期刊
GENE THERAPY
卷 19, 期 3, 页码 246-254出版社
NATURE PUBLISHING GROUP
DOI: 10.1038/gt.2011.102
关键词
self-inactivating; gamma-retrovirus; cGMP; process development; scale-up; bioreactor
类别
资金
- Cincinnati Children's Research Foundation
- German Academic Exchange Service (DAAD)
- European Union
- German Ministry for Research (BMBF)
- Medical Research Council
- Wellcome Trust
- Great Ormond Street Children's Charity
- MRC [G0501969] Funding Source: UKRI
- Great Ormond Street Hospital Childrens Charity [V1223] Funding Source: researchfish
- Medical Research Council [G0501969] Funding Source: researchfish
The need for gamma-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 x 10(7) 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. Gene Therapy (2012) 19, 246-254; doi:10.1038/gt.2011.102; published online 14 July 2011
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