title: A platform for high-throughput molecular characterization of recombinant monoclonal antibodies.
abstract: We describe quantitative characterization of a sample preparation platform for rapid and high-throughput analysis of recombinant monoclonal antibodies (MAbs) and their post-translational modifications. MAb capture, desalting and in situ reduction/alkylation were accomplished by sequential adsorption of analyte to solid phase beads (protein A, reverse-phase) suspended in microtiter plate wells. Following elution and rapid tryptic digestion in the presence of acid-labile surfactant (RapiGest), peptides were fractionated by stepwise elution from reverse-phase pipet tips and the fraction containing Fc N-glycopeptides isolated. Direct quantitative analysis of the relative abundance of peptide glycoforms by MALDI-TOF MS in linear mode closely correlated with normal phase HPLC analysis of fluorophore labeled N-glycans released by PNGaseF.
authors.affiliation: School of Engineering, University of Queensland, St. Lucia, Qld 4072, Australia.
authors.forename: Mark J
authors.initials: MJ
authors.lastname: Bailey
authors.forename: Andrew D
authors.initials: AD
authors.lastname: Hooker
authors.forename: Carolyn S
authors.initials: CS
authors.lastname: Adams
authors.forename: Shuhong
authors.initials: S
authors.lastname: Zhang
authors.forename: David C
authors.initials: DC
authors.lastname: James
mesh_terms: D000327:Adsorption; D000818:Animals; D000911:Antibodies, Monoclonal / Q000032:analysis* / Q000302:isolation & purification; D005591:Chemical Fractionation / Q000379:methods; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D006020:Glycopeptides / Q000302:isolation & purification; D006031:Glycosylation; D006801:Humans; D007074:Immunoglobulin G / Q000302:isolation & purification; D051379:Mice; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000032:analysis*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D000911:Antibodies, Monoclonal; D006020:Glycopeptides; D007074:Immunoglobulin G; D011994:Recombinant Proteins