酶法定点制备抗HER2抗体偶联药物的质量研究

Establishment of Quality Control Methods of Site-Specific Enzymatic Coupling Antibody-Drug Conjugate Targeting HER2

目的建立酶法定点偶联制备抗体药物(antibody drug conjugate,ADC)抗表皮生长因子受体-2(human epidermal growth factor receptor 2,HER2)单抗-澳瑞他汀(MMAE)的质控方法。方法建立适宜的方法对抗体偶联药物的纯度(分子大小变异体、电荷变异体)、偶联修饰位点和偶联比(antibodydrugratio,DAR)、结合活性和生物学活性、以及偶联过程工艺相关杂质[游离小分子药物和谷氨酰胺转移酶(microbial transglutaminase,mTG)残留]进行控制。结果体积排阻色谱(SEC)纯度主峰为(98.35±0.07)%,非还原十二烷基硫酸钠毛细管凝胶电泳法(CE-SDS)完整蛋白比例为(95.64±0.07)%,还原轻链比例为(31.15±0.19)%,重链比例为(67.87±0.06)%,全柱成像毛细管电泳(iCIEF)主峰比例为(51.05±0.7)%,偶联反应工艺偶联率95%以上,LC-MS测定的DAR为1.93(完整相对分子量),RP-HPLC测定的DAR为2.0,HC:Q298的连接子毒素(linker and drug,LD)修饰比例近100%,样品相对生物学活性(相对于参比品计算出的相对活性)平均值在(101±6.7)%,相对结合活性平均值为(95±3.8)%,RP-HPLC测定游离小分子含量均低于定量下限15 ng,ELISA法测定残留mTG含量均低于定量下限为0.165 ng·mL^(-1)。结论初步建立了酶法定点偶联制备HER2单抗-MMAE偶联药物质控方法,该质控方法具有保证产品安全、有效、质量可控的特点,为酶法定点偶联制备抗体偶联药物的质量研究提供了参考依据。

OBJECTIVE To establish quality control methods of an anti-human epidermal growth factor receptor 2(HER2)-MMAE(LD),a kind of enzymatic site-specific prepared antibody-drug conjugate(ADC).METHODS An orthogonal method was established to determine the purity(molecular size variants,charge variants),conjugation modification site,and antibody drug ratio(DAR),binding activity and biological activity,and process-related impurities(free small molecule drugs and MTG residues)in the coupling process.RESULTS The SEC peak monomer purity was(98.35±0.07)%,the NRCE-SDS monomer purity was(95.64±0.07)%,the RCE-SDS purity light chain ratio was(31.15±0.19)%,heavy chain ratio was(67.87±0.06)%,the iCIEF purity main peak ratio was(51.05±0.7)%,coupling reaction rate was above 95%,DAR were 1.93 and 2.0 respectively determined by LC-MS and RP-HPLC,LND1002 modification(HC:Q298)rate was nearly 100%,the average relative biological activities was(101±6.7)%,the average relative binding activity was(95±3.8)%,the free drug content was lower than 15 ng,and the residual mTG was lower than 0.165 ng·mL^(-1).CONCLUSION The methods for quality control of the product are cleveloped to ensure thequality safety and effectiveness,and provide a reference for the quality control of site-specific enzymatic coupling antibody-drug conjugates.