Monoclonal antibodies encounter many stresses during their entire production lifetime. Aggregation propensity of mAbs is influenced by gamut of other critical quality attributes (CQA) such as charge variants in addition to environmental factors. This study aims to elucidate any potential underlying correlation between charge and stability of mAbs. Semi-preparative cation exchange chromatography was used to isolate one acidic and three basic charge variants of mAb 1 (anti-VEGF mAb) and three acidic and one basic variant of mAb 2 (anti-HER2 mAb), respectively. Acidic variants were primarily identified as deamidated species while basic variants were formed due to incomplete lysine clipping on the C-terminus of mAb heavy chain. Separated variants were subjected to a battery of stresses commonly encountered during downstream purification of mAbs, such as thermal, mechanical, high salt and low pH stress. Size-based and charge based heterogeneities were monitored using analytical size exclusion chromatography and cation exchange chromatography, respectively. Dynamic light scattering was used to assess changes in hydrodynamic size upon stress application. The results clearly indicate that charge variants behave differently vis-à-vis the control mAb samples. For mAb 1, A1 (thermal and low pH stress) and B3 (thermal, mechanical, and salt stress) showed higher rate of degradation. Similarly, for mAb 2, A2 (thermal and low pH stress) and A3 (mechanical and salt stress) degraded faster. It was also noted that acidic variants increased significantly than basic variants upon stress. Thermal and mechanical stress caused higher visible degradation as observed by DLS. This knowledge is essential to carry out pooling decisions and warrants a case-by-case assessment of variants to ensure complete knowledge of the product in the QbD paradigm.

Impact of charge variants on degradation of monoclonal antibodies

ACS Spring 2022

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