Determination of recombinant Interferon-alpha 2 in E. coil periplasmic extracts by reversed-phase high-performance liquid chromatography

Autor(es)

Dias, Paulo V. S. ^[1] ; Arthuso, Fernanda S. ^[1] ; Oliveira, Joao E. ^[1] ; Suzuki, Miriam F. ^[1] ; Sousa, Jose M. ^[1] ; Ribela, Maria Teresa C. P. ^[1] ; Bartolini, Paolo ^[1] ; Soares, Carlos R. J. ^[1]

Afiliação do(s) autor(es)

^[1] CNEN SP, IPEN, Biotechnol Ctr, Sao Paulo - Brazil

• Tipo de documento: Artigo Científico
• Fonte: JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES; v. 1072, p. 193-198, JAN 1 2018.
• Citações Web of Science: 1

Resumo

Reversed-phase high-performance liquid chromatography (RP-HPLC) has been used to analyze Interferon alpha-2 (IFN-alpha 2) as a pure protein or as a pharmaceutical preparation: a method for analyzing periplasmic IFN-a2 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-alpha 2a and IFN-alpha 2b 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 similar to 1%, intra-day and inter-day quantitative determinations presented relative standard deviations always <= 5%, while the working sensitivity was of similar to 0.3 mu g of IFN-alpha 2 (RSD = 5%). The method proved to be suitable for detecting and quantifying also glycosylated and oxidized forms and N-methionylated IFN-alpha 2 molecules, it was, however, not able to distinguish between IFN-alpha 2a and IFN-alpha 2b. This rapid methodology allows the application of RP-HPLC as a powerful tool to monitor the production yield and quality of IFN-alpha 2 in osmotic shock fluids, right after, or even during the fermentation process. (AU)

• Processo FAPESP: 12/24345-4 - Avaliação do potencial de antagonistas de prolactina para o tratamento da obesidade e do Diabetes mellitus 2
• Beneficiário: Carlos Roberto Jorge Soares
• Modalidade de apoio: Auxílio à Pesquisa - Regular
• Processo FAPESP: 15/10928-6 - Desenvolvimento de processo de fermentação em biorreator para produção de interferon ±-2a secretado no espaço periplásmico de Escherichia coli
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• Processo FAPESP: 15/26058-0 - Estudo do perfil de N-glicosilação e dos sítios de ocupação em várias preparações de tireotropina humana (hTSH) hipofisária e recombinante
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• Modalidade de apoio: Auxílio à Pesquisa - Regular