Glycosylation is one of the most hotly debated phenomena in biotechnology, particularly for development of biosimilars. Regulators are still unsure how glycosylation patterns affect safety and efficacy.
The research group of Pauline Rudd, Ph.D., a professor at the National Institute for Bioprocessing Research and Training, is developing a database tool that could help.
GlycoBase, a database of fluorescently labeled glycans and polysaccharide fragments released from monoclonal antibodies and other glycoproteins, answers critical questions on glycosylation patterns and composition, as well as the similarity of glycosylations between two molecules. Because reactor conditions can have profound effects on glycosylation, GlycoBase could also find application in PAT and Quality by Design for processes involving glycosylated biotherapeutics.
A typical analysis begins with releasing glycans from the protein, labeling them with 2-aminobenzamide, followed by enzymatic digestion. The digest is then analyzed using HILIC (hydrophobic interaction LC), which is ideal for separations based on small differences in polarity. HILIC is also sensitive to polysaccharide structures and linkages, a capability mass detection lacks.
Finally, the fragments are identified by matching with entries in GlycoBase. The technique is extremely sensitive, as a nanogram of protein is sufficient to perform a full glycan sequence analysis, according to Dr. Rudd.
Dr. Rudd’s group has collaborated with many academic groups, which have cited GlycoBase extensively in the literature. Companies have also taken notice, among them Eli Lilly, Organon, Schering-Plough, and BD, who have licensed the database for their internal use.