title: Profiling N-linked oligosaccharides from IgG by high-performance anion-exchange chromatography with pulsed amperometric detection.
abstract: Understanding and characterizing protein therapeutic glycosylation is important with growing evidence that glycosylation impacts biological efficacy, pharmacokinetics and cellular toxicity. Protein expression systems and reactor conditions can impact glycosylation, leading to potentially undesirable glycosylation. For example, high-mannose species may be present, which are atypical of human antibody glycosylation. Their presence in the Fc domain has been linked to increased serum clearance of immunoglobulin G (IgG) antibodies. High-performance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD) is an effective tool for determining glycans present in glycoprotein therapeutics. We report an improved HPAE-PAD method for IgG oligosaccharide separation. The neutral glycans are well resolved, including separation of high-mannose species from typical human IgG glycans. Oligosaccharide identification was performed by comparison to known standards in conjunction with selective exoglycosidase digestion of both standards and released glycans. Retention times (RTs) of known glycans were compared with the retention times of maltose, maltotriose and maltotetraose standards to define a retention index value for each glycan. These retention indices were used to aid identification of glycans from an example monoclonal antibody sample of unknown glycosylation. Method ruggedness was evaluated across duplicate systems, analysts and triplicate column lots. Comparing two systems with different analysts and columns, retention time precision relative standard deviations (RSDs) were between 0.63 and 4.0% while retention indices precision RSDs ranged from 0.27 to 0.56%. The separation is orthogonal to capillary electrophoresis-based separation of labeled IgG oligosaccharides.
authors.affiliation: Thermo Fisher Scientific, 1214 Oakmead Parkway, Sunnyvale, CA 94085, USA jeff.rohrer@thermofisher.com.
authors.forename: Jeffrey S
authors.initials: JS
authors.lastname: Rohrer
authors.affiliation: Thermo Fisher Scientific, 1214 Oakmead Parkway, Sunnyvale, CA 94085, USA.
authors.forename: Lipika
authors.initials: L
authors.lastname: Basumallick
authors.affiliation: Thermo Fisher Scientific, 1214 Oakmead Parkway, Sunnyvale, CA 94085, USA.
authors.forename: Deanna C
authors.initials: DC
authors.lastname: Hurum
mesh_terms: D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D006031:Glycosylation; D006801:Humans; D006868:Hydrolysis; D007074:Immunoglobulin G / Q000097:blood / Q000737:chemistry*; D009844:Oligosaccharides / Q000737:chemistry / Q000302:isolation & purification*; D015203:Reproducibility of Results; D005644:alpha-L-Fucosidase / Q000737:chemistry*; D001616:beta-Galactosidase / Q000737:chemistry*
chemical_list: D007074:Immunoglobulin G; D009844:Oligosaccharides; D001616:beta-Galactosidase; D005644:alpha-L-Fucosidase
keywords: HPAE-PAD; HPLC; N-linked glycans; glycoprotein; monoclonal antibodies