Interlaboratory validation of ion chromatography for analysis of N-glycan profile of human erythropoietin
Objective To validate a method,ion chromatography(IC)coupled with pulsed amperometric detector(PAD),for the analysis of recombinant human erythropoietin(rhEPO)N-glycan profile in several laboratories.Methods The buffer solution of rhEPO sample was replaced by ultrafiltration with 25 mmol/L phosphate buffer solution,and the concentration was adjusted to 2 mg/mL.After enzymatic digestion with glycosidase F,the supernatant was precipitated with ethanol,and freeze-dried to obtain free N-glycan of rhEPO.The chromatographic conditions were as follows:The analytical column was Dionex CarboPac PA200,and the protection column was Dionex CarboPac PA200.Mobile phase A was 50 mmol/L sodium hydroxide solution,mobile phase B was 200 mmol/L sodium hydroxide solution,and mobile phase C was 250 mmol/L sodium acetate solution.The injection volume was 25 µL with the flow rate of 0.5 mL/min and the column temperature of 30℃,and gradient elution was performed.The retention time of the highest peak in the peak cluster and the percentage area of the peak cluster were obtained with the analysis software of the instrument.Three laboratories(L1-L3)were selected for joint validation of the method,including accuracy,precision,linearity,limit of detection(LOD)and limit of quantification(LOQ),as well as stability.Results The peaks of rhEPO N-glycan were clearly visible.The area percentage of each peak cluster of the samples with different concentrations in three laboratory ranged from 84％to 116％,and the accuracy of the method was good.The RSDs of the highest peak retention time in the peak clusters were all less than 5％,the RSDs of the percentage area were all less than 10％,and the reproducibility of the method was good.When the protein concentration was in the range of 1.0-3.0 mg/mL,the linearity was good,R2＞0.98.The LOD and LOQ were approximately 0.10mg/mL and 0.32 mg/mL respectively.The stability was good at 2-8 ℃ for 48 h.Conclusion The IC analysis methodology for rhEPO N-glycan profile was established,and the interlaboratory validation indicators were good,which will provide tech-nical support for improving the quality standard of rhEPO.
Human erythropoietin(hEPO)N-glycan profileIon chromatography(IC)Interlaboratory validation
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