摘要
目的:建立并验证应用离子交换色谱(IEC-HPLC)测定重组人血清白蛋白干扰素α2b融合蛋白(r IFNα2b-HSA)纯度的方法。方法:IEC-HPLC色谱条件:TSKgel Q-STAT色谱柱(3.0 mm×10 cm);流动相A液为Tris(HCl)缓冲液,B液为Tris(HCl)Na Cl缓冲液;柱温25℃;流速0.5 mL·min^-1;进样体积20μL;检测波长214 nm;梯度洗脱条件:0~3 min,100%A;3~18 min,100%A→0%A;18~21 min,0%A;21~22 min,0%A→100%A。对该方法的系统适用性、专属性、线性、精密度及定量限进行考察,并与反相色谱法、电泳法纯度测定结果进行比较。结果:系统适用性:IEC-HPLC法杂质与主峰的分离度>1.5,理论塔板数>20000;专属性:用Na OH溶液对供试品进行处理,主峰与杂质峰分离度良好;线性范围为0.05~1 mg·m L^-1(R2=0.9974);方法精密度(RSD)为0.52%(n=6);定量下限为0.0018 mg·m L^-1;用3种方法对3批样品进行纯度测定,结果均大于99.0%。结论:对该方法进行方法学研究,其结果均符合2015年版《中华人民共和国药典》三部《药品质量标准分析方法验证指导原则》相关要求,适用于r IFNα2b-HSA的纯度测定。
Objective:To establish and verify a technical method for determination of the purity of recombinant human serum albumin interferon α2 b fusion protein(rIFNα2 b-HSA) by ion exchange chromatography(IECHPLC). Method:The IEC-HPLC chromatographic systems were applied for the quantification of α2 b protein by TSK gel Q-STAT column(3.0 mm × 10 cm) with mobile phase A[Tris(HCl)] buffer and B[Tris(HCl) NaCl buffer] under the gradient elution conditions: 0-3 min, 100%A;3-18 min, 100%A → 0%A;18-21 min, 0%A;21-22 min, 0%A→ 100%A, and 20 μL of theα2 b sample was applied and detective on 25 ℃, 0.5 mL·min^-1 flow rate and 214 nm. The system applicability,specificity,linearity,precision and limit of quantification of this method were investigated,and compared with the results obtained by reversed-phase chromatography and electrophoresis. Results:System applicability:the separating degree of IEC-HPLC method for separation of impurities and main peak was higher than 1.5,the number of theoretical plates was higher than 20 000;Specificity:the sample was treated with NaOH solution,the main peak and impurity peak are well separated;the linear range was 0.05-1 mg·m L^-1(R2=0.997 4);method precision(RSD) was 0.52%(n=6);the limit of quantification was 0.001 8 mg·mL^-1. Three batches of samples were purified by 3 methods,and the purity was all higher than 99.0%. Conclusion:A methodological study of this method is performed,and the results meet the relevant requirements of the"Chinese Pharmacopoeia(2015 edition)" and"Guidelines for the Verification of Drug Quality Standard Analysis Methods",and this method is suitable for determination of rIFNα2 b-HSA purity.
作者
杜莹莹
武福军
韩浩
张亚男
刘志敏
DU Ying-ying;WU Fu-jun;HAN Hao;ZHANG Ya-nan;LIU Zhi-min(Shanxi Kangbao Biological Product Co.Ltd.,Changzhi 046011,China;Institute of Bioengineering,Academy of Military Medicine,Beijing 100071,China)
出处
《药物分析杂志》
CAS
CSCD
北大核心
2020年第8期1490-1493,共4页
Chinese Journal of Pharmaceutical Analysis
基金
国家“十一五”863目标导向性课题资助(009AA02Z108)。
