JP6997079B2 - Composition for medium - Google Patents
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- JP6997079B2 JP6997079B2 JP2018517040A JP2018517040A JP6997079B2 JP 6997079 B2 JP6997079 B2 JP 6997079B2 JP 2018517040 A JP2018517040 A JP 2018517040A JP 2018517040 A JP2018517040 A JP 2018517040A JP 6997079 B2 JP6997079 B2 JP 6997079B2
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Description
本発明は、遺伝子組み換えタンパク質の産生能力を高めることができる培地用組成物に関するものである。 The present invention relates to a composition for a medium capable of enhancing the ability to produce a recombinant protein.
バイオ医薬品（抗体医薬品）は、特異的、高活性でかつ副作用が少ないなどの従来の低分子医薬にはない効果を有しており、今後高い成長が見込まれている。また、再生医療に必要な細胞の培養（iPS細胞、ES細胞等）、酵素などの有用物質の生産等にも細胞培養が必要である。そのため企業間の開発競争は激化しており、製造や研究開発の一部外注化が進むと予想されている。
バイオ医薬品の多くが、翻訳後修飾が比較的ヒトに近い等の理由により動物細胞を用いて生産されている。この動物細胞で物質生産するには、動物細胞の増殖に必要な血清や血清中から分離したタンパク質成分を添加した培地がよく使われている。しかし、血清は生物材料であるために、マイコプラズマ、ウイルス、ＢＳＥの原因となる異常プリオンが混入している可能性がある。また、血清はロットごとに生物活性が異なり、品質のよいロットを選択するための検査が必要であるところ、この検査にもコストが発生する。そのため、最近、血清を含まない無血清培地に種々の成分を加えて細胞を増殖させる様々な方法が提案されている（特許文献１）。Biopharmacy (antibody drug) has effects that conventional small molecule drugs do not have, such as specificity, high activity, and few side effects, and high growth is expected in the future. In addition, cell culture is also necessary for cell culture (iPS cells, ES cells, etc.) necessary for regenerative medicine and production of useful substances such as enzymes. As a result, development competition between companies is intensifying, and it is expected that some manufacturing and R & D will be outsourced.
Many biopharmaceuticals are produced using animal cells because their post-translational modifications are relatively close to humans. In order to produce substances in these animal cells, a medium containing serum necessary for the growth of animal cells or a protein component separated from the serum is often used. However, since serum is a biological material, it may be contaminated with mycoplasma, viruses, and abnormal prions that cause BSE. In addition, the biological activity of serum differs from lot to lot, and a test for selecting a high-quality lot is required, and this test also incurs costs. Therefore, recently, various methods have been proposed in which various components are added to a serum-free medium containing no serum to proliferate cells (Patent Document 1).
そこで、細胞増殖自体の誘起または促進に不可欠な成分に加えて、培養速度を高い水準に維持する役割を果たす補完的な成分の添加が検討されてきた。例えば、大豆由来β－コングリシニン（特許文献２）、リン脂質（特許文献３）などが、報告されている。 Therefore, in addition to the components essential for inducing or promoting cell proliferation itself, the addition of complementary components that play a role in maintaining the culture rate at a high level has been studied. For example, soybean-derived β-conglycinin (Patent Document 2), phospholipid (Patent Document 3) and the like have been reported.
一方、バイオ医薬品生産等のために開発される無血清培地の最終目的は組み換えタンパク質生産を高い効率で行うことであるので、生産能力を上げることが望まれている。そのため、無血清培地の細胞増殖の改善だけでなく、タンパク質分泌促進因子を開発することも必要である。 On the other hand, since the ultimate purpose of the serum-free medium developed for the production of biopharmacy is to carry out the production of recombinant proteins with high efficiency, it is desired to increase the production capacity. Therefore, it is necessary not only to improve cell proliferation in serum-free medium, but also to develop protein secretagogue factors.
本発明は、上記のような状況に鑑み、無血清培地を用いた場合であっても、遺伝子組み換えタンパク質の産生能力を高めることができる方法及び組成物を提供することを課題とする。 In view of the above circumstances, it is an object of the present invention to provide a method and a composition capable of enhancing the production ability of a recombinant protein even when a serum-free medium is used.
本発明者らは、酵母抽出物を無血清培地に添加した培地で動物細胞を培養すると、組み換えタンパク質の生産能力を高めることができることを見出し、本発明に至った。 The present inventors have found that culturing animal cells in a medium obtained by adding a yeast extract to a serum-free medium can increase the production capacity of recombinant proteins, leading to the present invention.
すなわち本発明は、
（１）酵母抽出物を含む遺伝子組換えタンパク質産生促進剤、
（２）酵母抽出物が有機溶媒抽出物である、（１）のタンパク質産生促進剤、
（３） （１）又は（２）のタンパク質促進剤を含む動物細胞用培地、
（４）酵母又は酵母残渣から有機溶媒により抽出する工程を有する遺伝子組換えタンパク質産生促進剤の製造方法、
（５）有機溶媒がエタノールである、（４）の製造方法、
を提供するものである。That is, 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) Animal cell medium containing the protein promoter of (1) or (2),
(4) A method for producing a recombinant protein production promoter, which comprises 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.
本発明によると、組み換えタンパク質の産生を促進することができ、有用物質を効率よく製造することができる。 According to the present invention, the production of recombinant proteins can be promoted, and useful substances can be efficiently produced.
以下、本発明を詳細に説明する。
本発明の培地は、遺伝子組み換えタンパク質を生産するための培地であって、酵母抽出物を含むことを特徴とする。Hereinafter, the present invention will be described in detail.
The medium of the present invention is a medium for producing a recombinant protein, and is characterized by containing a yeast extract.
本願発明の組成物であるタンパク質産生促進剤は、以下のように得ることができる。本願発明の製造に用いられる酵母としては、パン酵母、ビール酵母、トルラ酵母（Candida utilis）などを挙げることができ、中でもトルラ酵母が望ましい。
酵母の培養方法は、特に限定されない。酵母の培養形式はバッチ培養、あるいは連続培養のいずれかが用いられる。培地には一般的に炭素源として、ブドウ糖、酢酸、エタノール、グリセロール、糖蜜、亜硫酸パルプ廃液等が用いられ、窒素源としては、尿素、アンモニア、硫酸アンモニウム、塩化アンモニウム、硝酸塩などが使用される。リン酸、カリウム、マグネシウム源も過リン酸石灰、リン酸アンモニウム、塩化カリウム、水酸化カリウム、硫酸マグネシウム、塩化マグネシウム等の通常の工業用原料でよく、その他亜鉛イオン、マンガンイオン、鉄イオン、銅イオン等の無機塩を添加する。その他、ビタミン、アミノ酸、核酸関連物質等を添加しても良い。カゼイン、酵母エキス、肉エキス、ペプトン等の有機物を添加しても良い。The protein production-promoting agent, which 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, beer yeast, and torula yeast (Candida utilis), and torula yeast is particularly preferable.
The yeast culture method is not particularly limited. As the yeast culture format, either batch culture or continuous culture is used. Generally, glucose, acetic acid, ethanol, glycerol, syrup, sulfite pulp waste liquid and the like are used as the carbon source for the medium, and urea, ammonia, ammonium sulfate, ammonium chloride, nitrate and the like are used as the nitrogen source. The source of phosphoric acid, potassium and magnesium may be ordinary industrial raw materials such as lime perphosphate, ammonium phosphate, potassium chloride, potassium hydroxide, magnesium sulfate and magnesium chloride, and other zinc ion, manganese ion, iron ion and copper. Add inorganic salts such as ions. In addition, 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.
さらに、本発明で用いられる酵母として、もっとも好ましくは、酵母エキス抽出残渣である。本願の酵母エキス抽出残渣とは、培養した酵母から酵母エキスなどを抽出した後の固形成分をいう。酵母エキス抽出法は、とくに制限がないが、一般的に、自己消化法、熱水抽出法、酵素抽出法、酸、若しくはアルカリ抽出法、又はこれらの組み合わせにより行う。これらの抽出法により抽出した後の酵母菌体や固形分を本願は、酵母エキス抽出残渣という。
当該残渣をドラムドライヤーで乾燥後、後述の方法により本願発明の組成物の製造を行うことができる。Further, the yeast used in the present invention is most preferably a yeast extract extraction residue. The yeast extract extraction residue of the present application refers to a solid component after extracting yeast extract or the like from 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. The yeast cells and solids after extraction by these extraction methods are referred to as yeast extract extraction residues in the present application.
After the residue is dried with a drum dryer, the composition of the present invention can be produced by the method described below.
酵母又は酵母残渣からの抽出工程は、酵母又は酵母残渣を蒸留水に懸濁、遠心をすることで、酵母又は酵母残渣を洗浄する工程を設けても良い。有機溶媒抽出、酵素処理した後有機溶媒抽出、超臨界抽出のいずれかの方法を用いる。例えば、有機溶媒抽出の場合、使用する溶剤としては、メタノール、エタノール、ｎ-プロパノール、ヘキサン、アセトン、塩素系溶剤がある。好ましくは炭素数1～３の低級アルコールであり、さらに好ましくは、エタノールである。そのため、目的に応じた含水エタノールが用いられる。水／エタノールの容積比は、通常０．０１／９９．９９～３０／７０、好ましくは０．５／９９．５～３０／７０である。この水分含量としては、原料中の水分量を考慮にいれても構わない。エタノール使用量は、特に限定ない。酵母又は酵母残渣に対して、１～３０倍程度のエタノールを使用する。 The extraction step from the yeast or the yeast residue may be provided with a step of washing the yeast or the yeast residue by suspending the yeast or the yeast residue in distilled water and centrifuging the yeast or the yeast residue. Either organic solvent extraction, enzymatic treatment followed by organic solvent extraction, or supercritical extraction is used. For example, in the case of organic solvent extraction, the solvent used includes methanol, ethanol, n-propanol, hexane, acetone, and a chlorine-based solvent. A lower alcohol having 1 to 3 carbon atoms is preferable, and ethanol is more preferable. 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 the water content, the water content in the raw material may be taken into consideration. The amount of ethanol used is not particularly limited. Use about 1 to 30 times as much ethanol as yeast or yeast residue.
抽出方法は、エタノールを加えて十分攪拌後、適当な温度および時間で反応を行う。温度は特に限定はないが、通常２０℃～８０℃であり、好ましくは５０℃～７０℃である。抽出時間は、特に限定はないが、通常は１時間以上、好ましくは３～９時間である。抽出方法は、攪拌、還流、浸漬、振盪、超音波抽出が挙げられる。抽出前に酵素処理をすると抽出時間の短縮が可能となる。酵素としては、細胞壁溶解酵素であるストレプトマイセス属由来のβグルカナーゼ「デナチームGEL」（ナガセケムテックス社製）、Taloromyces属由来のβグルカナーゼ「Filtrase BRX」（DSMジャパン社製）、「ツニカーゼFN」（天野エンザイム社製）がある。好ましくはツニカーゼFNである。 As for the extraction method, ethanol is added and the mixture is sufficiently stirred, and then the reaction is carried out 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 more, preferably 3 to 9 hours. Extraction methods include stirring, refluxing, dipping, shaking and ultrasonic extraction. If enzyme treatment is performed before extraction, the extraction time can be shortened. As enzymes, β-glucanase "Denateam GEL" (manufactured by Nagase ChemteX) derived from Streptomyces, which is a cell wall lytic enzyme, β-glucanase "Filtrase BRX" (manufactured by DSM Japan), "Tunicase FN" derived from Taloromyces. (Manufactured by Amano Enzyme). Tunicase FN is preferred.
有機溶媒抽出をした後は、中性脂質・エルゴステロール・遊離脂肪酸を除去するため、水を加える分別工程や冷却工程を加えても良い。それにより酸化しにくく経時的な劣化を改善することが可能となる。加水して調製するエタノール濃度は５０～９０％が良く、好ましくは７０％が良い。冷却工程は、冷却温度としては10℃以下が良い。特には5℃以下が望ましい。冷却時間は１～１０時間、好ましくは1時間程度が良い。 After the organic solvent extraction, a separation step of adding water or a cooling step may be added in order to remove neutral lipids, ergosterols and free fatty acids. As a result, it is difficult to oxidize and deterioration over time can be improved. The concentration of ethanol prepared by adding water is preferably 50 to 90%, preferably 70%. In the cooling process, the cooling temperature should be 10 ° C or lower. Especially, it is desirable that the temperature is 5 ° C or lower. The cooling time is preferably 1 to 10 hours, preferably about 1 hour.
なお、超臨界抽出は、酵母懸濁液に超臨界状態の二酸化炭素を接触させた後、減圧して大気圧に戻すことにより酵母懸濁液から二酸化炭素を分離する方法である。 The supercritical extraction is a method of separating carbon dioxide from the yeast suspension by contacting the yeast suspension with carbon dioxide in a supercritical state and then reducing the pressure to return to atmospheric pressure.
上述のようにして得られる酵母抽出物を濃縮する場合は、公知の濃縮装置を用いることが出来る。出来るだけ温度を低くすることが可能な減圧濃縮機を使用することが好ましい。 When concentrating the yeast extract obtained as described above, a known concentrator can be used. It is preferable to use a vacuum concentrator that can lower the temperature as much as possible.
さらに、賦形剤として、中鎖脂肪酸や臭気抑制のためシクロデキストリン・クラスターデキストリン等を添加して乾燥することも可能である。乾燥は、凍結乾燥、真空乾燥等の公知の乾燥法を用いることが出来る。 Further, it is also possible to add medium-chain fatty acid, cyclodextrin, cluster dextrin or the like as an excipient to suppress odor and dry the mixture. For drying, known drying methods such as freeze-drying and vacuum drying can be used.
本発明の組成物は、本発明の活性に必要な純度を得るため、又は、原料由来の臭気等に応じて、シリカゲルカラム等で純度を高めることもできる。純度を調製する工程は、前述までの各工程を適宜組み合わせることができる。 The composition of the present invention may be purified by a silica gel column or the like in order to obtain the purity required for the activity of the present invention or depending on the odor derived from the raw material. The steps for adjusting the purity can be appropriately combined with the above-mentioned steps.
上記のようにして得られる本願組成物は、酵母又は酵母残渣に含まれる脂質成分を多く含む組成物である。本願では、当該組成物をタンパク質産生促進剤として使用することが出来る。 The composition of the present application obtained as described above is a composition containing a large amount of lipid components contained in yeast or yeast residue. In the present application, the composition can be used as a protein production promoter.
このようにして得られた本願の組成物を動物培養用培地に添加することで動物細胞用培地を得ることができる。
本願組成物は、血清培地、低血清培地でも使用できるが、無血清培地の方がより好適に使用できる。培地の組成は、一般的な組成でよく、一般的に市販されている動物細胞用培地も使用することができる。また、宿主細胞に応じで適宜選択することができる。
本発明で用いる動物細胞としては、特に制限はないが、哺乳動物細胞に対して、特にCHO-K1、HEK293等に対して好適である。An animal cell medium can be obtained by adding the composition of the present application 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 generally commercially available medium for animal cells can also be used. Further, it can be appropriately selected depending on the host cell.
The animal cells used in the present invention are not particularly limited, but are suitable for mammalian cells, particularly CHO-K1, HEK293 and the like.
本発明の組成物を培地へ添加する方法は、任意である。通常、前述までの方法で得られた、本発明の組成物を噴霧乾燥、凍結乾燥等して使用する。また、乾燥品を使用しなくても良い。本願発明の組成物の培地への添加量は、精製度合に応じて適宜調整する。乾燥品を使用する場合の添加量は、通常、培地当たり、０．００１重量％～０．５重量％の濃度で添加する。 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 above-mentioned method is spray-dried, freeze-dried or the like before use. Moreover, it is not necessary to use a dried product. The amount of the composition of the present invention added to the medium is appropriately adjusted according to the degree of purification. When a dried product is used, the amount to be added is usually 0.001% by weight to 0.5% by weight per medium.
本発明の組成物を動物細胞用培地に添加し、培養することで、組換えタンパク質等の有用物質の発現が促進される。そのため、本願組成物を用いて培養した培地から目的とする有用物質を単離することにより、有用物質を製造することができる。動物細胞から得られる有用物質としては、抗体、酵素、ホルモンなどがある。 By adding the composition of the present invention to a medium for animal cells and culturing it, the expression of useful substances such as recombinant proteins is promoted. Therefore, a useful substance can be produced by isolating the target useful substance from the medium cultured using the composition of the present application. Useful substances obtained from animal cells include antibodies, enzymes, hormones and the like.
以下に実施例を用いて、本発明を具体的に説明する。
本発明はこれらに限定されるものではない。Hereinafter, the present invention will be specifically described with reference to Examples.
The present invention is not limited to these.
（酵母抽出物の製造例）
（酵母の培養）
キャンディダ・ウチリスＣＳ７５２９株（ＦＥＲＭＰ－３３４０）を予めＹＰＤ培地（酵母エキス１％、ポリペプトン２％、グルコース２％）を含む三角フラスコで種母培養し、これを３０Ｌ容発酵槽で、１８Ｌ培地に１～２％植菌した。培地組成は、グルコース４％、燐酸一アンモニウム０．３％、硫酸アンモニウム０．１６１％、塩化カリウム０．１３７％、硫酸マグネシウム０．０８％、硫酸銅１．６ｐｐｍ、硫酸鉄１４ｐｐｍ、硫酸マンガン１６ｐｐｍ、硫酸亜鉛１４ｐｐｍを用いた。培養条件は、ｐＨ４．０、培養温度３０℃、通気量１ｖｖｍ、撹拌６００ｒｐｍで行った。ｐＨのコントロールは、アンモニアで行った。１６時間菌体培養した後、培養液を回収し、遠心分離により集菌して、１８０ｇの湿潤酵母菌体を得た。
（酵母エキスの抽出）
菌体培養後の湿潤酵母菌体を蒸留水に懸濁して遠心分離を繰り返すことで洗浄した。洗浄後は湿潤菌体を蒸留水に再度懸濁する、又は、凍結乾燥、若しくは熱風乾燥させた乾燥菌体を蒸留水に懸濁する。２Ｎ ＮａＯＨでｐＨ１３に調整後、７０℃で２０分間攪拌し、酵母エキスを抽出した。酵母エキス抽出後の菌体残渣をドラムドライヤーで乾燥し、乾燥物１００ｇを酵母残渣として使用した。(Production example of yeast extract)
(Yeast culture)
Candida uchiris CS7529 strain (FERMP-3340) was cultivated in an Erlenmeyer flask containing YPD medium (yeast extract 1%, polypeptone 2%, glucose 2%) in advance, and this was placed in a 30 L fermenter in an 18 L medium. Inoculated 1-2%. The medium composition was glucose 4%, monoammonium phosphate 0.3%, ammonium sulfate 0.161%, potassium chloride 0.137%, magnesium sulfate 0.08%, copper sulfate 1.6 ppm, iron sulfate 14 ppm, manganese sulfate 16 ppm, 14 ppm 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. After culturing the cells for 16 hours, the culture broth was collected and collected by centrifugation to obtain 180 g of wet yeast cells.
(Extraction of yeast extract)
Wet yeast cells after cell culture were suspended in distilled water and washed by repeated centrifugation. After washing, the wet cells are suspended again in distilled water, or the dried cells that have been freeze-dried or hot-air dried are suspended in distilled water. After adjusting the pH to 13 with 2N NaOH, the mixture was stirred at 70 ° C. for 20 minutes to extract the yeast extract. The bacterial cell residue after extracting the yeast extract was dried with a drum dryer, and 100 g of the dried product was used as the yeast residue.
製造例１
上記酵母残渣１００ｇに対して９５％エタノールを２倍量となるように加え、６０℃、９時間抽出した。５℃に降温して１時間保持した後、ろ紙（ADVANTEC社、NO.5C）で濾過し不溶物を取り除いた。さらに、ろ過液をロータリーエバポレーターにて減圧濃縮乾固し、組成物５．５ｇを得た。Production Example 1
95% ethanol was added in a double amount to 100 g of the yeast residue, and the mixture was extracted at 60 ° C. for 9 hours. After lowering the temperature to 5 ° C. and holding for 1 hour, the insoluble material was removed by filtering with a filter paper (ADVANTEC, NO.5C). Further, the filtrate was concentrated to dryness under reduced pressure using a rotary evaporator to obtain 5.5 g of the composition.
得られた組成物をTLC分析した。
分析条件：
TLCガラスプレートシリカゲル600 F254 5×20cm
展開溶媒：クロロホルム：メタノール：水＝65:25:4
サンプル：左から製造例１
ホスファチジルコリン
ホスファチジルイノシトール
ホスファチジルエタノールアミン
発色：ニンヒドリンスプレーII
The resulting composition was TLC analyzed.
Analysis conditions:
TLC glass plate silica gel 600 F254 5 × 20cm
Developing solvent: Chloroform: Methanol: Water = 65: 25: 4
Sample: Production example 1 from the left
Phosphatidylcholine
Phosphatidylinositol
Phosphatidylethanolamine Color development: Ninhydrin Spray II
結果を図１に示す（検出されたスポット外縁に沿って略円を記載した）。本発明（製造例１）の組成物では、４つのスポットが確認された。また、ホスファチジルコリン（ＰＣ）では、全く検出されなかったが、ホスファチジルイノシトール（ＰＩ）とホスファチジルエタノールアミン（ＰＥ）においても、それぞれスポットが確認された。 The results are shown in FIG. 1 (abbreviated circles are shown along the outer edges of the detected spots). In the composition of the present invention (Production Example 1), four spots were confirmed. In addition, although it was not detected at all in phosphatidylcholine (PC), spots were also confirmed in phosphatidylinositol (PI) and phosphatidylethanolamine (PE), respectively.
製造例１で得られた組成物を培地に添加し、Gaussia Luciferase（GLuc）の発現量、Human IL-6（hIL6）の発現量を解析した。使用した培地は、ＲＰＭＩ１６４０（WAKO製 「189-02025」）に、製造例１で得られた組成物を０．０１％添加した無血清培地を用いた。
安定発現株の取得：ＣＨＯ－Ｋ１細胞を1×104 cells/mL/24 well plateに播種した。翌日、hIL6遺伝子（500ng/well）をトランスフェクションした。トランスフェクション3日目に、細胞を100mm dishに移して、細胞が８０％程度コンフルエントになってから、Ｇ４１８含有培地に切り替え、約１週間培養しながらセレクションを行った。その後、出来上がったコロニーをピックアップして96wellpateで培養し、活性確認を行い、活性があった細胞を更に24well plate、6well plateにスケールアップしてhIL6安定発現株を取得した。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. As the medium used, a serum-free medium obtained by adding 0.01% of the composition obtained in Production Example 1 to RPMI1640 (“189-02025” manufactured by WAKO) was used.
Acquisition of stable expression strain: CHO-K1 cells were seeded in 1 × 10 4 cells / mL / 24 well plate. The next day, the hIL6 gene (500 ng / well) was transfected. On the 3rd day of transfection, the cells were transferred to a 100 mm dish, and after the cells became about 80% confluent, the cells were switched to a G418-containing medium, and selection was performed while culturing for about 1 week. Then, the completed colonies were picked up and cultured at 96 well pate to confirm the activity, and the active cells were further scaled up to 24 well plate and 6 well plate to obtain a stable hIL6 expression strain.
（実施例１） 酵母抽出物添加無血清培地のGluc安定発現株に与える影響
GLuc安定発現株を前述のhIL6と同様の方法で取得し、5×105 cells/2 mL/6 well plateに播種した。翌日、PBS(-)で洗浄し、酵母抽出物含有培地2 mL／well添加し、48時間培養した。 48hr後、培養上清中のGluc活性を測定するとともに、セルカウント法にて細胞数を計測した。なお、結果はRLU/cells/dayで示している。結果を図２に示す。
取得した安定株3株(230株、232株、236株)で評価し、3株とも、細胞当たりのGluc活性は、RPMI1640/酵母抽出物群がRPMI1640/FBS群やCDM4CHO群よりも高い活性を示した。なお、RPMI1640/FBS群は、RPMI1640に、ウシ血清(FBS)を終濃度10%となるように添加して、CHO-K1細胞を培養する培地として用いた群である。CDM4CHO群は、CHO-K1細胞を培養する培地にCDM4CHO（GE Healthcare社製「HyClone」）のみを用いた群である。(Example 1) Effect of serum-free medium containing yeast extract on Gluc stable expression strains
A GLuc stable expression strain was obtained in the same manner as in hIL6 described above, and seeded in 5 × 10 5 cells / 2 mL / 6 well plates. The next day, the cells were washed with PBS (-), 2 mL / well of yeast extract-containing medium was added, and the cells were cultured for 48 hours. After 48 hours, the Gluc activity in the culture supernatant was measured, and the number of cells was measured by the cell counting method. The results are shown in RLU / cells / day. The results are shown in FIG.
The obtained stable strains (230 strains, 232 strains, and 236 strains) were evaluated, and the Gluc activity per cell of all 3 strains was higher in the RPMI1640 / yeast extract group than in the RPMI1640 / FBS group and CDM4CHO group. Indicated. The RPMI1640 / FBS group is a group in which bovine serum (FBS) is added to RPMI1640 so as to have a final concentration of 10% and used as a medium for culturing CHO-K1 cells. The CDM4CHO group is a group in which only CDM4CHO (“HyClone” manufactured by GE Healthcare) is used as a medium for culturing CHO-K1 cells.
（実施例2） 酵母抽出物添加無血清培地のhIL6安定発現株に与える影響
取得した安定株2株(HE11A1株、HE12A2株)で評価し、2株を5×105 cells/2 mL/6 well plateに播種した。翌日、PBS(-)で洗浄し、酵母抽出物含有培地2 mL/well添加し、48時間培養した。 48hr後、培養上清中のhIL6発現量を測定するとともに、セルカウント法にて細胞数を計測した。なお、結果はpg/cells/dayで示している。結果を図3に示す。
HE11A1株、HE12A2株とも、細胞当たりのIL-6産生量はRPMI1640/酵母抽出物群が、ほかの2群と比べて明らかに高い値を示した。 (Example 2) Effect of yeast extract-added serum-free medium on hIL6 stable expression strains Evaluated with the obtained 2 stable strains (HE11A1 strain, HE12A2 strain), and 2 strains were evaluated as 5 × 10 5 cells / 2 mL / 6 It was sown on a well plate. The next day, the cells were washed with PBS (-), 2 mL / well of yeast extract-containing medium was added, and the cells were cultured for 48 hours. After 48 hours, the expression level of hIL6 in the culture supernatant was measured, and the number of cells was measured by the cell counting method. The results are shown in pg / cells / day. The results are shown in Figure 3.
In both the HE11A1 strain and the HE12A2 strain, the IL-6 production per cell was clearly higher in the RPMI1640 / yeast extract group than in the other two groups.
以上説明してきたように、本発明によると、組み換えタンパク質の産生を促進することができ、有用物質を効率よく製造することができる。 As described above, according to the present invention, the production of recombinant proteins can be promoted, and useful substances can be efficiently produced.
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| WO2007043330A1 (en) | 2005-10-12 | 2007-04-19 | Asahi Breweries, Ltd. | Process for producing recombinant protein |
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| JP2001501830A (en) | 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 | Animal cell culture medium containing plant-derived nutrients |
| JP2002520014A (en) | 1998-07-10 | 2002-07-09 | 中外製薬株式会社 | Serum-free medium for animal cell culture |
| WO2007043330A1 (en) | 2005-10-12 | 2007-04-19 | Asahi Breweries, Ltd. | Process for producing recombinant protein |
| JP2010503397A (en) | 2006-09-13 | 2010-02-04 | アボット・ラボラトリーズ | Improved cell culture |
| JP2011200180A (en) | 2010-03-26 | 2011-10-13 | Nippon Menaade Keshohin Kk | Undifferentiation maintaining agent and proliferation promoter of stem cell |
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