| 王琰,张培培,姚尚辰,尹利辉,林兰,张彦民,胡昌勤.采用超滤前处理考马斯亮蓝法测定酶法工艺产品阿莫西林中残留蛋白的含量[J].中国药事,2020,34(1):39-46 |
| 采用超滤前处理考马斯亮蓝法测定酶法工艺产品阿莫西林中残留蛋白的含量 |
| Determination of Residual Proteins in Amoxicillin Produced through Enzymatic Process by Ultrafiltration Pretreatment Bradford Method |
| 投稿时间：2019-04-04 |
| DOI：10.16153/j.1002-7777.2020.01.006 |
| 中文关键词: 超滤 Bradford法 酶法工艺 阿莫西林 残留蛋白 含量测定 |
| 英文关键词: ultrafiltration Bradford method enzymatic process amoxicillin residual proteins determination |
| 基金项目:国家“重大新药创制”科技重大专项资助项目（编号2017ZX09101001-007） |
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| 中文摘要: |
| 目的：药品中外源性残留蛋白不仅影响药品质量，且严重威胁人用药安全。青霉素G酰基转移酶（简称PGA）是酶法工艺生产β-内酰胺抗生素过程中所用关键催化酶，有必要对其进行研究并严格控制终产品中的蛋白残留量。已有方法采用凝胶过滤法（GFC）对残留蛋白进行前处理，但该法存在富集效率低等诸多问题。本文探讨并尝试建立一种不同方式和原理的蛋白分离、富集方法，采用超滤前处理结合考马斯亮蓝（Bradford）法测定酶法工艺产品阿莫西林中残留蛋白的含量。方法：采用Amicon®Ultra-15型超滤管（15 mL，截留分子量为3 kDa）对阿莫西林供试液进行前处理，离心力为5000 g（7000rpm），对溶液进行净化并对蛋白进行浓缩富集，再采用Bradford法对浓缩液进行蛋白定量测定，检测波长为595 nm。结果：该法具有较好的专属性；蛋白浓度在1.0~100.0 μg·mL-1（r=0.9921）内校正曲线（lg A~lg C）的线性关系良好；回收率>72%（n=3×3）；Bradford法最低检出限为0.1 μg·mL-1，相当于0.0001%；Bradford法的精密度≤ 5.0%。结论：该方法准确、可靠，相比于已有方法，蛋白富集量更大、操作简便、设备要求低，可用于酶法工艺阿莫西林中残留蛋白的测定，对酶法工艺半合成抗生素中残留蛋白的质控及酶法工艺稳定性监测提供了一定技术指导。 |
| 英文摘要: |
| Objective: Exogenous residual proteins in drugs not only affect the quality of drugs, but also seriously threaten the safety of drug use. Penicillin G acylase (PGA) is a critical catalytic enzyme utilized in the amoxicillin production by the enzymatic process. It is necessary to study it and strictly control the content of residual proteins in the final products. The gel filtration method (GFC) was used to pretreat residual proteins but the method had many problems such as low enrichment efficiency. In this study, a different principle and method for separating and enriching proteins was explored and established. An ultrafiltration pretreatment combining with Bradford method was used for the determination of residual proteins in amoxicillin produced through enzymatic process. Methods: Amicon® Ultra-15 (15 mL, MWCO 3 kDa) was used for the pretreatment of amoxicillin test solution in terms of purification of solution and protein enrichment. Centrifugal force was 5000 g (7000 rpm). Then, Bradford method was used for the quantitation of residual proteins in concentrated solution. The detection wavelength was 595 nm. Results: This method had good specificity. The linearity of the calibration curve (lgA-lgC) was good within the concentration range of 1.0~100.0 μg·mL-1 (r=0.9921). The recovery rate was more than 72% (n=3×3). The limit of detection of Bradford method was 0.1 μg·mL-1 with an equivalence to 0.0001%. The precision of Bradford method was ≤ 5.0%. Conclusion: Compared to the other methods, the established method in this study is accurate, reliable, more protein-enriched, easy to be operated, low equipment requirements and suitable for the determination of residual proteins in amoxicillin produced by enzymatic process, which provides a technical guidance for the quality control of residual protein of antibiotics produced by enzymatic process and stability monitoring of enzymatic process. |
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