A novel toolbox for E. coli lysis monitoring

Abstract

The bacterium Escherichia coli is a well-studied recombinant host organism with a plethora of applications in biotechnology. Highly valuable biopharmaceuticals, such as antibody fragments and growth factors, are currently being produced in E. coli. However, the high metabolic burden during recombinant protein production can lead to cell death, consequent lysis, and undesired product loss. Thus, fast and precise analyzers to monitor E. coli bioprocesses and to retrieve key process information, such as the optimal time point of harvest, are needed. However, such reliable monitoring tools are still scarce to date. In this study, we cultivated an E. coli strain producing a recombinant single-chain antibody fragment in the cytoplasm. In bioreactor cultivations, we purposely triggered cell lysis by pH ramps. We developed a novel toolbox using UV chromatograms as fingerprints and chemometric techniques to monitor these lysis events and used flow cytometry (FCM) as reference method to quantify viability offline. Summarizing, we were able to show that a novel toolbox comprising HPLC chromatogram fingerprinting and data science tools allowed the identification of E. coli lysis in a fast and reliable manner. We are convinced that this toolbox will not only facilitate E. coli bioprocess monitoring but will also allow enhanced process control in the future.Electronic supplementary materialThe online version of this article (doi:10.1007/s00216-016-9907-z) contains supplementary material, which is available to authorized users.

Secretion of recombinant proteins from E. coli

“…One common approach is the growth of the culture on solid media and counting the colony forming units (CFU). However, this approach is not always available due to high instrument costs [215,244,245]. As it is possible for viable cells to lose the ability to form colonies, the number of viable cells might be underestimated in the CFU-counting method [244].…”

“…This method was performed in reports of nucleoside triphosphate pyrophosphohydrolase MazG [248] and catabolite repressor protein (CRP) production [218]. Moreover, protein leakage can also be detected using offline high pressure liquid chromatography (HPLC) techniques, but this is also time-consuming [215,245]. In a coupled assay, a tetrazolium salt is converted into a red formazan product that can be quantified.…”

“…However, DNA detection methods are prone to error due to loss or fragmentation of DNA while preparing the samples or due to its long persistence in a PCR-detectable form [215]. In Rajamanickam et al [245], nucleic acid impurities in supernatants were used as indicators for lysis. Whereas the latter method was found to be more sensitive, the RT-PCR correlation was better.…”

An automated data‐driven DSP development approach for glycoproteins from yeast

“…Ultraviolet (UV) chromatograms as fingerprints with chemometric tools and sound data science have been shown to contain process information, and we recently used this approach to develop a monitoring tool for USP bioprocesses . UV chromatograms have also been implemented for real‐time pooling decisions in DSP processes .…”

HPLC fingerprinting approach for raw material assessment and unit operation tracking for IVIG production from Cohn I+II+III fraction

“…Monolith technology was already implemented for characterization of transferrin and albumin in IgG concentrates, by utilizing conjoint LC where transferrin and albumin are eluting as baseline‐separated peaks and quantification of the analytes is established by the means of standard calibration curve . Recently, HPLC fingerprinting approach has been successfully implemented for making real‐time pooling decisions for process chromatography as well as for Escherichia coli lysis monitoring . By using UV chromatograms as fingerprints and chemometric techniques, real‐time decisions about critical process steps based on product quality attributes can be made.…”