SCIEX has produced an application note describing the N-glycosylation profiling of carbohydrates associated with therapeutic antibodies for faster, more cost-effective development of biologics.
Introduction
Glycan identification using traditional techniques is slow, requiring time- and labour-intensive sample preparation that can take a day or more. The goal of this application note was to demonstrate the advantages of the award-winning SCIEX fast glycan labelling and analysis technology for preparation and analysis of N-linked oligosaccharides.
Sample Preparation
Individual steps of the manual and automated approaches of the magnetic bead-mediated sample preparation protocol were performed using the SCIEX fast glycan labelling and analysis kit, which included three internal GU standards for instant, automated structural assignment.4 PNGase F was used for enzymatic release of the N-linked carbohydrates. This was followed by derivatization with a charged fluorophore (aminopyrene trisulfonate, APTS).
Results and Discussion
Results illustrate that by applying the Fast Glycan Labelling and Analysis Kit Assay for N-glycosylation analysis, sample preparation can be completed in as fast as 1 hour with excellent yield and a high level of reproducibility. Because the centrifugation steps during the sample preparation protocol are unnecessary, the process can be automated using liquid handling robots to provide a flexible and robust approach capable of reproducible sample processing.
Key Features
- Complete sample preparation in about one hour with excellent yield and high reproducibility.
- Magnetic bead clean-up reduces the presence of unbound dye, resulting in better characterization of complex sialylated structures.
- Sample preparation procedure can be applied manually to a single sample or to as many as 96 samples at a time using automated liquid handling platforms.
- A novel high-resolution gel buffer ensures excellent separation of target molecules.
- 5 minute separations with automated, immediate glycan identification.
- 32 Karat™ software automatically generates a report identifying all carbohydrate species with glycan image and monoisotopic mass information.
