Addressing a whole bioprocess in real-time using an optical biosensor-formation, recovery and purification of antibody fragments from a recombinant E. coli host

Abstract

The use of biosensor technology is described to address in real-time the production and subsequent purification of a bioactive recombinant protein product. The product, D1.3 Fv antibody fragment, was expressed in Escherichia coli and purified via two process routes, one for extracellular and one for intracellular product material. The cells were harvested by centrifugation in a solid bowl CARR Powerfuge and stored at -70 degrees C. Clarification of the supernatant was performed by depth filtration, followed by affinity chromatography for final purification of the extracellular product. To purify the intracellular product the harvested cells were resuspended and homogenised. Removal of debris in the CARR Powerfuge was followed by depth filtration and affinity chromatography. In this work we have shown the rapid determination of bioactive product levels, and the impact this has on improved accountability and confidence is demonstrated in process mass balances on the product using the data acquired during process operation.

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

“…For many applications of high‐pressure homogenization two passes (equivalent t H = 10 min) is used for adequate release with acceptably low formation of fine particles as cell debris or five passes (equivalent t H = 25 min) for near complete release with high levels of fine particles in disruptates (e.g., Boychyn et al, 2000; Bracewell et al, 2004; Levesley and Hoare, 1999; Salt et al, 1995). The corresponding exposure time for the same extent of protein release using focused acoustics is ∼3 and 7 min, respectively.…”

Impedimetric Dengue Biosensor based on Functionalized Graphene Oxide Wrapped Silica Particles

“…Applications in environmental and agricultural fields, and particularly for anti-terrorist activity and homeland security, are also rapidly increasing [10,11]. For example, optical biosensors have now the highest sensitivity, approaching theoretical limits of interface sensitivity, which is critical for detection of drug candidates, viruses or pathogens [12][13][14][15][16].…”

Soft sensors in bioprocessing: A status report and recommendations

“…multimers or fragments of the product or undesired chemical modifications of the product such as differently glycosylated or deamidated and oxidized forms). This makes direct measurement via a soft sensor challenging; success has been reported using biosensor [35] and Fourier transform infrared spectroscopy approaches [36]. Such methods usually only provide relatively coarse grain understanding of the product, while more detailed aspects of the product structure would be measured off‐line in a quality control laboratory often using mass spectrometry.…”