Establishment and verification of chromogenic substrate method for determination of recombinant human glucocerebrosidase activity
10.13200/j.cnki.cjb.004554
- VernacularTitle:重组人葡萄糖脑苷脂酶活性生色底物测定方法的建立及验证
- Author:
WANG Lvyin
- Publication Type:Journal Article
- Keywords:
Recombinant human glucocerebrosidase(rhGCase);
Enzyme replacement therapy(ERT);
Chromogenic substrate method;
Enzyme activity
- From:
Chinese Journal of Biologicals
2025;38(09):1072-1078
- CountryChina
- Language:Chinese
-
Abstract:
Objective To establish and validate a chromogenic substrate method for the determination of enzyme activity of recombinant human glucocerebrosidase(rhGCase) in order to evaluate the enzyme activity of rhGCase and other recombinant enzyme replacement therapy(ERT) products.Methods A chromogenic substrate method for detecting the enzyme activity of CAN103, a rhGCase drug independently developed in china, was established based on enzyme reaction kinetics theory.The concentration of substrate solution(0. 125, 0. 375, 1. 000, 2. 000, 3. 500, 6. 250, 8. 500, 10. 000, 12. 500 mmol/L), dilution multiple of enzyme(16 000, 4 000, 1 000, 500, 400, 300, 200, 100 times dilution), reaction time(2, 5, 10, 12, 15, 18, 20,30 min) and pH of dilution buffer(3. 0, 4. 0, 5. 0, 5. 5, 5. 9, 6. 2, 6. 5, 7. 0, 8. 0) were optimized. The method was verified for theprecision,accuracy,linearityrange,detectionrangeofp-nitrophenol,robustness,stabilityandspecificity.Theenzymeactivities of three batches of Imiglucerase for injection and three batches of Velaglucerase α for injection were detected by the established method.Results The optimum concentration of substrate was 15 mmol/L, dilution times of enzyme was 300 times, reaction time was 12 min, and pH of dilution buffer was 5. 9. The CVs of precision verification were less than 10%. The recovery rates of CAN103 at the relative concentrations of 50%, 75%, 100%, 150% and 200% were all in the range of 95% to 105%. The measured enzymatic reaction rates of sample solution at the relative concentrations of 1. 67-6. 67 μg/mL ranged from 95%to 200%, exhibiting a good linear relationship with the theoretical values, and the linear equation was Y = 0. 928 6 X + 0. 411 0,R~2= 0. 999 9. P-nitrophenol could be quantified accurately in the concentration range of 0. 01 to 0. 20 mmol/L. The CVs of robustness verification were less than 5%. The sample solution could be stored at 2-8 ℃ for 48 h. Dilution buffer and preparation buffer showed no effect on the detection results. The specific activities of Imiglucerase for injection of CW3060,CW3493 and CW4109 were 42. 32, 43. 08 and 40. 77 U/mg, and the specific activities of Velaglucerase α for injection of TVWE04A02, TVVA01A02 and TVVA01A01 were 42. 58, 41. 26 and 41. 41 U/mg, respectively.Conclusion The established chromogenic substrate method has good precision, accuracy and robustness, and can be used for the determination of enzyme activity of rhGCase for injection.
