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Received November 20, 2001; accepted February 4, 2002
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 reversedphase 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 Bchain 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.
KEY WORDS: AspB28 human insulin derivatives; deamidation; isomerization; isoAspB28 human insulin; AspB28, desPheB1-N-oxalylValB2 human insulin.
ABBREVIATIONS: insulin aspart, AspB28 human insulin; MW, molecular weight; RP-HPLC, reversed-phase high-performance liquid chromatography; AspB3 I, V8 fragment I of AspB3 insulin aspart; MW, molecular weight.
INTRODUCTION
The aim of treatment diabetes mellitus is to achieve normoglycemia 24 h per day and, thereby, to prevent or delay the development of late chronic complications (1,2). This is difficult to achieve with human insulin preparations, especially at meal times when insulin absorption is too slow due to the natural tendency of human insulin to self-associate to hexamers. In an attempt to make the absorption rate profile of injected insulin similar to the normal postprandial insulin response, fast-acting monomeric insulin analogues have been developed. Two are available for patients: AspB28 human insulin, called insulin aspart and marketed as NovoRapid- (Novo Nordisk, Bagsvaerd, Denmark) (3,4); and lyspro insulin (LysB28, ProB29), called lispro insulin and marketed as Humalog- (Eli Lilly and Company, Indianapolis, Indiana)...