title: Ultrafast and high-throughput N-glycan analysis for monoclonal antibodies.
abstract: Glycosylation is a critical attribute for development and manufacturing of therapeutic monoclonal antibodies (mAbs) in the pharmaceutical industry. Conventional antibody glycan analysis is usually achieved by the 2-aminobenzamide (2-AB) hydrophilic interaction liquid chromatography (HILIC) method following the release of glycans. Although this method produces satisfactory results, it has limited use for screening a large number of samples because it requires expensive reagents and takes several hours or even days for the sample preparation. A simple and rapid glycan analysis method was not available. To overcome these constraints, we developed and compared 2 ultrafast methods for antibody glycan analysis (UMAG) that involve the rapid generation and purification of glycopeptides in either organic solvent or aqueous buffer followed by label-free quantification using matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Both methods quickly yield N-glycan profiles of test antibodies similar to those obtained by the 2-AB HILIC-HPLC method. In addition, the UMAG method performed in aqueous buffer has a shorter assay time of less than 15 min, and enables high throughput analysis in 96-well PCR plates with minimal sample handling. This method, the fastest, and simplest as reported thus far, has been evaluated for glycoprofiling of mAbs expressed under various cell culture conditions, as well as for the evaluation of antibody culture clones and various production batches. Importantly the method sensitively captured changes in glycoprofiles detected by traditional 2-AB HILIC-HPLC or HILIC-UPLC. The simplicity, high speed, and low cost of this method may facilitate basic research and process development for novel mAbs and biosimilar products.
authors.affiliation: a Bioprocess Development, Merck Research Laboratories, Merck & Co., Inc. , Kenilworth , USA.
authors.forename: Xiaoyu
authors.initials: X
authors.lastname: Yang
authors.affiliation: a Bioprocess Development, Merck Research Laboratories, Merck & Co., Inc. , Kenilworth , USA.
authors.forename: Sunnie Myung
authors.initials: SM
authors.lastname: Kim
authors.affiliation: a Bioprocess Development, Merck Research Laboratories, Merck & Co., Inc. , Kenilworth , USA.
authors.forename: Richard
authors.initials: R
authors.lastname: Ruzanski
authors.affiliation: a Bioprocess Development, Merck Research Laboratories, Merck & Co., Inc. , Kenilworth , USA.
authors.forename: Yuetian
authors.initials: Y
authors.lastname: Chen
authors.affiliation: a Bioprocess Development, Merck Research Laboratories, Merck & Co., Inc. , Kenilworth , USA.
authors.forename: Sarath
authors.initials: S
authors.lastname: Moses
authors.affiliation: a Bioprocess Development, Merck Research Laboratories, Merck & Co., Inc. , Kenilworth , USA.
authors.forename: Wai Lam
authors.initials: WL
authors.lastname: Ling
authors.affiliation: a Bioprocess Development, Merck Research Laboratories, Merck & Co., Inc. , Kenilworth , USA.
authors.forename: Xiaojuan
authors.initials: X
authors.lastname: Li
authors.affiliation: a Bioprocess Development, Merck Research Laboratories, Merck & Co., Inc. , Kenilworth , USA.
authors.forename: Shao-Chun
authors.initials: SC
authors.lastname: Wang
authors.affiliation: a Bioprocess Development, Merck Research Laboratories, Merck & Co., Inc. , Kenilworth , USA.
authors.forename: Huijuan
authors.initials: H
authors.lastname: Li
authors.forename: Alexandre
authors.initials: A
authors.lastname: Ambrogelly
authors.affiliation: a Bioprocess Development, Merck Research Laboratories, Merck & Co., Inc. , Kenilworth , USA.
authors.forename: Daisy
authors.initials: D
authors.lastname: Richardson
authors.affiliation: a Bioprocess Development, Merck Research Laboratories, Merck & Co., Inc. , Kenilworth , USA.
authors.forename: Mohammed
authors.initials: M
authors.lastname: Shameem
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000032:analysis* / Q000737:chemistry; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D057166:High-Throughput Screening Assays; D006801:Humans; D011134:Polysaccharides / Q000032:analysis*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
chemical_list: D000911:Antibodies, Monoclonal; D011134:Polysaccharides
keywords: Antibody; N-glycan analysis; biosimilar; high throughput; ultrafast