Determination of recombinant Interferon-α2 in E. coli periplasmic extracts by reversed-phase high-performance liquid chromatography.
J Chromatogr B Analyt Technol Biomed Life Sci
; 1072: 193-198, 2018 Jan 01.
Article
em En
| MEDLINE
| ID: mdl-29179059
ABSTRACT
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 â¼1%, intra-day and inter-day quantitative determinations presented relative standard deviations always≤5%, while the working sensitivity was of â¼0.3µg 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.
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Base de dados:
MEDLINE
Assunto principal:
Proteínas Recombinantes
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Cromatografia Líquida de Alta Pressão
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Interferon-alfa
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Escherichia coli
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Cromatografia de Fase Reversa
Idioma:
En
Ano de publicação:
2018
Tipo de documento:
Article