SOLID-STATE STABILITY OF HUMAN INSULIN .1. MECHANISM AND THE EFFECT OF WATER ON THE KINETICS OF DEGRADATION IN LYOPHILES FROM PH 2-5 SOLUTIONS

Purpose. Previous studies have established that in aqueous solution at low pH human insulin decomposition proceeds through a cyclic anhydrid e intermediate leading to the formation of both deamidated and covalen t dimer products. This study examines the mechanism and kinetics of in sulin degradation in the amorphous solid state (lyophilized powders) a s a function of water content over a similar pH range. Methods. Soluti ons of 1.0 mg/mL insulin were adjusted to pH 2-5 using HCl, freeze-dri ed, then exposed to various relative humidities at 35 degrees C. The w ater content within the powders was determined by Karl Fischer titrati on, and the concentrations of insulin and its degradation products wer e 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 v ersus time were non-linear, asymptotically approaching non-zero appare nt plateau values, mathematically describable by a reversible, first-o rder kinetic model. The rate of degradation of insulin in the solid st ate was observed to increase with decreasing apparent pH ('pH') yieldi ng, at any given water content, solid-state 'pH'-rate profiles paralle l to the solution pH-rate profile. This 'pH' dependence could be accou nted for in terms of the fraction of the insulin A21 carboxyl in its n eutral form, with an apparent pKa of approximate to 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 degrees C involves rate-limiting intramolecular nucleophili c attack of the Asn(A21) C-terminal carboxylic acid onto the side-chai n amide carbonyl to form a reactive cyclic anhydride intermediate, whi ch further reacts with either water or an N-terminal primary amino gro up (e.g., Phe(B1) and Gly(A1)) of another insulin molecule to generate either deamidated insulin (AsPA21) or an amide-linked covalent dimer (e.g., [AsPA21-Phe(B1)] Or [Asp(A21)-Gly(A1)] respectively. The rate o f insulin degradation in lyophilized powders at 35 degrees C increases with water content at levels of hydration well below the suspected gl ass transition and approaches the rate in solution at or near the wate r 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 plastic ization effect of sorbed water. The formation of the cyclic anhydride intermediate at A21 occurs readily even in the glassy state, presumabl y due to the conformational flexibility of the A21 segment even under conditions in which the insulin molecules as a whole are largely immob ile.