Method for stabilizing bicarbonate-buffered culture medium

Abstract

One purpose of the present disclosure is to provide a method for stabilizing amino acids in a cell culture solution containing serum albumin. The present disclosure provides a method for stabilizing a bicarbonate-buffered culture medium, the method including: placing a bicarbonate-buffered culture medium in an inner space of a gas permeation-resistant container; and adjusting the pH value of the bicarbonate-buffered culture medium to a value less than 7.2. The bicarbonate-buffered culture medium contains serum albumin and also contains a glutamine-containing dipeptide, asparagine, or cystine.

Description

Method for stabilizing bicarbonate buffer medium

[0001] This disclosure relates to the field of cell culture. Specifically, this disclosure relates to a method for stabilizing amino acids and dipeptides in bicarbonate buffered medium. This disclosure also relates to a method for manufacturing bicarbonate buffered medium packages. This disclosure also relates to bicarbonate buffered medium packages.

[0002] Cell culture media provide cells with the nutrients necessary to maintain and / or proliferate them in vitro. The nutrient composition, pH, and osmotic pressure of cell culture media vary depending on the cell type, cell density, and the culture system used.

[0003] Glutamine (Gln) is the primary energy source for cultured cells, and therefore cell culture media typically contain Gln. It is known that the amount of Gln required for optimal growth of mammalian cell cultures is 3 to 10 times greater than the amount of other amino acids. Gln is unstable in the culture medium and degrades over time to form pyroglutamic acid and ammonia, which can be cytotoxic.

[0004] To avoid the formation of cellularly harmful substances such as ammonia due to the degradation of granulocytes (Gln), dipeptides containing granulocytes, such as alanylglutamine (AlaGln) and glycylglutamine (GlyGln), are used (Non-Patent Literature 1). It is believed that granulocytes containing dipeptides in the culture medium are broken down into individual amino acids by enzymes produced by cells and then utilized by the cells. Although granulocytes containing dipeptides have high heat resistance, they are known to be unstable in the presence of serum, and ammonia is produced (Non-Patent Literature 2). Similarly, in the presence of serum albumin, ammonia is produced from the culture medium containing granulocytes containing dipeptides. This is thought to be due to the degradation of serum albumin or the degradation of amino acids intermingled with serum albumin (Non-Patent Literature 3).

[0005] Several culture media for mammalian cells, including those from mice, rats, hamsters, pigs, cattle, goats, buffalo, dogs, and monkeys, are recommended to have a pH of 7.2 to 8.0 during storage (Non-Patent Literature 4).

[0006] Roth et al. , In Vitro Cellular & Developmental Biology, 24(7):696-698, 1988Christie, A. , & Butler, M. , J. Biotechnol. , 37(3):277-290, 1994 Kleijkers, S, H, M, et al. , Hum. Reprod. , 31(6):1192-1199, 2016 Comparative Embryo Culture Methods and Protocols, Humana Press, Eds. , Jason R. Herrick: 18, 37, 55, 107, 128, 147, 182, 256, 342, 2019

[0007] The inventors have found that glutamine-containing dipeptides in bicarbonate buffer medium are not sufficiently stable in the presence of serum albumin. Furthermore, the inventors have found that asparagine and cystine in the bicarbonate buffer medium are also not sufficiently stable. The inventors have conducted extensive research on methods to stabilize bicarbonate buffer medium containing serum albumin and the aforementioned amino acids (amino acid-based compound). As a result, the inventors have found that the stability of the amino acids is improved by setting the pH of the bicarbonate buffer medium containing serum albumin to a more acidic range than the recommended storage range of pH 7.2 to 8.0, or to a more acidic range, thereby completing the present invention.

[0008] One aspect of the present disclosure provides a method for stabilizing predetermined amino acids in a bicarbonate buffer medium containing serum albumin. One aspect of the present disclosure provides a bicarbonate buffer medium package containing serum albumin and predetermined amino acids exhibiting improved storage stability. One aspect of the present disclosure provides a method for manufacturing the bicarbonate buffer medium package. More specifically, the present disclosure provides the following inventions: [Item 1] A method for stabilizing a bicarbonate buffer medium, comprising placing the bicarbonate buffer medium in the internal space of a gas-permeable container and adjusting the pH of the bicarbonate buffer medium to less than 7.2, wherein the bicarbonate buffer medium comprises serum albumin and a dipeptide containing glutamine, asparagine, or cystine. [Item 2] The method according to item 1, wherein adjusting the pH of the bicarbonate buffer medium comprises passing carbon dioxide through the bicarbonate buffer medium. [Item 3] The method according to item 1 or item 2, wherein adjusting the pH of the bicarbonate buffer medium comprises adjusting the pH to 6.0 or more and less than 7.2. [Item 4] The method according to any one of items 1 to 3, wherein the bicarbonate buffer medium comprises the dipeptide, and the dipeptide comprises at least one selected from the group consisting of alanylglutamine (hereinafter also referred to as "AlaGln"), glycylglutamine (hereinafter also referred to as "GlyGln"), leucylglutamine (hereinafter also referred to as "LeuGln"), valylglutamine (hereinafter also referred to as "ValGln"), and isoleucylglutamine (hereinafter also referred to as "IleGln").

[0009] [Clause 5] A method for producing a bicarbonate buffer medium package, comprising: placing the bicarbonate buffer medium in the internal space of a gas-permeable container; adjusting the pH of the bicarbonate buffer medium to less than 7.2; and sealing the gas-permeable container, wherein the bicarbonate buffer medium comprises serum albumin and a dipeptide containing glutamine, asparagine, or cystine. [Clause 6] The production method according to Claim 5, wherein adjusting the pH of the bicarbonate buffer medium comprises passing carbon dioxide through the bicarbonate buffer medium. [Clause 7] The production method according to Claim 5 or Claim 6, wherein adjusting the pH of the bicarbonate buffer medium comprises adjusting the pH to 6.0 or more and less than 7.2. [Clause 8] The method for producing a product according to any one of Clauses 5 to 7, wherein the bicarbonate buffer medium comprises the dipeptide, and the dipeptide comprises at least one selected from the group consisting of alanylglutamine (AlaGln), glycylglutamine (GlyGln), leucylglutamine (LeuGln), valylglutamine (ValGln), and isoleucylglutamine (IleGln).

[0010] [Clause 9] A bicarbonate buffer medium package comprising a gas-permeable container and a bicarbonate buffer medium in the internal space of the gas-permeable container, wherein the pH of the bicarbonate buffer medium is less than 7.2, and the bicarbonate buffer medium comprises serum albumin and a dipeptide containing glutamine, asparagine, or cystine. [Clause 10] The package according to Claim 9, wherein the pH of the bicarbonate buffer medium is 6.0 or greater and less than 7.2. [Clause 11] The package according to Claim 9 or Claim 10, wherein the bicarbonate buffer medium comprises the dipeptide, wherein the dipeptide comprises at least one selected from the group consisting of alanylglutamine (AlaGln), glycylglutamine (GlyGln), leucylglutamine (LeuGln), valylglutamine (ValGln), and isoleucylglutamine (IleGln).

[0011] Figure 1 is a series of line graphs comparing the percentage change in content (%) of five types of inorganic salts (top), three types of energy substrates (middle), and 21 types of amino acids (bottom) over a predetermined storage period, for cell culture media without human serum albumin (HSA) (Figure 1A) and cell culture media containing HSA (Figure 1B). Figure 2 is a line graph showing the percentage change in content (%) of amino acids over a predetermined storage period in cell culture media with 21 types of amino acids and HSA added (left: pH 8.0, right: pH 7.0). The dashed line indicates a judgment criterion with a content of ±30% as an indicator. If the percentage change in content is in the range of 70% to 130%, it is evaluated as stable. Figure 3 is a graph showing the shelf life of amino acids (alanine (Ala) and AlaGln) in the cell culture medium (left figure: pH 8.0, right figure: pH 7.0), calculated from the results in Figure 2 based on the stability testing guidelines of the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) in Japan, the United States, and the EU. The dashed line indicates the judgment criterion, with a content of ±30% as the indicator. The gray area indicates the one-sided 95% confidence interval of the regression line. Figure 4 is a series of line graphs showing the rate of change in the content of 21 types of amino acids in the cell culture medium at a predetermined pH (pH 7 to pH 6). The dashed line indicates the judgment criterion, with a content of ±30% as the indicator. If the rate of change in content is in the range of 70% to 130%, it is evaluated as stable. Figure 5A is a line graph showing the change in concentration of glutamine (Gln) over time in the cell culture medium at a predetermined pH (pH 7 to pH 6). Figure 5B is a line graph showing the change in ammonia concentration over time in a series of cell culture media at different pH levels. Figure 6 is a graph showing the change in ammonia concentration calculated from the results in Figure 5B based on the ICH stability test guidelines. The dashed line indicates the ammonia concentration that is considered to adversely affect human embryo development. The gray area indicates the one-sided 95% confidence interval of the regression line. Figure 7A is a line graph showing the percentage change in amino acid content over a predetermined storage period in cell culture media supplemented with 21 types of amino acids and HSA (left: 5 mg / mL HSA, right: 1 mg / mL HSA). The dashed line indicates the judgment criterion with a content of ±30% as the indicator.Figure 7B is a graph showing the shelf life of amino acids in the cell culture medium, calculated from the results of Figure 7A based on the ICH stability test guidelines. The dashed line indicates the judgment criterion with a content of ±30% as an indicator. The gray area indicates the one-sided 95% confidence interval of the regression line. Figure 8A is a line graph showing the percentage change in amino acid content over a predetermined storage period in cell culture medium containing 21 types of amino acids including AlaGln and HSA (left figure: 1000 μM AlaGln, center figure: 398 μM AlaGln) and cell culture medium containing 21 types of amino acids including GlyGln and HSA instead of AlaGln (right figure: 1000 μM GlyGln). The dashed line indicates the judgment criterion with a content of ±30% as an indicator. Figure 8B is a graph showing the shelf life of amino acids in the cell culture medium, calculated from the results of Figure 8A based on the ICH stability test guidelines. The dashed line indicates the judgment criteria based on a content of ±30%. The gray area indicates the one-sided 95% confidence interval of the regression line. Figure 9A is a line graph showing the glutamine (Gln) concentration (left) and ammonia concentration (right) over a predetermined storage period in cell culture medium supplemented with 21 types of amino acids (dipeptides are AlaGln or GlyGln) and HSA. Figure 9B is a graph showing the change in ammonia concentration calculated from the results of Figure 9A (right) based on the ICH stability test guidelines. The dashed line indicates the ammonia concentration that is considered to adversely affect human embryo development. The gray area indicates the one-sided 95% confidence interval of the regression line.

[0012] (Definition) The term "bicarbonate buffer medium" refers to a culture medium having components and composition containing bicarbonates for culturing cells isolated from their origin. Bicarbonate buffer medium is also called bicarbonate medium. Bicarbonate buffer medium can be used, for example, to culture mammalian somatic cells, fertilized eggs, or embryos. Examples of bicarbonate buffered media for mammalian somatic cell culture include TCM199, BME, CMRL1066, MEM, DMEM, McCoy's 5a, Waymouth's MB752 / 1, Trowell's T8, Ham's F-10, Leiboviz' L-15, NCTC135, Ham's F-12, RPMI1640, Kane&Foote, α-MEM, Williams' E, MCDB104, IMDM, DMEM / F-12, RDF, e-RDF, BGJb, MCDB131, or NCTC109. Examples of bicarbonate buffered media for mouse embryo culture include KSOM. ＡＡ Examples of bicarbonate buffered media for human embryo culture include Global, CSC, G-TL, 1-Step, HFF99, or HiGROW OVIT.

[0013] The components and composition of an example bicarbonate buffer medium are shown in the table below. All numerical values ​​in the table are in μM. The notation "○" in the table indicates that the component is present in the culture medium.

[0014] All values ​​in the table are in units of μM.

[0015] All values ​​in the table are in units of μM.

[0016] All values ​​in the table are in units of μM.

[0017] The term "bicarbonate-buffered basal medium" means a culture medium (basal medium) having components and composition containing bicarbonate, substantially free from the additives described later in this disclosure. In one embodiment, it is a medium containing or essentially the same components and composition as the bicarbonate-buffered medium in this disclosure, except that it substantially does not contain the additives in this disclosure. The additives in this disclosure are a combination of serum albumin and a predetermined set of amino acids. The additives in this disclosure essentially consist of, for example, a combination of serum albumin and a predetermined set of amino acids.

[0018] The “predetermined amino acids” in this disclosure are glutamine-containing dipeptides, asparagine, or cystine, or combinations thereof. The predetermined amino acids in this disclosure include, for example, asparagine. The predetermined amino acids in this disclosure include, for example, cystine. The predetermined amino acids in this disclosure include, for example, the aforementioned dipeptide. The predetermined amino acids in this disclosure include, for example, a combination of asparagine and cystine. The predetermined amino acids in this disclosure include, for example, a combination of asparagine and the aforementioned dipeptide. The predetermined amino acids in this disclosure include, for example, a combination of cystine and the aforementioned dipeptide. The predetermined amino acids in this disclosure include, for example, a combination of asparagine, cystine, and the aforementioned dipeptide. Preferably, the predetermined amino acids in this disclosure include a combination of asparagine, cystine, and the aforementioned dipeptide.

[0019] In one embodiment, the bicarbonate buffer medium comprises serum albumin and a glutamine-containing dipeptide. In the above example, the bicarbonate buffer medium preferably further comprises either or both asparagine and cystine. In one embodiment, the bicarbonate buffer medium comprises serum albumin and asparagine. In the above example, the bicarbonate buffer medium preferably further comprises either or both a glutamine-containing dipeptide and cystine. In one embodiment, the bicarbonate buffer medium comprises serum albumin and cystine. In the above example, the bicarbonate buffer medium preferably further comprises either or both a glutamine-containing dipeptide and asparagine.

[0020] Examples of amino acids include isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), threonine (Thr or T), tryptophan (Trp or W), valine (Val or V), histidine (His or H), tyrosine (Tyr or Y), cysteine ​​(Cys or C), aspartic acid (Asp or D), asparagine (Asn or N), serine (Ser or S), glutamic acid (Glu or E), glutamine (Gln or Q), proline (Pro or P), glycine (Gly or G), alanine (Ala or A), and arginine (Arg or R). Amino acids are commercially available, for example, or can be produced according to known methods. Amino acids may have either L-forms or D-forms, or mixtures thereof. The amino acids are, for example, L-forms or mixtures in which the proportion of L-forms is higher than that of D-forms. The amino acids are, for example, D-forms or mixtures in which the proportion of D-forms is higher than that of L-forms. Preferably, the amino acids are L-forms or mixtures in which the proportion of L-forms is higher than that of D-forms. More preferably, the amino acids are L-forms. When the bicarbonate buffer medium according to this disclosure contains D-form amino acids (including when they are part of the dipeptide according to this disclosure), the content of the D-form amino acids can be determined considering the content of the L-form amino acids and the utilization efficiency of the D-form amino acids by cells.

[0021] "Asparagine" refers to 2-amino-3-carbamoylpropionic acid. The bicarbonate buffer medium according to this disclosure contains, for example, 1 to 2000 μM of asparagine. The asparagine concentration is, for example, 1 to 1000 μM, 1 to 800 μM, 1 to 600 μM, 1 to 400 μM, 1 to 200 μM, 1 to 100 μM, 5 to 1000 μM, 5 to 800 μM, 5 to 600 μM, 5 to 400 μM, 5 to 200 μM, 5 to 100 μM, 10 to 1000 μM, 10 to 800 μM, 10 to 600 μM, 10 to 400 μM, 10 to 200 μM, or 10 to 100 μM.

[0022] "Cystine" refers to 3,3'-dithiobis(2-aminopropionic acid). Cystine is a cysteine ​​dimer in which two cysteine ​​molecules are linked via a disulfide bond. Cystine is, for example, commercially available or can be produced according to known methods. Cystine may, for example, have one cysteine ​​molecule as the L-form and the other as the D-form. Cystine may, for example, have both cysteine ​​molecules as either the L-form or the D-form. Cystine may, for example, have a higher proportion of the L-form cysteine. Cystine may, for example, have a higher proportion of the D-form cysteine. The bicarbonate buffer medium according to this disclosure contains, for example, 1 to 500 μM of cystine. The cystine concentration is, for example, 1–500 μM, 1–250 μM, 1–150 μM, 1–100 μM, 5–500 μM, 5–250 μM, 5–150 μM, 5–100 μM, 10–500 μM, 10–250 μM, 10–150 μM, 10–100 μM, 25–500 μM, 25–250 μM, 25–150 μM, or 25–100 μM.

[0023] A "glutamine-containing dipeptide" refers to a dipeptide capable of releasing glutamine under predetermined conditions. A glutamine-containing dipeptide is, for example, a molecule in which one molecule of glutamine and one molecule of an amino acid are linked by a peptide bond. A glutamine-containing dipeptide releases glutamine, for example, in a bicarbonate buffer medium containing serum albumin. A glutamine-containing dipeptide contains glutamine at either the N-terminus, the C-terminus, or both. Such glutamine-containing dipeptides are, for example, commercially available or can be produced according to known methods.

[0024] The glutamine-containing dipeptide is, for example, one or a combination thereof selected from the group consisting of alanylglutamine (e.g., L-Alanyl-L-glutamine), glycylglutamine (e.g., L-Glycyl-L-glutamine), leucylglutamine (e.g., L-leucyl-L-glutamine), valylglutamine (e.g., L-valyl-L-glutamine), and isoleucylglutamine (e.g., L-isoleucyl-L-glutamine).

[0025] The bicarbonate buffer medium according to this disclosure contains, for example, 50 to 7000 μM of the dipeptide. The dipeptide concentration can be, for example, 50 to 5000 μM, 50 to 2500 μM, 50 to 2000 μM, 50 to 1500 μM, 50 to 1000 μM, 100 to 5000 μM, 100 to 2500 μM, 100 to 2000 μM, 100 to 1500 μM, 100 to 1000 μM, 150 to 5000 μM, 150 to 2500 μM, 150 to 2000 μM, 150 to 1500 μM, or 150 to 1000 μM. The concentrations are μM, 200-5000 μM, 200-2500 μM, 200-2000 μM, 200-1500 μM, 200-1000 μM, 250-5000 μM, 250-2500 μM, 250-2000 μM, 250-1500 μM, 250-1000 μM, 300-5000 μM, 300-2500 μM, 300-2000 μM, 300-1500 μM, or 300-1000 μM. When the dipeptide is glutamylglutamine (GlnGln), the dipeptide concentration is, for example, half of the above concentration range.

[0026] The term "serum albumin" refers to a type of globular protein in the blood of vertebrates, which accounts for the majority (e.g., about 60%) of the total protein in the blood. Serum albumin can be commercially available, for example, or prepared from mammalian blood according to known methods. The serum albumin according to this disclosure is, for example, prepared from mammalian blood. In one embodiment, the serum albumin is prepared from mammalian blood. The serum albumin is, for example, human serum albumin (HSA) or bovine serum albumin (BSA). The bicarbonate buffer medium according to this disclosure contains, for example, serum albumin at a concentration of 1 mg / mL to 50 mg / mL. The bicarbonate buffer medium according to this disclosure is, for example, 1-50 mg / mL, 1-40 mg / mL, 1-30 mg / mL, 1-20 mg / mL, 1-10 mg / mL, 2-50 mg / mL, 2-40 mg / mL, 2-30 mg / mL, 2-20 mg / mL, 2-10 mg / mL, 3-50 mg / mL, 3-40 mg / mL, 3-30 mg / mL, 3-20 mg / mL, 3-10 mg / mL, 4-50 mg / mL, 4-40 mg / mL, 4-30 mg / mL, 4-20 mg / mL, 4-10 mg / mL, 5-50 mg / mL, 5-40 mg / mL, 5-30 mg / mL, 5-20 mg / mL, or 5-10 mg / mL.

[0027] The pH of the bicarbonate buffer medium is measured in accordance with the 17th edition of the Japanese Pharmacopoeia. In this disclosure, the pH of the bicarbonate buffer medium is the value measured at 25 degrees Celsius. The pH of the bicarbonate buffer medium according to this disclosure can be determined, for example, from the color exhibited by a predetermined pH indicator (e.g., phenol red) when the bicarbonate buffer medium is used.

[0028] The pH of the bicarbonate buffer medium relating to this disclosure is less than 7.2, for example, 6.0 or more and less than 7.2, 6.1 or more and less than 7.2, 6.2 or more and less than 7.2, 6.3 or more and less than 7.2, 6.4 or more and less than 7.2, 6.5 or more and less than 7.2, 6.6 or more and less than 7.2, 6.7 or more and less than 7.2, 6.8 or more and less than 7.2, 6.9 or more and less than 7.2, 7.0 or more and less than 7.2, 7.1 or more and less than 7.2, 6.0 or more and 7.1 or less, 6.1 or more and 7.1 or less, 6.2 or more and 7.1 or less, 6.3 or more and 7.1 or less, 6.4 or more and 7.1 or less, 6.5 or more and 7.1 6.6 to 7.1, 6.7 to 7.1, 6.8 to 7.1, 6.9 to 7.1, 7.0 to 7.1, 6.0 to 7.0, 6.1 to 7.0, 6.2 to 7.0, 6.3 to 7.0, 6.4 to 7.0 Lower, 6.5 to 7.0, 6.6 to 7.0, 6.7 to 7.0, 6.8 to 7.0, 6.9 to 7.0, 6.0 to 6.9, 6.1 to 6.9, 6.2 to 6.9, 6.3 to 6.9, 6.4 to 6.9, 6.5 to 6.9, 6.6 to 6.9, 6.7 to 6.9, 6.8 to 6.9, 6.0 to 6.8, 6.1 to 6.8, 6.2 to 6.8, 6.3 to 6.8, 6.4 to 6.8, 6.5 to 6.8, 6 .6 or more and 6.8 or less, 6.7 or more and 6.8 or less, 6.0 or more and 6.7 or less, 6.1 or more and 6.7 or less, 6.3 or more and 6.7 or less, 6.4 or more and 6.7 or less, 6.5 or more and 6.7 or less, 6.6 or more and 6.7 or less, 6.0 or more and 6.6 or less, 6.1 The values ​​are 6.6 or less, 6.2 or less, 6.3 or less, 6.4 or less, 6.5 or less, 6.0 or less, 6.1 or less, 6.2 or less, 6.3 or less, 6.4 or less, 6.5 or less, 6.0 or less, 6.1 or less, 6.2 or less, 6.3 or less, 6.4 or less, 6.0 or less, 6.1 or less, 6.2 or less, 6.3 or less, 6.0 or less, 6.3 or less, 6.1 or less, 6.1 or less, 6.2 or less, 6.1 or less, or 6.0 or less. The bicarbonate buffer medium is preferably 6.4 or less and 7.0 or less, more preferably 6.5 or less and 6.9 or less, and even more preferably 6.6 or less and 6.8 or less.

[0029] The term "container" means a container capable of holding liquid. The containers relating to this disclosure are gas-permeable. The container has members that define its internal space. The container includes, for example, an outer wall or film that defines its internal space, an opening that connects the internal space to an external space, and a stopper or lid that can seal the opening. The container essentially consists of or consists solely of, for example, a film that defines its internal space and an opening that connects the internal space to an external space.

[0030] Gas-permeable containers are formed from materials such as glass, aluminum foil, polyethylene terephthalate (PET), polyethylene terephthalate copolymer (PETG), biaxially oriented polypropylene (OPP), ethylene vinyl alcohol copolymer (EVOH), polyvinylidene chloride (PVDC), and nylon (NY); multilayer materials formed by laminating these materials (e.g., PET / NY / EVOH); vapor-deposited materials formed by depositing metals such as amorphous carbon, silicon oxide, or aluminum, or inorganic materials such as PVDC onto these materials (e.g., K-coat films such as K-coat nylon (KON), K-coat PET (KPET), or K-coat OP (KOP)); or multilayer materials laminated with aluminum foil; or combinations thereof. Gas-permeable containers are formed from, for example, K-coat films or PETG.

[0031] "Gas permeability resistance" refers to its oxygen permeation rate [cm²]. ３ / m ２ This refers to a value of 24h·atm of 20 or less. A gas-permeable container has an oxygen permeation rate of, for example, 15 or less, 10 or less, or 5 or less. A gas-permeable container has an oxygen permeation rate of preferably 5 or less, more preferably 3 or less, even more preferably 2 or less, and even more preferably 1 or less. A gas-permeable container has an oxygen permeation rate of, for example, 1 or less. The oxygen permeation rate is measured according to JIS K7126-2 (isobaric method).

[0032] The “bicarbonate buffered medium package” relating to this disclosure comprises a gas-permeable container and the bicarbonate buffered medium relating to this disclosure in its internal space. The package, for example, has a label attached to the gas-permeable container indicating the components and composition of the bicarbonate buffered medium placed inside. The label includes, for example, a statement that the medium is stable or storable for a period of at least six months at a predetermined storage temperature.

[0033] (Method for stabilizing a bicarbonate buffer medium) One aspect of the present disclosure provides a method for stabilizing a bicarbonate buffer medium. The stabilization method comprises placing a bicarbonate buffer medium containing serum albumin and amino acids in the internal space of a gas-permeable container, and adjusting the pH of the bicarbonate buffer medium to less than 7.2, wherein the amino acids include a glutamine-containing dipeptide, asparagine, or cystine.

[0034] "Placing" the bicarbonate buffer medium means, for example, making the bicarbonate buffer medium present in a specific space. For example, the bicarbonate buffer medium can be made present in the internal space of a gas-permeable container by pouring it into the internal space of the gas-permeable container using a dispenser such as a pipette.

[0035] The stabilization method may further include, for example, preparing a bicarbonate buffer medium containing serum albumin and the amino acids by combining serum albumin, predetermined amino acids according to the disclosure, and a bicarbonate buffered basal medium. In this preparation step, the bicarbonate buffered basal medium containing the serum albumin and the amino acids may be mixed, for example. The serum albumin and amino acids combined with the bicarbonate buffer medium may be in solid form or liquid form. One embodiment provides a method for stabilizing a bicarbonate buffer medium, comprising preparing a bicarbonate buffer medium by combining serum albumin, predetermined amino acids according to the disclosure, and a bicarbonate buffered basal medium, placing the bicarbonate buffer medium containing the serum albumin and the amino acids in the internal space of a gas-permeable container, and adjusting the pH of the bicarbonate buffer medium to less than 7.2, wherein the amino acids include a glutamine-containing dipeptide, asparagine, or cystine.

[0036] The pH of the bicarbonate buffer medium can be adjusted to less than 7.2, for example, by passing carbon dioxide gas through the bicarbonate buffer medium placed in the internal space of the gas-permeable container. The pH of the bicarbonate buffer medium can be adjusted, for example, by adding a pH indicator that changes color depending on the pH to the bicarbonate buffer medium and using the color of the pH indicator as an indicator. The pH of the bicarbonate buffer medium can be adjusted, for example, by setting conditions for a predetermined pH in advance and passing carbon dioxide gas through the bicarbonate buffer medium placed in the internal space of the gas-permeable container according to the set conditions. The carbon dioxide gas is, for example, a gas containing 80% or more, 90% or more, 95% or more, 97.5% or more, 98% or more, 99% or more, 99.5% or more, or 99.9% or more carbon dioxide (for example, a mixed gas of carbon dioxide and nitrogen).

[0037] (Method for manufacturing a bicarbonate buffered medium package) One aspect of the present disclosure provides a method for manufacturing a bicarbonate buffered medium package. The manufacturing method includes placing a bicarbonate buffered medium containing serum albumin and amino acids in the internal space of a gas permeable container, adjusting the pH of the bicarbonate buffered medium to less than 7.2, and sealing the gas permeable container, wherein the amino acids include a dipeptide containing glutamine, asparagine, or cystine.

[0038] The descriptions of the step of placing the bicarbonate buffered medium and the step of adjusting the pH of the bicarbonate buffered medium in the above-described stabilization method are respectively applicable to the corresponding steps of this manufacturing method. In this manufacturing method, the step of placing the bicarbonate buffered medium and the step of adjusting the pH of the bicarbonate buffered medium are preferably performed, for example, in a sterile space. The sterile space is, for example, the internal space of a facility suitable for the manufacture of sterile pharmaceuticals by aseptic operation methods.

[0039] "Sealing" a container means applying a technical measure to the container that enables prevention of unwanted liquid and gas movement into and out of the container under general temperature and pressure conditions. For the technical measure for sealing, for example, known measures can be used. The technical measure for sealing includes, for example, plugging, covering, or adhering. The container can be sealed, for example, by plugging or covering. The plug or cover includes, for example, an O-ring. The container can be sealed, for example, by adhesion (heat fusion, use of an adhesive).

[0040] The bicarbonate buffered medium package produced by the manufacturing method exhibits improved stability. The bicarbonate buffered medium of the package is sealed in a gas permeation resistant container, but it may be technically difficult to completely prevent carbon dioxide gas from leaking out of the container into the external space. Therefore, the pH of the bicarbonate buffered medium of the package may exceed 7.2 at a specific time after production. Even in this case, by setting the pH to less than 7.2 during the production of the package, the predetermined amino acids present in the medium are stabilized compared to the predetermined amino acids in the bicarbonate buffered medium with a pH of 7.2 or higher during production. As a result, the bicarbonate buffered medium package according to the present disclosure exhibits improved stability. Therefore, the bicarbonate buffered medium package according to the present disclosure has the advantage of exhibiting long-term (e.g., 6 months) storage stability. In one example, when the bicarbonate buffered medium according to the present disclosure is stored at 5°C ± 3°C for 6 months, the predetermined amino acids according to the present disclosure are within the range of ±30% of the amino acid concentration at the time of production or indicated on the label and are stable.

[0041] The bicarbonate buffered medium package according to the present disclosure, for example, at any point from the time of production to the time of use, the bicarbonate buffered medium constituting the package has the pH specified in the claims. The bicarbonate buffered medium package according to the present disclosure preferably has, at the time of production, the bicarbonate buffered medium constituting the package having the pH specified in the claims. The bicarbonate buffered medium package according to the present disclosure preferably has, at any point up to 6 months after production, the bicarbonate buffered medium constituting the package having the pH specified in the claims.

[0042] The bicarbonate buffered medium of the bicarbonate buffered medium package produced by the manufacturing method can, for example, be directly used for cell culture. The bicarbonate buffered medium can, for example, immediately before use, be brought to a desired pH range (e.g., pH 7.3 - 7.5) under known cell culture conditions (e.g., 6% CO ２ incubator) with gas communication possible.

[0043] (Bicarbonate-buffered medium package) One aspect of the present disclosure provides a bicarbonate-buffered medium package. The package comprises a gas-permeable container and a bicarbonate-buffered medium containing serum albumin and amino acids in the internal space of the gas-permeable container, wherein the pH of the bicarbonate-buffered medium is less than 7.2, and the amino acids include a glutamine-containing dipeptide, asparagine, or cystine.

[0044] The term “contains” means that the enumerated elements and / or steps exist, and other elements and / or steps may also exist. The term “consists of” means that the enumerated elements and / or steps exist, and other elements and / or steps are excluded. The term “essentially consists of” means that the enumerated elements and / or steps exist, and other elements and / or steps may exist to the extent that they do not adversely affect the technical features relating to this disclosure. The term “substantially does not contain” as used herein does not exclude “not contain at all.”

[0045] Terms and descriptions used in this disclosure to refer to specific aspects or embodiments shall apply to other aspects or embodiments as appropriate, unless otherwise specified. Specific examples are described below, which illustrate preferred embodiments of the present invention and shall not in any way limit the invention described in the appended claims.

[0046] [Example] HiGROW IVF (Fuso Pharmaceutical Co., Ltd.), a bicarbonate buffer medium, contains amino acids (21 types), energy substrates (glucose, pyruvate, lactic acid), and inorganic salts (NaCl, KCl, CaCl). ２ MgSO ４ 7KH ２ PO ４) is included. When stored at 5°C ± 3°C for one year, there was no change in the concentrations of these components (Figure 1A). Human serum albumin (HSA: Fuso Pharmaceutical Co., Ltd.) was added to the culture medium HiGROW IVF (Fuso Pharmaceutical Co., Ltd.) to a final concentration of 5 mg / mL. When the culture medium (containing HSA) was stored at a predetermined temperature for 200 days, there were time-dependent changes in the concentrations of alanine (Ala), asparagine (Asn), cystine (CysCys), and alanylglutamine (AlaGln) (Figure 1B). The amino acid concentrations were analyzed according to the method described in "1.3 Amino Acid Analysis" below.

[0047] [Example 1] 1. Stability test of amino acids in culture media at pH 7.0 and pH 8.0 1.1 Preparation of culture media The pH of the culture media HiGROW IVF (Fuso Pharmaceutical Co., Ltd.) was adjusted to pH 7.0 or pH 8.0 with carbon dioxide. Then, HSA (Fuso Pharmaceutical Co., Ltd.) was added to each culture media to a final concentration of 5 mg / mL. Each culture media was filtered and sterilized using a Millipak 60 filter unit 0.22 μm (Merck Millipore), and 20 mL each was filled into PETG 20 mL serum vials (Thermo Fisher).

[0048] The culture medium prepared in Example 1 is shown below.

[0049] 1.2 Storage and Extraction of Culture Medium The prepared culture medium was stored at 5°C ± 3°C and extracted at 0, 2, and 3 months after preparation for amino acid analysis.

[0050] 1.3 Amino Acid Analysis (1) Preparation of Sample Solution 10 μL of the culture medium, 10 μL of water, and 30 μL of acetonitrile were mixed and centrifuged. The supernatant was collected and diluted 10-fold with Milli-Q water to obtain the sample solution. (2) Preparation of Standard Solution 10 μL of amino acid solution with the composition shown in the table below, 5 μL of 0.5 mM glutamine (Gln) solution, 10 μL of water, and 25 μL of acetonitrile were mixed and centrifuged. The supernatant was collected and diluted 10-fold with Milli-Q water to obtain the standard solution.

[0051]

[0052] (3) Analysis One μL of the sample solution prepared in (1) and 0.4, 0.8, 1.2, 1.6, and 2.0 μL of the standard solutions prepared in (2) were taken and analyzed using a high-performance liquid chromatograph mass spectrometer LCMS-8060NX (Shimadzu) under the following conditions. A calibration curve was created from the peak area of ​​the standard solutions, and the concentration of each amino acid in the culture medium (μmol / L) was calculated. <Liquid Chromatography Conditions> Column: Discovery HS F5-3 3μm, 2.1×150mm (Merck) Column temperature: Constant temperature around 40°C Mobile phase A: 0.1% formic acid / water Mobile phase B: 0.1% formic acid / acetonitrile Flow rate: 0.35 mL / min (Mobile phases A and B are used individually or mixed, and changed in steps) <Mass Spectrometer Conditions> Nebulizer gas flow rate: 3.0 L / min Dry gas flow rate: 10 L / min Heating gas flow rate: 10 L / min Interface temperature: 270°C DL temperature: 250°C Heat block temperature: 400°C Ionization mode: ESI CID gas pressure: 270 kPa <Method Package> Cell Culture Profiling (Shimadzu)

[0053] 1.4 Results and Discussion The results of "1.3 Amino Acid Analysis" are shown in Figure 2. The dashed line in Figure 2 indicates the judgment criteria based on a content of ±30%. If the rate of change in content is in the range of 70% to 130%, it is evaluated as stable. The left side of Figure 2 shows that when the pH of the cell culture medium is 8.0, alanine (Ala) and alanylglutamine (AlaGln) are evaluated as unstable around a storage period of 90 days. The right side of Figure 2 shows that when the pH of the cell culture medium is 7.0, both Ala and AlaGln are stable up to around a storage period of 120 days. It was suggested that cystine (CysCys) is a relatively unstable amino acid among the amino acids other than alanine and alanylglutamine.

[0054] The shelf life was calculated based on the stability testing guidelines of the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) (Figure 3). The results suggested that alanine (Ala) was stable for 62 days in a culture medium containing serum albumin (pH 8.0), while alanylglutamine (AlaGln) was stable for 91 days. In contrast, the shelf life of Ala in the same culture medium, except for the pH being 7.0, was 105 days, and the shelf life of AlaGln was 139 days.

[0055] Example 1 suggests that the stability of these amino acids in a culture medium containing serum albumin can be improved by making the pH of the cell culture medium more acidic than the recommended pH range (pH 7.2 to 8.0) during storage, and even more acidic than the recommended range.

[0056] [Example 2] 2. Stability test of amino acids in culture media at pH 7.00, pH 6.75, pH 6.50, pH 6.25, and pH 6.00 2.1 Preparation of culture media Culture media at pH 7.0, pH 6.75, pH 6.5, pH 6.25, and pH 6.0 (containing HSA at a final concentration of 5 mg / ml) were prepared in the same manner as in 1.1.

[0057] The culture medium prepared in Example 2 is shown below.

[0058] 2.2 Storage and Extraction of Culture Medium The prepared culture medium was stored at 5°C ± 3°C and extracted at 0, 1, 2, 6, 9, and 13 months after preparation for amino acid analysis. Ammonia analysis was also performed on the culture medium at 0, 6, and 13 months after preparation.

[0059] 2.3 Amino Acid Analysis (1) Preparation of Sample Solution 5 μL of the culture medium and 15 μL of a 6.7% sulfosalicylic acid solution containing 0.17 mmol / L DL-norvaline (internal standard) were mixed, and the mixture was subjected to centrifugation. 3 μL of the supernatant was mixed with 12 μL of 125 mmol / L NaOH-containing AccQ Tag Ultra borate buffer (Waters) and 6 μL of AccQ Tag Ultra derivatization reagent (Waters). The mixture was allowed to stand for 1 minute and then heated at 55°C for 10 minutes to obtain the sample solution. (2) Preparation of standard solutions The amino acid solutions with the compositions shown in the table below were serially diluted 3.5 times with Milli-Q water to prepare five concentrations of amino acid solutions: ×1, ×1 / 3.5, ×1 / 11.55, ×1 / 40.425, and ×1 / 141.4875. 390 μL of each concentration of amino acid solution was taken and mixed with 10 μL of 20% HSA. 5 μL of this mixture was treated in the same manner as the culture medium to prepare the standard solution.

[0060]

[0061] (3) Analysis One μL each of the sample solution and standard solution prepared in (1) and (2) was taken and tested using an ultra-high-performance liquid chromatograph ACQUITY UPLC (Waters) under the following conditions. A calibration curve was created from the peak area of ​​the standard solution, and the concentration of each amino acid in the culture medium (μmol / L) was calculated. Test Conditions Detector: UV absorbance spectrophotometer (measurement wavelength: 260 nm) Column: AccQ Tag Ultra C18 1.7 μm 2.1 × 100 mm (Waters) Column temperature: Constant temperature around 55°C Mobile phase A: 10% AccQ Tag Ultra Eluent A (Waters) / water Mobile phase B: AccQ Tag Ultra Eluent B (Waters) Flow rate: 0.7 mL / min (Mobile phases A and B are used individually or mixed, and the flow rate is gradually changed)

[0062] 2.4 Ammonia Analysis A 200 μmol / L ammonium chloride solution was serially diluted 2-fold with Milli-Q water to prepare ammonia standard solutions ranging from 200 to 12.5 μmol / L. 10 μL each of culture medium and standard solution were added to a 96-well plate. 150 μL of Shikaliquid NH3 Reagent 1 (Kanto Chemical) was added and mixed, and the mixture was allowed to react at room temperature for 5 minutes. Then, 50 μL of Shikaliquid NH3 Reagent 2 (Kanto Chemical) was added to the reaction solution and mixed. The absorbance at 450 nm was measured at 30-second intervals for 5 minutes using a microplate reader SPECTRAmax PLUS 384 (molecular device). The obtained data was analyzed using SoftMAX Pro (molecular device) and the Vmax value was calculated. A calibration curve was created from the Vmax values ​​of the standard solutions, and the ammonia concentration (μmol / L) in the culture medium was calculated.

[0063] 2.5 Results and Discussion The results of "2.3 Amino Acid Analysis" are shown in Figure 4. The left panel of Figure 4 shows that when the pH of the cell culture medium is 7.00, alanine (Ala), cystine (CysCys), alanylglutamine (AlaGln), and asparagine (Asn) are evaluated as unstable around a storage period of 120 days. Figure 4 shows that stability increases as the pH of the cell culture medium becomes more acidic (~pH 6.00) from 7.00.

[0064] Similar to Example 1, the shelf life of these amino acids was calculated based on the ICH stability testing guidelines. The results are shown in the table below. N.D.: Not decided (Validity period: >500 days)

[0065] These results indicate that the stability of these amino acids in a culture medium containing serum albumin is improved by making the culture medium more acidic (around pH 6.0) than the recommended storage range (pH 7.2–8.0).

[0066] The results of "2.3 Amino Acid Analysis" and "2.4 Ammonia Analysis" are shown in Figure 5. Figure 5 shows that the concentrations of glutamine (Figure 5A) and ammonia (Figure 5B) in the culture medium increase as the storage period lengthens, and the degree of increase decreases as the pH of the culture medium becomes more acidic. The increase in Gln concentration is thought to be the result of the decomposition of the dipeptide (AlaGln) in the culture medium, and the increase in ammonia concentration is thought to be the result of the decomposition of Gln in the culture medium.

[0067] When culturing human embryos, it is known that if the ammonia concentration in the culture medium reaches around 119 μM, it can adversely affect development (Virant-Klun, I., et al., Fertil. Steril., 2006). Based on the ICH stability testing guidelines, the shelf life was calculated (Figure 6). The results suggested that if the culture medium pH was 7.00, the ammonia concentration could exceed 119 μM after approximately 434 days of storage, and if the pH was 6.75, it could exceed this after approximately 1529 days.

[0068] Example 2 suggests that the ammonia concentration generated in a culture medium containing serum albumin can be suppressed to a level that adversely affects cell culture, particularly human embryo development, by making the pH more acidic than the recommended range for storing the cell culture medium (pH 7.2 to 8.0), and even more acidic than that recommended range.

[0069] [Example 3] 3. Comparison of 5 mg / mL HSA and 1 mg / mL HSA 3.1 Preparation of Culture Medium The pH of the culture medium HiGROW OVIT (Fuso Pharmaceutical Co., Ltd.) was adjusted to pH 6.75 with carbon dioxide. HSA was added to it to a final concentration of 5 mg / mL or 1 mg / mL. Each culture medium was filtered and sterilized using a Millipak 60 filter unit 0.1 μm (Merck Millipore), and 30 mL was filled into 30 mL PET culture bottles (Corning).

[0070] The culture medium prepared in Example 3 is shown below.

[0071] 3.2 Storage and Retrieval of Culture Medium The prepared culture medium was stored at 5°C ± 3°C and retrieved at 0, 2, 3, 6, and 13 months after preparation for amino acid analysis and ammonia analysis. 3.3 Amino Acid Analysis Performed in the same manner as 2.3. 3.4 Ammonia Analysis Performed in the same manner as 2.4.

[0072] 3.5 Results and Discussion The results of "3.3 Amino Acid Analysis" are shown in Fig. 7A. On the left of Fig. 7A, when the pH of the cell culture medium is 6.75 and the HSA concentration is 5 mg / ml, it is evaluated that alanine (Ala), alanylglutamine (AlaGln), cystine (CysCys), and asparagine (Asn) are not stable from 200 days to 300 days of storage period. On the right of Fig. 7A, when the HSA concentration decreases to 1 mg / ml, these amino acids are shown to be stable even beyond 400 days.

[0073] Based on the ICH stability test guidelines, the expiration date was calculated (Fig. 7B). As a result, when the HSA concentration in the cell culture medium (pH 6.75) is 5 mg / ml, it is suggested that the expiration date of Ala is 241 days, AlaGln is 285 days, CysCys is 207 days, and Asn is 231 days (left of Fig. 7B). In contrast, when the HSA concentration decreases to 1 mg / ml, it is suggested that the expiration date of these amino acids exceeds 5,000 days (right of Fig. 7B).

[0074] Example 3 shows that the stability of amino acids depends on the HSA concentration added to the cell culture medium. This result suggests that the decomposition of amino acids and the generation of ammonia during the storage of the cell culture medium are the result of amino acid decomposition by the action of HSA rather than the degradation of amino acids alone.

[0075] [Example 4] 4. Comparison of Types and Concentrations of Dipeptides Containing Glutamine 4.1 Preparation of Culture Medium KSOM ＡＡThree types of culture solutions were prepared by replacing the 1000 μmol / L Glutanine (GlyGln), 1000 μmol / L AlaGln, or 398 μmol / L AlaGln contained in the culture medium (Biggers JD., Reprod. Biomed. Online, 2002) with either 1000 μmol / L Glycyl-glutamine (GlyGln), 1000 μmol / L AlaGln, or 398 μmol / L AlaGln. The pH was then adjusted to 6.75 with carbon dioxide. HSA was added to each culture solution to a final concentration of 5 mg / mL. Each culture solution was filtered and sterilized using a 0.22 μm Stericup (Merck Millipore) filter, and 20 mL of each solution was packed into 20 mL PETG serum vials. Note that KSOM was used in this study. ＡＡ The culture medium did not contain 1 mg / mL Bovine Serum Albumin, and instead of 100 IU / mL penicillin and 5 μg / mL streptomycin, 10 mg / L gentamicin sulfate was used.

[0076] The culture medium prepared in Example 4 is shown below.

[0077] 4.2 Storage and Extraction of Culture Medium The prepared culture medium was stored at 5°C ± 3°C and extracted at 0, 1, 2, 3, 4, 5, and 6 months after preparation for amino acid analysis and ammonia analysis.

[0078] 4.3 Amino Acid Analysis The analysis was carried out in the same manner as in 2.3, except for the following points. For the analysis of GlyGln, 390 μL of 1000 μmol / L GlyGln solution was mixed with 10 μL of 20% HSA, and 5 μL of this solution was treated in the same manner as the culture medium in "2.3 (1)" to prepare the GlyGln standard solution, which was then tested using an ultra-high-performance liquid chromatograph (ACQUITY UPLC). The GlyGln content in the culture medium was calculated from the area value of the GlyGln standard solution.

[0079] 4.4 Ammonia Analysis The same procedure as in 2.5 was followed.

[0080] 4.5 Results and Discussion The results of "4.3 Amino Acid Analysis" are shown in Figure 8. The shelf life was calculated based on the ICH Stability Test Guidelines (Figure 8B). Figure 8A shows that in a cell culture medium with a pH of 6.75 and an HSA concentration of 5 mg / ml, when AlaGln was added at 1000 μM or 398 μM (left figure: 1000 μM, center figure: 398 μM), alanine (Ala), aspartic acid (Asp), asparagine (Asn), and cystine (CysCys) were not stable up to 180 days of storage. For Ala, the degree of instability was greater at higher AlaGln concentrations. Specifically, when the AlaGln concentration was 1000 μM, the shelf life of Ala was 23 days, Asp was 132 days, CysCys was 140 days, and Asn was 146 days (Figure 8B left). In the same cell culture medium except for the AlaGln concentration of 398 μM, the effective period for Ala was 42 days, for Asp 135 days, for CysCys 137 days, and for Asn 145 days (Figure 8B, center). The stability of the amino acids other than Ala, Asp, Asn, and CysCys, was not affected by the change in the concentration of added AlaGln, while only Ala was affected, suggesting that Ala is a degradation product of the dipeptide AlaGln.

[0081] The dipeptide shows that in cell culture media using GlyGln (1000 μM) instead of AlaGln (1000 μM), the concentration of Gly, a degradation product of GlyGln, tends to increase (Figure 8A, right panel).

[0082] As a result, when the cell culture medium pH was 6.75 and the HSA concentration was 5 mg / ml, the shelf life of Asp (131 days), CysCys (126 days), and Asn (138 days) in the culture medium to which 1000 μM of GlyGln was added was almost the same as the shelf life of the cell culture medium to which AlaGln was added instead of GlyGln, except that the shelf life of Gly was 193 days (Figure 8B, right).

[0083] The results of "4.3 Amino Acid Analysis" and "4.4 Ammonia Analysis" are shown in Figure 9. Figure 9A shows that the concentrations of glutamine (Figure 9A left) and ammonia (Figure 9A right) in the culture medium increase as the storage period lengthens, with the greatest increase observed for AlaGln 1000 μM (shelf life: 669 days) and the smallest increase observed for GlyGln 1000 μM (shelf life: 953 days).

[0084] Example 4 demonstrates that the stability of amino acids and the concentration of ammonia generated in a cell culture medium containing serum albumin and amino acids depend on the type and concentration of dipeptides added to the cell culture medium.

[0085] [Example 5] 5. Comparison of HSA products 5.1 Preparation of culture medium Similar to 4.1, KSOM containing 1000 μmol / L of AlaGln ＡＡ A culture medium was prepared. Then, two tubes were prepared with the pH adjusted to 6.75 using carbon dioxide, and two types of HSA (Manufacturer A and Manufacturer B) were added to each to achieve a final concentration of 5 mg / mL. The prepared solutions were filtered and sterilized using a 0.22 μm Stericup, and 20 mL of each was filled into 20 mL PETG serum vials.

[0086] The culture medium prepared in Example 5 is shown below.

[0087] 5.2 Storage and retrieval of culture medium: Performed in the same manner as in 4.2. 5.3 Amino acid analysis: Performed in the same manner as in 2.4. 5.4 Ammonia analysis: Performed in the same manner as in 2.5.

[0088] 5.5 Results and Discussion Based on the results of "5.3 Amino Acid Analysis," the shelf life of alanine (Ala) in cell culture solution A, which contained HSA and 21 types of amino acids from manufacturer A, was 23 days, while the shelf life of Ala in cell culture solution B, which contained HSA and 21 types of amino acids from manufacturer B, was 45 days. The shelf life of the amino acids that were unstable during a 180-day storage period is shown in the table below.

[0089]

[0090] The results of "5.4 Ammonia Analysis" showed that in cell culture medium A, the ammonia concentration exceeded 119 μM, which is considered to adversely affect human embryo development, at 669 days, whereas in cell culture medium B, it did not exceed this level until 769 days. These results indicate that amino acid degradation and the concentration of ammonia generated differ depending on the HSA product, and that in HSA products with a greater degree of amino acid degradation, the ammonia concentration generated exceeds 119 μM more quickly.

[0091] Since the HSA products from manufacturer A and manufacturer B are from the same animal species, it can be reasonably assumed that the amino acid sequence of serum albumin itself is the same. In these HSA products, the methods for purifying albumin from serum are different, so it is thought that the types and amounts of trace components, including bio-derived degrading enzymes, differ. Example 5 indirectly shows that the degradation of amino acids and ammonia generation in cell culture media containing HSA and amino acids, as shown in Examples 1 to 4, are not due to the degradation of amino acids alone, but rather to degrading enzymes derived from HSA.

Claims

1. A method for stabilizing a bicarbonate buffer medium, comprising placing the bicarbonate buffer medium in the internal space of a gas-permeable container and adjusting the pH of the bicarbonate buffer medium to less than 7.2, wherein the bicarbonate buffer medium contains serum albumin and a dipeptide containing glutamine, asparagine, or cystine.

2. The method according to claim 1, wherein adjusting the pH of the bicarbonate buffer medium includes passing carbon dioxide through the bicarbonate buffer medium.

3. The method according to claim 1 or 2, wherein adjusting the pH of the bicarbonate buffer medium includes adjusting the pH to 6.0 or more and less than 7.

2.

4. The method according to any one of claims 1 to 3, wherein the bicarbonate buffer medium contains the dipeptide, and the dipeptide contains at least one selected from the group consisting of alanylglutamine (hereinafter also referred to as "AlaGln"), glycylglutamine (hereinafter also referred to as "GlyGln"), leucylglutamine (hereinafter also referred to as "LeuGln"), valylglutamine (hereinafter also referred to as "ValGln"), and isoleucylglutamine (hereinafter also referred to as "IleGln").

5. A method for manufacturing a bicarbonate buffer medium package, comprising: placing the bicarbonate buffer medium in the internal space of a gas-permeable container; adjusting the pH of the bicarbonate buffer medium to less than 7.2; and sealing the gas-permeable container, wherein the bicarbonate buffer medium contains serum albumin and a dipeptide containing glutamine, asparagine, or cystine.

6. The manufacturing method according to claim 5, wherein adjusting the pH of the bicarbonate buffer medium includes passing carbon dioxide through the bicarbonate buffer medium.

7. The manufacturing method according to claim 5 or 6, wherein adjusting the pH of the bicarbonate buffer medium includes adjusting the pH to 6.0 or more and less than 7.

2.

8. The method for producing according to any one of claims 5 to 7, wherein the bicarbonate buffer medium contains the dipeptide, and the dipeptide contains at least one selected from the group consisting of AlaGln, GlyGln, LeuGln, ValGln, and IleGln.

9. A package comprising a bicarbonate buffer medium, comprising a gas-permeable container and a bicarbonate buffer medium in the internal space of the gas-permeable container, wherein the pH of the bicarbonate buffer medium is less than 7.2, and the bicarbonate buffer medium comprises serum albumin and a dipeptide containing glutamine, asparagine, or cystine.

10. The package according to claim 9, wherein the pH of the bicarbonate buffer medium is 6.0 or higher and less than 7.

2.

11. The package according to claim 9 or 10, wherein the bicarbonate buffer medium comprises the dipeptide, and the dipeptide comprises at least one selected from the group consisting of AlaGln, GlyGln, LeuGln, ValGln, and IleGln.

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