DEVELOPMENT OF SCALE-DOWN TECHNIQUES FOR INVESTIGATION OF RECOMBINANTESCHERICHIA-COLI FERMENTATIONS - ACID METABOLITES IN SHAKE FLASKS ANDSTIRRED BIOREACTORS

We have developed shake-flask screening conditions that are predictive of specific expression of the chimeric toxin, TGFalpha-PE40, by recom binant Escherichia coli JM109 in stirred bioreactors. When a nutrient- rich stirred bioreactor medium was used in shake flasks, neither the e xtent of growth nor the specific level of recombinant protein expressi on duplicated the performance in stirred bioreactor fermentations. Inc omplete oxidation of glucose and concomitant accumulation of organic a cid metabolites, as well as oxygen limitation and lack of pH control, were examined as contributors to the poorer performance in the flask. The medium buffering capacity, initial glucose level, and flask aerati on were evaluated to establish the limits of ''scale-down'' conditions for expression both in a complex nutrient medium (M101) similar to th at used in stirred bioreactors and in a defined (FM) medium. Acid meta bolites and ethanol were measured as indicators of carbon flow from gl ucose as well as indirect indicators of oxygen limitation. For the com plex M101 medium, optimal shake-flask performance in 250-mL, nonbaffle d flasks at 37-degrees-C occurred with 0.3x medium strength, supplemen tation with 0.3 M HEPES buffer (pH 7.5), and 10 mL of medium per flask . Cultures grown under these conditions produced a maximum density of 3.6 g of dry cell weight/L (as estimated by absorbance measurements at 600 nm) and maintained a pH near neutrality. Additionally, metabolite markers of anaerobic or microaerobic conditions, such as ethanol, lac tate, and pyruvate, were not detected, and specific expression of TGFa lpha-PE40 was comparable to stirred bioreactors induced for expression at various biomass levels. When culture parameters were controlled wi thin these limits, similar results were also observed in the defined F M medium.