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目的建立人源化叶酸受体α抗体的质控方法。方法应用基于表面等离子体共振(Surface plasmon resonance,SPR)技术的BIAcore3000系统测定人源化叶酸受体α单抗与重组人叶酸受体α的亲和力,从而反映该抗体的功能;采用ELISA法测定其抗原结合力;SDS-PAGE和SEC-HPLC测定其纯度;紫外分光光度法测定其蛋白含量;毛细管等电聚焦电泳法测定等电点;其他各项指标检测按《中国药典》三部(2005版)要求进行。结果人源化叶酸受体α单抗成品的平均相对百分效价为105%,RSD为6.0%。单抗成品及参考品与叶酸受体的结合力均存在量效关系,且符合四参数方程式:Y=(A-D)/[1+(X/C)B]+D,其曲线相关系数在0.98以上,成品经3次测定,相对抗原结合力平均值为97%,RSD为25%。还原SDS-PAGE分析显示,单抗成品的IgG重链和轻链百分比为98.2%;非还原SDS-PAGE分析显示,完整IgG百分比为96.7%;SEC-HPLC分析显示,单抗成品单体为99.1%,聚合体为0.9%。其他各项指标均符合《中国药典》三部(2005版)要求。结论已初步建立了人源化叶酸受体α抗体的质控方法,为该制品的质量控制奠定了基础。
Objective To establish a quality control method of humanized folate receptor α antibody. Methods The BIAcore3000 system based on surface plasmon resonance (SPR) was used to determine the affinity of humanized folate receptor α monoclonal antibody to recombinant human folate receptor α to reflect the function of the antibody. ELISA Antigen binding; SDS-PAGE and SEC-HPLC determination of its purity; UV spectrophotometry determination of its protein content; capillary isoelectric focusing electrophoresis determination of isoelectric point; other indicators of detection according to “Chinese Pharmacopoeia” three ) Requested. Results The average relative relative potency of the humanized folate receptor α monoclonal antibody was 105% with a RSD of 6.0%. The results showed that there was a dose-response relationship between the monoclonal antibody and reference substance and folate receptor binding, and the four-parameter equation was Y = (AD) / [1+ (X / C) B] + D with a curve correlation coefficient of 0.98 Above, the finished product was measured three times, the average relative antigen binding capacity was 97%, RSD was 25%. Reduced SDS-PAGE analysis showed that the percentages of IgG heavy chain and light chain were 98.2% for monoclonal antibody and 96.7% for non-reducing SDS-PAGE. SEC-HPLC analysis showed 99.1 %, Polymer 0.9%. Other indicators are in line with the “Chinese Pharmacopoeia” three (2005 version) requirements. Conclusion The quality control method of humanized folate receptor α antibody has been initially established, which lays the foundation for the quality control of this product.