title: Chemical and thermal stability of insulin: effects of zinc and ligand binding to the insulin zinc-hexamer.
abstract: OBJECTIVE
  To study the correlation between the thermal and chemical stability of insulin formulations with various insulin hexamer ligands.
  
  
  METHODS
  The thermal stability was investigated using differential scanning calorimetry (DSC) and near-UV circular dichroism (NUV-CD). The formation of chemical degradation products was studied with reversed-phase and size-exclusion chromatography and mass spectrometry.
  
  
  RESULTS
  An excellent correlation between the thermal stabilization by ligand binding and the deamidation of Asn(B3) was observed. The correlation between thermal stability and the formation of covalent dimer and other insulin related products was less clear. Zinc was found to specifically increase the deamidation and covalent dimer formation rate when the insulin hexamer was not further stabilized by phenolic ligand. Thiocyanate alone had no effect on the thermal stability of the insulin zinc-hexamer but significantly improved the chemical stability at 37 degrees C. At low temperatures thiocyanate induced a conformational change in the insulin hexamer. NUV-CD thermal scans revealed that this effect decreased with temperature; when the thermal denaturation temperature was reached, the effect was eliminated.
  
  
  CONCLUSIONS
  Thermal stability can be used to predict the rate of Asn(B3) deamidation in human insulin. Chemical degradation processes that do not rely on the structural stability of the protein do not necessarily correlate to the thermal stability.
authors.affiliation: Department of Pharmaceutics and Analytical Chemistry, The Danish University of Pharmaceutical Sciences, 2100 Copenhagen, Denmark. ksan@novonordisk.com
authors.forename: Kasper
authors.initials: K
authors.lastname: Huus
authors.forename: Svend
authors.initials: S
authors.lastname: Havelund
authors.forename: Helle B
authors.initials: HB
authors.lastname: Olsen
authors.forename: Marco
authors.initials: M
authors.lastname: van de Weert
authors.forename: Sven
authors.initials: S
authors.lastname: Frokjaer
mesh_terms: D002152:Calorimetry, Differential Scanning; D004355:Drug Stability; D006358:Hot Temperature; D007328:Insulin / Q000737:chemistry* / Q000378:metabolism; D008024:Ligands; D013861:Thiocyanates / Q000494:pharmacology; D015032:Zinc / Q000737:chemistry / Q000494:pharmacology*
chemical_list: D007328:Insulin; D008024:Ligands; D013861:Thiocyanates; D015032:Zinc; C031760:thiocyanate