目的: 建立和优化鉴别、纯度和效价测定等质量控制方法,提高和完善大肠埃希菌(Escherichia coli)、欧文氏菌(Erwinia carotovora)来源的门冬酰胺酶的质量标准。方法:考察了Xbridge protein BEH SEC(300 mm×7.8 mm,3.5 μm,200Å)和TSK G3000swxl(300 mm×7.8 mm,5 μm)2款色谱柱在纯度测定方法中的分离情况,并对流动相进行了优化;考察了效价测定方法中反应的量效关系和线性范围,探讨了斜率比模型和标准曲线法的效价计算方式。结果:以0.1 mol·L-1磷酸盐缓冲液(pH 6.7)为流动相,采用Xbridge protein BEH SEC色谱柱进行分离,主峰前后各杂质峰分离度有了较大改善。2种菌种来源的门冬酰胺酶效价反应剂量在2~30 U·mL-1范围内均呈直线反应,r均大于0.995,标准曲线测定方法更为简单、准确,样品采用高低双剂量可以有效消除实验误差,双剂量测定偏差可小于±2.0%。结论:与绝对法相比,相对法在效价测定的准确度和重现性方面都有较大提高;优化后的纯度测定方法提高了杂质峰的分离度,可以更好地避免因积分方式带来的结果误差。
Objective: To establish and optimize the method for determining the purity and potency of asparaginase(Escherichia coli) and asparaginase(Erwinia carotovora) and improve its quality standards. Methods: The separation effects of Xbridge protein BEH SEC column (300 mm×7.8 mm, 3.5 μm, 200Å) and TSK G3000swxl (300 mm×7.8 mm, 5 μm) were tested and the mobile phase was optimized for the purity method. The form of the dosage-effect relationship and the linear range of assay were examined, and meanwhile the feasibility of the slope ratio model and the standard curve model were evaluated. Results: Improved resolution between the impurity peak and the main peak were obtained using the Xbridge protein BEH SEC column. Besides, the assay results showed that, it was a linear response for both of the asparaginases in the dosage range of 2-30 U·mL-1 and the regression coefficient r could be greater than 0.995. Meanwhile,the standard curve measurement method was more accurate and simple, the sample using double dosage could effectively eliminate the experimental errors, and the measurement deviation could be less than ±2.0% between two dosages. Conclusion: Compared with the absolute method, the relative method has greatly improved the accuracy and reproducibility of potency determination. Better resolutions can be obtained by using the optimized purity determination method, which can better avoid the result error caused by the integration method.
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