College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing 211816, China.
School of Pharmaceutical Sciences, Nanjing Tech University, Nanjing 211816, China.
Int J Mol Sci. 2023 Oct 14;24(20):15184. doi: 10.3390/ijms242015184.
Charge variants, as an important quality attribute of mAbs, must be comprehensively characterized and monitored during development. However, due to their complex structure, the characterization of charge variants is challenging, labor-intensive, and time-consuming when using traditional approaches. This work combines on-line and off-line 2D-LC-MS to comprehensively characterize mAb charge variants and quickly offer precise instructions for process development. Six charge variant peaks of mAb 1 were identified using the developed platform. Off-line 2D-LC-MS analysis at the peptide level showed that the acidic peak P1 and the basic peaks P4 and P5 were caused by the deamidation of asparagine, the oxidation of methionine, and incomplete C-terminal K loss, respectively. On-line 2D-LC-MS at the intact protein level was used to identify the root causes, and it was found that the acidic peak P2 and the basic peak P6 were due to the glutathionylation of cysteine and succinimidation of aspartic acid, respectively, which were not found in off-line 2D-LC-MS because of the loss occurring during pre-treatment. These results suggest that process development could focus on cell culture for adjustment of glutathionylation. In this paper, we propose the concept of precision process development based on on-line 2D-LC-MS, which could quickly offer useful data with only 0.6 mg mAb within 6 h for precise instructions for process development. Overall, the combination of on-line and off-line 2D-LC-MS can characterize mAb charge variants more comprehensively, precisely, and quickly than other approaches. This is a very effective platform with routine operations that provides precise instructions for process development within hours, and will help to accelerate the development of innovative therapeutics.
电荷变异体作为单抗的一个重要质量属性，在开发过程中必须进行全面的表征和监测。然而，由于其复杂的结构，使用传统方法对电荷变异体进行表征既具有挑战性，又耗费人力和时间。本工作结合在线和离线二维液相色谱-质谱联用技术，全面表征单抗的电荷变异体，并快速为工艺开发提供准确的指导。使用开发的平台，鉴定了单抗 1 的 6 个电荷变异体峰。在肽水平的离线二维液相色谱-质谱联用分析表明，酸性峰 P1 和碱性峰 P4 和 P5 分别是由于天冬酰胺的脱酰胺、蛋氨酸的氧化和 C 末端 K 不完全丢失引起的。在完整蛋白水平的在线二维液相色谱-质谱联用用于鉴定根本原因，发现酸性峰 P2 和碱性峰 P6 分别是由于半胱氨酸的谷胱甘肽化和天冬氨酸的琥珀酰亚胺化引起的，这是由于预处理过程中发生的损失，在离线二维液相色谱-质谱联用中未发现。这些结果表明，工艺开发可以集中在细胞培养上，以调整谷胱甘肽化。在本文中，我们提出了基于在线二维液相色谱-质谱联用的精确工艺开发概念，该概念仅用 0.6 毫克单抗在 6 小时内即可快速提供有用数据，为工艺开发提供准确的指导。总之，在线和离线二维液相色谱-质谱联用可以比其他方法更全面、更准确、更快速地对单抗的电荷变异体进行表征。这是一个非常有效的平台，具有常规操作，可以在数小时内为工艺开发提供准确的指导，有助于加速创新疗法的开发。
