Oligosaccharide Release and MALDI-TOF MS Analysis of N-Linked Carbohydrate Structures from Glycoproteins
With the growth in the number of recombinant proteins being developed for use as human therapeutics, there also exists a corresponding need for new and highly sensitive analytical techniques to characterize these new pharmaceuticals. Many new assays were developed to monitor and characterize the heterogeneity that naturally exists in these complex molecules (1,2). For glycoprotein-based therapeutics, one major source of heterogeneity arises from the oligosaccharides present on the protein (3). This oligosaccharide heterogeneity is a natural outcome of the glycoprotein synthesis pathways in the host cells, such as Chinese hamster ovary cells (CHO;4) or baby hamster kidney cells (BHK;5). For some glycoproteins, e.g., erythropoietin (6) and rituximab (7), the distribution of oligosaccharides can affect the in vitro biological activity of the molecule. In these cases, it is imperative to have analytical methods in place that measure the oligosaccharide distribution during the drug development phase, as well as after regulatory approval for the market. Oligosaccharide profiling of glycoproteins has become a common tool in both academic laboratories, as well as in the commercial environment. Most techniques employ enzymatic digestion to release the N-linked oligosaccharides; however, there are various methods used for detection of the released oligosaccharides. These include analysis of fluorescently labeled oligosaccharides by capillary electrophoresis (8, 9, 10) or high-performance liquid chromatography (HPLC; 11,12), along with analyses of the unlabeled oligosaccharides by high-performance anion-exchange chromatography (HPAEC;13) and matrix-associated laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS;14, 15, 16).
KeywordsHPLC Manifold Citrate Fluoride Adduct
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