Stability of human insulin: effect of water content and added excipients on the degradation of lyophilized insulin at acidic pH

The use of proteins as pharmaceutically active drugs has increased because of advances in biotechnology. Since proteins cannot be orally administered they are usually injected. However, proteins can be unstable in solution and are sometimes marketed as solid. This study examines the effect of water, 'pH', and added excipients on the solid-state chemical stability of a model protein, human insulin. Solid-state amorphous proteins undergo the same type of chemical reactions as n solution. In both solution and solid-state between pH 3-5 the mechanism of degradation of human insulin, without added excipients, occurs at an asparagine C-terminal carboxylic acid, and involves unimolecular rate limiting formation of a reactive cyclic anhydride intermediate, which further reacts bimolecularly with either water or an N-terminal primary amine of insulin to generate the deamidated or an amide linked covalent dimer, respectively. The 'pH' dependent solid-state reaction rate is completely accounted for by the ionization of the C-terminal carboxylic acid, without invoking solubilization in liquid microenvironments. Intramolecular cyclization involving functional groups in close proximity is difficult to suppress in the solid-state. The overall rate of insulin solid-state degradation is governed by cyclic anhydride formation, and is reduced at most 10-fold at low water contents. Sorbed water accelerates protein unimolecular reactions by acting as a plasticizer increasing conformational flexibility, and providing a medium for proton transfer. Molecular mobility, required for bimolecular reactions, is dependent on the size of the reactant, water content and the state of the amorphous solid. At low water contents, solid-state bimolecular reactions involving water dominate over those involving larger molecules. The solid-state deamidation of insulin is favored over covalent dimerization at low water contents due to the higher mobility of water, but dimerization becomes increasingly prevalent at higher water contents. In a glassy solid, reactant mobility are reduced, but dramatically increase upon a water induced glass transition to a rubbery" collapsed solid. At pH 4.0 insulin reacts with reducing sugars via the Maillard reaction, and is very slow in a solid glassy matrix but dramatically increases upon a glass transition.