Lu Xin-An, He Ting, Han Zhihai, Ding Yanping, Zhao Liang, Liu Guanghua, De Smet Floris, Huang Xiaojun, Chen Danqing, Qi Feifei, Zhao Xiangyu
Immunochina Pharmaceuticals Co., Ltd, 100089, Beijing, China.
Department of Pulmonary and Critical Care Medicine, The Sixth Medical Center of PLA General Hospital, 100048, Beijing, China.
Cytotechnology. 2020 Oct 29;72(6):897-905. doi: 10.1007/s10616-020-00433-4.
The supercoiled circular (SC) topology form of plasmid DNA has been regarded to be advantageous over open circular or linearized analogue in transfection and expression efficiency, and therefore are largely demanded in the biopharmaceutical manufacturing. However, production of high-purity SC plasmid DNA would result in high manufacturing cost. The effect of SC proportion in plasmid DNA on the quality of packaged lentiviral vectors has never been reported. In this study, we established an efficient system for production of high-titer lentiviral vectors using suspension HEK293SF cells in serum-free media, and the lentiviral titer was not associated with the proportion of SC plasmid DNA. Plasmids DNA with different proportion of SC, open-circular, and linearized forms were prepared using the thermal denaturation method, and were transfected to adherent HEK293T or suspension HEK293SF cells for packaging of lentiviral vectors. The titer of lentiviral vectors from HEK293T cells, but not from HEK293SF cells, was significantly impaired when the proportion of SC plasmid DNA decreased from 60-80% to 30-40%. Further decrease of SC plasmid proportion to 3% led to a dramatic reduction of lentiviral titer no matter the packaging cell line was. However, lentiviral vectors from HEK293SF cells still showed a high titer even when the proportion of SC plasmid DNA was 3%. This study demonstrated that extremely high proportion of SC plasmid DNA was not required for packaging of high-titer lentiviral vector in HEK293SF cells, at least under our manufacturing process.
质粒DNA的超螺旋环状（SC）拓扑结构形式在转染和表达效率方面被认为优于开环或线性化类似物，因此在生物制药生产中需求量很大。然而，高纯度SC质粒DNA的生产会导致高昂的制造成本。SC质粒DNA比例对包装慢病毒载体质量的影响尚未见报道。在本研究中，我们建立了一种在无血清培养基中使用悬浮HEK293SF细胞生产高滴度慢病毒载体的高效系统，且慢病毒滴度与SC质粒DNA的比例无关。使用热变性方法制备了不同比例的SC、开环和线性化形式的质粒DNA，并将其转染至贴壁的HEK293T或悬浮的HEK293SF细胞中以包装慢病毒载体。当SC质粒DNA的比例从60 - 80%降至30 - 40%时，来自HEK293T细胞而非HEK293SF细胞的慢病毒载体滴度显著受损。无论包装细胞系如何，将SC质粒比例进一步降至3%会导致慢病毒滴度急剧下降。然而，即使SC质粒DNA的比例为3%，来自HEK293SF细胞的慢病毒载体仍显示出高滴度。本研究表明，至少在我们的生产过程中，在HEK293SF细胞中包装高滴度慢病毒载体不需要极高比例的SC质粒DNA。
