Consideration of conformational transitions and racemization during process development of recombinant glucagon-like peptide-1.

Physicochemical characterization of dry, excipient-free recombinant glucagon-like peptide-1 (rGLP-1) indicates the conformation and purity of the bulk peptide is dependent on the purification scheme and the in-process storage and handling. The recombinant peptide preparations were highly pure and consistent with the expected primary structure and bioactivity. However, variations in solubility were observed for preparations processed by different methods. The differences in solubility were shown to be due to conformational differences induced during purification. A processing scheme was identified to produce rGLP-1 in its native, soluble form, which exhibits FT-IR spectra, consistent with glucagon-like peptide-1 synthesized by solid-state peptide synthesis. rGLP-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 combination of enzymatic digestion using leucine aminopeptidase (which cleaves N-terminal L-amino acids >> D-amino acids) and matrix-assisted laser desorption ionization mass spectrometry was used to identify racemization as a degradation pathway. The racemization rate increased with increasing temperature and base concentration, but decreased with increasing peptide concentration. The racemized peptides were shown to be less bioactive than rGLP-1.