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.

Aqueous stability of recombinant human thrombopoietin as a function of processing schemes.

Preformulation studies conducted with recombinant human thrombopoietin (rhTPO), a 332 amino acid glycoprotein which stimulates platelet production, show distinctions in degradation profiles as a function of processing schemes. The stability-limiting degradation pathways change as a function of purification stage and method and are dependent upon the presence of contaminating protease. The stability-limiting degradation pathway of affinity-purified and in-process rhTPO preparations is primarily attributed to proteolysis initiated by a protease present as a fermentation contaminant. The proteolysis increases with increasing pH as a function of temperature. The degradation profiles for these preparations show that bioactivity initially increases and then decreases with increasing pH as a function of temperature. This is consistent with proteolysis to active forms which ultimately undergo degradation to less active forms. Similar studies conducted with rhTPO preparations purified by a combination of more conventional chromatographic steps show different stability-limiting degradation pathways and a different pH-stability profile when compared to affinity purified or in-process preparations. In this case, degradation is accompanied by decreases in activity under all conditions, consistent with the conversion to less active forms. These results illustrate the importance of preformulation and stability characterization of protein pharmaceuticals in support of both process and formulation development. Issues related to storage and handling of inprocess preparations differ from those with formulated product since the stability-limiting degradation pathways change as a function of purification stage.