基因工程tPA製劑之檢驗方法探討   全文下載

Evaluation on the Analytical Methods for Tissue Plasminogen Activator (tPA) in Pharmaceutical Formulations

組織纖維蛋白溶解?原活化因子（tissue plasminogen activator, tPA），為絲胺酸型蛋白?（fibrinolytic serine protease），係利用基因工程開發之血栓溶解劑（thrombolytic agents），臨床上已將tPA 應用治療於腦或心肌之梗塞（infarction）。藥典所收載之tPA 效價測定方法係採觀察血栓溶解時間（clot lysis）的方式來判定，唯此方法之血栓溶解時間並非一個定點，而是一個範圍，因此實驗誤差大，且操作亦極為繁瑣，並不適用於該製劑之例行品質監測檢驗。本研究目標在於應用新方法，以期能達到準確、快速適合於例行品質管制之用途。以毛細管電泳法（capillary zone electrophoresis）、分子篩高效液相層析法（SE-HPLC）、體外血栓溶解法（in vitro clot lysis）、酵素免疫分析法（ELISA）及tPA 酵素基質呈色法等進行tPA 製劑之相關分析，發現tPA 酵素以基質S-2288TM 作呈色反應，靈敏度高且於5-50 μg/mL 間具良好之線性關係，r2 達0.9988 以上，同日間之CV% 介於0.5-4.3%，在測試的幾種方法中，本法具準確、快速及經濟的原則，符合tPA 製劑之例行性檢驗的要求。"

Tissue plasminogen activator, tPA, is a thrombolytic agent for treatment of the brain or myocardium infarction. Clot lysis assay is a functional determination method of tPA described in the pharmacopeia. However, the accuracy of clot lysis assay is insufficient because the lysis-time is not determined by the end-point detection. Therefore, an alternative analytical method for the postmarketing quality survey of tPA pharmaceutical formulations is needed. This study was aimed to establish a rapid, precise and costsaving analytical method for tPA routine test. We have compared methods of capillary zone electrophoresis (CZE), size-exclusion high performance liquid chromatography (SE-HPLC), in vitro clot lysis, enzyme-linked immunosorbent assay (ELISA), and chromogenic substrate assay for the evaluation of recombinant tPA drug products. The results showed that S-2288TM, as substrate of chromogenic assay for tPA, exhibited a good linear relationship at tPA concentration of 5-50 μg/mL (correlation coefficient: 0.9988). The intraday precision ranged between 0.5% - 4.3%. Results obtained from our comparative study, suggested that the chromogenic substrate assay is among the best for tPA assay. The features of precision, rapidity and inexpensiveness of this method are suitable for a routine test for tPA in pharmaceutical formulations.