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 various therapeutic agents. A current impediment for performing quantitative glycomics is the shortage of widely available standard glycoproteins and isotopically labeled reagents to enable accurate quantitation. The issue with glycan quantitation was highlighted by inter-laboratory studies conducted by the Human Proteome Organization (HUPO) and the Association of Biomolecular Resource Facilities (ABRF). Both of these studies demonstrated errors greater than several hundred percent in the analysis of mid-to-low level glycans were compared across participating laboratories. The inability to accurately quantitate low level glycans is particularly worrisome since it is often glycans of low abundance that have the largest impact, as is seen with the therapeutic human intravenous immunoglobulin G (IVIg). The focus of this proposal is to develop a well-characterized standard glycoprotein with isotopically labeled glycans to enable the accurate, robust, and reproducible analysis of N-linked glycans at the relative and potentially absolute level. A monoclonal antibody (mAb) was selected because of the widespread use of these as therapeutic agents coupled with the need for glycan quantification by various regulatory agencies. Here, a known quantity of the isotopically labeled mAb (i-mAb) can be added directly to therapeutic mAb preparation, analyzed by any standard procedure that includes mass spectrometry, and glycan quantitation will be provided by comparing the ratios of the native to isotopically labeled ions. i-mAb is expected to be an excellent internal standard for all therapeutic mAbs because of the similarity in glycan structures and in the amino acid sequence flanking the glycosylation site that is found on most mAb-based therapeutics. Since i-mAb can be added directly to the sample before any processing and is virtually identical to the analyte mAb, this approach is expected to be capable of compensating for a wide range of systematic errors, such as differential losses during sample handling, matrix effects, operator errors/inequalities, instrumental drift/response, etc. It is further anticipated hat i-mAb will enable researchers at different locations to obtain comparable results despite using different instruments, which is not possible with current methodology.
Public Health Relevance Statement: Public Health Relevance: The accurate quantification of glycoprotein glycans is important for numerous fields ranging from the discovery of new diagnostic/prognostic markers to the development of various therapeutic agents. A current impediment for performing these analyses is the shortage of widely available reference standards to enable accurate quantitation. The focus of this proposal is to develop a well-characterized standard glycoprotein with isotopically labeled glycans to enable the accurate, robust, and reproducible analysis of N-linked glycans at the relative and potentially absolute level.
Project Terms: Amino Acid Sequence; base; Blinded; Cell Line; Cell Nucleus; Complex; Coupled; Development; Diagnostic; Embryo; Erythropoietin; Glycoconjugates; Glycolipids; Glycoproteins; Glycosaminoglycans; glycosylation; Half-Life; Heparin; Human; Immunoglobulin G; Inequality; Institution; instrument; Intravenous Immunoglobulins; Investigation; Ions; kidney cell; Label; Laboratories; Laboratory Study; Link; Location; Malignant Neoplasms; Mass Spectrum Analysis; Metabolic; Methodology; Monoclonal Antibodies; novel; novel diagnostics; Phase; Play; Polysaccharides; Preparation; Procedures; Process; Product Labeling; Production; prognostic; Prognostic Marker; Protein-Carbohydrate Interaction; Proteome; public health relevance; Reagent; Recombinant Proteins; Reference Standards; Relative (related person); Research Personnel; Resources; response; Role; Sampling; scale up; Scientist; Serum; Site; stability testing; Structure; System; Therapeutic; Therapeutic Agents; Therapeutic Monoclonal Antibodies; Therapeutic Uses
