Capillary electrophoresis with laser-induced fluorescence detection for detailed studies on N-linked oligosaccharide profile of therapeutic recombinant monoclonal antibodies.

Total N-linked oligosaccharide profiling method for recombinant monoclonal antibody (rmAb) using capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) and an approach for detailed structural analysis of N-linked oligosaccharide were developed. A CE-LIF method using 2-aminobenzoic acid (2-AA) as a fluorogenic reagent allowed sensitive detection of several minor peaks besides typical asialo-biantennary complex type oligosaccharides in the analysis of N-linked oligosaccharide from a commercial rmAb pharmaceutical, rituximab. These minor peaks were successfully assigned as sialo-biantennary complex type and high-mannose type oligosaccharides by comparison with the migration times of 2-AA derivatized oligosaccharides which were separately fractionated and determined by high-performance liquid chromatography (HPLC) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). In development of biopharmaceuticals, it is important to evaluate these minor oligosaccharides, because some of these minor glycans are likely to influence immunogenicity and clearance rate in vivo. The repetitive analysis using CE-LIF showed excellent precision in relative corrected peak areas. These results demonstrate that the present CE-LIF method is applicable for both structural characterization and quantitative profiling of N-linked oligosaccharides derived from rmAb pharmaceuticals. The present method will be a powerful tool for rapid, quantitative and exhaustive evaluation of N-linked oligosaccharides in various stages of rmAb pharmaceutical development such as clone selection, bioprocess control, and routine lot release testing to ensure product efficacy and consistency.