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| Content determination of sialic acid in recombinant human erythropoietin by high-performance anion-exchange chromatography-pulsed amperometric detection |
| LI Xiang YU Lei FAN Wenhong ZHOU Yong RAO Chunming |
| Division of Recombinant Biological Products, National Institutes for Food and Drug Control, Beijing 100050, China |
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Abstract Objective To develop a high-performance anion-exchange chromatography-pulsed amperometric detection (HPAEC-PAD) method to determine the content of sialic acid in recombinant human erythropoietin (rhEPO). Methods The rhEPO samples were prepared by changing rhEPO bulks to 5 mmol/L phosphate buffer (pH 7.0), and adjusted to 0.33 mg/mL. Taking 20 μL sample solution, and then 20 μL of 10 mU/μL neuraminidase was added, followed by incubation at 37℃ for 1 h. Finally, 50 μL 0.04 mmol/L ketodeoxynonulosonic acid (KDN) and 70 μL milli-Q water were added and mixed thoroughly followed by detection. CarboPac PA1 separation column and CarboPac PA100 guard column were used, with mobile phase being mixture of 150 mmol/L sodium hydroxide solution and 125 mmol/L sodium acetate solution, column temperature being 30℃, flow rate being 1.0 mL/min, and the data acquisition time being 15 min with isocratic elution. The sialic acid content was calculated based on the dose-effect relation of the ratio of peak area of sialic acid to KDN and the content of sialic acid. Results The concentrations of sialic acid standard solutions and the ratios of peak area of sialic acid to KDN showed a good linearity relationship between 0.0015 and 0.0075 μg/mL with R2 being higher than 0.99, the repeatability RSD was below 4%. The recovery rates of samples at low, medium and high concentration were 94.9%, 94.3% and 96.7%, respectively. Six batches of rhEPO were tested by the developed HPAEC-PAD method and the results had no significant differences compared with the results of resorcinol method (P = 0.7488). Conclusion This research has successfully established the HPAEC-PAD method to determine the content of sialic acid in rhEPO, with good linearity, repeatability and robustness, which can be used in routine determination of sialic acid in rhEPO.
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