一次性生物反应器悬浮培养HEK293细胞生产Ad-IFNγ 的工艺

Producing Ad-IFN gamma by suspension culture of HEK293 cells in a disposable bioreactor

摘要:

腺病毒载体是极具发展前景的基因治疗载体之一，为获得一条新型腺病毒规模化生产工艺，研究采用5 L振荡激流式一次性生物反应器悬浮培养HEK293细胞来生产重组腺病毒载体。细胞经种子链逐步扩增后，接种至AP10生物反应器，采用CD293无血清培养基流加培养悬浮HEK293细胞，细胞密度达约2.0×106个/mL时，以30 MOI (Multiplicity of infection) 感染重组Ad-IFNγ (Recombinant adenovirus-interferon gamma)，48 h后收获细胞，3次冻融裂解离心收获上清病毒粗产品。采用壳蛋白免疫法快速测定粗产品滴度。结果表明，采用振荡激流式一次性生物反应器，流加HEK293细胞悬浮培养6 d，密度可达2.0×106个/mL，Ad-IFNγ粗产量达1.49×1013 IFU (Infectious unit)，单细胞包装量达3 800 IFU/cell。采用阴离子交换层析法纯化重组腺病毒，回收率35.9%。建立了利用5 L激流式一次性生物反应器悬浮培养HEK293细胞生产重组腺病毒载体Ad-IFNγ的初步工艺。

关键词:重组腺病毒，HEK293细胞悬浮培养，一次性生物反应器，分离纯化，阴离子交换层析

Abstract:

Adenovirus vectors are promising delivery systems for gene therapy. We established a new process for clinic trial of recombinant adenovirus vectors using a novel disposable bioreactor. The suspension HEK293 cells were inoculated into a 5 L disposable bioreactor with parameters control of pH, DO, agitation and temperature. After 6 days of a fed-batch culture, the final cell density reached 2.0×106 cells/mL. The culture was infected with Ad-IFNγ at an MOI of 30. The harvest was performed at approximately 48 h post-infection and crude viral lysate was obtained after 3 freeze/thaw cycles and centrifugation. The maximum titers of crude viral lysate was 1.49×1013 Infectious units (IFU) and the bulk product specific was 3 800 IFU/cell. Purified Ad-IFNγ by anion-exchange chromatography and the final recovery of infectious unit reached 35.9%. The result demonstrates that an efficient and stable process of producing Ad-IFNγ using a disposable fed-batch bioreactor is established.

Key words:adenovirus vectors, suspension culture of HEK293 cell, disposable bioreactor, purification, anion-exchange chromatography