Poster (Scientific congresses and symposiums)
Quality control of insulin formulations: API , protamine and aggregates follow-up
Demelenne, Alice; Napp, Aurore; Lamalle, Caroline et al.
2017The 45th International Symposium on High Performance Liquid Phase Separations and Related Techniques (HPLC)
 

Files


Full Text
Poster HPLC 2017 AD.pdf
Publisher postprint (606.76 kB)
Request a copy

All documents in ORBi are protected by a user license.

Send to



Details



Keywords :
insulin; protamine; aggregation; capillary electrophoresis; size-exclusion chromatography
Abstract :
[en] The prevalence of diabetes is increasing every year and insulin preparations are mostly prescribed for treatment of both type 1 and type 2 diabetes. Since these biopharmaceutical formulations are expensive and require a prescription, they are an important target for counterfeiting in developing countries. Therefore, there is a need for fast and efficient methods for quality control of biopharmaceutical products such as insulin formulations. A fully validated micellar electrokinetic chromatography (MEKC) method was developed for quantification of human insulin and NPH insulin (insulin combined with protamine) in formulations. The BGE used was made of 50 mM ammonium acetate (pH 9.0), 20 mM Sodium dodecyl sulfate and 13 % (v/v) acetonitrile and samples were introduced at the short capillary end allowing a separation of insulin and two major excipients (phenol and m-cresol) within 3 minutes. This method was compared with HPLC method using a mobile phase composed of water with 0.1% formic acid / acetonitrile with 0.1 % formic acid in gradient mode. Secondly a MEKC method was also optimized to analyze protamine peptides in insulin formulations. The major protamine peptides could be separated using a BGE made of 100 mM phosphate buffer (pH 2) with 50 mM Thesit®. This method was used to check conformity of protamine in commercially available formulations. Finally, different approaches were investigated in order to follow insulin aggregation. Indeed, insulin is prone to unfold when submitted to denaturating factors as temperature, ionic strength, agitation and pH. An accumulation of unfolds protein in bloodstream results in a high tendency to aggregate and form amyloid fibrils. A deposit of those fibrils in the subcutaneous tissue leads to a complication called “insulin-derived amyloidosis”. On the other hand, during its production, insulin is often subjected to extreme conditions making aggregation, as well as protein stability, important parameters to be controlled during its quality control. In this study, insulin aggregates were generated after optimization of incubation conditions (pH, temperature, agitation…). Those aggregates were then analyzed by size-exclusion chromatography (SEC) and capillary electrophoresis (CE). We showed that capillary gel electrophoresis (CGE) is a promising technique to analyze covalent aggregates of insulin due to the fact that it separates the aggregates according to their size and not to their size/charge ratio. The use of a laser-induced fluorescence detector was also found attractive to enhance the sensitivity of the method.
Research center :
CIRM - Centre Interdisciplinaire de Recherche sur le Médicament - ULiège
Disciplines :
Pharmacy, pharmacology & toxicology
Author, co-author :
Demelenne, Alice ;  Université de Liège > Département de pharmacie > Analyse des médicaments
Napp, Aurore ;  Université de Liège > Département de pharmacie > Analyse des médicaments
Lamalle, Caroline
Servais, Anne-Catherine  ;  Université de Liège > Département de pharmacie > Analyse des médicaments
Fillet, Marianne ;  Université de Liège > Département de pharmacie > Analyse des médicaments
Language :
English
Title :
Quality control of insulin formulations: API , protamine and aggregates follow-up
Publication date :
21 June 2017
Event name :
The 45th International Symposium on High Performance Liquid Phase Separations and Related Techniques (HPLC)
Event organizer :
University of Prague
Event place :
Prague, Czechia
Event date :
Du 18 juin 2017 au 22 juin 2017
Audience :
International
Available on ORBi :
since 27 June 2017

Statistics


Number of views
233 (30 by ULiège)
Number of downloads
12 (10 by ULiège)

Bibliography


Similar publications



Contact ORBi