Simultaneous detection of nicotinamide adenine nucleotides and adenylate pool to quantify redox and energy states in mAb-producing CHO cells by capillary electrophoresis

Author: Jiaqi Wang, Chen Wang, Li Fan, Liang Zhao*, Wen-Song Tan.
Analytical and Bioanalytical Chemistry, 2019, 411(13): 2971-2979

Abstract
Chinese hamster ovary (CHO) cells are predominant in the production of therapeutic proteins to treat various diseases. Characterization and investigation of CHO cell metabolism in a quick and simple way could boost process and cell line development. Therefore, a method to simultaneously detect seven redox- and energy-related metabolites in CHO cells by capillary electrophoresis has been developed. An on-line focusing technique was applied to improve the peak shape and resolution by using a 50 μm× 44 cm uncoated fused silica capillary. Key parameters and their interactions were investigated by design of experiments (DoE) and optimized conditions were determined by desirability function as follows: 24 °C, 95 mM, and pH 9.4 of BGE. The method was validated to ensure sensitivity, linearity, and reproducibility. The limits of detection (LODs) ranged from 0.050 to 0.688 mg/L for seven metabolites, and correlation coefficients of linearity were all greater than 0.996. The relative standard deviations (RSD) of migration time and peak area were smaller than 0.872% and 5.5%, respectively, except for NADPH, and the recoveries were between 97.5 and 101.2%. The method was successfully applied to analyze the extracts from CHO cells under two different culture conditions.

通过毛细管电泳同时检测烟酰胺腺嘌呤核苷酸和腺苷酸池来量化生产单抗的CHO细胞的氧化还原和能量状态
中国仓鼠卵巢(CHO)细胞是生产用于治疗各种疾病的治疗性蛋白的主要宿主细胞。对CHO细胞代谢进行简单快速的表征和研究有利于促进过程开发和细胞系构建。因此，我们建立了一种毛细管电泳同时检测CHO细胞中7种氧化还原和能量代谢相关化合物的方法。采用50 μm× 44 cm的无涂层熔融二氧化硅毛细管对代谢物进行在线检测改善了峰形并提高了分辨率。采用实验设计软件(DoE)对关键参数及其相互作用进行了研究，通过期望函数确定BGE的最优条件为24℃，95 mM, pH 9.4。并对该方法进行验证，以确保其灵敏度、线性和重复性。7种代谢物的检出限(lod)范围为0.050 ~ 0.688 mg/L，线性相关系数均大于0.996。除NADPH外，其它六种代谢物的迁移时间和峰面积的相对标准偏差(RSD)分别小于0.872%和5.5%，加样回收率在97.5 ~ 101.2%之间。该方法成功应用于两种不同培养条件下CHO细胞提取物的分析。