靶向c-Met抗体偶联药物(RC108)在食蟹猴中PK生物分析方法学研究  

The PK bioanalysis method study of c-Met antibody-drug conjugate (RC108) in cynomolgus monkey

作　　者：周宏雨 王淑娟 刘志浩 马克 王凌 姜静[1,2] ZHOU Hong-yu;WANG Shu-juan;LIU Zhi-hao;MA Ke;WANG Ling;JIANG Jing(Binzhou Medical University,Yantai 264003,China;RemeGen Company Ltd.,Yantai 265503,China;Junke Zhengyuan Pharmaceutical Research Company Ltd.,Beijing 102206,China)

机构地区：[1]滨州医学院,山东烟台264003 [2]荣昌生物制药(烟台)股份有限公司,山东烟台265503 [3]军科正源(北京)药物研究有限责任公司,北京102206

出　　处：《药学学报》2023年第6期1663-1668,共6页Acta Pharmaceutica Sinica

基　　金：山东省自然科学基金项目(R2021MH220)。

摘　　要：抗体偶联药物(antibody-drug conjugate,ADC)有着低毒、高效的药物特点,在癌症治疗中发挥着重要作用。但是因其结构的复杂性,带来药代动力学(pharmacokinetic,PK)生物分析的困难。本研究建立一种应用配基结合分析法(ligand-binding assay,LBA)和液相色谱与串联质谱联用法(liquid chromatography-tandem mass spectrometry,LC-MS/MS)检测食蟹猴血浆中ADC (RC108)的分析方法,用于分析定量食蟹猴血浆中的总抗体、结合抗体和游离药物。基于LBA法,在96孔板预包被rabbit anti-RC108 Fab和mouse anti-MMAE m Ab分别作为总抗体和结合抗体的试剂,加入待测样品,随后依次加入检测试剂goat anti-human Ig G (H+L)-HRP和显色液四甲基联苯胺(tetramethylbenzidine,TMB),H2SO4终止反应后,酶标仪450 nm/630 nm波长处读取数据;LC-MS/MS分析方法定量单甲基澳瑞他汀E (MMAE)浓度,并参考相关法规进行方法学验证。对RC108药物定量总抗体、结合抗体和游离药物的分析方法得到了良好的准确度和精密度,并对选择性、稀释线性、钩状效应和稳定性等进行了验证,均符合生物分析的要求。最终通过LBA法和LC-MS/MS法建立了一种兼具高灵敏与高通量的测定食蟹猴血浆中受试物RC108浓度(总抗体、结合抗体、游离MMAE)的生物分析方法,为后续非临床研究食蟹猴的PK研究提供技术支撑。Antibody-drug conjugate(ADC)has the characteristics of low toxicity and high efficiency,and plays an important role in cancer treatment.However,due to the complexity of its structure,it brings difficulties in pharmacokinetic(PK)bioanalysis.This study established an analytical method for the detection of ADC(RC108)in cynomolgus monkey plasma by ligand-binding assay(LBA)and liquid chromatography tandem mass spectrometry(LC-MS/MS),which was used to analyze and quantify the total antibody,bound antibody and free drug in cynomolgus monkey plasma.Based on the LBA method,rabbit anti-RC108 Fab and mouse anti-MMAE(monomethyl auristatin E)mAb were pre-coated in 96-well plates as the total antibody and antibody binding reagents,respectively.The samples to be tested were added,and then the detection reagents were added in turn.Goat anti-human IgG(H+L)-HRP,chromogenic solution tetramethylbenzidine(TMB),H2SO4 terminate the reaction,read data at 450 nm/630 nm wavelength of microplate reader;LC-MS/MS analysis method quantifies MMAE concentration,and refer to relevant regulations for methodological validation.The analytical method for quantifying total antibody,bound antibody and free drug of RC108 drug obtained good accuracy and precision,and the selectivity,dilution linearity,hook effect,parallelism and stability were verified.Meet the requirements of biological analysis.Finally,a bioanalytical method for the determination of the concentration of the test substance RC108(total antibody,conjugated antibody,free MMAE)in cynomolgus monkey plasma with high sensitivity and high throughput was established by LBA and LC-MS/MS method.Subsequent non-clinical research on PK research in cynomolgus monkeys will provide technical support.

关 键 词：抗体偶联药物 配体结合分析法 液相色谱与串联质谱联用法 定量分析 

分 类 号：R917[医药卫生—药物分析学]