Improved insulin stability through amino acid substitution

Insulin analogs designed to decrease self-association and increaseabsorption rates from subcutaneous tissue were found to havealtered stability. Replacement of H^B10 with aspartic acid increasedstability while substitutions at B²⁸ and/or B²⁹ were eithercomparable to insulin or had decreased stability. The principalchemical degradation product of accelerated storage conditionswas a disulfidelinked multimer that was formed through a disulfideinterchange reaction which resulted from ß-eliminationof the disulfides. The maintenance of the native state of insulinwas shown to be important in protecting the disulfides fromreduction by dithiothreitol and implicitly from the disulfideinter change reaction that occurs during storage. To understandhow these amino acid changes alter chemical stability, the intramolecularconformational equilibria of each analog was assessed by equilibriumdenaturation. The Gibbs free energy of unfolding was comparedwith the chemical stability during storage for over 20 analogs.A significant positive correla tion (R²=0.8 and P < 0.0005)exists between the conformational stability and chemical stabilityof these analogs, indicating that the chemical stability ofinsulin's disulfides is under the thermodynamic control of theconformational equilibria.