Antibody-drug conjugates (ADCs) have become major biopharmaceutical drugs in the field of oncology. Traditional ADCs possess a stochastic distribution of cytotoxic payloads linked to several different amino acid residues of the antibody. This heterogeneous nature of stochastic ADCs results in a complex conjugation-site characterization. To improve upon traditional ADC technology, we have developed a chemical conjugation platform, termed AJICAP™, for site-specific modification of native antibodies using a class of IgG Fc-affinity reagents (Yamada et al., 2019). Here, we report further investigation focusing on peptide mapping of the AJICAP™-ADC to confirm the exact conjugation position of the first generation AJICAP™-ADC. Neutral pH pretreatment for peptide mapping prevented undesired PTMs such as succinimide ring hydrolysis. Mirroring comparison using the purified ADC visibly indicated that Lys248 in the Fc region was conjugated to the drug-linker. MS/MS analysis also provided evidence to support Lys248 conjugation. Finally, extracted ion-chromatogram methodology suggested the site-specificity of AJICAP™ conjugation. Purified ADCs by preparative HIC-HPLC showed clear visual results and more than 93% sequence coverage by a single enzymatic digestion. The analytical strategy described herein demonstrated a robust analytical methodology for revealing the conjugation site of ADCs.

中文翻译：

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通过化学偶联技术产生的抗体-药物偶联物的位点特异性证明：AJICAP第一代。

抗体-药物缀合物（ADC）已成为肿瘤学领域的主要生物药物。传统的ADC具有随机分布的细胞毒性有效载荷，该有效载荷与抗体的几个不同氨基酸残基相关。随机ADC的这种异质性导致复杂的共轭位点表征。为了改进传统ADC技术，我们开发了一种化学偶联平台，称为AJICAP™，可使用一类IgG Fc亲和试剂对天然抗体进行位点特异性修饰（Yamada等，2019）。在这里，我们报告了针对AJICAP™-ADC肽图定位的进一步研究，以确认第一代AJICAP™-ADC的确切缀合位置。用于肽图分析的中性pH预处理可防止发生不希望的PTM，如琥珀酰亚胺环水解。使用纯化的ADC的镜像比较明显表明，Fc区中的Lys248与药物接头偶联。MS / MS分析还提供了支持Lys248偶联的证据。最后，提取的离子色谱方法表明AJICAP™偶联的位点特异性。通过制备型HIC-HPLC纯化的ADC表现出清晰的视觉结果，并且通过一次酶消化即可获得超过93％的序列覆盖率。本文描述的分析策略展示了一种强大的分析方法，可用于揭示ADC的共轭位点。通过制备型HIC-HPLC纯化的ADC表现出清晰的视觉结果，并且通过一次酶切消化的序列覆盖率超过93％。本文描述的分析策略展示了一种强大的分析方法，可用于揭示ADC的共轭位点。通过制备型HIC-HPLC纯化的ADC表现出清晰的视觉结果，并且通过一次酶切消化的序列覆盖率超过93％。本文描述的分析策略展示了一种强大的分析方法，可用于揭示ADC的共轭位点。