PEPTIDE, CELL GROWTH PROMOTER, PROTEIN PRODUCTION PROMOTER, CULTURE MEDIUM, CELL GROWTH METHOD USING PEPTIDE, AND PROTEIN PRODUCTION METHOD USING PEPTIDE

Abstract

An object is to provide a synthetic culture medium not containing animal-derived components. In particular, an object is to provide a culture medium not containing animal-derived components but containing peptides that promote cell growth or contribute to promotion of protein production. A culture medium containing a peptide selected from the group consisting of Gly-Glu-Lys (GEK), Asp-Gly-Pro (DGP), Ala-Gly-Lys (AGK), Gly-Pro-Pro (GPP), Gly-Gly-Pro (GGP), Ala-Glu-Lys (AEK), Ala-Gly-Gly (AGG), Ala-Ser-Asn (ASN), and Glu-Gly-Lys (EGK) is provided.

Claims

1. A peptide which is selected from the group consisting of Gly-Glu-Lys (GEK), Asp-Gly-Pro (DGP), Ala-Gly-Lys (AGK), Gly-Pro-Pro (GPP), Gly-Gly-Pro (GGP), Ala-Glu-Lys (AEK), Ala-Gly-Gly (AGG), Ala-Ser-Asn (ASN), and Glu-Gly-Lys (EGK).

2. A cell growth promoter comprising one or more of the peptides according to claim 1.

3. A protein production promoter comprising one or more of the peptides according to claim 1.

4. A culture medium comprising the cell growth promoter according to claim 2.

5. A cell growth method using one or more of the peptides according to claim 1.

6. A protein production method using one or more of the peptides according to claim 1.

7. The protein production method according to claim 6, comprising batch culture or fed-batch culture.

8. A culture medium comprising the protein production promoter according to claim 3.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0016] FIG. 1 shows the relationship between the concentration of GEK and the viable cell count (absorbance) in a cell growth test.

[0017] FIG. 2 shows the relationship between the concentration of DGP and the viable cell count (absorbance) in the cell growth test.

[0018] FIG. 3 shows the relationship between the concentration of AGK and the viable cell count (absorbance) in the cell growth test.

[0019] FIG. 4 shows the relationship between the concentration of GPP and the viable cell count (absorbance) in the cell growth test.

[0020] FIG. 5 shows the relationship between the concentration of GGP and the viable cell count (absorbance) in the cell growth test.

[0021] FIG. 6 shows the relationship between the concentration of AEK and the viable cell count (absorbance) in the cell growth test.

[0022] FIG. 7 shows the relationship between the concentration of AGG and the viable cell count (absorbance) in the cell growth test.

[0023] FIG. 8 shows the relationship between the concentration of ASN and the viable cell count (absorbance) in the cell growth test.

[0024] FIG. 9 shows the relationship between the concentration of EGK and the viable cell count (absorbance) in the cell growth test.

[0025] FIG. 10 shows the relationship between the concentration of GGG and the viable cell count (absorbance) in the cell growth test.

[0026] FIG. 11 shows the viable cell count (absorbance) for each tripeptide in the cell growth test.

[0027] FIG. 12 shows time-dependent changes in the viable cell count (absorbance) for each tripeptide in the cell growth test.

[0028] FIG. 13 shows the viable cell count for each tripeptide in a cell growth test for 3 days.

[0029] FIG. 14 shows the cell viability for each tripeptide in the cell growth test for 3 days.

[0030] FIG. 15 shows the viable cell count for each tripeptide in a cell growth test for 5 days.

[0031] FIG. 16 shows the cell viability for each tripeptide in the cell growth test for 5 days.

[0032] FIG. 17 shows the viable cell count for each tripeptide in a cell growth test of one type of tripeptide.

[0033] FIG. 18 shows the cell viability for each tripeptide in the cell growth test of one type of tripeptide.

[0034] FIG. 19 shows the amount of protein produced for each tripeptide in the cell growth test of one type of tripeptide.

[0035] FIG. 20 shows the viable cell count for each combination of the tripeptides in a cell growth test of two types of tripeptides.

[0036] FIG. 21 shows the cell viability for each combination of the tripeptides in the cell growth test of two types of tripeptides.

[0037] FIG. 22 shows the amount of protein produced for each combination of the tripeptides in the cell growth test of two types of tripeptides.

[0038] FIG. 23 shows the viable cell count for each combination of the tripeptides in a cell growth test of three types of tripeptides.

[0039] FIG. 24 shows the cell viability for each combination of the tripeptides in the cell growth test of three types of tripeptides.

[0040] FIG. 25 shows the amount of protein produced for each combination of the tripeptides in the cell growth test of three types of tripeptides.

[0041] FIG. 26 shows the viable cell count of AGK cells and the amount of protein produced in a cell growth test with the addition of vitamins and nucleic acids.

[0042] FIG. 27 shows the relationship between the concentration and the viable cell count of GEK in a cell growth test using a total synthesis medium.

[0043] FIG. 28 shows the relationship between the concentration and the cell viability of GEK in the cell growth test using the total synthesis medium.

[0044] FIG. 29 shows the relationship between the concentration and the amount of protein produced by GEK in the cell growth test using the total synthesis medium.

[0045] FIG. 30 shows the viable cell count for each tripeptide in a cell growth test using a medium supplemented with vitamins and the like.

[0046] FIG. 31 shows the cell viability for each tripeptide in the cell growth test using the medium supplemented with vitamins and the like.

[0047] FIG. 32 shows the amount of protein produced by each tripeptide in a protein production test using a medium supplemented with vitamins and the like.

[0048] FIG. 33 shows the amount of protein produced by each tripeptide in the protein production test using the medium supplemented with vitamins and the like.

DESCRIPTION OF EMBODIMENTS Embodiments of the present invention will be specifically described below.

Peptide

[0049] A peptide of the present invention is selected from the group consisting of Gly-Glu-Lys (GEK), Asp-Gly-Pro (DGP), Ala-Gly-Lys (AGK), Gly-Pro-Pro (GPP), Gly-Gly-Pro (GGP), Ala-Glu-Lys (AEK), Ala-Gly-Gly (AGG), Ala-Ser-Asn (ASN), and Glu-Gly-Lys (EGK). In addition, the above-mentioned peptide can be a pharmaceutically acceptable salt, and amino acids that do not change the activity of the peptide can be chemically modified. Among them, Gly-Glu-Lys (GEK), Asp-Gly-Pro (DGP), Ala-Gly-Lys (AGK), Gly-Pro-Pro (GPP), and Gly-Gly-Pro (GGP) are preferable.

[0050] The term pharmaceutically acceptable salt includes: inorganic acid salts such as hydrochlorides, phosphates, and sulfates; inorganic base salts such as sodium salts, potassium salts, and calcium salts; organic acid salts such as sulfonates, succinates, and oxalates; and organic base salts such as alkylammonium salts.

[0051] The phrase amino acids that do not change the activity of the peptide can be chemically modified means chemical modification of amino acids that do not significantly change the activity of the peptide even when the amino acids are chemically modified, and examples thereof include C-terminal modification with an amide, an ester, an acyl group, or the like, and N-terminal modification with an acetyl group.

[0052] The above-mentioned proline (Pro (P)) may be hydroxyproline (Hyp) into which a hydroxyl group is introduced.

[0053] The above-mentioned tripeptides were intensively searched for by fractionating hundreds of peptides with various lengths, which are mainly contained in fish meat extracts and their enzyme decomposition products, under various conditions to identify which of them promotes animal cell growth and which of them promotes protein production, and then confirming their effects for each peptide.

[0054] The above-mentioned peptide can be obtained by a method of fractionation from fish meat extracts and their enzyme decomposition products, by chemical synthesis methods including peptide synthesis methods, or by means such as expression by a recombinant DNA method.

[0055] In the method of fractionation from fish meat extracts and their enzyme decomposition products or the like, fractionation and isolation are carried out by adjusting various conditions of gel filtration chromatography and normal phase/reverse phase HPLC. In the chemical synthesis method, a peptide having a specific sequence can be obtained with synthesized amino acids or by synthesizing chemically modified amino acids by a chemical reaction. In the recombinant DNA method, a desired peptide can be obtained by generating recombinant proteins containing a plurality of peptide sequences from recombinant cells, purifying these proteins, and thereafter decomposing by enzymatic treatment or chemical treatment.

Cell Growth Promoter

[0056] A cell growth promoter of the present invention contains one or more of peptides selected from the group consisting of Gly-Glu-Lys (GEK), Asp-Gly-Pro (DGP), Ala-Gly-Lys (AGK), Gly-Pro-Pro (GPP), Gly-Gly-Pro (GGP), Ala-Glu-Lys (AEK), Ala-Gly-Gly (AGG), Ala-Ser-Asn (ASN), and Glu-Gly-Lys (EGK). In addition, the cell growth promoter of the present invention promotes cell growth as compared to the case in which the one or more peptides are not contained.

[0057] The above-mentioned peptide can be a pharmaceutically acceptable salt, and amino acids that do not change the activity of the peptide can be chemically modified. The above-mentioned proline (Pro (P)) may be hydroxyproline (Hyp) into which a hydroxyl group is introduced.

[0058] Peptides are selected by appropriately combining one or more of the above-mentioned peptides.

[0059] Among them, in the case of one type, Gly-Glu-Lys (GEK), Asp-Gly-Pro (DGP), Ala-Gly-Lys (AGK), Gly-Pro-Pro (GPP), and Gly-Gly-Pro (GGP) are more preferable.

[0060] In the case of two types, examples of preferable combinations include: Asp-Gly-Pro (DGP) and Ala-Gly-Lys (AGK); Gly-Glu-Lys (GEK) and Ala-Gly-Lys (AGK); Asp-Gly-Pro (DGP) and Gly-Glu-Lys (GEK); Gly-Pro-Pro (GPP) and Ala-Gly-Lys (AGK); and Gly-Pro-Pro (GPP) and Gly-Glu-Lys (GEK).

[0061] In the case of three types, examples of preferable combinations include: a combination of Gly-Pro-Pro (GPP)+Asp-Gly-Pro (DGP)+Gly-Glu-Lys (GEK), a combination of Gly-Pro-Pro (GPP)+Asp-Gly-Pro (DGP)+Ala-Gly-Lys (AGK), and a combination of Gly-Pro-Pro (GPP)+Gly-Glu-Lys (GEK)+Ala-Gly-Lys (AGK).

Protein Production Promoter

[0062] A protein production promoter of the present invention contains one or more of peptides selected from the group consisting of Gly-Glu-Lys (GEK), Asp-Gly-Pro (DGP), Ala-Gly-Lys (AGK), Gly-Pro-Pro (GPP), Gly-Gly-Pro (GGP), Ala-Glu-Lys (AEK), Ala-Gly-Gly (AGG), Ala-Ser-Asn (ASN), and Glu-Gly-Lys (EGK). In addition, the protein production promoter of the present invention promotes protein production as compared to the case in which the one or more peptides are not contained.

[0063] The above-mentioned peptide can be a pharmaceutically acceptable salt, and amino acids that do not change the activity of the peptide can be chemically modified. The above-mentioned proline (Pro (P)) may be hydroxyproline (Hyp) into which a hydroxyl group is introduced.

[0064] Peptides are selected by appropriately combining one or more of the above-mentioned peptides.

[0065] Among them, in the case of one type, examples of preferable peptides include Gly-Glu-Lys (GEK), Asp-Gly-Pro (DGP), Ala-Gly-Lys (AGK), Gly-Pro-Pro (GPP), and Gly-Gly-Pro (GGP).

[0066] In the case of two types, examples of preferable combinations include: Asp-Gly-Pro (DGP) and Ala-Gly-Lys (AGK); Gly-Glu-Lys (GEK) and Ala-Gly-Lys (AGK); Asp-Gly-Pro (DGP) and Gly-Glu-Lys (GEK); Gly-Pro-Pro (GPP) and Ala-Gly-Lys (AGK); and Gly-Pro-Pro (GPP) and Gly-Glu-Lys (GEK).

[0067] In the case of three types, examples of preferable combinations include: a combination of Gly-Pro-Pro (GPP)+Asp-Gly-Pro (DGP)+Gly-Glu-Lys (GEK), a combination of Gly-Pro-Pro (GPP)+Asp-Gly-Pro (DGP)+Ala-Gly-Lys (AGK), and a combination of Gly-Pro-Pro (GPP)+Gly-Glu-Lys (GEK)+Ala-Gly-Lys (AGK).

Culture Medium

[0068] A culture medium of the present invention contains the above-mentioned cell growth promoter containing the above-mentioned peptides, or the above-mentioned protein production promoter containing the above-mentioned peptides.

[0069] The concentration of the peptides in the culture medium is appropriately set according to cells and culture conditions. In other words, regarding the amount of the peptides in the culture medium, a concentration that can maintain the survival of cells is the lower limit concentration, a concentration that maximizes the amount of cell growth and the amount of protein produced as compared to culture media to which the cell growth promoter or the protein production promoter is not added is the preferable concentration, and a maximum concentration that is not harmful as the composition of the culture medium is the upper limit concentration. An example of the concentration per one peptide is 0.1 mM to 50 mM, preferably 0.2 mM to 10 mM, and more preferably 0.5 mM to 5 mM with respect to the culture medium.

[0070] The culture medium can be appropriately blended with other components used in animal cell culture medium. Examples thereof include vitamins, nucleic acids, amino acids, inorganic salts, sugars, polyamines, carbohydrates, proteins, fatty acids, lipids, pH adjusters, zinc, copper, and selenium.

[0071] Examples of the vitamins include choline chloride, niacinamide, D-pantothenic acid hemicalcium salt, folic acid, cyanocobalamin, pyridoxal hydrochloride, riboflavin, biotin, myo-inositol, ascorbic acid, thiamine hydrochloride, and vitamin B12.

[0072] Examples of the nucleic acids include xanthine, hypoxanthine, uridine, guanine hydrochloride, inosine, guanosine, cytidine, thymidine, and adenine.

[0073] Examples of the 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, and arginine.

[0074] Examples of the inorganic salts include calcium chloride, magnesium sulfate, potassium chloride, sodium hydrogen carbonate, sodium chloride, and sodium dihydrogen phosphate monohydrate.

[0075] Examples of other ingredients include D-glucose, ?-lipoic acid, phenolsulfonphthalein (phenol red), sodium pyruvate, AlbuMax (registered trademark) II, human transferrin (holo), ammonium metavanadate, copper sulfate, manganese chloride, sodium selenate, ethanolamine, glutathione, methotrexate, and insulin. Furthermore, depending on the purpose, serum components such as fetal bovine serum may be contained, but are not contained when the intention is to exclude animal-derived components from the culture medium.

Cell Growth Method and Protein Production Method

[0076] A cell growth method and a protein production method of the present invention are performed by blending the peptide of the present invention in the above-mentioned culture medium to culture various animal cells.

[0077] The cell growth method and the protein production method are exemplified below, but are not limited thereto.

[0078] Animal cells are acclimated to serum-free flotation using a basal medium. One or more of peptides selected from the group consisting of Gly-Glu-Lys (GEK), Asp-Gly-Pro (DGP), Ala-Gly-Lys (AGK), Gly-Pro-Pro (GPP), Gly-Gly-Pro (GGP), Ala-Glu-Lys (AEK), Ala-Gly-Gly (AGG), Ala-Ser-Asn (ASN), and Glu-Gly-Lys (EGK) are added to the basal medium. At this time, vitamins, nucleic acids, sugars, polyamines, and amino acids, which are components with which the basal medium is supplemented, may be added.

[0079] Using a bioreactor, the animal cells acclimated to the basal medium are seeded in the basal medium to which the peptides have been added to perform cell growth and protein production.

Examples

[0080] Next, the present invention will be specifically described with reference to examples, but these examples are not intended to limit the present invention.

Measurement of Relationship Between Concentration of Each Peptide Solution and Viable Cell Count

[0081] Peptides having the sequences of Gly-Glu-Lys (GEK), Asp-Gly-Pro (DGP), Ala-Gly-Lys (AGK), Gly-Pro-Pro (GPP), Gly-Gly-Pro (GGP), Ala-Glu-Lys (AEK), Ala-Gly-Gly (AGG), Ala-Ser-Asn (ASN), and Glu-Gly-Lys (EGK), and Gly-Gly-Gly (GGG) are synthesized to prepare each of peptide solutions at 10-fold concentrations shown in Tables 1 to 10.

[0082] A cell suspension prepared such that CHO-K1 (RI KEN BioResource Research Center, cell number: RCB2330) was at 3?10.sup.4 cells/mL was seeded in a 96-well plate at 100 ?L/well and cultured in an incubator at 37? C. and 5% CO.sub.2 for 24 hours. As a culture medium, an MEM? culture medium (Gibco) containing 10% FBS was used.

[0083] After removing the culture medium from each well and washing with the MEM? culture medium (100 ?L), 90 ?L of a new MEMa culture medium was dispensed into each well, and each peptide solution (10 ?L) was added (total 100 ?L/well) such that final concentrations were within a range of 0 mM to 5 mM as shown in Tables 1 to 10 to perform culture for 5 days. A similar culture test was performed using a system containing neither peptide nor FBS as a comparative sample. After culturing for 5 days, 10 ?L/well of a viable cell count measurement reagent SF (Nacalai Tesque Inc.) was added, and a color reaction was carried out in an incubator at 37? C. and 5% CO.sub.2 for 2 hours to measure an absorbance at 450 nm with a plate reader. A reference wavelength was 630 nm. It was confirmed that the absorbance at 450 nm correlated with the cell number.

[0084] The above preparation, culture, and measurement were performed with n=3, and Tables 1 to 10 and FIGS. 1 to 10 show the absorbance at the concentration of each peptide solution.

TABLE-US-00001 TABLE 1 Test section Absorbance Standard GEK(mM) (450 nm) deviation SD 0 0.0000 0.0000 0.128 ?0.0903 0.0933 0.257 0.0657 0.1054 0.512 0.3020 0.1473 1.02 0.6377 0.1016 2.05 1.0050 0.1205 4.10 0.5757 0.0055 n = 3, average value

TABLE-US-00002 TABLE 2 Test section Absorbance Standard deviation DGP(mM) (450 nm) SD 0 0.0000 0.0000 0.138 0.0957 0.0779 0.278 0.2464 0.0453 0.555 0.4127 0.0450 1.11 0.5617 0.0947 2.22 0.7657 0.0716 4.44 0.4004 0.0580 n = 3, average value

TABLE-US-00003 TABLE 3 Test section Absorbance Standard deviation AGK(mM) (450 nm) SD 0 0.0000 0.0000 0.123 0.0180 0.0823 0.247 0.1440 0.0613 0.494 0.3360 0.1299 0.99 0.5380 0.0297 1.97 0.6613 0.0490 3.95 0.4693 0.0550 n = 3, average value

TABLE-US-00004 TABLE 4 Test section Absorbance Standard deviation GPP(mM) (450 nm) SD 0 0.0000 0.0000 0.137 0.1163 0.1072 0.276 0.2303 0.1138 0.550 0.4117 0.1168 1.10 0.6443 0.1129 2.20 0.5700 0.1032 4.40 0.3620 0.0721 n = 3, average value

TABLE-US-00005 TABLE 5 Test section Absorbance Standard deviation GGP(mM) (450 nm) SD 0 0.0000 0.0000 0.135 0.0420 0.0851 0.270 0.0966 0.0863 0.539 0.2946 0.1217 1.08 0.4653 0.0707 2.16 0.5483 0.1205 4.31 0.3286 0.0545 n = 3, average value

TABLE-US-00006 TABLE 6 Test section Absorbance Standard deviation AEK(mM) (450 nm) SD 0 0.0000 0.0000 0.129 0.0350 0.0250 0.259 0.1143 0.0840 0.516 0.1927 0.0850 1.03 0.2337 0.0570 2.06 0.5013 0.1066 4.13 0.4857 0.0873 n = 3, average value

TABLE-US-00007 TABLE 7 Test section Absorbance Standard deviation AGG(mM) (450 nm) SD 0 0.0000 0.0000 0.156 0.0277 0.0831 0.313 0.0274 0.0458 0.625 0.0967 0.0975 1.25 0.2064 0.1312 2.5 0.3357 0.1854 5 0.2080 0.0951 n = 3, average value

TABLE-US-00008 TABLE 8 Test section Absorbance Standard deviation ASN(mM) (450 nm) SD 0 0.0000 0.0000 0.138 ?0.0527 0.0550 0.276 ?0.0250 0.0273 0.552 ?0.0214 0.0506 1.10 ?0.0224 0.0129 2.21 0.1170 0.1066 4.42 0.3320 0.0070 n = 3, average value

TABLE-US-00009 TABLE 9 Test section Absorbance Standard deviation EGK(mM) (450 nm) SD 0 0.0000 0.0000 0.128 0.0383 0.0922 0.257 0.1123 0.0985 0.512 0.0893 0.0710 1.02 0.1503 0.0951 2.05 0.1876 0.0861 4.10 0.2373 0.0824 n = 3, average value

TABLE-US-00010 TABLE 10 Test section Absorbance Standard deviation GGG(mM) (450 nm) SD 0 0.0000 0.0000 0.156 0.0067 0.0080 0.313 0.0037 0.0056 0.625 0.0027 0.0066 1.25 ?0.0013 0.0036 2.5 0.0013 0.0031 5 0.0083 0.0006 n = 3, average value

[0085] From Tables 1 to 10 and FIGS. 1 to 10, it was found that, in the tested peptides except GGG, the addition of the peptide solution increased the cell number as compared to the case of no addition. In addition, it was found that the concentration of the peptide solution at which the cell number increased the most differed depending on the type of peptide.

[0086] Furthermore, the absorbance at the concentration of the peptide solution that increased the cell number the most is summarized in Table 11 and FIG. 11 for each peptide tested in order to compare and show the degree of cell growth promotion when the concentration of the peptide solution was optimized.

TABLE-US-00011 TABLE 11 Peptides Concentration Absorbance Standard deviation (concentration) (mM) (450 nm) SD GGG (5 mM) 5.0000 0.0083 0.0006 EGK (4.1 mM) 4.0997 0.2373 0.0824 ASN (4.4 mM) 4.4166 0.3320 0.0070 AGG (2.5 mM) 2.5000 0.3357 0.1854 AEK (2.1 mM) 2.0649 0.5013 0.1066 GGP (2.2 mM) 2.1566 0.5483 0.1205 GPP (1.1 mM) 1.1008 0.6443 0.1129 AGK (2.0 mM) 1.9745 0.6613 0.0490 DGP (2.2 mM) 2.2182 0.7657 0.0716 GEK (2.1 mM) 2.0488 1.0050 0.1205

[0087] It was found that, under the above-mentioned testing conditions, cell growth was promoted in the order of Gly-Glu-Lys (GEK), Asp-Gly-Pro (DGP), Ala-Gly-Lys (AGK), Gly-Pro-Pro (GPP), Gly-Gly-Pro (GGP), Ala-Glu-Lys (AEK), Ala-Gly-Gly (AGG), Ala-Ser-Asn (ASN), and Glu-Gly-Lys (EGK) as the sequences of the peptides.

Measurement of Relationship Between Number of Days of Cell Growth of Each Peptide and Cell Number

[0088] Peptides having the sequences of Gly-Pro-Pro (GPP), Asp-Gly-Pro (DGP), Gly-Glu-Lys (GEK), and Ala-Gly-Lys (AGK) were synthesized to prepare each peptide solution such that the concentration was a concentration at which the cell number increased the most within a range of 0 mM to 5 mM. In other words, Gly-Pro-Pro (GPP) was adjusted to 11 mM, Asp-Gly-Pro (DGP) was adjusted to 22 mM, Gly-Glu-Lys (GEK) was adjusted to 21 mM, and Ala-Gly-Lys (AGK) was adjusted to 20 mM, and an amount of 1/10 of the total culture medium volume was added to each well immediately before the cell culture test such that final concentrations were 1.1 mM, 2.2 mM, 2.1 mM, and 2.0 mM, respectively.

[0089] A cell suspension prepared such that CHO-K1 (RIKEN BioResource Research Center, cell number: RCB2330) was at 3?10.sup.4 cells/mL was seeded in a 96-well plate at 100 ?L/well and cultured in an incubator at 37? C. and 5% CO.sub.2 for 24 hours. As a culture medium, an MEM? culture medium (Gibco) containing 10% FBS was used.

[0090] After removing the culture medium from each well and washing with the MEM? culture medium (100 ?L), 90 ?L of a new MEM? culture medium was dispensed into each well, and each peptide solution (10 ?L) was added (total 100 ?L/well) to perform culture for 0 to 5 days. Every day, 10 ?L/well of a viable cell count measurement reagent SF (Nacalai Tesque Inc.) was added, and a color reaction was carried out in an incubator at 37? C. and 5% CO.sub.2 for 2 hours to measure an absorbance at 450 nm with a plate reader. A reference wavelength was 630 nm. It was confirmed that the absorbance at 450 nm correlated with the cell number. A similar culture test was performed using a system containing neither peptide nor FBS as a comparative sample.

[0091] The above preparation, culture, and measurement were performed with n=3, and Table 12 and FIG. 12 show the absorbance and the standard deviation of each peptide solution for each day.

TABLE-US-00012 TABLE 12 Day 0 1 2 3 4 5 OD450 (average value n = 3) 1.1 mM GPP 0.2023 0.5267 0.7083 0.8350 1.0227 0.9003 2.2 mM DGP 0.1607 0.4507 0.6840 0.7700 0.9313 0.9363 2.1 mM GEK 0.1613 0.4280 0.6043 0.7300 0.9023 0.9107 2.0 mM AGK 0.1757 0.4577 0.7440 0.7997 1.0263 1.0620 0% FBS 0.1970 0.3873 0.4227 0.2927 0.3200 0.2313 Standard deviation (SD, n = 3) 1.1 mM GPP 0.0169 0.0410 0.0421 0.0190 0.1292 0.0613 2.2 mM DGP 0.0183 0.0462 0.0541 0.0507 0.1180 0.1310 2.1 mM GEK 0.0046 0.0269 0.0172 0.0599 0.1270 0.1182 2.0 mM AGK 0.0081 0.0229 0.0471 0.0318 0.1376 0.1074 0% FBS 0.0010 0.0104 0.0150 0.0153 0.0140 0.0320

[0092] From Table 12 and FIG. 12, it was found that, under the above-mentioned testing conditions, the addition of the peptide solution increased the cell number as days passed as compared to the system without the addition.

Cell Growth Test of Each Peptide on Coating Agent for 3 Days

[0093] Peptides having the sequences of Gly-Glu-Lys (GEK) and Asp-Gly-Pro (DGP) were synthesized to prepare each peptide solution such that the concentration was a concentration at which the cell number increased the most within a range of 0 mM to 5 mM. In other words, Gly-Glu-Lys (GEK) was adjusted to 21 mM, and Asp-Gly-Pro (DGP) was adjusted to 22 mM, and an amount of 1/10 of the total culture medium volume was added to each well immediately before the cell culture test such that final concentrations were 2.1 mM and 2.2 mM, respectively.

[0094] Poly-L-lysine (Peptide Institute, Inc., Poly-L-Lysine Hydrochloride, code: 3075) was prepared to 0.1 mg/mL, and 200 ?L was dispensed into each well of a 24-well plate, which was left to stand in an incubator at 37? C. for 2 hours. After removing the residual liquid with an aspirator, rinsing was performed with distilled water, irradiation was performed with a UV lamp in a clean bench without a lid, and drying was performed overnight to sterilize. A cell suspension prepared such that CHO-K1 (RIKEN BioResource Research Center, cell number: RCB2330) was at 4?10.sup.4 cells/mL was seeded in a 24-well plate at 500 ?L/well and cultured in an incubator at 37? C. and 5% CO.sub.2 for 24 hours. As a culture medium, an MEM? culture medium (Gibco) containing 10% FBS was used.

[0095] After removing the culture medium from each well and washing with the MEM? culture medium (500 ?L), 450 ?L of a new MEM? culture medium was dispensed into each well, and each peptide solution (50 ?L) was added (total 500 ?L/well) to perform culture for 3 days. The cells were recovered to count the cell number. A similar culture test was performed using a system containing neither peptide nor FBS as a comparative sample.

[0096] The culture medium in each well was recovered in a 1.5 mL tube to be rinsed with 200 ?L of MEM?, and the rinsed liquid was also recovered in the same 1.5 mL tube. Thereafter, 100 ?L of trypsin was added and incubated for 3 minutes. After rinsing with 300 ?L of MEM? containing 10% FBS, the rinsed liquid was also recovered in the same 1.5 mL tube. After rinsing again with 200 ?L of MEMa containing 10% FBS, the rinsed liquid was also recovered in the same 1.5 mL tube.

[0097] A centrifugation operation was performed on the cells recovered in the 1.5 mL tube. The centrifugation conditions were 1,000 rpm, 10 minutes, and 4? C.

[0098] The supernatant was removed, and 300 ?L of Cold PBS (phosphate buffered saline) was added to perform a centrifugation operation under the same conditions. The present operation was repeated twice.

[0099] Suspension was carried out with 100 ?L of Binding Buffer, and 2 ?L of propidium iodide (PI) was added and stirred in the tube to cause a reaction for 15 minutes at room temperature and with light shielding. Thereafter, the viable cell count and the viability were measured with a flow cytometer.

[0100] The above preparation, culture, and measurement were performed with n=3. Table 13 and FIG. 13 show the viable cell count in each peptide solution, and Table 14 and FIG. 14 show the cell viability in each peptide solution.

TABLE-US-00013 TABLE 13 Cells cell count (?10.sup.4) 1 2 3 Average SD 0% FBS 0.59 0.88 0.64 0.71 0.16 2.2 mM Asp-Gly-Pro 11.69 10.47 13.82 11.99 1.70 2.1 mM Gly-Glu-Lys 17.94 19.72 20.39 19.35 1.26

TABLE-US-00014 TABLE 14 Viability 1 2 3 Average SD 0% FBS 13.1 15.3 15.3 14.6 1.3 2.2 mM Asp-Gly-Pro 77.9 75.6 82.0 78.5 3.2 2.1 mM Gly-Glu-Lys 94.7 95.1 93.8 94.5 0.7

[0101] From Table 13, Table 14, FIG. 13, and FIG. 14, it was found that the viable cell count increased and the cell viability was also higher in the peptide culture on a coating agent for 3 days under the above-mentioned testing conditions, as compared to the system to which the peptides were not added.

Cell Growth Test of Each Peptide on Coating Agent for 5 Days

[0102] Peptides having the sequences of Asp-Gly-Pro (DGP), Ala-Gly-Lys (AGK), Gly-Glu-Lys (GEK), and Gly-Gly-Gly (GGG) were synthesized to prepare each peptide solution such that the concentration was a concentration at which the cell number increased the most within a range of 0 mM to 5 mM. In other words, Asp-Gly-Pro (DGP) was adjusted to 22 mM, Ala-Gly-Lys (AGK) was adjusted to 20 mM, Gly-Glu-Lys (GEK) was adjusted to 21 mM, and Gly-Gly-Gly (GGG) was adjusted to 25 mM, and an amount of 1/10 of the total culture medium volume was added to each well immediately before the cell culture test such that final concentrations were 2.2 mM, 2.0 mM, 2.1 mM, and 2.5 mM, respectively. Poly-L-lysine (Peptide Institute, Inc., Poly-L-Lysine Hydrochloride, code: 3075) was prepared to 0.1 mg/mL, and 200 ?L was dispensed into each well of a 24-well plate, which was left to stand in an incubator at 37? C. for 2 hours. After removing the residual liquid with an aspirator, rinsing was performed with distilled water, irradiation was performed with a UV lamp in a clean bench without a lid, and drying was performed overnight to sterilize. A cell suspension prepared such that CHO-K1 (RIKEN BioResource Research Center, cell number: RCB2330) was at 4?10.sup.4 cells/mL was seeded in each well of a 24-well plate at 500 ?L/well and cultured in an incubator at 37? C. and 5% CO.sub.2 for 24 hours. As a culture medium, an MEM? culture medium (Gibco) containing 10% FBS was used.

[0103] After removing the culture medium from each well and washing with the MEM? culture medium (500 ?L), 450 ?L of a new MEM? culture medium was dispensed into each well, and each peptide solution (50 ?L) was added (total 500 ?L/well) to perform culture for 5 days. The cells were recovered to count the cell number. A similar culture test was performed using a system containing neither peptide nor FBS as a comparative sample.

[0104] The culture medium in each well was recovered in a 1.5 mL tube to be rinsed with 200 ?L of MEM?, and the rinsed liquid was also recovered in the same 1.5 mL tube. Thereafter, 100 ?L of trypsin was added and incubated for 3 minutes. After rinsing with 300 ?L of MEM? containing 10% FBS, the rinsed liquid was also recovered in the same 1.5 mL tube. After rinsing again with 200 ?L of MEM? containing 10% FBS, the rinsed liquid was also recovered in the same 1.5 mL tube.

[0105] A centrifugation operation was performed on the cells recovered in the 1.5 mL tube. The centrifugation conditions were 1,000 rpm, 10 minutes, and 4? C.

[0106] The supernatant was removed, and 300 ?L of Cold PBS (phosphate buffered saline) was added to perform a centrifugation operation under the same conditions. The present operation was repeated twice.

[0107] Suspension was carried out with 100 ?L of Binding Buffer, and 2 ?L of propidium iodide (PI) was added and stirred in the tube to cause a reaction for 15 minutes at room temperature and with light shielding. Thereafter, the viable cell count and the cell viability were measured with a flow cytometer.

[0108] The above preparation, culture, and measurement were performed with n=3. Table 15 and FIG. 15 show the viable cell count in each peptide solution, and Table 16 and FIG. 16 show the cell viability in each peptide solution.

TABLE-US-00015 TABLE 15 Cells cell count (?10.sup.4) 1 2 3 Mean SD 0% FBS 0.0070 0.0100 0.0030 0.0067 0.0035 2.2 mM Asp-Gly-Pro 15.8220 14.8590 14.1250 14.9353 0.8511 2.0 mM Ala-Gly-Lys 2.6860 3.0605 2.1185 2.6217 0.4743 2.1 mM Gly-Glu-Lys 15.7190 16.9875 17.0425 16.5830 0.7488 2.5 mM Gly-Gly-Gly 0.1360 0.0080 0.0065 0.0502 0.0743

TABLE-US-00016 TABLE 16 Viability 1 2 3 Mean SD 0% FBS 0.4 0.4 0.3 0.4 0.1 2.2 mM Asp-Gly-Pro 91.5 91.2 86.0 89.6 3.1 2.0 mM Ala-Gly-Lys 26.4 26.4 22.1 25.0 2.5 2.1 mM Gly-Glu-Lys 90.2 90.3 90.5 90.3 0.2 2.5 mM Gly-Gly-Gly 4.45 0.31 0.21 1.7 2.4

[0109] From Table 15, Table 16, FIG. 15, and FIG. 16, it was found that, except GGG, the cell number increased and the cell viability was also higher in the peptide culture on a coating agent for 5 days under the above-mentioned testing conditions, as compared to the system to which the peptides were not added.

Cell Growth Test and Protein Production Test on Each Peptide and Combination of Peptides on Coating Agent

[0110] Peptides having the sequences of Gly-Pro-Pro (GPP), Asp-Gly-Pro (DGP), Gly-Glu-Lys (GEK), and Ala-Gly-Lys (AGK) were synthesized to prepare each peptide solution such that the concentration was a concentration at which the cell number increased the most within a range of 0 mM to 5 mM. In other words, Gly-Pro-Pro (GPP) was adjusted to 11 mM, Asp-Gly-Pro (DGP) was adjusted to 22 mM, Gly-Glu-Lys (GEK) was adjusted to 21 mM, and Ala-Gly-Lys (AGK) was adjusted to 20 mM, and an amount of 1/10 of the total culture medium volume was added to each well immediately before the cell culture test such that final concentrations were 1.1 mM, 2.2 mM, 2.1 mM, and 2.0 mM, respectively. Poly-L-lysine (Peptide Institute, Inc., Poly-L-Lysine Hydrochloride, code: 3075) was prepared to 0.1 mg/mL, and 200 ?L was dispensed into each well of a 24-well plate, which was left to stand in an incubator at 37? C. for 2 hours. After removing the residual liquid with an aspirator, rinsing was performed with distilled water, irradiation was performed with a UV lamp in a clean bench without a lid, and drying was performed overnight to sterilize. A cell suspension prepared such that CHO DP-12 (ATCC, Cat. No. CRL-12445) was at 2?10.sup.4 cells/well and 500 ?L was seeded in a 24-well plate and cultured in an incubator at 37? C. and 5% CO.sub.2 for 24 hours. As a culture medium, a DMEM basal medium in which 200 nM of methotrexate and 2 ?g/mL of insulin were blended in a DMEM culture medium (Gibco) containing 10% FBS was used.

[0111] After removing the culture medium from each well and washing with the DMEM basal medium (500 ?L), 450 ?L of a new DMEM basal medium was dispensed into each well, and each peptide solution (50 ?L) was added (total 500 ?L/well) to perform culture for 5 days.

[0112] As peptides, in addition to single GPP, DGP, GEK, and AGK, combinations of GPP+GEK, GPP+AGK, DGP+GEK, GEK+AGK, DGP+AGK, GPP+GEK+AGK, GPP+DGP+AGK, and GPP+DGP+GEK were used. A similar culture test was performed using a system containing neither peptide nor FBS as a comparative sample.

[0113] In order to measure the amount of protein produced, 100 ?L of the culture medium supernatant was recovered in a 1.5 mL tube and diluted to quantitatively determine the amount of protein produced by an ELISA method.

[0114] In order to recover the cells, the culture medium in each well was recovered in a 1.5 mL tube to be rinsed with 200 ?L of the DMEM basal medium, and the rinsed liquid was also recovered in the same 1.5 mL tube. Thereafter, 100 ?L of trypsin was added and incubated for 3 minutes. After rinsing with 300 ?L of the DMEM basal medium containing 10% FBS, the rinsed liquid was also recovered in the same 1.5 mL tube. After rinsing again with 200 ?L of the DMEM basal medium containing 10% FBS, the rinsed liquid was also recovered in the same 1.5 mL tube.

[0115] A centrifugation operation was performed on the cells recovered in the 1.5 mL tube. The centrifugation conditions were 1,000 rpm, 10 minutes, and 4? C.

[0116] The supernatant was removed, and 300 ?L of Cold PBS (phosphate buffered saline) was added to perform a centrifugation operation under the same conditions. The present operation was repeated twice.

[0117] Suspension was carried out with 100 ?L of Binding Buffer, and 2 ?L of propidium iodide (PI) was added and stirred in the tube to cause a reaction for 15 minutes at room temperature and with light shielding. Thereafter, the viable cell count and the cell viability were measured with a flow cytometer.

[0118] The above preparation, culture, and measurement were performed with n=3. Table 17, FIG. 17, Table 20, FIG. 20, Table 23, and FIG. 23 show the viable cell count in each peptide solution. Table 18, FIG. 18, Table 21, FIG. 21, Table 24, and FIG. 24 show the cell viability in each peptide solution. Table 19, FIG. 19, Table 22, FIG. 22, Table 25, and FIG. 25 show the amount of protein produced in each peptide solution.

TABLE-US-00017 TABLE 17 Cells cell count (?10.sup.4) Average SD GPP 15.5502 0.8225 DGP 14.6333 0.9364 GEK 15.9345 0.4884 AGK 19.0887 0.9484 0% FBS 4.0250 0.4633

TABLE-US-00018 TABLE 18 Viability 1 2 3 Average SD GPP 88.9 87.9 87.9 88.2 0.6 DGP 86.3 87.3 87.2 86.9 0.6 GEK 86.5 87.4 86.7 86.9 0.5 AGK 89.0 88.5 88.1 88.5 0.5 0% FBS 57.8 59.5 57.9 58.4 1.0

TABLE-US-00019 TABLE 19 Amount of protein produced (mg/L) 1 2 3 Average SD GPP 5.78 5.31 6.50 5.86 0.60 DGP 5.37 4.90 5.44 5.24 0.29 GEK 6.13 4.59 6.06 5.59 0.87 AGK 7.36 6.61 6.78 6.92 0.39 0% FBS 3.95 4.47 4.63 4.35 0.36

TABLE-US-00020 TABLE 20 Cells cell count (?10.sup.4) Average SD GPP + GEK 9.1175 0.5391 GPP + AGK 9.4750 0.1945 DGP + GEK 8.9357 0.6300 GEK + AGK 9.1192 0.1442 DGP + AGK 9.8113 0.6386 0% FBS 1.0642 0.1651

TABLE-US-00021 TABLE 21 Viability 1 2 3 Average SD GPP + GEK 86.3 85.7 85.1 85.7 0.6 GPP + AGK 86.0 86.2 85.1 85.8 0.6 DGP + GEK 84.1 85.0 86.2 85.1 1.1 GEK + AGK 86.0 85.4 87.3 86.2 1.0 DGP + AGK 84.6 85.5 86.0 85.4 0.7 0% FBS 37.5 40.9 41.0 39.8 2.0

TABLE-US-00022 TABLE 22 Amount of protein produced (mg/L) 1 2 3 Average SD GPP + GEK 3.70 3.95 3.06 3.57 0.46 GPP + AGK 5.54 4.29 4.63 4.82 0.85 DGP + GEK 4.03 2.90 3.30 3.41 0.57 GEK + AGK 3.32 3.44 3.72 3.49 0.21 DGP + AGK 3.98 3.48 3.03 3.50 0.48 0% FBS 2.55 2.69 2.84 2.69 0.15

TABLE-US-00023 TABLE 23 Cells cell count (?10.sup.4) Average SD GPP + GEK + AGK 10.5085 0.9625 GPP + DGP + AGK 9.6858 1.0850 GPP + DGP + GEK 9.4232 1.3146 0% FBS 1.8085 0.4705

TABLE-US-00024 TABLE 24 Viability 1 2 3 Average SD GPP + GEK + AGK 86.2 86.2 86.4 86.3 0.1 GPP + DGP + AGK 86.4 87.5 86.9 86.9 0.6 GPP + DGP + GEK 84.4 85.4 84.8 84.9 0.5 0% FBS 40.7 44.8 45.6 43.7 2.6

TABLE-US-00025 TABLE 25 Amount of protein produced (mg/L) 1 2 3 Average SD GPP + GEK + AGK 4.23 3.90 4.17 4.10 0.18 GPP + DGP + AGK 4.12 4.21 4.07 4.13 0.07 GPP + DGP + GEK 3.06 3.30 3.98 3.45 0.48 0% FBS 2.87 3.16 3.41 3.15 0.27

[0119] From Tables 17 to 25 and FIGS. 17 to 25, it was found that the addition of a single peptide or a combination of two or more peptides under the above-mentioned testing conditions promoted cell growth and promoted protein production.

Cell Growth Test and Protein Production Test on Each PeptideEffects of Adding Vitamins and Nucleic Acids

[0120] A peptide having the sequence Ala-Gly-Lys (AGK) was synthesized to prepare 1.0 mM and 2.0 mM of peptide solutions.

[0121] A cell suspension prepared such that CHO DP-12 (ATCC, Cat. No. CRL-12445) was at 2?10.sup.4 cells/mL was seeded in a 24-well plate at 1?10.sup.4 cells/well (500 ?L) and cultured in an incubator at 37? C. and 5% CO.sub.2 for 24 hours. As a culture medium, a DMEM basal medium in which 200 nM of methotrexate and 2 ?g/mL of insulin were blended in a DMEM culture medium (Gibco) containing 10% FBS was used.

[0122] After removing the culture medium from each well and washing with the DMEM basal medium (500 ?L), each peptide solution (500 ?L) was dispensed to each well (total 500 ?L/well) to perform culture for 5 days.

[0123] After replacing each culture medium with an evaluation medium of the following test section, culture was performed in an incubator at 37? C. and 5% CO.sub.2 for 5 days.

<Evaluation Medium>

[0124] DMEM basal medium [0125] AGK (1 mM)+DMEM basal medium [0126] AGK (2 mM)+DMEM basal medium [0127] Medium supplemented with vitamins and nucleic acids (DMEM basal medium+vitamin+nucleic acid) [0128] AGK (1 mM)+medium supplemented with vitamins and nucleic acids [0129] AGK (2 mM)+medium supplemented with vitamins and nucleic acids

[0130] After 5 days of culture, the total amount of the culture medium in each well of the 24-well plate was recovered, a centrifugation operation (5,000 rpm, for 5 minutes) was performed, and the supernatant was separately recovered to measure the amount of produced protein by ELISA.

[0131] The composition of the above-mentioned vitamins and nucleic acids is shown in Table 26.

TABLE-US-00026 TABLE 26 (Final concentration Nucleic (Final concentration Vitamin of each component) acid of each component) Choline chloride 4.155 mg/L Xanthine 0.9 mg/L Niacinamide 0.276 mg/L Hypoxanthine 1.8 mg/L D-pantothenic acid 45.8 ?g/L Uridine 3.6 mg/L hemicalcium salt Folic acid 3.6 ?g/L Guanine 2.4 mg/L hydrochloride Cyanocobalamin 0.8 ?g/L Inosine 4.9 mg/L Pyridoxal hydrochloride 12.2 ?g/L Guanosine 3.8 mg/L Riboflavin 13.8 ?g/L Cytidine 0.4 mg/L Biotin 0.8 ?g/L Thymidine 0.5 mg/L Myo-inositol 0.73 mg/L Adenine 0.8 mg/L

[0132] The cells adhered to the wells after recovering the culture medium were detached by trypsin treatment and suspended again in the DMEM basal medium containing 10% FBS to measure the viable cell count and the viability by a trypan blue staining method using a cell counter.

[0133] The above preparation, culture, and measurement were performed with n=3. Table 27 shows the viable cell count, the cell viability, the amount of protein produced in each evaluation medium, and FIG. 26 shows the viable cell count and the amount of protein produced.

TABLE-US-00027 TABLE 27 Viable cell Standard Amount of count deviation Standard protein Standard (?10.sup.4cells/ (?10.sup.4cells/ Viability deviation produced deviation Test section well) well) (%) (%) (mg/L) (mg/L) Basal medium 2.61 0.43 33 10 4.68 0.4 1 mM AGK 11.97 2.00 65 8 7.5 0.6 2 mM AGK 11.73 1.53 65 2 7.4 0.4 Basal medium 1.17 0.43 17 2 4.36 0.3 (Vitamin + Nucleic acid) 1 mM AGK(Vitamin + 34.65 6.10 88 2 15.74 3.6 Nucleic acid) 2 mM AGK(Vitamin + 47.07 8.50 89 2 14.34 2.4 Nucleic acid)

[0134] From Table 27 and FIG. 26, it was found that, under the above-mentioned testing conditions, the addition of peptides to the basal medium increased both the viable cell count and the amount of protein produced, and the further addition of vitamins and nucleic acids further promoted both cell growth and protein production.

Cell Growth Test and Protein Production Test when Peptide was Added to Total Synthesis Medium

[0135] A peptide having the sequence of Gly-Glu-Lys (GEK) was synthesized to prepare peptide solutions at the concentrations of 0 mM, 2.6 mM, 5.1 mM, 10 mM, 20.5 mM, and 41 mM, and an amount of 1/10 of the total culture medium volume was added to each well immediately before the cell culture test such that final concentrations were 0 mM, 0.26 mM, 0.51 mM, 1.0 mM, 2.05 mM, and 4.1 mM, respectively. In the present test, CHO DP-12 (ATCC, Cat. No. CRL-12445) acclimated to an ASF104 basal medium in which 200 nM of methotrexate and 2 ?g/mL of insulin were blended in an ASF104 culture medium (Ajinomoto Co., Inc.), which is a total synthesis medium for CHO, was used. A cell suspension prepared such that a cell concentration was 4?10.sup.4 cells/mL was seeded in a 24-well plate at 450 ?L/well and cultured in an incubator at 37? C. and 5% CO.sub.2 for 24 hours. As a culture medium, an ASF104 basal medium in which 200 nM of methotrexate and 2 ?g/mL of insulin were blended in an ASF104 culture medium (Ajinomoto Co., Inc.), which is a total synthesis medium for CHO, was used.

[0136] After 24 hours, 50 ?L of the prepared peptide solution was added to each well to perform culture for 5 days. The cells were recovered after culture to count the viable cell count.

<Evaluation Medium>

[0137] ASF104 basal medium [0138] GEK (0.26 mM)+ASF104 basal medium [0139] GEK (0.51 mM)+ASF104 basal medium [0140] GEK (1.0 mM)+ASF104 basal medium [0141] GEK (2.05 mM)+ASF104 basal medium [0142] GEK (4.1 mM)+ASF104 basal medium

[0143] 100 ?L of the culture medium supernatant was collected in a 1.5 mL tube to quantitatively determine the amount of protein produced. The supernatant was diluted to measure the amount of protein produced by ELISA.

[0144] In order to analyze the cells, the culture medium in each well was recovered in a 1.5 mL tube to be rinsed with 200 ?L of PBS, and the rinsed liquid was also recovered in the same 1.5 mL tube. Thereafter, 100 ?L of 0.25% trypsin/EDTA was added to perform incubation for 1 minute. 100 ?L of a trypsin inhibitor was added to recover in the same 1.5 mL tube. After rinsing with 200 ?L of PBS to recover the rinsed liquid in the same 1.5 mL tube, a centrifugation operation was performed. Suspension was performed in 100 ?L of PBS to measure the viable cell count and the cell viability by a trypan blue staining method using a cell counter.

[0145] The above preparation, culture, and measurement were performed with n=3. Table 28 and FIG. 27 show the viable cell count in each peptide solution, Table 29 and FIG. 28 show the cell viability in each peptide solution, and Table 30 and FIG. 29 show the amount of protein produced.

TABLE-US-00028 TABLE 28 Viable cell count (?10.sup.6 cells/well) 1 2 3 Average SD Vehicle 1.4 1.5 1.2 1.3 0.2 0.26 mM GEK 1.6 1.5 1.6 1.6 0.0 0.51 mM GEK 1.5 1.7 1.6 1.6 0.2 1.0 mM GEK 1.3 1.8 1.4 1.5 0.3 2.05 mM GEK 1.9 2.2 1.4 1.9 0.4 4.1 mM GEK 1.8 1.8 1.5 1.7 0.1

TABLE-US-00029 TABLE 29 Viability (%) 1 2 3 Average SD Vehicle 95 96 97 96.0 1.0 0.26 mM GEK 96 99 96 97.0 1.7 0.51 mM GEK 95 98 97 96.7 1.5 1.0 mM GEK 97 97 96 96.7 0.6 2.05 mM GEK 96 96 98 96.7 1.2 4.1 mM GEK 97 98 96 97.0 1.0

TABLE-US-00030 TABLE 30 Amount of protein produced (mg/L) 1 2 3 Average SD Vehicle 11.4 11.9 12.8 12.0 0.7 0.26 mM GEK 12.2 12.6 12.7 12.5 0.2 0.51 mM GEK 12.8 13.2 12.6 12.9 0.3 1.0 mM GEK 12.4 12.8 14.1 13.1 0.9 2.05 mM GEK 17.5 17.7 15.6 17.0 1.1 4.1 mM GEK 13.9 13.2 12.8 13.3 0.6

[0146] From Tables 28 to 30 and FIGS. 27 to 29, it was found that, under the above-mentioned testing conditions, when a GEK peptide was added, the viable cell count increased, the cell viability was similarly high, and the amount of protein produced also tended to increase, indicating that cell growth was promoted and protein production was promoted, as compared to the basal medium in which the commercially available total synthesis medium was used.

Cell Growth Test and Protein Production Test on GEK and DGP in Floating Cell System

[0147] Peptides having the sequences of Gly-Glu-Lys (GEK) and Asp-Gly-Pro (DGP) were synthesized to prepare peptide solutions of 2.87 mM of Gly-Glu-Lys (GEK), and 1.55 mM of Asp-Gly-Pro (DGP).

[0148] 5 mL of each of a DMEM/F12 basal medium, a medium supplemented with vitamins and the like (DMEM/F12 basal medium+a component supplemented with vitamins and the like), and each peptide solution was added to each well of a 24-well (Deep well) cassette of Micro-24 Bioreactor System (manufactured by Nihon Pall Corporation) to perform culture overnight under the conditions of 37? C., pH 7, and a stirring speed of 650 rpm. On the next day, pH calibration was performed, and thereafter 2 mL of the cell suspension prepared at 3.5?10.sup.5 cells/mL was added to each well of the 24-well (Deep well) cassette, seeded such that the cell density of the floating cells was 1?10.sup.5 cells/mL (7 mL/well), and cultured in the following evaluation medium under culture conditions of 37? C., pH 7, a stirring speed of 650 rpm, and dissolved oxygen of 30%.

<Evaluation Medium>

[0149] DMEM/F12 basal medium [0150] Medium supplemented with vitamins and the like (basal medium+component supplemented with vitamins and the like) [0151] GEK (2.05 mM)+medium supplemented with vitamins and the like [0152] DGP (1.11 mM)+medium supplemented with vitamins and the like

[0153] As a culture medium, a DMEM/F12 basal medium in which 200 nM of methotrexate, 10 ?g/mL of insulin, 5.5 ?g/mL of transferrin, 6.7 ng/mL of sodium selenite, 10 ?L/mL of Anti-Clumping Agent, and 10 ?L/mL of 10% Pluronic F68 were blended in a DMEM/F12 culture medium (Gibco) was used.

[0154] As the floating cells used, serum-free floating CHO DP-12 obtained by acclimating CHO DP-12 (ATCC, Cat. No. CRL-12445) to serum-free flotation and subculturing using a 100 mL volume Erlenmeyer flask and with a shaking culture device (Custom Bio Shaker CO2-BR-43FL, TAITEC CORPORATION) under the culture conditions of 37? C., 5% CO.sub.2, and a stirring speed of 125 rpm was used.

[0155] Table 31 shows the above-mentioned component supplemented with vitamins and the like.

TABLE-US-00031 TABLE 31 (Final concentrationof Component each component) Vitamin Choline chloride 20.775 mg/L Niacinamide 1.38 mg/L D-pantothenic acid 229 ?g/L hemicalcium salt Folic acid 18 ?g/L Cyanocobalamin 4 ?g/L Pyridoxal hydrochloride 61 ?g/L Riboflavin 69 ?g/L Biotin 4 ?g/L Myo-inositol 3.65 mg/L Nucleic Xanthine 4.5 mg/L acid Hypoxanthine 9 mg/L Uridine 18 mg/L Guanine hydrochloride 12 mg/L Inosine 24.5 mg/L Guanosine 19 mg/L Cytidine 2 mg/L Thymidine 2.5 mg/L Adenine 4 mg/L Sugar Glucose 220 mg/L Polyamine Spermidine 1.75 mg/L Spermine 0.5 mg/L Putrescine 0.375 mg/L Amino acid Cystine 2.4 mg/L Asparagine 32.3 mg/L Aspartic acid 192.5 mg/L Serine 126.35 mg/L Glycine 85.35 mg/L Glutamine 2.45 mg/L Cysteine 22.55 mg/L Threonine 128 mg/L Glutamic acid 213.35 mg/L Alanine 185.15 mg/L Proline 30.5 mg/L Lysine HCl 277.85 mg/L Histidine 120.65 mg/L Arginine 216.15 mg/L Valine 167.15 mg/L Methionine 90.65 mg/L Tyrosine 18.35 mg/L Isoleucine 138.65 mg/L Leucine 246.85 mg/L Phenylalanine 120.15 mg/L tryptophan 38 mg/L

[0156] After the third day of culture, 200 p?L of the culture medium was recovered in a 1.5 mL tube from each well of the 24-well (Deep well) cassette. 50 ?L out of this was put in another 1.5 mL tube, and after adding 50 ?L of trypan blue to sufficiently suspend, the viable cell count and the viability were measured using a cell counter (Countess II, manufactured by Life Technologies Corporation). The above preparation, culture, and measurement were performed with n=2 or 3, and Table 32 and FIG. 30 show the viable cell count in each evaluation medium. In addition, Table 33 and FIG. 31 show the viability in each evaluation medium.

TABLE-US-00032 TABLE 32 Day 0 1 2 3 4 5 6 7 cell number (?10.sup.6cells/ml) basal medium 0.10 1.07 1.39 1.55 1.55 1.29 medium 0.10 1.23 1.67 2.18 2.35 2.11 supplemented with vitamins and the like GEK 2.05 mM 0.10 1.73 2.40 2.65 2.64 3.05 DGP 1.11 mM 0.10 1.15 2.52 2.58 3.50 3.01 standard deviation basal medium 0.00 0.15 0.33 0.48 0.81 0.33 medium 0.00 0.06 0.00 0.18 0.16 0.39 supplemented with vitamins and the like GEK 2.05 mM 0.00 0.36 0.41 0.29 0.27 0.10 DGP 1.11 mM 0.00 0.16 0.14 0.21 1.07 0.07

TABLE-US-00033 TABLE 33 Day 0 1 2 3 4 5 6 7 Viability (%) basal medium 98.0 94.00 88.33 83.67 74.67 68.33 medium 98.0 97.00 95.00 93.50 91.50 86.50 supplemented with vitamins and the like GEK 2.05 mM 98.0 97.00 97.00 95.33 84.33 79.00 DGP 1.11 mM 98.0 97.50 97.50 94.50 87.00 81.50 standard deviation basal medium 0.0 3.00 8.08 9.02 12.58 9.29 medium 0.0 0.00 2.83 2.12 4.95 0.71 supplemented with vitamins and the like GEK 2.05 mM 0.0 1.00 1.00 3.79 12.66 8.19 DGP 1.11 mM 0.0 0.71 2.12 0.71 0.00 0.71

[0157] From Table 32, FIG. 30, Table 33, and FIG. 31, it was found that the addition of peptides to the basal medium or the medium supplemented with vitamins and the like increased both the viable cell count and the viability under the above-mentioned testing conditions.

[0158] After the third day of culture, 150 ?L out of 200 ?L of the culture medium recovered from each well of the 24-well (Deep well) cassette was subjected to a centrifugation operation (5,000 rpm, for 5 minutes) in a 1.5 mL tube, and the supernatant was separately recovered to measure the amount of produced protein by ELISA.

[0159] Table 34 and FIG. 32 show the measured amount of protein produced.

TABLE-US-00034 TABLE 34 Amount of protein Day produced (mg/L) 3 4 5 6 7 Basal medium 18.64 27.76 30.61 35.40 35.89 Medium supplemented with 16.34 38.33 60.83 64.83 89.44 vitamins and the like GEK 2.05 mM 26.32 54.96 75.54 98.46 127.08 DGP 1.11 mM 19.86 43.99 63.65 82.54 104.75 Day Standard deviation 3 4 5 6 7 Basal medium 4.59 5.86 8.45 8.86 12.55 Medium supplemented with 1.14 2.61 5.64 9.90 15.29 vitamins and the like GEK 2.05 mM 6.02 8.53 11.17 7.64 5.38 DGP 1.11 mM 5.64 10.12 10.01 5.74 7.60

[0160] From Table 34 and FIG. 32, it was found that the addition of peptides to the basal medium or the medium enriched with vitamins and the like increased the amount of protein produced.

Protein Production Test on AGK and GPP in Floating Cell System

[0161] Peptides having the sequences of Ala-Gly-Lys (AGK) and Gly-Pro-Pro (GPP) were synthesized to prepare peptide solutions of 5.53 mM of Ala-Gly-Lys (AGK) and 6.16 mM of Gly-Pro-Pro (GPP).

[0162] 5 mL of each of a basal medium, a medium supplemented with vitamins and the like (basal medium +a component supplemented with vitamins and the like), and each peptide solution was added to each well of a 24-well (Deep well) cassette of Micro-24 Bioreactor System (manufactured by Nihon Pall Corporation) to perform culture overnight under the conditions of 37? C., pH 7, and 650 rpm. On the next day, pH calibration was performed, and thereafter 2 mL of the cell suspension prepared at 3.5?10.sup.5 cells/mL was added to each well of the 24-well (Deep well) cassette, seeded such that the cell density of the floating cells was 1?10.sup.5 cells/mL (7 mL/well), and cultured in the following evaluation medium under culture conditions of 37? C., pH 7, 650 rpm, and dissolved oxygen of 30%.

<Evaluation Medium>

[0163] Basal medium [0164] Medium supplemented with vitamins and the like (basal medium+component supplemented with vitamins and the like) [0165] AGK (3.95 mM)+medium supplemented with vitamins and the like [0166] GPP (4.40 mM)+medium supplemented with vitamins and the like

[0167] As a culture medium, a basal medium in which 200 nM of methotrexate, 10 ?g/mL of insulin, 5.5 ?g/mL of transferrin, 6.7 ng/mL of sodium selenite, 10 ?L/mL of Anti-Clumping Agent, and 10 ?L/mL of 10% Pluronic F68 were blended in a DMEM/F12 culture medium (Gibco) was used.

[0168] As the floating cells used, serum-free floating CHO DP-12 obtained by acclimating CHO DP-12 (ATCC, Cat. No. CRL-12445) to serum-free flotation and subculturing using a 100 mL volume Erlenmeyer flask and with a shaking culture device (Custom Bio Shaker CO2-BR-43FL, TAITEC CORPORATION) under the culture conditions of 37? C., 5% CO.sub.2, and a stirring speed of 125 rpm was used.

[0169] Table 31 shows the above-mentioned component supplemented with vitamins and the like.

[0170] After the third day of culture, 150 ?L of the culture medium was recovered in a 1.5 mL tube from each well of the 24-well (Deep well) cassette to perform a centrifugation operation (5,000 rpm, for 5 minutes), and the supernatant was recovered to measure the amount of produced protein by ELISA.

[0171] Table 35 and FIG. 33 show the measured amount of protein produced.

TABLE-US-00035 TABLE 35 Amount of protein Day produced (mg/L) 3 4 5 6 7 Basal medium 13.11 19.59 26.73 28.57 27.65 Medium supplemented with 13.96 28.00 43.26 51.30 63.31 vitamins and the like AGK 3.95 mM 17.74 31.36 49.43 65.58 73.46 GPP 4.40 mM 14.95 32.30 52.53 67.01 80.86 Day Standard deviation 3 4 5 6 7 Basal medium 2.60 6.39 7.17 11.01 10.42 Medium supplemented with 1.93 6.75 11.09 16.74 15.34 vitamins and the like AGK 3.95 mM 1.73 0.99 2.67 5.86 6.88 GPP 4.40 mM 0.94 1.34 5.68 13.16 11.46

[0172] From Table 35 and FIG. 33, it was found that the addition of peptides to the basal medium or the medium supplemented with vitamins and the like increased the amount of protein produced under the above-mentioned testing conditions.

[0173] In addition, in the protein production in the above-mentioned floating cell system, the CHO cells were serum-free floating, but may be acclimated by first, performing cell culture using only a serum culture medium, thereafter performing cell culture in half a serum culture medium and a serum-free culture medium, and finally performing cell culture using only a serum-free culture medium.

[0174] Furthermore, in the protein production test in the above-mentioned floating cell system, the CHO cells were used, but the culture medium containing the peptide of the present invention is also applicable to cell lines such as hybridomas, HEK293, COS, and Sf9 which are utilized to produce other substances.

[0175] The protein production method using the peptide of the present invention may include a step of fed-batch culture in which a culture medium is replenished during production, in addition to the above-mentioned batch culture.