National Organization for Research and Control of Biologicals, Egypt.
Analytical Chemistry Department, Faculty of Pharmacy, Cairo University, Egypt; Bioanalysis Research Group, School of Pharmacy, Newgiza University, Egypt.
J Pharm Biomed Anal. 2018 Feb 20;150:268-277. doi: 10.1016/j.jpba.2017.12.022. Epub 2017 Dec 13.
Antibody-drug conjugates (ADC) represent an emerging, novel class of biopharmaceuticals. The heterogeneity originating from the sophisticated structure requires orthogonal analytical techniques for quality and stability assessment of ADC to ensure safety and efficacy. In this study, the stability of Trastuzumab (recombinant humanized IgG1 mAb, targeting HER2 receptor) and its ADC with DM1 (anti-tubulin anticancer drug), Trastuzumab emtansine (T-DM1) were studied. SE-HPLC was used to monitor formation of aggregates and/or fragments of the monoclonal antibodies (mAb). Correlation with the results of reducing and non-reducing sodium dodecyl sulphate - polyacrylamide gel electrophoresis (SDS-PAGE) and dynamic light scattering (DLS) were performed to interpret the obtained results. RP-HPLC was used for assessment of the stability of DM1 in ADC while spectrophotometry was employed to determine drug antibody ratio (DAR) . The studied drugs were subjected to several stress conditions including pH, temperature, mechanical agitation and repeated freeze and thaw to generate possible degradation products and ensure suitability of the assay protocol. The degradation pattern and extent were demonstrated under the indicated stress conditions. The correlation between the results of SE-HPLC and those of SDS-PAGE and DLS ensured the validity of the orthogonal assay protocol and indicated aggregates that were not detected using SE-HPLC. Results showed clearly that T-DM1 is relatively less stable than its parent mAb. This was attributed to the presence of the drug-linker part that is attached to the mAb. RP-HPLC showed that the cytotoxic drug moiety is liable for degradation under the studied conditions resulting in alteration of DAR as well as formation of degradation products. This confirmed the need for more robust coupling chemistries for production of safe and effective ADC and highlighted the importance of orthogonal testing protocols for quality assessment. The assay protocol should be applicable for quality and stability assessment of various ADC.
抗体药物偶联物（ADC）代表了一类新兴的、新型的生物制药。其复杂结构所产生的异质性需要采用正交分析技术来评估 ADC 的质量和稳定性，以确保其安全性和有效性。在本研究中，研究了曲妥珠单抗（靶向 HER2 受体的重组人源化 IgG1 mAb）及其与 DM1（抗微管抗癌药物）偶联的 ADC、曲妥珠单抗emtansine（T-DM1）的稳定性。采用 SEC-HPLC 监测单克隆抗体（mAb）的聚集物和/或片段的形成。通过与还原和非还原十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）和动态光散射（DLS）的结果进行相关性分析，对获得的结果进行解释。采用 RP-HPLC 评估 ADC 中 DM1 的稳定性，采用分光光度法测定药物抗体比（DAR）。研究的药物经受了几种应激条件，包括 pH、温度、机械搅拌和反复冻融，以产生可能的降解产物，并确保测定方案的适用性。在所述应激条件下，对降解模式和程度进行了研究。SE-HPLC 与 SDS-PAGE 和 DLS 的结果之间的相关性确保了正交测定方案的有效性，并表明了 SE-HPLC 未检测到的聚集物。结果清楚地表明，T-DM1 比其母体 mAb 相对不稳定。这归因于存在与 mAb 连接的药物-接头部分。RP-HPLC 表明，在研究条件下，细胞毒性药物部分易于降解，导致 DAR 改变以及降解产物的形成。这证实了需要更稳健的偶联化学来生产安全有效的 ADC，并强调了正交测试方案对质量评估的重要性。该测定方案应适用于各种 ADC 的质量和稳定性评估。
