Online Deglycosylation Analysis
Improving and streamlining glycan analysis has also occupied a team of scientists at Agilent Technologies. “Our goal is to improve current workflows that use laborious enzyme-cleavage protocols of glycans followed by analytical characterization with mass spec and other time-consuming techniques,” said Kevin Killeen, Ph.D., director of molecular separations.
To improve throughput and simplify analysis, Agilent has built an integrated microfluidic LC/MS device for enzymatic deglycosylation with immobilized PNGase F, constructed using laser ablation and lamination technologies. It performs complete sample preparation and analysis of n-linked glycans of IgG mAb samples. The microfluidic workflow system telescopes the required time for analysis from hours to a few minutes, according to Agilent.
Glycans are released and purified from the antibody using an in situ porous graphitized carbon-trapping column. The deglycosylated recombinant antibodies are irreversibly trapped on the column, and the glycans are then eluted using a nanoseparation column with a formic acid gradient. Subsequently the sample undergoes electrospray ionization and identification by TOF-MS detection.
“We measure the amino form of the glycans instead of the hydroxyl form typically observed using in-solution cleavage,” said Dr. Killeen, claiming several benefits to this strategy. “This an important advantage in characterizing minor components of the mAb glycan profiles. We also can better characterize the heterogeneity of deglycosylated antibodies with our on-chip workflow.
“Our approach reduces multiday solution-phase enzyme cleavage of glycans and sample clean-up workflows for MALDI, LC/MS, or fluorescent-labeling analysis to minutes for the HPLC chip approach,” Dr. Killeen concluded.