WO2022137357A1 - ペプチド、細胞増殖促進剤、タンパク質産生促進剤、培地、該ペプチドを用いた細胞増殖方法、及び、該ペプチドを用いたタンパク質産生方法 - Google Patents

ペプチド、細胞増殖促進剤、タンパク質産生促進剤、培地、該ペプチドを用いた細胞増殖方法、及び、該ペプチドを用いたタンパク質産生方法 Download PDF

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WO2022137357A1
WO2022137357A1 PCT/JP2020/048023 JP2020048023W WO2022137357A1 WO 2022137357 A1 WO2022137357 A1 WO 2022137357A1 JP 2020048023 W JP2020048023 W JP 2020048023W WO 2022137357 A1 WO2022137357 A1 WO 2022137357A1
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Prior art keywords
gly
peptide
medium
pro
lys
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PCT/JP2020/048023
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English (en)
French (fr)
Japanese (ja)
Inventor
義則 保苅
名津子 奈良輪
啓太 青島
晴美 杉谷
亜矢 関根
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Maruhachi Muramatsu Inc
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Maruhachi Muramatsu Inc
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Priority to US18/267,509 priority Critical patent/US20240101599A1/en
Priority to KR1020237022069A priority patent/KR20230124607A/ko
Priority to PCT/JP2020/048023 priority patent/WO2022137357A1/ja
Priority to JP2022523975A priority patent/JP7166039B1/ja
Publication of WO2022137357A1 publication Critical patent/WO2022137357A1/ja
Priority to JP2022166686A priority patent/JP2023002665A/ja
Anticipated expiration legal-status Critical
Priority to JP2023178978A priority patent/JP7719520B2/ja
Priority to JP2023178979A priority patent/JP7644527B2/ja
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0802—Tripeptides with the first amino acid being neutral
    • C07K5/0804—Tripeptides with the first amino acid being neutral and aliphatic
    • C07K5/0806—Tripeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atoms, i.e. Gly, Ala
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0819—Tripeptides with the first amino acid being acidic
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0018—Culture media for cell or tissue culture

Definitions

  • the present invention relates to peptides.
  • the present invention relates to a novel tripeptide suitable for animal cell culture, a cell growth promoter containing the peptide, a protein production promoter, a medium, a cell proliferation method using the peptide, and a protein production method using the peptide. ..
  • mammalian-derived extracts such as fetal bovine serum are added in an amount of about 5% to 20% with respect to the medium, accounting for 75% to 95% of the cost of the medium, and lots of quality due to animal origin. There was a problem that there was a difference. Furthermore, since there is concern about correlation with mad cow disease, bovine spongiform encephalopathy, infectious spongiform encephalopathy, Kreuzfeld-Jakob disease, etc., a medium containing no animal-derived extract such as bovine fetal serum has been tried. However, the cell viability was significantly reduced in the early stage of culture, and another problem that long-term culture and mass culture were difficult occurred.
  • An object of the present invention is to provide a synthetic medium containing no animal-derived components.
  • it is an object to provide a medium containing a peptide that promotes cell proliferation and contributes to the promotion of protein production even if it does not contain animal-derived components.
  • the present inventors have found a peptide that promotes cell proliferation and contributes to the promotion of protein production, a cell proliferation promoting agent containing the peptide, and protein production containing the peptide.
  • a medium containing the accelerator, the peptide was found.
  • the peptides of the present invention are Gly-Glu-Lys (GEK), Asp-Gly-Pro (DGP), Ala-Gly-Lys (AGK), Gly-Pro-Pro (GPP), Gly-Gly-Pro ( It is characterized by being selected from the group consisting of GGP), Ala-Glu-Lys (AEK), Ala-Gly-Gly (AGG), Ala-Ser-Asn (ASN), and Glu-Gly-Lys (EGK). And.
  • the cell proliferation promoter of the present invention is characterized by containing one or more of the above peptides.
  • the protein production promoter of the present invention is characterized by containing one or more of the above peptides.
  • the medium of the present invention is characterized by containing the cell proliferation promoter or the protein production promoter.
  • the cell proliferation method of the present invention is characterized by using one or more of the above peptides.
  • the protein production method of the present invention is characterized in that one or more of the above peptides are used.
  • the peptide of the present invention can provide a cell proliferation promoter, a protein production promoter, a medium, a cell proliferation method, and a protein production method containing a chemically synthesized substance that does not contain animal-derived components. .. That is, it is possible to provide a cell proliferation promoting agent, a protein production promoting agent, and a medium having stable quality by having no concern about correlation with mad cow disease, cost reduction, and clarifying the component details. ..
  • the relationship between the concentration of GEK and the number of living cells (absorbance) in the cell proliferation test is shown.
  • the relationship between the concentration of DGP and the number of living cells (absorbance) in the cell proliferation test is shown.
  • the relationship between the concentration of AGK and the number of living cells (absorbance) in the cell proliferation test is shown.
  • the relationship between the concentration of GPP and the number of living cells (absorbance) in the cell proliferation test is shown.
  • the relationship between the concentration of GGP and the number of living cells (absorbance) in the cell proliferation test is shown.
  • the relationship between the concentration of AEK and the number of living cells (absorbance) in the cell proliferation test is shown.
  • the relationship between the concentration of AGG and the number of living cells (absorbance) in the cell proliferation test is shown.
  • the relationship between the concentration of ASN and the number of living cells (absorbance) in the cell proliferation test is shown.
  • the relationship between the concentration of EGK and the number of living cells (absorbance) in the cell proliferation test is shown.
  • the relationship between the concentration of GGG and the number of living cells (absorbance) in the cell proliferation test is shown.
  • the number of living cells (absorbance) of each tripeptide in the cell proliferation test is shown.
  • the time course of the viable cell number (absorbance) of each tripeptide in the cell proliferation test is shown.
  • the number of viable cells of each tripeptide in the cell proliferation test for 3 days is shown.
  • the cell viability of each tripeptide in the cell proliferation test for 3 days is shown. The number of viable cells of each tripeptide in the cell proliferation test for 5 days is shown. The cell viability of each tripeptide in the cell proliferation test for 5 days is shown. The number of living cells of each tripeptide in the cell proliferation test of one tripeptide is shown. The cell viability of each tripeptide in the cell proliferation test of one tripeptide is shown. The amount of protein produced by each tripeptide in the cell proliferation test of one tripeptide is shown. The number of viable cells of each tripeptide combination in the cell proliferation test of two kinds of tripeptides is shown. The cell viability of each tripeptide combination in the cell proliferation test of two tripeptides is shown.
  • the amount of protein produced by each tripeptide combination in the cell proliferation test of two tripeptides is shown.
  • the number of viable cells of each tripeptide combination in the cell proliferation test of 3 kinds of tripeptides is shown.
  • the cell viability of each tripeptide combination in the cell proliferation test of three tripeptides is shown.
  • the amount of protein produced by each tripeptide combination in the cell proliferation test of three tripeptides is shown.
  • the number of viable cells of AGK and the amount of protein produced in the cell proliferation test to which vitamins and nucleic acids were added are shown.
  • the relationship between the concentration of GEK and the number of living cells in the cell proliferation test using the fully synthetic medium is shown.
  • the relationship between the concentration of GEK and the cell viability in the cell proliferation test using the fully synthetic medium is shown.
  • the relationship between the concentration of GEK and the amount of protein produced in a cell proliferation test using a fully synthetic medium is shown.
  • the number of viable cells of each tripeptide in the cell proliferation test using a fortified medium such as vitamin is shown.
  • the cell viability of each tripeptide in the cell proliferation test using a fortified medium such as vitamin is shown.
  • the amount of protein produced by each tripeptide in a protein production test using a reinforced medium such as vitamin is shown.
  • the amount of protein produced by each tripeptide in a protein production test using a reinforced medium such as vitamin is shown.
  • the peptides of the present invention are 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 can be a pharmaceutically acceptable salt, and amino acids having the same activity of the peptide can be chemically modified.
  • Gly-Glu-Lys Gly-Glu-Lys (GEK), Asp-Gly-Pro (DGP), Ala-Gly-Lys (AGK), Gly-Pro-Pro (GPP), and Gly-Gly-Pro (GGP) preferable.
  • “Pharmaceutically acceptable salts” include inorganic acid salts such as hydrochlorides, phosphates and sulfates, inorganic base salts such as sodium salts, potassium salts and calcium salts, sulfonates, succinates and oxalic acids. Examples thereof include organic acid salts such as salts and organic base salts such as alkylammonium salts.
  • “Chemically modifying an amino acid whose peptide activity does not change” means chemically modifying an amino acid whose peptide activity does not change significantly even if the amino acid is chemically modified, and is a C-terminal with an amide, an ester, an acyl group, or the like. Modification, modification of the N-terminal with an acetyl group, and the like are exemplified.
  • the proline (Pro (P)) may be hydroxyproline (Hyp) into which a hydroxyl group has been introduced.
  • tripeptides are mainly composed of hundreds of peptides of various lengths contained in fish meat extracts and their enzymatic degradation products, and various peptides that promote animal cell proliferation and protein production. It was enthusiastically searched by fractionating and identifying under the conditions and confirming the effect for each peptide.
  • the peptide can be obtained by a method such as fractionation from an extract of fish meat or an enzymatic decomposition product thereof, a chemical synthesis method including a peptide synthesis method, or an expression by a recombinant DNA method.
  • a method of fractionating from an extract of fish meat or an enzymatic decomposition product thereof various conditions of gel filtration chromatography and normal-phase / reverse-phase HPLC are adjusted for fractionation and isolation.
  • a synthesized amino acid or a chemically modified amino acid can be synthesized by a chemical reaction to obtain a peptide having a specific sequence.
  • a recombinant protein containing a plurality of peptide sequences can be produced by a recombinant, and the protein can be purified and then degraded by enzymatic treatment, chemical treatment, or the like to obtain the desired peptide. can.
  • the cell growth promoter of the present invention is 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 from the group 1 Contains more than a species of peptide. In addition, it promotes cell proliferation as compared with the case where the peptide of one or more kinds is not contained.
  • the above peptide can be a pharmaceutically acceptable salt, and further, amino acids having the same peptide activity can be chemically modified.
  • the proline (Pro (P)) may be hydroxyproline (Hyp) into which a hydroxyl group has been introduced.
  • Peptides are selected by appropriately combining one or more of the above peptides.
  • Gly-Glu-Lys Gly-Glu-Lys (GEK), Asp-Gly-Pro (DGP), Ala-Gly-Lys (AGK), Gly-Pro-Pro (GPP), Gly-Gly-Pro (GGP) is more preferred.
  • Asp-Gly-Pro DGP
  • Ala-Gly-Lys AGK
  • Gly-Pro-Pro Gly-Glu-Lys
  • AGK Asp-Gly-Pro
  • Gly-Glu-Lys Gly-Pro-Pro (GPP) and Ala-Gly-Lys (AGK), Gly-Pro-Pro (GPP), Gly-Glu-Lys (GEK) and the like are preferred combinations. Illustrated.
  • the protein production promoter of the present invention is 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 from the group 1 Contains more than a species of peptide. In addition, it promotes protein production as compared with the case where it does not contain the one or more kinds of peptides.
  • the above peptide can be a pharmaceutically acceptable salt, and further, amino acids having the same peptide activity can be chemically modified.
  • the proline (Pro (P)) may be hydroxyproline (Hyp) into which a hydroxyl group has been introduced.
  • Peptides are selected by appropriately combining one or more of the above peptides.
  • Gly-Glu-Lys Gly-Glu-Lys (GEK), Asp-Gly-Pro (DGP), Ala-Gly-Lys (AGK), Gly-Pro-Pro (GPP), Gly-Gly-Pro (GGP) is exemplified as a preferred peptide.
  • Asp-Gly-Pro DGP
  • Ala-Gly-Lys AGK
  • Gly-Glu-Lys GEK
  • Ala-Gly-Lys AGK
  • Asp-Gly-Pro DGP
  • Gly-Glu-Lys Gly-Pro-Pro (GPP) and Ala-Gly-Lys (AGK), Gly-Pro-Pro (GPP), Gly-Glu-Lys (GEK) and the like are preferred combinations. Illustrated.
  • the medium of the present invention contains the cell proliferation promoter containing the peptide or the protein production promoter containing the peptide.
  • 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 maintain survival, and the amount of cell proliferation and protein production is the largest as compared with the medium to which no cell proliferation promoter or protein production promoter is added. Is the preferred concentration, and the maximum concentration that is not harmful to the composition of the medium is the upper limit concentration.
  • 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 with respect to the medium.
  • Other components used in the animal cell culture medium can be appropriately added to the medium.
  • examples thereof include vitamins, nucleic acids, amino acids, inorganic salts, sugars, polyamines, carbohydrates, proteins, fatty acids, lipids, pH adjusters, zinc, copper, selenium and the like.
  • 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.
  • nucleic acid examples include xanthine, hypoxanthine, uridine, guanine hydrochloride, inosine, guanosine, cytidine, thymidine, adenine and the like.
  • Amino acids include glycine, L-alanine, L-arginine hydrochloride, L-aspartin-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- Examples thereof include treonine, L-tryptophan, L-tyrosine disodium salt, L-valine, arginine and the like.
  • the inorganic salt include calcium chloride, magnesium sulfate, potassium chloride, sodium hydrogen carbonate, sodium chloride, sodium dihydrogen phosphate-monohydrate and the like.
  • Other components include D-glucose, ⁇ -lipoic acid, phenol sulfonphthaline (phenol red), sodium pyruvate, AlbuMax® II, human transferase (holo), ammonium metavanadate, copper sulfate, manganese chloride, Examples thereof include sodium selenate, ethanolamine, glutathione, methotrexate, and insulin.
  • serum components such as fetal bovine serum may be contained, but if there is an intention to remove the animal-derived components from the medium, they are not contained.
  • Cell proliferation method and protein production method The cell proliferation method and the protein production method of the present invention are carried out by blending the peptide of the present invention with the above-mentioned medium and culturing various animal cells.
  • the cell proliferation method and the protein production method are exemplified below, but are not limited thereto.
  • Basis medium is used to acclimate animal cells to serum-free suspension.
  • Gly-Glu-Lys 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 one or more peptides selected from the group consisting of Glu-Gly-Lys (EGK) are added to the basal medium. do.
  • vitamins, nucleic acids, sugars, polyamines, and amino acids, which are components that reinforce the basal medium may be added.
  • animal cells conditioned in the basal medium are seeded in the basal medium to which the peptide has been added, and cell proliferation and protein production are performed.
  • Gly-Glu-Lys 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) sequences were synthesized to synthesize peptides. Peptide solutions were prepared so as to have 10 times the concentration shown in Tables 1 to 10.
  • CHO-K1 (RIKEN BioResource Research Center, model number RCB2330) was inoculated on a 96-well plate at a cell suspension prepared at 3x10 4 cells / mL so as to be 100 ⁇ L / well, and 37 ° C., CO 25 %. The cells were cultured in an incubator for 24 hours. As the medium, a MEM ⁇ medium (gibco) containing 10% FBS was used. After removing the medium of each well and washing out with MEM ⁇ medium (100 ⁇ L), 90 ⁇ L of new MEM ⁇ medium was dispensed, and each peptide solution (10 ⁇ L) was added (total 100 ⁇ L / well), 0 mM to 5 mM.
  • MEM ⁇ medium gibco
  • the final concentrations shown in Tables 1 to 10 within the range of 1) were set, and the cells were cultured for 5 days.
  • a similar culture test was performed in a system containing neither peptide nor FBS as a comparative sample. After culturing for 5 days, 10 ⁇ L / well of the viable cell number measuring reagent SF (nacalai tesque) was added, and the color reaction was carried out in an incubator at 37 ° C. and CO 25% for 2 hours, and the absorbance at 450 nm was measured with a plate reader. ..
  • the reference wavelength was 630 nm. It has been confirmed that the absorbance at 450 nm correlates with the number of cells.
  • the peptide sequences are 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
  • Peptides having the sequences of Gly-Pro-Pro (GPP), Asp-Gly-Pro (DGP), Gly-Glu-Lys (GEK), and Ala-Gly-Lys (AGK) were synthesized and within the range of 0 mM to 5 mM. Peptide solutions were prepared so as to have the concentration that increases the number of cells most.
  • Gly-Pro-Pro 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.
  • DGP Asp-Gly-Pro
  • Gly-Glu-Lys Gly-Glu-Lys
  • AGK Ala-Gly-Lys
  • CHO-K1 (RIKEN BioResource Research Center, model number RCB2330) was seeded on a 96-well plate at a cell suspension prepared at 3x10 4 cells / mL so as to be 100 ⁇ L / well, and 37 ° C., CO 25 %. The cells were cultured in an incubator for 24 hours. As the medium, a MEM ⁇ medium (gibco) containing 10% FBS was used. After removing the medium of each well and washing out with MEM ⁇ medium (100 ⁇ L), 90 ⁇ L of new MEM ⁇ medium was dispensed, and each peptide solution (10 ⁇ L) was added (total 100 ⁇ L / well), 0 to 5. Cultured for days.
  • Poly-L-lysine (Peptide Institute, Poly-L-lysine hydrochloride, model number 3075) was prepared at 0.1 mg / mL, 200 ⁇ L was dispensed into each well of a 24-well plate, and incubator at 37 ° C. for 2 hours. It was left still. After removing the residual liquid with an aspirator, it was rinsed with distilled water, irradiated with a UV lamp in a clean bench without a lid, dried overnight and sterilized.
  • CHO-K1 (RIKEN BioResource Research Center, model number RCB2330) was seeded on a 24-well plate at 500 ⁇ L / well of a cell suspension prepared at 4x10 4 cells / mL at 37 ° C.
  • the cells were cultured in an incubator for 24 hours.
  • a MEM ⁇ medium (gibco) containing 10% FBS was used.
  • MEM ⁇ medium 500 ⁇ L
  • 450 ⁇ L of new MEM ⁇ medium was dispensed, and each peptide solution (50 ⁇ L) was added (total 500 ⁇ L / well) and cultured for 3 days. did.
  • the cells were collected and the number of cells was counted.
  • a similar culture test was performed in a system containing neither peptide nor FBS as a comparative sample.
  • the medium in each well was collected in a 1.5 mL tube, rinsed with 200 ⁇ L of MEM ⁇ , and the rinsed solution was also collected in the same 1.5 mL tube. Then, 100 ⁇ L of trypsin was added and the incubation was carried out for 3 minutes. Rinse was performed with 300 ⁇ L of MEM ⁇ containing 10% FBS, and the rinsed liquid was also collected in the same 1.5 mL tube. Rinse again with 200 ⁇ L of MEM ⁇ containing 10% FBS, and the rinsed liquid was also collected in the same 1.5 mL tube.
  • the cells collected in a 1.5 mL tube were centrifuged. Centrifugal conditions were 1000 rpm, 10 minutes and 4 ° C. The supernatant was removed, 300 ⁇ L of Cold PBS (phosphate buffered saline) was added, and centrifugation was performed under the same conditions. This operation was repeated twice.
  • the suspension was suspended in 100 ⁇ L of Binding Buffer, 2 ⁇ L of PI (propidium iodide) was added, the tube was stirred, and the mixture was reacted at room temperature and light shielding for 15 minutes. Then, the number of viable cells and the survival rate were measured with a flow cytometer.
  • Poly-L-lysine (Peptide Institute, Poly-L-lysine hydrochloride, model number 3075) was prepared at 0.1 mg / mL, 200 ⁇ L was dispensed into each well of a 24-well plate, and incubator at 37 ° C. for 2 hours. It was left still. After removing the residual liquid with an aspirator, it was rinsed with distilled water, irradiated with a UV lamp in a clean bench without a lid, dried overnight and sterilized.
  • CHO-K1 (RIKEN BioResource Research Center, model number RCB2330) was inoculated into each well of a 24-well plate at 500 ⁇ L / well of a cell suspension prepared at 4x10 4 cells / mL, and CO 2 at 37 ° C. The cells were cultured in a 5% incubator for 24 hours. As the medium, a MEM ⁇ medium (gibco) containing 10% FBS was used. After removing the medium of each well and washing out with MEM ⁇ medium (500 ⁇ L), 450 ⁇ L of new MEM ⁇ medium was dispensed, and each peptide solution (50 ⁇ L) was added (total 500 ⁇ L / well) and cultured for 5 days. did. The cells were collected and the number of cells was counted. A similar culture test was performed in a system containing neither peptide nor FBS as a comparative sample.
  • the medium in each well was collected in a 1.5 mL tube, rinsed with 200 ⁇ L of MEM ⁇ , and the rinsed solution was also collected in the same 1.5 mL tube. Then, 100 ⁇ L of trypsin was added and the incubation was carried out for 3 minutes. Rinse was performed with 300 ⁇ L of MEM ⁇ containing 10% FBS, and the rinsed liquid was also collected in the same 1.5 mL tube. Rinse again with 200 ⁇ L of MEM ⁇ containing 10% FBS, and the rinsed liquid was also collected in the same 1.5 mL tube.
  • the cells collected in a 1.5 mL tube were centrifuged. Centrifugal conditions were 1000 rpm, 10 minutes and 4 ° C. The supernatant was removed, 300 ⁇ L of Cold PBS (phosphate buffered saline) was added, and centrifugation was performed under the same conditions. This operation was repeated twice.
  • the suspension was suspended in 100 ⁇ L of Binding Buffer, 2 ⁇ L of PI (propidium iodide) was added, the tube was stirred, and the mixture was reacted at room temperature and light shielding for 15 minutes. Then, the number of living cells and the cell viability were measured with a flow cytometer.
  • Peptides having the sequences of Gly-Pro-Pro (GPP), Asp-Gly-Pro (DGP), Gly-Glu-Lys (GEK), and Ala-Gly-Lys (AGK) were synthesized and within the range of 0 mM to 5 mM. Peptide solutions were prepared so as to have the concentration that increases the number of cells most.
  • Gly-Pro-Pro 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.
  • DGP Asp-Gly-Pro
  • Gly-Glu-Lys Gly-Glu-Lys
  • AGK Ala-Gly-Lys
  • Poly-L-lysine (Peptide Institute, Poly-L-lysine hydrochloride, model number 3075) was prepared at 0.1 mg / mL, 200 ⁇ L was dispensed into a 24-well plate, and the mixture was allowed to stand in an incubator at 37 ° C. for 2 hours. .. After removing the residual liquid with an aspirator, it was rinsed with distilled water, irradiated with a UV lamp in a clean bench without a lid, dried overnight and sterilized.
  • CHO DP-12 (ATCC, model number CRL-12445) was seeded on a 24-well plate with a cell suspension prepared to 2x10 4 cells / well ⁇ 500 ⁇ L and incubated at 37 ° C.
  • DMEM basal medium containing 200 nM methotrexate and 2 ⁇ g / mL insulin in DMEM medium (gibco) containing 10% FBS was used. After removing the medium of each well and washing out with DMEM basal medium (500 ⁇ L), 450 ⁇ L of new DMEM basal medium is dispensed and each peptide solution (50 ⁇ L) is added (total 500 ⁇ L / well) for 5 days. It was cultured.
  • GPP + GEK As the peptide, a combination of GPP + GEK, GPP + AGK, DGP + GEK, GEK + AGK, DGP + AGK, GPP + GEK + AGK, GPP + DGP + AGK, and GPP + DGP + GEK was used in addition to the simple substance of GPP, DGP, GEK, and AGK.
  • a similar culture test was performed in a system containing neither peptide nor FBS as a comparative sample.
  • the medium in each well was collected in a 1.5 mL tube, rinsed with 200 ⁇ L of DMEM basal medium, and the rinsed solution was also collected in the same 1.5 mL tube. Then, 100 ⁇ L of trypsin was added and the incubation was carried out for 3 minutes. Rinse with 300 ⁇ L of DMEM basal medium containing 10% FBS, and the rinsed solution was also collected in the same 1.5 mL tube. Rinse again with 200 ⁇ L of DMEM basal medium containing 10% FBS, and the rinsed solution was also collected in the same 1.5 mL tube.
  • the cells collected in a 1.5 mL tube were centrifuged. Centrifugal conditions were 1000 rpm, 10 minutes and 4 ° C. The supernatant was removed, 300 ⁇ L of Cold PBS (phosphate buffered saline) was added, and centrifugation was performed under the same conditions. This operation was repeated twice.
  • the suspension was suspended in 100 ⁇ L of Binding Buffer, 2 ⁇ L of PI (propidium iodide) was added, the tube was stirred, and the mixture was reacted at room temperature and light shielding for 15 minutes. Then, the number of living cells and the cell viability were measured with a flow cytometer.
  • n 3
  • the number of viable cells in each peptide solution is shown in Table 17, FIG. 17, Table 20, FIG. 20, Table 23, and FIG. 23, and the cell viability in each peptide solution is shown. It is shown in Table 18, FIG. 18, Table 21, FIG. 21, Table 24, and FIG. 24, and the amount of protein produced in each peptide solution is shown in Table 19, FIG. 19, Table 22, FIG. 22, Table 25, and FIG. 25.
  • DMEM basal medium containing 200 nM methotrexate and 2 ⁇ g / mL insulin in DMEM medium (gibco) containing 10% FBS was used.
  • the medium of each well was removed, washed out with DMEM basal medium (500 ⁇ L), and then each peptide solution (500 ⁇ L) was dispensed into each well (total 500 ⁇ L / well) and cultured for 5 days.
  • compositions of the above vitamins and nucleic acids are shown in Table 26.
  • the cells adhering to the well after collection of the medium are exfoliated by trypsin treatment, suspended again in DMEM basal medium containing 10% FBS, and the number of viable cells and survival rate are determined by the trypan blue staining method at the cell counter. It was measured.
  • Peptides having the sequence of Gly-Glu-Lys were synthesized, peptide solutions having concentrations of 0 mM, 2.6 mM, 5.1 mM, 10 mM, 20.5 mM, and 41 mM were prepared, and each well was prepared immediately before the cell culture test. The final concentrations were 0 mM, 0.26 mM, 0.51 mM, 1.0 mM, 2.05 mM, and 4.1 mM, respectively, by adding 1/10 of the total medium volume of.
  • Gly-Glu-Lys peptide solutions having concentrations of 0 mM, 2.6 mM, 5.1 mM, 10 mM, 20.5 mM, and 41 mM were prepared, and each well was prepared immediately before the cell culture test. The final concentrations were 0 mM, 0.26 mM, 0.51 mM, 1.0 mM, 2.05 mM, and 4.1 mM, respectively, by adding 1/10 of the total medium volume of.
  • CHO DP-12 (ATCC, model number CRL-12445) was added to ASF104 basal medium containing 200 nM methotrexate and 2 ⁇ g / mL insulin in ASF104 medium (Ajinomoto), which is a completely synthetic medium for CHO. used.
  • ASF104 basal medium containing 200 nM methotrexate and 2 ⁇ g / mL insulin in ASF104 medium (Ajinomoto), which is a completely synthetic medium for CHO.
  • the medium in each well was collected in a 1.5 mL tube, rinsed with 200 ⁇ L PBS, and the rinsed solution was also collected in the same 1.5 mL tube. Then, 0.25% trypsin / EDTA 100 ⁇ L was added and the incubation was carried out for 1 minute. 100 ⁇ L of trypsin inhibitor was added and collected in the same 1.5 mL tube. The rinse was performed with 200 ⁇ L of PBS, and the rinsed solution was also collected in the same 1.5 mL tube and then centrifuged. The cells were suspended in 100 ⁇ L of PBS, and the number of viable cells and the cell viability were measured by the trypan blue staining method on a cell counter.
  • n 3
  • 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, and the amount of protein produced is shown. It is shown in Table 30 and FIG. 29.
  • DMEM / F12 basal medium, vitamin-reinforcing medium (DMEM / F12 basal medium + vitamin-reinforcing component), and peptide solutions are added to each well of the 24-well (Deep well) cassette of the Micro-24 bioreactor system (manufactured by Nippon Paul Co., Ltd.). 5 mL each was added, and the cells were cultured overnight at 37 ° C., pH 7, and a stirring speed of 650 rpm. The next day, pH calibration was performed, and then 2 mL of the cell suspension prepared to 3.5x10 5 cells / mL was added to each well of the 24-well (Deep well) cassette, and the cell density of the floating cells was 1x10 5 cells / mL.
  • the cells were sown at (7 mL / well), cultured at 37 ° C., pH 7, stirring speed 650 rpm, culture conditions of 30% dissolved oxygen, and cultured in the following evaluation medium.
  • the medium was 200 nM methotrexate, 10 ⁇ g / mL insulin, 5.5 ⁇ g / mL transferase, 6.7 ng / mL sodium selenate, 10 ⁇ L / mL Anti-Clumping Agent, 10 ⁇ L / in DMEM / F12 medium (gibco).
  • DMEM / F12 basal medium containing 10% Pluronic F68 in mL was used.
  • CHO DP-12 (ATCC, model number CRL-12445) was acclimatized to serum-free suspension, and a shaking type culture device (Custom Bio Shaker CO2-BR-43FL, Titec) was used to measure 100 mL. Using an Erlenmeyer flask, serum-free suspended CHO DP-12 subcultured under the culture conditions of 37 ° C., CO 25 %, and stirring speed of 125 rpm was used.
  • Table 31 shows the reinforcing components such as vitamins.
  • the cells were sown at (7 mL / well), cultured at 37 ° C., pH 7, 650 rpm, under culture conditions of 30% dissolved oxygen, and cultured in the following evaluation medium.
  • the medium is DMEM / F12 medium (gibco) with 200 nM methotrexate, 10 ⁇ g / mL insulin, 5.5 ⁇ g / A basal medium containing mL transferase, 6.7 ng / mL sodium selenate, 10 ⁇ L / mL Anti-Clumping Agent, and 10 ⁇ L / mL 10% Pluronic F68 was used.
  • CHO DP-12 (ATCC, model number CRL-12445) was acclimatized to serum-free suspension, and a shaking type culture device (Custom Bio Shaker CO2-BR-43FL, Titec) was used to measure 100 mL. Using an Erlenmeyer flask, serum-free suspended CHO DP-12 subcultured under the culture conditions of 37 ° C., CO 25 %, and stirring speed of 125 rpm was used.
  • CHO cells were suspended without serum. First, cell culture was performed using only the serum medium, and then cell culture was performed using the serum medium and the serum-free medium in half. Finally, the cells may be cultivated using only a serum-free medium for acclimatization.
  • the medium containing the peptide of the present invention is also applied to cell lines such as hybridoma, HEK293, COS, and Sf9 used for producing other substances. It is possible.
  • the method for producing a protein using the peptide of the present invention may include a step of fed-batch culture in which a medium is added during production.

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PCT/JP2020/048023 2020-12-22 2020-12-22 ペプチド、細胞増殖促進剤、タンパク質産生促進剤、培地、該ペプチドを用いた細胞増殖方法、及び、該ペプチドを用いたタンパク質産生方法 Ceased WO2022137357A1 (ja)

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PCT/JP2020/048023 WO2022137357A1 (ja) 2020-12-22 2020-12-22 ペプチド、細胞増殖促進剤、タンパク質産生促進剤、培地、該ペプチドを用いた細胞増殖方法、及び、該ペプチドを用いたタンパク質産生方法
JP2022523975A JP7166039B1 (ja) 2020-12-22 2020-12-22 ペプチド、細胞増殖促進剤、タンパク質産生促進剤、培地、該ペプチドを用いた細胞増殖方法、及び、該ペプチドを用いたタンパク質産生方法
JP2022166686A JP2023002665A (ja) 2020-12-22 2022-10-18 ペプチド、細胞増殖促進剤、タンパク質産生促進剤、培地、該ペプチドを用いた細胞増殖方法、及び、該ペプチドを用いたタンパク質産生方法
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