Wang Lingyu, Zeng Yajun, Gao Wenyuan, Shen Pengcheng, Han Ping, Zhang Zhongli
Department of Analytical Science and Development, Shanghai Henlius Biologics Co., Ltd., Shanghai, 201600, China.
BioDrugs. 2025 Apr 22. doi: 10.1007/s40259-025-00718-0.
Recombinant immunoglobulin G2 (IgG2) antibodies are effective neutralization agents or antagonists with high medical value because of their high specificity, long half-life, and absent effector functions. During biopharmaceutical process development, charge heterogeneity and bioactivity alternation related to charge variation should be investigated to understand the structure-activity relationship (SAR) in therapeutic antibodies. Isoelectric focusing can provide fine resolution for the charge heterogeneity profiling of IgG2, while ion exchange chromatography cannot achieve effective separation of IgG2 charge variants. In-depth investigation of charge heterogeneity requires a fractionation tool to enrich and isolate acidic and basic variants.
This study aims to investigate the structural origins and functional implications of charge heterogeneity in two therapeutic IgG2 antibodies, including an anti-receptor activator of nuclear factor κ-B ligand (RANKL) biosimilar of denosumab and an innovative anti-CD73 IgG2 (H-mab), using free-flow isoelectric focusing (FF-IEF).
Charge variants of denosumab and H-mab were fractionated using FF-IEF, followed by characterization of isolated IgG2 charge variants to establish the SAR between charge-related antibody modifications and bioactivities. The investigation incorporated in silico 3D modeling of the FcRn-IgG2 Fc complex and the CD73-Fab complex to facilitate the SAR interrogation of these two IgG2s.
The acidic charge variants of denosumab were driven primarily by N-glycan sialylation and deamidation in the Fc region, showing negligible effects on biological functions except for a reproducible reduction in FcRn binding affinity. In contrast, the basic charge variants of H-mab were associated with D-succinimide formation in the heavy chain complementarity-determining region 3 (CDR3), significantly impairing binding and enzymatic inhibition activities.
This study underscores the irreplaceable role of FF-IEF in both biosimilar and innovative therapeutic pipelines, highlighting the importance of monitoring charge heterogeneity and understanding SAR in therapeutic IgG2 antibodies.
重组免疫球蛋白G2（IgG2）抗体因其高特异性、长半衰期和无效应功能，是具有高医学价值的有效中和剂或拮抗剂。在生物制药工艺开发过程中，应研究与电荷变化相关的电荷异质性和生物活性改变，以了解治疗性抗体的构效关系（SAR）。等电聚焦可为IgG2的电荷异质性分析提供高分辨率，而离子交换色谱无法有效分离IgG2电荷变体。深入研究电荷异质性需要一种分级分离工具来富集和分离酸性和碱性变体。
本研究旨在使用自由流等电聚焦（FF-IEF）研究两种治疗性IgG2抗体（包括地诺单抗的抗核因子κB配体受体激活剂（RANKL）生物类似物和创新型抗CD73 IgG2（H-mab））中电荷异质性的结构起源和功能影响。
使用FF-IEF对地诺单抗和H-mab的电荷变体进行分级分离，随后对分离出的IgG2电荷变体进行表征，以建立与电荷相关的抗体修饰和生物活性之间的构效关系。该研究纳入了FcRn-IgG2 Fc复合物和CD73-Fab复合物的计算机三维建模，以促进对这两种IgG2的构效关系研究。
地诺单抗的酸性电荷变体主要由Fc区域的N-聚糖唾液酸化和脱酰胺作用驱动，除FcRn结合亲和力可重复降低外，对生物学功能的影响可忽略不计。相比之下，H-mab的碱性电荷变体与重链互补决定区3（CDR3）中的D-琥珀酰亚胺形成有关，显著损害结合和酶抑制活性。
本研究强调了FF-IEF在生物类似物和创新治疗药物研发流程中不可替代的作用，突出了监测治疗性IgG2抗体电荷异质性和理解构效关系的重要性。
