Peptide, cell proliferation promoter, protein production promoter, culture medium, cell proliferation method using said peptide, and protein production method using said peptide

Abstract

To provide a synthetic culture medium free from animal-derived components, specifically, to provide a culture medium that contains a peptide capable of promoting cell growth and contributing to the promotion of protein production even in the absence of animal-derived components.SOLUTION: Provided is a culture medium that contains a peptide selected from a 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).SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a peptide, and in particular to a novel tripeptide suitable for animal cell culture, a cell growth promoter, a protein production promoter, and a medium containing the peptide, a cell growth method using the peptide, and a protein production method using the peptide. [Background technology]

[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-related components are added for the purpose of growing the animal cells (Patent Documents 1 and 2).

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

[0004] In addition, the addition of fish meat extracts and fish meat-related components, which are enzyme decomposition products of fish meat, solved the problems of cost and the decrease in cell viability in the early stages of culture. However, the problem of lot-to-lot quality differences 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 enzyme decomposition conditions of the target fish, so there are various unknown risks when used as a culture medium, making it difficult to use safely. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. WO99 / 63058 [Patent Document 2] JP 2003-334068 A Summary of the Invention [Problem to be solved by the invention]

[0006] The present invention aims to provide a synthetic medium that does not contain any animal-derived components, and in particular, to provide a medium that promotes cell growth and contains peptides that contribute to the promotion of protein production, even if it does not contain any animal-derived components. [Means for solving the problem]

[0007] As a result of intensive research in light of the above circumstances, the inventors have discovered a peptide that promotes cell proliferation and contributes to promoting protein production, and have also discovered a cell proliferation promoter containing said peptide, a protein production promoter containing said peptide, and a culture medium containing said peptide.

[0008] That is, the peptide of the present invention is characterized in that it 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).

[0009] The cell proliferation promoting agent of the present invention is characterized by containing one or more of the above peptides.

[0010] The protein production promoter of the present invention is characterized by containing one or more of the above peptides.

[0011] The medium of the present invention is characterized by containing the above-mentioned cell growth promoter or the above-mentioned protein production promoter.

[0012] The cell proliferation method of the present invention is characterized by using one or more of the above peptides.

[0013] The protein production method of the present invention is characterized by using one or more of the above peptides. Effect of the Invention

[0014] The peptide of the present invention can provide a cell proliferation promoter, a protein production promoter, a culture medium, a cell proliferation method, and a protein production method that are free of animal-derived components and contain chemically synthesized substances. In other words, it is possible to provide a cell proliferation promoter, a protein production promoter, and a culture medium that are free of concerns about a correlation with mad cow disease, etc., are cost-effective, and have clear component details, resulting in stable quality. [Brief description of the drawings]

[0015] [Figure 1] 1 shows the relationship between GEK concentration and viable cell count (absorbance) in a cell proliferation test. [Diagram 2] This shows the relationship between DGP concentration and the number of viable cells (absorbance) in a cell proliferation test. [Diagram 3] 1 shows the relationship between AGK concentration and viable cell number (absorbance) in a cell proliferation test. [Figure 4] This shows the relationship between the concentration of GPP and the number of viable cells (absorbance) in a cell proliferation test. [Diagram 5] This shows the relationship between GGP concentration and the number of viable cells (absorbance) in a cell proliferation test. [Figure 6] 1 shows the relationship between the AEK concentration and the number of viable cells (absorbance) in a cell proliferation test. [Figure 7] 1 shows the relationship between the AGG concentration and the number of viable cells (absorbance) in a cell proliferation test. [Figure 8] 1 shows the relationship between the ASN concentration and the number of viable cells (absorbance) in a cell proliferation test. [Figure 9] 1 shows the relationship between EGK concentration and viable cell count (absorbance) in a cell proliferation test. [Figure 10] 1 shows the relationship between the concentration of GGG and the number of viable cells (absorbance) in a cell proliferation test. [Figure 11] The number of viable cells (absorbance) for each tripeptide in a cell proliferation test is shown. [Figure 12] The figure shows the time course of the viable cell count (absorbance) for each tripeptide in a cell proliferation test. [Figure 13] The viable cell counts for each tripeptide in a 3-day cell proliferation test are shown. [Figure 14] The cell viability of each tripeptide in a 3-day cell proliferation test is shown. [Figure 15] The viable cell counts for each tripeptide in a 5-day cell proliferation test are shown. [Figure 16] The cell viability of each tripeptide in a 5-day cell proliferation test is shown. [Figure 17] The figure shows the number of viable cells for each tripeptide in a cell proliferation test using one tripeptide. [Figure 18] The figure shows the cell viability of each tripeptide in a cell proliferation test using one tripeptide. [Figure 19] The figure shows the amount of protein produced by each tripeptide in a cell proliferation test using one type of tripeptide. [Figure 20] The figures show the number of viable cells for each combination of tripeptides in a cell proliferation test using two tripeptides. [Figure 21] 1 shows the cell viability for each combination of tripeptides in a cell proliferation test using two tripeptides. [Figure 22] The figure shows the amount of protein produced by each combination of tripeptides in a cell proliferation test using two types of tripeptides. [Figure 23] The figure shows the number of viable cells for each combination of tripeptides in a cell proliferation test using three tripeptides. [Figure 24]The figure shows the cell viability for each combination of tripeptides in a cell proliferation test using three tripeptides. [Diagram 25] The figure shows the amount of protein produced by each combination of tripeptides in a cell proliferation test using three types of tripeptides. [Figure 26] The figures show the number of viable cells and the amount of protein produced by AGK in a cell proliferation test in which vitamins and nucleic acids were added. [Figure 27] This shows the relationship between the concentration of GEK and the number of viable cells in a cell proliferation test using a completely defined medium. [Figure 28] 1 shows the relationship between GEK concentration and cell viability in a cell proliferation test using a completely defined medium. [Figure 29] 1 shows the relationship between the concentration of GEK and the amount of produced protein in a cell proliferation test using a completely synthetic medium. [Diagram 30] The figures show the number of viable cells for each tripeptide in a cell proliferation test using a vitamin-supplemented medium. [Diagram 31] The cell viability of each tripeptide in a cell proliferation test using a medium supplemented with vitamins and the like is shown. [Diagram 32] The amount of protein produced by each tripeptide in a protein production test using a medium supplemented with vitamins and the like is shown. [Diagram 33] The amount of protein produced by each tripeptide in a protein production test using a medium supplemented with vitamins and the like is shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] The embodiments of the present invention will be specifically described below.

[0017] (peptide) The 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). The peptide may be converted into a pharma- ceutically acceptable salt, and the amino acids that do not change the activity of the peptide may 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 preferred. Examples of "pharmacologically acceptable salts" include inorganic acid salts such as hydrochloride, phosphate, sulfate, etc.; inorganic base salts such as sodium salt, potassium salt, calcium salt, etc.; organic acid salts such as sulfonate, succinate, oxalate, etc.; organic base salts such as alkylammonium salt, etc. "Chemically modifying an amino acid without changing the activity of the peptide" means chemically modifying an amino acid with a group that does not significantly change the activity of the peptide, and examples of such modification 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 above tripeptides were discovered through extensive research, focusing on hundreds of peptides of various lengths contained mainly in fish meat extracts and their enzymatic hydrolyses, and identifying those that promote animal cell proliferation and protein production by fractionating them under various conditions, and then confirming the effects of each peptide.

[0019] The above peptides can be obtained by means of fractionation from fish meat extracts or their enzymatic hydrolysis products, chemical synthesis including peptide synthesis, or expression by recombinant DNA techniques. In the method of fractionating fish meat extracts or their enzymatic decomposition products, the various conditions of gel filtration chromatography and normal phase / reverse phase HPLC are adjusted for fractionation and isolation. In the chemical synthesis method, peptides with specific sequences can be obtained by synthesizing synthetic amino acids or chemically modified amino acids through chemical reactions. 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] (Cell proliferation promoter) The cell proliferation promoter of the present invention contains one or more 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). The cell proliferation promoter promotes cell proliferation compared to a case not containing the one or more peptides.

[0021] The peptide may be converted into a pharma- ceutical acceptable salt, and further, the amino acid may be chemically modified without changing the activity of the peptide. The proline (Pro(P)) may be hydroxyproline (Hyp) having a hydroxyl group introduced therein.

[0022] The peptides are selected by appropriately combining one or more of the above peptides. Among these, when only one type is used, Gly-Glu-Lys (GEK), Asp-Gly-Pro (DGP), Ala-Gly-Lys (AGK), Gly-Pro-Pro (GPP), and Gly-Gly-Pro (GGP) are more preferable. In the case of two types, preferred 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), Gly-Pro-Pro (GPP) and Gly-Glu-Lys (GEK), etc. In the case of three types, preferred combinations include Gly-Pro-Pro (GPP) + Asp-Gly-Pro (DGP) + Gly-Glu-Lys (GEK), Gly-Pro-Pro (GPP) + Asp-Gly-Pro (DGP) + Ala-Gly-Lys (AGK), and Gly-Pro-Pro (GPP) + Gly-Glu-Lys (GEK) + Ala-Gly-Lys (AGK).

[0023] (Protein production promoter) The protein production promoter of the present invention contains one or more 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), and promotes protein production compared to when the one or more peptides are not contained.

[0024] The peptide may be converted into a pharma- ceutical acceptable salt, and further, the amino acid may be chemically modified without changing the activity of the peptide. The proline (Pro(P)) may be hydroxyproline (Hyp) having a hydroxyl group introduced therein.

[0025] The peptides are selected by appropriately combining one or more of the above peptides. Among these, when only one type is used, preferred examples of peptides include Gly-Glu-Lys (GEK), Asp-Gly-Pro (DGP), Ala-Gly-Lys (AGK), Gly-Pro-Pro (GPP), and Gly-Gly-Pro (GGP). In the case of two types, preferred 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), Gly-Pro-Pro (GPP) and Gly-Glu-Lys (GEK), etc. In the case of three types, preferred combinations include Gly-Pro-Pro (GPP) + Asp-Gly-Pro (DGP) + Gly-Glu-Lys (GEK), Gly-Pro-Pro (GPP) + Asp-Gly-Pro (DGP) + Ala-Gly-Lys (AGK), and Gly-Pro-Pro (GPP) + Gly-Glu-Lys (GEK) + Ala-Gly-Lys (AGK).

[0026] (Culture medium) The medium of the present invention contains the cell growth promoter containing the peptide, or the protein production promoter containing the peptide.

[0027] The concentration of the peptide in the medium is appropriately set depending on the cells and culture conditions. That is, the lower limit of the amount of peptide in the medium is the concentration at which the cells can be maintained alive, the preferred concentration is the concentration at which the amount of cell growth or protein production is maximized compared to a medium to which a cell growth promoter or a protein production promoter is not added, and the upper limit is the maximum concentration at which the composition of the medium is not harmful. An example of the concentration per peptide is 0.1 mM to 50 mM, preferably 0.2 mM to 10 mM, more preferably 0.5 mM to 5 mM, relative to the medium.

[0028] The medium may contain other components used in animal cell culture media, such as vitamins, nucleic acids, amino acids, inorganic salts, sugars, polyamines, carbohydrates, proteins, fatty acids, lipids, pH adjusters, zinc, copper, and selenium. 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, and the like. Examples of nucleic acids include xanthine, hypoxanthine, uridine, guanine hydrochloride, inosine, guanosine, cytidine, thymidine, adenine, and the like. 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, and arginine. Examples of inorganic salts include calcium chloride, magnesium sulfate, potassium chloride, sodium hydrogen carbonate, sodium chloride, sodium dihydrogen phosphate monohydrate, and the like. Other components include D-glucose, α-lipoic acid, phenolsulfonphthalein (phenol red), sodium pyruvate, AlbuMax® 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 included depending on the purpose, but are not included when it is intended to remove animal-derived components from the medium.

[0029] (Methods for cell proliferation and protein production) The cell growth method and protein production method of the present invention are carried out by adding the peptide of the present invention to the above-mentioned medium and culturing various animal cells. Exemplary cell growth methods and protein production methods are provided below, but are not limited thereto.

[0030] The animal cells are adapted to serum-free suspension using a basal medium. One or more 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 that supplement the basal medium, may also be added. Using a bioreactor, animal cells acclimatized to the basal medium are seeded on the basal medium supplemented with the peptide, and cell growth and protein production are carried out. EXAMPLES

[0031] The present invention will now be described in detail with reference to examples, but the present invention is not limited to these examples.

[0032] (Measurement of the relationship between the concentration of each peptide solution and the number of viable cells) Peptides having the sequences 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), Glu-Gly-Lys (EGK), and Gly-Gly-Gly (GGG) were synthesized, and peptide solutions were prepared at 10-fold concentrations as shown in Tables 1 to 10. CHO-K1 (RIKEN BioResource Research Center, model number RCB2330) was cultured at 3x10 4The cell suspension adjusted to 100 cells / mL was seeded onto a 96-well plate at 100 μL / well and cultured for 24 hours in an incubator at 37°C and 5% CO2. MEMα medium (Gibco) containing 10% FBS was used as the medium. After removing the medium from each well and washing with MEMα medium (100 μL), 90 μL of fresh MEMα medium was dispensed and each peptide solution (10 μL) was added (total 100 μL / well) to the final concentration shown in Tables 1 to 10 within the range of 0 mM to 5 mM, and cultured for 5 days. As a comparison sample, a similar culture test was performed using a system containing neither peptide nor FBS. After culturing for 5 days, 10 μL / well of the viable cell count measurement reagent SF (Nacalai Tesque) was added, and the color reaction was performed for 2 hours in an incubator at 37°C and 5% CO2, and the absorbance at 450 nm was measured using a plate reader. The reference wavelength was 630 nm. It has been confirmed that the absorbance at 450 nm correlates with the cell number.

[0033] The above preparation, incubation, and measurement were carried out in triplicate, and the absorbance at each peptide solution concentration is shown in Tables 1 to 10 and Figures 1 to 10.

[0034] [Table 1]

[0035] [Table 2]

[0036] [Table 3]

[0037] [Table 4]

[0038] [Table 5]

[0039] [Table 6]

[0040] [Table 7]

[0041] [Table 8]

[0042] [Table 9]

[0043] [Table 10]

[0044] From Tables 1 to 10 and Figures 1 to 10, it was found that, for the peptides tested, except for GGG, the addition of peptide solution increased the cell count compared to the absence of addition. It was also found that the concentration of peptide solution at which the cell count increased most differed depending on the type of peptide.

[0045] Furthermore, to compare the degree of cell proliferation promotion when the peptide solution concentration was optimized, the absorbance at the peptide solution concentration that maximized the cell number for each peptide tested is summarized in Table 11 and Figure 11.

[0046] [Table 11]

[0047] Under the above test conditions, it was found that cell proliferation was promoted in the following order of peptide sequences: 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).

[0048] (Measurement of the relationship between cell proliferation days and cell number for each peptide) Peptides having the sequences Gly-Pro-Pro (GPP), Asp-Gly-Pro (DGP), Gly-Glu-Lys (GEK), and Ala-Gly-Lys (AGK) were synthesized, and peptide solutions were prepared so that the concentration that would increase the number of cells the most was within the range of 0 mM to 5 mM. That is, Gly-Pro-Pro (GPP) was prepared at 11 mM, Asp-Gly-Pro (DGP) at 22 mM, Gly-Glu-Lys (GEK) at 21 mM, and Ala-Gly-Lys (AGK) at 20 mM, and 1 / 10 of the total medium volume of each well was added immediately before the cell culture test, resulting in final concentrations of 1.1 mM, 2.2 mM, 2.1 mM, and 2.0 mM, respectively. CHO-K1 (RIKEN BioResource Research Center, model number RCB2330) was cultured at 3x10 4 The cell suspension adjusted to 100 cells / mL was seeded onto a 96-well plate at 100 μL / well and cultured for 24 hours in an incubator at 37°C and 5% CO2. MEMα medium (Gibco) containing 10% FBS was used as the medium. After removing the medium from each well and washing with MEMα medium (100 μL), 90 μL of fresh MEMα medium was dispensed and each peptide solution (10 μL) was added (total 100 μL / well) and cultured for 0 to 5 days. Every day, 10 μL / well of the viable cell count measurement reagent SF (Nacalai Tesque) was added, and the color reaction was allowed to occur for 2 hours in an incubator at 37°C and 5% CO2, and the absorbance at 450 nm was measured using a plate reader. The reference wavelength was 630 nm. It has been confirmed that the absorbance at 450 nm correlates with the cell number. As a comparison sample, a similar culture test was performed using a system that did not contain peptide or FBS.

[0049] The above preparation, culture, and measurement were performed in triplicate, n=3, and the absorbance and standard deviation of each peptide solution on each day are shown in Table 12 and FIG.

[0050] [Table 12]

[0051] From Table 12 and FIG. 12, it was found that under the above test conditions, the cell count increased over time by adding the peptide solution compared to the system without the addition of the peptide solution.

[0052] (Cell proliferation test on each peptide coating agent - 3 days -) Peptides having the sequences Gly-Glu-Lys (GEK) and Asp-Gly-Pro (DGP) were synthesized, and peptide solutions were prepared so that the concentration at which the number of cells was most increased was within the range of 0 mM to 5 mM. That is, Gly-Glu-Lys (GEK) was adjusted to 21 mM, and Asp-Gly-Pro (DGP) was adjusted to 22 mM, and 1 / 10 of the total medium volume of each well was added immediately before the cell culture test, resulting in final concentrations of 2.1 mM and 2.2 mM, respectively. Poly-L-lysine (Peptide Institute, Poly-L-lysine hydrochloride, model number 3075) was prepared to 0.1 mg / mL, and 200 μL was dispensed into each well of a 24-well plate and allowed to stand for 2 hours in an incubator at 37°C. After removing the remaining liquid with an aspirator, the plate was rinsed with distilled water and irradiated with a UV lamp in a clean bench without a lid on, overnight to dry and sterilize. CHO-K1 (RIKEN BioResource Research Center, model number RCB2330) was cultured at 4x10 4 The cell suspension adjusted to 500 cells / mL was seeded onto a 24-well plate at 500 μL / well and cultured for 24 hours in an incubator at 37°C and 5% CO2. MEMα medium (Gibco) containing 10% FBS was used as the medium. After removing the medium from each well and washing with MEMα medium (500 μL), 450 μL of fresh MEMα medium was dispensed and each peptide solution (50 μL) was added (total 500 μL / well) and cultured for 3 days. The cells were collected and counted. As a comparison sample, a similar culture test was performed using a system that did not contain peptide or FBS.

[0053] The medium in each well was collected in a 1.5 mL tube, rinsed with 200 μL of MEMα, and the rinsed liquid was also collected in the same 1.5 mL tube. Then, 100 μL of trypsin was added and incubated for 3 minutes. The well was rinsed with 300 μL of MEMα containing 10% FBS, and the rinsed liquid was also collected in the same 1.5 mL tube. The well was rinsed again with 200 μL of MEMα containing 10% FBS, and the rinsed liquid was also collected in the same 1.5 mL tube.

[0054] The cells collected in the 1.5 mL tube were centrifuged at 1000 rpm for 10 minutes at 4°C. The supernatant was removed, 300 μL of cold PBS (phosphate buffered saline) was added, and centrifugation was performed under the same conditions. This operation was repeated twice. The cells were suspended in 100 μL of binding buffer, 2 μL of PI (propidium iodide) was added, the tube was stirred, and the reaction was allowed to proceed at room temperature in the dark for 15 minutes. After that, the number of live cells and the survival rate were measured using a flow cytometer.

[0055] The above preparation, culture, and measurement were performed in triplicate. The number of live cells in each peptide solution is shown in Table 13 and FIG. 13, and the cell viability in each peptide solution is shown in Table 14 and FIG.

[0056] [Table 13]

[0057] [Table 14]

[0058] From Table 13, Table 14, Figures 13 and 14, it was found that in the peptide culture on the coating agent under the above test conditions for 3 days, the number of live cells increased and the cell survival rate was also high compared to the system without addition of peptide.

[0059] (Cell proliferation test on each peptide coating agent - 5 days -) Peptides having the sequences Asp-Gly-Pro (DGP), Ala-Gly-Lys (AGK), Gly-Glu-Lys (GEK), and Gly-Gly-Gly (GGG) were synthesized, and peptide solutions were prepared so that the concentration at which the number of cells was most increased was within the range of 0 mM to 5 mM. That is, Asp-Gly-Pro (DGP) was prepared at 22 mM, Ala-Gly-Lys (AGK) at 20 mM, Gly-Glu-Lys (GEK) at 21 mM, and Gly-Gly-Gly (GGG) at 25 mM, and 1 / 10 of the total medium volume of each well was added immediately before the cell culture test, and the final concentrations were 2.2 mM, 2.0 mM, 2.1 mM, and 2.5 mM, respectively. Poly-L-lysine (Peptide Institute, Poly-L-lysine hydrochloride, model number 3075) was prepared to 0.1 mg / mL, and 200 μL was dispensed into each well of a 24-well plate and allowed to stand for 2 hours in an incubator at 37°C. After removing the remaining liquid with an aspirator, the plate was rinsed with distilled water and irradiated with a UV lamp in a clean bench without a lid on, overnight to dry and sterilize. CHO-K1 (RIKEN BioResource Research Center, model number RCB2330) was cultured at 4x10 4 The cell suspension adjusted to 100 cells / mL was seeded into each well of a 24-well plate at 500 μL / well and cultured for 24 hours in an incubator at 37°C and 5% CO2. MEMα medium (Gibco) containing 10% FBS was used as the medium. The medium in each well was removed, and the wells were washed with MEMα medium (500 μL). Then, 450 μL of fresh MEMα medium was dispensed and each peptide solution (50 μL) was added (total 500 μL / well), and the wells were cultured for 5 days. The cells were collected and counted. As a comparison sample, a similar culture test was performed using a system that did not contain peptide or FBS.

[0060] The medium in each well was collected in a 1.5 mL tube, rinsed with 200 μL of MEMα, and the rinsed liquid was also collected in the same 1.5 mL tube. Then, 100 μL of trypsin was added and incubated for 3 minutes. The well was rinsed with 300 μL of MEMα containing 10% FBS, and the rinsed liquid was also collected in the same 1.5 mL tube. The well was rinsed again with 200 μL of MEMα containing 10% FBS, and the rinsed liquid was also collected in the same 1.5 mL tube.

[0061] The cells collected in the 1.5 mL tube were centrifuged at 1000 rpm for 10 minutes at 4°C. The supernatant was removed, 300 μL of cold PBS (phosphate buffered saline) was added, and centrifugation was performed under the same conditions. This operation was repeated twice. The cells were suspended in 100 μL of binding buffer, 2 μL of PI (propidium iodide) was added, the tube was stirred, and the reaction was allowed to proceed at room temperature in the dark for 15 minutes. After that, the number of live cells and cell viability were measured using a flow cytometer.

[0062] The above preparation, culture, and measurement were performed in triplicate. The number of live cells in each peptide solution is shown in Table 15 and FIG. 15, and the cell survival rate in each peptide solution is shown in Table 16 and FIG.

[0063] [Table 15]

[0064] [Table 16]

[0065] From Table 15, Table 16, Figures 15 and 16, it was found that in the peptide culture on the coating agent under the above test conditions for 5 days, the cell number increased and the cell survival rate was higher than in the system without addition of peptide, except for GGG.

[0066] (Cell proliferation tests and protein production tests on coating agents using each peptide and combinations of peptides) Peptides having the sequences Gly-Pro-Pro (GPP), Asp-Gly-Pro (DGP), Gly-Glu-Lys (GEK), and Ala-Gly-Lys (AGK) were synthesized, and peptide solutions were prepared so that the concentration that would increase the number of cells the most was within the range of 0 mM to 5 mM. That is, Gly-Pro-Pro (GPP) was prepared at 11 mM, Asp-Gly-Pro (DGP) at 22 mM, Gly-Glu-Lys (GEK) at 21 mM, and Ala-Gly-Lys (AGK) at 20 mM, and 1 / 10 of the total medium volume of each well was added immediately before the cell culture test, resulting in final concentrations of 1.1 mM, 2.2 mM, 2.1 mM, and 2.0 mM, respectively. Poly-L-lysine (Peptide Institute, Poly-L-lysine hydrochloride, model number 3075) was prepared to 0.1 mg / mL, dispensed in 200 μL portions into a 24-well plate, and left to stand in an incubator at 37°C for 2 hours. After removing the remaining liquid with an aspirator, the plate was rinsed with distilled water and irradiated with a UV lamp in a clean bench without a lid overnight to dry and sterilize. CHO DP-12 (ATCC, Cat. No. CRL-12445), 2x10 4 The cell suspension was prepared so that the cells / well was 500μL, and then seeded on a 24-well plate and cultured for 24 hours in an incubator at 37℃ and 5% CO2. The medium used was DMEM basal medium (Gibco) containing 10% FBS, 200nM methotrexate, and 2μg / mL insulin. The medium in each well was removed and washed with DMEM basal medium (500 μL), after which 450 μL of fresh DMEM basal medium was dispensed and each peptide solution (50 μL) was added (total 500 μL / well), followed by incubation for 5 days. The peptides used were GPP, DGP, GEK, and AGK alone, as well as combinations of GPP+GEK, GPP+AGK, DGP+GEK, GEK+AGK, DGP+AGK, GPP+GEK+AGK, GPP+DGP+AGK, and GPP+DGP+GEK. As a comparison sample, a similar culture test was performed using a system that did not contain peptides or FBS.

[0067] To measure the amount of produced protein, 100 μL of the culture supernatant was collected in a 1.5 mL tube, diluted, and the amount of produced protein was quantified by ELISA.

[0068] To collect the cells, the medium in each well was collected in a 1.5 mL tube, rinsed with 200 μL of DMEM basal medium, and the rinsed liquid was also collected in the same 1.5 mL tube. Then, 100 μL of trypsin was added and incubated for 3 minutes. The cells were rinsed with 300 μL of 10% FBS-containing DMEM basal medium, and the rinsed liquid was also collected in the same 1.5 mL tube. The cells were rinsed again with 200 μL of 10% FBS-containing DMEM basal medium, and the rinsed liquid was also collected in the same 1.5 mL tube.

[0069] The cells collected in the 1.5 mL tube were centrifuged at 1000 rpm for 10 minutes at 4°C. The supernatant was removed, 300 μL of cold PBS (phosphate buffered saline) was added, and centrifugation was performed under the same conditions. This operation was repeated twice. The cells were suspended in 100 μL of binding buffer, 2 μL of PI (propidium iodide) was added, the tube was stirred, and the reaction was allowed to proceed at room temperature in the dark for 15 minutes. After that, the number of live cells and cell viability were measured using a flow cytometer.

[0070] The above preparation, culture, and measurement were performed n=3, and the number of viable cells in each peptide solution is shown in Table 17, Figure 17, Table 20, Figure 20, Table 23, and Figure 23. The cell viability in each peptide solution is shown in Table 18, Figure 18, Table 21, Figure 21, Table 24, and Figure 24. The amount of protein produced in each peptide solution is shown in Table 19, Figure 19, Table 22, Figure 22, Table 25, and Figure 25.

[0071] [Table 17]

[0072] [Table 18]

[0073] [Table 19]

[0074] [Table 20]

[0075] [Table 21]

[0076] [Table 22]

[0077] [Table 23]

[0078] [Table 24]

[0079] [Table 25]

[0080] From Tables 17 to 25 and Figs. 17 to 25, it was found that under the above test conditions, the addition of a single peptide or a combination of two or more peptides promotes cell proliferation and protein production.

[0081] (Cell proliferation test and protein production test for each peptide - Effect of adding vitamins and nucleic acids -) A peptide having the sequence Ala-Gly-Lys (AGK) was synthesized, and 1.0 mM and 2.0 mM peptide solutions were prepared. CHO DP-12 (ATCC, Cat. No. CRL-12445), 2x10 4 The cell suspension was adjusted to 1x10 cells / mL. 4The cells were seeded in a 24-well plate at 500 μL / well and cultured for 24 hours in an incubator at 37°C and 5% CO2. The medium used was DMEM basal medium (Gibco) containing 10% FBS, 200 nM methotrexate, and 2 μg / mL insulin. The medium in each well was removed and washed with DMEM basal medium (500 μL), after which each peptide solution (500 μL) was dispensed into each well (total 500 μL / well) and cultured for 5 days.

[0082] After replacing the medium with the evaluation medium for each of the following test groups, the plants were cultured in an incubator at 37°C and 5% CO2 for 5 days. <Evaluation medium> ·DMEM basal medium ·AGK(1mM)+DMEM basal medium ·AGK(2mM)+DMEM basal medium Vitamin and nucleic acid supplemented medium (DMEM basal medium + vitamins + nucleic acids) ·AGK(1mM) + vitamin and nucleic acid supplemented medium ·AGK(2mM) + vitamin and nucleic acid supplemented medium

[0083] After 5 days of culture, the entire medium in each well of the 24-well plate was collected and centrifuged (5000 rpm, 5 minutes), and the supernatant was collected separately to measure the amount of produced protein by ELISA.

[0084] The composition of the above vitamins and nucleic acids is shown in Table 26.

[0085] [Table 26]

[0086] After medium collection, the cells adhering to the wells were detached by trypsin treatment and suspended again in DMEM basal medium containing 10% FBS, and the number of live cells and viability were measured by trypan blue staining using a cell counter.

[0087] The above preparation, culture, and measurement were performed in triplicate. The viable cell count, cell viability, and amount of protein produced in each evaluation medium are shown in Table 27, and the viable cell count and amount of protein produced are shown in FIG.

[0088] [Table 27]

[0089] From Table 27 and Figure 26, it was found that under the above test conditions, the addition of peptides to the basal medium increased both the number of live cells and the amount of protein produced, but the further addition of vitamins and nucleic acids further promoted both cell proliferation and protein production.

[0090] (Cell proliferation test and protein production test when peptides are added to a completely synthetic medium)

[0091] A peptide having a Gly-Glu-Lys (GEK) sequence was synthesized, and peptide solutions with concentrations of 0 mM, 2.6 mM, 5.1 mM, 10 mM, 20.5 mM, and 41 mM were prepared. Just before the cell culture test, 1 / 10 of the total medium volume of each well was added to give final concentrations of 0 mM, 0.26 mM, 0.51 mM, 1.0 mM, 2.05 mM, and 4.1 mM, respectively. For this test, CHO DP-12 (ATCC, model number CRL-12445) was used, which had been acclimatized to ASF104 basal medium (Ajinomoto), a fully synthetic medium for CHO, containing 200 nM methotrexate and 2 μg / mL insulin. The cell concentration was 4 x 10 4 The cell suspension adjusted to 100 cells / mL was seeded onto a 24-well plate at 450 μL / well and cultured for 24 hours in an incubator at 37°C and 5% CO2. The medium used was ASF104 basal medium, which is a fully synthetic medium for CHO, ASF104 medium (Ajinomoto), supplemented with 200 nM methotrexate and 2 μg / mL insulin.

[0092] After 24 hours, 50 μL of the prepared peptide solution was added to each well and cultured for 5 days. After culture, the cells were harvested and the number of viable cells was counted. <Evaluation medium> ASF104 basal medium ·GEK(0.26mM)+ASF104 basal medium ·GEK(0.51mM)+ASF104 basal medium ·GEK(1.0mM) +ASF104 basal medium ·GEK(2.05mM)+ASF104 basal medium ·GEK(4.1mM) +ASF104 basal medium

[0093] To quantify the amount of produced protein, 100 μL of the culture supernatant was collected in a 1.5 mL tube, diluted, and the amount of produced protein was measured by ELISA.

[0094] To analyze the cells, the medium in each well was collected in a 1.5 mL tube, rinsed with 200 μL of PBS, and the rinsed liquid was collected in the same 1.5 mL tube. Then, 100 μL of 0.25% trypsin / EDTA was added and incubated for 1 minute. 100 μL of trypsin inhibitor was added and collected in the same 1.5 mL tube. The cells were rinsed with 200 μL of PBS, and the rinsed solution was also collected in the same 1.5 mL tube and centrifuged. The cells were suspended in 100 μL of PBS, and the viable cell count and cell viability were measured by trypan blue staining using a cell counter.

[0095] The above preparation, culture, and measurement were performed in triplicate. The number of viable cells in each peptide solution is shown in Table 28 and FIG. 27. The cell viability in each peptide solution is shown in Table 29 and FIG. 28. The amount of produced protein is shown in Table 30 and FIG. 29.

[0096] [Table 28]

[0097] [Table 29]

[0098] [Table 30]

[0099] As can be seen from Tables 28 to 30 and Figures 27 to 29, under the above test conditions, when GEK peptides were added, the number of live cells increased, the cell viability was equally high, and the amount of protein produced tended to increase compared to a basal medium using a commercially available completely synthetic medium, thereby promoting cell proliferation and protein production.

[0100] (Cell proliferation and protein production assays of GEK and DGP in suspension cell lines) Peptides having the sequences Gly-Glu-Lys (GEK) and Asp-Gly-Pro (DGP) were synthesized, and peptide solutions of 2.87 mM Gly-Glu-Lys (GEK) and 1.55 mM Asp-Gly-Pro (DGP) were prepared.

[0101] 5 mL of DMEM / F12 basal medium, vitamin supplemented medium (DMEM / F12 basal medium + vitamin supplements), and each peptide solution were added to each well of a 24-well (deep well) cassette of a Micro-24 bioreactor system (manufactured by Nippon Pall Corporation), and cultured overnight at 37°C, pH 7, and agitation speed of 650 rpm. pH calibration was performed the next day, and then 3.5x10 5 Add 2 mL of cell suspension adjusted to 10 cells / mL to each well of a 24-well (deep well) cassette until the cell density of the suspended cells reaches 1 x 10 5 The cells were seeded at 7 cells / mL (7 mL / well) and cultured in the following evaluation medium under the following culture conditions: 37°C, pH 7, stirring speed 650 rpm, and dissolved oxygen 30%. <Evaluation medium> ·DMEM / F12 basal medium Vitamin supplemented medium (basal medium + vitamin supplements) ·GEK (2.05mM) + vitamin supplemented medium ·DGP (1.11mM) + vitamin supplemented medium The medium used was DMEM / F12 basal medium (Gibco) supplemented with 200 nM methotrexate, 10 μg / mL insulin, 5.5 μg / mL transferrin, 6.7 ng / mL sodium selenite, 10 μL / mL Anti-Clumping Agent, and 10 μL / mL 10% Pluronic F68.

[0102] The suspension cells used were serum-free suspended CHO DP-12 (ATCC, model number CRL-12445) that had been adapted to serum-free suspension and subcultured in a shaking culture device (Custom Bio Shaker CO2-BR-43FL, Taitec) using a 100 mL Erlenmeyer flask under culture conditions of 37°C, 5% CO2, and an agitation speed of 125 rpm.

[0103] The above vitamin and other supplementary ingredients are shown in Table 31.

[0104] [Table 31]

[0105] After the third day of culture, 200 μL of medium was collected from each well of the 24-well (deep well) cassette into a 1.5 mL tube. 50 μL of the medium was transferred to another 1.5 mL tube, and 50 μL of trypan blue was added and thoroughly suspended, after which the number of viable cells and viability were measured using a cell counter (Countess II, Life Technologies).

[0106] The above preparation, culture, and measurement were performed on n=2 or 3, and the viable cell count in each evaluation medium is shown in Table 32 and Figure 30. The survival rate in each evaluation medium is shown in Table 33 and Figure 31.

[0107] [Table 32]

[0108] [Table 33]

[0109] From Table 32, FIG. 30, Table 33, and FIG. 31, it was found that under the above test conditions, when peptides were added to the basal medium or the medium supplemented with vitamins, etc., both the number of viable cells and the survival rate increased.

[0110] From the third day of culture onwards, 200 μL of medium was collected from each well of the 24-well (deep well) cassette, and 150 μL was centrifuged (5000 rpm, 5 minutes) in a 1.5 mL tube, and the supernatant was collected separately and the amount of produced protein was measured by ELISA. The measured amounts of produced proteins are shown in Table 34 and FIG.

[0111] [Table 34]

[0112] From Table 34 and FIG. 32, it was found that the amount of produced protein increased when peptides were added to the basal medium or the medium enriched with vitamins, etc.

[0113] (Protein production test of AGK and GPP in suspension cell system) Peptides having the sequences Ala-Gly-Lys (AGK) and Gly-Pro-Pro (GPP) were synthesized, and peptide solutions of 5.53 mM Ala-Gly-Lys (AGK) and 6.16 mM Gly-Pro-Pro (GPP) were prepared.

[0114] 5 mL of basal medium, vitamin supplemented medium (basal medium + vitamin supplements), and each peptide solution were added to each well of a 24-well (deep well) cassette of a Micro-24 bioreactor system (manufactured by Nippon Pall Corporation), and cultured overnight at 37°C, pH 7, and 650 rpm. pH calibration was performed the next day, and then 3.5x105 Add 2 mL of cell suspension adjusted to 10 cells / mL to each well of a 24-well (deep well) cassette until the cell density of the suspended cells reaches 1 x 10 5 The cells were seeded at 7 cells / mL (7 mL / well) and cultured in the following evaluation medium under the following culture conditions: 37°C, pH 7, 650 rpm, and dissolved oxygen 30%. <Evaluation medium> Basal medium Vitamin supplemented medium (basal medium + vitamin supplements) ·AGK (3.95mM) + vitamin supplemented medium ·GPP(4.40mM) + vitamin supplemented medium The medium used was a basal medium containing DMEM / F12 medium (Gibco) supplemented with 200 nM methotrexate, 10 μg / mL insulin, 5.5 μg / mL transferrin, 6.7 ng / mL sodium selenite, 10 μL / mL Anti-Clumping Agent, and 10 μL / mL 10% Pluronic F68.

[0115] The suspension cells used were serum-free suspended CHO DP-12 (ATCC, model number CRL-12445) that had been adapted to serum-free suspension and subcultured in a shaking culture device (Custom Bio Shaker CO2-BR-43FL, Taitec) using a 100 mL Erlenmeyer flask under culture conditions of 37°C, 5% CO2, and an agitation speed of 125 rpm.

[0116] The above vitamin and other supplementary ingredients are shown in Table 31.

[0117] After the third day of culture, 150 μL of medium was collected from each well of the 24-well (deep well) cassette into a 1.5 mL tube and centrifuged (5000 rpm, 5 minutes). The supernatant was collected and the amount of produced protein was measured by ELISA. The measured amounts of produced proteins are shown in Table 35 and FIG.

[0118] [Table 35]

[0119] From Table 35 and FIG. 33, it was found that under the above test conditions, the amount of produced protein increased when peptides were added to the basal medium or the medium supplemented with vitamins, etc.

[0120] In the above-mentioned protein production in a suspension cell system, CHO cells are suspended in serum-free medium. Alternatively, the cells may be cultured first using only serum medium, then using half serum medium and half serum-free medium, and finally using only serum-free medium to culture the cells for acclimatization.

[0121] In addition, although CHO cells were used in the above-mentioned protein production test in a suspension cell system, the medium containing the peptide of the present invention can also be applied to cell lines such as hybridomas, HEK293, COS, and Sf9 that are used for producing other substances.

[0122] The method for producing a protein using the peptide of the present invention may include, in addition to the batch culture described above, a step of fed-batch culture in which the medium is replenished during production.

Claims

1. A cell proliferation promoter for culture media containing Gly-Glu-Lys (GEK) as an active ingredient.

2. A protein production promoter for culture media containing Gly-Glu-Lys (GEK) as an active ingredient.

3. A method for growing cells, except within the human body, comprising the step of adding the cell growth promoting agent for culture medium described in claim 1 to a culture medium.

4. A method for promoting protein production outside the human body, comprising the step of adding the medium protein production promoter described in claim 2 to a medium.

5. A method for promoting protein production except within the human body as described in claim 4, wherein the culture method used is batch culture or fed-batch culture.

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