Standardization of the first batch of national reference standard of lysozyme

LIU Li-sha; HE Dong-mei; LI Jing; DONG Xin; HE Lan-ying; ZHANG Dou-sheng; REN Li-ping; FAN Hui-hong

doi:10.20251/j.cnki.1003-3734.2026.05.008

Abstract：Objective:To establish the first batch of the national reference standard of lysozyme for content determination.Methods:The structure of the candidate lysozyme reference material was confirmed by HRMS,IR,UV spectroscopy and electrophoresis.Hygroscopicity,isoelectric point(pI),total nitrogen content,and HPLC purity were determined.A collaborative calibration was conducted by six laboratories from drug control institutes and lysozyme manufacturers using two methods:potency determination of the candidate material using the lysozyme potency assay method specified in the 6th volume of the second part of the Ministry of Health's Drug Standards;and content determination using the JP lysozyme hydrochloride assay method with JP lysozyme reference standard(Lot LYS03A)as the reference.Results:The structure of the candidate lysozyme material was confirmed.Hygroscopicity testing indicated it is extremely hygroscopic.The pI was determined to be 10.1,total nitrogen content was 16.8%,and HPLC purity was 94.0%.Collaborative calibration yielded a mean potency of 20 219.85 units·mg-1 with a 95%confidence interval of 18716.48 units·mg-1 to 21723.22 units·mg-1 and a mean content of 0.9765 mg(potency)·mg-1(anhydrous basis)with a 95%confidence interval of 0.9565 mg(potency)·mg-1 to 0.9966 mg(potency)·mg-1(anhydrous basis).Conclusion:Approved by the National Drug Reference Standards Committee,the first batch of candidate lysozyme reference material(batch 140878-202401)was assigned a value of 0.98 mg(potency)·mg-1(anhydrous basis),and authorized for use as the national reference standard for content determination in lysozyme raw materials and preparations.