Chromatography

Biography

Biography: Britta Eggenreich

Abstract

The bacterium Escherichia coli, is a well-studied recombinant host organism with a plethora of applications in biotechnology. High valuable biopharmaceuticals, such as recombinant enzymes, antibody fragments and growth factors, are currently being produced in E. coli. These molecules are usually produced intra-cellularly which is why cell disruption is required as the first step in the downstream process. For that purpose high pressure homogenization is the system of choice since it is scalable and can be run in continuous mode. However, it is crucial to determine cell disruption efficiency to: Avoid product loss in intact cells, but also to avoid unnecessary long disruption cycles and thus harm the product. Usually, cell disruption efficiency is evaluated either by determination of colony forming units (CFUs) or photometric measurements of nucleic acids and protein content in the lysate. However, these methods are both characterized by disadvantages, as CFUs can only be counted on the next day, resulting in great time delay, and photometric measurements are affected by matrix effects. In this study, we implemented a novel online tool based on UV chromatogram fingerprints and chemometric techniques to monitor cell disruption efficiency. We used: 1) Measurement of the total protein content in the supernatant, 2) determination of CFUs and 3) flow cytometry as reference analytics to validate this novel tool. Finally, we performed a design of experiments study, where we changed the factors concentration of biomass per ml buffer, number of homogenization cycles and pressure during homogenization to analyze and optimize the unit operation high pressure homogenization for a recombinant E. coli strain producing a highly valuable antibody fragment. Summarizing, we could nicely demonstrate the power of the novel online tool, which will certainly facilitate the evaluation of this crucial unit operation in the future.