抗体偶联药物抗HER2单抗-MCC-DM1质控方法的建立

Development of methods for quality control of an anti-human epidermal growth factor receptor-2-MCC-DM1

目的:建立抗体偶联药物(ADC)抗人表皮生长因子受体-2(HER2)单抗-马来酰亚胺基-环己烷-1-羧化物(MCC)-美登素衍生物(DM1)(trastuzumab-DM1,T-DM1)的质控方法。方法:利用人乳腺癌BT-474细胞增殖抑制实验测定T-DM1的生物学活性;利用生物分子间相互作用分析核心技术(biomolecular interaction analysis core-technology,BIAcore)测定其结合常数(KD);利用HER2抗原和抗DM1抗体双夹心酶联免疫法(ELISA)对T-DM1进行鉴别;利用液质联用法和紫外分光光度法测定药物抗体偶联比(DAR);利用反相高效液相色谱(RP-HPLC)法测定游离药物DM1的含量;利用十二烷基磺酸钠-毛细管凝胶电泳(capillary electrophoresis-sodium dodecyl sulfonate,CE-SDS)和分子排阻色谱(size exclusion-high performance liquid chromatography,SE-HPLC)分析纯度;利用成像毛细管等点聚焦电泳(i CIEF)分析电荷异质性,其他各项指标均符合现行版中国药典的要求及其他相关要求。结果:T-DM1生物学活性相对效价为(94.04±2.60)%,KD为(1.03±0.02)E-9 mol·L-1,RSD均小于5%;ELISA鉴别为阳性;液质联用法和紫外分光光度法测得的DAR分别为3.21及3.25;RP-HPLC法测定游离药物DM1的含量为(0.822 2±0.050 5)%,RSD小于10%;非还原CE-SDS主峰面积为(96.63±0.07)%,RSD为0.07%;SE-HPLC主峰面积为(97.65±0.005 8)%,RSD为0.005 8%;i CIEF分析可以将每个偶联数的抗体药物分开,达到很好的鉴别。结论:建立ADC中T-DM1质控方法具有保证产品安全、有效、质量可控的特点,为我国ADC的质量检测提供了参考依据。

Objective： To develop quality control methods of an anti-human epidermal growth factor receptor- 2-MCC-DM1 （ T-DM1 ）, a kind of antibody-drug conjugate （ ADC ）. Methods： The bioactivity of T-DM1 was evaluated by determining its cytotoxic effect on human breast cancer BT-474 cells, the binding constant （ KD ） was evaluated by biomolecular interaction analysis core-technology （ BIAcore ）, T-DM1 was identified by enzyme- linked immune （ ELISA ）, the determination of drug antibody ratio （ DAR ） was conducted by LC-MS and UV, the determination of free drug DM1 was conducted by reversed-phase-high performance liquid chromatography （ RP-HPLC ）, and purity was analyzed by capillary electrophoresis-sodium dodecyl sulfonate （ CE-SDS ） and size exclusion-high performance liquid chromatography （ SE-HPLC ）, and the charge heterogeneity was determined by imaged capillary isoelectric focusing （ iCIEF ）. Results： The relative potency of T-DM1 was （ 94.04 ± 2.60 ） %, and KD was （ 1.03 ± 0.02 ） E-9 mol· L^-1. It was identified as positive by ELISA. The DAR was to be 3.21 and 3.25 respectively by LC-MS and UV. The content of free drug DM1 was （ 0. 822 2 ± 0. 050 5 ） %. The main peak area percentage showed by non-reduced CE-SDS was （ 96.63 ± 0. 07 ） %, the main peak area percentage showed by SEC-HPLC was （ 97.65 ± 0. 005 8 ） %. Antibody-conjugated drugs with different DAR can be separated effectively by iCIEF. Other indicators were in line with the current edition of the Chinese pharmacopoeia and other related requirements. Conclusion： Methods for quality control of T-DM 1 is developed, which ensures the safety, effectiveness and quality controllability of the product and provides a reference for quality control of domestic ADC.