FabRICATOR® Xtra LALA for Ultrafast Microdroplet Digestion of Antibodies with Fc-silencing Mutations
Scientists at New Jersey Institute of Technology, Johnson & Johnson and collaborators have demonstrated a novel approach for mass spectrometry (MS)-based characterization of hinge-mutated antibodies. Combining the recently introduced FabRICATOR Xtra LALA enzyme with ultrafast microdroplet digestion allows for fully automated, high-throughput and efficient antibody characterization, with the entire sample preparation and MS analysis completed in around 1 minute per sample!
FabRICATOR Xtra LALA is an IgG-specific protease that digests antibodies which have been engineered to contain mutated hinges. It digests below the hinge, generating F(ab’)2 and Fc fragments. By leveraging an Agilent Jet Stream (AJS) ion source, antibody-enzyme mixtures can be sprayed into micron-sized droplets, with the resulting microdroplet environment serving to dramatically accelerate enzymatic reaction rates.
Figure 1. FabRICATOR Xtra LALA is an IgG-specific protease that digests antibodies which have been engineered to contain mutated hinges, including the LALA mutation. It digests below the hinge, generating F(ab’)2 and Fc fragments.
As monoclonal antibody (mAb)-based therapeutics continue to dominate biopharmaceutical pipelines, Fc-silencing mutations are increasingly engineered into candidates to optimize safety and efficacy. These mutations are highly effective at silencing immune activation, by reducing unwanted interactions with Fc-gamma receptors, however, they pose major analytical challenges because they can hinder some traditional enzymatic digestion methods required for MS-based analysis. FabRICATOR® (the IdeS enzyme) has, for a long time, been routinely utilized for middle-level characterization of antibodies, but does not efficiently digest Fc-mutated antibodies. The introduction of FabRICATOR Xtra LALA, therefore, represents a breakthrough approach to overcome a critical limitation in current mAb analytical workflows by enabling efficient and reliable digestion of mutated antibodies that are resistant to IdeS digestion!
FabRICATOR Xtra LALA has an expanded substrate specificity, which enables it to digest a variety of Fc-mutated antibodies. In this study, antibodies containing LAGA (L235A/G237A), LFLE (L234F/L355E), LALA-DS (L234A/L235A/D265S) and LALA-PG (L234A/L235A/P282G) mutations were all analysed using the described microdroplet digestion approach. Of the samples analyzed in this study, in many cases, digestion using IdeS was not achieved, while FabRICATOR Xtra LALA achieved digestion efficiencies of ~80% even for the highly challenging LALA-DS and LALA-PG mutated antibodies.
Digesting within microdroplets means that enzymatic reactions occur within milliseconds, compared to traditional in-solution digestion which often requires 30 minutes to several hours. In one particularly striking example from this study, efficient digestion of the typically highly resistant PD-L1 (LALA-PG) antibody was completed within 1 millisecond using the FabRICATOR Xtra LALA microdroplet approach, compared to 2.5 hours needed for equivalent digestion in-solution! This represents a 9-million-fold increase in reaction speed. In high-throughput tests, this approach was used to successfully analyze 94 samples on a 96-well plate within 104 minutes, maintaining excellent reproducibility (coefficient of variation ~5%) and minimal sample consumption.
FabRICATOR Xtra LALA has been shown to be a significant tool for middle-level analysis of Fc-mutated antibodies. In combination with a microdroplet digestion approach, it enables high-throughput, cost-effective, and robust antibody characterization. This is directly applicable to industrial biopharmaceutical development, where rapid screening of hundreds or thousands of engineered antibody variants may be required. The work described here, therefore, represents a major advance toward automated, ultrafast, and universal subunit analysis for both wild-type and heavily engineered antibody therapeutics.
Reference
Yang et al., 2025. Ultrafast Microdroplet Digestion of Antibodies with Fc-Silencing Mutations. Analytical Chemistry.
FabRICATOR®Xtra – Below hinge digestion of mutated IgG