Key Laboratory of Drug Metabolism and Pharmacokinetics, China Pharmaceutical University, Nanjing, 210009, China; State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing, 210009, China.
Shanghai Hengrui Pharmaceutical Co., Ltd, Shanghai, 200245, China.
Anal Chim Acta. 2017 Feb 22;955:67-78. doi: 10.1016/j.aca.2016.11.073. Epub 2016 Dec 2.
Trastuzumab-MCC-DM1 (T-DM1) is an antibody-drug conjugate (ADC) that consists of a monoclonal antibody (mAb) trastuzumab non-cleavably linked to a cytotoxic drug DM1. During production, the DM1 agents were conjugated to the lysine residues of the mAb in a non-specific manner, yielding a heterogeneous mixture of ADC molecules that differ with respect to both the number and the conjugation sites of DM1 per mAb molecule. Since drug conjugation sites of ADC can significantly impact properties such as stability and pharmacokinetic behaviors, a rapid and reliable approach for conjugation site analysis of ADCs is highly demanded. Herein, we have employed a signature ion fingerprinting approach to specifically determine lysine residues with DM1 conjugation, and developed a normalized peak area quantitation method to characterize the percentage of DM1-conjugated lysine for each putative site using a T-DM1 biosimilar as a model drug. With this integrative approach, 38 lysine residues were identified with DM1 conjugation among 90 possible sites. More interestingly, we found that the T-DM1 biosimilar exhibited a specific preference of DM1-conjugation for several lysine residues, and such preference was consistent among three production batches. A molecular modeling approach was subsequently utilized to analyze all the conjugation sites, and revealed an intriguing correlation of the conjugated residue's microenvironment with the conjugation level. In summary, our study introduced an approach that is widely applicable to ADCs of interest for conjugation site analysis. Moreover, it suggests the necessity of performing conjugation site analysis for product and process characterization and also for routine use in lot release and stability testing of manufactured ADCs.
曲妥珠单抗-MCC-DM1（T-DM1）是一种抗体药物偶联物（ADC），由单克隆抗体（mAb）曲妥珠单抗不可裂解地连接到细胞毒性药物 DM1 组成。在生产过程中，DM1 试剂以非特异性方式与 mAb 的赖氨酸残基结合，产生 ADC 分子的不均匀混合物，这些分子在 DM1 与 mAb 分子的结合数量和结合位点方面存在差异。由于 ADC 的药物结合位点会显著影响稳定性和药代动力学行为等特性，因此非常需要一种快速可靠的 ADC 结合位点分析方法。在此，我们采用特征离子指纹图谱方法特异性地确定具有 DM1 结合的赖氨酸残基，并开发了一种归一化峰面积定量方法，使用 T-DM1 生物类似药作为模型药物，对每个假定的结合位点的 DM1 结合的赖氨酸百分比进行特征化。通过这种综合方法，在 90 个可能的位点中鉴定出 38 个与 DM1 结合的赖氨酸残基。更有趣的是，我们发现 T-DM1 生物类似药对几个赖氨酸残基具有 DM1 结合的特定偏好，并且这种偏好在三个生产批次中是一致的。随后利用分子建模方法对所有结合位点进行了分析，结果表明，结合残基的微环境与结合水平之间存在有趣的相关性。总之，我们的研究介绍了一种广泛适用于 ADC 结合位点分析的方法。此外，它还表明，对于产品和工艺特性的表征以及制造 ADC 的批放行和稳定性测试的常规使用，进行结合位点分析是必要的。
