Determination of recombinant Interferon-α2 in E. coli periplasmic extracts by reversed-phase high-performance liquid chromatography
| Autor: | Fernanda dos Santos Arthuso, Paolo Bartolini, Carlos R. J. Soares, Paulo V.S. Dias, Maria Teresa C.P. Ribela, Miriam F. Suzuki, João E. Oliveira, José M. de Sousa |
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| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
Glycosylation Osmotic shock Clinical Biochemistry medicine.disease_cause Sensitivity and Specificity 01 natural sciences Biochemistry High-performance liquid chromatography Analytical Chemistry 03 medical and health sciences Phase (matter) Escherichia coli medicine Humans Chromatography High Pressure Liquid Chromatography Reverse-Phase Chromatography Chemistry 010401 analytical chemistry Interferon-alpha Reproducibility of Results Cell Biology General Medicine Periplasmic space Recombinant Proteins 0104 chemical sciences 030104 developmental biology Yield (chemistry) Periplasm Linear Models Fermentation Oxidation-Reduction Quantitative analysis (chemistry) |
| Zdroj: | Journal of Chromatography B. 1072:193-198 |
| ISSN: | 1570-0232 |
| Popis: | Reversed-phase high-performance liquid chromatography (RP-HPLC) has been used to analyze Interferon α-2 (IFN-α2) as a pure protein or as a pharmaceutical preparation: a method for analyzing periplasmic IFN-α2 directly in osmotic shock extract has, however, never been reported. This work describes an RP-HPLC methodology for the qualitative and quantitative analysis of human IFN-α2a and IFN-α2b directly in bacterial periplasmic extracts or in purified preparations. The analytical method has been set up and validated for accuracy, precision, linearity, sensitivity and specificity. A recovery test indicated an average bias of ∼1%, intra-day and inter-day quantitative determinations presented relative standard deviations always≤5%, while the working sensitivity was of ∼0.3μg of IFN-α2 (RSD=5%). The method proved to be suitable for detecting and quantifying also glycosylated and oxidized forms and N-methionylated IFN-α2 molecules, it was, however, not able to distinguish between IFN-α2a and IFN-α2b. This rapid methodology allows the application of RP-HPLC as a powerful tool to monitor the production yield and quality of IFN-α2 in osmotic shock fluids, right after, or even during the fermentation process. |
| Databáze: | OpenAIRE |
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