Determination of pegfilgrastim aggregates by size-exclusion high-performance liquid chromatography on a methacrylate-based column

摘要

A size-exclusion high-performance liquid chromatographic method using a methacrylate-based column was developed, validated and implemented for the determination of pegfilgrastim aggregates. The samples were directly injected into a TSKgeI G4000PWXL column (7.5 mm x 300 mm, 10 mu m, <500 A degrees) with a mobile phase of 100 mM phosphate, pH 2.5. Detection was made at 215 nm and analyses were run at a flow-rate of 0.6 ml/min at 10 degrees C. Vortex-mixing of samples produced oligomers, however, very high molecular weight aggregates were formed at high temperatures. The method exhibited linearity over the concentration range of 0.1-14 mg/m1 for pegfilgrastim monomer and high molecular weight aggregates with a correlation coefficient of greater than 0.99. The method was specific and sensitive, with a lower quantification limit of 0.1 mg/ml and a detection limit of 0.02 mg/mi. Over 1200 samples were analyzed by the present method without significant change in the column performance. (C) 2017 International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.