Presenting at Informa’s “Comparability for Biologics” meeting in Berlin, Parastoo Azadi, Ph.D., technical director at the Complex Carbohydrate Research Center at the University of Georgia, described the tools and techniques used for comparability studies of glycoproteins to assess batch-to-batch variability in glycosylation.
Analytical methods can differentiate between N-linked and O-linked glycosylation, provide data on the percent and sites of glycosylation, and identify what carbohydrates are present at which sites. They can yield both qualitative and quantitative information, with the demand for quantitation increasing and techniques and technology evolving to meet that demand.
“Quantification is still a black box for carbohydrates,” she said. The field is advancing beyond conventional MALDI-MS technology for generating molecular weight data to the more advanced MS-MS techniques that provide sequence information on glycan fractions.
Whereas HPLC of labeled oligosaccharides remains an analytical standard, new derivatization methods to modify and stabilize the carbohydrates for analysis, coupled with MS-MS techniques, “may enable us to do accurate quantitation in the next five years,” she predicted.
Even as they become more quantitative, analytical techniques for characterizing carbohydrates also need to evolve beyond one-sample-at-a-time analysis and enable high-throughput profiling as well as structural analysis and identification of glycans in complex mixtures.
The nonprofit Complex Carbohydrate Research Center not only performs basic research on the synthesis, structural characterization, and medical aspects of glycosylated compounds and polysaccharides, it also operates an analytical services facility and publishes the results of its analyses.
In a study presented at the Society for Glycobiology’s annual conference, scientists described the glycobiological characterization of human biglycan, a proteoglycan component of the extracellular matrix present in a variety of tissue types. Altered biglycan expression has been associated with medical conditions such as osteoporosis, osteoarthritis, corneal diseases, and atherosclerosis.
Dr. Azadi and colleagues characterized the glycan portion of human recombinant biglycan using SDS-PAGE, HPLC, and MS techniques. LC/MS-MS analysis following 18O labeling revealed that two potential N-linked glycosylation sites in the non-proteoglycan form of biglycan are fully glycosylated. The researchers used ESI-MS-MS analysis to identify all major glycosylation of the biglycan proteoglycan.