CONSIDERATION OF CONFORMATIONAL TRANSITIONS AND RACEMIZATION DURING PROCESS-DEVELOPMENT OF RECOMBINANT GLUCAGON-LIKE PEPTIDE-1

Physicochemical characterization of dry, excipient-free recombinant gl ucagon-like peptide-1 (rGLP-1) indicates the conformation and purity o f the bulk peptide is dependent on the purification scheme and the in- process storage and handling. The recombinant peptide preparations wer e highly pure and consistent with the expected primary structure and b ioactivity. However, variations in solubility were observed for prepar ations processed by different methods. The differences in solubility w ere shown to be due to conformational differences induced during purif ication. A processing scheme was identified to produce rGLP-1 in its n ative, soluble form, which exhibits FT-IR spectra, consistent with glu cagon-like peptide-1 synthesized by solid-state peptide synthesis. rGL P-1 was also found to undergo base-catalyzed amino acid racemization. Racemization can impact the yield and impurity profile of bulk rGLP-1, since the peptide is exposed to alkali during its purification. A com bination of enzymatic digestion using leucine aminopeptidase (which cl eaves N-terminal L-amino acids much greater than D-amino acids) and ma trix-assisted laser desorption ionization mass spectrometry was used t o identify racemization as a degradation pathway. The racemization rat e increased with increasing temperature and base concentration, but de creased with increasing peptide concentration. The racemized peptides were shown to be less bioactive than rGLP-1.