title: Determination of pegfilgrastim aggregates by size-exclusion high-performance liquid chromatography on a methacrylate-based column.
abstract: 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 TSKgel G4000PWXL column (7.5 mm × 300 mm, 10 μm, <500 A°) 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 °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/ml 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/ml. Over 1200 samples were analyzed by the present method without significant change in the column performance.
authors.affiliation: Medical Cellular & Molecular Research Center, Golestan University of Medical Sciences, Gorgan, Iran; AryaTinaGene Biopharmaceutical Company, Gorgan, Iran.
authors.forename: Majid
authors.initials: M
authors.lastname: Shahbazi
authors.affiliation: AryaTinaGene Biopharmaceutical Company, Gorgan, Iran.
authors.forename: Elnaz
authors.initials: E
authors.lastname: Tamaskany Zahedy
authors.affiliation: AryaTinaGene Biopharmaceutical Company, Gorgan, Iran.
authors.forename: Shiva
authors.initials: S
authors.lastname: Kiumarsi
authors.affiliation: AryaTinaGene Biopharmaceutical Company, Gorgan, Iran.
authors.forename: Mojtaba
authors.initials: M
authors.lastname: Hadi Soltanabad
authors.affiliation: AryaTinaGene Biopharmaceutical Company, Gorgan, Iran.
authors.forename: Saleh
authors.initials: S
authors.lastname: Shahbazi Azar
authors.affiliation: Medical Cellular & Molecular Research Center, Golestan University of Medical Sciences, Gorgan, Iran; AryaTinaGene Biopharmaceutical Company, Gorgan, Iran; Neuroscience Research Center, Golestan University of Medical Sciences, P.O.Box: 49175-553, Gorgan, Iran. Electronic address: haminhplc@yahoo.com.
authors.forename: Hossein
authors.initials: H
authors.lastname: Amini
mesh_terms: D002850:Chromatography, Gel / Q000295:instrumentation / Q000379:methods*; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation / Q000379:methods*; D000069585:Filgrastim; D016179:Granulocyte Colony-Stimulating Factor / Q000032:analysis* / Q000737:chemistry; D008689:Methacrylates / Q000737:chemistry*; D008970:Molecular Weight; D011092:Polyethylene Glycols; D011994:Recombinant Proteins / Q000032:analysis / Q000737:chemistry; D015203:Reproducibility of Results; D013696:Temperature
chemical_list: D008689:Methacrylates; D011994:Recombinant Proteins; D016179:Granulocyte Colony-Stimulating Factor; C455861:pegfilgrastim; D011092:Polyethylene Glycols; D000069585:Filgrastim
keywords: Aggregation; Filgrastim; Methacrylate-based column; PEGylation; Pegfilgrastim; Size-exclusion HPLC