UHPLC/MS Analysis of Oxidized Filgrastim Variants on BIOshell™ A400 Protein C4

UHPLC/MS Analysis of Oxidized Filgrastim Variants on BIOshell™ A400 Protein C4


column BIOshell A400 Protein C4, 10 cm x 2.1 mm I.D., 3.4 µm particles (66825-U)
column temp. 75 C
mobile phase [A] water (0.1% v/v difluoroacetic acid); [B] acetonitrile (0.1% v/v difluoroacetic acid)
gradient 50 to 53% B in 10 min
flow rate 0.2 ml/min
sample oxidized filgrastim, 100 µg/mL, 0.1% hydrogen peroxide (aq)
injection 1 µl
detector MS, ESI(+), TIC, 1000-20000 m/z


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 amino acid side chains like methionine side chains (sulfhydryl oxidation to the sulfoxide). In a sample of filgrastim that had been stressed with dilute (0.1%) hydrogen peroxide, five oxidized variants can be resolved on a BIOshell A400 Protein C4 column. Using mass spectrometry (MS), one can also determine the number of oxidation events that each analyte was subjected to by looking at the mass shift between the native filgrastim molecular weight and the molecular weight of each analyte (determined by deconvolution of the total ion current (TIC) chromatogram).  
Categories Analytical Chromatography, Proteins, Peptides
Featured Industry Life Science and Biopharma
Legal Information BIOshell is a trademark of Sigma-Aldrich Co. LLC
Other Notes The oxidized filgrastim sample was prepared by resuspending a 0.98 mg filgrastim standard to 0.98 mg/mL with water, then diluting to 100 μg/mL with 0.1% v/v hydrogen peroxide. The sample was allowed to equilibrate for five hours at room temperature prior to assay.
suitability application for UHPLC


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