1. HCDC of E. coli under Fed- Batch conditions Whiffin Cooney Cord-Ruwisch High cell density cultures Chemostat and Batch culture have different advantages and problems Productivity R is higher in chemostat Maintaining sterility is easier in batch. Contamination from inside = highly productive mutants reverting to wild strain For typical industry processes batch is preferred although it is more difficult to control and has lower. Batch is the only option for : seondary metabolites

2. Process Control examples ExampleMeasure What Act By Why ChemostatpHAdding NaOH Avoid Contamination Anaerobic digestion H2Adjusting loading rate / recycle of methanogens / adding NaOH Avoid acidification SNDDO, OURAir supplyAvoid over/ underoxidation HCDCStarvation levelFeedingAvoid acetate build-up in cells

3. What is HCDC High Cell density Cultures are essential for maximum productivity of recombinant protein production (e.g. pharmaceuticals) After producing the biomass to the highest possible density (e.g. OD 50) the expression of the recombinant product (e.g. on mulitple plasmid copies) is induced. To produce OD 50 (about 50 g/L of dry mass) more than 100 g of substrate (e.g. sugar) is needed.  Substrate inhibition (almost “jam”).

4. What is HCDC High Cell density Cultures are essential for maximum productivity of recombinant protein production (e.g. pharmaceuticals) After producing the biomass to the highest possible density (e.g. OD 50) the expression of the recombinant product (e.g. on mulitple plasmid copies) is induced. To produce OD 50 (about 50 g/L of dry mass) more than 100 g of substrate (e.g. sugar) is needed.  Substrate inhibition (jam).

5. Why Fed-batch for HCDC? To add the full amount of substrate needed at the beginning  substrate inhibition Solution: After an initial batch with less substrate, add substrate sequentially (Fed batch) S Time Initial Batch Fed-batch The fedbatch has no outflow Feed is added from a concentrate (syrup) Arrows show S- addition

6. Need for precise feed addition Too little glucose addition slows down the process too much Too high glucose concentrations  build up acetate even at high DO concentrations when sugars are saturating S Time Initial Batch Controlled sugar addit. Acetate buildup interferes with growth and expression of recombinant proteins Glucose should be administered on demand (avoid over saturation and strong substrate limitation)

7. Why does acetate build up? TCA 24 6 10 3 2020 2020 8282 8282 0101 0101 2020 2020 ETC, OUR 8*2 0 Glycolysis faster than TCA / ETC / OUR Hence acetate (8 2 ) builds up. Metabolic overflow Crabtree effect. TCA bottleneck Abbreviations: TCA- Tricarboxylic Acid Cycle. OUR = oxygen uptake rate, ETC = Electron Transport Chain, 24-6 = glucose, 10-3= pyruvate

8. Strategies to administer glucose on demand Administering feed ON DEMAND requires feedback loop (measuring glucose demand – then increasing or decreasing glucose flow rate to reactor). No demand if glucose is saturating. A number of strategies are possible to evaluate DEMAND for glucose: Strategy 1: Glucose stat: measure glucose and only increase feedrate when glucose is less than saturating levels. Problems: No online glucose sensor, need for very precise measurements at very low [glucose ](close to ks value of E.coli)

9. Strategies to administer glucose on demand Strategy2: Acetate stat: measure acetate online and lower glucose feedrate as soon as acetate builds up. Problem: No online acetate sensor Strategy 3: Monitor glucose saturation from changes in OUR in response to feedrate changes. (“Starvostat”) BTW. E.coli can take up acetate excreted acetate under glucose starvation.

10. How to determine glucose demand of the culture ? Experience from our lab class (OUR) shows that a starving culture responds to the addition of substrate by increasing OUR. If there is not effect by adding extra substrate  culture was S-saturated.  Culture can provide information to operator whether it is substrate saturated or limited.

11. Keep DO steady state by constant glucose feed rate Do a step increase in feedrate No decrease in DO (hence no increase in OUR)  go back to old feedrate, as culture is feed saturated (no demand) Decrease in DO  culture was limited  shows a higher capacity for OUR, ETC and TCA and demands more glucose without risk of acetate buildup Monitor glucose demand of the culture Feed rate Time Glucose feedrate D.O. Sign of demand

12. Keep testing for glucose demand. If the culture shows glucose demand, increase the feedrate by say 2% and keep testing for further glucose demand. As long as the culture will grow, its demand will increase S Time Glucose feedrate D.O. Feedback control to keep culture on the border of substrate saturation Problem with this approach: If culture is saturated the glucose step increase  acetate buildup. Better approach?

13. Method published by V. Whiffin et al. Measure OUR-1 Interrupt feed for 30 sec. Measure OUR-2 If OUR-2 < OUR-1,(=starving) then increase FR by 1 %, else decrease FR by 1% Keep new FR for 2 min Resume from the top

14. Keep testing for glucose demand. If the culture shows glucose demand, increase the feedrate by say 2% and keep testing for further glucose demand. As long as the culture will grow, its demand will increase S Time Glucose feedrate OUR Feedback control to keep culture on the border of substrate saturation Problem with this approach: If culture is saturated the glucose step increase  acetate buildup. Better approach?