抗体偶联药物抗HER²单抗-MCC-DM1质控方法的建立

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(sizeexclusion-high performance liquid chromatography, SE-HPLC)分析纯度;利用成像毛细管等点聚焦电泳(iCIEF)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(0.8222±0.0505)%, 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±0.0058)%, RSD为0.0058%; iCIEF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 enzymelinked 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. 8222±0. 0505)%. 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. 0058)%. 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-DM1 is developed, which ensures the safety, effectiveness and quality controllability of the product and provides a reference for quality control of domestic ADC.