Insulin aggregation assessment by capillary gel electrophoresis without sodium dodecyl sulfate: Comparison with size-exclusion chromatography

TALANTA

卷:199

Article

Demelenne, A.¹  Napp, A.¹  Bouillenne, F.²  Crommen, J.¹  Servais, A. -C.¹  Fillet, M.¹ 

[1] Univ Liege, CIRM, Dept Pharm, Lab Anal Med, Ave Hippocrate 15,B36 3,Tower 4, B-4000 Liege, Belgium

[2] Univ Liege, Ctr Prot Engn CIP, Lab Enzymol & Prot Folding, Allee Six Aout 11,B6a, B-4000 Liege, Belgium

关键词: Acidic pH SEC;    Neutral pH SEC;    CGE;    Sodium deoxycholate;    Dimers;    Pharmacopoeia;   

DOI  :  10.1016/j.talanta.2019.02.074

来源: Elsevier

【 摘 要 】

Size-exclusion chromatography (SEC) is a method of choice for the analysis of protein aggregates in pharmaceuticals. The United States and European Pharmacopoeias currently use a SEC method with an acidic pH mobile phase to assess the content of aggregates in insulin formulations. In this article, we analyzed aggregated human insulin samples and demonstrated that both methods under neutral conditions, namely neutral pH SEC (nSEC) and capillary gel electrophoresis (CGE), yield to similar aggregate content contrary to SEC under acidic conditions (aSEC). aSEC showed polymeric complexes that were not observed in nSEC and CGE. During method development, the effect on SEC profiles of arginine and acetonitrile were highlighted. In CGE, the effect of SDS on disruption of non-covalent insulin aggregates was confirmed and the benefit of sodium deoxycholate addition in sieving gel was discussed. The three methods were applied to the analysis of an insulin formulation and similar results to those obtained for human insulin as raw material were observed. Finally, the CGE method was used to study the stability of human insulin under different storage conditions. In view of the obtained results one may question the relevance of the current pharmacopoeia method to study insulin aggregates by emphasizing the importance of the mobile phase composition and pH in SEC. The new CGE method developed is an easy method for studying non-covalent aggregates of insulin, which could be applied to other proteins.

【 授权许可】

Free