Multi-Site N-glycan mapping study 1: Capillary electrophoresis – laser induced fluorescence

An international team that included 20 independent laboratories from biopharmaceutical companies, universities, analytical contract laboratories and national authorities in the United States, Europe and Asia was formed to evaluate the reproducibility of sample preparation and analysis of N-glycans using capillary electrophoresis of 8-aminopyrene-1,3,6-trisulfonic acid (APTS)-labeled glycans with laser induced fluorescence (CE-LIF) detection (16 sites) and ultra highperformance liquid chromatography (UHPLC, 12 sites; results to be reported in a subsequent publication). All participants used the same lot of chemicals, samples, reagents, and columns/capillaries to run their assays. Migration time, peak area and peak area percent values were determined for all peaks with >0.1% peak area. Our results demonstrated low variability and high reproducibility, both, within any given site as well across all sites, which indicates that a standard N-glycan analysis platform appropriate for general use (clone selection, process development, lot release, etc.) within the industry can be established

I. Water-driven chemoselective reactions of squarate derivatives with amino acids and peptides: Mechanism and applications. II. Biocompatible hydrogels: Transferring bioinert chemistry from surfaces to 3-dimensional materials

Chemoselective organic reactions in entirely aqueous environment are useful for interfacing chemistry with life sciences, including immobilization of peptides and mammalian cells on surfaces, and running designed reaction in living systems. This dissertation describes a fundamentally new class of highly chemoselective reaction employing rationally designed squarate derivatives - molecules with abiotic structures - and N-terminal cysteine residue of peptides (or proteins) in an entirely aqueous solution at neutral pH. The mechanistic study of this chemoselective ligation revealed that the reaction not only proceeds in water, but also promoted by the hydrogen bonding of water. By fine tuning the reactivity of squarate derivative and controlling the pH of the solution, a methodology had been established for immobilizing peptides and mammalian cells using only the N-terminus cysteine for conjugation to surface bound squarates. This highly resolved control of selectivity enables the immobilization and biological studies of peptides and protein containing internal cysteines. Few other potential application of this reaction were also presented including a new class of labeling reagent for peptides, proteins and mammalian cells that have long-term stability in water and has potential for the development of a biotin probe that can survive the complex biological environment. Mimics of the ligand for mediating mammalian cell adhesion, Agr-Gly-Asp (RGD) tripeptide, were also constructed based on a squarate core structure. The synthesis of this new class of non-peptidic molecules is highly efficient - consisting of 1-5 steps. Inhibition assays suggest that this class of molecules block the specific binding site on integrin membrane protein for the natural ligand RGD. Cell adhesion assays were conducted on bioinert self-assembled monolayers (SAMs) of alkanethiols on gold film to reduce the high signal-to-noise ratio usually associated with traditional fibronectin based assay. Exploring the transfer of the bioinert anti-biofouling surface chemistry of the mannitol-terminated self-assembled monolayer into three-dimensional materials, a novel hydrogel composed of mannitol-derivatized acrylamide monomers was synthesized. This polymeric material prevents mammalian cell adhesion more effectively than other hydrogel materials based on monomers such as glycerol and acrylamide. This result validates the hypothesis that assembly of molecules known as Kosmotropes, which render the folding structure of proteins, also exhibit bioinertness

Mapping the Energetic Epitope of an Antibody/Interleukin-23 Interaction with Hydrogen/Deuterium Exchange, Fast Photochemical Oxidation of Proteins Mass Spectrometry, and Alanine Shave Mutagenesis

Epitope mapping the specific residues of an antibody/antigen interaction can be used to support mechanistic interpretation, antibody optimization, and epitope novelty assessment. Thus, there is a strong need for mapping methods, particularly integrative ones. Here, we report the identification of an energetic epitope by determining the interfacial hot-spot that dominates the binding affinity for an anti-interleukin-23 (anti-IL-23) antibody by using the complementary approaches of hydrogen/deuterium exchange mass spectrometry (HDX-MS), fast photochemical oxidation of proteins (FPOP), alanine shave mutagenesis, and binding analytics. Five peptide regions on IL-23 with reduced backbone amide solvent accessibility upon antibody binding were identified by HDX-MS, and five different peptides over the same three regions were identified by FPOP. In addition, FPOP analysis at the residue level reveals potentially key interacting residues. Mutants with 3–5 residues changed to alanine have no measurable differences from wild-type IL-23 except for binding of and signaling blockade by the 7B7 anti-IL-23 antibody. The M5 IL-23 mutant differs from wild-type by five alanine substitutions and represents the dominant energetic epitope of 7B7. M5 shows a dramatic decrease in binding to BMS-986010 (which contains the 7B7 Fab, where Fab is fragment antigen-binding region of an antibody), yet it maintains functional activity, binding to p40 and p19 specific reagents, and maintains biophysical properties similar to wild-type IL-23 (monomeric state, thermal stability, and secondary structural features)