熱原動物替代試驗方法-單核球活化試驗法之建立   全文下載

Establishment of Monocyte Activation Test (MAT) as Alternative In-Vitro Pyrogens Test Method

熱原（Pyrogen）來自於細菌、病毒、真菌及其相關代謝物等，進入人體後可能驅動先天免疫系統活化，導致發燒、發炎、休克甚至多重器官衰竭。因此，針對無菌製劑及注射液等，中華藥典均訂有熱原試驗規定，以避免熱原污染之產品流入市面。傳統實驗兔熱原試驗（Rabbit Pyrogen Test）為常用之熱原檢測方法，為配合衛生福利部於「藥品查驗登記審查準則」增列「熱原試驗應以非活體動物替代方式優先」，推動替代（Replacement）、減少（Reduction）、精緻化（Refinement）之3R政策，部分產品得以細菌內毒素檢驗法（Bacterial Endotoxin Test, BET）替代，惟BET仍具侷限性，僅能檢測革蘭氏陰性菌之脂多醣（Lipopolysaccharide, LPS）。為此，本研究擬評估單核球活化試驗法（Monocyte Activation Test, MAT）用於檢測檢品中潛在熱原之可行性，依據中華藥典「單核球活化試驗法（通則3011）」進行方法A之預實驗，使用市售MAT套組對細菌內毒素標準品進行測試、分析該方法於檢品（分別使用2種抗蛇毒血清）之干擾因子試驗、偵測非內毒素熱原（Non-Endotoxin Pyrogen, NEP）對此系統之適用性及偵測系統之干擾試驗。結果顯示，MAT適用於抗蛇毒血清之熱原檢測，後續以此方法進行常規檢驗，測試結果熱原含量分別為小於16 IU/mL及小於2 IU/mL。未來將持續評估不同蛇種的抗蛇毒血清與人體血清蛋白等的適用性，以提供國內醫藥品製造廠建立非活體熱原替代試驗法之參考。

Bacteria, viruses, fungi and their related metabolites are potential origins of pyrogens. Pyrogens may stimulate the innate immune system, leading to fever, inflammation, shock, and even multiple organ failure. Therefore, for sterile preparations and injectable solutions, the Taiwan Pharmacopeia (TWP) has established pyrogen test regulations to prevent products contaminated with pyrogens from entering the market. The rabbit pyrogen test (RPT) is commonly used for pyrogen detection. However, in response to the 3R policy promoted by the Ministry of Health and Welfare to prioritize nn vitro pyrogen-test methods as alternatives for animal test”, RPT has been replaced by the bacterial endotoxin test (BET) in certain products. However, BET still has limitations, such as it can only detect lipopolysaccharides (LPS) in Gram-negative bacteria. To address this issue, this study evaluated the feasibility of the MAT for detecting potential pyrogens in pharmaceutical products, and conducted a preparatory testing of Method A based on the“Monocyte activation test (General Chapter 3011)”in the TWP. A commercial MAT kit was used to test the bacterial endotoxin standard, analyze the interference factors, and evaluate the applicability of this method for detecting non-endotoxin contaminants and interference in the detection system. This study has accomplished both preparatory testing and routine testing evaluations of two venom antisera using the commercial MAT kit. The results showed that the MAT is applicable for venom antisera, and the pyrogen contents of two test samples was less than 16 IU/mL and less than 2 IU/mL, respectively. In the future, the applicability of different types of venom antiserum will be evaluated to provide reference for domestic pharmaceutical manufacturers to establish non-animal alternatives for pyrogen testing.