Antibody Oxidation
Analysis of oxidation at the Subunit Level
Oxidation of antibodies is a key quality attribute that may affect therapeutic antibody functionality and needs close quantification and characterization. It can result in reduction of the in vivo half-life, efficacy, and stability of the mAb. FabRICATOR digestion generates antibody subunits ideal for the study of antibody oxidation. The robustness of the digestion allows monitoring of antibody subunit oxidation in regulated environments.
Subunit Oxidation Analysis using FabRICATOR
Figure 1. Classical middle down workflow for mAb analysis using LC/MS
The oxidation of an antibody causes a +16 Da size difference and at the same time the charge of the molecule or domain is changed. The characterization and monitoring of the oxidization of antibodies is typically done on LC/MS or RP-HPLC systems. The cysteine protease FabRICATOR (IdeS) specifically cuts IgG into F(ab’)2 and Fc/2 fragments, which can be reduced into Fc/2, Fd' and LC fragments (Fig. 1).
Digestion with FabRICATOR can be done in a range of buffers, including formulation buffers, making sample preparation quick and easy. It is a robust and fast digestion that can be easily transferred to different types of antibodies without the need for optimization.
Antibody Subunit Separation of Oxidation Peaks
Researchers at Merck induced oxidation on monoclonal antibodies and monitored the oxidation levels by looking at FabRICATOR digested antibody subunits (An et al. 2014). The oxidation was induced by treating samples with the chemical tert-butylhydroperoxide (tBHP) or light exposure. The results from tBHP and light exposure of 0.5X ICH revealed shifts of +16 Da and +32 Da in the Fc/2 subunit, indicating oxidation of one or two methionines respectively. A separate oxidized peak (+16 Da) was observed for the Fd fragment while no oxidation was observed for the LC fragment (Fig. 2). The authors concluded that FabRICATOR is an important tool for rapid and easy characterization of antibody oxidation. They also highlighted the advantages of the exceptional site specificity and activity the enzyme has toward IgG subclasses.
Validated QC Assay for Subunit Oxidation Monitoring
In the final example, Sokolowska and colleagues at Janssen Research and Development qualified and validated a subunit LC-MS method for quality control and stability testing of the oxidation status of commercial antibodies. While the implementation of LC-MS methods in commercial QC labs is challenging due to the lack of options for fully GMP compliant systems, Sokolowska et al. successfully developed an automated LC-MS method requiring minimum analyst training. They combined FabRICATOR with IgGZERO to yield deglycosylated subunits after treatment with DTT. Using RP-HPLC coupled to QTOF, methionine oxidation patterns could be monitored for stability testing and commercial product release. Together with automated data processing using validated GMP compliant software, they show the workflow in action in a QC environment.
