JP2018023365A - Aav調製物からの混入ウイルスの除去 - Google Patents
Aav調製物からの混入ウイルスの除去 Download PDFInfo
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- JP2018023365A JP2018023365A JP2017125458A JP2017125458A JP2018023365A JP 2018023365 A JP2018023365 A JP 2018023365A JP 2017125458 A JP2017125458 A JP 2017125458A JP 2017125458 A JP2017125458 A JP 2017125458A JP 2018023365 A JP2018023365 A JP 2018023365A
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Abstract
Description
本発明は、ウイルス学及び遺伝子療法の分野に関する。特に、本発明は、混入している桿状のウイルス、例えば、バキュロウイルス等を、本質的に球状のウイルス、例えば、アデノ随伴ウイルス遺伝子療法ベクター等の調製物から除去するための方法に関する。
遺伝子療法において使用するためのウイルスベクターの生産では、安全上の理由で複製欠損性ウイルスが使用される。複製欠損性ウイルスは、in vivoでヒト細胞に感染し、その細胞に導入遺伝子を移入することができるが、細胞において自ら複製することができない。一般には、複製欠損性ウイルスは、例えば、rep遺伝子及びcap遺伝子等の、ウイルスの複製のために重要なウイルス遺伝子を欠失させることによって実現される。これにより、対象の遺伝子産物(複数可)をウイルス遺伝子の代わりに組み込むことも可能になる。宿主細胞においてウイルス粒子を生成するために、複製欠損性ウイルスから欠失しているウイルス遺伝子を別々に、例えば、ヘルパーウイルスを用意することによってもたらす必要がある。
本発明の簡単な説明
第1の態様では、本発明は、バキュロウイルスビリオンの集団からパルボウイルスビリオンの集団を分離するための方法であって、パルボウイルスビリオンの集団及びバキュロウイルスビリオンの集団を含む試料を、パルボウイルスビリオンのみが通過できるフィルターで濾過するステップを含む方法に関する。フィルターは30〜70nm、より好ましくは30〜40nm、さらにより好ましくは32〜38nm、最も好ましくは(約)35nmの公称孔径を有することが好ましい。好ましい実施形態では、パルボウイルスはアデノ随伴ウイルスであり、バキュロウイルスはアウトグラファ・カリフォルニカ(Autographa californica)マルチカプシド(multicapsid)核多角体病ウイルス(AcmNPV)であることがより好ましい。
「ウイルス(virus)」又は「複数のウイルス(viruses)」という用語は、本明細書で使用される場合、天然に存在するウイルス又は遺伝子操作によって改変されたウイルス(すなわち、いわゆる組換えウイルス)だけでなく、ウイルス粒子、すなわち、感染性ウイルスと非感染性ウイルスの両方、国際公開第96/11272号によるパピローマウイルス(papillomavirus)様粒子等のウイルス様粒子(「VLP」)、並びに核酸を含有するが空であってもよいカプシド、及びその一部、特に、1つ又は複数の、好ましくは数個のサブユニット又はカプソメア、特にいくつかのカプソメアが会合して又は組み合わさってカプシドの少なくともおよそ50%、好ましくは少なくとも80%、特におよそ90%を構成するようなカプシドも包含する。混合物から除去されるウイルスは、特に、本質的に球状ではない桿状構造を有するが、医薬製品であるウイルスは、本質的に球状、好ましくは正二十面体の形状である。さらに、「ウイルス」という用語は、そのウイルスのビリオンの集団、好ましくはウイルスの均一な集団を指し得ることが理解される。したがって、「パルボウイルス」という用語は、パルボウイルスビリオンの集団、好ましくはパルボウイルスビリオンの均一な集団を指し得る。
組換えrAAVは、昆虫細胞において、昆虫細胞にAAVベクター及びrep機能及びcap機能を有する3つの組換えバキュロウイルスを感染させる、バキュロウイルスベースの発現系を用いて生産することができる。感染させた昆虫細胞を培養すると、AAV非構造タンパク質遺伝子及びAAV構造タンパク質遺伝子が発現され、導入遺伝子DNAが複製され、組換えAAV粒子(rAAV粒子)がパッケージングされ、アセンブリングされる。rAAV粒子は、両末端がITR領域と隣接する対象の遺伝子産物(複数可)(導入遺伝子(複数可))を、一本鎖DNAの形態で含有する。同時に、組換えバキュロウイルスはこれらの細胞において複製され、このうちのいくらかは、一般に、数日後に感染細胞の溶解及び死をもたらす。生じたウイルス(バキュロウイルス及びrAAV粒子)は細胞培養上清に部分的に放出される、又は、溶解した細胞内に残る。この理由で、一般に、細胞を、当業者に公知の細胞破壊方法、例えば、交互に凍結融解することを用いて、又は、ウイルスの本質的に完全な放出を実現するために例えばトリプシンを用いた酵素による加水分解によって、又は界面活性剤溶解によって破壊する。
Urabeら、2002(Hum.Gene Ther 13(16):1935−1943)によって以前に記載されている通り、昆虫細胞に3つの組換えバキュロウイルスを感染させた後に、アデノ随伴ウイルスベクター(AAV)を生産した。
感染させた3日後に細胞培養物を界面活性剤により溶解させ、その後、9U/mLのベンゾナーゼ(Benzonase)(Merck)を添加することによってヌクレアーゼ処理し、製造者の推奨でインキュベートした。
バキュロウイルスの滴定についての結果が図2に示されている。
Claims (15)
- バキュロウイルスビリオンの集団からパルボウイルスビリオンの集団を分離するための方法であって、パルボウイルスビリオンの集団及びバキュロウイルスビリオンの集団を含む試料をフィルターで濾過するステップを含み、前記フィルターが30〜70nmの公称孔径を有する、方法。
- パルボウイルスがアデノ随伴ウイルスである、請求項1に記載の方法。
- バキュロウイルスがアウトグラファ・カリフォルニカマルチカプシド核多角体病ウイルス(AcmNPV)である、請求項1又は2に記載の方法。
- フィルターがウイルスフィルター又は限外濾過膜である、請求項1〜3のいずれか一項に記載の方法。
- フィルターがサーフェスフィルター及び/又はデプスフィルターである、請求項1〜4のいずれか一項に記載の方法。
- フィルターが、銅アンモニア再生セルロース、ポリフッ化ビニリデン、ポリスルホン、ポリテトラフルオロエチレン、ポリプロピレン、修飾若しくは非修飾ポリエーテルスルホン、酢酸セルロース、硝酸セルロース、ポリアミド又は再生セルロースからなる群から選択される材料を含む、請求項1〜5のいずれか一項に記載の方法。
- 前記フィルターが30〜40nmの公称孔径を有する、請求項1〜6のいずれか一項に記載の方法。
- 前記フィルターが32〜38nmの公称孔径を有する、請求項1〜7のいずれか一項に記載の方法。
- フィルターが、銅アンモニア再生セルロース、好ましくは銅アンモニア再生セルロース中空糸を含み、より好ましくはフィルターがプラノバ35膜又はウルチポアDV50フィルターである、請求項6〜8のいずれか一項に記載の方法。
- 前記試料を、2つ以上のフィルターを使用する濾過に供する、請求項1〜9のいずれか一項に記載の方法。
- 濾過するステップの前の前記試料が、密度勾配、予備濾過、クロマトグラフィーステップ、好ましくはアフィニティークロマトグラフィー及び/又はイオン交換クロマトグラフィー、並びにこれらの方法の組み合わせからなる群から選択される方法を使用して予備精製される、請求項1〜10のいずれか一項に記載の方法。
- 予備濾過が、孔径が70〜200nmのフィルターによって実施される、請求項11に記載の方法。
- 前記試料のpHが6〜10、好ましくは7〜9、より好ましくは7.5〜8.5、最も好ましくは約8である、請求項1〜12のいずれか一項に記載の方法。
- ウイルスフィルター表面1cm2当たり試料1〜200ml、好ましくは1cm2当たり80〜120mlが濾過される、請求項1〜13のいずれか一項に記載の方法。
- パルボウイルスの少なくとも85%が溶出し、バキュロウイルスの力価が少なくとも5log低下する、請求項1〜14のいずれか一項に記載の方法。
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JP2023506472A (ja) * | 2019-12-12 | 2023-02-16 | イー・エム・デイー・ミリポア・コーポレイシヨン | 透析濾過緩衝液を用いて増強したウイルス濾過 |
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