JOAO EZEQUIEL DE OLIVEIRA
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Resumo IPEN-doc 26740 Human bone morphogenetic protein (hBMP)-2 characterization by physical chemical, immunological and biological assays2019 - SUZUKI, M.F.; OLIVEIRA, J.E.; DAMIANI, R.; LIMA, E.R.; AMARAL, K.C.; SILVA, F.M.; BARTOLINI, P.Commercial preparations of human-met-BMP-2 (GenScript) and of CHO-derived hBMP-2 (Infuse-Medtronic) provided a complete characterization of this protein, which belongs to the “transforming growth factors β” superfamily, via SDS-PAGE, Western blotting, reversed-phase HPLC, high-performance size-exclusion chromatography and MALDI-TOF-MS. E.coli-derived met-hBMP-2 has shown a large presence of dimer (MM= 26,054 Da), versus a theoretic value of 26,072 Da. More complex was the distribution of the CHO-derived product, whose exact MM has never been reported due to variable glycosylation: via MALDI-TOF-MS a dimer (28,732 Da) and a large amount of monomer (14,377 Da) were found. A novel method based on RP-HPLC was also validated for hBMP-2 qualitative and quantitative analysis directly in ongoing culture media. The classical “in vitro” bioassay, via alkaline phosphatase induction in murine myoblastic cells C2C12, confirmed that hBMP-2 bioactivity is mostly related to the dimer, being ∼6-fold higher for the CHO-derived glycosylated form. Considering that hBMP-2 is a highly effective osteoinductors, plays an important role during bone regeneration and repair, as well as during embryonic development, and presents an extremely high aggregate value, we believe that these data pave the way to the characterization of this important factor when obtained by DNA recombinant techniques in different host cells.Resumo IPEN-doc 24397 Carbohydrate composition and site-occupancy determination in pituitary and recombinant preparations of human thyrotropin2017 - BARTOLINI, PAOLO; 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.Human thyrotropin (hTSH) is a glycoprotein with three potential glycosylation sites: two in the -subunit and one in the -subunit. Carbohydrate site-occupancy is frequently neglected in glycoprotein characterization, even if related to folding, trafficking, initiation of inflammation, host defence and congenital disorders of glycosylation (CDG). For the first-time N-glycoprofiling analysis was applied to site-occupancy determination of two native pituitary hTSH, in comparison with three CHO-derived preparations of hTSH, a widely used biopharmaceutical. A single methodology provided: (i) average N-glycan mass; (ii) mass fraction of each monosaccharide and of sulfate; (iii) percent carbohydrate. The results indicate that occupancy (65–87%) and carbohydrate mass (12–19%) can be 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). The number of moles of fucose per mole of hTSH was found 2.5-fold higher in the pituitary preparations. One of these native preparations, presenting the smallest glycan mass, lowest occupancy, GalNAc, sulfate, Gal and sialic acid contents, also presented the lowest in vivo bioactivity and circulatory half-life. This methodology, extremely important for comparing a recombinant biopharmaceutical to its native equivalent, can be applied to any physiologically or clinical relevant glycoprotein.Resumo IPEN-doc 22311 N-glycoprofiling analysis in a simple glycoprotein model: glycosylated human prolactin2014 - CAPONE, MARCOS V.; SUZUKI, MIRIAM F.; OLIVEIRA, JOAO E.; SOARES, CARLOS R.; BARTOLINI, PAOLOResumo IPEN-doc 21356 Molecular cloning, characterization and phylogenetic analysis of pirarucu (Arapaima gigas) FSH and LH beta-subunits2015 - BARTOLINI, PAOLO; CARVALHO, ROBERTO F.; SEVILHANO, THAIS C. dos A.; OLIVEIRA, JOAO E.; GARCEZ, RIVIANEArtigo IPEN-doc 20704 N-glycoprofiling analysis in a single glycoprotein model: a comparison between recombinant and pituitary glycosylated human prolactin2015 - CAPONE, MARCOS V.N.; SUZUKI, MIRIAM F.; OLIVEIRA, JOAO E.; DAMIANI, RENATA; SOARES, CARLOS R.J.; BARTOLINI, PAOLOResumo IPEN-doc 20256 Effects of butyrate and manganese on productivity, sialylation, N-glycosylation site occupancy and biological properties of CHO-derived thyrotropin2014 - DAMIANI, RENATA; OLIVEIRA, JOAO E.; ALMEIDA, BEATRIZ E.; SANT'ANA, PATRICIA M.; DALMORA, SERGIO L.; BARTOLINI, PAOLO; RIBELA, MARIA T.C.P.