Protein production promoter, culture medium containing said protein production promoter, and method for promoting protein production using said protein production promoter

Abstract

The present invention addresses the main problem of providing a synthetic culture medium that does not contain animal-derived components. In particular, the present invention addresses the problem of providing a culture medium containing, at a low concentration (at most about 2,000 μM), a peptide that contributes to promoting protein production even when animal-derived components are not contained.　Provided is a protein production promoter comprising, as an active ingredient, a peptide mixture at a total concentration of at most 2,000 μM, wherein the peptide mixture is composed of two peptides selected from the group consisting of Gly-Glu-Lys (GEK), Asp-Gly-Pro (DGP), Ala-Gly-Lys (AGK), Ala-Glu-Lys (AEK), Ala-Gly-Gly (AGG), Ala-Ser-Asn (ASN), Gly-Pro-Pro (GPP), Gly-Gly-Pro (GGP), and Glu-Gly-Lys (EGK), and is a combination with which protein production is further promoted by mixing the selected two peptides.

Description

Protein production promoter, medium containing said protein production promoter, and method for promoting protein production using said protein production promoter

[0001] The present invention relates to a protein production promoter containing a tripeptide mixture suitable for animal cell culture, a medium containing the protein production promoter, and a method for promoting protein production using the protein production promoter.

[0002] When culturing animal cells to obtain a native protein produced by the animal cells, or when culturing animal cells into which a gene encoding a desired protein has been introduced to produce a desired protein, in addition to nutritional components such as vitamins, amino acids, salts, and sugars, mammalian extracts such as fetal bovine serum and fish meat-related components are added for the purpose of growing the animal cells (Patent Documents 1 and 2).

[0003] However, mammalian-derived extracts such as fetal bovine serum are added to the medium in an amount of approximately 5% to 20%, accounting for 75% to 95% of the cost of the medium, and there are problems in that the quality varies from lot to lot because of the animal origin. Furthermore, due to concerns about a correlation with mad cow disease, bovine spongiform encephalopathy, transmissible spongiform encephalopathy, Creutzfeldt-Jakob disease, etc., attempts have been made to develop media that do not contain mammalian-derived extracts such as fetal bovine serum. However, this resulted in a significant decrease in cell viability in the early stages of culture, making long-term culture or large-scale culture difficult.

[0004] Furthermore, the addition of fish meat extracts or fish meat-related components, which are enzymatically decomposed fish meat, has resolved issues such as cost and reduced cell viability in the early stages of culture. However, the problem of lot-to-lot quality variations due to animal origin remains. Furthermore, the details of the fish meat-related components are unknown, and the components vary depending on the type, part, and enzymatic decomposition conditions of the target fish. Therefore, there are various unknown risks when using the fish meat-related components as a culture medium, making it difficult to use safely.

[0005] It was then discovered that specific tripeptides can be used as protein production promoters (Patent Document 3). However, these tripeptides are formulated at relatively high concentrations (on the order of mM), which poses a cost problem. Furthermore, the protein production curves (protein production versus concentration) of many of the specified tripeptides plateau or begin to decrease above a certain concentration (for example, around 2500 μM), which is expected to result in an overconfluent environment, i.e., an unfavorable cell growth environment, and therefore, there are problems in that protein production may not necessarily be promoted efficiently.

[0006] International Publication No. WO99 / 63058 Japanese Patent Application Laid-Open No. 2003-334068 International Publication No. WO2022 / 137357

[0007] The main objective of the present invention is to provide a synthetic culture medium that does not contain any animal-derived components, and in particular, to provide a culture medium that does not contain any animal-derived components but contains a peptide that contributes to promoting protein production at a low concentration (approximately 2000 μM or less). Methods for solving the problem

[0008] In view of the above circumstances, the present inventors have conducted extensive research and found a peptide mixture that contributes to the promotion of protein production, a protein production promoter containing the peptide mixture, and a culture medium containing the peptide mixture. Specifically, the inventors have found a peptide mixture in which the actual measured protein production amount of the mixed peptides is greater than the sum of the protein production amounts of the individual peptides.

[0009] The protein production promoter of the present invention comprises a peptide mixture as an active ingredient, the peptide mixture consisting of two peptides selected from the group consisting of Gly-Glu-Lys (GEK), Asp-Gly-Pro (DGP), Ala-Gly-Lys (AGK), Ala-Glu-Lys (AEK), Ala-Gly-Gly (AGG), Ala-Ser-Asn (ASN), Gly-Pro-Pro (GPP), Gly-Gly-Pro (GGP), and Glu-Gly-Lys (EGK), and the two selected peptides are a combination that further promotes protein production when mixed.

[0010] The medium of the present invention is characterized in that it contains the protein production promoter, and the peptide mixture contained in the protein production promoter has a final concentration in the medium of 2000 μM or less in total.

[0011] The method for promoting protein production of the present invention is characterized by using the above-mentioned protein production promoter.

[0012] The protein production promoter of the present invention can provide a protein production promoter, culture medium, and protein production method that are free of animal-derived components and contain chemically synthesized substances. In other words, it is possible to provide a protein production promoter and culture medium that are free from concerns about a correlation with mad cow disease and the like, are cost-effective, and have clear component details, resulting in stable quality. Furthermore, the protein production promoter of the present invention can reduce the amount of peptide used and achieve a greater synergistic effect between peptides.

[0013] 1 shows the relationship between the concentration of GEK and the amount of protein produced (sometimes referred to herein as "protein production amount" or "antibody production amount") in a protein production test. 2 shows the relationship between the concentration of DGP and the amount of protein produced in a protein production test. 3 shows the relationship between the concentration of AGK and the amount of protein produced in a protein production test. 4 shows the relationship between the concentration of AEK and the amount of protein produced in a protein production test. 5 shows the relationship between the concentration of AGG and the amount of protein produced in a protein production test. 6 shows the relationship between the concentration of ASN and the amount of protein produced in a protein production test. 7 shows the relationship between the concentration of GPP and the amount of protein produced in a protein production test. 8 shows the relationship between the concentration of GGP and the amount of protein produced in a protein production test. 9 shows the relationship between the concentration of EGK and the amount of protein produced in a protein production test. 10 shows the relationship between the amount of protein produced for each peptide (200 μM) in a protein production test. 11 shows the relationship between the measured values ​​and calculated values ​​for a combination of two peptide mixtures (200 μM each peptide) and the amount of protein produced in a protein production test. 1 shows the relationship between the synergistic effect (ratio of actual value to calculated value) of the amount of protein produced by combining two types of peptide mixtures in a protein production test.

[0014] The embodiments of the present invention will be specifically described below.

[0015] (Protein Production Promoting Agent) The protein production promoting agent of the present invention contains a peptide mixture as an active ingredient and promotes protein production compared to one that does not contain the peptide mixture. Furthermore, the peptide mixture is a combination of peptides that further promotes protein production by mixing them together. In this specification, "protein production" may also be referred to as "antibody production."

[0016] The peptide mixture consisted of Gly-Glu-Lys (GEK, sometimes designated herein as "a"), Asp-Gly-Pro (DGP, sometimes designated herein as "b"), Ala-Gly-Lys (AGK, sometimes designated herein as "c"), Ala-Glu-Lys (AEK, sometimes designated herein as "d"), Ala-Gly-Gly (AGG, sometimes designated herein as "e"), Ala-Ser-Asn (AS), and Ala-Ser-Asn (AS). The peptide mixture is composed of two peptides selected from the group consisting of Gly-Pro-Pro (GPP, sometimes referred to herein as "g"), Gly-Gly-Pro (GGP, sometimes referred to herein as "h"), and Glu-Gly-Lys (EGK, sometimes referred to herein as "i"), and the two selected peptides are a combination that further promotes protein production when mixed. That is, the peptide mixture is selected from 36 combinations of two of the nine tripeptides a, b, c, d, e, f, g, h, and i.

[0017] The peptides can be converted into pharmaceutically acceptable salts, and amino acids that do not change the activity of the peptide can be chemically modified. Examples of "pharmaceutically acceptable salts" include inorganic acid salts such as hydrochloride, phosphate, and sulfate; inorganic base salts such as sodium salt, potassium salt, and calcium salt; organic acid salts such as sulfonate, succinate, and oxalate; and organic base salts such as alkylammonium salts. "Chemically modifying an amino acid that does not change the activity of the peptide" means chemically modifying an amino acid with a compound that does not significantly change the activity of the peptide even if the amino acid is chemically modified. Examples include modification of the C-terminus with an amide, ester, or acyl group, and modification of the N-terminus with an acetyl group. The proline (Pro(P)) may be converted to hydroxyproline (Hyp) by introducing a hydroxyl group.

[0018] The tripeptides that make up the above peptide mixture were thoroughly explored by fractionating and identifying those that promote protein production under various conditions, focusing on the hundreds of peptides of various lengths contained mainly in fish meat extracts and their enzymatic hydrolysates, and then confirming their effects for each peptide.

[0019] The peptides can be obtained by fractionation from fish meat extracts or their enzymatic hydrolysates, chemical synthesis including peptide synthesis, or expression using recombinant DNA techniques. In the method of fractionation from fish meat extracts or their enzymatic hydrolysates, fractionation and isolation are performed by adjusting various conditions for gel filtration chromatography or normal-phase / reverse-phase HPLC. In the chemical synthesis method, synthetic amino acids or chemically modified amino acids are synthesized by chemical reactions to obtain peptides with specific sequences. In the recombinant DNA method, recombinant proteins containing multiple peptide sequences are produced by recombinant organisms, and the proteins are purified and then decomposed by enzymatic or chemical treatment to obtain the desired peptides.

[0020] The peptide mixture is preferably contained in the protein production promoter so that the total final concentration in the medium is 2000 μM or less. Other examples of upper limits for the final concentration include 1500 μM or less, 1000 μM or less, and 500 μM or less. Such upper limits for the final concentration are set to facilitate the achievement of a synergistic effect by mixing peptides. This is because the protein production curves (protein production amount versus peptide concentration) for many of the identified peptides plateau or begin to decrease above a certain concentration (e.g., around 2500 μM), which is expected to result in an overconfluent state, i.e., an unfavorable cell growth environment, making it difficult to achieve a synergistic effect. Furthermore, the peptide mixture need only contain an amount of active ingredient sufficient to promote protein production. The lower limit for the total final concentration in the medium is preferably 50 μM or more, and examples include 100 μM or more and 200 μM or more.

[0021] The amounts of the two types of peptides contained in the peptide mixture may be equal (50:50) or may be different, as long as mixing the peptides promotes protein production, for example, in a mixing ratio of 10:90 to 90:10.

[0022] (Culture Medium) The culture medium of the present invention contains the protein production promoter containing the peptide mixture.

[0023] The concentration of the peptide mixture in the medium is appropriately set depending on the cells and culture conditions. That is, the minimum concentration of the peptide mixture in the medium is the concentration at which cells can be maintained viable, and the preferred concentration is the concentration at which the protein production is increased compared to a medium without a protein production promoter and at which the synergistic effect between the peptides in the peptide mixture is enhanced. Here, a synergistic effect between the peptides in the peptide mixture was evaluated as being observed when the measured protein production amount of the peptide mixture was higher than the calculated total protein production amount of each peptide. As described above, the upper limit of the total final concentration of the peptide mixture in the medium is preferably 2000 μM or less, 1500 μM or less, 1000 μM or less, or 500 μM, and the lower limit of the final concentration is preferably 50 μM or more, 100 μM or more, or 200 μM or more.

[0024] The medium can be appropriately blended with other components used in animal cell culture media. Examples include vitamins, nucleic acids, amino acids, inorganic salts, sugars, polyamines, carbohydrates, proteins, fatty acids, lipids, pH adjusters, zinc, copper, selenium, etc. Examples of vitamins include choline chloride, niacinamide, D-pantothenic acid hemicalcium salt, folic acid, cyanocobalamin, pyridoxal hydrochloride, riboflavin, biotin, myo-inositol, ascorbic acid, thiamine hydrochloride, vitamin B12, etc. Examples of nucleic acids include xanthine, hypoxanthine, uridine, guanine hydrochloride, inosine, guanosine, cytidine, thymidine, adenine, etc. Examples of amino acids include glycine, L-alanine, L-arginine hydrochloride, L-asparagine monohydrate, L-aspartic acid, L-cysteine ​​hydrochloride monohydrate, L-cystine dihydrochloride, L-glutamic acid, L-glutamine, L-histidine hydrochloride monohydrate, L-isoleucine, L-leucine, L-lysine hydrochloride, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine disodium salt, L-valine, arginine, etc. Examples of inorganic salts include calcium chloride, magnesium sulfate, potassium chloride, sodium bicarbonate, sodium chloride, sodium dihydrogen phosphate monohydrate, etc. Other components include D-glucose, α-lipoic acid, phenolsulfonephthalein (phenol red), sodium pyruvate, AlbuMax (registered trademark) II, human transferrin (holo), ammonium metavanadate, copper sulfate, manganese chloride, sodium selenate, ethanolamine, glutathione, methotrexate, insulin, etc. Furthermore, serum components such as fetal bovine serum may be added depending on the purpose, but will not be added if there is an intention to remove animal-derived components from the medium.

[0025] (Method for Promoting Protein Production) The method for promoting protein production of the present invention is carried out by adding a protein production promoter containing the peptide mixture of the present invention to the medium described above and culturing various animal cells. Examples of the method for promoting protein production are shown below, but are not limited thereto.

[0026] The animal cells used may or may not be adapted to serum-free suspension. A protein production promoter containing the peptide mixture of the present invention is added to a basal medium. At this time, components that reinforce the basal medium, such as vitamins, nucleic acids, sugars, polyamines, and amino acids, may also be added. In an adherent culture system using a cell culture dish or plate, or a suspension culture system using a bioreactor, animal cells are seeded in a basal medium supplemented with peptides to produce proteins.

[0027] The present invention will now be described in detail with reference to examples, but these examples are not intended to limit the scope of the present invention.

[0028] (Evaluation Test 1: Measurement of the Relationship Between the Concentration of Each Peptide Solution and the Amount of Protein Produced) Prior to the evaluation of a mixture of multiple peptides, each peptide was evaluated individually. Peptides having the sequences Gly-Glu-Lys (GEK, a), Asp-Gly-Pro (DGP, b), Ala-Gly-Lys (AGK, c), Ala-Glu-Lys (AEK, d), Ala-Gly-Gly (AGG, e), Ala-Ser-Asn (ASN, f), Gly-Pro-Pro (GPP, g), Gly-Gly-Pro (GGP, h), and Glu-Gly-Lys (EGK, i) were synthesized, and peptide solutions were prepared at the concentrations shown in Tables 1 to 9 (10 μM, 50 μM, 100 μM, 250 μM, 500 μM, and 1000 μM). For dissolving and diluting each peptide, DMEM basal medium (Gibco) containing 200 nM methotrexate and 2 μg / mL insulin was used. CHO DP-12 (ATCC, model number CRL-12445) was used at 3 × 10 4The cell suspension, adjusted to 100 cells / mL, was seeded into a 96-well plate at 100 μL / well and cultured for 24 hours in a 37°C, 5% CO2 incubator. The culture medium used was DMEM basal medium (Gibco) containing 10% FBS, 200 nM methotrexate, and 2 μg / mL insulin. After removing the medium from each well, pre-prepared peptide solutions (100 μL) were added (total 100 μL / well) and cultured for 5 days. A similar culture test was performed using a system containing neither peptide nor FBS as a control. After 5 days of culture, 10 μL of the culture supernatant from each well was collected in a 1.5 mL tube, diluted, and the amount of protein produced was quantified by ELISA. The quantified values ​​were corrected by subtracting the blank value.

[0029] The above culture experiment was performed n=3, and the mass of protein produced at each concentration of each peptide solution is shown in Tables 1 to 9 and Figures 1 to 9. In Figures 1 to 9, the horizontal axis represents peptide concentration (µM), and the vertical axis represents protein production mass (mg / L).

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[0036]

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[0038]

[0039] Tables 1 to 9 and Figures 1 to 9 show that for the tested peptides, the addition of a single peptide solution increases the amount of protein produced compared to when no peptide solution is added (the point where the concentration is zero). Figures 1 to 9 also confirm that within the peptide concentration range tested, the protein production curve (protein production amount versus peptide concentration) does not plateau or decrease.

[0040] (Evaluation Test 2: Measurement of the Relationship between the Type of Two Peptide Mixtures and the Protein Production Mass) Thirty-six peptide mixtures were prepared by combining two types of each of the nine synthesized peptides, Gly-Glu-Lys (GEK, a), Asp-Gly-Pro (DGP, b), Ala-Gly-Lys (AGK, c), Ala-Glu-Lys (AEK, d), Ala-Gly-Gly (AGG, e), Ala-Ser-Asn (ASN, f), Gly-Pro-Pro (GPP, g), Gly-Gly-Pro (GGP, h), and Glu-Gly-Lys (EGK, i), so that each peptide was 200 μM, for a total of 400 μM.

[0041] A test for measuring the amount of protein produced was carried out under the same conditions as in Evaluation Test 1.

[0042] The above culture experiment was performed in triplicate. Table 10 and Figure 10 show the protein production amounts for each peptide at 200 μM alone, and Table 11 and Figure 11 show the protein production amounts for the two-peptide mixture. In the figures, a, b, c, etc. indicate the types of peptides mixed, and the actual values ​​are those measured in Evaluation Test 2, while the calculated values ​​are the sum of the antibody production amounts (protein production amounts) for each peptide at 200 μM alone for 36 combinations of two peptides. Furthermore, the ratio of the actual values ​​to the calculated values ​​(= actual value / calculated value × 100 (%)) is shown in Table 11 and Figure 12.

[0043]

[0044]

[0045] 10, under the above test conditions, when the peptide concentration was 200 μM, it was found that protein production was promoted in the following order of peptide sequences: Gly-Pro-Pro (GPP, g), Gly-Gly-Pro (GGP, h), Gly-Glu-Lys (GEK, a), and Ala-Gly-Lys (AGK, c). However, it should be noted that the values ​​must be evaluated taking into account the standard deviation.

[0046] Furthermore, Table 11, Figures 11 and 12 show that, in the case of peptide mixtures in which two peptides were selected from nine specific peptides, protein production was promoted synergistically in many peptide mixtures under the conditions of this evaluation test. That is, by mixing two selected peptides in a concentration range in which the rising protein production curve does not plateau or begin to decline, protein production was promoted more than when only one peptide was mixed, and a smaller total amount of peptide mixture than when only one peptide was mixed was able to achieve an equivalent protein production promotion effect. It was also shown that, under the conditions of this evaluation test, in some combinations of peptides from among Gly-Pro-Pro (GPP, g), Gly-Gly-Pro (GGP, h), and Gly-Glu-Lys (GEK, a), which individually produced high protein production, the value of actual value / calculated value x 100 (%) was clearly less than 100%, even when standard deviation was taken into account. Specifically, the peptide mixtures of ag, ah, ai, and gh were used. Since the amount of protein produced by each peptide was high at a concentration of 200 μM, it is believed that the synergistic effect of promoting protein production was relatively small.

[0047] In the protein production in the above-mentioned adherent culture cell system, CHO cells that have not been acclimatized to serum-free suspension were used. However, CHO cells that have been acclimatized to serum-free suspension may also be used by first culturing the cells using only serum medium, then culturing the cells half in serum medium and half in serum-free medium, and finally culturing the cells using only serum-free medium.

[0048] Furthermore, in the above-mentioned protein production test in an adherent culture cell system, CHO DP-12 cells were used, but the medium containing the peptide of the present invention can also be applied to cell lines such as CHO-K1, hybridomas used for substance production, HEK293, COS, and Sf9.

[0049] The method for producing a protein using the peptide of the present invention may include, in addition to the above-mentioned adherent culture, batch culture in suspension culture, and fed-batch culture in which the medium is replenished during production.

[0050] Furthermore, the tripeptides and combinations of tripeptides that make up the peptide mixture of the present invention are designed with a particular focus on promoting protein production (antibody production). It should be noted that amino acids with high cellular requirements are not selected for peptide formation. This is because the properties of amino acids do not necessarily remain the same even when peptided, and even short sequences such as tripeptides can have significantly different properties depending on the sequence order.

[0051] The present invention can be embodied in the following aspects: [Item 1] A protein production promoter comprising a peptide mixture as an active ingredient, wherein the peptide mixture consists of two peptides selected from the group consisting of Gly-Glu-Lys (GEK), Asp-Gly-Pro (DGP), Ala-Gly-Lys (AGK), Ala-Glu-Lys (AEK), Ala-Gly-Gly (AGG), Ala-Ser-Asn (ASN), Gly-Pro-Pro (GPP), Gly-Gly-Pro (GGP), and Glu-Gly-Lys (EGK), and wherein mixing of the two selected peptides further promotes protein production. [Item 2] A culture medium comprising the protein production promoter according to Item 1, wherein the peptide mixture contained in the protein production promoter has a total final concentration of 2000 μM or less in the culture medium. [Item 3] A method for promoting protein production, using the protein production promoter according to Item 1 above.

Claims

1. A protein production promoter comprising a peptide mixture as an active ingredient, wherein the peptide mixture consists of two peptides selected from the group consisting of Gly-Glu-Lys (GEK), Asp-Gly-Pro (DGP), Ala-Gly-Lys (AGK), Ala-Glu-Lys (AEK), Ala-Gly-Gly (AGG), Ala-Ser-Asn (ASN), Gly-Pro-Pro (GPP), Gly-Gly-Pro (GGP), and Glu-Gly-Lys (EGK), and wherein the two selected peptides are a combination that further promotes protein production when mixed.

2. A medium containing the protein production promoter according to claim 1, wherein the final concentration of the peptide mixture contained in said protein production promoter in the medium is 2000 μM or less in total.

3. A method for promoting protein production using the protein production promoter according to claim 1.

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