Analysis of N-Glycans Released from Proteins of Therapeutic and Clinical Significance Using Capillary Electrophoresis and Liquid Chromatography Coupled to Mass Spectrometry

N-linked glycosylation is a prevalent post-translational modification which modulates the physical, chemical and biological properties of proteins. Glycosylation is important to monitor in both disease and biotherapeutic products.;A growing part of the biopharmaceutical sector are monoclonal antibody (mAb) based therapeutics, typically of the immunoglobulin G (IgG) class which contain a conserved glycosylation site at asparagine-297 in the crystallizable fragment (Fc) region. This site can be occupied by 32 possible glycan structures (in human serum IgG) that affect the function of the molecule. Therefore due to the vast microheterogeneity that can arise, biopharmaceutical products must be analyzed during and after manufacturing to verify the glycan chains or monosaccharides present. One approach to this analysis is by using capillary zone electrophoresis (CZE) with laser induced fluorescence (LIF) to monitor the abundance of individual monosaccharides from released and hydrolyzed N-glycan chains on the mAb biotherapeutic. This quantitative approach was applied to nine biotherapeutic monoclonal antibodies. The original procedure, as obtained from the literature, was further optimized, using sample clean-up protocols and dilution, to reduce noise present due to the sensitivity of the fluorescence detector and ultimately to improve the separation of the monosaccharide peaks. The resulting data showed that the therapeutics contained high levels of fucosylation with variable levels of galactosylation consistent with human glycosylation.;To investigate alterations in sialylated glycans that are associated with disease, a previously developed fluoride-mediated negative ion microfluidic chip LC-MS method can be applied; however, the glycosidic bond between the sialic acid and underlying glycan is labile and easily lost during mass spectrometric ionization. To minimize the charge on the sialic acids and therefore, to enable the application of our fluoride-mediated method, a charge neutralization reaction such as methylation or methyl amidation of the carboxylic acid can be used. A variety of reagents were tested to methylate the carboxylic acid on the sialic acid; however, none of the methylation procedures showed completed conversion of the carboxylic acids to methyl esters. A methyl amidation approach was also undertaken with (7-azabenzotriazol-1-yloxy)trispyrrolidinophosphonium hexafluorophosphate (PyAOP). This approach proved to be promising as it showed complete conversion for both linkages (α(2,3) and α(2,6)) on disialyllacto- N-tetraose, a disialylated milk sugar. When the procedure was further applied to a disialylated, complex N-glycan, an incomplete reaction was seen indicating the further need for optimization of the reaction conditions. After further optimization, PyAOP will be applied to investigate glycosylation on alpha-1 acid glycoprotein, a heavily sialylated glycoprotein involved in immune response and implicated in disease progression.