Analysis and characterization of aggregation of a therapeutic Fc-fusion protein
| Autor: | Szilan Fodor, Izydor Apostol, Tian Wang, Suminda Hapuarachchi, Xinzhao Grace Jiang, Kenneth Chen, Gang Huang |
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| Rok vydání: | 2013 |
| Předmět: |
Recombinant Fusion Proteins
Clinical Biochemistry Pharmaceutical Science Salt (chemistry) Peptide Protein aggregation medicine.disease_cause Mass Spectrometry Analytical Chemistry Freezing Drug Discovery Protein purification Escherichia coli medicine Moiety Sulfhydryl Compounds Spectroscopy Inclusion Bodies chemistry.chemical_classification biology Protein Stability Temperature Hydrogen-Ion Concentration Immunoglobulin Fc Fragments Fc fusion chemistry Biochemistry biology.protein Salts Protein G Chromatography Liquid |
| Zdroj: | Journal of Pharmaceutical and Biomedical Analysis. 72:59-64 |
| ISSN: | 0731-7085 |
| Popis: | Protein aggregation was observed in a purification intermediate of a therapeutic Fc-fusion protein stored at -30 °C, even though the protein was stable at 4 and -80 °C. The protein was expressed in Escherichia coli as an inclusion body, refolded, and purified using chromatography columns. To study the nature of this aggregation, a series of experiments were conducted to investigate factors that contributed to the protein instability during freezing. We found that the presence of free thiols in the protein is the intrinsic cause. The free thiol cross-linking sites were determined to be at the peptide moiety of the Fc-fusion protein using LC-MS. Partially frozen accompanied by the elevated pH and increased salt and protein concentrations were identified as extrinsic factors that facilitated the aggregation. These results provided important insights into purification process improvement and solution storage of this Fc-fusion protein. |
| Databáze: | OpenAIRE |
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