Development of Monolithic Column Materials for the Separation and Analysis of Glycans

Abstract

Monolithic column materials offer great advantages as chromatographic media in bioseparations and as solid-supports in biocatalysis. These single-piece porous materials have an interconnected ligament structure that limits the void volume inside the column, thus increasing the efficiency without sacrificing the permeability. The preparation of monolithic materials is easy, reproducible and has available a wide range of chemistries to utilize. Complex, heterogeneous and isobaric glycan structures require preparation methods that may include glycan release, separation and enrichment prior to a comprehensive and site-specific glycosylation analysis. Monolithic column materials aid that demand, as shown by the results reported by the research works presented in this review. These works include selective capture of glycans and glycoproteins via their interactions with lectins, boronic acids, hydrophobic, and hydrophilic/polar functional groups on monolith surfaces. It also includes immobilization of enzymes trypsin and PNGase F on monoliths to digest and deglycosylate glycoproteins and glycopeptides, respectively. The use of monolithic capillary columns for glycan separations through nano-liquid chromatography (nano-LC) and capillary electrochromatography (CEC) and coupling these columns to MS instruments to create multidimensional systems show the potential in the development of miniaturized, high-throughput and automated systems of glycan separation and analysis.

Nanotechnology in Glycomics: Applications in Diagnostics, Therapy, Imaging, and Separation Processes

“…Glycans can be classified into several categories based on the bond between a glycan and a protein: N ‐glycans (via –NH 2 group to asparagine), O ‐glycans (via –OH group to serine, threonine, or hydroxylated amino acids), and less‐abundant forms of glycans, such as C ‐glycans (C—C bond via tryptophan) and the quite unusual S ‐glycans (C—S bond via cysteine) . Moreover, glycans can be branched by the formation of biantennary, triantennary, and more complex antennary structures …”

“…There are other options for glycan enrichment, such as reverse‐phase mode, electrostatic mode (for glycans containing negatively charged sialic acids), electrostatic repulsion hydrophilic interactions, and affinity capture based on protein A, which is applicable for the selective enrichment of antibodies with the subsequent glycoprofiling of glycans present especially in the Fc fragment of IgGs …”

Use of monolithic supports for high‐throughput protein and peptide separation in proteomics

“…In this way monolith can be tuned for a specific application in proteomics depending on the nature of the nanoparticle . The incorporation of gold nanoparticles with immobilized Erythrina cristagalli lectin and concanavalin A was employed to improve binding capacity. This monolith casted into pipette tip offered a possibility for design of HTP strategies.…”

Selective capture of glycoproteins using lectin-modified nanoporous gold monolith

“…The conventional packed columns with uniform size porous particles have been traditionally used in these chromatographic separations. A new generation of separation media called monolithic materials has become an interesting option due to their design that allows faster, more efficient and versatile separations of glycans, glycopeptides and glycoproteins [29]. Monolithic columns are usually prepared in situ fused with silica capillary tubes by co-polymerization of cross-linking and functional monomers together with porogens and initiators.…”