SOLID-STATE STABILITY OF HUMAN INSULIN .2. EFFECT OF WATER ON REACTIVE INTERMEDIATE PARTITIONING IN LYOPHILES FROM PH 2-5 SOLUTIONS - STABILIZATION AGAINST COVALENT DIMER FORMATION

Previous studies have established that at low pH human insulin decompo sition proceeds through a two-step mechanism involving rate-limiting i ntramolecular formation of a cyclic anhydride intermediate at the C-te rminal Asn(A21) followed by intermediate partitioning to various produ cts, most notably desamido insulin and covalent dimers, in both aqueou s solution and in the amorphous (lyophilized) solid state. This study examines the product distribution resulting from insulin degradation i n lyophilized powders as a function of water content and the phase beh avior of the solid (glassy versus rubbery) between pH 3 and 5. in amor phous solids at low water content (glassy state), the cyclic anhydride intermediate of insulin reacts predominantly with water to form deami dated insulin, whereas the intermolecular reaction with another insuli n molecule to form a covalent dimer accounts for less than or equal to 15% of the total degradation. Increasing water content reduces the gl ass transition temperature of insulin to <35 degrees C, and covalent d imer formation becomes increasingly favored relative to deamidation. A n increase in solid-state pH also favors dimerization as deprotonation of the terminal amino groups of insulin renders them more nucleophili c. Covalent dimerization was almost totally suppressed by incorporatio n into a glassy matrix of trehalose, which both minimizes molecular mo bility and physically separates the insulin molecules. The kinetics an d product distribution of human insulin in lyophilized powders between pH 3 and 5 illustrate the differential sensitivities of various solid -state reaction types to the effects of water activity and solid-phase behavior. The intramolecular cyclization at the Asn(A21) position req uires only short-range conformational flexibility and thus is only mod estly restricted even in the grassy state. On the other hand, the comp eting bimolecular reactions involving either water or another molecule of insulin combining with the intermediate anhydride are dependent on molecular mobility of the reactants, in accord with predictions of fr ee volume theory. In the glassy state, deamidation (reaction with wate r) is favored because of the restricted molecular mobility of proteins in rigid matrices. Increasing plasticization with increasing water co ntent favors covalent aggregate formation because of the higher depend ence of protein mobility on free volume within the solid matrix.