Verification of the new proposed version of pyrogen result judgment algorithm / 中国药学杂志

OBJECTIVE: To verify the new proposd version of pyrogen result judgment algorithm (NPRJA) and validate practicality, accuracy and reliability. METHODS: Six hundred and fifty-four batches of pyrogen test data from our laboratory in near three years were used; 3 648 rabbit body temperature fluctuations qualified in preview were statistically analyzed. Only single rabbit having lever was used to judge the pyrogen qualified or not; using logic anlysis method determined the NPRJA. RESULTS: According to the existing Chinese Pharmacopoeia volume III, suspicious rate of pyrogenic results was 28.0%, but the suspicious rate was only 10.4% in NPRJA. The new model can reduce over 60% of the repeat test. However, all failed samples were not leaked in new determiner. In preflight warm qualified rabbits, the temperature fluctuations were rather more than reach 0.4℃ within the first 3 h, they had more than 8% in whole rabbits. CONCLUSION: In pyrogen test rabbit own body temperature appears fluctuation, single rabbit warming≥ 0.6℃ serves as one of the criteria for re-examination of the sample, its results will lead to raising re-examination rate! The new proposed version of pyrogen result judgment algorithm was used to threshold of the sum of temperature rise of all tested rabbits, that not only guarantees the no changing of accuracy in existing present pyrogen test results, also significantly reduces the amount of animals and experimental repetitions. Thereby reducing the amount of experimental and labor costs. Meanwhile, it might be convenient and practical, as will as a worthy utilization and promoting patterns.

Comparative evaluation of pyrogens tests in pharmaceutical products

A comparison of methodologies for detection of pyrogens in pharmaceutical products was performed. The rabbit pyrogen test was optimized and the dose-response curve was obtained for the 2nd International Standard for bacterial endotoxins, establishing 13.81 EU/mL/kg as the concentration of endotoxin necessary to induce a temperature rise of 0.5ºC. The 0.5ºC cut-off was shown to give results that were more compatible with the pyrogenic doses for humans. The Limulus amoebocyte lysate test (LAL) was standardized with gel-clot and chromogenic endpoints, and used for the comparative evaluation of pharmaceutical products showing good agreement. The use of beta-glucan-reactive and non-reactive LAL reagents identified some products with false-positive results. The interference test was carried out and the specifications validated for some new products as the maximum valid dilution. The results emphasized the importance and limitations of the assays recommended for the evaluation of purity and quality control of parenteral medicinal products, improving the existing methodologies in the context of reduction and replacement in the use of animal models.

Realizou-se a comparação de metodologia para avaliação de pirogênios em produtos farmacêuticos. Otimizou-se o teste da hipertermia em coelhos elaborando a curva dose-resposta com o 2º Padrão Internacional de endotoxinas bacterianas, com base na qual determinou-se a concentração de 13,81 UE/mL por kg de peso corporal, necessária para produzir aumento de temperatura de 0,5ºC. Observou-se que o limite de 0,5ºC forneceu resultados comparáveis com as doses pirogênicas para o homem. Padronizou-se o teste do lisado de amebócitos do Limulus (LAL) com determinação do ponto final cromogênico e por geleificação, que foram utilizados para a avaliação de produtos farmacêuticos obtendo-se resultados concordantes. Avaliaram-se as respostas de reagentes LAL reativos e não-reativos a beta-glicanos, observando diferenças que poderiam reprovar amostras com base em resultados falso-positivos. Executou-se o teste de interferências, validou-se o procedimento e estabeleceu-se a máxima diluição válida para produtos farmacêuticos sem especificações farmacopéicas. Os resultados enfatizam a importância e as limitações dos ensaios preconizados para avaliação da pureza e controle da qualidade de produtos farmacêuticos parenterais, contribuindo para aprimorar as metodologias existentes no contexto da redução e substituição dos modelos animais.