caa a22 4500 605499713 CHVBK 20210128100552.0 cr unu---uuuuu 210128e20151001xx s 000 0 eng 10.1007/s00253-015-6799-3 doi (NATIONALLICENCE)springer-10.1007/s00253-015-6799-3 Enhancing the selective extracellular location of a recombinant E. coli domain antibody by management of fermentation conditions [Elektronische Daten] [Ioannis Voulgaris, Gary Finka, Mark Uden, Mike Hoare] The preparation of a recombinant protein using Escherichia coli often involves a challenging primary recovery sequence. This is due to the inability to secrete the protein to the extracellular space without a significant degree of cell lysis. This results in the release of nucleic acids, leading to a high viscosity, difficulty to clarify, broth and also to contamination with cell materials such as lipopolysaccharides and host cell proteins. In this paper, we present different fermentation strategies to facilitate the recovery of a V H domain antibody (13.1kDa) by directing it selectively to the extracellular space and changing the balance between domain antibody to nucleic acid release. The manipulation of the cell growth rate in order to increase the outer cell membrane permeability gave a small ~1.5-fold improvement in released domain antibody to nucleic acid ratio without overall loss of yield. The introduction during fermentation of release agents such as EDTA gave no improvement in the ratio of released domain antibody to nucleic acid and a loss of overall productivity. The use of polyethyleneimine (PEI) during fermentation waswith the aim to (a) permeabilise the outer bacterial membrane to release selectively domain antibody and (b) remove selectively by precipitation nucleic acids released during cell lysis. This strategy resulted in up to ~4-fold increase in the ratio of domain antibody to soluble nucleic acid with no reduction in domain antibody overall titre. In addition, a reduction in host cell protein contamination was achieved and there was no increase in endotoxin levels. Similar results were demonstrated with a range of other antibody products prepared in E. coli. The Author(s), 2015 Periplasmic release nationallicence Polyethyleneimine nationallicence Fermentation nationallicence Domain antibody nationallicence E. coli nationallicence Voulgaris Ioannis BioPharm R&D, BioPharm Process Research, GlaxoSmithKline R&D, SG1 2NY, Stevenage, UK aut Finka Gary BioPharm R&D, BioPharm Process Research, GlaxoSmithKline R&D, SG1 2NY, Stevenage, UK aut Uden Mark BioPharm R&D, BioPharm Process Research, GlaxoSmithKline R&D, SG1 2NY, Stevenage, UK aut Hoare Mike The Advanced Centre for Biochemical Engineering, Department of Biochemical Engineering, University College London, WC1H 0AH, London, UK aut Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/20(2015-10-01), 8441-8453 0175-7598 99:20<8441 2015 99 253 https://doi.org/10.1007/s00253-015-6799-3 text/html Onlinezugriff via DOI BK010053 XK010053 XK010000 Metadata rights reserved Springer special CC-BY-NC licence nationallicence 1 research-article jats NATIONALLICENCE NATIONALLICENCE NL-springer NATIONALLICENCE 856 40 https://doi.org/10.1007/s00253-015-6799-3 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Voulgaris Ioannis BioPharm R&D, BioPharm Process Research, GlaxoSmithKline R&D, SG1 2NY, Stevenage, UK aut NATIONALLICENCE 700 1- Finka Gary BioPharm R&D, BioPharm Process Research, GlaxoSmithKline R&D, SG1 2NY, Stevenage, UK aut NATIONALLICENCE 700 1- Uden Mark BioPharm R&D, BioPharm Process Research, GlaxoSmithKline R&D, SG1 2NY, Stevenage, UK aut NATIONALLICENCE 700 1- Hoare Mike The Advanced Centre for Biochemical Engineering, Department of Biochemical Engineering, University College London, WC1H 0AH, London, UK aut NATIONALLICENCE 773 0- Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/20(2015-10-01), 8441-8453 0175-7598 99:20<8441 2015 99 253