JP7093087B2 - 遺伝子組み換えヒト甲状腺刺激ホルモンを含む組成物および遺伝子組み換えヒト甲状腺刺激ホルモンを生産する方法 - Google Patents
遺伝子組み換えヒト甲状腺刺激ホルモンを含む組成物および遺伝子組み換えヒト甲状腺刺激ホルモンを生産する方法 Download PDFInfo
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Images
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Description
本発明の目的は、本発明の方法により生産される遺伝子組み換えヒト甲状腺刺激ホルモンを含む、再発甲状腺がんの治療または診断のための組成物を提供することである。
[課題を解決するための手段]
上記の目的を達成するため、本発明は、活性成分として遺伝子組み換えヒト甲状腺刺激ホルモン(rhTSH)を含む、再発甲状腺がんの診断または治療のための組成物を提供するものであり、rhTSHは、rhTSHを産生する細胞株を35℃から40℃の培養温度で培養すること、培養細胞数が細胞3×106から2×107個/mLに達した時に培養温度を29℃から34℃の範囲に下げて細胞株を培養すること、および培養液からrhTSHを得ることを含む流加培養により得られる。
[発明の効果]
遺伝子組み換えヒトTSH(rhTSH)発現ベクターの調製および形質転換
本発明の発明者らは、rhTSHを高収率で生産するための発現ベクターを調製した。
rhTSHを高収率で発現する単細胞クローンの調製
例1で調製したpAD-rhTSH発現ベクターから、rhTSHを高収率で発現することができる細胞を以下に示すように調製した。
rhTSHを生産する細胞培養法の確立
上記の例2で選択した3つのRCBクローンについて、rhTSHを生産する最適な細胞培養法を確立した。現在Genzymeという会社からのみ市販されているrhTSHであるタイロゲン(登録商標)は溶液状態で比較的不安定であることが知られており、その生産には灌流培養が使用されている。他の培養法と比べて、灌流培養は比較的生産性が低く、そのプロセスは複雑でコストがかかる。Genzymeのタイロゲン(登録商標)の灌流培養による生産収率は20から30mg/Lであることが知られている。一方、流加培養の場合、生産性は灌流培養に比べて比較的高く、プロセスは複雑ではなく、培養プロセスにおいてrhTSHを高品質に維持できるという利点もある。従って、rhTSHを生産する有益な培養条件の確立が試みられてきた。そのタンパク質の溶液形態での安定性が、様々な要因、たとえば温度、pH、細胞生存率、培養期間等により影響を受けることが知られているので、Genzymeのタイロゲン(登録商標)(以下「対照群」と呼ぶ)の品質と比較することにより流加培養の最適な条件を確立した。
rhTSHを生産する最適温度を確立するために、様々な温度条件で細胞を培養した。
上記の例3.1では、rhTSHを生産する温度条件を確立した。しかし、温度を下げる時の生細胞密度のrhTSHの生産に対する影響を確認するため、以下の実験を実施した。
rhTSHを生産する最適な培養期間を確立するために、生細胞密度、細胞増殖率、およびrhTSHの生産性を、上述したように18日間の培養期間で確認した。
従来のrhTSHタンパク質精製法
性腺刺激ホルモンは、脊椎動物の脳下垂体前葉から分泌される糖タンパク質ポリペプチドホルモンであり、卵胞刺激ホルモン(FSH)、黄体ホルモン(LH)、ヒト絨毛性性腺刺激ホルモン(hCG)、およびTSHなどが挙げられ、それらは同じαサブユニットと特異的なβサブユニットを有する。従って、一般的に、rhTSHタンパク質の精製は性腺刺激ホルモンの精製法と同じような方法で実施される。従って、従来の精製法により精製したタンパク質の精製収率と純度を、本発明により精製したタンパク質のものと比較するため、rhTSHタンパク質を従来の精製法で精製した。
rhTSHタンパク質の高収率精製法の確立
一般的に、rhTSHタンパク質は幅広いPI値を持ち、3つのN-グリコシル化部位が存在するため不純物を除去するのは簡単ではない。そのため、rhTSHタンパク質を効率的に精製する精製条件を確立した。
精製したrhTSHタンパク質の品質分析
rhTSHタンパク質生産方法の確立の主な目的は、流加培養条件下でタンパク質の生産性を向上させることである。本発明による条件下で、rhTSHの生産のため18日間細胞を培養した時、タンパク質の生産性は非常にすぐれていたが、タンパク質の品質は低下するという問題があった。従って、12日間培養したrhTSHタンパク質を等電点電気泳動プロファイル(IEF)およびペプチドマッピングに付し、市販されている対照群と比較した。
精製したrhTSHタンパク質の生理活性の確認
上述の方法により精製したrhTSHタンパク質のin vitroおよびin vivo活性を確認するため、以下の実験を実施した。
rhTSHタンパク質のin vitro活性を確認するため、TSH/TSHRのシグナル伝達経路により誘導されるセカンドメッセンジャーであるcAMPの発現を測定した。
rhTSHタンパク質のin vivo活性を確認するため、rhTSHを動物モデルに投与した時の血中のT4量を測定した。
以下に、本願の実施態様を付記する。
[1] 遺伝子組み換えヒト甲状腺刺激ホルモン(rhTSH)を活性成分として含む、再発甲状腺がんの診断または治療のための組成物であって、
1)rhTSHを産生する細胞株を35℃から40℃の培養温度で培養する工程;
2)培養細胞数が細胞3×10 6 から2×10 7 個/mLに達した時に、前記培養温度を29℃から34℃の範囲に下げることにより前記細胞株を培養する工程;および
3)培養液からrhTSHを得る工程
を含む流加培養により得られる、組成物。
[2] 前記ヒト甲状腺刺激ホルモンが、配列番号1で表されるアミノ酸配列を有するポリペプチドと配列番号2で表されるアミノ酸配列を有するポリペプチドを含む、[1]に記載の組成物。
[3] 工程1)のrhTSHを産生する前記細胞株が、rhTSHのαサブユニットをコードする遺伝子とそのβサブユニットをコードする遺伝子を発現する発現ベクターを含む、[1]に記載の組成物。
[4] 前記細胞株が不死のハイブリドーマ細胞、NS/O骨髄腫細胞、293細胞、チャイニーズハムスター卵巣細胞、HeLa細胞、CapT細胞、およびCOS細胞から成る群より選択されるいずれか1つである、[3]に記載の組成物。
[5] 工程1)の前記培養温度が36℃から38℃の範囲である、[1]に記載の組成物。
[6] 前記培養温度が37℃である、[5]に記載の組成物。
[7] 工程2)において前記培養温度を下げる時、前記細胞数が細胞5×10 6 から1×10 7 個/mLの範囲である、[1]に記載の組成物。
[8] 前記細胞数が細胞6×10 6 から8×10 6 個/mLの範囲である、[7]に記載の組成物。
[9] 前記細胞数が細胞6×10 6 個/mLである、[8]に記載の組成物。
[10] 工程2)の前記培養温度が31℃から33℃の範囲である、[1]に記載の組成物。
[11] 前記培養温度が33℃である、[10]に記載の組成物。
[12] 前記組成物が放射性ヨウ素治療において抗がん療法用補助剤として用いられる、[1]に記載の組成物。
[13] 1)rhTSHを産生する細胞株を35℃から40℃の培養温度で培養する工程;
2)培養細胞数が細胞3×10 6 から2×10 7 個/mLに達した時に、前記培養温度を29℃から34℃の範囲に下げることにより前記細胞株を培養する工程;および
3)培養液からrhTSHを得る工程
を含む、遺伝子組み換えヒト甲状腺刺激ホルモンを流加培養により生産する方法。
[14] 前記ヒト甲状腺刺激ホルモンが、配列番号1で表されるアミノ酸配列を有するポリペプチドと配列番号2で表されるアミノ酸配列を有するポリペプチドを含む、[13]に記載の方法。
[15] 工程1)のrhTSHを産生する前記細胞株が、rhTSHのαサブユニットをコードする遺伝子とそのβサブユニットをコードする遺伝子を発現する発現ベクターを含む、[13]に記載の方法。
[16] 前記細胞株が不死のハイブリドーマ細胞、NS/O骨髄腫細胞、293細胞、チャイニーズハムスター卵巣細胞、HeLa細胞、CapT細胞、およびCOS細胞から成る群より選択されるいずれか1つである、[13]に記載の方法。
[17] 工程1)の前記培養温度が36℃から38℃の範囲である、[13]に記載の方法。
[18] 前記培養温度が37℃である、[17]に記載の方法。
[19] 工程2)において前記培養温度を下げる時、前記細胞数が細胞5×10 6 から1×10 7 個/mLの範囲である、[13]に記載の方法。
[20] 前記細胞数が細胞6×10 6 から8×10 6 個/mLの範囲である、[19]に記載の方法。
[21] 前記細胞数が細胞6×10 6 個/mLである、[20]に記載の方法。
[22] 工程2)の前記培養温度が31℃から33℃の範囲である、[13]に記載の方法。
[23] 前記培養温度が33℃である、[22]に記載の方法。
Claims (9)
- 1)rhTSHを産生するチャイニーズハムスター卵巣(CHO)細胞株を35℃から40℃の培養温度で培養する工程;
2)培養細胞数が細胞5×106から1×107個/mLに達した時に、前記培養温度を29℃から34℃の範囲に下げることにより前記細胞株を培養する工程;ならびに
3)(i)抗-性腺刺激ホルモン抗体に結合した樹脂を用いたアフィニティークロマトグラフィーにより一次精製液を得る工程、
(ii)前記一次精製液をフィルターで深層ろ過することにより二次精製液を得る工程、
(iii)前記二次精製液をダイアフィルトレーションすることにより三次精製液を得る工程、および
(iv)前記三次精製液をアニオン交換クロマトグラフィーにより精製することにより、宿主細胞タンパク質(HCP)が100ppm未満のrhTSHを得る工程
を含む、培養液からrhTSHを得る工程
を含む、遺伝子組み換えヒト甲状腺刺激ホルモンを流加培養により高収率および高純度で生産する方法。 - 前記ヒト甲状腺刺激ホルモンが、配列番号1で表されるアミノ酸配列を有するポリペプチドと配列番号2で表されるアミノ酸配列を有するポリペプチドを含む、請求項1に記載の方法。
- 工程1)のrhTSHを産生する前記細胞株が、rhTSHのαサブユニットをコードする遺伝子とそのβサブユニットをコードする遺伝子を発現する発現ベクターを含む、請求項1に記載の方法。
- 工程1)の前記培養温度が36℃から38℃の範囲である、請求項1に記載の方法。
- 前記培養温度が37℃である、請求項4に記載の方法。
- 工程2)において前記培養温度を下げる時の前記培養細胞数が細胞6×106から8×106個/mLの範囲である、請求項1に記載の方法。
- 前記細胞数が細胞6×106個/mLである、請求項6に記載の方法。
- 工程2)の前記培養温度が31℃から33℃の範囲である、請求項1に記載の方法。
- 前記培養温度が33℃である、請求項8に記載の方法。
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