RENATA DAMIANI
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Resumo IPEN-doc 11433 Comparative studies of pituitary (NIDDK, USA) and recombinant (Thyrogen and IPEN) human thyroid stimulating hormone (hTSH) for what concerns cabohydrate structures and charge heterogeneity2006 - OLIVEIRA, J.E.; LOUREIRO, R.F.; CARVALHO, C.M.; DAMIANI, R.; BARTOLINI, P.; RIBELA, M.T.C.P.Resumo IPEN-doc 12453 Influence of reduced Co2 environment on the secretion yield, potency and glycan structures of recombinant thyrotropin (hTSH) from CHO cells2007 - RIBELA, MARIA T.C.P.; OLIVEIRA, JOAO E.; DAMIANI, RENATA; CARVALHO, CRISTIANE M.; LHOTA, GABRIELE; VORAUER-UHL, KAROLA; BARTOLINI, PAOLOA consistent increase of — 60%, in the secretion yield of CHO-derived hTSH was observed by changing cell 'culture CO2 conditions from 5% CO2 to air enviroment (0.03 % CO2). The quality of the product obtained under both conditions was analysed for what concerns N-glycan structures, charge isomers and biological activity in comparison with a well known commercial preparation (Thyrogen). The N-glycans identified in the three preparations were of the complex type, presenting di-, tri- and tetraantennary structures, with variable level of sialylation. Considering the latter characteristic, which is directly related to in vivo bioactivity, the three preparations have practically an identical percentage (86-88%) of sialylated structures, with some difference in percentage of di- and tii- sialylated glycan. Monosialylated glycans (N2G2S1, N2G1S1 and N2G2S1F) represent the three most abundant structures with 68-69% of all identified forms in the three preparations. The main difference was found in terms of antennarity with 8-10% more N2 structures for hTSH-IPEN produced in the absence of CO2 (-0O2) and 7-9 % more N3 structures for hTSH-IPEN (+CO2) and Thyrogen. Also for what concerns the total percentage of neutral glycans (12- 14 %), the three preparations are practically identical. No remarkable difference in charge isomers was also observed between the three preparations, the isoelectric focusing (IEF) profiles showing six well visible bands in the 5.39 - 7.35 pI range, the three major bands focusing at pI 5.85, 6.20 and 6.63. A considerably different distribution, with more acidic forms was observed, though, for two native pituitary preparations of hTSH. When analyzed via a simple and precise single-dose bioassay, a slightly significant difference (p<0.02) in activity was found between the two IPEN preparations. hTSH-IPEN (+ CO2) potency was non significantly different from that of Thyrogen, both being 1.6-1.8-fold more potent than the native pituitary reference preparation. We can conclude that, at least in the case of CHO-derived hTSH, different production processes may not greatly affect its glycan structures, charge isomer distribution or biological activity.Resumo IPEN-doc 12452 A practical RP-HPLC method for recombinant human thyrotropin (hTSH) laboratory scale purification2007 - DAMIANI, RENATA; OLIVEIRA, JOAO E.; CARVALHO, CRISTIANE M.; PERONI, CIBELE N.; BARTOLINI, PAOLO; RIBELA, MARIA T.C.P.Resumo IPEN-doc 12451 Physico-chemical characterization of alpha and beta subunit of recombinant human glycoprotein hormones: hTSH and hLH2007 - CARVALHO, CRISTIANE M.; OLIVEIRA, JOAO E.; DAMIANI, RENATA; ALMEIDA, BEATRIZ E.; CECCHI, CLAUDIA R.; BARTOLINI, PAOLO; RIBELA, MARIA T.C.P.Resumo IPEN-doc 14160 Indentity and integraty of 'alfa' and 'beta' - subunit of human thyrotropin prepared by prolonged acetic acid treatment2009 - ALMEIDA, BEATRIZ E.; CARVALHO, CRISTIANE M.; DAMIANI, RENATA; OLIVEIRA, JOAO E.; BARTOLINI, PAOLO; RIBELA, MARIA T.C.P.Alpha- and beta- subunits, prepared by efficiently dissociating, during 16 hours, a recombinant thyrotropin (hTSH) preparation with 0.4 M acetic acid and isolating them by RP-HPLC, were analysed for what concerns their identity and integrity. Identity was evaluated by MALDI-TOF mass spectrometry (MALDI-TOF MS). A relative molecular mass of 14021 and of 15851 was obtained for a-hTSH and (3-hTSH respectively. These values agree with those obtained by analyzing the preparation before dissociation, a difference of -1.8% for a and +1.3% for (3 being observed. Integrity of the subunits was evaluated by their capacity of self reassembling and of restoring the in vivo bioactivity of the hormone. When a-hTSH and I3-hTSH subunits were incubated together in 0.2 M sodium phosphate buffer, pH 7.0, at 25°C and under gentle shaking, a complete reassociation occurred after 4 days, forming an heterodimer. In an in vivo mouse bioassay, the T4 levels of the animals treated with the reassociated hormone were non-significantly different (p> 0.05) from those obtained when the original preparation was administered (2.71± 0.63 pg/dL versus 2.84± 0.23 pg/dL, n=6, respectively). In conclusion, subunits prepared by prolonged acetic acid treatment maintain their original molecular mass and can perfectly restore the biological activity of the reassociated heterodimers.