Fast UHPLC Analysis of SILu™ Lite SigmaMAb Universal Antibody Standard IdeS Fragments on BIOshell™ A400 Protein C4

Fast UHPLC Analysis of SILu™ Lite SigmaMAb Universal Antibody Standard IdeS Fragments on BIOshell™ A400 Protein C4


sample preparation Deglycosylation of the monoclonal antibody standard was per protocol for an immobilized EndoS2 glycosidase (Genovis Inc). (Proteolysis was per protocol for an immobilized IdeS potease (Genovis Inc). Reduction following proteolysis was by 10 mM dithiobutylamine in neutral buffer.)
column BIOshell A400 Protein C4, 10 cm x 2.1 mm I.D., 3.4 µm particles (66825-U)
column temp. 80 °C
mobile phase [A] 70:30, (0.1% TFA in water):(0.1% TFA in acetonitrile) ; [B] 50:50, (0.1% TFA in water):(0.1% TFA in acetonitrile)
gradient 15 to 100% B in 6 min
flow rate 0.2 mL/min
pressure 600 psi (initial)
sample 1 g/L antibody standard
injection 0.5 µL
detector UV, 215 nm


Analysis Note Proteolysis of IgG by IdeS yields three large fragments amenable to further characterization. The three fragments are well separated in minimal time at high temperature on BIOshell A400 Protein C4. Deglycosylation does result in greater retention of the the singly clipped Fc fragment, as would be predicted. The initial mobile phase conditions were selected to ensure adequate retention so as to yield good peak shape for the singly clipped Fc fragment (peak 1).

Both top-down and bottom-up approaches to protein characterization have their advantages and limitations. One such limitation of the top-down approach, particularly with large proteins, is the need for state-of-the-art MS resolution, and interpretation of the complexity of the generated data. One popular approach to address the experimental constraints of a top-down strategy is to employ an intermediate or combined approach that can be termed a middle-down and/or middle-up approach. Instead of dealing with the intact mass of the large protein, or all the peptides of a bottom-up approach, the protein sample is first cleaved into just a few fragments which upon separation can be handled individually. This has been made popular in the case of monoclonal antibody (mAb) characterization, by use of the protease IdeS.1 This protease cleaves IgG into two fragments: the two antigen binding domains held together by disulfide bonds [F(ab)2’] and the two Fc domains. Upon disulfide reduction the F(ab)2’ fragment yields the constituent Fd’ domain and the light chain. Instead of characterizing the intact IgG, the researcher now has three smaller IgG fragments to analyze (Fd’, Fc, and the light chain). BIOshell A400 Protein C4 resolves all three of these very well in minimal time, with standard mobile phases for protein or peptide reversed-phase chromatography. Elevated column temperature is essential to obtaining the best peak shape. In the example shown, a native mAb standard (Sigma p/n MSQC4) was compared to a sample that had been deglycosylated (IgG is glycosylated on the Fc domain). Both native and deglycosylated mAb were digested by IdeS and then reduced with dithiobutylamine. It’s not surprising that the deglycosylated Fc fragment does show slightly greater retention. Good peak shape typically requires adequate retention, and this is evident in the elution time of the Fc fragment.

1.Pawel-Rammingen, et. al. IdeS, a novel streptococcal cysteine proteinase with unique specificity for immunoglobulin G. EMBO J, 2002, 21(7), 1607.
Categories Analytical Chromatography, Proteins
Featured Industry Life Science and Biopharma
Legal Information SILu is a trademark of Sigma-Aldrich Co. LLC, BIOshell is a trademark of Sigma-Aldrich Co. LLC
suitability application for UHPLC


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