Enhancing the selective extracellular location of a recombinant E. coli domain antibody by management of fermentation conditions

• Published in: Applied microbiology and biotechnology 2015-10, Vol.99 (20), p.8441-8453
• Main Authors: Voulgaris, Ioannis, Finka, Gary, Uden, Mark, Hoare, Mike
• Format: Article
• Language: English
• Subjects: Acids; Analysis; Antibodies; Antibodies - genetics; Antibodies - metabolism; Biomedical and Life Sciences; Biotechnological Products and Process Engineering; Biotechnology; cell growth; Cellular biology; Culture Media - chemistry; E coli; EDTA (chelating agent); Endotoxins; Escherichia coli; Escherichia coli - drug effects; Escherichia coli - genetics; Escherichia coli - growth & development; Escherichia coli - metabolism; extracellular space; Fermentation; Genetic recombination; Glycerol; Influence; Life Sciences; lipopolysaccharides; membrane permeability; Microbial Genetics and Genomics; Microbiology; Nucleic acids; Permeability; Permeability - drug effects; Physiological aspects; Polyethyleneimine - metabolism; Protein expression; Protein Transport; Proteins; Recombinant proteins; Recombinant Proteins - genetics; Recombinant Proteins - secretion; Studies; Viscosity; Yeast
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s00253-015-6799-3

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 (13.1 kDa) 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 was with 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.