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(Reference retrieved automatically from Web of Science through information on FAPESP grant and its corresponding number as mentioned in the publication by the authors.)

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

Full text
Author(s):
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]
Total Authors: 8
Affiliation:
[1] CNEN SP, IPEN, Biotechnol Ctr, Sao Paulo - Brazil
Total Affiliations: 1
Document type: Journal article
Source: JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES; v. 1072, p. 193-198, JAN 1 2018.
Web of Science Citations: 1
Abstract

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)

FAPESP's process: 12/24345-4 - Evaluation of the potential of prolactin antagonists to the treatment of obesity and Diabetes mellitus 2
Grantee:Carlos Roberto Jorge Soares
Support type: Regular Research Grants
FAPESP's process: 15/10928-6 - Development of fermentation in a bioreactor for the production of interferon ±-2a secreted into the periplasmic space of Escherichia coli
Grantee:Paulo Victor Sarmento Dias
Support type: Scholarships in Brazil - Master
FAPESP's process: 15/26058-0 - Glycoprofiling and N-glycosylation site occupancy of different human thyrotropin (hTSH) preparations of pituitary or recombinant origin
Grantee:Carlos Roberto Jorge Soares
Support type: Regular Research Grants