Establishment of a method for determining the key parameters of hydrolysis kinetics of acid α-glucosidase for injection by ion chromatography
10.16438/j.0513-4870.2024-0495
- VernacularTitle:离子色谱法测定注射用阿糖苷酶α糖原水解动力学关键参数
- Author:
Xin-yue HU
1
;
Jia-hao KONG
2
;
Yue SUN
1
;
Lü-yin WANG
1
;
Xiao-ming ZHANG
1
;
Ping LÜ
1
;
Cheng-gang LIANG
1
;
Jing LI
1
Author Information
1. National Institutes for Food and Drug Control, State Key Laboratory of Drug Regulatory Science, NHC Key Laboratory of Research on Quality and Standardization of Biotech Products, NMPA Key Laboratory for Quality Research and Evaluation of Biological Products, NMPA Key Laboratory for Quality Research and Evaluation of Chemical Drugs, Beijing
2. Beijing Institute of Petrochemical Technology, Beijing
- Publication Type:Research Article
- Keywords:
acid α-glucosidase for injection;
ion chromatography;
pulse amperometric detector;
Michaelis-Menten equation;
enzyme kinetics;
on-line automation
- From:
Acta Pharmaceutica Sinica
2024;59(12):3361-3366
- CountryChina
- Language:Chinese
-
Abstract:
The Dionex CaboPacTM PA10 BioLCTM Analyical 2 mm × 250 mm column was used with a protective column (Dionex CaboPacTM PA10 BioLCTM Guard 2 mm × 50 mm). 100 mmol·L-1 sodium hydroxide solution was used as eluent; the flow rate was 0.25 mL·min-1. Sample tray temperature: 35 ℃. The pulse amperometric detector was adopted, and the waveform was Gold CWE, Ag-AgCl RE, Carbo, Quad. The samples were cultured with 8 concentrations of glycogen substrates (0.31, 1.25, 2.5, 5, 10, 20, 30, and 40 mg·mL-1). D-Glucose concentrations were measured at 5 different time points (T0, T1, T2, T3 and T4). The glucose concentration from T1 to T4 minus the glucose concentration at T0. The reaction rate was calculated at different glycogen substrate concentrations. These reaction rates are plotted against substrate concentrations using Michaelis-Menten equation. The kinetic parameters were expressed as Vmax (nmol·mg-1·min-1) and Km (mg·mL-1). The RSD of glucose standard curve R2 (n = 6, linear range: 1.25-500 μmol·L-1) was 0.1% and the RSD (n = 6) of the slope of the standard curve was 2.2%. The mean limit of quantitation was 0.14 μmol·L-1, and the mean limit of detection was 0.05 μmol·L-1. The RSD of Km and Vmax were 4.4% and 4.6% respectively in three separate experiments. The durability of the method was good. The method was developed for the on-line automatic determination of the hydrolysis kinetics of acid α-glucosidase (GAA) for injection by ion chromatography. The method has good precision, repeatability and durability, and can be used for the determination of glycogen hydrolysis kinetics of GAA for injection, and could reference value for the enzyme kinetics evaluation of recombinant enzyme replacement therapy.
