Gao Li, Li Xiaohong, Zhang Mengxin, Li Bin, Miao Xiuxiu, Yu Chao, Guo Wei, Zhang Cuiqiao, He Xiangyuan, Huang Kaisheng, Qian Zichen
Department of Process Development, RemeGen Co., Ltd, No.56 Beijing Middle Road, Yantai, 264006, China.
Bioprocess Biosyst Eng. 2025 Jul 15. doi: 10.1007/s00449-025-03200-x.
Chinese hamster ovary (CHO) cells are the most widely used host for the commercial production of recombinant therapeutic proteins. The rapidly growing demand for large quantities of biologics at controllable cost-of-goods requires continuous cell engineering and process optimization of the CHO host cells. MicroRNAs (miRNAs) have been shown to enhance recombinant protein production in CHO cells. While studies have demonstrated that transient overexpression of certain miRNAs can increase recombinant protein yields, systematic comparisons of different miRNA overexpression forms (primary, precursor, and mature) remain limited. Furthermore, their application in stable cell line development, particularly for difficult-to-express proteins, has yet to be thoroughly explored. This study evaluated three miRNA overexpression strategies: primary miRNAs (pri-miRNAs), precursor miRNAs (pre-miRNAs), and flanked mature miRNAs (incorporating the mature sequence plus reverse complementary and loop sequences), to enhance the expression of difficult-to-express proteins in stable CHO cell lines. Notably, these miRNA constructs were built-in with the gene of interest (GOI) on the same vector to simplify stable cell line generation. Our results indicate that the pre-miRNA overexpression strategy is the most effective. Overexpression of premiR-92a, premiR-200a, premiR-483, and premiR-106b significantly increased the expression level of a bispecific antibody (BsAb) and an Fc-fusion protein without compromising product quality. Further clone evaluation of the premiR-92a and premiR-483 overexpression groups revealed an improved proportion of high-productivity and stable clones. In conclusion, this study demonstrates that integrating pre-miRNA expression cassettes into therapeutic protein vectors for co-expression is a valuable and effective engineering strategy for developing a robust stable CHO expression platform.
中国仓鼠卵巢（CHO）细胞是重组治疗性蛋白商业化生产中使用最广泛的宿主。以可控的商品成本对大量生物制品的快速增长需求，要求对CHO宿主细胞进行持续的细胞工程改造和工艺优化。微小RNA（miRNA）已被证明可提高CHO细胞中重组蛋白的产量。虽然研究表明某些miRNA的瞬时过表达可提高重组蛋白产量，但不同miRNA过表达形式（初级、前体和成熟）的系统比较仍然有限。此外，它们在稳定细胞系开发中的应用，特别是对于难以表达的蛋白，尚未得到充分探索。本研究评估了三种miRNA过表达策略：初级miRNA（pri-miRNA）、前体miRNA（pre-miRNA）和侧翼成熟miRNA（包含成熟序列加上反向互补和环序列），以增强稳定CHO细胞系中难以表达蛋白的表达。值得注意的是，这些miRNA构建体与感兴趣的基因（GOI）构建在同一载体上，以简化稳定细胞系的产生。我们的结果表明，pre-miRNA过表达策略是最有效的。pre-miR-92a、pre-miR-200a、pre-miR-483和pre-miR-106b的过表达显著提高了双特异性抗体（BsAb）和Fc融合蛋白的表达水平，且不影响产品质量。对pre-miR-92a和pre-miR-483过表达组的进一步克隆评估显示，高生产力和稳定克隆的比例有所提高。总之，本研究表明，将pre-miRNA表达盒整合到治疗性蛋白载体中进行共表达，是开发强大的稳定CHO表达平台的一种有价值且有效的工程策略。
