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Chemical and Thermal Stability of Insulin: Effects of Zinc and Ligand Binding to the Insulin Zinc-Hexamer

Pharmaceutical Research volume 23, pages 2611–2620 (2006)Cite this article

Abstract

Purpose

To study the correlation between the thermal and chemical stability of insulin formulations with various insulin hexamer ligands.

Materials and 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 AsnB3 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°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 AsnB3 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.

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Abbreviations

CD:

circular dichroism

C p :

excess heat capacity

DSC:

differential scanning calorimetry

MALDI-TOF:

matrix-assisted laser desorption ionization mass spectrometry

PABA:

para-aminobenzoic acid

RP-HPLC:

reversed-phase high-performance liquid chromatography

SEC:

size-exclusion chromatography

T m :

transition midpoint

4H3N:

4-hydroxy-3-nitrobenzoic acid

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Acknowledgment

We thank Frantisek Hubálek for assistance with mass spectrometry measurements and valuable discussions of the results.

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Affiliations

  1. Department of Pharmaceutics and Analytical Chemistry, The Danish University of Pharmaceutical Sciences, 2100, Copenhagen, Denmark

    Kasper Huus, Marco van de Weert & Sven Frokjaer

  2. Diabetes Protein Engineering, Novo Nordisk A/S, 2880, Bagsvaerd, Denmark

    Kasper Huus, Svend Havelund & Helle B. Olsen

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  1. Kasper Huus
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  2. Svend Havelund
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  4. Marco van de Weert
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Correspondence to Kasper Huus.

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Huus, K., Havelund, S., Olsen, H.B. et al. Chemical and Thermal Stability of Insulin: Effects of Zinc and Ligand Binding to the Insulin Zinc-Hexamer. Pharm Res 23, 2611–2620 (2006). https://doi.org/10.1007/s11095-006-9098-y

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  • DOI: https://doi.org/10.1007/s11095-006-9098-y

Key words

  • chemical stability
  • deamidation
  • differential scanning calorimetry
  • dimerization
  • insulin
  • protein stability
  • thermal stability