目的 建立一种基于半胱氨酸偶联方式的抗体偶联药物抗Delta样蛋白3(DLL3)单抗-MPVP-PBD的质控方法。方法 本实验利用转基因细胞法,以转染了DLL3的HEK 293T细胞系为靶细胞测定抗DLL3单抗-MPVP-PBD的生物学活性;利用ELISA法测定结合活性;利用CE-SDS和SEC-UPLC测定大小异质性;利用iCIEF分析电荷异质性;分别利用HIC-HPLC和LC-MS对药物抗体偶联比(drug-to-antibody ratio,DAR)进行测定;利用RP-HPLC测定游离小分子药物含量。结果 抗DLL3单抗-MPVP-PBD的生物学活性EC₅₀平均值为(4.02±0.22)ng·mL^-1,RSD为5.47%;结合活性EC₅₀平均值为(12.67±1.09)ng·mL^-1,RSD为8.60%;非还原CE-SDS 完整IgG峰面积百分比为(25.58±0.15)%、HHL为(36.56±0.24)%、轻链为(14.61±0.13)%等,RSD均小于2%;还原CE-SDS 重链和轻链峰面积之和为(96.97±0.31)%,RSD为0.32%;SEC-UPLC主峰面积为(98.68±0.05)%,多聚体(1.32±0.05)%,RSD均小于5%;iCIEF 主峰面积为(68.19±0.68)%,主峰等电点(8.28±0.01),RSD均小于2%;LC-MS测定DAR为2.08(完整相对分子质量水平)和1.73(轻重链水平);HIC-HPLC测定DAR为(1.86±0.03),0-药物为(19.49±0.28)%,1-药物为(11.21±0.20)%,2-药物为(48.64±0.68)%,3-药物为(5.21±0.40)%,4-药物为(15.44±1.19)%,RSD均小于10%; LC-MS测定了偶联位点占有率;RP-HPLC测定游离小分子药物为(23.20±0.82)%,RSD为3.53%。结论 本实验初步建立了抗体偶联药物抗DLL3单抗-MPVP-PBD的质控方法,该质控方法具有保证产品安全、有效、质量可控的特点,可作为其常规检测方法,为我国相关抗体偶联药物的质量检测提供了参考依据。

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.