Characterization of IgG N-glycans employing a microfluidic chip that integrates glycan cleavage, sample purification, LC separation, and MS detection.
A novel polymeric microfluidic device with an on-chip enzyme reactor has been developed for the characterization of recombinant glycoproteins. The enzyme reactor chip packed with PNGase F-modified solid support material was combined with a microfluidic glycan cleanup chip and a commercially available HPLC-chip to perform glycoprotein deglycosylation, protein removal, glycan capture, glycan LC separation, and nanoelectrospray into a time-of-flight mass spectrometry (TOF-MS) system. With this integrated chip, the combined sample preparation and sample analysis time was reduced from multiple hours to less than 10 min. A once tedious and time-consuming glycan analysis workflow is now integrated into an HPLC-chip device. Glycan profiling analysis has been achieved with as little as 100 ng of monoclonal antibody. Furthermore, a single chip was shown to retain activity and perform equivalently for over 250 replicate glycan profiles from a recombinant antibody.
Bynum
Maggie A
MA
Agilent Laboratories, Agilent Technologies, Santa Clara, California 95051, USA. maggie_bynum@agilent.com
Yin
Hongfeng
H
Felts
Katherine
K
Lee
Yvonne M
YM
Monell
Craig R
CR
Killeen
Kevin
K
D000911:Antibodies, Monoclonal / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004800:Enzymes, Immobilized / Q000378:metabolism; D046210:Microfluidic Analytical Techniques / Q000379:methods*; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000378:metabolism; D011134:Polysaccharides / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D000911:Antibodies, Monoclonal; D004800:Enzymes, Immobilized; D011134:Polysaccharides; D011994:Recombinant Proteins; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase