A novel toolbox for E. coli lysis monitoring

Vignesh Rajamanickam,David Wurm,Christoph Herwig,Oliver Spadiut,Christoph Slouka

doi:10.1007/s00216-016-9907-z

Vignesh Rajamanickam, David Wurm + Show 3 more

Open Access

https://doi.org/10.1007/s00216-016-9907-z

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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.

Highlights

Escherichia coli is one of the most popular host organisms for recombinant protein production (e.g., [1, 2])
We developed a novel toolbox based on UV chromatograms as fingerprints to identify E. coli cell lysis
UV chromatographic data at 260 nm were arranged as chromatogram fingerprints as shown in Electronic Supplementary Material (ESM) Fig. S1

Summary

Introduction

Escherichia coli is one of the most popular host organisms for recombinant protein production (e.g., [1, 2]). Strong induction of recombinant protein production results in great cell stress and high metabolic burden, potentially leading to cell death and lysis [3]. Flow cytometry (FCM) is the predominant method to monitor and quantify E. coli cell death. UV spectroscopy coupled to high pressure liquid chromatography (HPLC) is implemented for real-time monitoring in downstream processes [8]. We hypothesized that UV chromatographic data of E. coli bioprocess samples contain information about impurity release and lysis events and can be used in upstream processing. We followed the impurity pattern of nucleic acids at 260 nm as marker for cell lysis along different E. coli bioprocesses. We combined UV chromatographic data with chemometric methods to identify lysis which may be used to define the optimal time point of harvest

Materials and methods

Discussion

Compliance with ethical standards