Mass Spectrometry Center of Excellence, Analytical Sciences, WuXi Biologics, 288 Fute Zhong Road, Waigaoqiao Free Trade Zone, Shanghai 200131, China.
Mass Spectrometry Center of Excellence, Analytical Sciences, WuXi Biologics, 288 Fute Zhong Road, Waigaoqiao Free Trade Zone, Shanghai 200131, China.
J Pharm Biomed Anal. 2024 Jun 15;243:116098. doi: 10.1016/j.jpba.2024.116098. Epub 2024 Mar 7.
Antibody-drug conjugates (ADCs) are a heterogeneous mixture of conjugated species with varied drug loadings. Depending on conjugation sites, linkers and drugs can exhibit different stability as influenced by the solvent-accessibility and local charge, resulting in different ADC efficacy, pharmacokinetics, and toxicity. Conjugation site analysis is critical for ADC structural characterization to assure product quality and consistency. It enables early conjugation studies at site-specific levels, confirms the absence of unexpected products to support conjugation process development, and aids in ensuring lot-to-lot consistency for comparability studies. Peptide mapping using liquid chromatography-tandem mass spectrometry is the industry standard method for analyzing conjugation sites. However, some concerns remain for this approach as the large and hydrophobic drug moieties often result in poor MS/MS fragmentation quality and impede the identification of conjugation sites. Additionally, the ionization discrepancy between conjugated and unconjugated peptides can lead to a relatively large bias for site occupancy calculation. In this work, we present a simple drug deconjugation-assisted peptide mapping method to identify and quantify the drug conjugation for ADCs with protease-cleavable linkers. Papain-based drug deconjugation was used to remove the highly hydrophobic drug moiety, which significantly improved the quantitation accuracy of conjugation level and the fragmentation quality. Sample preparation conditions were optimized to avoid introducing artificial modifications, allowing the tracking of initial sample status and subsequent changes of quality attributes during process development and stability assessment. This method was applied to analyze thermally-stressed ADC samples to monitor changes of site-specific conjugation levels, DAR, succinimide hydrolysis of the linker, and various PTMs. We believe this is an effective and straightforward tool for conjugation site analysis while simultaneously monitoring multiple quality attributes for ADCs with protease-cleavable linkers.
抗体偶联药物（ADCs）是一种缀合物种的不均匀混合物，具有不同的药物载量。根据缀合位点、连接子和药物的不同，其稳定性会受到溶剂可及性和局部电荷的影响，从而导致 ADC 功效、药代动力学和毒性的不同。对于 ADC 结构特征的分析，缀合位点分析是至关重要的，以确保产品质量和一致性。它能够在特定的位点进行早期的缀合研究，确认不存在意外的产物，以支持缀合过程的开发，并有助于确保在可比性研究中批次间的一致性。使用液相色谱-串联质谱法进行肽图分析是分析缀合位点的行业标准方法。然而，这种方法仍然存在一些问题，因为大的疏水性药物部分通常会导致较差的 MS/MS 碎片化质量，并阻碍缀合位点的鉴定。此外，缀合肽和未缀合肽之间的离子化差异会导致对位点占有率计算的相对较大偏差。在这项工作中，我们提出了一种简单的药物去缀合辅助肽图分析方法，用于鉴定和定量具有蛋白酶可切割连接子的 ADC 中的药物缀合。木瓜蛋白酶基药物去缀合用于去除高度疏水性的药物部分，这显著提高了缀合水平的定量准确性和碎片化质量。优化了样品制备条件，以避免引入人工修饰，允许在工艺开发和稳定性评估过程中跟踪初始样品状态和随后的质量属性变化。该方法应用于分析受热应激的 ADC 样品，以监测特定位点的缀合水平、药物抗体比（DAR）、连接子的琥珀酰亚胺水解以及各种 PTM 的变化。我们相信，这是一种用于具有蛋白酶可切割连接子的 ADC 的缀合位点分析的有效且直接的工具，同时还可以监测多个质量属性。
