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).

6-pentyl-α-pyrone production byTrichoderma harzianum: The influence of energy dissipation rate and its implications on fungal physiology

“…Although the DO critical tension for maximum 6PP production is not known, a value of 35% of air saturation was chosen based on the determined values reported for other fungal metabolites, such as pneumocandin B 0 , penicillin, and amyloglucosidase (20%, 25%, and 40%, respectively) produced in submerged fermentation by Glarea lozoyensis, Penicillium chrysogenum, and A. oryzae (Amanullah et al, 2002;Henriksen et al, 1997;Pollard et al, 2002). Calculations using a model of a pellet under diffusional limitation (Cui et al, 1998), which took into account the oxygen concentration in the bulk, pellet porosity, and oxygen consumption rate, showed that even pellets with core diameters similar to our largest aggregates (<360 mm) would not be oxygen limited.…”

Scale up of a viscous fungal fermentation: Application of scale‐up criteria with regime analysis and operating boundary conditions

“…Each seed stage was run at 258C for approximately 3-5 days and, to ensure consistent results in the production fermentations, the transfer criteria to the next stage were based equally on the three parameters: cell mass, respiration, and residual carbohydrate. Pilot scale fermenters of 0.07 m 3 , 0.8 m 3 , and 19 m 3 were used as described by Junker et al (1988), Nienow et al (1994), and Pollard et al (2002b). Large-scale manufacturing was completed using a 57-m 3 fermenter, similar to that defined by Kelly and Humphrey (1998).…”

“…The soluble production medium was primarily composed of fructose, proline, and yeast extract as described by Connors et al (2000). Several midcycle additions of carbohydrates and amino acids were made to extend the production phase and to control unwanted pneumocandin analogs, as described in Pollard et al (2002b). The fermentation was controlled at pH 5.2 using sulfuric acid and sodium hydroxide, with temperature maintained at 258C.…”

“…The seed train incorporated two flask stages followed by two fermenter stages as described by Pollard et al (2002b). All seed stages used a medium primarily composed of yeast extract, cottonseed flour (Pharmamedia, Traders Protein, Lubbock, TX), and glucose (Connors et al, 2000), and the sources of raw materials were the same throughout the scale-up work.…”

Asymmetric reduction of α, β-unsaturated ketone to (R) allylic alcohol byCandida chilensis

“…Fermenter scales of 30, 70, 280, 800, and 19,000 L were used as described previously in Junker et al (2002), and Pollard et al (2002b). The defined yeast medium was pH adjusted to 4.8 and sterilized in situ at 122-1248C for 45 min.…”