转染
生物反应器
病毒载体
效价
细胞培养
生物
重组DNA
胎牛血清
载体(分子生物学)
质粒
病毒
病毒学
生物医学工程
医学
生物化学
基因
遗传学
植物
作者
Johannes C.M. van der Loo,William Swaney,Elke Grassman,Austen Terwilliger,Tomoyasu Higashimoto,Axel Schambach,Christopher Baum,Adrian J. Thrasher,David A. Williams,Diana Nordling,Lilith Reeves,Punam Malik
出处
期刊:Gene Therapy
[Springer Nature]
日期:2011-07-14
卷期号:19 (3): 246-254
被引量:31
摘要
The need for γ-retroviral (gRV) vectors with a self-inactivating (SIN) design for clinical application has prompted a shift in methodology of vector manufacturing from the traditional use of stable producer lines to transient transfection-based techniques. Herein, we set out to define and optimize a scalable manufacturing process for the production of gRV vectors using transfection in a closed-system bioreactor in compliance with current good manufacturing practices (cGMP). The process was based on transient transfection of 293T cells on Fibra-Cel disks in the Wave Bioreactor. Cells were harvested from tissue culture flasks and transferred to the bioreactor containing Fibra-Cel in the presence of vector plasmid, packaging plasmids and calcium-phosphate in Dulbecco's modified Eagle's medium and 10% fetal bovine serum. Virus supernatant was harvested at 10–14 h intervals. Using optimized procedures, a total of five ecotropic cGMP-grade gRV vectors were produced (9 liters each) with titers up to 3.6 × 107 infectious units per milliliter on 3T3 cells. One GMP preparation of vector-like particles was also produced. These results describe an optimized process for the generation of SIN viral vectors by transfection using a disposable platform that allows for the generation of clinical-grade viral vectors without the need for cleaning validation in a cost-effective manner.
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