Zhao Fang, Wan Yuxiang, Nie Lei, Jiao Jingyu, Gao Dong, Sun Yan, Chen Zhenhua, Shi Yingting, Yang Jiayu, Pan Jianyang, Wang Haibin, Qu Haibin
Pharmaceutical Informatics Institute, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, China.
Hisun BioPharmaceutical Co., Ltd., Hangzhou, China.
Biotechnol J. 2023 Jul;18(7):e2200616. doi: 10.1002/biot.202200616. Epub 2023 May 6.
Controlling the process of CHO cell fed-batch culture is critical for biologics quality control. However, the biological complexity of cells has hampered the reliable process understanding for industrial manufacturing. In this study, a workflow was developed for the consistency monitoring and biochemical marker identification of the commercial-scale CHO cell culture process through H NMR assisted with multivariate data analysis (MVDA). Firstly, a total of 63 metabolites were identified in this study object in H NMR spectra of the CHO cell-free supernatants. Secondly, multivariate statistical process control (MSPC) charts were used to evaluate process consistency. According to MSPC charts, the batch-to-batch quality consistency was high, indicating the CHO cell culture process at the commercial scale was well-controlled and stable. Then, the biochemical marker identification was provided through orthogonal partial least square discriminant analysis (OPLS-DA) based S-line plots during the cell logarithmic expansion, stable growth, and decline phases. Identified biochemical markers of the three cell growth phases were as follows: L-glutamine, pyroglutamic acid, 4-hydroxyproline, choline, glucose, lactate, alanine, and proline were of the logarithmic growth phase; isoleucine, leucine, valine, acetate, and alanine were of the stable growth phase; acetate, glycine, glycerin, and gluconic acid were of the cell decline phase. Additional potential metabolic pathways that might influence the cell culture phase transitions were demonstrated. The workflow proposed in this study demonstrates that the combination of MVDA tools and H NMR technology is highly appealing to the research of the biomanufacturing process, and applies well to provide guidance in future work on consistency evaluation and biochemical marker monitoring of the production of other biologics.
控制CHO细胞补料分批培养过程对于生物制品的质量控制至关重要。然而，细胞的生物学复杂性阻碍了对工业制造过程的可靠理解。在本研究中，开发了一种工作流程，通过核磁共振氢谱（¹H NMR）辅助多元数据分析（MVDA），对商业规模的CHO细胞培养过程进行一致性监测和生化标志物鉴定。首先，在CHO细胞无细胞上清液的¹H NMR谱中，本研究对象共鉴定出63种代谢物。其次，使用多元统计过程控制（MSPC）图来评估过程一致性。根据MSPC图，批次间质量一致性较高，表明商业规模的CHO细胞培养过程得到了良好控制且稳定。然后，通过基于正交偏最小二乘判别分析（OPLS-DA）的S线图，在细胞对数生长期、稳定生长期和衰退期进行生化标志物鉴定。三个细胞生长阶段鉴定出的生化标志物如下：L-谷氨酰胺、焦谷氨酸、4-羟基脯氨酸、胆碱、葡萄糖、乳酸、丙氨酸和脯氨酸属于对数生长期；异亮氨酸、亮氨酸、缬氨酸、乙酸盐和丙氨酸属于稳定生长期；乙酸盐、甘氨酸、甘油和葡萄糖酸属于细胞衰退期。还展示了可能影响细胞培养阶段转变的其他潜在代谢途径。本研究提出的工作流程表明，MVDA工具和¹H NMR技术的结合对生物制造过程的研究极具吸引力，并且很好地适用于为未来其他生物制品生产的一致性评估和生化标志物监测工作提供指导。
