Determination of Antioxidants and Their Degradation Products in Recombinant Exendin-4-FC Fusion Protein Injection by HPLC
10.13748/j.cnki.issn1007-7693.20223808
- VernacularTitle:HPLC测定重组Exendin-4-FC融合蛋白注射液中抗氧剂及其降解物
- Author:
Zehua LU
1
;
Sulong JI
1
;
Shuaihu LIU
1
;
Li WANG
1
;
Yan GAO
1
;
Zhiqiang SHEN
2
;
Jingyan LI
1
;
Bin WANG
2
Author Information
1. Henan Shengming Biotechnology Research Co., Ltd., Xinxiang 453000, China
2. Hualan Genetic Engineering Co., Ltd., Xinxiang 453000, China
- Publication Type:Journal Article
- Keywords:
recombinant Exendin-4-FC fusion protein injection ；antioxidants； HPLC
- From:
Chinese Journal of Modern Applied Pharmacy
2024;41(1):112-118
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVE :To establish a method for determining the content of 11 antioxidants and their degradation products in recombinant Exendin-4-FC fusion protein injection by HPLC.
METHODS
The protein was precipitated with saturated ammonium sulfate. After centrifugation, the supernatant was transferred to a C18 solid phase extraction cartridge activated by methanol. Then the cartridge was eluted with 4 mL of methanol and 5 mL of ethyl acetate respectively, and the eluent was diluted with methanol-ethyl acetate(2∶3) mixed solvent and passed through a 0.22 µm PTFE hydrophobic filter. It was analyzed by HPLC and quantified by external standard method. Chromatographic conditions: Kinetex® XB-C18 100Å (100 mm×4.6 mm, 2.6 µm)column, the detection wavelength was 230 nm, the column oven was 30 ℃, the injection volume was 5 µL and the flow rate was 0.4 mL·min–1, mobile phase was 0.1% formic acid-methanol(A)-0.1% formic acid aqueous solution(B), the running time was 45 min.
RESULTS
The 11 target substances showed a good linear relationship in the range of 2.5−35 μg·mL–1 with R2 ≥0.99. At three different concentration(25, 10, 5 μg·mL–1) of spiked samples, the average recovery rates of 11 antioxidants ranged from 88.1% to 106.5%, with RSDs in the range of 0.10%–9.05%. The RSDs of 6 repeatable samples was 2.01%–4.77%, which of 12 intermediate precision samples was 2.58%–9.75%. The positive/inverted samples of three batches of recombinant Exendin-4-FC fusion protein injection were detected at 0 month, 3 months and 6 months(25 ℃), and the results showed that there was no antioxidant and its degradation leaching in all batches of samples at different detection points.
CONCLUSION
The method has good specificity, high accuracy and precision, good solution stability, high durability and can be used for the content detection of antioxidants in drugs.
