of one or more dipeptides has a free amino group. In another embodiment, one or more dipeptides are alanyltyrosine and / or alanylcysteine.

[0030] In other embodiments, the cell culture medium prepared according to this method is concentrated as a 2× or greater than 2× formulation, as discussed elsewhere in this application. In certain embodiments, one or more small peptides or dipeptides, such as alanyltyrosine and / or alanylcysteine, are present in the cell culture medium prepared according to this method.

[0031] For example, the concentrations of small peptides or dipeptides in some cell culture media or concentrated media or concentrates are approximately 0.5 g / L to 30 g / L, 0.5 g / L to 25 g / L, 0.5 g / L to 20 g / L, 0.5 g / L to 16 g / L, 0.5 g / L to 10 g / L, 0.5 g / L to 5 g / L, 0.5 g / L to 4 g / L, 1 g / L to 30 g / L, 1 g / L to 20 g / L, 1 g / L to 16 g / L, 1 g / L to 10 g / L, 1 g / L to 5 g / L, 2.5 g / L to 30 g / L, 2.5 g / L to 20 g / L, 2.5 g / L to 16 g / L, and 2.5 g / L to 10 g / L. The concentration may be g / L, approximately 2.5 g / L to approximately 5 g / L, or approximately 2.5 g / L to approximately 4.5 g / L, approximately 5 g / L to approximately 30 g / L, approximately 5 g / L to approximately 25 g / L, approximately 5 g / L to approximately 20 g / L, approximately 5 g / L to approximately 16 g / L, approximately 5 g / L to approximately 10 g / L, approximately 5 g / L to approximately 5 g / L, approximately 5 g / L to approximately 4 g / L, etc.

[0032] Another aspect provides a composition comprising a cell culture medium containing a small peptide or dipeptide as described herein, and at least one of the above-mentioned cells. In one embodiment, the at least one cell is a CHO cell.

[0033] Another aspect relates to kits for use in cell culture. A kit may comprise one or more containers containing a cell culture medium containing small peptides or dipeptides as described herein. The kit may optionally comprise at least one additional component selected from at least one growth factor, at least one animal tissue extract, at least one animal organ extract, at least one animal gland extract, at least one enzyme, at least one protein, at least one vitamin, at least one cytokine, at least one lipid, at least one trace element, at least one extracellular matrix component, at least one buffer, at least one antibiotic, and at least one viral inhibitor. The kit may also comprise one or more cells or cell types.

[0034] Another aspect relates to a method for producing viruses or VLPs using cell culture media containing small peptides or dipeptides as described herein. Specifically, the method comprises (a) a step of bringing cells (e.g., mammalian cells) into contact with a virus under conditions suitable for promoting viral infection of cells; and (b) a step of culturing the cells in the culture medium described herein under conditions suitable for promoting viral production by the cells. In one embodiment, the cells producing the virus or VLP are mammalian cells such as CHO cells, or insect cells, or plant cells, or fungal cells. In another embodiment, a non-mammalian virus or VLP is manipulated to be able to infect mammalian host cells, such as human cells.

[0035] In another aspect, the disclosure provides a method for producing polypeptides, such as immunoglobulins or fragments thereof, using cell culture media containing small peptides or dipeptides as described herein. Specifically, the method includes culturing cells genetically engineered to produce polypeptides in a dipeptide-containing culture medium under conditions suitable for polypeptide expression by the cells. In one embodiment, the cells are mammalian cells, such as CHO cells.

[0036] Accordingly, one aspect of the present invention relates to a cell culture medium, concentrated feed, or concentrated feed supplement comprising at least one small peptide, wherein the peptide comprises at least two amino acids, at least one of which is cysteine ​​or tyrosine. Another aspect of the present invention relates to the above cell culture medium, concentrated feed, or concentrated feed supplement, wherein at least one of the remaining amino acids of the small peptide is selected from the group consisting of alanine, glycine, serine, valine, proline, aspartic acid, and glutamic acid. In yet another aspect of the present invention, at least one of the remaining amino acids of the small peptide is alanine or glycine. Another aspect of the present invention relates to the cell culture medium, concentrated feed, or concentrated feed supplement described above, wherein at least one of the small peptides is a dipeptide selected from the group consisting of X-tyrosine, X-cysteine, tyrosine-X, and cysteine-X, or salts thereof, where X is selected from the group consisting of alanine, glycine, serine, valine, proline, aspartic acid, and glutamic acid, and / or the N-terminal amino acid of one or more small peptides has a free amino group. In one aspect, X is alanine or glycine.

[0037] In some aspects of the present invention, the short peptide may be a dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide, or decapeptide; or the short peptide may be at least about 2 to 10 amino acids long; or in some aspects, the short peptide is at least about 10 amino acids long. In some aspects, the short peptide may contain 2, 3, 4, 5, or 6 amino acids.

[0038] In other contexts, cell culture media are liquids.

[0039] In any one of the above cell culture media, the cell culture medium, concentrated feed, or concentrated feed supplement is a dry powder or granular dry powder.

[0040] In a further aspect, any one of the above cell culture media may contain carbohydrates and amino acids or salts thereof. In one embodiment, the carbohydrate is a hexose sugar. In another embodiment, the amino acid or salt thereof is one or more of arginine, asparagine, aspartic acid, cysteine, glutamic acid, histidine, isoleucine, lysine, methionine, phenylalanine, proline, hydroxyproline, serine, threonine, tryptophan, tyrosine, and valine. In a further embodiment, the medium further contains vitamins, salts, buffers, or inorganic elements.

[0041] In some aspects described above, the cell culture medium, concentrate, or concentrate supplement may be free of lipids, hydrolysates or parts thereof, or growth factors; in other aspects described above, the cell culture medium, concentrate, or concentrate supplement may be free of proteins; therefore, in some preferred aspects, the cell culture medium, concentrate, or reconstituted medium containing small peptides may be serum-free, protein-free, and / or hydrolysate-free.

[0042] In certain aspects, the cell culture medium, concentrated feed, or concentrated feed supplement is concentrated as a 2× or greater than 2× formulation. In preferred aspects, the above cell culture medium, concentrated feed, or concentrated feed supplement contains one or more dipeptides, the dipeptides may be either alanyltyrosine and / or alanylcysteine ​​and / or alanylcystine dimer. In preferred aspects, one or more dipeptides may be present at a concentration of about 1 g / L to about 16 g / L; or one or more dipeptides may be present at a concentration of about 2.5 g / L to about 8.5 g / L. In one aspect, the liquid stored at 2–8°C remains precipitate-free for more than 12 months.

[0043] The present invention also relates to a method for culturing cells, comprising the steps of bringing the cells into contact with a cell culture basin medium under conditions that support the cell culture, and replenishing the cell culture basin medium with the concentrated feed or concentrated medium. Replenishing may be performed during fed-batch culture; or in addition to an existing supply schedule; or replenishing may be performed sequentially, as opposed to being performed incrementally by bolus addition. In some cases, the cell culture basin medium may be replenished with concentrated feed or concentrated medium from day 2 onward; or the replenishment of the cell culture basin medium may be performed using a single concentrated feed or using multiple concentrated feeds; or the replenishment may be performed from day 0, day 1, day 2, day 3, day 4, or day 5 after the start of cell culture, and thereafter daily until day 13 or day 14; or each replenishment may be about 1-10% or about 5-20% of the total starting volume of the cell culture basin medium.

[0044] In all aspects of the methods used herein, cells may be manipulated cells; or recombinant cells; or plant cells; or any one of animal cells, plant cells, insect cells, bird cells, yeast cells, algal cells, or fish cells. Animal cells may be mammalian, insect, bovine, primate cells, or pluripotent stem cells. In one context, animal cells may be mammalian cells; mammalian cells include keratinocytes, cervical epithelial cells, bronchial epithelial cells, tracheal epithelial cells, renal epithelial cells, and retinal epithelial cells), as well as established cell lines and their strains (e.g., 293 fetal kidney cells, BHK cells, HeLa cervical epithelial cells, and PER-C6 retinal cells, MDBK(NBL-1) cells, 911 cells, CRFK cells, MDCK cells, CHO cells, BeWo cells, Chang cells, Detroit 562 cells, HeLa 229 cells, HeLa S3 cells, Hep-2 cells, KB cells, LS180 cells, LS174T cells, NCI-H-548 cells, RPMI 2650 cells, SW-13 cells, T24 cells, WI-28 VA13, 2RA cells, WISH cells, BS-CI cells, LLC-MK2 cells, clone M-3 cells, 1-10 cells, RAG cells, TCMK-1 cells, Y-1 cells, LLC-PK1 cells, PK(15) cells, GH1 cells, GH3 cells, L2 cells, LLC-RC Fibroblasts derived from any tissue or organ (including but not limited to the heart, liver, kidney, colon, intestine, esophagus, stomach, nervous tissue (brain, spinal cord), lung, vascular tissue (arteries, veins, capillaries), lymphoid tissue (lymphatic glands, pharyngeal tonsils, tonsils, bone marrow, and blood), spleen), as well as fibroblasts and fibroblast-like cell lines (e.g., CHO cells, TRG-2 cells, IMR-33 cells, Don cells, GHK-21 cells, citrullinocytes, Dempsey cells, Detroit 551 cells, Detroit 510 cells, Detroit 525 cells, Detroit 529 cells, Detroit 532 cells, Detroit 539 cells, Detroit 548 cells, Detroit 573 cells, HEL299 cells, IMR-90 cells, MRC-5 cells, WI-38 cells, WI-26 cells, MiCl1 cells, CHO cells, CV-1 cells, COS-1 cells, COS-3 cells, COS-7 cells, Vero cells, DBS-FrhL-2 cells, BALB / 3T3 cells, F9 cells, SV-T2 Cells, M-MSV-BALB / 3T3 cells, K-BALB cells, BLO-11 cells, NOR-10 cells, C3H / IOTI / 2 cells, HSDM1C3 cells, KLN205 cells, McCoy cells, mouse L cells, strain 2071 (mouse L) cells, LM strain (mouse L) cells, L-MTK - (Mouse L) cells, NCTC clones 2472 and 2555, SCC-PSA1 cells, Swiss / 3T3 cells, Indian muntjac cells, SIRC cells, C II Cells, and may be Jensen cells, Sp2 / 0, NSO, NS1 cells, or manipulated versions thereof. In one context, mammalian cells may be CHO cells.

[0045] In all aspects of the methods used herein, cells can produce immunoglobulins or fragments thereof; cells can produce immunoglobulins or fragments thereof in amounts greater than 3000 mg / L.

[0046] Alternatively, in all aspects of the methods used herein, cells may produce viruses or virus-like particles (VLPs); the viruses may be recombinant viruses. The viruses may be, but are not limited to, adenoviruses, lentiviruses, baculoviruses, Sendai viruses, vaccinia viruses, or engineered derivatives thereof. In the aspects described herein, VLPs may contain nucleic acids, or they may contain RNA.

[0047] In yet another aspect, the present invention also relates to a method for preparing a cell culture medium, concentrate feed, or cell culture supplement, comprising the step of mixing a carbohydrate and at least one amino acid or a salt thereof with one or more small peptides, wherein each of the one or more small peptides comprises cysteine ​​or tyrosine. In one embodiment, the amino acids of the one or more small peptides other than cysteine ​​or tyrosine (or referred to as the remaining amino acids) may be selected from the group consisting of alanine, glycine, serine, valine, proline, aspartic acid, arginine, glutamine, or glutamic acid. In one preferred aspect, the amino acid other than cysteine ​​or tyrosine is alanine or glycine. In another set, the remaining amino acids may also be one or more of arginine, asparagine, aspartic acid, cysteine, glutamic acid, histidine, isoleucine, lysine, methionine, phenylalanine, proline, hydroxyproline, serine, threonine, tryptophan, tyrosine, and valine. In another preferred aspect of the above method, one or more dipeptides may be selected from X-tyrosine, X-cysteine, tyrosine-X, or cysteine-X, or salts thereof, where X is selected from alanine, glycine, serine, valine, proline, aspartic acid, arginine, or glutamic acid, and the N-terminal amino acid of one or more dipeptides has a free amino group. In the most preferred aspect, X is alanine or glycine. In the most preferred aspect, the carbohydrate may be glucose. In addition, the cell culture medium may further contain vitamins, salts, buffers, or inorganic elements; or it may not contain lipids, hydrolysates or parts thereof, or growth factors; or it may not contain proteins; or the cell culture medium may be concentrated as a 2× or greater than 2× formulation; or one or more dipeptides may be alanyltyrosine and / or alanylcysteine; or one or more dipeptides may be present in the cell culture medium at a concentration of about 1 g / L to about 16 g / L; or preferably, one or more dipeptides may be present at a concentration of about 2.5 g / L to about 8.5 g / L.In most cases, cell culture media stored at 2–8°C remain precipitate-free for more than 12 months.

[0048] The present invention also relates to a method for analyzing cell culture media, concentrated feeds, or reconstituted media, the method comprising the step of determining the presence or absence of short peptides containing cysteine ​​or tyrosine in the media. In one aspect, the cell culture media, concentrated feed, or reconstituted media may be serum-free, protein-free, and hydrolyzate-free, and the analysis may be, but not limited to, mass spectrometry (LCMS), capillary electrophoresis, and HPLC.

[0049] The present invention also relates to a composition comprising the above-mentioned cell culture medium, concentrated feed, or reconstituted medium, and cells.

[0050] The present invention also relates to a method for producing recombinant proteins in a cell culture medium, comprising the steps of: contacting cells with the cell culture medium, concentrated feed or reconstituted medium; and culturing the cells under conditions suitable for cell proliferation and / or expression of the recombinant protein.

[0051] The present invention also relates to a kit for culturing cells in vitro, comprising one or more containers, wherein the first container comprises a medium containing at least one of the above-mentioned dipeptides, the medium supporting cell growth and / or recombinant protein expression during culture. Cell culture may be in suspension culture or in adherent culture. Growth may be high-density growth.

[0052] The present invention also relates to a method for producing a virus or viral particle in the above-mentioned cell culture medium, concentrated feed, or reconstituted medium, the method comprising the step of culturing recombinant cells in the cell culture medium, wherein the medium supports the growth of the cells under conditions suitable for the expression of the virus or viral particle.

[0053] The present invention also relates to recombinant proteins produced by the above method; or viruses or viral particles produced by the above method; or the use of the above cell culture medium, concentrate, or reconstituted medium for producing viruses or recombinant proteins; or culture medium, concentrate, or reconstituted medium prepared by the above method. [Invention 1001] A cell culture medium comprising at least one small peptide, wherein the peptide comprises at least two amino acids, at least one of which is cysteine ​​or tyrosine. [Invention 1002] The cell culture medium of the present invention 1001, wherein at least one of the remaining amino acids of the small peptide is selected from the group consisting of alanine, glycine, serine, valine, proline, aspartic acid, and glutamic acid. [Invention 1003] The cell culture medium of the present invention 1002, wherein at least one of the remaining amino acids of the small peptide is alanine or glycine. [Invention 1004] The cell culture medium of the present invention 1001, wherein at least one of the small peptides is a dipeptide selected from the group consisting of X-tyrosine, X-cysteine, tyrosine-X, and cysteine-X, or salts thereof, and X is selected from the group consisting of alanine, glycine, serine, valine, proline, aspartic acid, and glutamic acid, and / or one or more of the small peptides have a free amino group as their N-terminal amino acid. [Invention 1005] A cell culture medium according to the present invention 1001, wherein X is alanine or glycine. [Invention 1006] A liquid cell culture medium according to any of the inventions 1001 to 1005. [Invention 1007] A cell culture medium according to any of the present invention 1001 to 1005, which is a dried powder or granular dried powder. [Invention 1008] A cell culture medium according to any of the present invention 1001 to 1007, further comprising carbohydrates and amino acids or salts thereof. [Invention 1009] A cell culture medium according to the present invention 1008, wherein the carbohydrate is a hexose. [Invention 1010] A cell culture medium according to the present invention 1008 or 1009, wherein the amino acid or salt thereof is one or more of arginine, asparagine, aspartic acid, cysteine, glutamic acid, histidine, isoleucine, lysine, methionine, phenylalanine, proline, hydroxyproline, serine, threonine, tryptophan, tyrosine, and valine. [Invention 1011] A cell culture medium according to any of the present invention 1001 to 1010, further comprising vitamins, salts, buffers, or inorganic elements. [Invention 1012] A cell culture medium according to any of the present invention 1001 to 1011, which does not contain lipids, hydrolysates or parts thereof, or growth factors. [Invention 1013] A cell culture medium according to any of the present invention 1001 to 1012, which does not contain protein. [Invention 1014] A cell culture medium according to any of the present invention 1001 to 1013, which is concentrated as a 2× or greater than 2× formulation. [Invention 1015] A cell culture medium according to any one of the present invention 1004 to 1014, wherein one or more of the dipeptides are alanyltyrosine and / or alanylcysteine ​​and / or alanylcystine dimer. [Invention 1016] A cell culture medium according to any one of the present invention 1004-1006 or 1008-1014, wherein one or more of the dipeptides are present at a concentration of about 1 g / L to about 16 g / L. [Invention 1017] A cell culture medium according to any one of the present invention 1004-1006 or 1008-1014, wherein one or more of the dipeptides are present at a concentration of about 2.5 g / L to about 8.5 g / L. [Invention 1018] A cell culture medium according to any of the present invention 1006 or 1008-1017, wherein the liquid stored at 2-8°C remains precipitate-free for more than 12 months. [Invention 1019] A method for culturing cells, The steps include bringing the cell culture medium and the cells into contact under conditions that support the culture of the cells, and A step of supplementing the cell culture basal medium with any of the concentrated feed or concentrated medium according to invention 1001 to 1018. A method that includes this. [Invention 1020] The method of the present invention 1019, wherein the cell culture basal medium is supplemented with the concentrated feed or the concentrated medium from the second day onward. [Invention 1021] The method of the present invention 1020, wherein the concentrated supply or concentrated medium is added to the cell culture basal medium starting from day 0, day 1, day 2, day 3, day 4, or day 5 after the start of cell culture, and thereafter the replenishment is carried out daily until day 13 or day 14. [Invention 1022] The method according to any of the present invention 1019 to 1021, wherein the supplementation to the cell culture basal medium is approximately 1-10% or approximately 5-20% of the total starting volume of the cell culture basal medium. [Invention 1023] The method according to any one of items 1019 to 1022 of the present invention, wherein the cells are animal cells, plant cells, insect cells, bird cells, yeast cells, algal cells, or fish cells. [Invention 1024] The method of the present invention 1022, wherein the cells produce immunoglobulin or a fragment thereof. [Invention 1025] The method of the present invention 1024, wherein the cells produce immunoglobulin or a fragment thereof in a concentration greater than 3000 mg / L. [Invention 1026] A method for preparing a cell culture medium, comprising the step of mixing a carbohydrate and at least one amino acid or a salt thereof with one or more small peptides, wherein each of the one or more small peptides comprises cysteine ​​or tyrosine. [Invention 1027] The method of the present invention 1026, wherein one or more amino acids of the small peptide, other than cysteine ​​or tyrosine, are selected from alanine, glycine, serine, valine, proline, aspartic acid, arginine, glutamine, or glutamic acid. [Invention 1028] The method of the present invention 1027, wherein one or more of the amino acids of the small peptide, other than cysteine ​​or tyrosine, are alanine or glycine. [Invention 1029] The method of the present invention 1026, wherein one or more of the small peptides are one or more dipeptides selected from X-tyrosine, X-cysteine, tyrosine-X, or cysteine-X, or salts thereof, where X is selected from alanine, glycine, serine, valine, proline, aspartic acid, arginine, or glutamic acid, and one or the N-terminal amino acid of the dipeptide has a free amino group. [Invention 1030] The method of the present invention 1029, wherein X is alanine or glycine. [Invention 1031] Any method of the present invention 1026 to 1030, wherein the carbohydrate is glucose. [Invention 1032] A method according to any one of the present invention 1026 to 1031, wherein at least one of the aforementioned amino acids or a salt thereof is one or more of arginine, asparagine, aspartic acid, cysteine, glutamic acid, histidine, isoleucine, lysine, methionine, phenylalanine, proline, hydroxyproline, serine, threonine, tryptophan, tyrosine, and valine. [Invention 1033] The method according to any one of the present invention 1026 to 1032, wherein the cell culture medium further comprises vitamins, salts, buffers, or inorganic elements. [Invention 1034] The method according to any one of the present invention 1026 to 1033, wherein the cell culture medium does not contain lipids, hydrolysates or parts thereof, or growth factors. [Invention 1035] The method according to any of the present invention 1026 to 1034, wherein the cell culture medium does not contain protein. [Invention 1036] The method of any one of the present invention 1026 to 1035, wherein the cell culture medium is concentrated as a 2× or greater than 2× formulation. [Invention 1037] A method according to any one of the present invention 1029 to 1036, wherein one or more of the dipeptides are alanyltyrosine and / or alanylcysteine. [Invention 1038] The method according to any one of the present invention 1029 to 1037, wherein one or more of the dipeptides are present in the cell culture medium at a concentration of about 1 g / L to about 16 g / L. [Invention 1039] A method according to any one of the present invention 1029 to 1037, wherein one or more of the dipeptides are present at a concentration of about 2.5 g / L to about 8.5 g / L. [Invention 1040] The method according to any one of the present invention 1029 to 1039, wherein the cell culture medium stored at 2 to 8°C remains precipitate-free for more than 12 months. [Invention 1041] A method for analyzing a cell culture medium, concentrated feed, or reconstituted medium, comprising the step of determining the presence or absence of a short peptide containing cysteine ​​or tyrosine in the medium. [Invention 1042] The method of the present invention 1041 for analyzing serum-free, protein-free, and hydrolyzate-free cell culture media, concentrated feeds, or reconstituted media, performed by a method selected from the group including mass spectrometry (LCMS), capillary electrophoresis, and HPLC. [Invention 1043] The method of the present invention 1041 for analyzing a cell culture medium, concentrated feed, or reconstituted medium that is serum-free, protein-free, and hydrolyzate-free, wherein the short peptide contains two, three, four, five, or six amino acids. [Invention 1044] The present invention 1041 method for analyzing a culture medium, wherein the short peptide is a dipeptide. [Invention 1045] The method of the present invention 1023, wherein the CHO cells produce a virus. [Invention 1046] The method of the present invention 1045, wherein the virus is a recombinant virus or a virus-like particle (VLP). [Invention 1047] The method of the present invention 1046, wherein the virus is an adenovirus, lentivirus, baculovirus, sendai virus, vaccinia virus, or a modified derivative thereof. [Invention 1048] The method of the present invention 1046, wherein the VLP possesses nucleic acid. [Invention 1049] The method of the present invention 1048, wherein the nucleic acid is RNA. [Invention 1050] The cell culture medium of the present invention 1009, wherein the hexose is selected from the group consisting of glucose, galactose, fructose, and maltose. [Invention 1051] Cell culture media, concentrated feeds, or reconstituted media containing small peptides containing cysteine ​​or tyrosine, being serum-free, protein-free, and / or hydrolyzate-free. [Invention 1052] A cell culture medium, concentrated feed, or reconstituted medium according to the present invention 1051, wherein the small peptide contains 2, 3, 4, 5, or 6 amino acids. [Invention 1053] A cell culture medium, concentrated feed, or reconstituted medium according to the present invention 1001, which supports the production of recombinant proteins. [Invention 1054] The cell culture medium, concentrated feed, or reconstituted medium of the present invention 1053, wherein the recombinant protein is an immunoglobulin or a fragment thereof. [Invention 1055] A composition comprising a cell culture medium, concentrated feed, or reconstituted medium according to the present invention 1001, and cells. [Invention 1056] A method for producing recombinant proteins in cell culture medium, The step of bringing cells into contact with the cell culture medium, concentrated feed, or reconstituted medium of the present invention 1001; and The step of culturing the cells under conditions suitable for cell proliferation and / or expression of the recombinant protein. A method that includes this. [Invention 1057] A kit for in vitro cell culture comprising one or more containers, wherein the first container comprises a medium of the present invention 1001 comprising at least one dipeptide, and the medium supports the growth of the cells in culture and / or the expression of recombinant proteins. [Invention 1058] The kit of the present invention 1057, wherein the culture is either a suspension culture or an adherent culture. [Invention 1059] The kit of the present invention 1057, wherein the aforementioned propagation is high-density propagation. [Invention 1060] A method for producing a virus or viral particles in a cell culture medium, concentrated feed, or reconstituted medium of the present invention 1001, comprising the step of culturing recombinant cells in a cell culture medium of the present invention 1001, wherein the medium supports the proliferation of the cells under conditions suitable for the expression of the virus or viral particles. [Invention 1061] Recombinant protein produced by the method of Invention 1056. [Invention 1062] A virus or viral particle produced by the method of the present invention 1060. [Invention 1063] Use of cell culture media, concentrated feeds, or reconstituted media of the present invention 1001 for producing viruses or recombinant proteins. [Invention 1064] A culture medium, concentrated feed, or reconstituted medium prepared by the method of Invention 1026. [Invention 1065] A cell culture medium according to the present invention 1001 or 1064, wherein the small peptide is one of a dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide, or decapeptide. [Invention 1066] A cell culture medium according to the present invention 1001 or 1064, wherein the small peptide has a length of at least about 2 to 10 amino acids. [Invention 1067] A cell culture medium according to the present invention 1001 or 1064, wherein the small peptide is at least about 10 amino acids long. [Invention 1068] The method of the present invention 1022, wherein the above supplementation is performed during fed-batch culture. [Brief explanation of the drawing]

[0054] The accompanying drawings included herein, which constitute part thereof, illustrate certain aspects of the present invention and, together with the written description, help to illustrate certain principles of the present invention. [Figure 1] The graph shows the viable cell density (×10⁶ cells / mL) (shown as a curve) and IgG titer (mg / L) (shown as a bar) of CHO cells grown in (i) cell culture basal medium supplemented with glucose supply (CD FortiCHO®), and (ii) CD FortiCHO® + aa (concentrated amino acid mixture containing low levels of cysteine ​​and tyrosine, and glucose) + DP (dipeptide) supply Schedule 2 (FP2), or (iii) CD FortiCHO® + aa (concentrated amino acid mixture containing low levels of cysteine ​​and tyrosine, and glucose) + DP (dipeptide) supply Schedule 3 (FP3). See examples. [Figure 2] An exemplary profile chromatogram of a solution containing the Ala-Tyr dipeptide is shown, with the Ala-Tyr dipeptide indicated by a peak at 6.172 min (lower panel). [Figure 3] An exemplary profile chromatogram of a solution containing the Ala-Cys dipeptide is shown, with the Ala-Cys dipeptide indicated by a peak at 6.631 min (lower panel). [Figure 4] An exemplary profile extract ion chromatogram of a solution containing the Ala-Tyr dipeptide standard is shown, indicated by the peak at 8.8 min. [Figure 5] An exemplary profile extract ion chromatogram of a solution containing an Ala-Cys dimer standard is shown, indicated by the peak at 9.6 min. [Figure 6] Exemplary profile total ion chromatograms of exemplary feed samples containing Ala-Cys dimers and Ala-Tyr dipeptides are shown. [Figure 7] Exemplary profile extract ion chromatograms of exemplary feed samples containing Ala-Tyr dipeptide and Ala-Cys dimer are shown. [Modes for carrying out the invention]

[0055] Detailed explanation The following will describe various exemplary embodiments in detail. The following detailed descriptions are provided to help the reader better understand the specific embodiments, features, and aspects of the invention, and should not be construed as limiting the scope of the invention.

[0056] 1. Definition To make the present invention easier to understand, certain terms are first defined. Further definitions will be provided throughout the detailed description.

[0057] As used herein, “cell culture” or “culture” refers to the maintenance of cells in an artificial (e.g., in vitro) environment. However, the term “cell culture” is a general term and may be used to encompass not only individual prokaryotic (e.g., bacterial) cells or eukaryotic (e.g., animal, plant, and fungal) cells, but also tissues, organs, organ systems, or whole organisms. It should be understood that the terms “tissue culture,” “organ culture,” “organ system culture,” or “organ-type culture” may sometimes be used interchangeably with the term “cell culture.”

[0058] As used herein, “culture” refers to the maintenance of cells in an artificial environment under conditions favorable for proliferation, differentiation, or sustained survival, whether in an active or quiescent state. Therefore, “culture” may be used interchangeably with “cell culture” or any of its synonyms described above.

[0059] As used herein, “cell culture medium,” “culture medium,” or “medium” (and in each case, the plural “media”) refers to a nutritional composition that supports the culture and / or proliferation of cells. A cell culture medium may be a complete formulation, i.e., a cell culture medium that does not require supplementation to culture cells; an incomplete formulation, i.e., a cell culture medium that requires supplementation; or a medium that can be supplemented if it is an incomplete formulation; or, if it is a complete formulation, a medium that can improve culture or culture outcomes. Unless otherwise indicated by context, the terms “cell culture medium,” “culture medium,” or “medium” (and in each case, the plural “media”) refer to an unadapted cell medium that has not been incubated with cells. Thus, the terms “cell culture medium,” “culture medium,” or “medium” (and in each case, the plural “media”) are distinguished from “used” or “adapted” media, which may contain many of the original components of the medium, as well as various cellular metabolites and secreted proteins.

[0060] As used herein, “small peptide” refers to a chain of 2 to 6 amino acids linked by one or more peptide bonds or equivalent bonds, with at least one amino acid being tyrosine or cysteine. Preferably, amino acids in the small peptide other than tyrosine or cysteine ​​exhibit good solubility at neutral pH. Therefore, in one embodiment, the amino acids in the small peptide other than tyrosine or cysteine ​​are selected from alanine, glycine, serine, valine, proline, glutamic acid, aspartic acid, glutamine, and arginine. In another embodiment, the N-terminal amino acid of the small peptide has a free amino group. In one embodiment of the present invention, the small peptide may contain a free amino group such as ethanolamine, or a non-amino acid or similar compound containing a free carboxyl group.

[0061] As used herein, “extract” typically refers to a composition comprising a concentrated preparation of a component or subgroup of a substance, formed by processing the substance either mechanically (e.g., by pressurization) or chemically (e.g., by distillation, precipitation, enzymatic action, or high-salt treatment).

[0062] As used herein, the term “component” refers to any compound that may be used in a cell culture medium to maintain or promote the growth of cells, whether of chemical or biological origin. The terms “component,” “nutrient,” and “component” may be used interchangeably and are all intended to refer to such compounds. Typical components that may be used in a cell culture medium include amino acids, salts, metals, sugars, carbohydrates, lipids, nucleic acids, hormones, vitamins, fatty acids, and proteins. Other components that promote or maintain cell culture ex vivo may be selected by those skilled in the art according to specific needs.

[0063] A “1× formulation” refers to any aqueous solution containing some or all of the components found in a cell culture medium at their active concentrations. A “1× formulation” can also refer to, for example, a cell culture medium or any subgroup of components for that medium. The concentration of a component in a 1× solution is approximately the same as the concentration of that component found in a cell culture formulation used to maintain or culture cells in vitro. A cell culture medium used for in vitro culture of cells is, by definition, a 1× formulation. If many components are present, each component in a 1× formulation has a concentration approximately equal to the concentration of those components in the cell culture medium. For example, RPMI-1640 culture medium contains, among other components, 0.2 g / L L-arginine, 0.05 g / L L-asparagine, and 0.02 g / L L-aspartic acid. A “1× formulation” of these amino acids contains these components in approximately the same concentration in solution. Therefore, when referring to a “1× formulation,” it is intended that each component in the solution has the same or approximately the same concentration as found in the described cell culture medium. The concentrations of components in 1× formulations of cell culture media are well known to those skilled in the art. See Cell Culture Technology for Pharmaceutical and Cell-Based Therapies, 42-50 (Sadettin Ozturk and Wei-Shou Hu eds., Taylor and Francis Group 2006), which is incorporated herein by reference in its entirety. However, the osmotic pressure and / or pH may differ in the 1× formulation compared to the culture medium, especially if fewer components are present in the 1× formulation.

[0064] The term "10× formulation" is intended to refer to a solution in which each component in the solution is concentrated approximately 10 times more than the same component in the cell culture medium. For example, the 10× formulation of RPMI-1640 culture medium may contain, among other components, 2.0 g / L L-arginine, 0.5 g / L L-asparagine, and 0.2 g / L L-aspartic acid (compared to the 1× formulation above). The "10× formulation" may contain many additional components at concentrations approximately 10 times higher than those found in the 1× culture medium. As is readily apparent, the "5× formulation," "25× formulation," "50× formulation," "100× formulation," "500× formulation," and "1000× formulation" indicate solutions containing components at approximately 5 times, 25 times, 50 times, 100 times, 500 times, and 1000 times higher concentrations, respectively, compared to the 1× cell culture medium. Here again, the osmotic pressure and pH of the culture medium formulation and concentrated solution may differ. The formulation may contain components or ingredients at concentrations of 1× for a specific cell culture protocol, but at concentrations such as 2×, 2.5×, 5×, 6.7×, 9×, 12× for different culture protocols or different basal media.

[0065] A dimer consists of two dipeptides. Therefore, for example, the Ala-Cys dimer constitutes Ala-Cys-Cys-Ala, where cys-cys are linked by a disulfide bond. Ala-Cys-Cys-Ala can also be called N,N',-di-L-alanyl-L-cysteine.

[0066] 1. Small peptides This disclosure relates to the use of small peptides, including dipeptides, in cell culture media. The applicants have found that the preparation of cell culture media containing desired concentrations of specific amino acids, such as tyrosine and cysteine, remains impossible due to the low solubility of tyrosine and the low stability of cysteine. The solubility and stability issues of tyrosine and cysteine ​​have been addressed by using lower concentrations of tyrosine or cysteine ​​than the desired concentration in aqueous cell culture media, but this makes them rate-limiting amino acids for optimal cell growth and / or protein production because the nutrients are used during cell culture. While using lower concentrations of tyrosine and cysteine ​​than the desired concentration can result in acceptable aqueous storage life, solubility, and stability, achieving the desired productivity may require adding larger volumes of medium during cell culture replenishment, for example in a fed-batch culture system. Furthermore, this is undesirable because it also requires readjustment of the system's stoichiometric equilibrium, pH, and / or osmotic pressure.

[0067] Problems associated with large-scale replenishment can be addressed by using concentrated feed supplements. A desirable concentrated feed supplement is a nutritionally complex and stoichiometrically balanced nutrient additive that replenishes nutrient-depleted culture systems in fed-batch culture systems and, in addition, and preferably, possesses auto-pH equilibrium and auto-osmotic equilibrium characteristics. However, due to the low solubility of tyrosine or the low stability of cysteine, concentrated feed compositions containing high levels of cysteine ​​and tyrosine have been difficult to prepare. For this reason, cysteine ​​or tyrosine generally elutes from the solution over time. Therefore, the synthesis of stable, single-part pH-neutral concentrated feeds with known compositions has been difficult. To circumvent this problem, acidic or alkaline multi-part concentrated feeds have been prepared.

[0068] The applicants have discovered that free amino acids, tyrosine, and cysteine ​​in cell culture media can be replaced with dipeptides such as alanyl-tyrosine, glycyl-tyrosine, alanyl-cysteine ​​(forming a disulfide dimer, [AlaCys]2), or glycyl-cysteine ​​(forming a disulfide dimer, [GlyCys]2), or other small peptides containing one or more tyrosine or cysteine. Cell culture media containing these dipeptides or other small peptides provide cells with sufficient concentrations of tyrosine and / or cysteine ​​to support maximum cell proliferation and / or protein production, while avoiding solubility and stability issues associated with free tyrosine and / or cysteine.

[0069] The small peptide of the present invention has 2 to 6 amino acids, at least one of which is cysteine ​​or tyrosine, and the remaining amino acids may be any amino acids. Preferably, the amino acids in the small peptide other than tyrosine or cysteine ​​exhibit good solubility characteristics at neutral pH. In one embodiment, the amino acids in the small peptide other than tyrosine or cysteine ​​are selected from alanine, glycine, serine, valine, proline, glutamic acid, or aspartic acid. In another embodiment, the N-terminal amino acid of the small peptide has a free amino group. In one embodiment of the present invention, the small peptide (2, 3, 4, 5, or 6 amino acids) may contain a free amino group such as ethanolamine, or a non-amino acid containing a free carboxyl group, or a similar compound that can contribute to the peptide bond. In another embodiment, the small peptide may contain cysteine ​​dimerized via a sulfide bond. Although the small peptides of the present invention are defined as having a length of 2 to 6 amino acids for cysteine-cysteine ​​dimerization (cys-cys), compositions may also exist that yield longer peptides due to spontaneous cysteine-cysteine ​​dimerization that may occur in the culture medium or feed of the present invention. Such compounds are still soluble even at high concentrations and are therefore entirely within the scope of the present invention.

[0070] In other embodiments, the small peptide is a dipeptide having two amino acids and represented by the formula: X-tyrosine, X-cysteine, tyrosine-X, or cysteine-X, or a salt thereof, where X is any amino acid, and the N-terminal amino acid of the dipeptide has a free amino group. Thus, this dipeptide is different from the N-acyl dipeptide disclosed in U.S. Patent No. 5,534,538, which requires an acyl group covalently bonded to the amino group of the N-terminal amino acid. According to U.S. Patent No. 5,534,538, it is the presence of the acyl group that confers excellent stability properties to the dipeptide. Preferably, X is an amino acid having good solubility properties. In one embodiment, X is alanine or glycine. In another embodiment, X is serine, valine, proline, glutamic acid, or aspartic acid.

[0071] In one embodiment, the small peptide is a dipeptide having two amino acids, and one or more dipeptides of the present invention are selected from X-tyrosine, X-cysteine, tyrosine-X, and cysteine-X, or salts thereof, where X is any amino acid, and the N-terminal amino acid of the dipeptide has a free amino group. In one embodiment, X is alanine or glycine. In another embodiment, X is any amino acid, a derivative of an amino acid, or a non-amino acid having an amino group such as ethanolamine. In one aspect of this embodiment, preferred amino acids are selected from the group: serine, valine, proline, aspartic acid, arginine, glutamine, or glutamic acid.

[0072] In another embodiment, the small peptide is a tripeptide having three amino acids, including the following tripeptides: XX-tyrosine, XX-cysteine, X-tyrosine-X, X-cysteine-X, tyrosine-XX, cysteine-XX, or salts thereof, where X is any amino acid. In one embodiment, the N-terminal amino acid of the tripeptide has a free amino group. Preferably, X is an amino acid having good solubility properties. In one embodiment, X is alanine or glycine. In another embodiment, X is serine, valine, proline, glutamic acid, glutamine, arginine, or aspartic acid. In a particular aspect of the present invention, the above composition containing cysteine ​​and tyrosine in the small peptide is used to increase protein production in a cell line. In a preferred aspect of the present invention, the increased protein is an antibody produced at a higher titer than in a medium without the small peptide. Higher antibody production means concentrations of approximately 0.1 g / L to 10 g / L, approximately 0.1 g / L to 5 g / L, approximately 0.1 g / L to 2.5 g / L, approximately 0.1 g / L to 1 g / L, preferably approximately 1 g / L to 10 g / L, and more preferably approximately 2 g / L to 8 g / L.

[0073] Depending on the cells and their intended use, one or more small peptides or dipeptides, such as in cell culture media, feeds, concentrated media, or concentrated feeds, are optimally present at stoichiometrically equilibrated concentrations to optimize cell culture performance. For example, the concentrations of one or more small peptides or dipeptides in some cell culture media or concentrated feeds are approximately 0.5 g / L to 30 g / L, 0.5 g / L to 25 g / L, 0.5 g / L to 20 g / L, 0.5 g / L to 16 g / L, 0.5 g / L to 10 g / L, 0.5 g / L to 5 g / L, 0.5 g / L to 4 g / L, 1 g / L to 30 g / L, 1 g / L to 20 g / L, 1 g / L to 16 g / L, 1 g / L to 10 g / L, 1 g / L to 5 g / L, 2.5 g / L to 30 g / L, 2.5 g / L to 20 g / L, 2.5 g / L to 16 g / L, and 2.5 g / L to 10 g / L. The concentrations may be g / L, approximately 2.5 g / L to approximately 5 g / L, or approximately 2.5 g / L to approximately 4.5 g / L, approximately 5 g / L to approximately 30 g / L, approximately 5 g / L to approximately 25 g / L, approximately 5 g / L to approximately 20 g / L, approximately 5 g / L to approximately 16 g / L, approximately 5 g / L to approximately 10 g / L, approximately 5 g / L to approximately 5 g / L, approximately 5 g / L to approximately 4 g / L, etc. In some culture medium formulations or prototype culture medium formulations, one or more small peptides and / or one or more dipeptides may be present in the culture medium or prototype medium at concentrations of approximately 2.5 g / L to approximately 8.5 g / L. In addition, some aspects of the present invention provide a method for preparing an "auto-pH medium" or feed powder that automatically reaches a desired pH upon rehydration / reconstitution with a solvent. In accordance with the present invention, such culture media or feeds may be in powder form (dry powder (DPM), high-performance powder (APM), or advanced granulation technology (AGT)) or liquid form.

[0074] The animal cell culture medium or animal cell culture supply prepared by this method has a pH of preferably about 6-8 or about 7-8 or about 6.0-6.3 (for insect cells), more preferably about 7-7.5 or about 7.2-7.4, and most preferably about 7.0 upon reconstitution; the plant cell culture medium or plant cell culture supply prepared by this method has a pH of preferably about 4-8, preferably about 4.5-7, 5-6, or 5.5-6, or preferably about 6.0-6.3 upon reconstitution. Naturally, the optimal pH of a given culture medium to be used for a particular cell type can also be empirically determined by those skilled in the art using methods known in the art.

[0075] 2. Cell culture medium Cell culture media consist of many components, and these components differ from one culture medium to another. As described above, cell culture media may be a complete formulation, i.e., a cell culture medium that does not require supplementation to culture cells, or an incomplete formulation, i.e., a cell culture medium that requires supplementation, or a supplement that can supplement an incomplete formulation, or, if it is a complete formulation, a supplement that can improve culture or culture results.

[0076] Generally, cell culture media contain solutes dissolved in a solvent. These solutes provide osmotic force, maintaining osmotic equilibrium across the cell membrane (or wall). In addition, solutes provide nutrients to the cells. Some nutrients may be chemical fuels for cellular function; some may be raw materials for cellular use in anabolism; some may be mechanisms such as enzymes or carriers that promote cellular metabolism; and some may be binders that bind to and buffer components for cellular use, or bind to or sequester harmful cellular products.

[0077] Depending on the cells and their intended use, the components of the cell culture medium are optimally present at equilibrated concentrations to optimize the performance of the cell culture. Performance is measured according to one or more desired characteristics, such as cell number, cell mass, cell density, O2 consumption, consumption of culture components such as glucose or nucleotides, production of biomolecules, secretion of biomolecules, formation of waste products or by-products such as metabolites, and effects on indicator or signaling molecules. Therefore, each or selected component is preferably optimized to an effective concentration for the intended purpose.

[0078] The culture medium or supplement of the present invention may be obtained in a dry form requiring only the addition of a solvent such as water. Preferably, the dry form powder is prepared by at least one method selected from the group consisting of milling, impact, extrusion and cutting or crushing, wet granulation, high shear granulation, bread granulation, and fluidized bed agglutination. The dry form includes, but is not limited to, dry powder form (DPM), agglutination (AGT®) form, high-performance powder medium (APM), or other suitable dry form. Preferably, once water is added, dissolution should occur immediately, and the resulting solution can be filtered and added directly to cells without pH adjustment. The reconstituted medium or concentrated supplement may be prepared in variable bulk quantities and may be sterilized in particular by ionization or ultraviolet irradiation.

[0079] a. Carbohydrates Cell culture media typically contain carbohydrates, amino acids, salts, trace elements, and vitamins. For mammalian cells, the primary carbohydrate used in cell culture media is glucose, routinely supplemented at 5–25 nM. (See Cell Culture Technology for Pharmaceutical and Cell-Based Therapies, 51 (Sadettin Ozturk and Wei-Shou Hu eds., Taylor and Francis Group 2006)). In addition to glucose, any hexose, such as galactose, fructose, or mannose, or combinations thereof, may also be used. Furthermore, mammals may also use glutamine as a primary energy source. Glutamine is often present at higher concentrations than other amino acids (2–8 mM). However, as mentioned above, glutamine can spontaneously decompose to form ammonia, and certain cell lines produce ammonia more rapidly, which is toxic. Therefore, glutamic acid and glutamine dipeptides are used as substitutes for glutamine to reduce the accumulation of toxic ammonia in cell culture media.

[0080] b. Amino acids Amino acids are essential in cell culture media for maintaining the metabolic function of cultured cells. Cell culture media typically contain essential amino acids (i.e., amino acids not normally synthesized in vivo by mammals) and certain non-essential amino acids. Non-essential amino acids are typically included in cell culture media when the cell line cannot synthesize them or cannot produce sufficient amounts to support maximum growth. Exemplary amino acids include L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-glutamic acid, L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine, and L-valine.

[0081] c. Salt Salts are added to cell culture media to maintain an isotonic state and prevent osmotic imbalances. The osmotic pressure of standard mammalian cell culture media is approximately 300 mOsm / kg, although many cell lines can tolerate fluctuations of approximately 10% of this value. The osmotic pressure of some insect cell cultures tends to be higher than 300 mOsm / kg, which may be 0.5%, 1%, 2 - 5%, 5 - 10%, 10 - 15%, 15 - 20%, 20 - 25%, or 25 - 30% higher than 300 mOsm / kg. The salts most commonly used in cell culture media include Na + , K + , Mg 2+ , Ca 2+ , Cl - , S04 2- , P04 3- , and HC03 - (e.g., CaCl2, KCl, NaCl, NaHC03, Na2HP04). Thus, the desired osmotic pressure of a cell culture medium for culturing a particular cell type can also be empirically determined by one skilled in the art using methods known in the art.

[0082] d. Inorganic elements As described in US 2005 / 0287666, which is hereby incorporated by reference in its entirety, other inorganic elements that are present in trace amounts in serum can be included in cell culture media. These include Mn, Cu, Zn, Mo, Va, Se, Fe, Ca, Mg, Si, and Ni. Less frequently, other inorganic elements that have been added to cell culture media include Al, Ag, Ba, Br, Cd, Co, Cr, F, Ge, J, Rb, and Zr. Many of these elements are involved in enzyme activity. They can be provided in the form of salts such as CaCl2, Fe(N03)3, MgCl2, MgS04, MnCl2, NaCl, NaHC03, Na2HP04, as well as ions of trace elements such as selenium, vanadium, and zinc. These trace elements can be in various forms, preferably Na2Se0 3、These elements can be supplied in the form of salts such as NH4V03. These inorganic salts and trace elements are commercially available, for example, from Sigma (Saint Louis, Missouri).

[0083] e. Vitamins Vitamins are typically used by cells as cofactors. While the vitamin requirements of each cell line vary considerably, extra vitamins are generally needed when the cell culture medium contains little serum or when cells are grown at high density. Exemplary vitamins include biotin, choline chloride, folic acid, i-inositol, nicotinamide, and D-Ca. ++ - Pantothenic acid, pyridoxal, riboflavin, thiamine, pyridoxine, niacinamide, A, B6, B 12 It contains C, D3, E, K, and p-aminobenzoic acid (PABA).

[0084] f. Serum Serum, the supernatant of coagulated blood, can be used in cell culture media to provide components that promote cell proliferation and / or productivity. These serum components include adhesion factors, micronutrients (e.g., trace elements), growth factors (e.g., hormones, proteases), and protective elements (e.g., antitoxins, antioxidants, antiproteases). Serum is available from a variety of animal sources, including cattle or horses. When included in cell culture media, serum is typically added at a concentration of 5–10%. Certain cell culture media are serum-free.

[0085] g. Growth factors To promote cell proliferation in the absence of serum or in serum-reduced media, one or more of the following polypeptides may be added to the cell culture medium: for example, fibroblast growth factor (FGF) including acidic FGF and basic FGF, insulin, insulin-like growth factor (IGF), epidermal growth factor (EGF), nerve growth factor (NGF), platelet-derived growth factor (PDGF), and any cytokines such as transforming growth factors (TGF) including TGFα and TGFβ, interleukin 1, 2, 6, granulocyte-stimulating factor, and leukemia suppressor (LIF).

[0086] In certain embodiments, the cell culture medium does not contain growth factors. In protein-free media, insulin may be replaced with zinc or a zinc-containing compound, as described in WO 98 / 08934, which is incorporated herein by reference in its entirety.

[0087] h. Lipids One or more lipids may also be added to the cell culture medium. Serum typically contains lipids such as fatty acids (e.g., linoleic acid, linolenic acid, arachidonic acid, palmitoleic acid, oleic acid, polyenoic acid, and / or fatty acids with 12, 14, 16, 18, 20, or 24 carbon atoms, where each carbon atom is branched or unbranched), phospholipids, lecithin (phosphatidylcholine), and cholesterol. Alternatively, one or more of these lipids may be included as supplements in serum-free media. Phosphatidic acid and lysophosphatidic acid promote the growth of certain anchorage-dependent cells, such as MDCK, mouse epithelium, and other kidney cell lines, while phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol promote the growth of human fibroblasts in serum-free media. Ethanolamine and cholesterol have also been shown to promote the growth of certain cell lines. In certain embodiments, the cell culture medium is lipid-free.

[0088] i. Carrier proteins One or more carrier proteins, such as bovine serum albumin (BSA) or transferrin, may also be added to the cell culture medium. Carrier proteins can be useful for transporting certain nutrients or trace elements. BSA is typically used as a lipid carrier, such as linoleic acid and oleic acid, which are insoluble in aqueous solution. In addition, BSA can also serve as a carrier for certain metals, such as Fe, Cu, and Ni. In protein-free formulations, non-animal alternatives to BSA, such as cyclodextrin, can be used as lipid transporters. Transferrin is involved in the transport of iron across the cell membrane. In some cases, human serum albumin may be required for culturing cells that are desirable for products produced for downstream therapeutic applications (e.g., in xenobiotic (XF) cultures). In other examples, recombinant human serum albumin may be used in cell culture media for culturing cells. In certain cases, to provide an animal-free (AOF) culture of cells, recombinant human serum albumin may be derived from plant, algal, or fungal sources such as rice, corn, potato, wheat, and even yeast. In protein-free formulations, transferrin may be substituted with ferric and / or ferrous salts, as described in WO 98 / 08934, which is incorporated herein by reference in whole, or with hydroxypyridine derivatives, as described in US 2007 / 0254358, which is incorporated herein by reference in whole. In addition, in protein-free formulations, insulin may be substituted with zinc, vanadium, or other suitable ferrous salts.

[0089] j. Adhesion proteins To help promote the adhesion of scaffold-dependent cells to the substrate, one or more adhesion proteins, such as fibronectin, laminin, and pronectin, may also be added to the cell culture medium.

[0090] k. Buffer Cell culture media may optionally contain one or more buffers. Suitable buffers include, but are not limited to, N-[2-hydroxyethyl]-piperazine-N'-[2-ethanesulfonic acid] (HEPES), MOPS, MES, phosphoric acid, bicarbonate, and other buffers suitable for use in cell culture applications. A suitable buffer provides buffering capacity without substantial cytotoxicity to the cultured cells. The selection of a suitable buffer is within the scope of common practice in the art of cell culture.

[0091] l. Polyanionic compounds or polycationic compounds Polyanionic or polycationic compounds may prevent cell aggregation and promote cell proliferation in suspension. See WO 98 / 08934, which is incorporated herein by reference in its entirety. Exemplary polyanionic compounds include polysulfonated or polysulfated compounds such as heparin, dextran sulfate, heparan sulfate, dermatan sulfate, chondroitin sulfate, pentoyl polysulfate, and proteoglycans.

[0092] In addition to the small peptides or dipeptides described herein, the cell culture medium includes one or more components such as those mentioned above. In one embodiment, the cell culture medium comprises one or more small peptides or one or more dipeptides as described above, and optionally one or more of the following components: ethanolamine, D-glucose, HEPES, insulin, cytokine (e.g., IL-6), heparin, dextran sulfate, linoleic acid, lipoic acid, phenol red, PLURONIC® F68, putrescine, sodium pyruvate, transferrin, L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-glutamic acid, L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, biotin, choline chloride, D-Ca ++- Pantothenic acid, folic acid, i-inositol, niacinamide, pyridoxine, riboflavin, thiamine, vitamin B 12 , one or more calcium salts, Fe(NO3)3, KCl, one or more magnesium salts, one or more manganese salts, NaCl, NaHCO3, Na2HP04, one or more selenium salts, one or more vanadium salts, and one or more zinc salts. In one embodiment, one or more dipeptides are selected from X-tyrosine, X-cysteine, tyrosine-X, and cysteine-X, or salts thereof, where X is any amino acid and the N-terminal amino acid of the dipeptide has a free amino group. In one embodiment, X is alanine or glycine. In another embodiment, X is serine, valine, proline, aspartic acid, or glutamic acid.

[0093] The media described herein may be 1× formulations, or may be concentrated to a higher concentration than 1× formulations, such as 2×, 5×, 10×, 20×, 50×, 500×, or 1000× medium formulations, as long as the solubility of the individual components allows. If the individual medium components are prepared as separate concentrated solutions, an appropriate (sufficient) amount of each concentrate is mixed with a diluent to prepare a 1× medium formulation. Typically, the diluent used is water, but other solutions including aqueous buffer, aqueous saline, or other aqueous solutions may also be used.

[0094] The media described herein may also be prepared in different forms, such as dry powder media, granular preparations (which require the addition of water but do not require other treatments such as pH adjustment (pHing)), liquid media, or medium concentrates.

[0095] 3. Serum-free culture medium Potential problems associated with serum, including batch-to-batch variability, high protein content, risk of contamination (e.g., viruses, mycoplasma, prions), limited availability, and high cost, have driven the development of serum-free media. Furthermore, improved levels of recombinant protein expression can be obtained from cells grown in serum-free media compared to the expression levels observed in cells grown in serum-supplemented media (Battista, PJ et al., Am. Biotech Lab. 12: 64-68 (1994)).

[0096] In these serum-free media, serum may be replaced with a distinct hormone or with a mixture of hormones such as HITES or ITES, containing hydrocortisone, insulin, transferrin, ethanolamine, and selenite. Alternatively, the serum-free medium may contain growth factor extracts from endocrine glands, such as epidermal growth factor or fibroblast growth factor. The serum-free medium may also contain other components as serum substitutes, including purified proteins (animal or recombinant), peptones, amino acids, inorganic salts, and animal or plant hydrolysates (or parts thereof).

[0097] Serum-free media may be of known or unknown composition. In known-composition media, the identity and quantities of the constituent components are known, whereas in unknown-composition media, the opposite is true. Therefore, known-composition media are designed, for one thing, to reduce the risk of contamination and to reduce batch-to-batch variability. Known-composition supplements that can be added to cell culture media include growth factors, hormones, carrier proteins, and / or adhesion factors. In a preferred embodiment, a basal medium used with a medium or feed containing small peptides is a known-composition medium. In another preferred embodiment, the concentrated cell culture medium or concentrated feed of the present invention containing small peptides is also a known-composition composition. In yet another preferred embodiment, the concentrated feed or concentrated medium of the present invention containing small peptides containing cysteine ​​and tyrosine is a single partial feed and of known composition. In another aspect, all of the above compositions containing small peptides containing cysteine ​​and tyrosine automatically equilibrate pH and automatically equilibrate osmotic pressure. In another aspect, all of the above compositions, including small peptides containing cysteine ​​and tyrosine, are stoichiometrically equilibrated.

[0098] 4. Protein-free culture medium Serum-free media contain small amounts of protein compared to cell culture media containing serum. However, serum-free media may still contain one or more of various animal-derived components, including albumin, fetuin, various hormones, and other proteins. The presence of proteins makes the purification of recombinant proteins difficult, time-consuming, and expensive, and can lead to a decrease in the yield and / or purity of the product. Therefore, in one embodiment, the cell culture medium is protein-free.

[0099] Protein-free media can be obtained by methods known in the art, such as by removing any residual proteins from serum-free media. Removing such proteins from cell culture media may impair the medium's ability to support cell proliferation; however, other components may be added to the medium to mitigate the effects of protein removal. For example, as mentioned above, cyclodextrin can replace BSA, and iron salts or hydroxypyridine derivatives can replace transferrin. In other cases, animal tissues or plant hydrolysates (or parts thereof) are used to supplement protein-free media.

[0100] 5. Fed-batch culture Fed-batch cell culture is typically used in the industrial production of biomolecules such as proteins to increase cell concentration and extend culture life, aiming for high product concentration and volume productivity. Fed-batch culture involves the controlled addition of one or more nutrients to a basal medium in the form of a feed that may contain nutrients readily utilized by cells, such as glucose and amino acids. Nutrients help control cell culture growth by preventing nutrient depletion and byproduct accumulation, and by controlling key parameters such as pH, osmotic pressure, and CO2 concentration within levels that promote cell growth for optimal product expression or minimize cell death. See Cell Culture Technology for Pharmaceutical and Cell-Based Therapies, 349-386 (Sadettin Ozturk and Wei-Shou Hu eds., Taylor and Francis Group 2006). Even in this case, fed-batch culture often results in high concentrations of inhibitory metabolites and high osmotic pressure that are ultimately incompatible with cell viability.

[0101] Basal media are typically used to maintain cell cultures and may contain many components, including amino acids, vitamins, organic and inorganic salts, sugars, and other components, each present in amounts that support cell culture in vitro. Basal media useful for prokaryotic cell cultures, including bacterial and archaeal cultures, viral cultures, plant cell cultures, insect cell cultures, and mammalian cell cultures can be used with small peptides. Examples of basal media include Eagle basal medium (BME), Eagle minimal essential medium (EMEM), Dulbecco's modified Eagle medium (DMEM), Glasgow modified Eagle medium (GMEM), Jocklic modified Eagle medium, α-modified Eagle medium, Roswell Park Memorial Institute (RPMI) medium, Fisher medium, Leibovitz L-15 medium, Trowell T-8 medium, Williams medium E, Biggers medium, Connaught Medical Research Laboratories (CMRL) 1066 medium, Ham F10 medium, Ham F12 medium, Iskov modified Dulbecco medium (IMDM), MCDB 104, MCDB 110, MCDB 153, medium 199, NCTC 135 medium, and Weymouth medium MB 752 / 1. For CHO cells, preferred basal media include CD CHO, CD OptiCHO®, and CD FortiCHO® (all from Life Technologies, Corp., Carlsbad, CA). Preferred concentrated feed supplements for CHO cells include, but are not limited to, CHO CD EfficientFeed® A (Invitrogen Cat. No. A1023401), CHO CD EfficientFeed® B (Invitrogen Cat. No. A1024001), CHO CD EfficientFeed® Kit (Invitrogen Cat. No. A1024101), and CD EfficientFeed® C AGT (Invitrogen Cat. No. A1327501, Life Technologies Corp., Carlsbad, CA).

[0102] In fed-batch culture, cells are typically grown in batches using basal medium up to a certain point in time. Then, medium supplements (concentrated feeds) containing concentrated solutions of one or more nutrients are added to provide nutrients, minimizing volume increases or dilution of the culture. When medium supplements are added to basal medium, this improves cell culture, for example, by more rapid cell growth, shorter doubling times, higher achievable cell densities, or higher production or yield of biomolecules such as proteins, including antibodies or other proteins of therapeutic interest.

[0103] The cell culture medium or concentrated feed of the present invention, suitable for use in supplementing basal media, contains one or more small peptides comprising one or more dipeptides as described herein. The cell culture medium or concentrated feed containing cysteine ​​and tyrosine may optionally be used in combination with another feed, for example, an amino acid concentrated mixture which may comprise one or more of the following components: adenine, ethanolamine, carbohydrate source (such as hexoses such as glucose, mannose, galactose, fructose, or even combinations thereof), heparin, buffer, hydrocortisone, lipoic acid, phenol red, phosphoethanolamine, putrescine, pyrubic acid Sodium phosphate, triiodothyronine, thymidine, L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-glutamic acid, L-glutamine, glycine, L-histidine, L-hydroxyproline, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, N-acetylcysteine, biotin, choline chloride, D-Ca ++ - Pantothenic acid, folic acid, i-inositol, niacinamide, pyridoxine, riboflavin, thiamine, vitamin B 12Pluronic F68, recombinant insulin, calcium salts, CuSiO4, FeSiO4, FeCl3, Fe(NO3)3, KCl, magnesium salts, manganese salts, sodium acetate, NaCl, NaHCO3, Na2HP04, Na2SiO4, selenium salts, silicon salts, molybdenum salts, vanadium salts, nickel salts, tin salts, ZnCl2, ZnSiO4, or other zinc salts.

[0104] Therefore, concentrated amino acid mixtures used to supplement basal media may consist of stoichiometrically equilibrated mixtures of amino acids, which may or may not contain cysteine ​​and tyrosine, and may optionally contain one or more of the following: carbon sources, vitamins, and trace elements, and may be known compositions in that they may not contain any lipids, hydrolysates, or growth factors. In some cases, concentrated amino acid mixtures may contain animal-derived hydrolysates or a portion of hydrolysates, such as plant hydrolysates. For example, any commercially available concentrated amino acid mixture can be used, such as CHO CD EfficientFeed® A (Invitrogen Cat. No. A1023401), CHO CD EfficientFeed® B (Invitrogen Cat. No. A1024001), CHO CD EfficientFeed® Kit (Invitrogen Cat. No. A1024101), or CD EfficientFeed® C AGT (Invitrogen Cat. No. A1327501, Life Technologies Corp., Carlsbad, CA).

[0105] In one embodiment, the cysteine ​​and tyrosine-containing cell culture medium or concentrated feed of the present invention, suitable for use in supplementing basal media, is protein-free. In another embodiment, the cell culture medium or concentrated feed of the present invention, suitable for use in supplementing basal media, is protein-free and, in addition, does not contain lipids, hydrolysates or any part thereof, or growth factors, and is therefore considered to have a known composition (CD). In one aspect of this embodiment, the cysteine ​​and tyrosine-containing cell culture medium or concentrated feed of the present invention may optionally contain one or more of the carbon source, vitamins, and trace elements, and may further be of a known composition in that it does not contain any lipids, hydrolysates, or growth factors.

[0106] 6. Cell The culture media containing cysteine ​​and tyrosine-containing small peptides or dipeptides described herein can also be used to culture a variety of cells. Cells grown using the culture media and supplies described herein may originate from any prokaryotes, including archaea, algae, yeast, fungi, plants, insects, animals, preferably mammals, and most preferably mice or humans. In one embodiment, the medium is used to culture plant cells, or eukaryotic cells including mammalian cells, fish cells, insect cells, amphibian cells, or animal cells such as avian cells.

[0107] Mammalian cells that can be cultured using the media described herein include primary epithelial cells (e.g., keratinocytes, cervical epithelial cells, bronchial epithelial cells, tracheal epithelial cells, renal epithelial cells, and retinal epithelial cells), as well as established cell lines and their strains (e.g., 293 fetal kidney cells, BHK cells, HeLa cervical epithelial cells, and PER-C6 retinal cells, MDBK(NBL-1) cells, 911 cells, CRFK cells, MDCK cells, CHO cells, BeWo cells, Chang cells, Detroit 562 cells, HeLa 229 cells, HeLa S3 cells, Hep-2 cells, KB cells, LS180 cells, LS174T cells, NCI-H-548 cells, RPMI 2650 cells, SW-13 cells, T24 cells, WI-28 cells) VA13, 2RA cells, WISH cells, BS-CI cells, LLC-MK2 cells, clone M-3 cells, 1-10 cells, RAG cells, TCMK-1 cells, Y-1 cells, LLC-PK1 cells, PK(15) cells, GH1 cells, GH3 cells, L2 cells, LLC-RC Fibroblasts derived from any tissue or organ (including but not limited to the heart, liver, kidney, colon, intestine, esophagus, stomach, nervous tissue (brain, spinal cord), lung, vascular tissue (arteries, veins, capillaries), lymphoid tissue (lymphatic glands, pharyngeal tonsils, tonsils, bone marrow, and blood), spleen), as well as fibroblasts and fibroblast-like cell lines (e.g., CHO cells, TRG-2 cells, IMR-33 cells, Don cells, GHK-21 cells, citrullinocytes, Dempsey cells, Detroit 551 cells, Detroit 510 cells, Detroit 525 cells, Detroit 529 cells, Detroit 532 cells, Detroit 539 cells, Detroit 548 cells, Detroit 573 cells, HEL299 cells, IMR-90 cells, MRC-5 cells, WI-38 cells, WI-26 cells, MiCl1 cells, CHO cells, CV-1 cells, COS-1 cells, COS-3 cells, COS-7 cells, Vero cells, DBS-FrhL-2 cells, BALB / 3T3 cells, F9 cells, SV-T2 Cells, M-MSV-BALB / 3T3 cells, K-BALB cells, BLO-11 cells, NOR-10 cells, C3H / IOTI / 2 cells, HSDM1C3 cells, KLN205 cells, McCoy cells, mouse L cells, strain 2071 (mouse L) cells, LM strain (mouse L) cells, L-MTK - (Mouse L) cells, NCTC clones 2472 and 2555, SCC-PSA1 cells, Swiss / 3T3 cells, Hoedica cells, SIRC cells, C II This includes cells, as well as Jensen cells, Sp2 / 0, NSO, NS1 cells, or their derivatives.

[0108] Cells cultured according to the methods disclosed herein may be normal cells, diseased cells, transformed cells, mutant cells, somatic cells, genetically engineered cells, germ cells, stem cells, progenitor cells, or embryonic cells, any of which may be established or transformed cell lines, or may be obtained from natural sources. Cells may be used for experimental purposes or for the production of useful components. In some cases, the cultured cells themselves are the product and are useful for cells in cell therapy. Cells may also be cultured for protein production, including antibody production, small RNA production (such as miRNA or siRNA), virus or VLP (virus-like particle) production, DNA or viral vector preparation and isolation, nucleic acid production, vitamin production, desired metabolites, biofuel synthesis, and the like. In one embodiment, the culture medium described herein is used to culture Chinese hamster ovary (CHO) cells. CHO cells are classified as both epithelial cells and fibroblasts derived from Chinese hamster ovaries. The cell line (CHO-K1) (Kao, F.-T. And Puck, TT, Proc. Natl. Acad. Sci. USA 60: 1275-1281 (1968)), which originated from Chinese hamster ovaries, has been cultured for many years. Most biopharmaceutical companies currently produce proteins in CHO cells due to its many advantages, including the accurate post-translational modifications such as human-like glycosylation patterns and a low risk of human viral infection.

[0109] 7. Cell Culture Cells supported by the culture media described herein can be cultured according to experimental conditions determined by the researcher. The following examples demonstrate at least one functional set of culture conditions useful for culturing certain mammalian cells. However, it should be understood that the optimal plating and culture conditions for a given animal cell type can be determined by those skilled in the art using only routine experiments. With regard to routine monolayer culture conditions using the cell culture media described herein, cells can be plated onto the surface of the culture vessel without adhesion molecules. Alternatively, the vessel can be pre-coated with natural, recombinant, or synthetic adhesion molecules or peptide fragments (e.g., collagen, fibronectin, vitronectin, laminin, etc., or their natural or synthetic fragments), which are commercially available, for example, from Life Technologies, Corp. (Carlsbad, CA), R&D Systems, Inc. (Rochester, Minnesota), Genzyme (Cambridge, Massachusetts), and Sigma (St. Louis, Missouri). Cells can also be seeded in or on natural or synthetic three-dimensional support substrates, such as pre-formed collagen gels or synthetic biomolecular materials. For suspension culture, cells are typically suspended in the culture media described herein and introduced into a culture vessel that facilitates cell culture in suspension, such as a spinner flask, perfusion apparatus, or bioreactor. See Cell Culture Technology for Pharmaceutical and Cell-Based Therapies, 156-174 (Sadettin Ozturk and Wei-Shou Hu eds., Taylor and Francis Group 2006). In some cases, some level of agitation of the medium and suspended cells is necessary. Agitation can be kept to a minimum to avoid denaturation of medium components and shearing of cells during culture.

[0110] The cell seeding density for each experimental condition can be optimized with respect to the specific culture conditions used. For routine monolayer culture in plastic culture vessels, 1–5 × 10⁶ is appropriate. 5 cells / cm 2 A starting seeding density of 10 is considered preferable, and for suspension culture, a higher seeding density (e.g., 5-20 × 10) is preferable. 5 Individual cells / ml may be used.

[0111] Mammalian cells are typically cultured in a cell incubator preferably at about 37°C, although this may be in the range of 30°C to 39°C. Non-mammalian cells may have other preferred temperatures for culture. Culture of mammalian and non-mammalian cells may be carried out stepwise, for example, at one temperature for optimal cell proliferation and at another temperature for optimal protein / peptide / fragment or virus production. The incubator atmosphere may be humidified and may contain about 3–10% carbon dioxide from the air, more preferably about 5–10%, and most preferably about 3–8%, although the culture of certain cell lines may require as much as 20% carbon dioxide from the air to obtain optimal results. The culture medium pH may be within a preferred range depending on the cell type, for example, about 6–8.5, preferably about 7.1–7.6, or preferably about 7.1–7.4, or more preferably about 7.1–7.3, or preferably about 6–6.3 for insect cells.

[0112] Cells in closed culture or batch culture are approximately 1.5–2.0 × 10⁴ 6 Once a density of cells / ml is reached, a complete medium change (i.e., replacing consumed medium with fresh medium) should be performed. Cells in perfusion culture (e.g., in a bioreactor or fermenter) receive fresh medium on a continuous recirculation basis.

[0113] 8. Virus production In addition to cell culture in suspension or monolayer culture, this medium can also be used in methods for producing viruses from mammalian cells. Such methods include (a) a step of contacting cells (e.g., mammalian cells) with a virus under conditions suitable for promoting viral infection of cells; and (b) a step of culturing the cells in a cell culture medium containing a small peptide or dipeptide as described herein under conditions suitable for promoting viral production by the cells. Cells can be contacted with the virus before, during, or after culturing in the culture medium. Optimal methods for infecting mammalian cells with viruses are well known in the art and are familiar to those skilled in the art. Virus-infected mammalian cells cultured in the culture medium described herein may be expected to exhibit higher viral titers (e.g., 2, 3, 5, 10, 20, 25, 50, 100, 250, 500, or 1000 times higher titers) than cells cultured in cell culture media other than those described herein.

[0114] These methods can be used to produce a variety of mammalian viruses or viruses, virus-like particles, and viral vectors adapted to infect mammalian cells, including but not limited to adenoviruses and their derivatives, adeno-associated viruses and their derivatives, retroviruses and their derivatives, lentiviruses and their derivatives, insect viruses such as baculoviruses and their derivatives, and Sendai viruses and their derivatives. After culturing infected cells in the culture media described herein, the used media containing viruses, viral vectors, viral particles, or their components (proteins and / or nucleic acids (DNA and / or RNA)), which may be recombinant viruses, can be used for a variety of purposes, including vaccine production, production of inhibitory RNA molecules such as miRNA and siRNA, production of viral vectors for use in cell transfection or gene therapy, infection of animals or cell cultures, and research on viral proteins and / or nucleic acids. Alternatively, viruses, viral vectors, viral particles, or their components may optionally be isolated from the culture media used by techniques for protein isolation and / or nucleic acid isolation that are well known to those skilled in the art.

[0115] In one embodiment, cells produce VLPs. A "VLP" or "virus-like particle" is a medium for delivering one or more compounds into a cell, including biomolecules such as lipids, carbohydrates, proteins, and nucleic acids. Other compounds that can be delivered using VLPs include dyes (e.g., fluorescent dyes), labels (e.g., fluorescent or radioactive labels), and drugs (e.g., antibiotics or antivirals). VLPs generally contain at least one viral protein. Typically, the viral protein surrounds the compound. However, in certain cases, the compound to be delivered can be associated with the VLP by means other than encapsulation within the VLP. For example, the compound can be bound to a viral protein (e.g., covalently or noncovalently) or incorporated into the outer shell, if present. In one aspect, VLPs can be associated with various types of nucleic acids (e.g., heterogeneous nucleic acids), such as DNA, RNA, both RNA and DNA, or RNA / DNA hybrids, or derivatives known in the art. Examples of VLPs include viral particle products produced using VIRAPOWER® adenovirus and lentiviral vector kits (see, for example, Invitrogen Corporation, cat. no. K4930-00, K4940-00, K4950-00, K4955-00, K4960-00, K4965-00, K4967-00, and K4985-00).

[0116] Viruses that may be used to prepare VLPs include, for example, phages (e.g., T even-numbered phages (e.g., T4 phage), T odd-numbered phages (e.g., T7 phage), bacteriophages φ29, λ phage, etc.), baculoviruses, adenoviruses, adeno-associated viruses, lentiviruses (e.g., Moloney mouse leukemia virus, HIV1, HTLV-III, etc.), Sendai virus, poxvirus, and alphaviruses (e.g., Semryki forest virus, Sindbis virus, etc.). Further examples of viruses that may be used to prepare VLPs, and methods for preparing VLPs, are described elsewhere in this specification.

[0117] 9. Recombinant protein production This culture medium can also be used in a method for producing recombinant proteins from the above-mentioned cells, preferably mammalian cells, and especially mammalian cells grown in suspension. Because this culture medium provides rapid, high-density suspension culture of mammalian cells, this method facilitates improved recombinant protein production. Protein means any of the following: full-length protein, protein fragment, peptide, cleaved protein product, cross-linked protein product, tagged peptide, or protein. Protein also means all types of native and modified proteins, including recombinant proteins, mutant proteins, engineered proteins, chimeric proteins, glycoproteins, lipoproteins, active proteins, and processed proteins. Proteins may be expressed naturally by cells or cell lines, or cell lines may be engineered to express them using standard genetic engineering methods known in the art, including but not limited to transfection, transduction, and electroporation. The resulting protein or peptide can be purified or isolated to a desired level of purity. Proteins, peptides, or fragments thereof that can be produced or expressed using the culture medium and / or feed composition of the present invention include, but are not limited to, extracellular proteins such as laminin, fibronectin, and integrins; enzymes such as caspases, proteases, subtilisin, kinases, RNAse, and DNAse; peptide hormones such as insulin and PTHrP; intracellular proteins including membrane proteins, receptors, nucleoproteins, and endoplasmic reticulum proteins; antibodies; antibody fragments such as antibody heavy or light chains; antigen-binding sites or motifs; and chimeric antibodies. Chimeric antibodies may be species / species chimeric or class / class chimeric. The expressed proteins or polypeptides that can be produced using the composition and culture medium / feed of the present invention may be human or mammalian protein sequences expressed in non-animal cell lines such as plant cells to produce exogenous activator-free animal-derived proteins for downstream therapeutic applications.

[0118] A method for producing polypeptides according to the present invention comprises the step of culturing genetically engineered cells (e.g., mammalian cells) to produce polypeptides in a small peptide or dipeptide-containing cell culture medium described herein, under conditions suitable for polypeptide expression by cells. Optimal methods for genetically engineering mammalian cells to express polypeptides of interest are well known in the art and therefore well known to those skilled in the art. See, for example, Cell Culture Technology for Pharmaceutical and Cell-Based Therapies, 15-40 (Sadettin Ozturk and Wei-Shou Hu eds., Taylor and Francis Group 2006). Cells can be genetically engineered before culturing in the medium of the present invention, or can be transfected with one or more exogenous nucleic acid molecules after being cultured in the medium. Genetically engineered cells can be cultured in the culture medium as a monolayer culture or more preferably as a suspension culture according to the methods described above. After cell culture, the polypeptide of interest can optionally be purified from the cells and / or used culture medium according to protein isolation techniques well known to those skilled in the art.

[0119] 10. Detection of small peptides in culture medium or concentrated feed The small peptides and dipeptides described herein can be detected using any technique known in the art for detecting amino acids and / or small peptides, including but not limited to acid hydrolysis, liquid chromatography, capillary electrophoresis (Brown et al., J. Chrom. (1994) A, 661: 279-285), HPLC (van Wandelen et al., J. Chrom. (1997) A, 763: 11-22), or mass spectrometry. Acid hydrolysis of peptides for chromatographic analysis of amino acid compositions and concentrates is well known in the art. By comparing the chromatographic profiles of amino acid peaks before and after acid hydrolysis, the composition and concentration of amino acids in the culture medium, feed, or supplement, and by extension, the small peptides, can be indicated. For example, if tyrosine is present in a small peptide in the culture medium, its concentration will increase in the acid hydrolysate of the culture medium sample (e.g., tyrosine peak height and / or peak region) compared to the peak height / region of the same culture medium sample before acid hydrolysis.

[0120] As an example, alanyltyrosine and alanylcysteine ​​dipeptides described herein were detected using the methodology described in van Wandelen et al., J. Chrom. (1997) A, 763: 11-22, which includes HPLC (High Performance Liquid Chromatography) separation of a 6-aminoquinolyl-N-hydroxysuccinimidylcarbamate (AQC) derivatized amino acid mixture on an AccQ-Tag Ultra® (Waters Corp., Milford, MA) column (2.1 × 100 mm, 1.7 μm). The detector parameters were set as follows: wavelength mode: single wavelength; wavelength: 260 nm; sampling rate: 20 (points / second); time constant: 0.4000 (seconds). Using these parameters in an exemplary run, the alanyltyrosine dipeptide sample had a peak elution time of approximately 6.172 minutes immediately after the internal standard (AABA) (see Figure 2), and in another exemplary run, the alanylcysteine ​​dipeptide sample had a peak elution rate of 6.631 minutes between lysine and tyrosine (see Figure 3).

[0121] Therefore, this AccQ-Tag® Ultra (Waters Corp., Milford, MA) pre-column derivatization method using ultra-high-performance liquid chromatography (UPLC) provides a method for testing whether any sample medium or feed supplement contains small peptides containing cysteine ​​or tyrosine, such as alanyltyrosine or alanylcysteine, which are dipeptides. The same method can also be used to detect other small peptides, including other dipeptides of interest.

[0122] In addition, the dipeptides described herein can also be quantified by liquid chromatography and mass spectrometry (LC / MS). Separation was performed using a reversed-phase liquid chtomography column (Acquity UPLC® HSS T3 1.8-μm, inner diameter 2.1-mm × 150-mm at 40°C) connected to a quadrupole time-flight mass spectrometer (Waters® SYNAPT® HDMS® system, Milford, MA). A total fluorine-substituted carboxylic acid (e.g., perfluoropentanoic acid) was used as an ion-pairing agent for optimal separation of polar compounds (Jun Qu, Yiming Wang, Guan Luo, Zhuping Wu, and Chengdui Yang, Anal. Chem., 2002, 74, 2034-2040; the entire work is incorporated herein by reference). Mobile phase A consisted of 0.1% formic acid and 0.05% perfluoropentanoic acid in water, and mobile phase B consisted of 0.1% formic acid and 0.05% perfluoropentanoic acid in 80% acetonitrile. All reagents were LC / MS grade. The ultrahigh performance liquid chromatography (UPLC) gradient was linear over 1–45% of mobile phase B at a flow rate of 0.4 mL / min for 15 minutes. Mass spectra were collected every 0.5 seconds in the range of 70–1000 Da in continuum, positive electrospray (+ES), and V mode. Protonated L-alanyl-L-cysteine ​​dimers and protonated L-alanyl-L-tyrosine ions were extracted from the total ion chromatogram as 383.105 (±0.03) Da and 253.118 (±0.03) Da, respectively. The retention times for the protonated L-alanyl-L-cysteine ​​dimer and the protonated L-alanyl-L-tyrosine ion were 9.60 (±0.03) minutes and 8.75 (±0.03) minutes, respectively (Figures 4-7).

[0123] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. In case of any conflict, this specification shall prevail, including the definitions. It will be readily apparent to those skilled in the art that other appropriate modifications and adaptations to the methods and applications described herein are evident and can be made without departing from the scope of the invention or any embodiment thereof. In addition, the materials, methods and examples are illustrative and not intended to be limiting. All publications, patent applications, patents, and other references cited herein are incorporated by reference in their entirety. [Examples]

[0124] Example 1: Preparation of dipeptide-containing culture medium Exemplary cell culture media containing the dipeptides alanyltyrosine (AlaTyr) and alanylcysteine ​​(forming the disulfide dimer, [AlaCys]2) were prepared. Specifically, alanyltyrosine and alanylcysteine ​​were added as dry powders to an aqueous cell culture basal medium containing a mixture of glucose and concentrated amino acids, and mixed until dissolved. Alanyltyrosine was added to the cell culture medium at a concentration of approximately 4.0 g / L. Alanylcysteine ​​was added to the cell culture medium at a concentration of approximately 3.0 g / L. The solubility of the dipeptides observed in water was approximately 15.4 g / L for AlaTyr, >100 g / L for AlaCys, and approximately 8.7 g / L for the [AlaCys]2 dimer. For comparison, the solubility of L-tyrosine in water is approximately 0.38 g / L at 20°C. Free L-cysteine ​​is readily oxidized to cystine. While L-cysteine ​​hydrochloride is fairly stable in acidic aqueous solutions, in neutral or alkaline aqueous solutions it is also converted to L-cystine by aerobic oxidation. The solubility of L-cystine in water was approximately 0.11 g / L at 25°C.

[0125] Example 2: Flow-type test Cells were grown in the basal cell culture medium CD FortiCHO® (Invitrogen Cat. No. A-1148301 and Custom Stock A-11437DK; Life Technologies Corp., Carlsbad, CA) and glucose (CD FortiCHO® + glucose; Figure 1: curved black squares). TIFF0007869825000001.tif8128 and forward slash bar TIFF0007869825000002.tif7128), or a concentrated amino acid mixture containing low levels of cysteine ​​or tyrosine, a mixture of glucose + dipeptide (DP) culture medium or feed as described in Example 1 (CD FortiCHO(trademark) + aa + DP feed FP2 and FP3; see Figure 1: (FP2) curved black circle TIFF0007869825000003.tif7128 and white bar TIFF0007869825000004.tif6128;(FP3) Black diamond-shaped curve TIFF0007869825000005.tif6128 and backward slash bar An integrated fed-batch assay was performed using IgG-producing CHO cells supplemented with one of the following (TIFF0007869825000006.tif6128). In Figure 1, "aa" refers to a mixture of glucose and a concentrated amino acid mixture containing low levels of cysteine ​​and tyrosine.

[0126] Other cell culture basal media tested were CD OptiCHO® and CD CHO, both from Life Technologies Corp., Carlsbad, CA (data not shown). The preferred medium used was CD FortiCHO®. Exemplary mixtures of concentrated amino acids include, for example, CHO CD EfficientFeed® A (Invitrogen Cat. No. A1023401), CHO CD EfficientFeed® B (Invitrogen Cat. No. A1024001), CHO CD Efficient Feed® Kit (Invitrogen Cat. No. A1024101), and CD Efficient Feed® C AGT (Custom Stock A-11525SA, Life Technologies Corp., Carlsbad, CA). The cysteine ​​and tyrosine-containing small peptides in the cell culture medium or supplement feed of the present invention are designed to be used with any exemplary cell culture basal medium and any exemplary stoichiometrically equilibrated concentrated amino acid mixture (aa) which optionally contains glucose, vitamins, trace elements, etc., suitable for the growth of a desired cell type, as can be determined by those skilled in the art.

[0127] Cells were grown in a 500 mL DasGip bioreactor with a pH control setpoint of 7.0 ± 0.05 and a pO2 control setpoint of 30%. CHO cells supplemented with glucose were automatically supplied with 3 g / L of glucose whenever the glucose level reached 2 g / L. CHO cells supplemented with dipeptides (DP) were subjected to two supply schedules. In the first supply schedule (see Figure 1, red curve and bars: FP2), CHO cells were supplied with 2% dipeptide-containing medium daily from day 4 to day 13. In the second supply schedule (see Figure 1, green curve and bars: FP3), CHO cells were supplied with 2% dipeptide-containing medium daily from day 5 to day 14.

[0128] Cells grown in CD FortiCHO® medium and supplemented with glucose exhibited IgG productivity (1600 mg / L) comparable to or better than that of a fed-add process based on CD OptiCHO® (Life Technologies Corp., Carlsbad, CA) or CD CHO (Life Technologies Corp., Carlsbad, CA) (data not shown). CHO cells grown in CD FortiCHO® and supplemented with concentrated amino acid and dipeptide-containing medium from Example 1 showed improved viability at 12 days and doubled in productivity, reaching approximately 3200 mg / L by day 15 compared to approximately 1600 mg / L for CD FortiCHO® + glucose at day 15 (Figure 1). Of the two supply schedules for the CD FortiCHO® + aa + dipeptide (DP) supply, both worked well and showed comparable levels of IgG production. The FP3 profile exhibited better-maintained glucose levels and more consistent levels of IgG production compared to the FP2 profile, while the FP2 profile promoted higher peak cell density. Notably, the dipeptide-containing cell culture medium resulted in enhanced cell density and productivity levels with significantly reduced volume supply rates, offering yet another advantage of using the dipeptide-containing cell culture medium described herein.

[0129] Liquid cell culture media or feed solutions containing the dipeptides alanyltyrosine and alanylcysteine ​​can be stored at 2–8°C and remain precipitate-free for over 10 months, thus demonstrating significantly longer liquid stability than predicted for such concentrated solutions.

[0130] All patents, patent applications, and published references cited herein are incorporated herein by reference in their entirety. While the present invention has been described in detail with reference to its preferred embodiments, it will be understood by those skilled in the art that various modifications in form and detail can be made in the present invention without departing from the scope of the invention as encompassed by the appended claims.

Claims

1. A cell culture supplement or cell culture medium for use in the culture of mammalian cells, comprising an alanine-cysteine ​​dipeptide and in the form of a dry powder, The cell culture supplement or cell culture medium wherein the alanine-cysteine ​​dipeptide is N,N'-di-L-alanyl-L-cystine containing two cysteine ​​residues linked by a disulfide bond.

2. The cell culture supplement or cell culture medium according to claim 1, further comprising an alanine-tyrosine dipeptide.

3. The dried powder is an aggregated powder, a dry powder medium (DPM), or a high-performance powder medium (APM), and the cell culture medium is ethanolamine, D-glucose, HEPES, insulin, cytokines, heparin, dextran sulfate, linoleic acid, lipoic acid, phenol red, PLURONIC® F68, putrescine, sodium pyruvate, transferrin, L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-glutamic acid, L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, biotin, choline chloride, D-Ca ++ - Pantothenic acid, folic acid, i-inositol, niacinamide, pyridoxine, riboflavin, thiamine, vitamin B 12 , one or more calcium salts, Fe(NO 3 ) 3 KCl, one or more magnesium salts, one or more manganese salts, NaCl, NaHCO3 3 kaNa 2 HP0 4 The cell culture supplement or cell culture medium according to claim 1 or 2, further comprising one or more components comprising one or more selenium salts, one or more vanadium salts, and one or more zinc salts.

4. The cell culture supplement or cell culture medium according to any one of claims 1 to 3, wherein the dried powder requires only the addition of a solvent such as water to provide a reconstituted supplement or culture medium.

5. The cell culture supplement or cell culture medium according to claim 4, wherein the dry powder is configured such that dissolution occurs immediately after water is added, and the resulting reconstituted supplement or medium can be filtered and added directly to cells without pH adjustment.

6. The cell culture medium according to any one of claims 1 to 5, wherein the cell culture medium further comprises carbohydrates.

7. The cell culture medium according to claim 6, wherein the carbohydrate is a hexose.

8. The cell culture medium according to claim 7, wherein the hexose is glucose.

9. A cell culture medium according to any one of claims 6 to 8, further comprising essential amino acids and non-essential amino acids.

10. A method for forming a reconstituted cell culture supplement or cell culture medium by adding water to a dried powder of a cell culture supplement or cell culture medium according to any one of claims 1 to 9.

11. The method according to claim 10, wherein the dry powder dissolves immediately after water is added, and the resulting liquid can be filtered and directly added to cells without pH adjustment.

12. A method for forming a reconstituted cell culture medium according to claim 10 or 11, wherein, if alanine-tyrosine dipeptide is present, alanine-cysteine ​​dipeptide is present at a concentration of 1 g / L to 16 g / L each.

13. A method for forming a reconstituted cell culture medium according to any one of claims 10 to 12, wherein, if alanine-tyrosine dipeptide is present, alanine-cysteine ​​dipeptide is present at a concentration of 2.5 g / L to 8.5 g / L, respectively.

14. The use of a cell culture medium according to any one of claims 1 to 9 for culturing mammalian cells, wherein the mammalian cells are not embryonic cells.

15. The use according to claim 14, comprising the step of culturing mammalian cells in a suspension to produce recombinant protein, wherein the use promotes enhanced production of recombinant protein.

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