NEW! We’re Launching FabRICATOR Xtra LALA Lyophilized!
We’re excited to share that we are launching a new member of the SmartEnzymes™ family that will facilitate middle-level analysis of hinge-mutated IgG!
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. FabRICATOR Xtra LALA is active on a variety of antibodies with mutated hinge regions and enables middle-level LC-MS characterization of critical quality attributes of hinge-mutated antibody-based therapeutics.
- Efficient below hinge digestion of hinge-mutated antibodies, including LALA
- Versatile – digests a variety of hinge-mutated antibodies
- Enables middle-level analysis of hinge-mutated antibody therapeutics
Analysis of Chain Pairing Variants in a Bispecific IgG4
Subunit analysis by LC-MS is a powerful approach to assess chain pairing variants in bispecific antibodies. By digesting the bsAb into scFc and F(ab’)2 fragments, the correct pairing of both the heavy and light chains can be analyzed in a single fragment (the F(ab’)2) without having to account for the complexity and heterogeneity introduced by Fc glycosylation. Many bsAb formats are heavily engineered and often contain Fc silencing mutations. While these modifications ensure correct assembly of the antibody and allow for control over Fc effector functions, they also make subunit analysis using traditional tools, such as IdeS, more challenging.
Here, we demonstrate analysis of a bispecific human IgG4 antibody, talquetamab, which contains the Fc silencing mutations F234A and L235A in the lower hinge. FabRICATOR Xtra LALA efficiently digested this antibody into scFc and F(ab’)2 fragments which were easily separated by reversed-phase chromatography. MS analysis of the F(ab’)2 fragment demonstrates correct assembly of the bsAb and the absence of any chain pairing-related impurities (Fig. 1).
Figure 1.Subunit analysis of a bispecific human IgG4. A bispecific antibody, talquetamab (hIgG4-FALA), was digested using FabRICATOR Xtra LALA (1 unit per µg IgG, PBS, 37°C) for 1 h followed by reversed-phase LC-MS analysis on a Waters™ BioAccord™ LC-MS system equipped with a Waters™ BioResolve™ RP mAb column (2.1 x 50 mm). scFc and F(ab’)2 fragments were chromatographically separated (top panel) and the deconvoluted spectrum of the F(ab’)2 subunit (bottom panel) demonstrates correct assembly of the bsAb with regards to both the heavy and the light chains. The orange arrows indicate the masses where light chain pairing variants would be detected, further demonstrating their absence. The asterisks correspond to species where either one, or both, of the antibody heavy chains have been digested after residue 236.
FabRICATOR Xtra – Below Hinge Digestion of Hinge-mutated IgG