Here we report the design and production of an antibody-fluorophore conjugate (AFC) as a non-toxic model of an antibody-drug conjugate (ADC). This AFC is based on the conjugation of dansyl sulfonamide ethyl amine (DSEA )-linker maleimide on interchain cysteines of trastuzumab used as a reference antibody. The resulting AFC was first characterized by routine analytical methods (SEC, SDS-PAGE, CE-SDS, HIC and native MS), resulting in similar chromatograms, electropherograms and mass spectra to those reported for hinge Cys-linked ADCs. IdeS digestion of the AFC was then performed, followed by reduction and analysis by liquid chromatography coupled to mass spectrometry analysis. Dye loading and distribution on light chain and Fd fragments were calculated, as well as the average dye to antibody ratio (DAR ) for both monomeric and multimeric species. In addition, by analyzing the Fc fragment in the same run, full glycoprofiling and demonstration of the absence of additional conjugation was easily achieved. As for naked antibodies and Fc-fusion proteins, IdeS proteolytic digestion may rapidly become a reference analytical method at all stages of ADC discovery, preclinical and clinical development. The method can be routinely used for comparability assays, formulation, process scale-up and transfer, and to define critical quality attributes in a quality-by-design approach.

中文翻译：

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抗体-药物偶联物模型在IdeS蛋白水解消化后通过LC-MS快速表征。

在这里，我们报告抗体-荧光团缀合物（AFC）的设计和生产，作为抗体-药物缀合物（ADC）的无毒模型。该AFC是基于在用作参考抗体的曲妥珠单抗的链间半胱氨酸上缀合的丹磺酰胺磺胺乙胺（DSEA）-连接基马来酰亚胺。首先通过常规分析方法（SEC，SDS-PAGE，CE-SDS，HIC和天然MS）对所得的AFC进行表征，得到的色谱图，电泳图和质谱与铰链半胱氨酸连接的ADC相似。然后进行AFC的IdeS消化，然后进行还原，并通过液相色谱和质谱分析进行分析。计算了染料在轻链和Fd片段上的负载和分布，以及单体和多聚体物种的平均染料与抗体之比（DAR）。此外，通过在同一轮中分析Fc片段，可以轻松实现完整的糖谱分析和不存在其他偶联的证明。至于裸抗体和Fc融合蛋白，在ADC发现，临床前和临床开发的所有阶段，IdeS蛋白水解消化都可能迅速成为参考分析方法。该方法可常规用于可比性测定，配方，工艺放大和转移，并通过按质量设计的方法定义关键的质量属性。临床前和临床开发。该方法可以常规用于可比性测定，配方，工艺放大和转移，并可以通过按质量设计方法定义关键的质量属性。临床前和临床开发。该方法可以常规用于可比性测定，配方，工艺放大和转移，并可以通过按质量设计方法定义关键的质量属性。