Zhang Xintao, Tang Hongping, Sun Ya-Ting, Liu Xuping, Tan Wen-Song, Fan Li
The State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, 200237, China.
Appl Microbiol Biotechnol. 2015 Aug;99(16):6643-52. doi: 10.1007/s00253-015-6617-y. Epub 2015 May 7.
C-terminal lysine variants are commonly observed in monoclonal antibodies (mAbs) and found sensitive to process conditions, especially specific components in culture medium. The potential roles of media arginine (Arg) and lysine (Lys) in mAb heavy chain C-terminal lysine processing were investigated by monitoring the lysine variant levels under various Arg and Lys concentrations. Both Arg and Lys were found to significantly affect lysine variant level. Specifically, lysine variant level increased from 18.7 to 31.8 % when Arg and Lys concentrations were increased from 2 to 10 mM. Since heterogeneity of C-terminal lysine residues is due to the varying degree of proteolysis by basic carboxypeptidases (Cps), enzyme (basic Cps) level, pH conditions, and product (Arg and Lys) inhibition, which potentially affect the enzymatic reaction, were investigated under various Arg and Lys conditions. Enzyme level and pH conditions were found not to account for the different lysine variant levels, which was evident from the minimal variation in transcription level and intracellular pH. On the other hand, product inhibition effect of Arg and Lys on basic Cps was evident from the notable intracellular and extracellular Arg and Lys concentrations comparable with Ki values (inhibition constant) of basic Cps and further confirmed by cell-free assays. Additionally, a kinetic study of lysine variant level during the cell culture process enabled further characterization of the C-terminal lysine processing.
C末端赖氨酸变体在单克隆抗体（mAb）中普遍存在，并且发现其对工艺条件敏感，尤其是培养基中的特定成分。通过监测不同精氨酸（Arg）和赖氨酸（Lys）浓度下的赖氨酸变体水平，研究了培养基中Arg和Lys在mAb重链C末端赖氨酸加工过程中的潜在作用。发现Arg和Lys均显著影响赖氨酸变体水平。具体而言，当Arg和Lys浓度从2 mM增加到10 mM时，赖氨酸变体水平从18.7%增加到31.8%。由于C末端赖氨酸残基的异质性是由于碱性羧肽酶（Cps）不同程度的蛋白水解作用引起的，因此在不同的Arg和Lys条件下研究了可能影响酶促反应的酶（碱性Cps）水平、pH条件和产物（Arg和Lys）抑制作用。从转录水平和细胞内pH的最小变化可以明显看出，酶水平和pH条件不能解释不同的赖氨酸变体水平。另一方面，Arg和Lys对碱性Cps的产物抑制作用从细胞内和细胞外显著的Arg和Lys浓度与碱性Cps的Ki值（抑制常数）相当中可以明显看出，并通过无细胞试验进一步得到证实。此外，对细胞培养过程中赖氨酸变体水平的动力学研究能够进一步表征C末端赖氨酸的加工过程。
