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

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

Conditions

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 1,000 psi initial (69 bar initial)
injection 1 µL
detector UV, 215 nm

Description

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
Other Notes Monoclonal antibody SO57 (0.5 mg) was digested by IdeS protease according to Genovis (www.genovis.com) 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 us outside the USA.
suitability application for HPLC

Materials

     
Related Links