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 containing the peptide, a protein production promoter, a medium, 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, mammalian extracts such as fetal bovine serum are added to culture media at a rate of approximately 5% to 20%, accounting for 75% to 95% of the cost of the culture media, and there are problems with the quality of the extracts being animal-derived. Furthermore, due to concerns about a correlation with mad cow disease, bovine spongiform encephalopathy, transmissible spongiform encephalopathy, Creutzfeldt-Jakob disease, etc., attempts have been made to develop media that do not contain mammalian extracts such as fetal bovine serum. However, this resulted in a significant decrease in cell viability early in the culture, making long-term and large-scale culture difficult.

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

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

[0006] An object of the present invention is to provide a synthetic culture medium that does not contain any animal-derived components, and in particular, to provide a culture medium that contains peptides that promote cell growth and contribute to the promotion of protein production, even without containing any animal-derived components. [Means for solving the problem]

[0007] In light of the above circumstances, the present inventors conducted extensive research and discovered a peptide that promotes cell proliferation and contributes to promoting protein production, as well as 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 promoter 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. [Effects of the Invention]

[0014] The peptides 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 from concerns about a correlation with mad cow disease, etc., are cost-effective, and have stable quality due to the detailed components being clearly defined. [Brief explanation of the drawings]

[0015] [Figure 1] 1 shows the relationship between GEK concentration and viable cell count (absorbance) in a cell proliferation test. [Figure 2] This shows the relationship between DGP concentration and viable cell count (absorbance) in a cell proliferation test. [Figure 3] 1 shows the relationship between AGK concentration and viable cell count (absorbance) in a cell proliferation test. [Figure 4] 1 shows the relationship between GPP concentration and viable cell count (absorbance) in a cell proliferation test. [Figure 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 concentration of AEK and the number of viable cells (absorbance) in a cell proliferation test. [Figure 7] 1 shows the relationship between AGG concentration and viable cell count (absorbance) in a cell proliferation test. [Figure 8] 1 shows the relationship between the concentration of ASN 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] This 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 graph shows the time course of the number of viable cells (absorbance) for each tripeptide in a cell proliferation test. [Figure 13] The figures show the number of viable cells for each tripeptide in a 3-day cell proliferation test. [Figure 14] The cell viability of each tripeptide in a 3-day cell proliferation test is shown. [Figure 15] The figures show the number of viable cells for each tripeptide in a 5-day cell proliferation test. [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 figure shows the number of viable cells for each combination of tripeptides in a cell proliferation test using two tripeptides. [Figure 21] The figure shows the cell viability of each combination of tripeptides in a cell proliferation test using two tripeptides. [Figure 22] The figure shows the amount of protein produced for each combination of tripeptides in a cell proliferation test using two 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 of each tripeptide combination in a cell proliferation test using three tripeptides. [Figure 25] The figure shows the amount of protein produced by each combination of tripeptides in a cell proliferation test using three tripeptides. [Figure 26] The figures show the number of viable AGK cells and the amount of protein produced in a cell proliferation test with the addition of vitamins and nucleic acids. [Figure 27] 1 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 defined medium. [Figure 30] The figures show the number of viable cells for each tripeptide in a cell proliferation test using a medium supplemented with vitamins and the like. [Figure 31] The cell viability of each tripeptide in a cell proliferation test using a medium supplemented with vitamins and the like is shown. [Figure 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. [Figure 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 INVENTION

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

[0017] (peptide) The peptides of the present invention are 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 peptides may be converted into pharmaceutically acceptable salts, and amino acids may be chemically modified without affecting the activity of the peptide. Among these, 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 "pharmaceutically 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 compound that does not significantly change the activity of the peptide, and examples of such compounds 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 the result of extensive research, focusing on the hundreds of peptides of various lengths contained primarily in fish meat extracts and their enzymatic hydrolysates. Those that promote animal cell proliferation and protein production were identified by fractionating them under various conditions, and the effects of each peptide were confirmed.

[0019] The above peptides can be obtained by fractionation from fish meat extracts or their enzymatic hydrolysates, by chemical synthesis including peptide synthesis, or by expression using recombinant DNA technology. Methods for fractionating fish extracts or their enzymatic hydrolysis products involve adjusting various conditions for gel filtration chromatography and normal-phase / reverse-phase HPLC to fractionate and isolate peptides. Chemical synthesis involves chemically synthesizing synthetic or chemically modified amino acids to obtain peptides with specific sequences. Recombinant DNA methods involve producing recombinant proteins containing multiple peptide sequences using recombinant organisms. After purifying these proteins, the desired peptides can be obtained by decomposing them using enzymes or chemical treatments.

[0020] (Cell proliferation promoter) The cell proliferation-promoting agent 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 cell proliferation compared to a composition that does not contain the one or more peptides.

[0021] The peptides may be converted into pharmaceutically acceptable salts, and the amino acids may be chemically modified without changing the activity of the peptides. The proline (Pro(P)) may be converted into hydroxyproline (Hyp) by introducing a hydroxyl group.

[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 preferred. 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), and Gly-Pro-Pro (GPP) and Gly-Glu-Lys (GEK). 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 comprises 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 a composition that does not contain the one or more peptides.

[0024] The peptides may be converted into pharmaceutically acceptable salts, and the amino acids may be chemically modified without changing the activity of the peptides. The proline (Pro(P)) may be converted into hydroxyproline (Hyp) by introducing a hydroxyl group.

[0025] The peptides are selected by appropriately combining one or more of the above peptides. Among these, when 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), and Gly-Pro-Pro (GPP) and Gly-Glu-Lys (GEK). 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 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 cells can be maintained viable, the preferred concentration is the concentration at which the amount of cell growth or protein production is maximized compared to a medium to which no cell growth promoter or protein production promoter has been added, and the upper limit is the maximum concentration that is not harmful to the medium composition. An example of the concentration per peptide is 0.1 mM to 50 mM, preferably 0.2 mM to 10 mM, and more preferably 0.5 mM to 5 mM, relative to the medium.

[0028] The medium can 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, and sodium dihydrogen phosphate monohydrate. Other components include D-glucose, α-lipoic acid, phenolsulfonephthalein (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 added depending on the purpose, but they should not be added if it is intended to remove animal-derived components from the medium.

[0029] (Method 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. Examples of cell growth methods and protein production methods are given 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 a basal medium. At this time, supplemental components of the basal medium, such as vitamins, nucleic acids, sugars, polyamines, and amino acids, 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. [Example]

[0031] The present invention will now be described in detail with reference to examples, but these examples are not intended to limit the scope of the present invention.

[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 3 x 10 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% CO. MEM alpha medium (Gibco) containing 10% FBS was used. The medium was removed from each well and washed with MEMα medium (100 μL). 90 μL of fresh MEMα medium was then dispensed into each well, and 10 μL of each peptide solution was added (total 100 μL / well) to achieve final concentrations ranging from 0 mM to 5 mM as listed in Tables 1 to 10. The cells were then cultured for 5 days. A similar culture test was also performed using a control system containing neither peptide nor FBS. After 5 days of culture, 10 μL of viable cell count reagent SF (Nacalai Tesque) was added per well. The color reaction was allowed to develop for 2 hours in an incubator at 37°C and 5% CO2. 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] Tables 1 to 10 and Figures 1 to 10 show that, for the tested peptides, except for GGG, the addition of peptide solution increased the cell count compared to when no peptide solution was added. Furthermore, it was found that the concentration of peptide solution that maximized the cell count varied 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), Glu-Gly-Lys (EGK).

[0048] (Measurement of the relationship between cell proliferation days and cell number for each peptide) Peptides with the sequences Gly-Pro-Pro (GPP), Asp-Gly-Pro (DGP), Gly-Glu-Lys (GEK), and Ala-Gly-Lys (AGK) were synthesized and prepared in a concentration range of 0 to 5 mM to maximize cell numbers. Specifically, 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. Just before cell culture testing, 1 / 10 of the total medium volume was added to each well to achieve 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 3 x 10 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% CO. MEM alpha medium (Gibco) containing 10% FBS was used. The medium was removed from each well and washed with MEMα medium (100 μL). 90 μL of fresh MEMα medium was then dispensed into each well, and 10 μL of each peptide solution was added (total 100 μL / well). The cells were cultured for 0–5 days. Each day, 10 μL of viable cell count reagent SF (Nacalai Tesque) was added per well. The color reaction was allowed to develop for 2 hours in an incubator at 37°C and 5% CO2. 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. A similar culture test was performed using a system containing neither peptide nor FBS as a control.

[0049] The above preparation, incubation, and measurement were carried out in triplicate, and the absorbance and standard deviation of each peptide solution for 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 addition of peptide solution increased the cell count over the course of days compared to the system without addition.

[0052] (Cell proliferation test on each peptide coating agent - 3 days -) Peptides with the sequences Gly-Glu-Lys (GEK) and Asp-Gly-Pro (DGP) were synthesized and prepared in a concentration range of 0 to 5 mM to maximize cell numbers. Specifically, Gly-Glu-Lys (GEK) was prepared at 21 mM, and Asp-Gly-Pro (DGP) was prepared at 22 mM. Immediately before cell culture testing, 1 / 10 of the total medium volume was added to each well to give 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 at 0.1 mg / mL, and 200 μL was dispensed into each well of a 24-well plate. The plate was then 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 sterilized overnight by irradiating the plate with a UV lamp in a clean bench without the lid on. 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% CO. MEM alpha medium (Gibco) containing 10% FBS was used. The medium was removed from each well and washed with MEMα medium (500 μL). Then, 450 μL of fresh MEMα medium was dispensed into each well, and each peptide solution (50 μL) was added (total 500 μL / well). The cells were then cultured for 3 days. The cells were then harvested and counted. A similar culture test was also performed using a system containing neither peptide nor FBS as a control.

[0053] The medium in each well was collected in a 1.5 mL tube and rinsed with 200 μL of MEMα. The rinsed solution was also collected in the same 1.5 mL tube. 100 μL of trypsin was then added and incubated for 3 minutes. The well was rinsed with 300 μL of MEMα containing 10% FBS, and the rinsed solution was also collected in the same 1.5 mL tube. The well was again rinsed with 200 μL of MEMα containing 10% FBS, and the rinsed solution 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 the mixture was centrifuged under the same conditions. This procedure was repeated twice. The cells were suspended in 100 μL of binding buffer, and 2 μL of PI (propidium iodide) was added. The tube was vortexed and incubated at room temperature in the dark for 15 minutes. The viable cell count and viability were then measured using a flow cytometer.

[0055] The above preparation, culture and measurement were carried out in triplicate. The number of viable cells in each peptide solution is shown in Table 13 and FIG. 13, and the cell survival rate in each peptide solution is shown in Table 14 and FIG.

[0056] [Table 13]

[0057] [Table 14]

[0058] Tables 13 and 14 and Figures 13 and 14 show that in the three-day peptide culture on the coating agent under the above test conditions, the number of viable cells increased and the cell survival rate was higher than in the system without peptide addition.

[0059] (Cell proliferation test on each peptide coating agent - 5 days -) Peptides with the sequences Asp-Gly-Pro (DGP), Ala-Gly-Lys (AGK), Gly-Glu-Lys (GEK), and Gly-Gly-Gly (GGG) were synthesized and prepared in a concentration range of 0 to 5 mM to maximize cell numbers. Specifically, 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. Just before cell culture testing, 1 / 10 of the total medium volume was added to each well to achieve final concentrations of 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 at 0.1 mg / mL, and 200 μL was dispensed into each well of a 24-well plate. The plate was then 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 sterilized overnight by irradiating the plate with a UV lamp in a clean bench without the lid on. CHO-K1 (RIKEN BioResource Research Center, model number RCB2330) was cultured at 4x10 4 The cell suspension, adjusted to 500 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% CO. MEM alpha medium (Gibco) containing 10% FBS was used. The medium was removed from each well and washed with MEMα medium (500 μL). Then, 450 μL of fresh MEMα medium was dispensed into each well, and each peptide solution (50 μL) was added (total 500 μL / well). The cells were then cultured for 5 days. The cells were then harvested and counted. A similar culture test was also performed using a system containing neither peptide nor FBS as a control.

[0060] The medium in each well was collected in a 1.5 mL tube and rinsed with 200 μL of MEMα. The rinsed solution was also collected in the same 1.5 mL tube. 100 μL of trypsin was then added and incubated for 3 minutes. The well was rinsed with 300 μL of MEMα containing 10% FBS, and the rinsed solution was also collected in the same 1.5 mL tube. The well was again rinsed with 200 μL of MEMα containing 10% FBS, and the rinsed solution 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 the mixture was centrifuged under the same conditions. This procedure was repeated twice. The cells were suspended in 100 μL of binding buffer, and 2 μL of PI (propidium iodide) was added. The tube was vortexed and incubated at room temperature in the dark for 15 minutes. The viable cell count and cell viability were then measured using a flow cytometer.

[0062] The above preparation, culture, and measurement were carried out in triplicate. The number of viable cells in each peptide solution is shown in Table 15 and FIG. 15, and the cell viability in each peptide solution is shown in Table 16 and FIG.

[0063] [Table 15]

[0064] [Table 16]

[0065] Tables 15 and 16 and Figures 15 and 16 show that, with the exception of GGG, the cell number increased and the cell survival rate was higher in peptide cultures on the coating agent under the above test conditions for 5 days compared to systems without peptide addition.

[0066] (Cell proliferation tests and protein production tests on coating agents using each peptide and a combination of peptides) Peptides with the sequences Gly-Pro-Pro (GPP), Asp-Gly-Pro (DGP), Gly-Glu-Lys (GEK), and Ala-Gly-Lys (AGK) were synthesized and prepared in a concentration range of 0 to 5 mM to maximize cell numbers. Specifically, 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. Just before cell culture testing, 1 / 10 of the total medium volume was added to each well to achieve 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 at 0.1 mg / mL and dispensed in 200 μL aliquots into a 24-well plate. The plate was then 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 the lid on overnight to dry and sterilize it. CHO DP-12 (ATCC, Cat. No. CRL-12445) 2x10 4 The cell suspension, prepared at 500 μL per well, was seeded onto a 24-well plate and cultured for 24 hours in an incubator at 37°C and 5% CO. The culture 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). Then, 450 μL of fresh DMEM basal medium was dispensed into each well, 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 control, a similar culture test was performed using a system containing neither peptide nor 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 recover the cells, the medium in each well was collected into a 1.5 mL tube and rinsed with 200 μL of DMEM basal medium. The rinsed solution was also collected into the same 1.5 mL tube. 100 μL of trypsin was then added and incubated for 3 minutes. The cells were rinsed with 300 μL of 10% FBS-containing DMEM basal medium, and the rinsed solution was also collected into the same 1.5 mL tube. The cells were again rinsed with 200 μL of 10% FBS-containing DMEM basal medium, and the rinsed solution was also collected into 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 the mixture was centrifuged under the same conditions. This procedure was repeated twice. The cells were suspended in 100 μL of binding buffer, and 2 μL of PI (propidium iodide) was added. The tube was vortexed and incubated at room temperature in the dark for 15 minutes. The viable cell count and cell viability were then measured using a flow cytometer.

[0070] The above preparation, culture, and measurement were performed in triplicate. 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] Tables 17 to 25 and Figures 17 to 25 show that under the above test conditions, cell proliferation and protein production are promoted by adding a single peptide or a combination of two or more peptides.

[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 onto a 24-well plate at 500 μL / well and cultured for 24 hours in an incubator at 37°C and 5% CO. The medium used was DMEM basal medium (Gibco) containing 10% FBS, 200 nM methotrexate, and 2 μg / mL insulin. The medium was removed from each well 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 cells 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 resuspended in DMEM basal medium containing 10% FBS. The number of viable cells and viability were measured using a cell counter and the trypan blue staining method.

[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 Figure 26.

[0088] [Table 27]

[0089] Table 27 and Figure 26 show that under the above test conditions, adding peptides to the basal medium increases both the number of viable cells and the amount of protein produced, but adding vitamins and nucleic acids further promotes both cell growth and protein production.

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

[0091] A peptide with the Gly-Glu-Lys (GEK) sequence was synthesized and peptide solutions were prepared at concentrations of 0 mM, 2.6 mM, 5.1 mM, 10 mM, 20.5 mM, and 41 mM. Just before cell culture testing, 1 / 10 of the total medium volume was added to each well to achieve final concentrations of 0 mM, 0.26 mM, 0.51 mM, 1.0 mM, 2.05 mM, and 4.1 mM. For this test, CHO DP-12 (ATCC, product number CRL-12445) cells were used, which had been adapted to ASF104 basal medium (Ajinomoto), a fully synthetic medium for CHO cells, supplemented with 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% CO. The culture medium used was ASF104 basal medium (Ajinomoto), a fully synthetic medium for CHO cells, 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 the culture, the cells were harvested and the number of viable cells was counted. <Evaluation medium> ASF104 basal medium GEK (0.26 mM) + ASF104 basal medium GEK (0.51 mM) + ASF104 basal medium GEK (1.0 mM) + ASF104 basal medium GEK (2.05 mM) + ASF104 basal medium GEK (4.1 mM) + 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 from each well was collected into a 1.5 mL tube, rinsed with 200 μL of PBS, and the rinsed solution was collected into the same 1.5 mL tube. 100 μL of 0.25% trypsin / EDTA was then added and incubated for 1 minute. 100 μL of trypsin inhibitor was added and the cells were collected into 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 using a cell counter by trypan blue staining.

[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 Figure 27, the cell viability in each peptide solution is shown in Table 29 and Figure 28, and the amount of protein produced is shown in Table 30 and Figure 29.

[0096] [Table 28]

[0097] [Table 29]

[0098] [Table 30]

[0099] Tables 28 to 30 and Figures 27 to 29 show that under the above test conditions, when GEK peptides were added, the number of viable cells increased, the cell survival rate was equally high, and the amount of protein produced also tended to increase, compared to a basal medium using commercially available completely synthetic medium, thereby promoting cell proliferation and protein production.

[0100] (Cell proliferation test and protein production test of GEK and DGP in suspension cell system) 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 in a Micro-24 bioreactor system (manufactured by Nippon Pall Corporation). The cells were cultured overnight at 37°C, pH 7, and agitation speed of 650 rpm. pH calibration was performed the next day, and then 3.5 x 10 5 Add 2 mL of the cell suspension prepared at 1 x 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 mL / well and cultured in the following evaluation medium under the following 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 supplement ingredients) GEK (2.05mM) + Vitamin supplemented medium DGP (1.11mM) + vitamin supplemented medium The culture 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 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 100 mL Erlenmeyer flasks under the following conditions: 37°C, 5% CO2, and an agitation speed of 125 rpm.

[0103] The above supplementary ingredients such as vitamins 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. The viable cell count and viability were then measured using a cell counter (Countess II, Life Technologies).

[0106] The above preparation, culture, and measurement were performed on 2 or 3 patients, and the viable cell counts in each evaluation medium are shown in Table 32 and Figure 30. The survival rates in each evaluation medium are shown in Table 33 and Figure 31.

[0107] [Table 32]

[0108] [Table 33]

[0109] From Table 32, Figure 30, Table 33 and Figure 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, 150 μL of the 200 μL of medium collected from each well of the 24-well (deep well) cassette was centrifuged (5000 rpm, 5 minutes) in a 1.5 mL tube, and the supernatant was collected separately to measure the amount of produced protein by ELISA. The measured amounts of produced protein are shown in Table 34 and FIG.

[0111] [Table 34]

[0112] From Table 34 and Figure 32, it was found that the amount of protein produced increased when peptides were added to a basal medium or a 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 in a Micro-24 bioreactor system (manufactured by Nippon Pall Corporation). The cells were cultured overnight at 37°C, pH 7, and 650 rpm. pH calibration was performed the next day, and then 3.5 x 105 Add 2 mL of the cell suspension prepared at 1 x 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 mL / well and cultured at 37°C, pH 7, 650 rpm, and dissolved oxygen of 30% in the following evaluation medium. <Evaluation medium> Basal medium Vitamin supplemented medium (basal medium + vitamin supplement ingredients) 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 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 100 mL Erlenmeyer flasks under the following conditions: 37°C, 5% CO2, and an agitation speed of 125 rpm.

[0116] The above vitamins 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 protein produced 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 cultured half in serum medium and half in serum-free medium, and finally cultured only in serum-free medium for acclimatization.

[0121] Furthermore, 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 used for the production of other substances, such as hybridomas, HEK293, COS, and Sf9.

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

Claims

1. A cell proliferation promoter for culture media that contains a peptide consisting of Ala-Gly-Lys (AGK) as an active ingredient and is free of amyloid β42 peptide aggregates and fibrin-Aβ42 coaggregates.

2. A protein production promoter for culture media that contains a peptide consisting of Ala-Gly-Lys (AGK) as an active ingredient and is free of amyloid β42 peptide aggregates and fibrin-Aβ42 coaggregates.

3. A method for promoting cell proliferation, excluding culture in the human body, comprising a step of culturing cells in a culture medium that does not contain amyloid β42 peptide aggregates and fibrin-Aβ42 coaggregates, using the cell proliferation promoter for culture medium described in claim 1.

4. A method for promoting protein production, excluding culture in the human body, comprising a step of culturing cells in a medium that does not contain amyloid β42 peptide aggregates and fibrin-Aβ42 coaggregates, using the protein production promoter for medium described in claim 2.

5. A method for promoting protein production excluding in vivo culture, comprising a step of culturing cells in a medium that does not contain amyloid β42 peptide aggregates and fibrin-Aβ42 coaggregates according to claim 4, wherein the culturing step includes batch culture or fed-batch culture.

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