Dimerization of a PACAP peptide analogue in DMSO via asparagine and aspartic acid residues.

To optimize the stability of a peptide development candidate for the treatment of type II diabetes, formulation studies were initiated in organic solvents and compared to results obtained in aqueous solutions. Stability was assessed by reversed phase liquid chromatography (RPLC) and electrospray ionization mass spectrometry (ESI-MS). Previous studies had shown deamidation and hydrolysis to be the primary mechanisms of degradation in aqueous formulations. Surprisingly, the use of an organic solvent did not decrease the rate of degradation and, as presented here, produced degradation products including dimers. We propose here that deamidation can readily occur in polar anhydrous organic solvents such as DMSO and that the dimer forms through intermolecular nucleophilic attack of an amino acid side chain on a stabilized cyclic imide intermediate.

High-pressure refolding of bikunin: efficacy and thermodynamics.

Bikunin is a glycosylated protein that aggregates extensively during mammalian cell culture, resulting in loss of activity, loss of native secondary structure, and the formation of nonnative disulfide bonds. We investigated the use of high hydrostatic pressure (1000-3000 bar) for the refolding of bikunin aggregates. The refolding yield obtained with pressure-modulated refolding at 2000 bar was 70 (+/-5%) by reverse-phase chromatography (RP-HPLC), significantly higher than the value of 55 (+/-6%) (RP-HPLC) obtained with traditional guanidine HCl "dilution-refolding." In addition, we determined the thermodynamics of pressure-modulated refolding. The change in volume for the transition of aggregate to monomer DeltaV(refolding) was calculated to be -28 (+/-5) mL/mole. Refolding was accompanied by a loss of hydrophobic exposure, resulting in a positive contribution to the DeltaV(refolding). These findings suggest that the disruption of electro-static interactions or the differences in size of solvent-free cavities between the aggregate and the monomer are the prevailing contributions to the negative DeltaV(refolding).