The criticality of high-resolution N-linked carbohydrate assays and detailed characterization of antibody effector function in the context of biosimilar development.

Accurate measurement and functional characterization of antibody Fc domain N-linked glycans is critical to successful biosimilar development. Here, we describe the application of methods to accurately quantify and characterize the N-linked glycans of 2 IgG1 biosimilars with effector function activity, and show the potential pitfalls of using assays with insufficient resolution. Accurate glycan assessment was combined with glycan enrichment using lectin chromatography or production with glycosylation inhibitors to produce enriched pools of key glycan species for subsequent assessment in cell-based antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity effector function assays. This work highlights the challenges of developing high-quality biosimilar candidates and the need for modern biotechnology capabilities. These results show that high-quality analytics, combined with sensitive cell-based assays to study in vivo mechanisms of action, is an essential part of biosimilar development.

High-field NMR studies of molecular recognition and structure-function relationships in antimicrobial piscidins at the water-lipid bilayer interface.

High magnetic field solid-state NMR was performed on amphipathic cationic antimicrobial peptides from fish to characterize their secondary structure and orientation in hydrated phospholipid bilayers. High-resolution distance and orientational restraints on 13C- and 15N-labeled amidated piscidins 1 and 3 provided site-specific information establishing alpha-helicity and an orientation parallel to the membrane surface. Few membrane-bound natural peptides with this topology have been structurally studied at high resolution in the presence of hydrated lipid bilayers. This orientation was foreseen since the partitioning of amphipathic cationic antimicrobial peptides at the water-bilayer interface allows for favorable peptide-lipid interactions, and it may be related to the mechanism of action. The enhanced resolution obtained at 900 MHz evidences a determinant advantage of ultra-high-field NMR for the structural determination of multiple-labeled peptides and proteins.