Solid-State Stability of Human Insulin I. Mechanism and the Effect of Water on the Kinetics of Degradation in Lyophiles from pH 2–5 Solutions

Abstract

Purpose. Previous studies have established that in aqueous solution at low pH human insulin decomposition proceeds through a cyclic anhydride intermediate leading to the formation of both deamidated and covalent dimer products. This study examines the mechanism and kinetics of insulin degradation in the amorphous solid state (lyophilized powders) as a function of water content over a similar pH range.

Methods. Solutions of 1.0 mg/mL insulin were adjusted to pH 2–5 using HC1, freeze-dried, then exposed to various relative humidities at 35°C. The water content within the powders was determined by Karl Fischer titration, and the concentrations of insulin and its degradation products were determined by HPLC. Degradation kinetics were determined by both the initial rates of product formation and insulin disappearance.

Results. Semi-logarithmic plots of insulin remaining in lyophilized powders versus time were non-linear, asymptotically approaching non-zero apparent plateau values, mathematically describable by a reversible, first-order kinetic model. The rate of degradation of insulin in the solid state was observed to increase with decreasing apparent pH (‘pH’) yielding, at any given water content, solid-state ‘pH’-rate profiles parallel to the solution pH-rate profile. This ‘pH’ dependence could be accounted for in terms of the fraction of the insulin A21 carboxyl in its neutral form, with an apparent pKa of ≈4, independent of water content. Aniline trapping studies established that the mechanism of degradation of human insulin in lyophilized powders between pH 3–5 and at 35°C involves rate-limiting intramolecular nucleophilic attack of the Asn_A21 C-terminal carboxylic acid onto the side-chain amide carbonyl to form a reactive cyclic anhydride intermediate, which further reacts with either water or an N-terminal primary amino group (e.g., Phe_B1, and Gly_Al) of another insulin molecule to generate either deamidated insulin (Asp_A21) or an amide-linked covalent dimer (e.g., [Asp_A21-Phe_B1] or [Asp_A21-Gly_A1]), respectively. The rate of insulin degradation in lyophilized powders at 35°C increases with water content at levels of hydration well below the suspected glass transition and approaches the rate in solution at or near the water content (20–50%) required to induce a glass transition.

Conclusions. The decomposition of human insulin in lyophilized powders between pH 3–5 is a water induced solid-state reaction accelerated by the plasticization effect of sorbed water. The formation of the cyclic anhydride intermediate at A21 occurs readily even in the glassy state, presumably due to the conformational flexibility of the A21 segment even under conditions in which the insulin molecules as a whole are largely immobile.