Conclusion
Until today, N-Linked glycan characterization workflows were tedious and time-consuming, making them unsuitable for real-time
bioprocessing applications. A microfluidic LC–MS chip approach is described that integrates and automates all analysis steps:
glycan cleavage, capture and purification, chromatographic separation, and MS detection. This approach reduces the previously
lengthy incubation time for deglycosylation with PNGase F from many hours to 6 s. It eliminates reaction steps, including
acidification, labeling, or derivatizing the free glycans used with traditional LC and MS approaches. Sample cleanup and labeling
steps common to LIF detection, and acidification and desalting steps common to MALDI-TOF-MS are not needed. Rapid TOF-MS detection
following deglycosylation makes it possible to measure -glycosylamine intermediates directly. Compared with measuring free
reducing end glycans, this approach provided the advantages of simpler chromatographic peaks, isomer detection, and enhanced
sensitivity.
In total, the integrated microfluidic LC–MS chip reduces analysis time from antibody injection to LC–MS results to 10 min
and is completely automated once sample is introduced. It was also shown to separate isomers quickly and efficiently. The
chip has the advantage of being able to characterize very complex glycan profiles. Low-level, unknown and nonfucosylated glycans
were easily identified. Using a different LC–MS chip configuration, it is possible to analyze the deglycosylated antibody.
The authors believe that this microfludic LC–MS chip-based workflow will facilitate fast and accurate glycan characterization
in all stages of glycoprotein development and processing.
Maggie Bynum* is a scientist at Agilent Technologies' Agilent Laboratories, 5301 Stevens Creek Blvd, Santa Clara, CA 95051, tel. 408.553.3152,
fax 408.553.2161, maggie_bynum@agilent.com
. Tomasz Baginski is a scientist, and Rodney Keck is a senior scientist and senior group leader at Genentech. Kevin Killeen is a director at Agilent Technologies' Agilent
Laboratories.
*To whom all correspondence should be addressed.
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