Multisite N-Glycan Mapping Study Using Orthogonal Methods: CE and UPLC
Presented by Melissa Schwartz
In the past decade many biopharmaceuticals have been approved for clinical use. Over half of these new generation drugs contain glycoproteins as active ingredient, such as monoclonal antibodies or other recombinant glycoproteins, which are mostly produced in mammalian cell cultures. The linked carbohydrate moieties not only affect their physicochemical properties and thermal stability, but also their receptor binding activity, circulating half-life and, last but not least, their immunogenicity. An international team, including 20 independent laboratories from biopharmaceutical companies, universities, analytical contract laboratories and national authorities in the United States, Europe and Asia, was formed to evaluate the precision and robustness of sample preparation and analysis of 2-aminobenzamide (2-AB)- and 8-aminopyrene-trisulfonic acid (APTS)-labeled N-glycans using two orthogonal bioseparation techniques: capillary electrophoresis with laser-induced fluorescence (CE-LIF) and ultrahigh performance liquid chromatography (UPLC). All participants used the same lot of chemicals, samples, reagents, and columns/capillaries to run their assays. After a predefined system suitability test to assure that systems were performing optimally, the laboratories analyzed 6 different samples including a pre-labeled homooligomer ladder standard, 3 pre-labeled glycan standards (containing only high mannose and complex type afucosylated and fucosylated glycans), released and pre-labeled glycan samples and 3 parallel prepared glycoprotein test articles. Migration time, peak area and peak area percent values were determined for all peaks with >0.1% peak area. Statistical analysis was performed following the ISO 5725-2 guideline principles. The data provided by each laboratory was critically evaluated to identify outliers. Glucose Unit (GU) values were determined for all peaks for both separation methods. In conclusion, the results validate the appropriate use of sample preparation and bioanalytical methods for the biopharmaceutical industry in support of clone selection, process development, regulatory submissions and lot release of therapeutic antibodies or other glycosylated biotherapeutics.
Ákos Szekrényes, Horváth Laboratory of Bioseparation Sciences, Debrecen, Hungary; SungAe Shur Park, Amgen Inc., Thousand Oaks, CA USA; Marcia Santos, Beckman Coulter, Inc., Brea, CA USA; Bill Warren, Waters Corporation, Milford, MA USA; Michael Kimzey, ProZyme, Inc., Hayward, CA USA; Eoin Cosgrave, Waters Corporation, Milford, MA USA; Ted Haxo, ProZyme, Inc., Hayward, CA USA; Shiva Pourkaveh, ProZyme, Inc., Hayward, CA USA; Zoltán Szabó, ProZyme, Inc., Hayward, CA USA; Zoran Sosic, Biogen Idec Inc., Cambridge, MA USA; Pamela Feng, Biogen Idec Inc., Cambridge, MA USA; Csaba Váradi, Horváth Laboratory of Bioseparation Sciences, Debrecen, Hungary; Jean-Bernard Falmagne, Analis s.a/n.v, Suarlee, Belgium; Preeti Sejwal, Bristol-Myers Squibb, Princeton, NJ USA; David Michels, Genentech, a Member of the Roche Group, South San Francisco, CA USA; Gordon Freckleton, ImClone Systems, A Wholly -owned Subsidiary of Eli Lilly, Branchburg, NJ USA; Melissa Hamm, Merck & Co. Inc., West Point, PA USA; Anastasiya Manuilov, Pfizer, Inc., Andover, MA USA; Toni Duffy, Pfizer, Inc., Andover, MA USA; Melissa Schwartz, Boehringer Ingelheim Fremont Inc., Fremont, CA USA; Jiann-Kae Lou, Regeneron Pharmaceuticals, Tarrytown, NY USA; Pui-King Leung, ImmunoGen, Inc., Waltham, MA USA; Jonathan van Dyck, Seattle Genetics, Inc., Bothell, WA USA; Marcell Olajos, Gedeon Richter, Plc., Budapest, Hungary; Bernd Moritz, Hoffmann-La Roche Ltd., Basel, Switzerland; Ron Kowle, Eli Lilly and Company, Indianapolis, IN 46285, USA; Gao Kai, National Institutes for Food and Drug Control, Beijing, China; Wang Wenbo, National Institutes for Food and Drug Control, Beijing, China; Jo Wegstein, ProZyme, Inc., Hayward, CA USA; András Guttman, Horváth Laboratory of Bioseparation Sciences, Debrecen, Hungary