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.

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Objective To prepare the 1st batch of national standard of recombinant trypsin in order to standardize and improve the quality of recombinant trypsin.Methods Enzyme-substrate identification,HPLC identification,N-terminal sequencing and TOF-MS were used to confirm the property and structure of recombinant trypsin;the purity was determined by HPLC;according to the methods in Chinese Pharmacopoeia(Volume Ⅲ 3603,2020 edition),the specific activity of the candidate standard was determined,and the stability and uniformity were investigated.Results The structure of recombinant trypsin was confirmed,and the specific activity of the 1 st batch of national standard of recombinant trypsin was 5 169 U/mg,containing 80% β-trypsin and 9% α-trypsin.The RSD of purity of α,β-trypsin and retention time of 12 candidate standards were all less than 2.0%.The purity of α,β-trypsin showed no obvious decrease stored at 25 ℃ and relative humidity(RH) 80% for 10 d,while the purity of β-trypsin decreased slightly and the purity of α-trypsin increased slightly stored at 40℃ and RH 80% for 10 d.The purity of β-trypsin decreased slightly when exposed to light(4 000 lx) for 10 d.Conclusion The national standard of recombinant trypsin with accurate structure and high purity was prepared,which can be used for system suitability test of the purity determination.