A platform for high-throughput molecular characterization of recombinant monoclonal antibodies.
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.
Bailey
Mark J
MJ
School of Engineering, University of Queensland, St. Lucia, Qld 4072, Australia.
Hooker
Andrew D
AD
Adams
Carolyn S
CS
Zhang
Shuhong
S
James
David C
DC
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
D000911:Antibodies, Monoclonal; D006020:Glycopeptides; D007074:Immunoglobulin G; D011994:Recombinant Proteins