FabRICATOR digestion generates antibody subunits and is used to study antibody oxidation. The robustness of the digestion allows monitoring of antibody subunit oxidation in regulated environments.
FabRICATOR (IdeS) is a cysteine protease that specifically cuts IgG into F(ab’)2 and Fc/2 fragments, that can be further reduced into Fc/2, Fc and LC fragments. The reaction is fast, 30 min incubation at 37°C, and robust and the method can easily be transferred to different types of antibodies and does not require optimization. Further, the enzyme is active in a range of buffers, including formulation buffers, which makes the sample preparation easy.
Figure 1. Graphical illustration of FabRICATOR digestion of IgG followed by reduction into Fc/2, Fd and LC fragments.
Oxidation of antibodies is a key quality attribute that may affect therapeutic antibody functionality and needs close quantification and characterization. The oxidation causes a +16 Da size difference and at the same time the charge of the molecule or domain is changed. For this reason, LC/MS or RP-HPLC can be used to study and monitor oxidation on antibodies.
Researchers at Merck have induced oxidation on monoclonal antibodies and used FabRICATOR to digest antibodies into subunits to monitor oxidation levels (An et al. 2014). Oxidation was induced in the antibody using chemicals, tBHP, and light, 0.5X ICH, and the results revealed shifts in the LC separation that correlated to +16 Da or +32 Da difference, indicating oxidation of one or two amino acids (Figure 2). This study highlight the subunit level of study allows an easy study of antibody oxidation. The authors conclude that FabRICATOR is an important tool for rapid and easy characterization of antibody oxidation.
Figure 2. Oxidation of mAb as studied using FabRICATOR digestion, and induction of oxidation using chemical, tBHP, and light, 0.5X ICH.
A similar approach has been published by researchers from LFB, but focusing on the oxidation levels of IVIG preparations, consisting of pooled IgGs (Leblanc et al. 2014). The authors conclude that the FabRICATOR based method could detect methionine oxidation subunits in a complex sample with high precision.
FabRICATOR is available as a lyophilized enzyme, immobilized on spin columns or in 96-well plate format for convenient sample preparation.
An, Y. et al., 2014. A new tool for monoclonal antibody analysis: application of IdeS proteolysis in IgG domain-specific characterization. mAbs, 6(4), pp.879–893. Download the paper here
Leblanc, Y. et al., 2014. LC-MS analysis of polyclonal IgGs using IdeS enzymatic proteolysis for oxidation monitoring. Journal of chromatography. B, Analytical technologies in the biomedical and life sciences, 961, pp.1–4.