基于DOE设计的IgG2型抗表皮生长因子受体单抗抗体依赖的细胞吞噬作用生物学活性报告基因检测法的建立及验证

Development and verification of a reporter gene determination assay for ADCP potency of IgG2 anti-EGFR monoclonal antibody based on DOE

目的通过实验设计(Design of Experiment,DOE)与单因素分析(one factor at a time,OFAT)相结合的方式,建立测定Ig G2型抗表皮生长因子受体(epidermal growth factor receptor,EGFR)单抗抗体依赖的细胞吞噬作用(antibody dependent cellular phagocytosis,ADCP)生物学活性的报告基因法,并进行验证。方法以Jurkat/NFAT-Re/FcγRⅡa稳转细胞株为效应细胞,A431细胞株为靶细胞,利用JMP软件采取DOE与OFAT相结合的方式,以上下渐近线比值(D/A)为统计量,对实验中7个关键因素进行条件筛选及优化,建立测定Ig G2型抗EGFR单抗ADCP生物学活性的报告基因法,并按照《中国药典》三部/四部(2020版)通则<9401>进行验证。用建立的方法测定Ig G2型抗EGFR单抗注射液生物学活性。结果经过3轮试验,建立了测定Ig G2型抗EGFR单抗ADCP生物学活性的报告基因法,该方法存在量效关系,符合四参数回归方程y=(A-D)/[1+(x/C)^(B)]+D,确定了7个关键条件的耐用范围,即效应细胞密度为(1.25~3.75)×10~4个/孔,效应细胞与靶细胞密度比值为1.0~2.0,靶细胞孵育时间为20~40 min,给药孵育时间为15~30 min,总作用时间为5.5~6.5 h,显色时间为5~30 min,显色剂为荧光素酶检测系统(Bright-Glo)。该方法专属性良好;对5个效价水平进行6次独立试验,线性回归方程相关系数(r)=0.9945,斜率为1.02;5个效价水平相对效价分别为(62.15±1.38)%、(78.53±2.82)%、(99.12±3.95)%、(123.27±4.59)%和(155.22±7.04)%,相对偏倚为-2.9%~0.2%,几何变异系数(generalized cross-validation,GCV)为2.2%~4.6%;在64%~156%范围内具有良好的线性、相对准确度和精密度。Ig G2型抗EGFR单抗生物学活性3次测定结果均值为(101.5±2.8)%。结论本研究建立的报告基因法可用于Ig G2型抗EGFR单抗ADCP生物学活性测定。

Objective To develop and verify a reporter gene assay for the determination of antibody dependent cellular phagocytosis(ADCP)potency of Ig G2 monoclonal antibody(m Ab)against epidermal growth factor receptor(EGFR)by combining Design of Experiment(DOE)and one factor at a time(OFAT).Methods The Jurkat/NFAT-Re/FcγRⅡa stably transformed cell line was used as effector cells,while the A431 cell line as the target cells.The JMP software was used to optimize the seven key factors in the experiment by combining DOE and OFAT analysis,while the ratio of upper and lower asymptotes(D/A)was used as the statistic,and the reporter gene method was developed to evaluate the ADCP potency of Ig G2 anti-EGFR m Ab.The method was verified according to the general chapter<9401>of Chinese Pharmaco-poeia(Ⅲ/Ⅳvolume,2020 edition)and used to determine the biological potency of Ig G2 anti-EGFR m Ab injection.Results After three rounds of experiments,the reporter gene method to evaluate the ADCP potency of Ig G2 anti-EGFR m Ab was developed.The method showed a dose-response relationship and was consistent with the four-parameter regression equa-tion y=(A-D)/[1+(x/C)~B]+D.The range of seven key conditions was determined:the density of effector cells was(1.25-3.75)×10~4 cells/well,the density ratio of effector cells to target cells was 1.0-2.0,the incubation time of target cells was 20-40 min,the incubation time of administration was 15-30 min,the total time was 5.5-6.5 h,and the color time was 5-30 min with luciferase detection system(Bright-Glo)as the color agent.The method had good specificity.Six independent tests were run for the five potency levels,with the correlation coefficient r of 0.9945 and the linear regression equation slope of 1.02.The relative potency of five potency levels respectively was(62.15±1.38)%,(78.53±2.82)%,(99.12±3.95)%,(123.27±4.59)%and(155.22±7.04)%,the range of relative biases was-2.9%-0.2%,and the range of generalized cross-validation(GCV)was 2.2%-4.6%.The method had good linearity,relative accuracy and precision in the range of 64%-156%.The mean value of the potency of IgG2 anti-EGFR m Ab in three tests was(101.5±2.8)%.Conclusion The reporter gene assay developed in this study can be used to evaluate the ADCP potency of IgG2 anti-EGFR mAb.