The use of Genovis enzymes has gained widespread use within the developers and producers of antibody based therapeutics. Due to the extraordinary specificity and robustness of the enzymes they are becoming a vital tool for the ever-increasing demand of analytical characterization of these large complex biomolecules.
Due to the artifact free sample preparation, ease of use and simple data interpretation FabRICATOR (IdeS) has become a valuable tool for routine characterization of mAb based bioteherapeutics. FabRICATOR was described in a recent paper by Merck Research Laboratories for domain characterization of therapeutic antibody products. IgG1, IgG2, IgG4 and Fc-fusion proteins were investigated using FabRICATOR domain analytics.
FabRICATOR digestion generates antibody subunits and have been used to study antibody micro variations such as oxidation. The specific digestion coupled to mild reaction conditions allows for easy monitoring of antibody subunit oxidation.
In many applications antibody fragments are desired over the entire antibody. Fc fragments may cause unwanted interactions in cell and tissue based applications and assays, both in vitro and in vivo, and thus F(ab’)2 or Fab fragments are preferred. Antibody subunit fragmentation significantly improves the information retrieved in antibody characterization with LC-MS. Fabricator is an excellent enzymatic tool in generating antibody subunit fragments. The enzyme cleaves IgG at one specific site yielding perfect F(ab’)2 and Fc fragments.
FabRICATOR is used in the subunit domain mapping / middle down approach.
As FabRICATOR only has one cleavage site regardless of incubation time, the amino acid sequence in each of the antibody fragments is exactly the same. 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.