RENATA DAMIANI
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Resumo IPEN-doc 16159 International collaborative study to establish the 2nd WHO International Standard of Human Recombinant Fillicle Stimulating Hormone for Bioassai2010 - OLIVEIRA, J.E.; ALMEIDA, B.E.; DAMIANI, R.; BARTOLINI, P.; RIBELA, M.T.C.P.The aim of the International Collaborative Study proposed by the National Institute for Biological Standards and Control (WHO) is to calibrate a new preparation of human follicle stimulating hormone (hFSH) relative to the 1st International Standard for bioassay of recombinant-hFSH (WHO 92/642), which is almost exhausted and requires to be replaced. The candidate preparation and four other samples, which correspond to this preparation stored at elevated temperatures (K, L, M, N), were analyzed qualitatively and quantitatively in our laboratory, by reversed-phase high-performance liquid chromatography (RPHPLC). The candidate content determined in six assays (8.59 ± 0.41 μg/ampoule) was 20% lower, when compared with the current standard. Based on this, an average value of 110 IU FSH per ampoule could be assigned to this preparation. The main isoform of the candidate preparation showed a lower retention time (22.5 ± 0.83 min) relative to WHO 92/642 (22.8 ± 0.81 min) and also to an internal reference preparation (22.6 ± 0.86 min). These differences, though, were not significant (p>0.05). Practically identical contents were also determined for K, L, M and N ampoules (8.44 ± 0.20; 8.56 ± 0.33; 8.57 ± 0.38; 8.69 ± 0.30 μg/ampoule, respectively). No significant differences in FSH retention times between the candidate preparation and the ampoules stored at elevated temperatures were observed.Resumo IPEN-doc 18703 Quantitative analysis of different preparations of human thyrotropin (hTSH): a comparison between RP-HPLC and the in vivo bioassay based on thyroxine stimulaton mice2012 - ALMEIDA, B.E.; DAMIANI, R.; OLIVEIRA, J.E.; RIBELA, M.T.C.P.; BARTOLINI, P.Dissertação IPEN-doc 14055 Expressao estavel de tireotrofina humana (r-hTSH) em celulas de mamifero (CHO) que expressam 'alfa'2,6-sialiltransferase2009 - DAMIANI, RENATAUma linhagem celular de CHO, previamente modificada geneticamente pela introdução de cDNA da 2,6 sialiltransferase de rato, gerou, pela primeira vez, um hTSH recombinante com sialilação humanizada (hlsr-hTSH), mais similar ao hormônio nativo, com 61% de ligação de ácido siálico na conformação 2,3 e 39%, na conformação 2,6. O clone mais produtivo, quando submetido à amplificação gênica com 8 M de metotrexato, apresentou um nível de secreção de aproximadamente 2 g de hTSH/106 células/dia, nível este útil para a purificação e caracterização do produto. A massa molecular relativa do heterodímero e das subunidades e do hlsr-hTSH purificado, determinada por espectrometria de massa MALDI-TOF, e a hidrofobicidade relativa, determinada por RP-HPLC, não apresentaram diferenças significativas com relação às preparações de hTSH recombinante derivadas de células CHO sem a modificação devida ao gene da 2,6 sialiltransferase. Entretanto, algumas diferenças foram observadas na composição dos N-glicanos, com mais estruturas tri- e tetra-sialiladas no hlsr-hTSH. O hlsr-hTSH mostrou-se eqüipotente (p>0,05) à preparação comercial de r-hTSH (Thyrogen) e 1,5 vezes mais potente do que a preparação nativa de hTSH (p<0,001), quando analisado por um bioensaio in vivo baseado na capacidade do TSH de estimular a liberação de T4.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 11858 Practical reversed-phase high-performance liquid chromatography method for laboratory-scale purification of recombinant human thyrotropin2007 - OLIVEIRA, JOAO E.; DAMIANI, RENATA; BARTOLINI, PAOLO; RIBELA, MARIA T.C.P.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.