ELISA法测定大鼠血浆中抗体偶联药物浓度及药代动力学研究

Determination of antibody-drug conjugate in rat plasma by ELISA and study of its pharmacokinetics

目的:建立检测大鼠血浆中抗体偶联药物(ADC006M)[重组抗人表皮生长因子受体2(HER2)结构域Ⅱ人源化单克隆抗体-单甲基澳瑞他汀E(MMAE)偶联剂]浓度的酶联免疫吸附分析法(ELISA),并用于研究单次静脉注射的药代动力学特征。方法:基于ELISA的检测原理,在96孔板上预先包被抗MMAE抗体,加入待测样品,用HRP标记的抗体B1G7(B1G7-HRP)进行检测,加入TMB底物显色,终止反应后,在酶标仪450 nm/630 nm波长处读取吸收度(A),并参考相关法规进行方法学验证。同时,单次静脉注射给予SD大鼠5 mg·kg^(-1)ADC006M测定其血浆浓度,对测定结果进行曲线拟合并计算药代动力学参数。结果:确定方法的线性范围为31.25~2000 ng·mL^(-1),定量限为31.25 ng·mL^(-1);方法批内、批间准确度RE分别在-14.6%~12.6%和-13.1%~-0.7%,批内、批间精密度RSD均≤12.6%;方法的选择性(基质效应)、特异性、稀释线性与钩状效应以及各项稳定性考察结果均良好。通过对SD大鼠血浆样品进行浓度测定,获得ADC006M药代动力学参数,雌鼠、雄鼠的C_(max)分别为(114218±17845)μg·L^(-1)和(119900±12259)μg·L^(-1),AUC_(0-∞)分别为(90546±6481)μg·L^(-1)·d^(-1)和(89648±13735)μg·L^(-1)·d^(-1),t_(1/2)分别为(1.46±0.33)d和(1.70±0.28)d。结论:本文建立的检测大鼠血浆中ADC006M浓度的ELISA方法,特异性、准确度、精密度、稳定性等均符合生物制药临床前药代动力学研究指导原则要求,ADC006M在大鼠体内的药代动力学特征不存在性别差异,其与同类ADC药物的代谢特征基本一致,具有较好的药代动力学特性。

Objective:To establish an enzyme-linked immunosorbent assay(ELISA)for the detection of the concentration of antibody-drug conjugate[ADC006 M,recombinant anti-human epidermal growth factor receptor 2(HER2)domainⅡhumanized monoclonal antibody-monomethylauristatin E(MMAE)conjugate]in rat plasma,and to study its pharmacokinetic characteristics of single intravenous injection.Methods:Based onthe test principle of ELISA,96-well plate was pre-coated with anti-MMAE antibody,after adding the samples,detected with HRP-labeled antibody B1 G7(B1 G7-HRP).Then,TMB substrate solution was added for color display.After the reaction was quenched,the absorption(A)was read at double wavelength of 450 nm/630 nm by using a microplate reader.With reference to the relevant regulations,the method had been validated.Meanwhile,SD rats were given 5 mg·kg^(-1) ADC006 M intravenously to determine the plasma concentration,and the curve fitting was performed tocalculate the pharmacokinetic parameters.Results:The linear range of the method was 31.25-2000 ng·mL^(-1),with lower limit of quantitation(LLOQ)of 31.25 ng·mL^(-1).The accuracy RE of intra-assay and inter-assay were-14.6%to 12.6%and^(-1)3.1%to-0.7%.The intra-and inter-precision RSD were both≤12.6%,respectively.The selectivity(matrix effect),specificity,dilution linearity,hook effect,and stability of the method were all good.The pharmacokinetic parameters of ADC006 M were determined by plasma concentration of SD rats,the C_(max) was(114218±17845)μg·L^(-1) for female and(119900±12259)μg·L^(-1) for male,respectively,AUC_(0-∞)was(90546±6481)μg·L^(-1)·d^(-1)and(89648±13735)μg·L^(-1)·d^(-1),t_(1/2) was(1.46±0.33)d and(1.70±0.28)d,respectively.Conclusion:An ELISA method was established to detect the concentration of ADC006 M in the plasma of SD rats,its specificity,accuracy,precision,stability and so on all conform to the requirements of the research guidelines of biological preclinical pharmacokinetics.There was no gender difference in the pharmacokinetic characteristics of ADC006 M in SD rats.It is basically consistent with the metabolic characteristics of similar ADC drugs and has great pharmacokinetic characteristics.