Department of Chemical Engineering, Indian Institute of Technology, Hauz Khas, New Delhi, 110016, India.
Sci Rep. 2021 Jan 29;11(1):2487. doi: 10.1038/s41598-020-79541-2.
The present study investigates the impact of charge variants on bevacizumab's structure, stability, and biological activity. Five basic and one acidic charge variants were separated using semi-preparative cation exchange chromatography using linear pH gradient elution with purity > 85%. Based on the commercial biosimilar product's composition, two basic variants, one acidic and the main bevacizumab product, were chosen for further investigation. Intact mass analysis and tryptic peptide mapping established the basic variants' identity as those originating from an incomplete clipping of either one or both C-terminal lysine residues in the heavy chain of bevacizumab. Based on peptide mapping data, the acidic variant formation was attributed to deamidation of asparagine residue (N84), oxidation of M258, and preservation of C-terminal lysine residue, located on the heavy chain of bevacizumab. None of the observed charge heterogeneities in bevacizumab were due to differences in glycosylation among the variants. The basic (lysine) variants exhibited similar structural, functional, and stability profiles as the bevacizumab main product. But it was also noted that both the variants did not improve bevacizumab's therapeutic utility when pooled in different proportions with the main product. The acidic variant was found to have an equivalent secondary structure with subtle differences in the tertiary structure. The conformational difference also translated into a ~ 62% decrease in biological activity. Based on these data, it can be concluded that different charge variants behave differently with respect to their structure and bioactivity. Hence, biopharmaceutical manufacturers need to incorporate this understanding into their process and product development guidelines to maintain consistency in product quality.
本研究考察了电荷变异体对贝伐珠单抗结构、稳定性和生物学活性的影响。使用线性 pH 梯度洗脱的半制备阳离子交换层析分离了五种碱性和一种酸性电荷变异体，纯度>85%。根据商业类似物产品的组成，选择了两种碱性变异体、一种酸性变异体和主要的贝伐珠单抗产品进行进一步研究。完整质量分析和胰蛋白酶肽图谱确定了碱性变异体的身份，它们源自贝伐珠单抗重链中一个或两个 C 末端赖氨酸残基的不完全剪切。根据肽图数据，酸性变异体的形成归因于天冬酰胺残基（N84）的脱酰胺、M258 的氧化和位于贝伐珠单抗重链上的 C 末端赖氨酸残基的保留。贝伐珠单抗中观察到的任何电荷不均一性都不是由于变异体之间的糖基化差异所致。碱性（赖氨酸）变异体表现出与贝伐珠单抗主要产品相似的结构、功能和稳定性特征。但也注意到，当这两种变异体以不同比例与主要产品混合时，并没有改善贝伐珠单抗的治疗效果。发现酸性变异体具有等效的二级结构，但在三级结构上存在细微差异。构象差异也导致生物学活性降低约 62%。基于这些数据，可以得出结论，不同的电荷变异体在其结构和生物活性方面表现不同。因此，生物制药制造商需要将这种理解纳入其工艺和产品开发指南中，以保持产品质量的一致性。
