高效分子排阻色谱法测定抗毒素/抗血清类制品纯度  

Determination of Purity of Antitoxin/Antiserum Products by High Performance Size Exclusion Chromatography

作　　者：周勤 徐明明 赵沁 段徐华 吴利红 邵泓 陈钢 ZHOU Qin;XU Mingming;ZHAO Qin;DUAN Xuhua;WU Lihong;SHAO Hong;CHEN Gang(Shanghai Institute for Food and Drug Control,NMPA Key Laboratory for Quality Control of Therapeutic Monoclonal Antibodies,Shanghai 201203,China)

机构地区：[1]上海市食品药品检验研究院,国家药品监督管理局治疗类单抗质量控制重点实验室,上海201203

出　　处：《中国现代应用药学》2021年第12期1484-1489,共6页Chinese Journal of Modern Applied Pharmacy

基　　金：上海市科学技术委员会科研计划项目(18DZ2202000)。

摘　　要：目的建立高效分子排阻色谱法(high performance size exclusion chromatography,HPSEC)测定抗毒素/抗血清类制品纯度,并应用该方法对52批样品进行测定。方法采用TOSOH TSKgelG3000SWxl色谱柱,以含1%异丙醇,0.2 mol·L^(−1)磷酸盐缓冲液(pH 7.0)为流动相,流速0.6 mL·min^(−1),检测波长280 nm,进样体积20μL,柱温室温,按面积归一化法计算各组分相对百分含量。结果选取抗毒素类和抗血清类制品各1批,在蛋白质浓度为1.2~24 mg·mL^(−1)时,主峰F(ab’)2、大分子杂质峰和小分子杂质峰峰面积均与相应蛋白质浓度呈良好线性关系(n=5,r>0.9990),重复性RSD均在0.4%~0.9%(n=6),主成分F(ab’)2定量限分别为0.1μg和0.2μg,检出限分别为0.02μg和0.1μg,制备的供试品溶液在室温条件下12 h内各组分稳定性良好。结论建立的HPSEC可用于抗毒素/抗血清类制品纯度的测定。OBJECTIVE To establish a high performance size exclusion chromatography(HPSEC)method for the purity determination of antitoxin/antiserum products and to be applied to determine the purity of 52 samples.METHODS TOSOH TSKgelG3000SWxl column was used,the 0.2 mol·L^(−1)phosphate buffer(pH 7.0)containing 1%isopropanol were used as mobile phase.The flow rate was 0.6 mL·min^(−1),the detection wavelength was 280 nm,the injection volume was 20μL,and the column was kept at room temperature.The relative percentage of each component was calculated by peak area normalization.RESULTS One batch of antitoxin and one batch of antiserum were selected,in the protein concentration ranges from 1.2−24 mg·mL^(−1),the peak areas of F(ab’)2,high molecular weight impurities and small molecular weight impurities had good linear relationship with the corresponding protein concentration(n=5,r>0.9990),repeatability RSD was 0.4%−0.9%(n=6).The limits of quantitation of F(ab’)2 were 0.1μg and 0.2μg,the limits of detection of F(ab’)2 were 0.02μg and 0.1μg,the sample solution was stable at room temperature within 12 h.CONCLUSION The HPSEC can be applied to determine the purity of antitoxin and antiserum products.

关 键 词：高效分子排阻色谱法 抗毒素/抗血清类制品 纯度 

分 类 号：R917.101[医药卫生—药物分析学]