title: 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.
authors.affiliation: IMRC for Bioprocessing, The Advanced Centre for Biochemical Engineering, Department of Biochemical Engineering, University College London, Torrington Place, London, WC1E 7JE, UK. d.bracewell@ucl.ac.uk
authors.forename: D G
authors.initials: DG
authors.lastname: Bracewell
authors.forename: R A
authors.initials: RA
authors.lastname: Brown
authors.forename: M
authors.initials: M
authors.lastname: Hoare
mesh_terms: D019149:Bioreactors / Q000382:microbiology*; D015374:Biosensing Techniques / Q000295:instrumentation / Q000379:methods*; D018929:Cell Culture Techniques / Q000295:instrumentation / Q000379:methods*; D049109:Cell Proliferation; D004926:Escherichia coli / Q000502:physiology*; D007118:Immunoassay / Q000295:instrumentation / Q000379:methods*; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D009862:Online Systems; D055095:Optics and Photonics
chemical_list: D007128:Immunoglobulin Fragments