Bioreactor Product Glycosylation Profile Assessment by the A2P2 System
In the case of sample preparation for a glycosylation profile assay, if the starting material is a cell culture sample, the process includes affinity protein cleanup, enzymatic release of glycans from the protein, chemical labeling of the released glycans with fluorescent tags, and subsequent HILIC chromatography column injection and analysis. As depicted in Figure 8c , the autosampler receives manually collected or automatically delivered bioreactor cell-free samples in vials or through the FTV. The autosampler needle takes the sample and injects it into the affinity capture column for cell culture matrix removal. The autosampler needle then delivers PNGase F to the capture column for on-column glycan release. The released glycans are transferred to the FTV, mixed with 2-AA, and directed to the 80°C reaction coil, where the fluorescent labeling reaction takes place. The fluorescently labeled glycans are then conveyed back to the FTV, ready for normal-phase chromatographic separation and quantitation.