Döring Sarah, Weller Michael G, Reinders Yvonne, Konthur Zoltán, Jaeger Carsten
Federal Institute of Material Testing and Research (BAM), 12489 Berlin, Germany.
Leibniz-Institut für Analytische Wissenschaften-ISAS-e.V., 44139 Dortmund, Germany.
Antibodies (Basel). 2025 Jan 7;14(1):3. doi: 10.3390/antib14010003.
This review describes mass spectrometry (MS)-based approaches for the absolute quantification of therapeutic monoclonal antibodies (mAbs), focusing on technical challenges in sample treatment and calibration. Therapeutic mAbs are crucial for treating cancer and inflammatory, infectious, and autoimmune diseases. We trace their development from hybridoma technology and the first murine mAbs in 1975 to today's chimeric and fully human mAbs. With increasing commercial relevance, the absolute quantification of mAbs, traceable to an international standard system of units (SI units), has attracted attention from science, industry, and national metrology institutes (NMIs). Quantification of proteotypic peptides after enzymatic digestion using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) has emerged as the most viable strategy, though methods targeting intact mAbs are still being explored. We review peptide-based quantification, focusing on critical experimental steps like denaturation, reduction, alkylation, choice of digestion enzyme, and selection of signature peptides. Challenges in amino acid analysis (AAA) for quantifying pure mAbs and peptide calibrators, along with software tools for targeted MS data analysis, are also discussed. Short explanations within each chapter provide newcomers with an overview of the field's challenges. We conclude that, despite recent progress, further efforts are needed to overcome the many technical hurdles along the quantification workflow and discuss the prospects of developing standardized protocols and certified reference materials (CRMs) for this goal. We also suggest future applications of newer technologies for absolute mAb quantification.
本综述介绍了基于质谱（MS）的治疗性单克隆抗体（mAb）绝对定量方法，重点关注样品处理和校准中的技术挑战。治疗性单克隆抗体对于治疗癌症、炎症性疾病、感染性疾病和自身免疫性疾病至关重要。我们追溯了它们从杂交瘤技术和1975年的首个鼠源单克隆抗体发展到如今的嵌合单克隆抗体和全人源单克隆抗体的历程。随着商业相关性的不断提高，可溯源至国际单位制（SI单位）的单克隆抗体绝对定量已引起科学界、工业界和国家计量机构（NMI）的关注。尽管针对完整单克隆抗体的方法仍在探索中，但使用高效液相色谱-串联质谱（HPLC-MS/MS）对酶解后的蛋白型肽段进行定量已成为最可行的策略。我们综述了基于肽段的定量方法，重点关注诸如变性、还原、烷基化、消化酶选择和特征肽段选择等关键实验步骤。还讨论了用于定量纯单克隆抗体和肽段校准物的氨基酸分析（AAA）中的挑战，以及用于靶向MS数据分析的软件工具。每章中的简短解释为新手提供了该领域挑战的概述。我们得出结论，尽管最近取得了进展，但仍需要进一步努力克服定量工作流程中的许多技术障碍，并讨论为此目标制定标准化方案和有证参考物质（CRM）的前景。我们还提出了新技术在单克隆抗体绝对定量中的未来应用。
