两种毛细管电泳方法分析重组人促红素异构体含量的比较

Analysis of recombinant human erythropoietin isoform content by two capillary electrophoresis

目的全柱成像毛细管等电聚焦电泳(whole column imaging detection-capillary isoelectric focusing electrophoresis,WCID-CIEF)及毛细管区带电泳法(capillary zone electrophoresis,CZE)检测分析重组人促红素(recombinant human erythropoietin,rhEPO)异构体含量。方法 5批rhEPO原液同时经WCID-CIEF法(采用4 mol/L尿素,0. 35%甲基纤维素,4%两性电解质混合溶液作为样品缓冲液,聚焦电压为3 k V,预聚焦时间1 min,聚焦时间7 min,成像波长280 nm)及CZE法(采用0. 01 mol/L NaCl、tricine、NaAC和7 mol/L Urea及2. 5 mmol/L腐胺混合溶液作为电泳缓冲液,pH 5. 55,进样压力3. 45 kPa,进样时间10 s,分离电压15. 4 kV,分离时间80 min,检测波长214 nm)检测分析异构体含量。结果 WCID-CIEF及CZE法测定5批rhEPO原液均有5~8条异构体条带,异构体含量差异无统计学意义(P> 0. 05),具有明显的相关性(r为0. 999),各峰面积占比相差总和小于3%。结论虽然WCIDCIEF谱图及CZE谱图的含义略有不同,但两种毛细管电泳方法的分析结果基本相同,均可满足rhEPO异构体的质量控质需求。

Objective To analyze recombinant human erythropoietin(rh EPO)isoform content by whole column imaging detection-capillary isoelectric focusing electrophoresis(WCID-CIEF)and capillary zone electrophoresis(CZE).Methods The contents of isoforms in five batches of bulk of rh EPO were determined by WCID-CIEF and CZE respectively.In WCID-CIEF,the mixture of 4 mol/L urea,0.35%methylcellulose and 4%pharmalyte was used as sample buffer,while the focusing voltage was set as 3 k V,the prefocusing time as 1 min,the focusing time as 7 min,and the detection wavelength as 280 nm.However,in CZE,the mixture of 0.01 mol/L sodium chloride,0.01 mol/L tricine,0.01 mol/L sodium acetate,7 mol/L urea and 2.5 mmol/L putrescine was used as running buffer(pH 5.55),while the pressure for sample injection was set as 3.45 kPa,while the time for injection as 10 s,separation voltage as 15.4 kV,the separation time as 80 min,and detection wavelength as 214 nm.Results Both WCID-CIEF and CZE showed 5~8 isoforms in five batches of bulk of rh EPO.The statistic results showed no significant difference between the contents of isoforms determined by the two methods(P>0.05).However,the determination results by the two methods showed significant correlation(r=0.999),and the difference in the percentages of the corresponding peak areas was less than3%.Conclusion Though the meanings of WCID-CIEF and CZE spectra are slightly different,the determination results by the two methods are basically in agreement,both of which meet the requirement for quality control of rhEPO isoforms.