單株抗體Durvalumab檢驗方法之評估研究   全文下載

The Assessment of Testing Methods of Durvalumab Monoclonal Antibody Preparations

單株抗體（monoclonal antibody）製劑多以基因工程技術由細胞培養而成，製程繁複且可能有蛋白質轉譯後修飾變異之風險，本研究擬建立癌細胞程序死亡配體programmed death-ligand 1（PD-L1）抑制劑durvalumab之分析方法。本研究運用新興技術建立單株抗體製劑品質檢驗研究評估體系，除以液相層析四極柱飛行時間串聯式質譜儀（liquid chromatography-quadrupole-time of flight mass spectrometer, LC-Q-TOF）與毛細管電泳分析儀分析durvalumab之物理化學特性，亦建立生物活性檢驗方法。結果顯示，durvalumab之主要醣化結構為G0F，次要醣化結構為G1F、G0及G2F。重鏈（heavy chain, HC）與輕鏈（light chain, LC）之分子量分別為50,908.95 Da與23,570.99 Da，以胰蛋白酶（trypsin）進行前處理後，比對HC與LC胺基酸序列涵蓋率達100%，並確認16對雙硫鍵鍵結位置。還原與非還原態之毛細管電泳分析（CE-SDS），結果顯示其純度分別為99.99%與92.52%，電位異質性分析顯示主要波峰之面積為67.53%，酸性波峰之面積總和為23.35%，皆符合原廠規定。生物活性分析方法結果顯示，本實驗方法之可重複性為5.95%且中間精密度為8.81%，不同durvalumab濃度下反應之R^2值為0.996且回收率介於86.77%-117.76%。本研究所建立之durvalumab單株抗體檢驗分析方法，可供品質管制之參考，未來亦可運用於產品上市後品質監測之參考方法，確保國人用藥安全。

Precision medicine products, such as monoclonal antibody, were manufactured from cell cultured by genetic engineering technology, the processes were complicated and variations of post-translational modification would increase risk factors. The objectives of this study were to establish the measuring methods of programmed death-ligand 1 (PD-L1) inhibitor durvalumab, one of the monoclonal antibodies established on genetic engineering technology. The physical and chemical properties were based on the liquid chromatography-quadrupole time-of-flight instrument (LC-Q-TOF) and capillary electrophoresis analyzer, the evaluation of bioactivity platform was performed. The results showed that the main glycosylation of durvalumab was G0F, followed by G1F, G0 and G2F. The molecular mass of heavy chain (HC) and light chain (LC) were 50,908.95 and 23,570.99 Da, repectively. The analytical coverage rate of amino acid sequence was 100%, and the position of 16 disulfide bond bridges had been completed and qualified. Reduced and non-reduced capillary electrophoresis (CE-SDS) results showed that purities were 99.99% and 92.52%, respectively, and the charge isoforms analysis showed the areas of main peak was 67.53% and total area of acid wave peaks was 23.35%. The results of the biological activity analysis displayed that the titer was 92.51% (compared to standard), repeatability was 5.95%, and the intermediate precision was 8.81%. The R2 value of the reaction under different durvalumab concentrations was 0.996, and the recovery rate was between 86.77% and 117.76%. The analytical method built by this project could be a reference method for quality control of this department. It could also be applied to product post-market monitoring to ensure the medication safety of people.