Desenvolvimento e validação de método analítico in vitro para determinação de potência de produtos farmacêuticos com o ativo eritropoietina
Data
2024-06-28
Autores
Camargo, Bruno Taborda Paes 
Orientadores
Lopes, Patrícia Santos
Tipo
Dissertação de mestrado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
O hormônio glicoproteico, eritropoietina humana (EPO) atua sobre as células predecessoras de eritrócitos estimulando sua proliferação, diferenciação, maturação e inibindo a apoptose, aumentando assim a produção de eritrócitos. A EPO é utilizada clinicamente para o tratamento de anemias associadas à falência renal crônica, tratamento de anemias associadas ao câncer, infecção por HIV, pré e pós-operatório, artrite reumatoide e transplante de medula óssea. Com o avanço da biotecnologia e o surgimento de técnicas de DNA recombinante, a EPO pode ser produzida em grande escala em Células de Ovário de Hamster Chinês (CHO) para a produção de medicamentos. O teste de potência da EPO é descrito ainda hoje, em compêndios oficiais, como um teste realizado em animais (in vivo), porém com o crescente cuidado ético no uso de animais em experimentos, os gastos com biotérios e a variabilidade dos resultados, iniciou-se o desenvolvimento de um método alternativo (in vitro). Utilizando uma linhagem celular não aderente predecessora de eritrócitos (TF-1), a metodologia em desenvolvimento é baseada em uma técnica colorimétrica e avalia a viabilidade e se as células estão metabolicamente ativas após estímulo com a EPO. Foram testadas variações analíticas para a técnica colorimétrica sendo que algumas dessas variações apresentaram resultados atisfatórios com curva de concentração resposta apresentando correlação forte para uma curva polinomial de segunda ordem. Foram realizadas em paralelo análises de espectrometria de massa e microscopia confocal para confirmar e avaliar a ação da EPO sobre a TF-1. A espectrometria de massa avaliou a regulação de proteínas intracelulares mediadas pela EPO e os resultados mostraram que 4 proteínas só puderam ser identificadas após a TF-1 receber estímulo da EPO, sendo estas proteínas de núcleo celular e estruturais. A Microscopia confocal avaliou a morfologia celular em cultivos com diferentes concentrações de EPO e foi possível visualizar que morfologicamente às células ficaram apresentaram modificações conforme a concentração de EPO aumentou na cultura. O método em desenvolvimento mostrou-se promissor, respondendo positivamente a avaliação de atividade celular, entregando uma curva de concentração resposta dependente da concentração de EPO utilizada no estímulo das células além dos ensaios de espectrometria e microscopia confirmarem que a linhagem celular responde ao estímulo do hormônio. Na sequência do trabalho, foi realizada a validação da metodologia desenvolvida, que entregou resultados dentro dos parâmetros estabelecidos para seletividade, linearidade, precisão, exatidão. A faixa de trabalho e os limites de detecção e de quantificação também puderam ser determinados. A metodologia in vitro proposta se mostra uma alternativa para substituição da metodologia in vivo atualmente descrita em compêndios oficiais.
The glycoprotein hormone, human erythropoietin (EPO) acts on erythrocyte predecessor cells by stimulating their proliferation, differentiation, maturation and inhibiting apoptosis, thus increasing erythrocyte production. EPO is used clinically for the treatment of anemias associated with chronic renal failure, treatment of anemias associated with cancer, HIV infection, preand postoperatively, rheumatoid arthritis and bone marrow transplantation. With the advancement of biotechnology and the emergence of recombinant DNA techniques, EPO can be produced on a large scale in Chinese Hamster Ovary Cells (CHO) cells for drug production. The EPO potency test is still described today, in official compendiums, as a test performed on animals (in vivo), but with the growing ethical care in the use of animals in experiments, the expenses with vivariums and the variability of results, it began the development of an alternative method (in vitro) has been developed. Using a predecessor cell line of erythrocytes (TF1), the methodology under development is based on a colorimetric technique and evaluates the viability and whether the cells are metabolically active after stimulation with EPO. Twelve analytical variations were tested for the colorimetric technique, and 6 of these variations presented satisfactory results with a concentrationresponse curve showing a strong correlation with a secondorder polynomial curve. Mass spectrometry and confocal microscopy analyzes were performed in parallel to confirm and evaluate the action of EPO on TF1. Mass spectrometry evaluated the regulation of intracellular proteins mediated by EPO and the results showed that 4 proteins could only be identified after TF1 received EPO stimulation, these being cell nucleus and structural proteins. Confocal microscopy evaluated cell morphology in cultures with different concentrations of EPO and it was possible to visualize that morphologically the cells became more stable as the concentration of EPO increased. The method under development showed to be promising, responding positively to the evaluation of cellular activity, delivering a concentration response curve dependent on the concentration of EPO used in cell stimulation, in addition to spectrometry and microscopy assays confirming that the cell lineage responds to the hormone stimulus. Following the work, the developed methodology was validated, which provided results within the expected configurations for selectivity, linearity, precision and accuracy. The working range and limits of detection and quantification can also be specific. The proposed in vitro methodology shows an alternative to replace the in vivo methodology currently described in official compendia.
The glycoprotein hormone, human erythropoietin (EPO) acts on erythrocyte predecessor cells by stimulating their proliferation, differentiation, maturation and inhibiting apoptosis, thus increasing erythrocyte production. EPO is used clinically for the treatment of anemias associated with chronic renal failure, treatment of anemias associated with cancer, HIV infection, preand postoperatively, rheumatoid arthritis and bone marrow transplantation. With the advancement of biotechnology and the emergence of recombinant DNA techniques, EPO can be produced on a large scale in Chinese Hamster Ovary Cells (CHO) cells for drug production. The EPO potency test is still described today, in official compendiums, as a test performed on animals (in vivo), but with the growing ethical care in the use of animals in experiments, the expenses with vivariums and the variability of results, it began the development of an alternative method (in vitro) has been developed. Using a predecessor cell line of erythrocytes (TF1), the methodology under development is based on a colorimetric technique and evaluates the viability and whether the cells are metabolically active after stimulation with EPO. Twelve analytical variations were tested for the colorimetric technique, and 6 of these variations presented satisfactory results with a concentrationresponse curve showing a strong correlation with a secondorder polynomial curve. Mass spectrometry and confocal microscopy analyzes were performed in parallel to confirm and evaluate the action of EPO on TF1. Mass spectrometry evaluated the regulation of intracellular proteins mediated by EPO and the results showed that 4 proteins could only be identified after TF1 received EPO stimulation, these being cell nucleus and structural proteins. Confocal microscopy evaluated cell morphology in cultures with different concentrations of EPO and it was possible to visualize that morphologically the cells became more stable as the concentration of EPO increased. The method under development showed to be promising, responding positively to the evaluation of cellular activity, delivering a concentration response curve dependent on the concentration of EPO used in cell stimulation, in addition to spectrometry and microscopy assays confirming that the cell lineage responds to the hormone stimulus. Following the work, the developed methodology was validated, which provided results within the expected configurations for selectivity, linearity, precision and accuracy. The working range and limits of detection and quantification can also be specific. The proposed in vitro methodology shows an alternative to replace the in vivo methodology currently described in official compendia.