RESUMO
We report a case study of an IgG1 with a unique basic charge variant profile caused by C-terminal proline amidation on either one or two heavy chains. The proline amidation was sensitive to copper ion concentration in the production media during cell culture: the higher the Cu ( 2+) ion concentration, the higher the level of proline amidation detected. This conclusion was supported by the analysis of samples that revealed direct correlation between the proline amidation level observed from peptide maps and the level of basic peaks measured by imaged capillary isoelectric focusing and a pH gradient ion-exchange chromatography method. The importance of these observations to therapeutic antibody production is discussed.
Assuntos
Amidas/metabolismo , Anticorpos Monoclonais/química , Cobre/farmacologia , Imunoglobulina G/química , Amidas/química , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/metabolismo , Células CHO , Cromatografia por Troca Iônica , Cobre/análise , Cricetinae , Meios de Cultura/química , Humanos , Imunoglobulina G/genética , Imunoglobulina G/metabolismo , Focalização Isoelétrica , Mapeamento de Peptídeos , Prolina/química , Força Próton-MotrizRESUMO
PURPOSE: This work examines the cause of aggregation of an Fc-fusion protein formulated in sorbitol upon frozen storage for extended periods of time at -30 degrees C. MATERIALS AND METHODS: We designed sub-ambient differential scanning calorimetry (DSC) experiments to capture the effects of long-term frozen storage. The physical stability of formulation samples was monitored by size exclusion high performance liquid chromatography (SE-HPLC). RESULTS: DSC analysis of non-frozen samples shows the expected glass transitions (Tg') at -45 degrees C for samples in sorbitol and at -32 degrees C in sucrose. In time course studies where sorbitol formulations were stored at -30 degrees C and analyzed by DSC without thawing, two endothermic transitions were observed: a melting endotherm at -20 degrees C dissipated over time, and a second endotherm at -8 degrees C was seen after approximately 2 weeks and persisted in all later time points. Protein aggregation was only seen in the samples formulated in sorbitol and stored at -30 degrees C, correlating aggregation with the aforementioned melts. CONCLUSIONS: The observed melts are characteristic of crystalline substances and suggest that the sorbitol crystallizes over time. During freezing, the excipient must remain in the same phase as the protein to ensure protein stability. By crystallizing, the sorbitol is phase-separated from the protein, which leads to protein aggregation.