Many types of analyses are needed in down-stream processing; ranging from immediate validation of protein sequence during synthesis to determinations of product purity during fractionation and finally assessing protein conformation at the end of the process.  Clearly �just in time� availability of analytical data is important.  Three types of measurements will be described; all based on immune selection.  A mass linked immuno-selective assay method for sequence analysis will be discussed that exploits rapid immunosorbent selection of a protein from a process stream, online trypsin digestion within a min, and MS analysis of the trypsin digest in several minutes.  Sequence coverage often approaches 95%.  Purity analysis in contrast uses immunosorbent subtraction of the product protein to determine residual impurities during fractionation.   Up to 300 assays in series have been achieved at 30 second intervals before the immunosorbent must be recycled.  Conformational analysis assays were based on the use of discontinuous epitope sensing, polyclonal antibodies to assess protein conformation in frontal immunoaffinity chromatography (FIC) assays.  Improperly folded protein elutes prematurely in FIC assays; being separated from native conformers.

Monitoring protein structure and purity in downstream processing

245th National Meeting (2013)

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