Nguyen Jennifer B, Liu Sophia, Howie Dylan A, Oberholtzer Zachary R, Ong Eric T, Rao Ramya, Prinston Jethro E, Dikiy Igor, Wu Jikang, Wu Zhijie, Zhao Yimeng, Li Meinuo, Molden Rosalynn, Molina Guido, Provoncha Kathleen, Sandu Cristinel, Qiu Haibo, Li Ning, Matousek William, Rosconi Michael P, Pyles Erica A
Protein Biochemistry, Regeneron Pharmaceuticals, Inc., Tarrytown, New York, USA.
Analytical Chemistry, Regeneron Pharmaceuticals, Inc., Tarrytown, New York, USA.
Protein Sci. 2025 Apr;34(4):e70079. doi: 10.1002/pro.70079.
Charge heterogeneity is an important quality attribute of therapeutic antibodies, and a detailed understanding of charge heterogeneity arising from post-translational modifications (PTMs) is required by regulatory agencies during drug development. Among antibody therapeutics, the bispecific antibody with two distinct Fab domains targeting distinct antigens provides additional complexity to the charge profile. In this study, charge variant species were enriched from three bispecific antibodies (bsAbs) each containing one anti-CD3 binding arm designed with differential affinity to CD3. The charge heterogeneity corresponding to each anti-CD3 arm within each enriched fraction was evaluated using a domain-specific, digestion-assisted imaged capillary isoelectric focusing (icIEF) method known as DiCE. Through fractionation, we observed that the anti-CD3 arm of each bispecific antibody exhibited different distributions of acidic variants, even when the anti-CD3 arms were identical based on primary sequence. Reduced peptide mapping was performed on specific fractions to identify unique site-specific PTMs that were uncovered or enriched through fractionation. In each case, the bispecific arm that was most susceptible to PTMs exhibited a more basic isoelectric point. Conformational stability analysis of each bispecific antibody using differential scanning calorimetry suggested that the more basic Fab arm tended to be correlated with a lower melting temperature, although it is unclear the extent to which PTMs on the basic arm may contribute to reduced conformational stability. Overall, these results provide additional evidence that each of the two arms of a bispecific antibody may exhibit differential susceptibility to post-translational modification and that this susceptibility is likely correlated with subtle differences in overall bispecific antibody structure, which is influenced by electrostatic properties inherent to the primary sequence. Future studies to obtain high-resolution structures of full-length bispecific antibodies by crystallography or cryo-electron microscopy may help to elucidate the driving force for susceptibility to PTMs in bispecific antibodies.
电荷异质性是治疗性抗体的一个重要质量属性，在药物开发过程中，监管机构要求详细了解由翻译后修饰（PTM）产生的电荷异质性。在抗体治疗药物中，具有两个靶向不同抗原的不同Fab结构域的双特异性抗体使电荷分布情况更加复杂。在本研究中，从三种双特异性抗体（bsAb）中富集了电荷变体，每种双特异性抗体均含有一个针对CD3设计的具有不同亲和力的抗CD3结合臂。使用一种称为DiCE的结构域特异性、酶解辅助成像毛细管等电聚焦（icIEF）方法，对每个富集级分中与每个抗CD3臂相对应的电荷异质性进行了评估。通过分级分离，我们观察到，即使基于一级序列抗CD3臂相同，每种双特异性抗体的抗CD3臂也表现出不同的酸性变体分布。对特定级分进行了还原肽图谱分析，以鉴定通过分级分离发现或富集的独特的位点特异性PTM。在每种情况下，最易发生PTM的双特异性臂表现出更高的等电点。使用差示扫描量热法对每种双特异性抗体进行的构象稳定性分析表明，碱性更强的Fab臂往往与更低的解链温度相关，尽管尚不清楚碱性臂上的PTM在多大程度上可能导致构象稳定性降低。总体而言，这些结果提供了额外的证据，表明双特异性抗体的两条臂中的每一条可能对翻译后修饰表现出不同的敏感性，并且这种敏感性可能与双特异性抗体整体结构中的细微差异相关，而这种差异受一级序列固有的静电性质影响。未来通过晶体学或冷冻电子显微镜获得全长双特异性抗体高分辨率结构的研究，可能有助于阐明双特异性抗体对PTM敏感性的驱动力。
