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.