High-Throughput Analysis and Automation for Glycomics Studies

Abstract

This review covers advances in analytical technologies for high-throughput (HTP) glycomics. Our focus is on structural studies of glycoprotein glycosylation to support biopharmaceutical realization and the discovery of glycan biomarkers for human disease. For biopharmaceuticals, there is increasing use of glycomics in Quality by Design studies to help optimize glycan profiles of drugs with a view to improving their clinical performance. Glycomics is also used in comparability studies to ensure consistency of glycosylation both throughout product development and between biosimilars and innovator drugs. In clinical studies there is as well an expanding interest in the use of glycomics—for example in Genome Wide Association Studies—to follow changes in glycosylation patterns of biological tissues and fluids with the progress of certain diseases. These include cancers, neurodegenerative disorders and inflammatory conditions. Despite rising activity in this field, there are significant challenges in performing large scale glycomics studies. The requirement is accurate identification and quantitation of individual glycan structures. However, glycoconjugate samples are often very complex and heterogeneous and contain many diverse branched glycan structures. In this article we cover HTP sample preparation and derivatization methods, sample purification, robotization, optimized glycan profiling by UHPLC, MS and multiplexed CE, as well as hyphenated techniques and automated data analysis tools. Throughout, we summarize the advantages and challenges with each of these technologies. The issues considered include reliability of the methods for glycan identification and quantitation, sample throughput, labor intensity, and affordability for large sample numbers.

Comparison of procainamide and 2-aminobenzamide labeling for profiling and identification of glycans by liquid chromatography with fluorescence detection coupled to electrospray ionization–mass spectrometry

“…Glycan release, 2-AB labeling, procainamide labeling, and labeled glycan purification were performed on a Hamilton Microlab STARlet liquid-handling robotic platform [10].…”

Ultrafast and high-throughput N-glycan analysis for monoclonal antibodies

“…30,31 Glycoprofiling is critical to understand antibody structure-activity relationships, to assess novel drug candidates in early development, and to ensure product comparability and consistency during the commercial product manufacture. For biosimilar antibodies, demonstration that the glycan profiles are equivalent or similar to the reference product is required for regulatory approval.…”

“…Development of rapid and high throughput glycoprofiling assays is highly desired and critical to support high sample loads. 30,31 Glycan analysis for N-linked glycoproteins usually involves the release of the oligosaccharide chains from the protein backbone by enzymatic cleavage with peptide N-glycosidase (PNGase F). 28,[32][33][34] Released glycans are normally labeled with a fluorescent tag such as 2-aminobenzamide (2-AB), and then CONTACT Xiaoyu Yang xiaoyu.yang@merck.com quantitated by hydrophilic interaction liquid chromatography (HILIC)-HPLC.…”

Chromatographic Monoliths for High-Throughput Immunoaffinity Isolation of Transferrin from Human Plasma

“…[13] To meet the demand of determining normal and aberrant biological glycosylation patterns of individual and total glycoproteins from body fluids and tissues in different human populations, methods for glycan analysis of large number of samples (so called high-throughput methods) are being extensively developed in recent years. [14][15][16][17] Transferrin (Tf) is a glycoprotein that transports iron to cells and in humans has two N-glycosylation sites -at asparagine 432 and asparagine 630. Tf N-glycans are mostly of biantennary complex type with terminal N-acetylneuraminic (sialic) acid.…”