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HPLC Analysis of Reduced Monoclonal Antibody Fragments on BIOshell™ A400 Protein C4

HPLC Analysis of Reduced Monoclonal Antibody Fragments 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. 90 C
mobile phase [A] 80:20, (0.1% TFA in water):(0.1% TFA in acetonitrile) ; [B] 50:50, (0.1% TFA in water):(0.1% TFA in acetonitrile)
gradient 30 to 70%B in 12 min
flow rate 0.3 ml/min
pressure 1000 psiinitial (69 bar)
injection 1 µl
detector UV, 215 nm


Analysis Note Digesting an antibody into a few large fragments facilitates a middle-down approach for subsequent structural characterization. The IdeS enzyme, from Streptococcus pyogenes and commercialized as FabRICATOR, cleaves IgG in the hinge region resulting in two fragments: F(ab′)2 and scFc. Reduction of F(ab′)2 dissociates it into its component polypeptides, Fd′ and LC. This application demonstrates the baseline resolution of the three fragments scFC, LC, and Fd′, in reasonable time with excellent peak shape.
Categories Analytical Chromatography, Forensics and Toxicology, Proteins, proteins, Antibodies, Forensics And Toxicology Antibodies
Featured Industry Life Science and Biopharma
Legal Information BIOshell is a trademark of Sigma-Aldrich Co. LLC
Other Notes Monoclonal antibody SO57 (0.5 mg) was digested by IdeS protease according to Genovis ( FragIT kit protocol. The digest was then exposed to 10 mM dithiobutylamine for one hour, at pH 7, before being diluted 1:5 with mobile phase A prior to injection; final protein conc. <0.3 g/L.
Genovis FragIT kit is available from Sigma, outside the USA.
suitability application for HPLC


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