目的: 建立重组C因子检测注射用重组Ⅲ型人源化胶原蛋白溶液细菌内毒素含量的方法。方法: 通过采用生物梅里埃重组C因子试剂盒、龙沙重组C因子试剂盒,分别对3批注射用重组Ⅲ型人源化胶原蛋白溶液进行了测定,并进行方法验证。结果: 2种试剂盒的标准曲线浓度点≥ 3个,线 性 r >0.980;阴性对照的ΔRFU值小于标准曲线最低点的ΔRFU值;细菌内毒素含量均< 6 EU · mL-1,重复性良好,细菌内毒素的回收率在50%~200%,符合《中华人民共和国药典》标准要求。结论: 注射用重组Ⅲ型人源化胶原蛋白溶液中细菌内毒素含量采用2个试剂盒测定的结果分别为<0.05 EU · mL-1 和<0.005 EU · mL-1,符合产品细菌内毒素的标准要求。该方法可用于注射用重组Ⅲ型人源化胶原蛋白溶液中细菌内毒素含量的测定。本文为采用重组C因子法测定重组蛋白产品中内毒素的相关研究提供参考。
Objective: To establish a recombinant C-factor method to detect the content of bacterial endotoxin in recombinant type Ⅲ humanized collagen solution for injection. Methods: Three batches of injectable recombinant type Ⅲ humanised collagen solutions were determinedand method validation by using the BIOMERIEUX recombinant C-factor kit and the lonza recombinant C-factor kit,respectively. Results: The results showed that about the two kits the concentration points of the standard curve were ≥ 3, and the linear correlation coefficient was r > 0.980, the ΔRFU value of the negative control was smaller than that of the lowest point of the standard curve; the bacterial endotoxin contents were all < 6 EU · mL-1. The reproducibility was good, the recoveries of bacterial endotoxin were in the range of 50%-200%, which was in accordance with the standard requirements of the ChP. Conclusion: The bacterial endotoxin content in the solution of recombinant type Ⅲ humanized collagen solution for injection is determined by the two kits. The results were < 0.05 EU · mL-1 and < 0.005 EU · mL-1, which are in accordance with the standard requirements for bacterial endotoxin in the product, respectively. The method can be used for the determination of bacterial endotoxin content in the solution of recombinant type Ⅲ humanized collagen solution for injection. This study provides a reference for the research related to the determination of endotoxin in recombinant protein products using recombinant C-factor method.
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