Step change in the efficiency of centrifugation through cell engineering: co‐expression of Staphylococcal nuclease to reduce the viscosity of the bioprocess feedstock

Abstract

Cell engineering to enable step change improvements in bioprocessing can be directed at targets other than increasing product titer. The physical properties of the process suspension such as viscosity, for example, have a major impact on various downstream processing unit operations. The release of chromosomal DNA during homogenization of Escherichia coli and its influence on viscosity is well-recognized. In this current article we demonstrate co-expression of Staphylococcus aureus nuclease in E. coli to reduce viscosity through auto-hydrolysis of nucleic acids. Viscosity reduction of up to 75% was achieved while the particle size distribution of cell debris was maintained approximately constant (d(50) = 0.5-0.6 microm). Critically, resultant step change improvements to the clarification performance under disc-stack centrifugation conditions are shown. The cell-engineered nuclease matched or exceeded the viscosity reduction performance seen with the addition of exogenous nuclease removing the expense and validation issues associated with such additions to a bioprocess. The resultant material dramatically altered performance in scale-down mimics of continuous disc-stack centrifugation. Laboratory scale data indicated that a fourfold reduction in the settling area of a disc-stack centrifuge can be expected due to a less viscous process stream achieved through nuclease co-expression with a recombinant protein.

Assessment of the manufacturability of Escherichia coli high cell density fermentations

“…Previously described effects include the impact of competing contaminants on chromatography performance 40 and the impact of viscosity on subsequent downstream unit operations, such as the reduction of viscosity from 2.4 mPas to 0.8 mPas (threefold reduction), leading to a fourfold reduction in the settling area required in the disc-stack centrifuge. 36 …”

“…36 Cell lysis due to culture ageing may often allow the release of intracellular materials, contributing to an increase in the viscosity of the feed stream as the fermentation progresses. This effect as well as that of the effect of shear on viscosity is shown in Figure 5.…”

IMAC capture of recombinant protein from unclarified mammalian cell feed streams

“…By contrast, proteases directly affect product stability and affinity tag cleavage (Gao et al, 2011). Contamination with hDNA significantly affects feed viscosity (Balasundaram et al, 2009), and therefore, column pressure drop as well as needing to meet stringent regulatory standards in final preparations (Nissom, 2007). In our process, cell damage was predicted to occur during column passage, especially as the cells traversed the relatively high shear regions of the frit.…”

Measuring E. coli and bacteriophage DNA in cell sonicates to evaluate the CAL1 reaction as a synthetic biology standard for qPCR

“…40 mL of this LB inoculum was used to inoculate 360 mL chemically defined media [2] in a 5L shake-flask to OD 600  = 2.5 (Fig. 1D), typical of the end-point of seed train cultivation used to provide inoculum for growth in bioreactors.…”