CHEMICAL-STABILITY OF INSULIN .4. MECHANISMS AND KINETICS OF CHEMICAL-TRANSFORMATIONS IN PHARMACEUTICAL FORMULATION

Insulin decomposes by a multitude of chemical reactions [1-3]. It deamidates at two different residues by entirely different mechanisms. In acid, deamidation at Asn(A21) is intramolecularly catalyzed by the protonated C-terminal, whereas above pH 6 an intermediate imide formation at residue Asn(B3) leads to isoAsp and Asp derivatives. The imide formation requires a large rotation around the alpha-carbon/peptide carbonyl carbon bond at B3, corresponding to a 10 angstrom movement of the B-chain N-terminal. The main determinant for the rate of B3 deamidation, as well as for the ratio between the two products formed, is the local conformational structure, which is highly influenced by various excipients and the physical state of the insulin. An amazing thermolysin-like, autoproteolytic cleavage of the A-chain takes place in rhombohedral insulin crystals, mediated by a concerted catalytic action by several, inter-hexameric functional groups and Zn2+. Intermolecular, covalent cross-linking of insulin molecules occurs via several mechanisms. The most prominent type of mechanism is aminolysis by the N-terminals, leading to isopeptide linkages with the A-chain side-chain amides of residues Gln(A15), Asn(A18) and Asn(A21). The same type of reaction also leads to covalent cross-linking of the N-terminal in protamine with insulin. Disulfide exchange reactions, initiated by lysis of the A7-B7 disulfide bridge, lead mainly to formation of covalent oligo- and polymers. Activation energy (E(a)) for the neutral deamidation and the aminolysis reactions was found to be 80 and 119 KJ/mol, respectively.