GlycoSense: a rapid and simple method for glycosylation detection and monitoring

Session: 
S3.3 Glycomics
Code: 
OL3.3.3
Location (hall): 
Galactose
Start/end time: 
Monday, July 1, 2019 - 17:45 to 18:00
Loretta
Yang

Loretta Yang1, Matthew Saunders1, Sheng-Cheng Wu2, Lu Meng2, Christian Gerner-Smidt2, Robert Woods3

1Lectenz Bio, San Diego, United States, 2Lectenz Bio, Athens, United States, 3Complex Carbohydrate Research Center, University of Georgia, Athens, United States

Background:
There is an unmet need for rapid, cost-effective, robust, and easy to use methods for monitoring protein glycosylation during glycoprotein production whether in cell culture or for in vitro glycoengineering. Lectenz Bio has developed an innovative technology called GlycoSense™ to analyze glycoproteins using flow cytometry and multiplex microspheres (beads).  This process analytical technology (PAT) does not supplant full glycoprofiling by traditional methods, but provides unique information that can guide process development and advance academic research.

Methods:
The GlycoSense™ method detects binding between glycans and glycan-specific reagents that are conjugated to spectrally-unique beads. By combining the individual bead-based reagents into a multiplex array, a profile of key glycan features can be obtained in a few minutes on a basic cytometer.  In addition to established carbohydrate-detection reagents (antibodies and lectins), the GlycoSense™ approach can employ the engineered proteins (known as Lectenz®), developed by Lectenz Bio.  Lectenz® are derived from catalytically inactivated glycan-processing enzymes that have been engineered into high affinity glycan binding reagents with well-defined specificities.  The conversion of such enzymes into affinity reagents is facilitated by computationally-guided directed evolution.  Lectenz® are being developed for a variety of other glycan detection and enrichment applications including affinity chromatography and Western blot.

Results:
We illustrate the performance of the GlycoSense™ method using glycoprotein standards and demonstrate its utility in glycoprotein cell culture, and in in vitro glycoengineering by monitoring changes in glycosylation patterns upon treatment of glycoproteins with glycosidases.

Conclusion:
The GlycoSense™ method offers a unique ability to rapidly detect and monitor changes in glycosylation using only a commonplace benchtop flow cytometer, without the need to purify the glycoprotein analyte or to release the glycans enzymatically.

Fluorescently labeled fetuin treated with sialidase for 1 hour, followed by galactosidase for 2 hours. Error bars are standard deviation values from triplicate time points, background subtracted and normalized to the highest signal for each bead.

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