Biotherapeutic’s higher order structure (HOS) is a critical determinant of its functional properties and conformational relevance. Here, we evaluated two covalent labeling methods: diethylpyrocarbonate (DEPC)-labeling and fast photooxidation of proteins (FPOP), in conjunction with mass spectrometry (MS), to investigate structural modifications for the new class of immuno-oncological therapy known as bispecific antigen-binding biotherapeutics (BABB). The evaluated techniques unveiled subtle structural changes occurring at the amino acid residue level within the antigen-binding domain under both native and thermal stress conditions, which cannot be detected by conventional biophysical techniques, e.g., near-ultraviolet circular dichroism (NUV-CD). The determined variations in labeling uptake under native and stress conditions, corroborated by binding assays, shed light on the binding effect, and highlighted the potential of covalent-labeling methods to effectively monitor conformational changes that ultimately influence the product quality. Our study provides a foundation for implementing the developed techniques in elucidating the inherent structural characteristics of novel therapeutics and their conformational stability.

中文翻译：

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通过共价标记和质谱绘制双特异性抗原结合生物治疗药物的构象变化

生物治疗药物的高阶结构 (HOS) 是其功能特性和构象相关性的关键决定因素。在这里，我们评估了两种共价标记方法：焦碳酸二乙酯 (DEPC) 标记和蛋白质快速光氧化 (FPOP)，与质谱 (MS) 相结合，以研究称为双特异性抗原的新型免疫肿瘤治疗的结构修饰-结合生物治疗药物（BABB）。评估的技术揭示了在天然和热应激条件下抗原结合域内氨基酸残基水平发生的微妙结构变化，这是传统生物物理技术（例如近紫外圆二色性（NUV-CD））无法检测到的。在天然和应激条件下标记摄取的确定变化，通过结合测定得到证实，揭示了结合效应，并强调了共价标记方法有效监测最终影响产品质量的构象变化的潜力。我们的研究为实施已开发的技术来阐明新型疗法的固有结构特征及其构象稳定性奠定了基础。