RENATA DAMIANI
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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.Artigo 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.Artigo IPEN-doc 20113 Reversed-phase performance liquid chromatography as an alternative to animal bioassay for human thyrotropin potency determination2014 - ALMEIDA, B.E.; DAMIANI, R.; OLIVEIRA, J.E.; DALMORA, S.L.; TORJESEN, P.A.; BARTOLINI, P.; RIBELA, M.T.C.P.Artigo IPEN-doc 11548 High-level secretion of growth hormone by retrovirally transduced primary human keratinocytes2006 - PERONI, CIBELE N.; CECCHI, CLAUDIA R.; DAMIANI, RENATA; SOARES, CARLOS R.J.; RIBELA, MARIA T.C.P.; ARKATEN, ROSANGELA R.; BARTOLINI, PAOLOA gene therapy clinical trial for treatment of growth hormone (GH) deficiency has not been reached yet, but several strategies using different gene transfer methodologies and animal models have been developed and showed successful results. We have set up an ex vivo gene therapy protocol using primary human keratinocytes transduced with an efficient retroviral vector (LXSN) encoding the human (hGH) or mouse GH (mGH) genes. These stably modified cells presented high in vitro expression levels of hGH (7 μg/106 cells/d) and mGH (11 μg/106 cells/d) after selection with geneticin. When the hGH-secreting keratinocytes were grafted onto immunodeficient dwarf mice (lit/scid), hGH levels in the circulation were about 0.2–0.3 ng/mL during a 12-d assay and these animals presented a significant body weight increase (p<0.01) compared to the control. Substitution of conventional grafting methodologies with organotypic raft cultures revealed a peak value of up to 20 ng mGH/mL in the circulation of grafted lit/scid mice at 1 h postimplantation, followed by a rapid decline to baseline (≈2 ng/mL) within 24 h. One week after grafting, however, the cultured excised implants still presented approx 45% of their original in vitro secretion efficiency. Further studies are being carrier out to identify the main factor(s) that still constitute one of the major impediments to the success of this promising model of cutaneous gene therapy.Artigo IPEN-doc 13175 Influence of a reduced COsub(2) environment on the secretion yield potency and N-glycan structures of recombinant thyrotropin from CHO cells2008 - OLIVEIRA, JOAO E.; DAMIANI, RENATA; VORAUER-UHL, KAROLA; BARTOLINI, PAOLO; RIBELA, MARIA T.C.P.Artigo IPEN-doc 14267 Stable expression of a human-like sialylated recombinant thyrotropin in a Chinese hamster ovary cell line expressing α2,6-sialyltransferase2009 - 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).Artigo IPEN-doc 16425 A pilot study on potency determination of human follicle-stimulating hormone: A comparison between reversed-phase high-performance liquid chromatography method and the vivo bioassay2011 - ALMEIDA, B.E.; OLIVEIRA, J.E.; DAMIANI, R.; DALMORA, S.L.; BARTOLINI, P.; RIBELA, M.T.C.P.Artigo IPEN-doc 17740 Qualitative and quantitative reversed-phase high performance liquid chromatographic analysis of glycoprotein hormones in the presence of a large excess of human serum albumin2012 - ALMEIDA, B.E.; OLIVEIRA, J.E.; DAMIANI, R.; DALMORA, S.L.; BARTOLINI, P.; RIBELA, M.T.C.P.