注射用美珀珠单抗关键质量属性的全面表征

Comprehensive characterization of critical quality attributes of meplazumab for injection

摘要

目的 全面表征注射用美珀珠单抗的一级结构、翻译后修饰、二硫键连接方式、纯度、高级结构及Fc段功能等关键质量属性, 为其质量控制与临床应用提供依据。方法 采用超高效液相色谱质谱联用测定完整分子量、肽图覆盖率、翻译后修饰及二硫键连接方式; 采用亲水相互作用色谱荧光检测质谱联用分析N糖型; 采用尺寸排阻色谱(SEC)、毛细管电泳(CE-SDS)及分析型超速离心(AUC)评估纯度与聚集状态; 采用圆二色谱分析二级结构; 采用差示扫描量热法(DSC)测定热稳定性; 采用表面等离子共振(SPR)技术测定Fc段与C1q、Fcγ受体及FcRn的亲和力。结果 美珀珠单抗完整分子量实测值与理论值一致, 氨基酸序列覆盖率达100%。翻译后修饰分析显示, 重链C端赖氨酸缺失比例为99.7%, Fc区甲硫氨酸M274氧化比例为11.9%, 重链互补决定区附近天冬氨酸D102异构化比例为18.1%。共鉴定出18对二硫键, 连接方式符合IgG2亚型特征。N糖型以G0F、G1F和G2F为主。SEC与CE-SDS测得单体纯度>99.5%, AUC在天然状态下测得单体比例为95.5%。二级结构以β折叠为主, DSC显示3个特征熔融温度。SPR分析表明, 美珀珠单抗与FcRn具有高亲和力(K_D=3.35×10^-8 mol/L)。结论 系统阐明了美珀珠单抗的关键质量特征, 确证其一级结构与高级结构的正确性, 并鉴定出M274氧化与D102异构化为需重点监控的潜在关键质量属性。研究结果为其质量控制标准建立及临床转化提供了关键理化依据, 同时为IgG2亚型抗体药物的深度表征积累了研究经验。

Abstract

Objective To comprehensively characterize the critical quality attributes of meplazumab for injection, including primary structure, post-translational modifications, disulfide bond linkages, purity, higher-order structure, and Fc region functionality, thereby providing a scientific basis for its quality control and clinical application. Methods Ultra-performance liquid chromatography-mass spectrometry was employed to determine the intact molecular weight, peptide mapping coverage, post-translational modifications, and disulfide bond linkages. Hydrophilic interaction chromatography coupled with fluorescence detection and mass spectrometry was used for N-glycan profiling. Size exclusion chromatography (SEC), capillary electrophoresis-sodium dodecyl sulfate (CE-SDS), and analytical ultracentrifugation (AUC) were applied to assess purity and aggregation status. Circular dichroism spectroscopy was utilized to analyze secondary structure, while differential scanning calorimetry (DSC) was performed to evaluate thermal stability. Surface plasmon resonance (SPR) technology was employed to determine the binding affinity of the Fc region to C1q, Fcγ receptors, and FcRn. Results The measured intact molecular weight of meplazumab was consistent with the theoretical value, and the amino acid sequence coverage reached 100%. Post-translational modification analysis revealed a C-terminal lysine truncation of 99.7% on the heavy chain, oxidation of methionine M274 in the Fc region at 11.9%, and isomerization of aspartic acid D102 near the heavy chain complementarity-determining region at 18.1%. A total of 18 disulfide bonds were identified, with linkage patterns consistent with the IgG2 subtype characteristics. The N-glycan profile was predominantly composed of G0F, G1F, and G2F glycoforms. SEC and CE-SDS demonstrated monomer purity exceeding 99.5%, while AUC under native conditions revealed a monomer content of 95.5%. The secondary structure was predominantly β-sheet, and DSC exhibited three characteristic melting temperatures. SPR analysis confirmed high binding affinity of meplazumab to FcRn (K_D=3.35×10^-8 mol/L). Conclusion This study systematically elucidates the critical quality attributes of meplazumab, confirming the correctness of its primary and higher-order structures. Oxidation of M274 and isomerization of D102 are identified as potential critical quality attributes requiring enhanced monitoring. These findings provide essential physicochemical evidence to support quality standard establishment and clinical translation of meplazumab, while also contributing valuable research experience for the in-depth characterization of IgG2 subtype antibody therapeutics.

关键词

单克隆抗体 / 美珀珠单抗 / IgG2型 / 质量表征 / 翻译后修饰 / 二硫键 / 糖基化 / 关键质量属性

Key words

monoclonal antibody / meplazumab / IgG2 subtype / quality characterization / post-translational modifications / disulfide bonds / glycosylation / critical quality attributes

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基金资助

临港国家实验室科研任务项目(LGL-2612-11)