标准研讨

门冬酰胺酶效价和纯度测定方法研究*

展开
  • 1.中国食品药品检定研究院, 北京102629;
    2.化学药品质量研究与评价重点实验室, 北京 102629
第一作者 Tel:(010)53851530; E-mail:wangyue07@nifdc.org.cn
**Tel:(010)53851585; E-mail:shenghuayaoshi@126.com

修回日期: 2021-05-08

  网络出版日期: 2024-06-21

基金资助

*北京市自然科学基金(7194337)

Study on the methods of purity and potency of asparaginase*

Expand
  • 1. National Institutes for Food and Drug Control, Beijing 102629, China;
    2. NMPA Key Laboratory for Quality Research and Evaluation of Chemical Drugs, Beijing 102629, China

Revised date: 2021-05-08

  Online published: 2024-06-21

摘要

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。

本文引用格式

王悦, 陈欣桐, 李京, 范慧红 . 门冬酰胺酶效价和纯度测定方法研究*[J]. 药物分析杂志, 2022 , 42(1) : 156 -165 . DOI: 10.16155/j.0254-1793.2022.01.18

Abstract

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.

参考文献

[1] BATOOL T, MAKKY EA, JALAL M, et al. A comprehensive review on L-asparaginase and its applications[J].Appl Biochem Biotechnol, 2016, 178(5):900
[2] AVRAMIS VI, PANOSYAN EH. Pharmacokinetic/pharmacodynamic relationships of asparaginase formulations: the past, the present and recommendations for the future[J].Clin Pharmacokinet, 2005, 44(4):367
[3] 罗学群. 不同类型左旋门冬酰胺酶的特点及临床应用[J].中国小儿血液与肿瘤杂志, 2013, 18(1):1
LUO XQ. The characteristics and clinical application of different types of L-asparaginase[J].J China Pediatr Blood Cancer, 2013, 18(1):1
[4] ASSELIN B, RIZZARI C. Asparaginase pharmacokinetics and implications of therapeutic drug monitoring[J].Leuk Lymphoma, 2015, 56(8):2273
[5] 谢鹏. 注射用门冬酰胺酶效价测量的不确定度评定[J].广东轻工职业技术学院学报, 2011, 35(1):18
XIE P. Uncertainty evaluation of asparaginase potency measurement for injection[J].J Guangdong Ind Polytech, 2011, 35(1):18
[6] 中华人民共和国药典2015年版. 二部[S].2015: 45
ChP 2015. Vol Ⅱ[S].2015: 45
[7] EP 9.2[S].2019: 4353
[8] VLUND A. Application of the four-parameter logistic model to bioassay: comparison with slope ratio and parallel line models[J].Biometrics, 1978, 34(3):357
[9] ZENATTI PP, MIGITA NA, CURY NM, et al. Low bioavailability and high immunogenicity of a new brand of E. coli L-asparaginase with active host contaminating proteins[J].EBioMedicine, 2018, 30: 158
[10] 吴敬, 赖龙生, 刘景晶, 等. 重组L-门冬酰胺酶Ⅱ的提取纯化工艺[J].中国医药工业杂志, 2000, 31(2):50
WU J, LAI LS, LIU JJ, et al. Study on extraction and purification technique of recombinant L-asparaginase Ⅱ[J].Chin J Pharm, 2000, 31(2):50
[11] 谢育媛, 孙允芳, 郭江红. 注射用门冬酰胺酶蛋白质杂质研究[J].中国药师, 2016, 19(8):1486
XIE YY, SUN YF, GUO JH. Analysis of protein impurities in asparaginase for injection[J].China Pharm, 2016, 19(8):1486
[12] VANCOILIE G, VERGAELEN M, HOOGENBOOM R. Ultra-high performance size-exclusion chromatography in polar solvents[J].J Chromatogr A, 2016, 1478: 43
[13] 周翔, 何义飞, 陶翎, et al. 门冬酰胺酶反相高效液相色谱分析方法的初步研究[J].药物生物技术, 2017, 24(5):406
ZHOU X, HE YF, TAO L, et al. Preliminory research on RP-HPLC analysis of L-asparaginase[J].Pharm Biotechnol, 2017, 24(5):406
[14] BECKETT A, GERVAIS D. What makes a good new therapeutic L-asparaginase?[J].World J Microbiol Biotechnol, 2019, 35(10):152
[15] FIGUEIREDO L, COLE PD, DRACHTMAN RA. Asparaginase Erwinia chrysanthemi as a component of a multi-agent chemotherapeutic regimen for the treatment of patients with acute lymphoblastic leukemia who have developed hypersensitivity to E. coli-derived asparaginase[J].Expert Rev Hematol, 2016, 9(3):227
文章导航

/