Quality Control of Anti-delta-like Protein 3 Antibody-MPVP-PBD
LI Meng1, SUN Liang1, ZHU Lei2, YU Chuan-fei1, WANG Wen-bo1, WU Gang1, ZHANG Rong-jian1, CUI Yong-fei1, WANG Lan1*
1. Key Laboratory of the Ministry of Health for Research on Quality and Standardization of Biotech Products, National Institutes for Food and Drug Control, Beijing 100050, China; 2. The PLA Rocket Force Characteristic Medical Center, Beijing 100088, China
Abstract：OBJECTIVE To establish a quality control method for anti-DLL3 monoclonal antibody-MPVP-PBD based on cysteine conjugation. METHODS The cell line HEK 293T transfected with DLL3 was used as the target cells to determine the biological activity against of anti-DLL3 McAb-MPVP-PBD, and the binding activity was determined by ELISA. Size heterogeneity was measured by CE-SDS and SEC-UPLC, and charge heterogeneity was analyzed by iCIEF. Drug-to-antibody ratio (DAR) was determined by HIC-HPLC and LC-MS, respectively. The contents of free small molecular drugs were determined by RP-HPLC. RESULTS The average biological activity EC50 of anti-DLL3 McAb-MPVP-PBD was (4.02±0.22) ng·mL-1 and RSD was 5.47%. The average binding activity EC50 was (12.67±1.09) ng·mL-1 and RSD was 8.60%. Peak area percentages of intact IgG, HHL and LC from non-reduced CE-SDS analysis were (25.58±0.15)%, (36.56±0.24)% and (14.61±0.13)% respectively, RSD was less than 2%. The sum of peak area percentages of HC and LC from reduced CE-SDS analysis was (96.97±0.31)% and RSD was 0.32%. The area percentages of main peak and aggregate from SEC-UPLC analysis were (98.68±0.05)% and (1.32±0.05)% respectively, with RSD less than 5%. The main peak area of iCIEF was (68.19±0.68)%, and the isoelectric point of the main peak was (8.28±0.01), RSD was less than 2%. The DAR determined by LC-MS was 2.08 (intact molecular weight level) and 1.73 (light and heavy chain level). The contents of 0-drug, 1-drug, 2-drug, 3-drug and 4-drug were (19.49±0.28)%, (11.21±0.20)%, (48.64±0.68)%, (5.21±0.40)% and (15.44±1.19)% from HIC-HPLC analysis respectively(RSD＜10%), by which the DAR was calculated as (1.86±0.03). The occupation ratio of conjugation sites was determined by LC-MS. Free small molecular drug was (23.20±0.82)% from RP-HPLC analysis and RSD was 3.53%. CONCLUSION The quality control methods of anti-DLL3 monoclonal antibody-MPVP-PBD for antibody-conjugated drugs are preliminarily established in this study. This method has the potential to ensure product safety, effectiveness and quality controllability. They can be used as a routine detection methods and provide a reference for the quality detection of related antibody-conjugated drugs in China.
李萌, 孙亮, 朱磊, 于传飞, 王文波, 武刚, 张荣建, 崔永霏, 王兰. 以Delta样蛋白3为靶点的抗体偶联药物质量研究[J]. 中国药学杂志, 2019, 54(24): 2018-2027.
LI Meng, SUN Liang, ZHU Lei, YU Chuan-fei, WANG Wen-bo, WU Gang, ZHANG Rong-jian, CUI Yong-fei, WANG Lan. Quality Control of Anti-delta-like Protein 3 Antibody-MPVP-PBD. Chinese Pharmaceutical Journal, 2019, 54(24): 2018-2027.
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2019, Vol. 54 
