Rapid Lectin-Based gold nanoparticle assay for glycoanalysis of therapeutic antibodies

 

Germarie Sánchez-Pomales, Michael J. Tarlov and Rebecca A. Zangmeister

 

Biochemical Science Division, Material Measurements Laboratory,

National Institute of Standards and Technology

 

 

Glycosylation, the enzymatic process by which carbohydrates are attached to proteins, can result in heterogeneity in the composition of the attached glycans. This heterogeneity can significantly affect the safety and efficacy profiles of protein drug products.  It is therefore essential for the biopharmaceutical industry to characterize, monitor and control the glycosylation of these products. Rapid assays based on lectin (i.e. multivalent carbohydrate binding protein) binding present a simple, fast and inexpensive alternative for glycosylation screening of therapeutic proteins, amenable to in-process control monitoring. A major disadvantage of this approach is poor lectin access to the glycans, which are often buried within the native conformation of the glycoprotein.  Therefore, development of more efficient lectin-based glycosylation assays should include a strategy to make the oligosaccharides more sterically accessible. To address this challenge, we adsorbed glycoproteins on gold nanoparticles (NPs). The rationale behind this method is that once the glycoprotein is adsorbed to the NPs, the glycans will extend out into solution and become more accessible to lectin-based analysis. This feature was exploited by developing a colorimetric assay where agglomeration of the NPs occurred due to the interaction between the target species immobilized on the NPs surface (i.e. glycoprotein), and the recognition molecule (i.e. lectin). By combining the sugar-binding specificity and the cross-linking capabilities of lectins, the non-specific adsorption of glycoproteins to gold surfaces, and the unique optical reporting properties of gold nanoparticles, we were able to interrogate, in just a few minutes, the glycosylation pattern of the model glycoprotein RNase B, polyclonal human immunoglobulin G, and the therapeutic monoclonal antibody, Rituxan®. This assay provides advantages over currently used glycoanalysis methods in terms of short analysis time, simplicity of the conjugation method, convenience of simple spectroscopic detection, and feasibility of providing complete glycan characterization of the protein drug product by using a variety of binding lectins.