Onitsuka Masayoshi, Kadoya Yukinori, Omasa Takeshi
Graduate School of Technology, Industrial and Social Sciences, Tokushima University, 2-1 Minamijosanjima-cho, Tokushima 770-8506, Japan.
Graduate School of Advanced Technology and Science, Tokushima University, 2-1 Minamijosanjima-cho, Tokushima 770-8506, Japan.
J Biosci Bioeng. 2019 Jun;127(6):752-757. doi: 10.1016/j.jbiosc.2018.11.015. Epub 2018 Dec 21.
Aggregation of therapeutic antibodies is one of the most important issues to be resolved in manufacturing processes because of reduced efficacy and immunogenicity. Despite aggregation studies in vitro, little is known about the aggregation mechanism in cell culture processes. In this study, we investigated the process of aggregate formation of IgG1 antibodies during the culture of Chinese hamster ovary (CHO) cells to determine how aggregation occurs. A recombinant CHO cell line was cultivated in a bioreactor, and purified IgG1 from daily culture supernatants was analyzed by size exclusion chromatography. We found a linear correlation between the peak plots of IgG1 by-products, dimeric and aggregated IgG1, and integrated viable cell density, indicating that these by-products were secreted from CHO cells at a constant secretion rate. In addition, aggregate formation was not reproduced in pseudo-culture experiments, and the solution structures of intracellular and extracellular IgG1 aggregates were similar. These results support the concept of secretory leakage of IgG1 by-products. Secreted aggregates appeared to be in an alternatively folded state, which can pass through the protein quality control system in mammalian cells.
由于疗效降低和免疫原性问题，治疗性抗体的聚集是制造过程中需要解决的最重要问题之一。尽管进行了体外聚集研究，但对于细胞培养过程中的聚集机制仍知之甚少。在本研究中，我们研究了中国仓鼠卵巢（CHO）细胞培养过程中IgG1抗体聚集体的形成过程，以确定聚集是如何发生的。将重组CHO细胞系在生物反应器中培养，并通过尺寸排阻色谱法分析每日培养上清液中纯化的IgG1。我们发现IgG1副产物、二聚体和聚集IgG1的峰图与活细胞密度积分之间存在线性相关性，表明这些副产物以恒定的分泌速率从CHO细胞中分泌出来。此外，在模拟培养实验中未重现聚集体形成，并且细胞内和细胞外IgG1聚集体的溶液结构相似。这些结果支持了IgG1副产物分泌泄漏的概念。分泌的聚集体似乎处于另一种折叠状态，能够通过哺乳动物细胞中的蛋白质质量控制系统。
