JOAO EZEQUIEL DE OLIVEIRA

JOAO EZEQUIEL DE OLIVEIRA

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Human bone morphogenetic protein‑2 (hBMP‑2) characterization by physical–chemical, immunological and biological assays
2020 - SUZUKI, MIRIAM F.; OLIVEIRA, JOAO E.; DAMIANI, RENATA; LIMA, ELIANA R.; AMARAL, KLEICY C.; SANTOS, ANDERSON M. de S.; MAGALHÃES, GERALDO S.; FAVERANI, LEONARDO P.; PEREIRA, LUIS A.V.D.; SILVA, FABIANA M.; BARTOLINI, PAOLO
Commercially available preparations of methionyl-human BMP-2 and CHO-derived hBMP-2, which belongs to the transforming growth factor β (TGF-β) superfamily, were used for a complete characterization. This protein is an extremely efficient osteoinductor that plays an important role during bone regeneration and embryonic development. Characterization was carried out via SDS-PAGE and Western blotting, followed by reversed-phase HPLC, sizeexclusion HPLC and MALDI-TOF-MS. The classical in vitro bioassay, based on the induction of alkaline phosphatase activity in C2C12 cells, confirmed that hBMP-2 biological activity is mostly related to the dimeric form, being ~ 4-fold higher for the CHO-derived glycosylated form when compared with the E. coli counterpart. The E. coli-derived methBMP- 2 has shown, by MALDI-TOF-MS, a large presence of the bioactive dimer. A more complex molecular mass (MM) distribution was found for the CHO-derived product, whose exact MM has never been reported because of its variable glycosylation. A method based on RP-HPLC was set up, allowing a quantitative and qualitative hBMP-2 determination even directly on ongoing culture media. Considering that hBMP-2 is highly unstable, presenting moreover an extremely high aggregate value, we believe that these data pave the way to a necessary characterization of this important factor when synthesized by DNA recombinant techniques in different types of hosts.
N-Glycoprofiling analysis for carbohydrate composition and site-occupancy determination in a poly-glycosylated protein: human thyrotropin of different origins
2017 - RIBELA, MARIA T.C.P.; DAMIANI, RENATA; SILVA, FELIPE D.; LIMA, ELIANA R.; OLIVEIRA, JOAO E.; PERONI, CIBELE N.; TORJESEN, PETER A.; SOARES, CARLOS R.; BARTOLINI, PAOLO
Human thyrotropin (hTSH) is a glycoprotein with three potential glycosylation sites: two in the -subunit and one in the -subunit. These sites are not always occupied and occupancy is frequently neglected in glycoprotein characterization, even though it is related to folding, trafficking, initiation of inflammation and host defense, as well as congenital disorders of glycosylation (CDG). For the first time N-glycoprofiling analysis was applied to the site-occupancy determination of two native pituitary hTSH, in comparison with three recombinant preparations of hTSH, a widely used biopharmaceutical. A single methodology provided the: (i) average N-glycan mass; (ii) mass fraction of each monosaccharide and of sulfate; and (iii) percent carbohydrate. The results indicate that the occupancy (65%–87%) and carbohydrate mass (12%–19%) can be up to 34%–57% higher in recombinant hormones. The average glycan mass is 24% lower in pituitary hTSH and contains ~3-fold fewer moles of galactose (p < 0.005) and sialic acid (p < 0.01). One of the two native preparations, which had the smallest glycan mass together with the lowest occupancy and GalNAc, sulfate, Gal and sialic acid contents, also presented the lowest in vivo bioactivity and circulatory half-life. The methodology described, comparing a recombinant biopharmaceutical to its native equivalent, can be applied to any physiologically or clinical relevant glycoprotein.
Improved bioprocess with CHO-hTSH cells on higher microcarrier concentration provides higher overall biomass and productivity for rhTSH
2011 - VENTINI, DANIELLA C.; DAMIANI, RENATA; SOUSA, ALVARO P.B.; OLIVEIRA, JOAO E. de; PERONI, CIBELE N.; RIBELA, MARIA T.C.P.; BARTOLINI, PAOLO; TONSO, ALDO; SOARES, CARLOS R.J.; PEREIRA, CARLOS A.
Two-step chromatographic purification of recombinant human thyrotrophin and its immunological, biological, physico-chemical and mass spectral characterization
2005 - MENDONCA, F.; OLIVEIRA, J.E.; BARTOLINI, P.; RIBELA, M.T.C.P.
Determination of Chinese hamster ovary cell-derived recombinant thyrotropin by reversed-phase liquid chromatography
2003 - OLIVEIRA, J.E.; MENDONCA, F.; PERONI, C.N.; BARTOLINI, P.; RIBELA, M.T.C.P.
Influence of a reduced COsub(2) environment on the secretion yield potency and N-glycan structures of recombinant thyrotropin from CHO cells
2008 - OLIVEIRA, JOAO E.; DAMIANI, RENATA; VORAUER-UHL, KAROLA; BARTOLINI, PAOLO; RIBELA, MARIA T.C.P.
Stable expression of a human-like sialylated recombinant thyrotropin in a Chinese hamster ovary cell line expressing α2,6-sialyltransferase
2009 - DAMIANI, RENATA; OLIVEIRA, JOAO E.; VORAUER-UHL, KAROLA; PERONI, CIBELE N.; VIANNA, ELIZABETH G.; BARTOLINI, PAOLO; RIBELA, MARIA T.C.P.
A CHO cell line, previously genetically modified by the introduction of rat α2,6-sialyltransferase cDNA, generated for the first time a human-like sialylated recombinant hTSH (hlsr-hTSH) more similar to the native hormone, with 61% of α2,3- and 39% of α2,6-linked sialic acid residues. The best clone, when submitted to gene amplification with up to 8 μM methotrexate, presented a secretion level of ∼2 μg hTSH/106 cells/day, useful for product purification and characterization. The relative molecular masses (Mr) of the heterodimer and of the α- and β-subunits of purified hlsr-hTSH, determined by MALDI-TOF mass spectrometry, and the relative hydrophobicities, determined by RP-HPLC, were not remarkably different from those presented by two r-hTSH preparations secreted by normal CHO cells. Some differences were observed, though, in N-glycan composition, with more tri- and much more tetra-sialylated structures in hlsr-hTSH. When analyzed via an in vivo bioassay based on hTSH-induced T4 release in mice, hlsr-hTSH was shown to be equipotent (p > 0.05) with the commercial preparation of r-hTSH (Thyrogen), and 1.6-fold more potent than native hTSH (p < 0.001).
Enhancement of human thyrotropin synthesis by sodium butyrate addition to serum-free CHO cell culture
2013 - DAMIANI, RENATA; ALMEIDA, BEATRIZ E.; OLIVEIRA, JOAO E.; BARTOLINI, PAOLO; RIBELA, MARIA T.C.P.