Development of Isotopically Labeled Glycoproteins for use as Internal Standards

DESCRIPTION provided by applicant The ability to accurately quantitate the glycan chains attached to glycoproteins has wide ranging implications Numerous studies over the past 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 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