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.
Sensitive Detection of Antifoam
According to Mark Schenerman, Ph.D., of MedImmune, “Antifoam C is widely used in the pharma and food industries, and while there are no known risks associated with its use, its presence is often monitored.”
The compound, whose active ingredient is poly-dimethylsiloxane, is a silicon-containing material used to control foaming in industrial and manufacturing processes.
Although it is generally removed by subsequent processing, quantitative demonstration of this can be a challenge. Developing a sensitive detection method has proved challenging due to its lack of spectroscopic absorbability. Elemental silicon (Si), however, can be detected using inductively coupled mass spectrometry, a sensitive approach.
“This technology has come a long way in the last 10 years,” Dr. Schenerman said. It uses an ionized gas, or plasma, and any material introduced into the plasma will itself be ionized and can be identified in the mass spectrometer. The procedure can measure Si at parts per trillion. It is widely used in trace-element identification throughout the industrial world.
MedImmune demonstrated that this approach to the quantification of Antifoam C is highly specific and reproducible. A number of different diluents and spiked solutions were monitored with a high degree of accuracy. Thus this method is suitable for determination of Antifoam C levels in production samples. It should be noted that this technique is applicable to the detection of any compound that has a unique and identifiable atom as part of its structure.