JOAO EZEQUIEL DE OLIVEIRA

JOAO EZEQUIEL DE OLIVEIRA

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Determination of recombinant Interferon-α2 in E. coli periplasmic extracts by reversed-phase high-performance liquid chromatography
2018 - 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.
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 ∼0.3 μg 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.
Biological activity of different batches of equine chorionic gonadotropin as determined by reversed-phase high-performance liquid chromatography and in vivo assay
2017 - ALVAREZ, RAFAEL H.; NATAL, FABIO L.N.; ALMEIDA, BEATRIZ E.; OLIVEIRA, JOAO E.; MELO, ALFREDO J.F.; RIBELA, MARIA T.C.; BARTOLINI, PAOLO
Aims: To evaluate the physicochemical profile of commercial batches of eCG, in order to find if differences can be related to their biological activity. Study Design: Commercial eCG was analyzed by reversed-phase high-performance liquid chromatography (RP-HPLC) and in vivo bioassay. Place and Duration of Study: Department of Biotechnology (IPEN-CNEN) and Animal antibody production Laboratory (Animal Science Institute), between June 2013 and April 2014. Methodology: Two commercial eCG batches for veterinary use (I and II) and an eCG official International Standard from the World Health Organization (WHO) were analyzed by RP-HPLC. Additionally, two experiments were designed to validate the biological activity. In experiment 1, groups of prepubertal 21–25 day old Wistar female rats (n = 6/group) received the equivalent to 0 UI (saline) and 10 IU of eCG of each one of these preparations. Autopsy was performed 48 h later and ovaries were removed and weighed. The experiment 2 was designed to determine whether increasing the dose of less active eCG batches could increase the ovarian response. Therefore, groups of prepubertal rats (n = 6/group) were treated with 10 and 30 IU eCG from batch II, while eCG from WHO (10 IU) and saline were control. The evaluation of ovarian response was done similar to Experiment 1. Differences among treatments were analyzed by one-way ANOVA. Results: Results of RP-HPLC showed differences in the main tR peak profile (tR 26.7) of the standard WHO compared with eCG batches I and II. In experiment 1, the average ovarian weight of rats treated with eCG from WHO (60.0 ± 12.1 mg) was higher (P < .01) than saline (23.1 ± 1.6 mg) and batches I (37.6 ± 1.4 mg) and II (31.0 ± 4.3 mg). In experiment 2, the ovarian weight of rats treated with 30 IU of eCG of batch II (45.7 ± 4.1 mg) was higher (P < .01) than saline (32.6 ± 1.4 mg) and significantly lower (P = .05) than 10 UI of the standard WHO (63.3 ± 8.1 mg). Conclusion: The low ovarian response to eCG treatments can be related to differences in the physicochemical profile of eCG batches and RP-HPLC is a fast and reliable tool for detecting these differences.
Effect of cold stress on physicochemical characteristics and biological activity of equine chorionic gonadotropin
2016 - ALVAREZ, R.H.; NATAL, F.L.N.; ALMEIDA, B.E.; OLIVEIRA, J.E.; BARTOLINI, P.; MELO, A.J.F.; DUARTE, K.M.R.; RIBELA, M.T.C.
The purpose of this study was to evaluate if freezing-thawing and cooling processes affect the structural properties and biological activity of commercial equine chorionic gonadotropin (eCG). First, the structure profile of diluted eCG underwent none, one or three cycles of freezing-thawing was analysed by reverse phase high-performance liquid chromatography (RP-HPLC). In a second experiment, groups of prepuberal rats were treated with sterile water for injection USP or eCG that underwent none, one or three cycles of freezing-thawing to assess the increase of ovarian weigh. Finally, groups of prepubertal gilts were treated with diluted eCG immediately after reconstitution (T1), after refrigeration for six months (T2) and after freezing and subsequently thawing for one (T3) or three (T4) cycles. The control group (T5) received sterile water for injection USP without eCG. Ovulation was induced with human chorionic gonadotropin (hCG), administered 72 h after the eCG. Gilts were slaughtered five days after the hCG injection and ovaries were recovered and analysed for the presence of corpora lutea. Data were analysed by ANOVA and Fisher’s exact tests. In the analyses by RP-HPLC, the retention times of cold stressed eCG were similar to unstressed control. The mean ovarian weight of rats treated with cold stressed and unstressed eCG was statistically higher than water control (P < 0.05). Lastly, significantly more gilts ovulated in groups T1, T2, T3 and T4 than in the control T5 (P < 0.05). It was concluded that freezing-thawing, as well as cooling over a period of up to six months, did not significantly change the structural properties or biological activity of eCG.
Quality control of recombinant human growth hormone directly in osmotic shock fluids
1997 - DALMORA, S.; OLIVEIRA, J.E.; AFFONSO, R.; RIBELA, M.T.C.P.; ARKATEN, R.R.; BARTOLINI, P.
RP-HPLC qualitative and quantitative analysis of glycoprotein hormones in the presence of high amounts of human serum albumin: hLH, hCG, hFSH and hTSH
2011 - ALMEIDA, B.E.; OLIVEIRA, J.E.; DAMIANI, R.; DALMORA, S.L.; BARTOLINI, P.; RIBELA, M.T.C.P.
Charge isomer distribution of different pituitary and recombinant human thyrotropin (hTSH) preparations
2010 - DAMIANI, R.; OLIVEIRA, J.E.; ALMEIDA, B.E.; BARTOLINI, P.; RIBELA, M.T.C.P.
International collaborative study to establish the 2nd WHO International Standard of Human Recombinant Fillicle Stimulating Hormone for Bioassai
2010 - OLIVEIRA, J.E.; ALMEIDA, B.E.; DAMIANI, R.; BARTOLINI, P.; RIBELA, M.T.C.P.
Physico-chemical quality control of human chrionic gonadotropin preparations
2010 - ALMEIDA, B.E.; OLIVEIRA, J.E.; DAMIANI, R.; DALMORA, S.L.; BARTOLINI, P.; RIBELA, M.T.C.P.
Reversed-phase higg-performance liquid chromatography characterization of human menopausal gonadotropin preparations
2010 - ALMEIDA, B.E.; OLIVEIRA, J.E.; DAMIANI, R.; SPIGOTI, G.; DALMORA, S.L.; BARTOLINI, P.; RIBELA, M.T.C.P.
Approaches to maximizing the periplasmic expression in Escherichia coli of the human growth hormone gene from a vector containing the lambda Psub(L)
2002 - SOARES, C.R.J.; OLIVEIRA, J.E.; SOUSA, J.M.; ARKATEN, R.R.; BARTOLINI, P.