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.

Engineering novel VPAC2-selective agonists with improved stability and glucose-lowering activity in vivo.

A previously described VPAC2-selective agonist, BAY 55-9837 (peptide HSDAVFTDNYTRLRKQVAAKKYLQSIKNKRY), had several limitations with respect to its potential as an insulin secretagogue for the treatment of type 2 diabetes. These limitations were primarily poor stability in aqueous buffer and short duration of action in vivo. In this report, we describe a series of novel analogs of BAY 55-9837 that were designed around the likely degradation mechanisms and structure-activity relationship of this peptide with a view to overcoming its limitations. These analogs were tested for improved liquid stability and retention of VPAC2-selective binding and activation, as well as prolonged activity in vivo. Although several degradation mechanisms were possible based on the degradation pattern, it was determined that deamidation at the two asparagines (N9 and N28) was the major instability determinant. Changing these two asparagines to glutamines did not negatively affect VPAC2-selective binding and activation. The double glutamine mutein analog, BAY(Q9Q28), retained full VPAC2 activity and selectivity while displaying no significant degradation when stored at 40 degrees C for 4 weeks. This is in contrast to BAY 55-9837, which showed greater than 80% degradation when stored at 40 degrees C for 2 weeks. A cysteine was added to the C terminus of BAY(Q9Q28), followed by site-specific cysteine conjugation with a 22- or 43-kDa polyethylene glycol (PEG) to yield BAY(Q9Q28C32)PEG22 or BAY(Q9Q28C32)PEG43, respectively. These PEGylated peptides retain the ability to selectively bind and activate the VPAC2 receptor and have prolonged glucose-lowering activity in vivo.

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).