CRISTIANE MOREIRA DE CARVALHO

CRISTIANE MOREIRA DE CARVALHO

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Comparative studies of pituitary (NIDDK, USA) and recombinant (Thyrogen and IPEN) human thyroid stimulating hormone (hTSH) for what concerns cabohydrate structures and charge heterogeneity
2006 - OLIVEIRA, J.E.; LOUREIRO, R.F.; CARVALHO, C.M.; DAMIANI, R.; BARTOLINI, P.; RIBELA, M.T.C.P.
Influence of reduced Co2 environment on the secretion yield, potency and glycan structures of recombinant thyrotropin (hTSH) from CHO cells
2007 - RIBELA, MARIA T.C.P.; OLIVEIRA, JOAO E.; DAMIANI, RENATA; CARVALHO, CRISTIANE M.; LHOTA, GABRIELE; VORAUER-UHL, KAROLA; BARTOLINI, PAOLO
A 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.
A practical RP-HPLC method for recombinant human thyrotropin (hTSH) laboratory scale purification
2007 - DAMIANI, RENATA; OLIVEIRA, JOAO E.; CARVALHO, CRISTIANE M.; PERONI, CIBELE N.; BARTOLINI, PAOLO; RIBELA, MARIA T.C.P.
Physico-chemical characterization of alpha and beta subunit of recombinant human glycoprotein hormones: hTSH and hLH
2007 - CARVALHO, CRISTIANE M.; OLIVEIRA, JOAO E.; DAMIANI, RENATA; ALMEIDA, BEATRIZ E.; CECCHI, CLAUDIA R.; BARTOLINI, PAOLO; RIBELA, MARIA T.C.P.
Analysis and characterization of different preparations of recombinant human follicie stimulating hormone (hFSH) and of subunits
2006 - RIBELA, MARIA T.C.P.; LOUREIRO, RENAN F.; OLIVEIRA, JOAO E.; CARVALHO, CRISTIANE M.; PERONI, CIBELE N.; BARTOLINI, P.
Efficient separation of the two subunits of human glycoprotein hormones by high performance liquid chromatography
2006 - LOUREIRO, R.F.; OLIVEIRA, J.E.; CARVALHO, C.M.; BARTOLINI, P.; RIBELA, M.T.C.P.
Reversed-phase high performance liquid chromatography (RP-HPLC) analysis and hydrophobicity studies of recombinant human pituitary hormones synthesized in E. coli and CHO cells
2005 - RIBELA, M.T.C.P.; CARVALHO, C.M.; HELLER, S.R.; LOUREIRO, R.F.; OLIVEIRA, J.E.; OZAKI, N.A.; PERONI, C.N.; SOARES, C.R.J.; SOUZA, J.M.; UEDA, E.K.; BARTOLINI, P.
The synthesis and laboratory production of human growth hormone (hGH) and prolactin (hPRL) have been carried out in genetically modified E. coli, while those of thyroid stimulating hormone (hTSH) and of two analogs antagonists of hPRL (G129RhPRL and S179D-hPRL) in stably transfected CHO cells. Human follicle stimulating hormone (hFSH) and luteinizing hormone (hLH) have not been synthesized yet in our laboratory but their HPLC analytical methodologies are under development. For the purpose of studying and improving synthesis and bioreaction yields and, at the same time, planning and following all subsequent purification steps, novel RP-HPLC methods have been set up for each hormone. For hGH and hPRL, isocratic RP-HPLC methods have been developed that can qualitatively and quantitatively analyze the two hormones directly in osmotic shock fluids, already during fermentation. For hTSH, hFSH and hLH, RP-HPLC gradient elutions have been set for the analysis of these hormones in their purified form and in CHO conditioned medium. For these three glycoproteins hydrophobicities have been compared and the following order established: hLH>hTSH>hFSH. An analogous hydrophobicity index has been also determined for hPRL and its analogs, being G129R-hPRL>hPRL>S179D-hPRL. Still concerning hFSH, for the first time it has been possible to optimize RP-HPLC elution conditions that are able to preserve its undissociated heterodimeric structure. Thanks to this tool it was thus possible to carry out a comparative study on pituitary, urinary and CHO-derived hFSH preparations, revealing differences that are probably due to the carbohydrate moiety, as already observed for hTSH. Classical highperformance size exclusion chromatography (HPSEC) together with MALDI-TOF-MS analysis was also employed along with these studies, to complement physico-chemical characterization of our proteins of interest.
Indentity and integraty of 'alfa' and 'beta' - subunit of human thyrotropin prepared by prolonged acetic acid treatment
2009 - 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.
Preparacao e caracterizaco das subunidades alfa e beta dos hormonios glicoproteicos humanos recombinantes: foliculotrofina, luteotrofina, tireotrofina e sua comparacao com os produtos hipofisarios
2008 - MAGEIKA, CRISTIANE M. de C.
Neste trabalho é descrito um método prático e eficiente para dissociar, em subunidades α e β, quantidades pequenas (da ordem de microgramas) dos hormônios foliculotrofina (hFSH), luteotrofina (hLH) e tireotrofina (hTSH) humana, nativos e recombinantes. A dissociação destes hormônios foi conseguida incubando-os, durante 16 horas, a 37ºC, com diferentes concentrações de ácido acético: 3M, 5M e 0,4M respectivamente para o hFSH, hLH e hTSH. Nestas condições, uma eficiência de dissociação acima de 98% foi obtida. Esta eficiência foi calculada com base nas determinações de massa dos heterodímeros e das subunidades, realizadas por MALDI-TOF-MS. Uma separação rápida e quantitativa das subunidades, com rendimentos da ordem de 80-90%, foi conseguida por cromatografia líquida de alta eficiência em fase reversa (RP-HPLC) em uma coluna C4. As subunidades foram caracterizadas quanto à pureza, hidrofobicidade, massa molecular e distribuição de carga por HPLC de exclusão molecular e fase reversa, SDS-PAGE e focalização isoelétrica. Quando analisadas quanto à hidrofobicidade, as subunidades mostraram-se aproximadamente iguais, enquanto as subunidades β dos três heterodímeros apresentaram a seguinte escala de hidrofobicidade: β-hFSH < β-hTSH < β-hLH. Com relação à massa molecular relativa (Mr), as subunidades α e β do hFSH apresentaram as maiores Mr enquanto as subunidades do hLH as menores. A distribuição dos isômeros de carga das subunidades dos três hormônios ocorreu em uma região ácida, para o hFSH, em uma região básica, para o hLH e em uma região intermediária, para o hTSH. As subunidades α dos três hormônios, quando analisadas via SDS-PAGE, apresentaram praticamente a mesma mobilidade eletroforética, enquanto as subunidades β apresentaram diferentes taxas de migração (mR), sendo mR β-hFSH < mR β-hTSH < mR β-hLH. Diferenças relativas à massa molecular, hidrofobicidade, migração eletroforética e distribuição de carga foram encontradas entre as preparações recombinantes e hipofisárias dos três hormônios. O método descrito é suave, prático e flexível e pode ser adaptado à dissociação de outras glicoproteínas heterodiméricas recombinantes ou nativas. Permite não só estudos e caracterização direta de cada subunidade, como também detectar a presença de subunidades livres em preparações farmacêuticas, que são contaminantes indesejáveis, sendo, portanto, uma ferramenta extremamente útil para o controle de qualidade de produtos farmacêuticos.
Efficient isolation of the subunits of recombinant and pituitary glycoprotein
2009 - CARVALHO, C.M.; OLIVEIRA, J.E.; ALMEIDA, B.E.; UEDA, E.K.M.; TORJESEN, P.A.; BARTOLINI, P.; RIBELA, M.T.C.P.