Mass spectrometry is one of the key analytical tools for characterizing therapeutic monoclonal antibodies (MAbs). Mass spectrometry in conjugation with HPLC is commonly used for studying the primary structure as well as post translation modifications (PTMs) and glycan structures of these large biomolecules. The large size of MAbs (150 kD) together with post translational modifications (PTMs) makes the analytical characterization of these biological therapeutics especially challenging.
The classical workflow for MS analysis of monoclonal antibodies (MAbs) involves an initial deglycosylation step where glycans are removed enzymatically or chemically and analyzed separately. This is followed by intact mass analysis and reduced domain analysis.
Fig 1. Classical middle down workflow when analyzing monoclonal antibodies with LC/MS
Further reduction in fragment size would be beneficial in terms of higher resolution. This has promoted the use of enzymes for additional cleavage of the MAb. The use of papain, pepsin or endoprotease Lys C have been reported for creating fragments less than 30 kD. A major drawback using these enzymes is the unspecific cleavage that occurs thereby introducing additional heterogeneity which to some extent counteracts the objective of increased resolution of individual fragments.
Fig 2. Mass spectra of F(ab')2 fragments of Trastazumab® generated either by pepsin (top) or FabRICATOR (bottom).
FabRICATOR protease, a recombinantely modified IdeS from Streptococcus pyogenes, solves the problems above by having an extraordinary specificity for one single cleavage site in the lower hinge region. Additionally the enzyme works right out of the box and no optimization of reaction conditions is needed. The reaction is fast (30 minutes) and yield is essentially 100%.
Fig 3. Middle down work flow using FabRICATOR enzyme and subsequent reduction. Sample preparation time approximately 2 hours.
By using the workflow depicted in Figure 3 a homogenous set of antibody fragments are obtained. As FabRICATOR only have one cleavage site regardless of incubation time, the amino acid sequence in each of the antibody fragments are exactly the same (assuming it is a MAb and no mutations has occurred). All fragments are below 30 kD which allows for mass spectrometric determination of major glycans, degree of fucosylation and many of the post translation modifications. As ionization efficiency is mostly related to the protein sequence, intensities can be considered quantitative which allows for rapid glycan content determination ( Chevreaux et al).
Monoisotopically resolved peaks of antibody fragments using high resolution MS
High resolution instruments e.g Q-TOFS now have the possibility for monoisotopically resolved peaks of the LC, Fd' and Fc/2 fragments after FabRICATOR cleavage and reduction ( Jabs et al). This enables rapid amino acid confirmation without proteolytic trypsin digestion. A review by Beck et al also highlights the advantages of using the FabRICATOR middle down strategy to MAb characterization - especially highlighting the:
The rapid sample preparation (less than 2 hours for entire analysis including digestion and LC/MS)
Low material consumption
N-Glycan profiling site by site
If present - Fab glycans can be resolved separately
Degree of fucosylation and galactosylation can be determined
Additionally, fragmentation technologies like ETD may allow for amino acid sequence confirmation of significant parts of the antibody using the middle down / sub unit fragmentation approach.