Davami Fatemeh, Eghbalpour Farnaz, Barkhordari Farzaneh, Mahboudi Fereidoun
Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran.
Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran ; Islamic Azad University of Arak, Arak, Iran.
Avicenna J Med Biotechnol. 2014 Jul;6(3):147-55.
Transient Gene Expression (TGE) gained popularity over the last decade as a rapid method for the production of milligram to gram quantities of recombinant proteins for preclinical studies in biophama industry. Thereby, the optimization of the TGE technique for Chinese hamster ovary (CHO) as the dominant host for the production of biotherapeutics is of great interest to reach the values for Human Embryo Kidney-293 (HEK-293) cells in terms of transfection efficiencies and production titers. TGE efficiencies are cell line and vector dependant.
In transfection efficiency optimization experiments, different starting cell densities, different amounts of plasmid DNA and PEI transfection reagent were investigated to achieve the best conditions leading to maximum transfection efficiencies. Furthermore, in order to investigate the effect of peptone feeding on transfection efficiency, three different sources of peptones with the greatest effect in the CD DG44 basal media were selected; Casein Tryptone N1, Soy petone A2SC and Soy peptone E110.
The transfection strategy performed here was able to make an outstanding increase in transfection efficiency of CHO DG44 cell line transfected with pTracer-SV40-mutated t-PA plasmid from 3.6% in our starting non-optimized condition to 66.93% in finally optimized situation. Moreover, peptone feeding strategy used here was successful to increase volumetric productivities up to 37%. In addition, the amounts of both PEI and plasmid DNA were reduced up to 66% and 25%, respectively compared to our previous protocol.
Here we described an optimization process for TGE in suspension-adapted CHO cells based on Polyethylenimine (PEI)/DNA concentration, DNA: PEI ratio, starting cell densities and peptone feeding strategy.
在过去十年中，瞬时基因表达（TGE）作为一种快速生产毫克至克级重组蛋白的方法，在生物制药行业的临床前研究中受到广泛关注。因此，优化TGE技术以用于中国仓鼠卵巢（CHO）细胞（作为生物治疗药物生产的主要宿主），对于在转染效率和生产滴度方面达到人胚肾293（HEK - 293）细胞的水平具有重要意义。TGE效率取决于细胞系和载体。
在转染效率优化实验中，研究了不同的起始细胞密度、不同量的质粒DNA和PEI转染试剂，以实现导致最大转染效率的最佳条件。此外，为了研究蛋白胨补料对转染效率的影响，在CD DG44基础培养基中选择了三种效果最佳的不同来源的蛋白胨；酪蛋白胰蛋白胨N1、大豆蛋白胨A2SC和大豆蛋白胨E110。
此处实施的转染策略能够使转染pTracer - SV40 - 突变型t - PA质粒的CHO DG44细胞系的转染效率显著提高，从我们起始未优化条件下的3.6%提高到最终优化情况下的66.93%。此外，此处使用的蛋白胨补料策略成功地将体积生产率提高了37%。此外，与我们之前的方案相比，PEI和质粒DNA的用量分别减少了66%和25%。
在此，我们描述了一种基于聚乙烯亚胺（PEI）/DNA浓度、DNA:PEI比例、起始细胞密度和蛋白胨补料策略的悬浮适应型CHO细胞中TGE的优化过程。
