人生长激素Fc融合蛋白电荷异质性全柱成像毛细管等电聚焦电泳分析方法的建立及验证  

Development and verification of whole-column imaging capillary isoelectric focusing electrophoresis analysis method for charge heterogeneity of human growth hormone Fc fusion protein

作　　者：马琳琳 刘莹 俞露 朱秋媚 刘涵 毕江坤 张凯宁 MA Linlin;LIU Ying;YU Lu;ZHU Qiumei;LIU Han;BI Jiangkun;ZHANG Kaining(Recombinant Protein Drug Laboratory,Changchun Institute of Biological Products Co.,Ltd.,Changchun 130012,Jilin Province,China)

机构地区：[1]长春生物制品研究所有限责任公司重组蛋白药物研究室,吉林长春130012

出　　处：《中国生物制品学杂志》2023年第6期700-706,713,共8页Chinese Journal of Biologicals

基　　金：吉林省科技发展计划项目(20160204034YY)。

摘　　要：目的建立全柱成像毛细管等电聚焦电泳(image capillary isoelectric focusing,iCIEF)方法分析重组人生长激素(recombinant human growth hormone,rhGH)Fc融合蛋白(Fc-rhGH)的电荷异质性,并对方法进行验证。方法通过对目标蛋白浓度、助溶剂(尿素)浓度、聚焦时间的优化建立Fc-rhGH的iCIEF分析方法。采用该方法与传统平板等电聚焦电泳(isoelectric focusing,IEF)同时分析目标蛋白,并比较分析结果;对建立的方法进行特异性、准确性、精密性、定量限(limit of quantitation,LOQ)、耐用性验证。结果优化后的方法为采用8 mol/L尿素、0.35%甲基纤维素(methyl cellulose,MC)、4%两性电解质、0.5%等电点标记物混合溶液为样品缓冲液,聚焦条件为:1500 V 1 min,3000 V 5.5 min。IEF不适用于分析Fc-rhGH原液的电荷异质性。采用优化的iCIEF进行分析,目标蛋白能明显区别于非相关蛋白,且空白试剂基线平稳;准确性验证回收率在90%~110%之间,线性范围为0.25~0.75 mg/mL(目标上样量的50%~150%);重复性验证中各异构体pI的RSD均小于0.3%,峰面积百分比RSD均小于5%;定量限为0.04 mg/mL;方法的样品保存时间耐用性、两性电解质pharmalyte 3-10耐用性和MC耐用性良好。采用该方法分析Fc-rhGH理化对照品的电荷异质性,可有效分离对照品的8个电荷异质体,pI范围在5.9~6.4。结论建立的iCIEF法具有良好的特异性、准确性、精密性、耐用性,比传统平板IEF更适合高效分析Fc-rhGH的电荷异质性,对Fc-rhGH及其他Fc融合蛋白的质量控制具有重要意义。Objective To develop and verify a whole-column image capillary isoelectric focusing(iCIEF)electrophoresis method to analyze the charge heterogeneity of recombinant human growth hormone Fc fusion protein(Fc-rhCH).Methods The iCIEF analysis method of Fc-rhGH was developed by optimizing the target protein concentration,cosolvent(urea)concentration and focusing time.The target protein was simultaneously analyzed by this method and traditional flat plate isoelectric focusing(IEF)electrophoresis,and the results were compared;The specificity,accuracy,precision,limit of quantitation(LOQ)and durability of the developed method were verified.Results The optimized method was using the mixed solution of 8 mol/L urea,0.35%methyl cellulose(MC),4%amphoteric electrolyte and 0.5%isoelectric point marker as the sample buffer,and the focusing condition was 1500 V 1min,3000 V 5.5 min.IEF was not suitable for analyzing the charge heterogeneity of Fc-rhGH solution.Using the optimized iCIEF for analysis,the target protein was significantly different from the unrelated protein,and the baseline of blank reagent was stable;The recovery rate of accuracy verification was within 90%~110%,and the linear range was 0.25~0.75 mg/mL(50%~150%of the target loading volume);The RSD of each isomer pl in the repeatability verification was less than 0.3%,and the RSD of peak area percentage was less than 5%;The LOQ was 0.04 mg/ml.The sample storage time durability,amphoteric electrolyte pharmalyte 3-10 durability and MC durability of this method were good.Using this method to analyze the charge heterogeneity of Fc-rhGH physicochemical reference substance,eight charge heterogeneities of the reference substance were effectively separated,and the pl ranged from 5.9 to 6.4.Conclusion The developed iCIEF method had good specificity,accuracy,precision and durability,and was more suitable for efficient analysis of charge heterogeneity of Fc-rhGH than traditional flat plate IEF,which was of great significance for the quality control of Fc-rhGH and other Fc fusion

关 键 词：全柱成像毛细管等电聚焦电泳 重组人生长激素Fc融合蛋白 电荷异质性 

分 类 号：R977.1[医药卫生—药品]