Investigation of the Physico-Chemical Properties that Enable Co-Formulation of Basal Insulin Degludec with Fast-Acting Insulin Aspart

Abstract

Purpose: To study the self-association states of insulin degludec and insulin aspart alone and combined in pharmaceutical formulation and under conditions simulating the subcutaneous depot.

Methods: Formulations were made of 0.6 mM degludec at 3 and 5 Zn/6 insulin monomers, and 0.6 mM aspart (2 Zn/6 insulin monomers). Self-association was assessed using size-exclusion chromatography (SEC) monitored by UV and orthogonal reverse-phase chromatography.

Results: Simulating pharmaceutical formulation, degludec eluted as dihexamers, whereas aspart eluted as hexamers and monomers. Combining degludec at low zinc with aspart increased dihexamer content, indicating hybrid hexamer formation. At high zinc concentration, however, there was no evidence of this. Simulating the subcutaneous depot by removing preservative, degludec eluted as multihexamers and aspart as monomers. Aspart was incorporated into the multihexamer structures when combined with degludec at low zinc, but there was no such interaction with high-zinc degludec. SEC using progressively diluted concentrations of phenol and m-cresol showed that dissociation of aspart into monomers occurs before the formation of degludec multihexamers.

Conclusion: Insulins degludec and aspart can be combined without forming hybrid hexamers, but this combinability is dependent on zinc and preservative concentration, and requires that degludec is fully dihexameric before addition of aspart.

Fig. 1

SEC simulating pharmaceutical formulation (method 1). Elution curves comparing insulin analogues eluted alone or in combination, with insulin degludec formulated at two different zinc (Zn) concentrations (n zinc ions per 6 insulin monomers). When mixed together in a 70:30 ratio, the proportion of insulin eluting as dihexamers is increased beyond 70% at the lower (b), but not the higher (d), zinc concentration. AU, absorbance unit; SEC, size-exclusion chromatography.

Fig. 2

SEC simulating depot environment (method 2). Elution curves comparing insulin analogs eluted alone or in combination with insulin degludec formulated at two different zinc (Zn) concentrations (n zinc ions per 6 insulin monomers). When mixed together in a 70:30 ratio, the proportion of insulin aspart eluting as monomers is reduced at the lower zinc concentration (b) due to incorporation into multihexamers. At the higher zinc concentration (d), insulin aspart elutes almost entirely in the monomeric fraction, and insulin degludec elutes in the multihexameric fraction, which shows slight leftward shift, indicating the formation of larger multihexamers. AU, absorbance unit; SEC, size-exclusion chromatography.

Fig. 3

SEC simulating depot environment (method 3), with varied pharmaceutical preservative concentrations (phenol and m-cresol). The percent of insulin aspart eluting as hexamers declines quickly as preservative concentration decreases, while insulin degludec becomes multihexameric only at very low phenol and m-cresol concentration. SEC, size-exclusion chromatography; Zn, zinc.

Fig. 4

After injection, insulin degludec and insulin aspart undergo different changes. Insulin aspart hexamers rapidly dissociate into monomers that are readily absorbed into the circulation over a short time. Insulin degludec dihexamers associate to form soluble multihexamers from which monomers are slowly released to be absorbed into the circulation over a prolonged time. IAsp, insulin aspart; IDeg, insulin degludec.