C-terminal lysine clipping and Fc receptor binding using SmartEnzymes
Researchers at LFB Biotechnologies in Paris, France have carried out a thorough analysis and characterization of the impact of C-terminal lysine clipping to Fc-receptor binding using a range of SmartEnzymes from Genovis.
The scientists separated an IgG1 antibody using SCX separation and purified the fractions without C-terminal lysines K0, with 1 C-terminal lysine K1 and with both lysines intact K2. The purified fractions were characterized for any further differences using FabRICATOR digestion and middle-level analysis. This approached enabled the researchers to study multiple post-translational modifications such as charge variants, oxidations and Fc glycosylation in a simple and robust way. The characterization revealed that the lysine heterogeneity was the main differentiator and all other PTMs were distributed between the fractions.
The fractions K0, K1 and K2 were purified using a FcγRIIIa receptor affinity assay and the fractions were studied using LC-MS. This time the antibodies were digested using FabALACTICA for above hinge digestion in combination with GlycINATOR to trim down the glycan heterogeneity and leaving the core GlcNAc with or without the core fucose. This approach revealed that the three fractions originating from the affinity column originated from the presence of core fucose at the core of the Fc-glycan. The impact of the core fucose on FcγRIIIa receptor affinity is well described in literature, but this analytical approach with both above hinge digestion and GlycINATOR trimming of glycans has never been performed previously.
In conclusion, the impact of the C-terminal lysine on Fc receptor binding was studied using both affinity purification and surface plasmon resonance. The data showed that the lysine content does not alter the binding and interaction with neither the activating FcγRIIIa receptor or the neonatal Fc-recptor, important for circulation of antibodies. This paper highlights the versatility of the SmartEnzymes for specific digestion above and under the hinge for middle-level analysis as well as Fc glycan trimming for core fucosylation analysis.
GlycINATOR – An IgG specific endoglycosidase that enable fast and robust analysis of core-fucosylation.
FabRICATOR – The widely used protease for middle-level antibody analytics using below hinge digestion and reduction
FabALACTICA – Specific above hinge digestion generating intact Fab and Fc fragments and used for homogenous Fab generation and paired Fc glycan analysis.