Absolute Quantitation of the N-Linked Glycans from Biotheraputic IgGs

• Published in: Journal of biomolecular techniques Vol. 30; no. Suppl; p. S3
• Main Authors: Orlando, Ron, Popov, Marla, Libert, Ben, Boyes, Barry
• Format: Journal Article
• Language: English
• Published: United States Association of Biomolecular Resource Facilities 01.12.2019
• Subjects: Poster Abstracts
• ISSN: 1524-0215; 1943-4731

The ability to accurately quantitate the glycan chains attached to glycoproteins has wide-ranging implications. Numerous studies over the past 40 years have demonstrated that abnormal glycosylation occurs in virtually all types of human cancers and demonstrate the potential of using glycan markers in either a diagnostic or a prognostic manner. The glycosylation on recombinant protein therapeutics is also known to have profound effects, with one of the better-known examples being the increased serum half-life of erythropoietin (EPO) resulting from glycoengineering. Hence, the quantification of glycoprotein glycans play important roles from the discovery of new diagnostic/prognostic markers to the development of therapeutic agents. The focus of this presentation is the evaluation of an isotopically labeled IgG as an internal standard for the relative and absolute quantitation of N-linked glycans attached to human IgGs. We have developed a HILIC-MRM protocol that permits us to monitor 36 glycoforms attached to the conserved N-linked glycosylation site in the FC region of human IgGs. This procedure can be applied to the analysis of IgGs in cell culture media without the need for extensive sample cleanup and involves minimal sample processing. Essentially, the sample is spiked with an internal standard consisting of an isotopically labeled IgG. The material is then reduced, alkylated, digested with trypsin, and analyzed by HILIC-MRM. The internal standard allows for both absolute and relative quantitation across the multiple samples.