Bioreactor Product Quality Assessment using MAM by the A2P2 System
A cell-free cell culture sample must undergo affinity purification, denaturation, reduction, alkylation, buffer exchange, and enzymatic digestion before analysis. Occasionally, a secondary enzyme is required to fully investigate product quality attributes. As shown inFigure 8d , 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 then transports the purified sample into one of the intermediate vials and sequentially adds denaturation and reduction reagents. Or, as an alternative approach, the reagents can be directly added to the FTV to streamline the process. The mixture is then delivered to and incubated inside the 40°C reaction coil, in either the stopped-flow mode or the preferred continuous-flow mode. After the protein is denatured and reduced, it is transferred to the FTV, where the alkylation reagent is added either directly or using a new intermediate vial. The mixture is then directed through the 40°C reaction coil for incubation to complete protein alkylation and is subsequently buffer exchanged using the desalting column. The desalted, reduced, and alkylated protein returns to the FTV, where it awaits mixing with an enzyme and incubation in the 30°C reaction coil. The digested protein is then ready for LC injection and mass spectrometry detection.