NEW FabDELLO – The SmartEnzyme for Mutated IgG

November 18, 2021 | New Product, News |

We introduce FabDELLO, a protease that specifically digests human IgG1 at a single site above the hinge, generating intact Fab and Fc fragments within two hours with no need for reducing conditions. The enzyme is active on antibodies with mutated hinge regions and enables middle-level LC-MS for characterization of critical quality attributes.
 

Mutated hinge regions are common modifications of therapeutic antibody candidates to reduce effector functions. The mutations may affect the antibody digestion efficiency of enzymes with high substrate specificity. For example, the highly specific FabRICATOR (IdeS) digestion efficiency is negatively impacted on antibodies containing the common LALA mutation.
 

 
FabDELLO acts specifically at an exposed lysine residue above the hinge and enables the popular middle-level LC-MS analysis of antibodies with mutated hinge regions. As an example, the commercially available IgG1 risankizumab, with a LALA mutation in the lower hinge region, was digested with FabDELLO and analyzed by reversed-phase LC-MS. The digestion resulted in generation of homogenous Fab and Fc fragments. The fragments were reduced with 20 mM DTT, for middle-level LC-MS analysis. This generated homogenous Fc/2, light chain (LC) and Fd fragments. The defined peaks observed after digestion with FabDELLO show homogenous fragment generation and robust performance of the enzyme.
 

 
Figure 1. The commercial IgG1 risankizumab, with a LALA mutation in the lower hinge, was digested with FabDELLO and analyzed by reversed-phase LC-MS on a Waters™ BioAccord™ system equipped with a Waters™ BioResolve™ RP mAb column (2.1 x 50 mm). UV 280 chromatogram of subunits analyzed non-reduced (left panel) and reduced (right panel) and deconvoluted spectra of the subunits are shown.
 


 


FabDELLO – Above hinge digestion of 8 x 100 or 96 x 100 µg human IgG1.