Dasgupta Anindya, Tinch Stuart, Szczur Kathleen, Ernst Rebecca, Shryock Nathaniel, Kaylor Courtney, Lewis Kendall, Day Eric, Truong Timmy, Swaney William
Division of Experimental Hematology and Cancer Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USA.
Methods Mol Biol. 2020;2086:27-60. doi: 10.1007/978-1-0716-0146-4_3.
In clinical gene transfer applications, lentiviral vectors (LV) have rapidly become the primary means to achieve permanent and stable expression of a gene of interest or alteration of gene expression in target cells. This status can be attributed primarily to the ability of the LV to (1) transduce dividing as well as quiescent cells, (2) restrict or expand tropism through envelope pseudo-typing, and (3) regulate gene expression within different cell lineages through internal promoter selection. Recent progress in viral vector design such as the elimination of unnecessary viral elements, split packaging, and self-inactivating vectors has established a significant safety profile for these vectors. The level of GMP compliance required for the manufacture of LV is dependent upon their intended use, stage of drug product development, and country where the vector will be used as the different regulatory authorities who oversee the clinical usage of such products may have different requirements. As such, successful GMP manufacture of LV requires a combination of diverse factors including: regulatory expertise, compliant facilities, validated and calibrated equipments, starting materials of the highest quality, trained production personnel, scientifically robust production processes, and a quality by design approach. More importantly, oversight throughout manufacturing by an independent Quality Assurance Unit who has the authority to reject or approve the materials is required. We describe here the GMP manufacture of LV at our facility using a four plasmid system where 293T cells from an approved Master Cell Bank (MCB) are transiently transfected using polyethylenimine (PEI). Following transfection, the media is changed and Benzonase added to digest residual plasmid DNA. Two harvests of crude supernatant are collected and then clarified by filtration. The clarified supernatant is purified and concentrated by anion exchange chromatography and tangential flow filtration. The final product is then diafiltered directly into the sponsor defined final formulation buffer and aseptically filled.
在临床基因转移应用中，慢病毒载体（LV）已迅速成为在靶细胞中实现目的基因永久稳定表达或改变基因表达的主要手段。这种地位主要归因于慢病毒载体能够：（1）转导分裂细胞和静止细胞；（2）通过包膜假型化限制或扩大嗜性；（3）通过内部启动子选择调节不同细胞谱系中的基因表达。病毒载体设计方面的最新进展，如去除不必要的病毒元件、拆分包装和自失活载体，为这些载体建立了显著的安全特性。慢病毒载体生产所需的GMP合规水平取决于其预期用途、药品开发阶段以及载体使用的国家，因为监督此类产品临床使用的不同监管机构可能有不同要求。因此，成功的慢病毒载体GMP生产需要多种因素的结合，包括：监管专业知识、合规设施、经过验证和校准的设备、最高质量的起始材料、训练有素的生产人员、科学稳健的生产工艺以及质量源于设计的方法。更重要的是，在整个生产过程中需要由有权拒收或批准材料的独立质量保证部门进行监督。我们在此描述我们设施中使用四质粒系统进行慢病毒载体的GMP生产，其中来自已批准主细胞库（MCB）的293T细胞使用聚乙烯亚胺（PEI）进行瞬时转染。转染后，更换培养基并添加核酸酶以消化残留的质粒DNA。收集两次粗上清液收获物，然后通过过滤进行澄清。澄清的上清液通过阴离子交换色谱和切向流过滤进行纯化和浓缩。然后将最终产品直接透析到申办者定义的最终配方缓冲液中并无菌灌装。
