Pilot-scale process development and scale up for antifungal production.
A pilot-scale fermentation was developed for an antifungal compound produced by a filamentous fungus. Replacement of galactose with lactose (20-fold cost savings) and a threefold phosphate reduction (15 to 5 g/L) improved productivity 2.5-fold. Addition of supplements--glycine, cobalt chloride, and trace elements--resulted in a further twofold productivity increase, greater process robustness, and less foaming which reduced antifoam addition tenfold (30 to <3 mL/L). Mid-cycle lactose limitations were addressed by raising initial lactose levels (40 to 120 g/L) resulting in another twofold productivity increase. Overall, peak titers increased tenfold from 45 +/- 9 to 448 +/- 39 mg/L, and productivities improved from 3 to 25 mg/L day. Despite its high productivity, process scale up was challenged by high broth viscosity (5,000-6,000 cP at 16.8 s(-1)). Gassed power requirements at the 600 L scale (4.7 kW/1,000 L) exceeded available power at the 15,000 L scale (3.0 kW/1,000 L), and broth transfer to the downstream isolation facility was hindered. Mid-cycle broth dilution with up to five 10 vol% additions of 12 wt% lactose solution or whole medium-reduced viscosity three- to fivefold (1,000-1,500 cP at 16.8 s(-1)), gassed power within scale-up limits (2.5 kW/1,000 L), and peak titer by up to 45%. The process was scaled up to the 15,000 L working volume based on constant aeration rate (vvm) and peak impeller tip speed, raising superficial velocities at similar shear. This strategy maximized mass transfer rates at target gassed power per unit volume levels, and along with controlled broth viscosity, precluded multiple dilution additions. A final titer of 333 mg/L with one dilution addition was achieved, somewhat lower than expected, likely owing to inhibition from some unmeasured volatile compound (not believed to be carbon dioxide) during an extended period of high back-pressure in the early production phase.
Fermentation Development and Operations, Merck Research Laboratories, Rahway, NJ, USA. beth_junker@merck.com
Beth
B
Junker
Andre
A
Walker
Michelle
M
Hesse
Michael
M
Lester
Diane
D
Vesey
Jens
J
Christensen
Bruce
B
Burgess
Neal
N
Connors
D000935:Antifungal Agents / Q000302:isolation & purification* / Q000378:metabolism*; D001203:Ascomycota / Q000254:growth & development / Q000378:metabolism; D001698:Biomedical Engineering; D019149:Bioreactors; D003470:Culture Media; D005285:Fermentation; D005690:Galactose / Q000378:metabolism; D005990:Glycerol / Q000378:metabolism; D007785:Lactose / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism; D010865:Pilot Projects; D014783:Viscosity
D000935:Antifungal Agents; D003470:Culture Media; D010710:Phosphates; D007785:Lactose; D005990:Glycerol; D005690:Galactose