Insulin aspart (AspB28 human insulin) derivatives formed in pharmaceutical solutions

Abstract

Purpose: To isolate and identify the main insulin aspart (AspB28 human insulin) derivatives formed in pharmaceuticals (pH 7.4 at 5 degrees C), to estimate rates of formation, and to determine their biologic potencies.

Methods: Insulin aspart derivatives have been isolated by reversed-phase high-performance liquid chromatography (RP-HPLC), and identified by RP-HPLC, peptide mapping, amino acid analysis, mass spectrometry. and N-terminal amino acid sequence analysis.

Results: The main derivatives formed were isoAspB28, isoAspB3, AspB3, and desPheB1-N-oxalyl-ValB2 insulin aspart. At 5 degrees C, the rate constants were 0.00028/month for isoAspB28 and isoAspB3, 0.00024/month for AspB3, and 0.00013/month for desPheB1-N-oxalyl-ValB2 derivatives of insulin aspart. Unexpectedly, the rate of isomerization of B28 was high compared to the rate of B3 deamidation at both 5 degrees C and 45 degrees C. The N-terminal and especially the C-terminal of the B-chain are highly flexible, which may explain the high rate of isoAspB28 formation and that deamidation of AsnB3 occurs. All the derivatives had full in vivo biologic potencies.

Conclusion: Except for isoAspB28 insulin aspart, the main derivatives formed in pharmaceuticals of insulin aspart and human insulin at pH 7.4 are similar. They are all fully active in vivo. In proteins, flexibility of the polypeptide chain seems more important than sequence in the formation of succinimides.