Artigo de peri??dico
Determination of recombinant Interferon-??2 in E. coli periplasmic extracts by reversed-phase high-performance liquid chromatography
Registro en:
1570-0232
1072
10.1016/j.jchromb.2017.11.023
66.188
56.25
Autor
DIAS, PAULO V.S.
ARTHUSO, FERNANDA S.
OLIVEIRA, JOAO E.
SUZUKI, MIRIAM F.
SOUSA, JOSE M.
RIBELA, MARIA T.C.P.
BARTOLINI, PAOLO
SOARES, CARLOS R.J.
Resumen
Reversed-phase high-performance liquid chromatography (RP-HPLC) has been used to analyze Interferon ??-2
(IFN-??2) as a pure protein or as a pharmaceutical preparation: a method for analyzing periplasmic IFN-??2
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-??2a and IFN-??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 ???1%, intra-day and
inter-day quantitative determinations presented relative standard deviations always ??? 5%, while the working
sensitivity was of ???0.3 ??g of IFN-??2 (RSD =5%). The method proved to be suitable for detecting and quantifying
also glycosylated and oxidized forms and N-methionylated IFN-??2 molecules, it was, however, not able to
distinguish between IFN-??2a and IFN-??2b. This rapid methodology allows the application of RP-HPLC as a
powerful tool to monitor the production yield and quality of IFN-??2 in osmotic shock fluids, right after, or even
during the fermentation process. Funda????o de Amparo ?? Pesquisa do Estado de S??o Paulo (FAPESP) Conselho Nacional de Desenvolvimento Cient??fico e Tecnol??gico (CNPq) FAPESP: 12/24345-4; 15/26058-0; 15/10928-6 CNPq: PQ 300473/2009-5; PQ 305756/ 2014-1; DT 310512/2010-7; 479455/2011-2