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

By: Hillel Brandes, and Stacy Shollenberger, Reporter US Volume 34.1

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 (Product No. 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 show slightly greater retention. Good peak shape typically requires adequate retention, and this is evident in the elution time of the Fc fragment.

Fast UHPLC Analysis of Lite SigmaMAb Universal Antibody Standard IdeS Fragments

Figure 1. Fast UHPLC Analysis of Lite SigmaMAb Universal Antibody Standard IdeS Fragments on BIOshell A400 Protein C4




  1. Pawel-Rammingen, et. al. 2002. IdeS, a novel streptococcal cysteine proteinase with unique specificity for immunoglobulin G. EMBO J, 21(7):1607


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