利用Crispr/Cas9技术改造CHO细胞株及其表达单克隆抗体的工艺参数优化

Chinese hamster ovary (CHO) cells are used to produce protein based therapeutics including monoclonal antibodies, cytokines, vaccines and fusion proteins in bioreactors. CHO cells are the most popular host cells. In this study, we illuminate CHO cells are modified by Crispr/Cas9 technology and the high productivity of monoclonal antibodies produced by modified CHO cells in DASGIP bioreactors. . Crispr/Cas9 system has been developed as the latest genome editing technology. Compared with traditional homologous recombination and newly developed ZFNS and TALENs, Crispr/Cas9 shows superiorities such as higher genome editing efficiency and disrupting multiple genes simultaneously. SgRNAs could be designed to target any DNA sequences which lead Crispr/Cas9 a time-saving and labor-saving process. This new technology has been evaluated as one of top 10 breakthroughs in 2013.. CHO cells are the most common expression hosts for therapeutical recombinant proteins. α-1,6 fucosytransferase (FUT8) is responsible for adding fucose to antibodies N-Glycans. Fucose could block the Fc region binding to Fc receptors and lead to lower antibody dependent cell mediated cytotoxicity (ADCC) effect. For CHO-GS expression system, the basal glutamine synthetase gene (GS) expression from the endogenous GS gene decreases the CHO-GS system selection. In our research, we would perform genome editing of FUT8 gene and GS gene in CHO-K1 cell line using Crispr/Cas9 technology, exploring the applications on modification of CHO cells by means of Crispr/Cas9 technology.. DASGIP is a parallel bioreactor system for the cultivation of microbial, plant, animal and human cells utilize industry-standard benchtop glass and single-use bioreactors. DASGIP bioreactors' best-in-class configurable control systems and state-of-the-art bioprocess analyzers deliver unparalleled functionality coupled with user-friendly design for optimal bioprocessing. DASGIP bioreactors are applied to cultivate modified recombinant CHO cells. According to the data of metabolic analysis by Nova through the bioprocess and dynamic model of CHO cell metabolism, process parameters are optimized to improve the expression of monoclonal antibodies. According to comparisons of monoclonal antibodies quality and quantity produced by modified CHO cells, We further demonstrate the effectiveness of the different cultivation methods including batch and fed-batch to the process of modified recombinant CHO cells in DASGIP bioreactors. . The purpose of this proposal is to apply Crispr/Cas9 technology on modification of CHO cells and pruduce high-expression and high-quality monoclonal antibodies using DASGIP mammalian cells parallel bioreactor system. The results of this research will be helpful to the further application and research for the biopharmaceutical pilot and academic research.

CHO细胞是表达生产单克隆抗体的主要宿主细胞，提高CHO细胞的表达效率具有巨大的经济价值，一直是被关注的热点。本项目主要研究Crispr/Cas9基因编辑技术在CHO细胞改造领域中的应用，利用Crispr/Cas9技术构建FUT8基因缺陷型细胞株，生产完全无岩藻糖的单克隆抗体；利用Crispr/Cas9技术进一步构建GS基因缺陷型细胞株，提高CHO细胞表达单克隆抗体的效率；运用DASGIP平行生物反应器培养改造重组CHO细胞，通过代谢分析，结合代谢反应动力学模型，优化培养工艺参数，实现高密度培养，提高单克隆抗体的表达效率。本项目的意义在于提供了Crispr/Cas9技术在CHO细胞株改造方面的研究思路，探索构建具有自主知识产权的CHO细胞株，进一步提高单克隆抗体的高效率高质量表达，为我国生物技术药物的工业生产提供可靠的理论基础和技术支撑。

单克隆抗体药物治疗在生物制药领域占据了很大的比例，CHO细胞是表达生产单克隆抗体的主要宿主细胞，提高CHO细胞的表达效率具有巨大的经济价值，一直是被关注的热点。本研究通过CRISPR/Cas9技术成功使CHO-S细胞中合成岩藻糖的关键基因α-1,6-岩藻糖转移酶（FUT8）基因沉默，获得了生产高ADCC活性抗体药物的FUT8-/--CHO-S细胞株，构建anti-Her2单抗的稳转细胞株并进行工艺培养研究。.选择FUT8基因的外显子9作为靶向基因设计sgRNA，将sgRNA插入到模式质粒pX330中，转染CHO-S细胞，通过LCA富集FUT8敲除的细胞，经过两轮的有限稀释，通过DNA测序鉴定、FITC-LCA膜结合表型鉴定等方法确定获得了岩藻糖敲除的单细胞克隆。利用改造的FUT8-/-细胞瞬时转染表达抗Her2抗体，HILIC-HPLC方法对抗体的糖型进行分析，确定抗体的岩藻糖完全被去除；利用Biacore检测抗体与FcγRⅢa抗原的亲和力，与野生型CHO-S细胞表达的抗体相比亲和力增强了7倍；利用乳酸脱氢酶释放法检测抗体的ADCC作用，对于SK-BR-3细胞和HepG2细胞来说，其ADCC杀伤作用分别提高了24.7倍和8.8倍。.通过电穿孔法、抗生素加压和有限稀释等方法成功构建并筛选出表达anti-HER2单抗的稳转细胞株Her-FUT8-/--CHO-S，FITC-LCA标记实验验证了该稳转细胞株无法合成含有岩藻糖的糖链，HILIC-HPLC分析证明其表达的抗体中无岩藻糖修饰。通过工艺优化，筛选得到具有较高生产潜力的细胞株、培养基和补料，在摇瓶培养中细胞生长11天，最高密度达到19.4 × 106 cells/mL，抗体产量为581.5 mg/L。生物反应器中对优化的培养条件进行工艺验证，结果表明，细胞的培养周期延长到13天，无岩藻糖anti-HER2抗体的表达量与摇瓶相比提高了37%（797.17 mg/L），抗体中无岩藻糖修饰， ADCC活性与野生型抗体相比增加了14倍。.研究结果表明，本研究改造的FUT8-/--CHO-S细胞株具有开发成工业细胞株的潜力，本研究的数据和结果为生物药物工业生产中开发新型细胞株的研究奠定理论基础。