Blue Stream offers a comprehensive panel of services for the glycosylation characterization of glycoproteins; including site identification of glycan attachment, characterization of the glycan itself, microheterogeneity determination of glycosylation of the glycoprotein, N-Linked Oligosaccharide profile, Sialic Acid Quantification, Monosaccharide Analysis, and complete comparability programs which may be used for manufacturing/process changes, and for comparisons of Originator vs. Follow-on Biologic products.
Our N-Linked Oligosaccharide profiling method provides a relative chromatographic quantification via HPLC and subsequent identification via MS. The assay control may be a Sponsor-supplied reference standard or one provided by Blue Stream, which demonstrates that the glycan hydrolysis, derivitization, and chromatographic separation are all occurring within method performance parameters. Subsequent analysis of the chromatographic peaks via LC/MS or LC/MS/MS will yield additional data to confirm the identity of the glycan, and allow deconstruction of the glycan for additional information on its branch and sub-unit structure.
Blue Stream may also utilize LC/MS or LC/MS/MS Peptide Maps to provide glycosylation characterization information in addition to amino acid sequence, disulfide bond mapping, and PTM determination. These Peptide Mapping methods will yield actual site of glycosylation, identification of the glycan, glycan structural analysis, site of sialylation, and may provide insights into the heterogeneity of the glycosylation as well.
The best combination of methods for the determination of glycosylation microheterogeneity of a glycoprotein is the use of an oligosaccharide profile method in-line with MSn. The site(s) of the glycan variability would be determined via LC/MS Peptide Mapping studies. However, to achieve a complete picture of glycosylation of a protein, or to complete a glycosylation characterization program, several complementary methods must be used. Blue Stream provides a full range of glycosylation characterization services, suitable for Sponsor sample characterization, comparability, and Lot Release Programs.
Glycosylation Characterization methods:
- N-Linked Oligosaccharide Profile
- Monosaccharide Analysis
- Sialic Acid Determination
- Peptide Mapping (LC/MS, LC/MS/MS)
- Glycan Site Identification
- Glycan Structural Analysis