title: Pilot-scale process sensitivity studies for the scaleup of a fungal fermentation for the production of pneumocandins.
abstract: The filamentous fungus Glarea lozoyensis produces a novel, pharmaceutically important pneumocandin (B(0)) that is used to synthesize a lipopeptide which demonstrates cidal activity against clinically relevant pathogens. A range of unwanted pneumocandin analogs are also produced by the organism. To maintain the unwanted impurities to acceptable levels upon scaleup, a good understanding of the impact of chemical and physical environment on the cell physiology is required, which benefits downstream processing. Pilot-scale studies were performed to determine the impact of dissolved oxygen, temperature, pH, and carbon dioxide on the process. Experiments included multiple fermenters (up to seven) at 0.07 and 0.8 m(3) scale using single source medium sterilization and inoculum. Gas blending was used to separate effects of dissolved oxygen from agitation. The process was significantly influenced by dissolved oxygen level. The critical dissolved oxygen tension (C(crit)) for growth was below 2% air saturation. The C(crit) for production of pneumocandin B(0) was 20% air saturation, with a significant reduction of the specific production rate below this value. In contrast, low dissolved oxygen levels produced a substantial increase of pneumocandins B(1), B(5), and E(0), while high dissolved oxygen levels produced a disproportionate increase of D(5). This sensivity to dissolved oxygen was independent of agitation within a power range of 2-15 kW/m(3). Broth viscosity was impacted below 10% dissolved oxygen, suggesting an effect on morphology. The process was shown to be sensitive to temperature but relatively insensitive to pH and carbon dioxide (in the exhaust gas) within the ranges studied. This scaledown analysis explained phenomena seen at pilot scale and helped define operating boundary conditions for successful scale up to 19 m(3).
authors.affiliation: Biocatalysis and Fermentation Development, Bioprocess R&D Laboratories, Merck & Co., PO Box 2000, Rahway, NJ 97065, USA. david_pollard@merck.com
authors.forename: D J
authors.initials: DJ
authors.lastname: Pollard
authors.forename: T F
authors.initials: TF
authors.lastname: Kirschner
authors.forename: D
authors.initials: D
authors.lastname: Hernandez
authors.forename: G
authors.initials: G
authors.lastname: Hunt
authors.forename: R
authors.initials: R
authors.lastname: Olewinski
authors.forename: P M
authors.initials: PM
authors.lastname: Salmon
mesh_terms: D000900:Anti-Bacterial Agents / Q000096:biosynthesis*; D019149:Bioreactors / Q000382:microbiology*; D002245:Carbon Dioxide / Q000378:metabolism*; D054714:Echinocandins; D005285:Fermentation; D005658:Fungi / Q000254:growth & development / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D010100:Oxygen / Q000378:metabolism*; D010455:Peptides; D010456:Peptides, Cyclic / Q000096:biosynthesis*; D013696:Temperature
chemical_list: D000900:Anti-Bacterial Agents; D054714:Echinocandins; D010455:Peptides; D010456:Peptides, Cyclic; C079465:pneumocandin B(0); D002245:Carbon Dioxide; D010100:Oxygen