Detecting cell lysis using viscosity monitoring in E. coli fermentation to prevent product loss

Abstract

Monitoring the physical or chemical properties of cell broths to infer cell status is often challenging due to the complex nature of the broth. Key factors indicative of cell status include cell density, cell viability, product leakage, and DNA release to the fermentation broth. The rapid and accurate prediction of cell status for hosts with intracellular protein products can minimise product loss due to leakage at the onset of cell lysis in fermentation. This article reports the rheological examination of an industrially relevant E. coli fermentation producing antibody fragments (Fab'). Viscosity monitoring showed an increase in viscosity during the exponential phase in relation to the cell density increase, a relatively flat profile in the stationary phase, followed by a rapid increase which correlated well with product loss, DNA release and loss of cell viability. This phenomenon was observed over several fermentations that a 25% increase in broth viscosity (using induction-point viscosity as a reference) indicated 10% product loss. Our results suggest that viscosity can accurately detect cell lysis and product leakage in postinduction cell cultures, and can identify cell lysis earlier than several other common fermentation monitoring techniques. This work demonstrates the utility of rapidly monitoring the physical properties of fermentation broths, and that viscosity monitoring has the potential to be a tool for process development to determine the optimal harvest time and minimise product loss. © 2016 The Authors. Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers, 32:1069-1076, 2016.

Keywords: E. coli; cell lysis; fermentation process monitoring; product leakage; viscosity.

Figure 1

Characterization of cell lysis in an E. coli (Fab') fermentation. (a) Optical density at 600 nm (absorbance units (AU), in triplicate) and total Fab' concentration (mg/mL, induplicate). Induction time was at 38 h, using IPTG. (b) Capacitance profile (viable cells/mL, online continuous measurement) and shear viscosity (Pa s, single measurement, held at steady state for 10 s). Capacitance was calibrated offline with flow cytometry data (in triplicate).

Figure 2

Analytical characterization of cell lysis in an E. coli (Fab’) fermentation. (a) Intracellular Fab' concentration and leakage of Fab' to extracellular space (postinduction, mg/mL, in duplicate). (b) Cytotoxicity (based on lactate dehydrogenase (LDH) release to extracellular space, %, in triplicate) and DNA release (postinduction, mg/mL, in triplicate).

Figure 3

Flow cytometry plots for BOX (bis‐oxonol) and PI (propidium iodide) stains. For each plot, UL quadrant denotes dead cells and cell fragments, UR quadrant denotes PI stained cells (nonviable), LL quadrant denotes viable polarized cells, and LR quadrant denotes viable cells that have been stained by BOX (depolarized membrane). (a) Sample was taken in mid‐exponential phase, (b) sample was taken in mid‐stationary phase at the onset of cell lysis (36 h postinduction), and (c) sample was taken in late stationary/decay phase (57 h postinduction). Samples were measured in triplicate.

Figure 4

Scanning Electron Microscopy (SEM) images at x10,000 magnification. (a) SEM image of an E. coli fermentation sample showing predominantly viable cells in early stationary phase. (b) SEM image of an E. coli fermentation sample in late stationary/decay phase, showing; (1) healthy cells, (2) swollen cells, and (3) shells of lysed cells.

Figure 5

Viscometry flow curves of E. coli cell broth at various times during the fermentation, over a shear rate range 100–1000 s⁻¹. Induction time was at 38 h, using IPTG. The viscometry measurements were carried out at 25°C using 50 mm parallel plates. An increase in shear thinning behavior is evident as the fermentation proceeded (flow behavior index was greater than 0.95 for all samples). Single viscometry measurements were recorded at each shear rate, held at steady state for 10 s.

Figure 6

Effect of product leakage (mg/mL, measured in duplicate) and DNA release (mg/mL, measured in triplicate) on viscosity increase (Pa s, single measurement, held at steady state for 10 s) in postinduction culture for three fermentation runs.