BEATRIZ ELANE DE ALMEIDA

BEATRIZ ELANE DE ALMEIDA

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Biological activity of different batches of equine chorionic gonadotropin as determined by reversed-phase high-performance liquid chromatography and in vivo assay
2017 - ALVAREZ, RAFAEL H.; NATAL, FABIO L.N.; ALMEIDA, BEATRIZ E.; OLIVEIRA, JOAO E.; MELO, ALFREDO J.F.; RIBELA, MARIA T.C.; BARTOLINI, PAOLO
Aims: To evaluate the physicochemical profile of commercial batches of eCG, in order to find if differences can be related to their biological activity. Study Design: Commercial eCG was analyzed by reversed-phase high-performance liquid chromatography (RP-HPLC) and in vivo bioassay. Place and Duration of Study: Department of Biotechnology (IPEN-CNEN) and Animal antibody production Laboratory (Animal Science Institute), between June 2013 and April 2014. Methodology: Two commercial eCG batches for veterinary use (I and II) and an eCG official International Standard from the World Health Organization (WHO) were analyzed by RP-HPLC. Additionally, two experiments were designed to validate the biological activity. In experiment 1, groups of prepubertal 21–25 day old Wistar female rats (n = 6/group) received the equivalent to 0 UI (saline) and 10 IU of eCG of each one of these preparations. Autopsy was performed 48 h later and ovaries were removed and weighed. The experiment 2 was designed to determine whether increasing the dose of less active eCG batches could increase the ovarian response. Therefore, groups of prepubertal rats (n = 6/group) were treated with 10 and 30 IU eCG from batch II, while eCG from WHO (10 IU) and saline were control. The evaluation of ovarian response was done similar to Experiment 1. Differences among treatments were analyzed by one-way ANOVA. Results: Results of RP-HPLC showed differences in the main tR peak profile (tR 26.7) of the standard WHO compared with eCG batches I and II. In experiment 1, the average ovarian weight of rats treated with eCG from WHO (60.0 ± 12.1 mg) was higher (P < .01) than saline (23.1 ± 1.6 mg) and batches I (37.6 ± 1.4 mg) and II (31.0 ± 4.3 mg). In experiment 2, the ovarian weight of rats treated with 30 IU of eCG of batch II (45.7 ± 4.1 mg) was higher (P < .01) than saline (32.6 ± 1.4 mg) and significantly lower (P = .05) than 10 UI of the standard WHO (63.3 ± 8.1 mg). Conclusion: The low ovarian response to eCG treatments can be related to differences in the physicochemical profile of eCG batches and RP-HPLC is a fast and reliable tool for detecting these differences.
Effect of cold stress on physicochemical characteristics and biological activity of equine chorionic gonadotropin
2016 - ALVAREZ, R.H.; NATAL, F.L.N.; ALMEIDA, B.E.; OLIVEIRA, J.E.; BARTOLINI, P.; MELO, A.J.F.; DUARTE, K.M.R.; RIBELA, M.T.C.
The purpose of this study was to evaluate if freezing-thawing and cooling processes affect the structural properties and biological activity of commercial equine chorionic gonadotropin (eCG). First, the structure profile of diluted eCG underwent none, one or three cycles of freezing-thawing was analysed by reverse phase high-performance liquid chromatography (RP-HPLC). In a second experiment, groups of prepuberal rats were treated with sterile water for injection USP or eCG that underwent none, one or three cycles of freezing-thawing to assess the increase of ovarian weigh. Finally, groups of prepubertal gilts were treated with diluted eCG immediately after reconstitution (T1), after refrigeration for six months (T2) and after freezing and subsequently thawing for one (T3) or three (T4) cycles. The control group (T5) received sterile water for injection USP without eCG. Ovulation was induced with human chorionic gonadotropin (hCG), administered 72 h after the eCG. Gilts were slaughtered five days after the hCG injection and ovaries were recovered and analysed for the presence of corpora lutea. Data were analysed by ANOVA and Fisher’s exact tests. In the analyses by RP-HPLC, the retention times of cold stressed eCG were similar to unstressed control. The mean ovarian weight of rats treated with cold stressed and unstressed eCG was statistically higher than water control (P < 0.05). Lastly, significantly more gilts ovulated in groups T1, T2, T3 and T4 than in the control T5 (P < 0.05). It was concluded that freezing-thawing, as well as cooling over a period of up to six months, did not significantly change the structural properties or biological activity of eCG.
Physical-chemical and biological characterization of different preparations of equine chorionic gonadotropin
2016 - ALVAREZ, RAFAEL H.; NATAL, FABIO L.N.; RIBELA, MARIA T.C.P.; ALMEIDA, BEATRIZ E. de; OLIVEIRA, JOAO E. de; BARTOLINI, PAOLO
Ovarian stimulation with commercial preparations of equine chorionic gonadotropin (eCG) produces extremely variable responses in domestic animals, ranging from excessive stimulation to practically no stimulation, when applied on the basis of their declared unitage. This study was conducted to analyze four commercial preparations from different manufacturers via reversed-phase HPLC (RP-HPLC) in comparison with a reference preparation and an official International Standard from the World Health Organization. The peaks obtained by this qualitative and quantitative physical–chemical analysis were compared using an in vivo bioassay based on the ovarian weight gain of prepubertal female rats. The RP-HPLC data showed one or two peaks close to a main peak (tR = 27.9 min), which were related to the in vivo bioactivity. Commercial preparations that have this altered peak showed very little or no in vivo activity, as demonstrated by rat ovarian weight and in peripubertal gilts induced to ovulate. Overall, these findings indicate that RP-HPLC can be a rapid and reliable tool to reveal changes in the physicochemical profile of commercial eCG that is apparently related to decreased biological activity of this hormone.
International collaborative study to establish the 2nd WHO International Standard of Human Recombinant Fillicle Stimulating Hormone for Bioassai
2010 - 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.
Quantitative analysis of different preparations of human thyrotropin (hTSH): a comparison between RP-HPLC and the in vivo bioassay based on thyroxine stimulaton mice
2012 - ALMEIDA, B.E.; DAMIANI, R.; OLIVEIRA, J.E.; RIBELA, M.T.C.P.; BARTOLINI, P.
Determinação de potência de diferentes preparações de foliculotrofina, luteotrofina e tireotrofina: comparação entre a quantificação por cromatografia líquida em fase reversa e por bioensaio in vivo
2013 - ALMEIDA, BEATRIZ E. de
Com a intenção de estabelecer métodos físico-químicos como uma alternativa ao bioensaio in vivo para determinação de atividade biológica, o conteúdo de hFSH, hTSH e hLH de diferentes preparações, nativas e recombinantes, foi determinado por cromatografia líquida de alta eficiência em fase reversa (RP-HPLC) e comparado ao dado obtido pelo clássico bioensaio in vivo em camundongos ou ratos (BA). Para estes hormônios foi encontrada uma relação linear entre os dois métodos: hFSH BAUI = 0,9925 RP-HPLCUI - 1,3165, r = 0,9371, p < 0,001, n = 24; hTSH BAμg = 0,9790 RP-HPLCμg - 0,052, r = 0,8725 , p < 0,001, n = 14; hLH BAUI = 0,8771 RP-HPLCUI + 12,41; r = 0,9786, p < 0,01, n = 5. Para outras nove preparações de hFSH e onze preparações de hTSH foi determinada a diferença média (d) entre a bioatividade predita pela RP-HPLC através destas equações e da média das bioatividades obtidas com os dois métodos. Para o hLH não foi possível determinar esta diferença em virtude das poucas amostras disponíveis. No caso do hFSH, d ± DP = -2,11 ± 3,49 % sendo a precisão de 1,16% e no caso do hTSH, d ± DP = -2,01 ± 5,56 % com precisão de 1,68%. Amostras parcialmente alteradas apresentaram diferentes graus de atividade de hFSH, hTSH e hLH que puderam ser preditas por RP-HPLC com uma aceitável concordância com os bioensaios in vivo. Estes resultados demonstraram que o emprego de um ensaio físico-químico sem o uso de animais, tal como a RP-HPLC, é uma alternativa viável ao uso do bioensaio in vivo para a determinação da potência de hFSH e hTSH, reduzindo assim o número de animais em geral utilizados para assegurar a qualidade e eficácia de um produto farmacêutico.
Reversed-phase performance liquid chromatography as an alternative to animal bioassay for human thyrotropin potency determination
2014 - ALMEIDA, B.E.; DAMIANI, R.; OLIVEIRA, J.E.; DALMORA, S.L.; TORJESEN, P.A.; BARTOLINI, P.; RIBELA, M.T.C.P.
A pilot study on potency determination of human follicle-stimulating hormone: A comparison between reversed-phase high-performance liquid chromatography method and the vivo bioassay
2011 - ALMEIDA, B.E.; OLIVEIRA, J.E.; DAMIANI, R.; DALMORA, S.L.; BARTOLINI, P.; RIBELA, M.T.C.P.