Ultra scale-down studies of the effect of flow and impact conditions duringE. coli cell processing

Abstract

The ability to recover cells from a fermentation broth in an intact form can be an important criterion for determining the overall performance of a recovery and purification sequence. Disruption of the cells can lead to undesired contamination of an extracellular product with intracellular components and vice versa loss of intracellular products may occur. In particular, the value of directed location of a product in the periplasmic space of say Escherichia coli (E. coli) would be diminished by such premature non-selective cell disruption. Several options exist for cell recovery/removal; namely centrifugation, in batch or continuous configuration, filtration or membrane operations, and in selected cases expanded beds. The choice of operation is dependant on many variables including the impact on the overall process sequence. In all cases, the cells are exposed to shear stresses of varying levels and times and additionally such environments exist in ancillary operations such as pumping, pipe flow, and control valves. In this study, a small-scale device has been designed to expose cells to controlled levels of shear, time and impact in a way that seeks to mimic those effects that may occur during full-scale processes. The extent of cell breakage was found to be proportional to shear stress. An additional level of breakage occurred due to the jet impacting on the collecting surface. Here it was possible to correlate the additional breakage with the impact velocity, which is a function of the distance that the jet travels before meeting the collection surface and the initial jet velocity.

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

“…During fermentation, as the limit of the periplasm is reached, cells begin to lose viability and leak the Fab' product and other intracellular content to the fermentation broth. Apart from product loss in late stage fermentation, the remaining viable cells become more fragile and break fourfold more than those harvested at an earlier stage, if subjected to the equivalent shear level of that in an industrial centrifuge . As cell lysis occurs in late stage fermentation, leakage of product to the fermentation broth also acts to increase the broth viscosity, as large quantities of chromosomal DNA and other intracellular content are released simultaneously into the broth .…”

An ultra scale‐down approach to study the interaction of fermentation, homogenization, and centrifugation for antibody fragment recovery from rec E. coli

“…Similar effects might be reproduced during focused acoustics (Li et al, 2012; Wenger et al, 2008). During disruption by either mechanism there will broadly be two parallel processes occurring the first being the breakage of the cells to release intracellular components and cell ghosts (e.g., Chan et al, 2006) and the second being attrition or degradation of the released components including the cell ghosts and the poly nucleic acids. The extent of both processes is likely to be determined by the power dissipation and the mean residence time in the zone of highest power dissipation.…”

Biomedical application of plasmid DNA in gene therapy: A new challenge for chromatography

“…The main options for harvesting large volumes of culture are continuous centrifugation or microfiltration (Kong et al 2008). Previous studies have shown that E. coli cells harvested with an intermittent-discharge, continuous flow disc-stack centrifuge are subjected to stresses that are sufficiently high to cause lysis, especially if cells grown in a defined medium (Chan et al 2006). Typically, the centrifugation method used had a strong influence on sc pDNA yield and enhances in productivity could be obtained by an integrated approach between fermentation and centrifugation conditions.…”