Minor changes in the quality of biologically manufactured monoclonal antibodies (mAbs) can affect their bioactivity, efficacy and safety. One of the most important variation concerns the N-glycosylation pattern, which has direct impact on an anti-tumor mechanism called antibody-dependent cell cytotoxicity (ADCC). Thus, careful engineering and production of the mAbs is expected to enhance protein-receptor binding and ADCC. The specific aim of this study is to evaluate the influence of terminal carbohydrates within the Ab Fc fragment on the interaction with CD16a receptor in native and label free conditions.
Here, we apply native electrospray ionization mass spectrometry (ESI-MS) to study mAb binding to a recombinant CD16a extracellular domain. The mAb molecules comprise the variants with minimal and maximal galactosylation as well as α2,3 and α2,6-sialic acid isomers. Direct ESI-MS titration allows us to determine the solution-phase antibody-receptor equilibria and by using a temperature-controlled nanoelectrospray source the thermal stability of the complex is examined. Additionally, the system is validated with a protein-cell assay using genetically modified human natural killer (NK) cell line expressing high affinity CD16a.
Based on these, we prove that galactosylation of the fucosylated Fc fragment increased the CD16a binding affinity by 1.5-fold when compared with non-galactosylated variant. The α2,6-sialylation has no significant effect on binding affinity, whereas the α2,3-sialylation decreases it by 1.72-fold. In line with expectation, the galactoslylated and α2,6-sialylated mAb:CD16a complex exhibit higher thermal stability when measured using a temperature gradient from 20 to 50˚C. A similar binding pattern is observed based on indirect staining approach using NK cells.
The results of our study deliver a profound insight into Ab:CD16a equilibria and shed new light on the Fc fragment N-glycosylation chemistry. Moreover, a new perspective for native ESI-MS approach in analysis of highly heterogeneous proteins is proposed.