Extreme scale-down of expanded bed adsorption: Purification of an antibody fragment directly from recombinant E. coli culture.
Scale-down is a methodology that combines the use of very small volumes of process fluid in dedicated devices to predict accurately the behaviour of process-scale biotechnological unit operations and for the production of comparable material for use in further devices which, taken together, facilitate the mimic of a complete full-scale process. This article provides the rationale behind the development of a small-scale mimic and demonstrates the use of a highly scaled-down expanded bed to predict hydrodynamic, kinetic, and adsorptive performance using less than 5-mL sample volumes. Data acquired on a specially developed 1.9 mm ID column was compared with that obtained in a standard 25 mm ID column. A homogenised E. coli system expressing an antibody fragment (F(ab)) adsorbed onto an rProtein A matrix was used to characterise the full adsorptive performance. Breakthrough curve studies using BSA in buffer were used to characterise binding kinetics. Performance at the two scales was comparable both in terms of expansion, axial dispersion, binding isotherms, and elution behaviour of the antibody fragment. The eluted F(ab) material was further purified by ion exchange chromatography to demonstrate the similarity between the profile of the product material obtained at both scales. The high level of scale-down (approximately 200-fold) provides for rapid process evaluation early in development, where material is at a premium and where a fast appreciation of the likely merits of one process strategy will lead to greater confidence in process selection and more robust flowsheets.
Willoughby
Nik
N
IMRC for Bioprocessing, The Advanced Centre for Biochemical Engineering, University College London, Torrington Place, London WC1E 7JE, United Kingdom. n.willoughby@ucl.ac.uk
Martin
Phillip
P
Titchener-Hooker
Nigel
N
D000327:Adsorption; D000906:Antibodies / Q000302:isolation & purification*; D002845:Chromatography / Q000295:instrumentation / Q000379:methods*; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004867:Equipment Design; D004926:Escherichia coli / Q000235:genetics; D005285:Fermentation; D007700:Kinetics; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000302:isolation & purification*
D000906:Antibodies; D011994:Recombinant Proteins