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WO2017195789A1 - Composition pour milieu de culture - Google Patents

Composition pour milieu de culture Download PDF

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Publication number
WO2017195789A1
WO2017195789A1 PCT/JP2017/017581 JP2017017581W WO2017195789A1 WO 2017195789 A1 WO2017195789 A1 WO 2017195789A1 JP 2017017581 W JP2017017581 W JP 2017017581W WO 2017195789 A1 WO2017195789 A1 WO 2017195789A1
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WO
WIPO (PCT)
Prior art keywords
yeast
composition
medium
cells
serum
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Application number
PCT/JP2017/017581
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English (en)
Japanese (ja)
Inventor
美玉 小野
亮輔 宮内
亮 岩切
Original Assignee
興人ライフサイエンス株式会社
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Publication date
Application filed by 興人ライフサイエンス株式会社 filed Critical 興人ライフサイエンス株式会社
Priority to JP2018517040A priority Critical patent/JP6997079B2/ja
Publication of WO2017195789A1 publication Critical patent/WO2017195789A1/fr

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    • 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
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the present invention relates to a medium composition that can enhance the production ability of a recombinant protein.
  • Biopharmaceuticals have effects not found in conventional low-molecular drugs such as specific, high activity and few side effects, and are expected to grow at a high rate in the future.
  • cell culture is also necessary for the culture of cells necessary for regenerative medicine (iPS cells, ES cells, etc.) and the production of useful substances such as enzymes. Therefore, development competition between companies is intensifying, and it is expected that some outsourcing of manufacturing and R & D will progress.
  • Many biopharmaceuticals are produced using animal cells because post-translational modifications are relatively similar to humans. In order to produce substances in these animal cells, a medium supplemented with serum necessary for the growth of animal cells and protein components separated from the serum is often used.
  • the final purpose of the serum-free medium developed for biopharmaceutical production and the like is to perform recombinant protein production with high efficiency, it is desired to increase the production capacity. Therefore, it is necessary not only to improve cell growth in serum-free medium, but also to develop a protein secretion promoting factor.
  • an object of the present invention is to provide a method and a composition capable of increasing the production ability of a recombinant protein even when a serum-free medium is used.
  • the present inventors have found that when animal cells are cultured in a medium in which a yeast extract is added to a serum-free medium, the ability to produce recombinant proteins can be increased, leading to the present invention.
  • the present invention (1) a recombinant protein production promoter containing a yeast extract, (2) The protein production promoter of (1), wherein the yeast extract is an organic solvent extract, (3) A medium for animal cells containing the protein promoter of (1) or (2), (4) A method for producing a recombinant protein production promoter comprising a step of extracting from yeast or yeast residue with an organic solvent, (5) The production method of (4), wherein the organic solvent is ethanol, Is to provide.
  • the medium of the present invention is a medium for producing a genetically modified protein, and is characterized by containing a yeast extract.
  • the protein production promoter that is the composition of the present invention can be obtained as follows.
  • Examples of the yeast used in the production of the present invention include baker's yeast, brewer's yeast, and Torula yeast (Candida utilis). Among these, Torula yeast is preferable.
  • the yeast culture method is not particularly limited. As the culture format of yeast, either batch culture or continuous culture is used. In general, glucose, acetic acid, ethanol, glycerol, molasses, sulfite pulp waste liquid and the like are used as the carbon source in the medium, and urea, ammonia, ammonium sulfate, ammonium chloride, nitrate and the like are used as the nitrogen source.
  • Phosphoric acid, potassium, and magnesium sources may be ordinary industrial raw materials such as lime perphosphate, ammonium phosphate, potassium chloride, potassium hydroxide, magnesium sulfate, magnesium chloride, and other zinc ions, manganese ions, iron ions, copper Add inorganic salt such as ions.
  • vitamins, amino acids, nucleic acid-related substances and the like may be added.
  • Organic substances such as casein, yeast extract, meat extract and peptone may be added.
  • yeast extract residue is most preferable as the yeast used in the present invention.
  • the yeast extract extraction residue of this application means the solid component after extracting yeast extract etc. from the cultured yeast.
  • the yeast extract extraction method is not particularly limited, but is generally performed by an autolysis method, a hot water extraction method, an enzyme extraction method, an acid or alkali extraction method, or a combination thereof.
  • yeast cells and solids after extraction by these extraction methods are referred to as yeast extract extraction residues.
  • the composition of the present invention can be produced by the method described below.
  • the extraction step from yeast or yeast residue may be provided with a step of washing the yeast or yeast residue by suspending the yeast or yeast residue in distilled water and centrifuging.
  • Either organic solvent extraction, enzyme treatment, organic solvent extraction, or supercritical extraction is used.
  • the solvent used includes methanol, ethanol, n-propanol, hexane, acetone, and a chlorinated solvent.
  • Preferred is a lower alcohol having 1 to 3 carbon atoms, and more preferred is ethanol. Therefore, hydrous ethanol according to the purpose is used.
  • the volume ratio of water / ethanol is usually 0.01 / 99.99 to 30/70, preferably 0.5 / 99.5 to 30/70. As this moisture content, the moisture content in the raw material may be taken into consideration.
  • the amount of ethanol used is not particularly limited. About 1 to 30 times as much ethanol as yeast or yeast residue is used.
  • the extraction method is to add ethanol and stir well, and then react at an appropriate temperature and time.
  • the temperature is not particularly limited, but is usually 20 ° C. to 80 ° C., preferably 50 ° C. to 70 ° C.
  • the extraction time is not particularly limited, but is usually 1 hour or longer, preferably 3 to 9 hours. Examples of the extraction method include agitation, reflux, immersion, shaking, and ultrasonic extraction. If the enzyme treatment is performed before the extraction, the extraction time can be shortened.
  • ⁇ -glucanase “Denateam GEL” derived from Streptomyces, which is a cell wall lytic enzyme (manufactured by Nagase ChemteX), ⁇ -glucanase “Filtrase BRX” derived from Taloromyces genus (manufactured by DSM Japan), “Tunicase FN” (Amano Enzyme). Tunicase FN is preferred.
  • a separation step of adding water and a cooling step may be added. Thereby, it is difficult to oxidize and it is possible to improve deterioration over time.
  • the ethanol concentration prepared by adding water is 50 to 90%, preferably 70%.
  • the cooling temperature is preferably 10 ° C. or lower. In particular, 5 ° C or less is desirable.
  • the cooling time is 1 to 10 hours, preferably about 1 hour.
  • the supercritical extraction is a method of separating carbon dioxide from the yeast suspension by bringing the yeast suspension into contact with carbon dioxide in a supercritical state and then reducing the pressure to atmospheric pressure.
  • a known concentrating device When concentrating the yeast extract obtained as described above, a known concentrating device can be used. It is preferable to use a vacuum concentrator capable of reducing the temperature as much as possible.
  • medium-chain fatty acids or cyclodextrins / cluster dextrins or the like as excipients to suppress odor and dry them.
  • a known drying method such as freeze drying or vacuum drying can be used.
  • composition of the present invention can be purified with a silica gel column or the like in order to obtain the purity necessary for the activity of the present invention or according to the odor or the like derived from the raw material.
  • the steps for preparing the purity can be appropriately combined with the steps described above.
  • the present composition obtained as described above is a composition containing a large amount of lipid components contained in yeast or yeast residue.
  • the composition can be used as a protein production promoter.
  • An animal cell culture medium can be obtained by adding the composition of the present invention thus obtained to an animal culture medium.
  • the composition of the present application can be used in a serum medium or a low serum medium, but a serum-free medium can be used more preferably.
  • the composition of the medium may be a general composition, and a commercially available medium for animal cells can also be used. Moreover, it can select suitably according to a host cell.
  • the animal cell used in the present invention is not particularly limited, but is suitable for mammalian cells, particularly CHO-K1, HEK293, and the like.
  • the method of adding the composition of the present invention to the medium is arbitrary. Usually, the composition of the present invention obtained by the method described above is used by spray drying, freeze drying or the like. Moreover, it is not necessary to use a dry product.
  • the amount of the composition of the present invention added to the medium is appropriately adjusted according to the degree of purification. In the case of using a dry product, the addition amount is usually 0.001% by weight to 0.5% by weight per medium.
  • composition of the present invention to a medium for animal cells and culturing promote the expression of useful substances such as recombinant proteins. Therefore, a useful substance can be manufactured by isolating the target useful substance from the culture medium cultured using the composition of the present application.
  • useful substances obtained from animal cells include antibodies, enzymes and hormones.
  • Candida utilis CS7529 strain (FERMP-3340) was pre-cultured in an Erlenmeyer flask containing YPD medium (yeast extract 1%, polypeptone 2%, glucose 2%) in advance, and this was cultured in an 18L medium in a 30L fermentor. 1-2% inoculated.
  • Medium composition is 4% glucose, 0.3% monoammonium phosphate, 0.161% ammonium sulfate, 0.137% potassium chloride, 0.08% magnesium sulfate, 1.6 ppm copper sulfate, 14 ppm iron sulfate, 16 ppm manganese sulfate, 14 ppm of zinc sulfate was used.
  • the culture conditions were pH 4.0, culture temperature 30 ° C., aeration rate 1 vvm, and stirring 600 rpm.
  • the pH was controlled with ammonia.
  • the culture solution was collected and collected by centrifugation to obtain 180 g of wet yeast cells.
  • yeast extract extraction The wet yeast cells after cell culture were suspended in distilled water and washed by repeated centrifugation. After washing, the wet cells are resuspended in distilled water, or freeze-dried or hot-air dried dry cells are suspended in distilled water. After adjusting to pH 13 with 2N NaOH, the mixture was stirred at 70 ° C. for 20 minutes to extract the yeast extract.
  • the cell residue after extraction of the yeast extract was dried with a drum dryer, and 100 g of the dried product was used as the yeast residue.
  • FIG. 1 substantially a circle is described along the detected spot outer edge.
  • PC phosphatidylcholine
  • PE phosphatidylethanolamine
  • the composition obtained in Production Example 1 was added to the medium, and the expression level of Gaussia Luciferase (GLuc) and the expression level of Human IL-6 (hIL6) were analyzed.
  • the medium used was a serum-free medium obtained by adding 0.01% of the composition obtained in Production Example 1 to RPMI 1640 (“189-02025” manufactured by WAKO). Acquisition of stable expression strain: CHO-K1 cells were seeded on 1 ⁇ 10 4 cells / mL / 24 well plate. The next day, the hIL6 gene (500 ng / well) was transfected. On the third day of transfection, the cells were transferred to a 100 mm dish.
  • the cells were switched to a G418-containing medium, and selection was performed while culturing for about 1 week. Thereafter, the completed colonies were picked up and cultured at 96 wellpate, and the activity was confirmed. The active cells were further scaled up to 24 well plate and 6 well plate to obtain hIL6 stable expression strain.
  • Example 1 Effect of serum-free medium supplemented with yeast extract on Gluc stable expression strain
  • a GLuc stable expression strain was obtained in the same manner as hIL6 described above and seeded in 5 ⁇ 10 5 cells / 2 mL / 6 well plate did. The next day, the cells were washed with PBS ( ⁇ ), added with 2 mL / well of yeast extract-containing medium, and cultured for 48 hours. After 48 hours, the Gluc activity in the culture supernatant was measured, and the number of cells was counted by the cell count method. The results are shown in RLU / cells / day. The results are shown in FIG.
  • the RPMI1640 / FBS group is a group in which bovine serum (FBS) is added to RPMI1640 at a final concentration of 10% and used as a medium for culturing CHO-K1 cells.
  • the CDM4CHO group is a group using only CDM4CHO ("HyClone" manufactured by GE Healthcare) as a medium for culturing CHO-K1 cells.
  • Example 2 Effect of yeast extract-added serum-free medium on hIL6 stable expression strain Evaluated with 2 stable strains obtained (HE11A1 strain, HE12A2 strain), and 2 strains were 5 ⁇ 10 5 cells / 2 mL / 6 Seeded on well plate. The next day, the cells were washed with PBS ( ⁇ ), added with 2 mL / well of yeast extract-containing medium, and cultured for 48 hours. After 48 hours, the expression level of hIL6 in the culture supernatant was measured, and the number of cells was counted by the cell count method. The results are shown in pg / cells / day. The results are shown in Figure 3. In both HE11A1 and HE12A2 strains, the production of IL-6 per cell was clearly higher in the RPMI1640 / yeast extract group than in the other two groups.
  • the production of a recombinant protein can be promoted, and a useful substance can be produced efficiently.

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  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Cell Biology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

L'objectif de la présente invention concerne une composition qui, lorsqu'elle est ajoutée à un milieu de culture sans sérum, un milieu de culture sérique ou analogue, peut augmenter l'activité pour produire une protéine recombinante de gène. On a découvert que si des cellules animales sont cultivées dans un milieu de culture préparé par addition d'un extrait de levure, en particulier une composition extraite du résidu de levure au moyen d'un solvant organique, à un milieu de culture sans sérum, l'aptitude à produire une protéine recombinante peut être augmentée. En particulier, on obtient une souche stable et la composition selon la présente invention est ajoutée à la souche stable, ce qui permet d'augmenter l'aptitude à la production d'une protéine recombinante.
PCT/JP2017/017581 2016-05-10 2017-05-09 Composition pour milieu de culture WO2017195789A1 (fr)

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JP2018517040A JP6997079B2 (ja) 2016-05-10 2017-05-09 培地用組成物

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JP2016-094556 2016-05-10

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2020162536A (ja) * 2019-03-29 2020-10-08 興人ライフサイエンス株式会社 組み換えタンパク質産生増加のための培地用組成物
WO2025058049A1 (fr) * 2023-09-15 2025-03-20 テーブルマーク株式会社 Composition pour milieu de base pour culture de cellules animales

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0249579A (ja) * 1988-02-29 1990-02-19 Kyowa Hakko Kogyo Co Ltd 培地
JP2001501830A (ja) * 1996-10-10 2001-02-13 ライフ テクノロジーズ，インコーポレイテッド 植物由来栄養素を含む動物細胞培養培地
JP2002520014A (ja) * 1998-07-10 2002-07-09 中外製薬株式会社 動物細胞培養用無血清培地
JP2010503397A (ja) * 2006-09-13 2010-02-04 アボット・ラボラトリーズ 細胞培養の改善
JP2011200180A (ja) * 2010-03-26 2011-10-13 Nippon Menaade Keshohin Kk 幹細胞の未分化維持剤及び増殖促進剤
WO2015030479A1 (fr) * 2013-08-30 2015-03-05 한미약품 주식회사 Procédé pour la production en masse de dérivé du facteur de coagulation sanguine vii humain

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2907503B2 (ja) * 1990-07-06 1999-06-21 オリエンタル酵母工業株式会社 酵母エキスの製造方法
JP4096009B2 (ja) * 2005-10-12 2008-06-04 アサヒビール株式会社 組換えタンパクの製造方法

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0249579A (ja) * 1988-02-29 1990-02-19 Kyowa Hakko Kogyo Co Ltd 培地
JP2001501830A (ja) * 1996-10-10 2001-02-13 ライフ テクノロジーズ，インコーポレイテッド 植物由来栄養素を含む動物細胞培養培地
JP2002520014A (ja) * 1998-07-10 2002-07-09 中外製薬株式会社 動物細胞培養用無血清培地
JP2010503397A (ja) * 2006-09-13 2010-02-04 アボット・ラボラトリーズ 細胞培養の改善
JP2011200180A (ja) * 2010-03-26 2011-10-13 Nippon Menaade Keshohin Kk 幹細胞の未分化維持剤及び増殖促進剤
WO2015030479A1 (fr) * 2013-08-30 2015-03-05 한미약품 주식회사 Procédé pour la production en masse de dérivé du facteur de coagulation sanguine vii humain

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2020162536A (ja) * 2019-03-29 2020-10-08 興人ライフサイエンス株式会社 組み換えタンパク質産生増加のための培地用組成物
WO2025058049A1 (fr) * 2023-09-15 2025-03-20 テーブルマーク株式会社 Composition pour milieu de base pour culture de cellules animales

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JPWO2017195789A1 (ja) 2019-03-14

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