Degradation of growth hormone releasing factor analogs in neutral aqueous solution is related to deamidation of asparagine residues

The incubation of a solution of the human growth hormone releasing factor analog, Leu27 hGRF(1-32)NH2 at pH 7.4 and 37 degrees, resulted in extensive degradation of the sample. The major degradation products were identified as the peptides beta-Asp8, Leu27 hGRF(1-32)NH2 and alpha-Asp8, Leu27 hGRF(1-32)NH2, produced by deamidation of the Asn8 residue. When tested as growth hormone (GH) secretagogues in cultured bovine anterior pituitary cells, beta-Asp8, Leu27 hGRF(1-32)NH2 was estimated to be 400-500 times less potent than the parent Asn8 peptide, while alpha-Asp8, Leu27 hGRF(1-32)NH2 was calculated to be 25 times less potent than the parent Asn8 peptide. Three additional analogs of Leu27 hGRF(1-32)NH2 containing either Ser or Asn at positions 8 and 28 were prepared and evaluated for their GH releasing activity and stability in aqueous phosphate buffer (pH 7.4, 37 degrees). Based on disappearance kinetics, Leu27 hGRF(1-32)NH2 had a half-life of 202 h while the other analogs had the following half-lives: Leu27, Asn28 hGRF(1-32)NH2 (150 h); Ser8, Leu27, Asn28 hGRF(1-32)NH2 (746 h); and Ser8, Leu27 hGRF(1-32)NH2 (1550 h). After 14 days, incubated samples of the Asn8 analogs lost GH releasing potency, while the Ser8 analogs retained full potency. The potential for loss of biological activity brought about by deamidation of other engineered peptides and proteins should be considered in their design.