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

HPLC Analysis of 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 initial)
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 is a Streptococcal protease (commercialized as FabRICATOR) that cleaves IgG in the hinge region resulting in two fragments: F(ab′)2 and scFc.
Categories Analytical Chromatography, Antibodies, Proteins
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 diluted 1:5 with mobile phase A, before 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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