HPLC Analysis of Native and Oxidized Filgrastim on BIOshell™ A400 Protein C4 Column

HPLC Analysis of Native and Oxidized Filgrastim on BIOshell™ A400 Protein C4 Column


column BIOshell A400 Protein C4, 10 cm x 2.1 mm I.D., 3.4 µm particles (66825-U)
column temp. 60 C
mobile phase [A] Water (0.1% trifluoroacetic acid); [B] Acetonitrile (0.1% trifluoroacetic acid)
gradient 55 to 70% B in 5 min.
flow rate 0.2 ml/min
pressure 590 psi (41 bar)
sample Native Filgrastim, 98 µg/mL, WaterOxidized Filgrastim, 98 µg/mL, 0.1% Hydrogen peroxide
injection 1 µl
detector UV, 215 nm


Analysis Note Filgrastim is a 175 amino acid polypeptide produced by recombinant DNA technology in genetically engineered Escherichia coli cells. This protein is used as a biosimilar to granulocyte colony-stiµlating factor (GCSF) to treat neutropenia, a disease characterized by a low concentration of neutrophils in a person′s blood due to chemotherapy, bone marrow transplantation, or genetic predisposition. Common impurities found in this biosimilar are oxidized filgrastim variants which are produced by the oxidation of methionine side chains (sulfhydryl oxidation to the sulfoxide). In a sample of filgrastim that had been stressed with dilute (0.1%) hydrogen peroxide, four oxidized variants can be resolved from native (i.e. unoxidized) filgrastim on a BIOshell A400 Protein C4 column. The top chromatogram shows native filgrastim and the bottom chromatogram shows filgrastim after oxidative stressing.
Categories Analytical Chromatography, Proteins, Peptides
Featured Industry Life Science and Biopharma
Legal Information BIOshell is a trademark of Sigma-Aldrich Co. LLC
Other Notes Oxidized filgrastim sample was prepared by mixing 50 μL of a 980 μg/mL filgrastim standard solution with 450 μL of 0.1% hydrogen peroxide and allowing the sample to equilibrate at room temperature for five hours prior to assay.
suitability application for HPLC


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