Department of Bioprocess Engineering, KRIBB School of Biotechnology, Korea University of Science and Technology (UST), 217 Gajeong-ro, Yuseong-gu, Daejeon 34113, Korea.
Bioprocess Engineering Center, KRIBB, 30 Yeongudanji-ro Ochang-eup, Cheongwon-gu, Cheongju-si 28116, Korea.
Int J Mol Sci. 2021 Nov 16;22(22):12344. doi: 10.3390/ijms222212344.
Transient gene expression is a suitable tool for the production of biopharmaceutical candidates in the early stage of development and provides a simple and rapid alternative to the generation of stable cell line. In this study, an efficient transient gene expression methodology using DC-Chol/DOPE cationic liposomes and pDNA in Chinese hamster ovary suspension cells was established through screening of diverse lipoplex formation conditions. We modulated properties of both the liposome formation and pDNA solution, together called complexation solutions. Protein expression and cellular cytotoxicity were evaluated following transfection over the cell cultivation period to select the optimal complexation solution. Changes in hydrodynamic size, polydispersity index, and ζ potential of the liposomes and lipoplexes were analyzed depending on the various pH ranges of the complexation solutions using dynamic light scattering. The transfer of lipoplexes to the cytosol and their conformation were traced using fluorescence analysis until the early period of transfection. As a result, up to 1785 mg/L and 191 mg/L of human Fc protein and immunoglobulin G (bevacizumab), respectively, were successfully produced using acidic liposome formation and alkaline pDNA solutions. We expect that this lipoplex formation in acidic and alkaline complexation solutions could be an effective methodology for a promising gene delivery strategy.
瞬时基因表达是在开发的早期阶段生产生物制药候选物的合适工具，为稳定细胞系的产生提供了一种简单、快速的替代方法。在这项研究中，通过筛选各种脂质体形成条件，建立了一种使用 DC-Chol/DOPE 阳离子脂质体和 pDNA 在中华仓鼠卵巢悬浮细胞中的高效瞬时基因表达方法。我们调节了脂质体形成和 pDNA 溶液的性质，这两者一起称为复合物溶液。在细胞培养期间进行转染后，评估蛋白质表达和细胞毒性，以选择最佳的复合物溶液。使用动态光散射分析取决于复合物溶液的各种 pH 范围，分析脂质体和脂质体的流体力学大小、多分散指数和 ζ 电位的变化。使用荧光分析跟踪脂质体向细胞质的转移及其构象，直到转染的早期阶段。结果，使用酸性脂质体形成和碱性 pDNA 溶液成功生产了高达 1785 mg/L 的人 Fc 蛋白和 191 mg/L 的免疫球蛋白 G（贝伐单抗）。我们期望这种在酸性和碱性复合物溶液中的脂质体形成是一种有前途的基因传递策略的有效方法。
