GLYCAN ARRAY FABRICATION UTILIZING SURFACTANT VESICLES FOR CHARACTERIZING LECTIN BINDING
Monique A. Makos, Lenea H. Stocker, Philip DeShong, Rebecca A. Zangmeister
For quality assurance and control purposes there is a need to quantify changes observed in glycan (carbohydrate moiety) structure during the production of monoclonal antibody drug therapies. Lectins, proteins with highly specific carbohydrate binding functionalities, are ubiquitous in nature and are capable of differentiating between similar glycan structures. There is great potential for using these natural proteins to monitor glycosylation; however, at present only a subset of lectin-carbohydrate binding interactions have been studied. Characterizing the binding of lectins has been hindered, in part, by their structural diversity. In addition, monovalent carbohydrate interactions with lectins are weak; thus, lectins typically possess two or more binding sites. To successfully characterize a particular lectin, analytical tools must be capable of monitoring various carbohydrate spacing situations. Although the importance of carbohydrate density on lectin binding is recognized, as yet there exists no one technique for quantifying this relationship.
The aim of this project is to develop a technology that can rapidly characterize the unique binding properties of unknown lectins. This will lead to the discovery of a diverse lectin library that can detect slight changes in carbohydrate structure during the manufacturing of monoclonal antibody drug products. Our approach is to use catanionic surfactant vesicles to control carbohydrate density. We have fabricated vesicles modified with glycoconjugates via hydrophobic insertion of the hydrocarbon tail into the vesicle membrane. The carbohydrate-functionalized vesicles spontaneously form in water, remain stable at room temperature for months, and can easily be prepared with carbohydrates on their outer surface in a controlled range of densities. The vesicles will be used to generate a glycan array with varying carbohydrate densities. The binding of a particular lectin to the glycan array will be monitored and quantified using a biotin-avidin fluorescence sandwich assay. This platform will provide a high-throughput tool for characterizing unknown lectins that can be used by the biomanufacturing industry to measure changes in the glycan structure of glycoproteins observed during production.