Scale-up and manufacturing of clinical-grade self-inactivating γ-retroviral vectors by transient transfection.
Scale-up and manufacturing of clinical-grade self-inactivating γ-retroviral vectors by transient transfection.
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作者：
临床应用对具有自失活（SIN）设计的γ-逆转录病毒（gRV）载体的需求促使载体制造方法从传统使用稳定生产线转向基于瞬时转染的技术。在此，我们着手定义和优化一种可扩展的制造工艺，用于在符合现行良好生产规范 (cGMP) 的封闭系统生物反应器中使用转染来生产 gRV 载体。该过程基于 Wave 生物反应器中 Fibra-Cel 盘上 293T 细胞的瞬时转染。从组织培养瓶中收获细胞，并在载体质粒、包装质粒和磷酸钙存在下，在Dulbecco改良Eagle培养基和10%胎牛血清中转移至含有Fibra-Cel的生物反应器中。每隔 10-14 小时收获一次病毒上清液。使用优化的程序，总共产生了五个共亲性 cGMP 级 gRV 载体（每个 9 升），在 3T3 细胞上滴度高达每毫升 3.6×107 感染单位。还生产了一种载体样颗粒的 GMP 制剂。这些结果描述了使用一次性平台转染生成 SIN 病毒载体的优化过程，该过程允许以经济高效的方式生成临床级病毒载体，而无需进行清洁验证。
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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影响因子：
5.1
作者：
Kraunus, J;Schaumann, DHS;Baum, C
通讯作者：
Baum, C
DOI：
10.1073/pnas.93.21.11382
发表时间：
1996-10-15
影响因子：
11.1
作者：
Naldini, L;Blomer, U;Verma, IM
通讯作者：
Verma, IM
影响因子：
5.4
作者：
Dull, T;Zufferey, R;Naldini, L
通讯作者：
Naldini, L
影响因子：
5.1
作者：
Herbst, F.;Ball, C. R.;Glimm, H.
通讯作者：
Glimm, H.
DOI：
10.1073/pnas.87.23.9202
发表时间：
1990-12-01
影响因子：
11.1
作者：
GREZ, M;AKGUN, E;OSTERTAG, W
通讯作者：
OSTERTAG, W