High Cell Density Cultivation Process for the Expression of Botulinum Neurotoxin a Receptor Binding Domain

• Published in: Toxins 2022-04, Vol.14 (4), p.281
• Main Authors: Ben David, Alon, Papir, Yoel, Hazan, Ophir, Redelman, Moses, Diamant, Eran, Barnea, Ada, Torgeman, Amram, Zichel, Ran
• Format: Article
• Language: English
• Subjects: Acetic acid; Animals; Antigens; Batch culture; Binding; Binding sites; Botulinum toxin type A; Botulinum Toxins, Type A - metabolism; Botulism; Botulism - prevention & control; Cell Count; Cell culture; Cell density; Clostridium botulinum; Cultivation; Culture Media - metabolism; Density; Domains; E coli; Escherichia coli; Fermentation; Flasks; Food contamination; Growth conditions; high cell density cultivation; Mice; Neurotoxins; Nutrients; Protein expression; Proteins; Receptors; recombinant protein expression; Recombinant Proteins - metabolism; subunit vaccine; Supplements; Toxins; Vaccines
• ISSN: 2072-6651; 2072-6651
• DOI: 10.3390/toxins14040281

The receptor-binding domain of botulinum neurotoxin (H fragment), is a promising botulism vaccine candidate. In the current study, fermentation strategies were evaluated to upscale H fragment expression. A simple translation of the growth conditions from shake flasks to a batch fermentation process resulted in limited culture growth and protein expression (OD of 11 and volumetric protein yields of 123 mg/L). Conducting fed-batch fermentation with rich media and continuous nutrient supplementation significantly improved culture growth (OD of 40.3) and protein expression (1093 mg/L). A further increase in H fragment yield was achieved by high cell density cultivation (HCDC). The bacterium was grown in a defined medium and with a combined bolus/continuous feed of nutrients to maintain desired oxygen levels and prevent acetate accumulation. The final OD of the process was 260, and the volumetric yield of the H fragment was 2065 mg/L, which reflects improvement by an order of magnitude. Purified H fragments, produced by HCDC, exhibited typical biochemical and protective characteristics in mice. Taken together, the advancements achieved in this study promote large-scale production of the H fragment in for use in anti-botulism vaccines.