Subunit Analysis of mAbs


More recently, middle-level approaches have grown in popularity. Antibody subunit analysis has become a widely accepted alternative to the bottom-up and top-down analytical strategies, providing rapid characterization of therapeutic antibodies and related products. A classical mass spec workflow for the analysis of mAbs may involve an initial deglycosylation step. The glycans can be removed chemically or enzymatically with enzymes such as IgGZERO or GlycINATOR, and then analyzed separately. This is followed by intact mass analysis and reduced domain analysis (Fig. 1).


Figure 1. Classical middle down workflow for mAb analysis using LC/MS.

Subunit vs Traditional Enzymes for LC-MS

An additional decrease in the size of the antibody fragments is beneficial for higher resolution in the mass spectrometry experiment. This can be achieved by enzymatic digestion of the protein backbone to generate smaller antibody fragments, traditionally using trypsin or the endoprotease Lys C.

Such enzymes require a time consuming laborious process and optimizations for each project or molecule (Fig. 2). An additional drawback with these enzymes is the unspecific cleavage introducing additional heterogeneity, which offsets the objective of increased resolution for the individual subunits.

NMR studies with FabALACTICA

The cysteine protease FabALACTICA (IgdE) digests human IgG1 at one specific site just above the hinge, generating a homogenous pool of intact Fab and Fc fragments. It is active in a broad range of pH and salt concentrations and does not require any reducing reagent or co-factors. Thereby, as opposed to many other generally used proteases, FabALACTICA does not need any optimization to avoid overdigestion. This makes FabALACTICA an attractive enzyme in e.g. LC-MS analysis under reducing and non-reducing conditions, crystallization and NMR studies of higher order structure (HOS) of antibodies, characterization of bi- or multispecific antibodies, monovalent binding studies, and intact Fc glycan profiling.


Critical quality attributes of a mAb can be assessed at precise atomic-level with HOS analysis after two-dimensional nuclear magnetic resonance spectroscopy (2D-NMR). A workflow for obtaining homogenous Fab and Fc fragments from FabALACTICA Immobilized, ideal for the evaluation of HOS in the chimeric mAb infliximab, was described in the poster "FabALACTICA generates pure and homogeneous mAb subunits that facilitate 2D-NMR spectroscopy analysis". The high quality signal and resolution of the spectra from the generated Fab and Fc fragments resulted in an ability to observe the structural changes between Fab and Fc, the difference between an oxidized and non-oxidized sample and compare the HOS of the originator and a biosimilar by looking at the atomic resolution.

Figure 3. a) Schematic overview of the sample prep process including digestion with FabALACTICA Immobilized and separation by affinity chromatography where the flow through was collected and analyzed. Ox-originator was generated by treating the originator with 0.3% H2O2 at 37°C, 2.5 h. b) Non-reducing SDS-PAGE showing the fractions of the Fab and Fc fragments generated by digestion with FabALACTICA Immobilized.