重组抗IgE单克隆抗体天冬氨酸异构化程度分析方法的建立

Development of aspartate isomerization assay for recombinant anti-IgE monoclonal antibodies

目的:建立一种检测重组抗IgE单克隆抗体(单抗)轻链互补决定区中天冬氨酸异构化程度的疏水作用层析(hydrophobic interaction chromatography,HIC)方法。方法:采用木瓜蛋白酶酶切抗IgE单抗,产生抗原结合片段和可结晶片段作为HIC方法的样品。筛选不同品牌及官能团的色谱柱,优化流动相的组成及洗脱梯度,研究样品于5℃和-20℃条件下的稳定性;同时用强制破坏的方式考察该方法的稳定性检测能力。并对该方法的专属性、精密度、线性和准确度进行验证。结果:采用TSKgel Butyl-NPR色谱柱、流动相甘油含量为5%时,色谱图的分离度较好。样品在进样器(5℃)中放置24 h或在-20℃中放置48 h可保持稳定。该方法能检出强制破坏样品的各成分含量随时间的变化,且专属性良好,重复性及中间精密度均符合规定,抗原结合片段各峰的线性决定系数均≥0.990且准确度均在90%~110%。结论:建立的HIC方法可有效分析单抗制品天冬氨酸异构化程度。

Objective:To establish a hydrophobic interaction chromatography(HIC)method to monitor aspartate isomerization level of recombinant anti-IgE monoclonal antibody complementary-determining region.Methods:The anti-IgE monoclonal antibody was digested by papain to generate fragments of antigen binding(Fab)and fragment of crystallizable as samples for HIC.The chromatographic columns of different brands with different functional groups were screened and the composition of mobile phase and elution gradient were optimized.Samples were analyzed under 5℃and-20℃to verify their stability.Stability-indicating ability of the method was examined with stressed antibody samples.Specificity,precision,linearity,and accuracy of the method were validated.Results:Resolution of HIC was optimal with TSKgel Butyl-NPR column and 5%glycerin.The sample was stable for 24 h in instrument auto-sampler(5℃)and 48 h at-20℃,respectively.This method was able to detect the change of different contents with time in stressed antibody samples.The method had good specificity.The repeatability and inter-precision met requirements.The linear coefficients of determination were≥0.990 for all Fab peaks and the accuracies of the method for detecting the peaks were 90%-110%.Conclusion:The HIC method developed can effectively detect aspartate isomerization in monoclonal antibodies.