1. High cell density culture system for production of live Salmonella typhimurium vaccines High cell density culture system for production of live Salmonella typhimurium vaccines Kaltima Phichai Chiang Mai Rajabhat University THAILAND

2. Introduction Live Salmonella typhimurium vaccines (STM1) is a deletion mutant strain of S. typhimurium of great interest as a live vaccine candidate for protection of poultry from infection with Salmonella spp. Live Salmonella typhimurium vaccines (STM1) is a deletion mutant strain of S. typhimurium of great interest as a live vaccine candidate for protection of poultry from infection with Salmonella spp. The development of a production technology for this live vaccine required the understanding of the nutritional requirements of the strain and its application to a high density culture system for cost-effective manufacture. The development of a production technology for this live vaccine required the understanding of the nutritional requirements of the strain and its application to a high density culture system for cost-effective manufacture.

3. High Cell Density Culture Systems Avenues for investigation Avenues for investigation 1.Production Strain 2.Culture medium 3.The plumbing These principles are equally applicable to eukaryotic expression systems. These principles are equally applicable to eukaryotic expression systems.

4. HCDC for Salmonella typhimurium Frozen seed vial (1 mL) Seed flask (100-500 mL) Seed fermenter (5-50 L) Production fermenter (100-1000 L) Blending with cryoprotectant and dispensing Freeze-drying Distribution Cell concentration (crossflow filtration) Storage Quality control

5. Objectives To develop a thorough understanding of the nutritional requirements of the strain To develop a thorough understanding of the nutritional requirements of the strain To use this knowledge in the formulation of a minimal medium which will support high cell density growth in a fed-batch bioreactor process. To use this knowledge in the formulation of a minimal medium which will support high cell density growth in a fed-batch bioreactor process.

6. Methodology The nutritional requirements of Salmonella typhimurium aroA mutant were initially determined with a chemically defined medium. The fermentation was further improved by mass balance of the medium and a change in fed batch operation strategy.

7. Medium formulation Closed loop strategy Closed loop strategy Chemically defined medium Chemically defined medium Components tested one-at-a-time Components tested one-at-a-time Growth monitored by total viable count (TVC) and optical density (OD) Growth monitored by total viable count (TVC) and optical density (OD) Shake flask culture Shake flask culture –1 L flasks + 200 mL –37°C –200 rpm

8. 2.6±0.2 × 10 9 cfu/mL 3.7±0.3 × 10 9 cfu/mL

9. Medium formulation

10. 2.7±0.7 × 10 9 cfu/mL 2.1±0.2 × 10 10 cfu/mL

11. Bioreactor plumbing Three options for mode of operation 1. Batch 2. Continuous culture 3. Fed batch

12. Batch Culture After seeding a liquid medium with an inoculum of living cells nothing (except gas) is added to the culture or removed from it as growth proceeds. After seeding a liquid medium with an inoculum of living cells nothing (except gas) is added to the culture or removed from it as growth proceeds. In this reaction, the concentrations of nutrients, cells, and products vary with time. In this reaction, the concentrations of nutrients, cells, and products vary with time.

13. Batch Culture T pH AIR DOT EXHAUST

14. Batch Culture Batch Culture

15. Advantages of batch culture systems: Simple to operate Established technology Risk of contamination is not great Moderate yield Disadvantages of batch culture systems: Limited productivity (greater portion of downtime)

16. Continuous Culture (CSTR) Continuous Culture (CSTR) F x f x s f s c if c i V T pH AIR DOT

17. Continuous Culture Continuous Culture Advantages of continuous culture systems: Potential for increase in total product yield Increased productivity due to reduced downtime Disadvantages of continuous culture systems: Increased complexity of operation Low product concentration (dilute product stream) Increased risk of contamination Issues of culture stability

18. Something in between: Fed Batch Culture The culture is grown in batch mode until just before onset of the stationary phase. A feed stream is introduced under under the control of: –Predetermined schedule (linear, exponential, or modeled) –Feedback loop (DOT, pH, nutrient conc.)

19. Fed batch culture system Fed batch culture system T pH PRODUCTION VESSEL AIR T SETPOINT MEDIUM STORAGE CONTROLLER MEDIUM PUMP

20. Profiles of Batch and Fed Batch Culture Profiles of Batch and Fed Batch Culture

21. Fed Batch Culture Systems Fed Batch Culture Systems Advantages of fed batch culture systems: Greater productivity than batch and continuous systems Simpler operation than continuous systems Culture stability can be controlled Disadvantages of fed batch culture systems : Greater operating complexity than batch systems Requires a thorough understanding of the nutritional requirements of the organism

22. Results

23. Results Fed-batch culture resulted in higher cell densities than batch culture with about a two-fold increase in dry cell weight. This was due to the extended growth phase that resulted from the gradual addition of glucose to the medium compared to batch culture. Fed-batch culture resulted in higher cell densities than batch culture with about a two-fold increase in dry cell weight. This was due to the extended growth phase that resulted from the gradual addition of glucose to the medium compared to batch culture.

24. 2.7±0.7 × 10 9 cfu/mL 2.1±0.2 × 10 10 cfu/mL 3.3±0.3 × 10 10 cfu/mL

25. Putting it all together: Medium design and plumbing Putting it all together: Medium design and plumbing Salmonella is known to be auxotrophic for a number of amino acids Amino acid supplement –Tyrosine, Phenylalanine, Tryptophan –Cysteine, Methionine –Glutamic acid, Leucine –Need to direct metabolism towards growth

26. Typical growth kinetics and stirrer speed for fed-batch culture of STM1 in CDM with 5 pulses of aromatic amino acids solution. Arrows indicate when aromatic amino acids were pulsed into the fermenter

27. Average growth kinetics for fed-batch culture of STM1 in CDM with three different growth rate set points

28. Maximum dry cell weight, viable cell count, feed time, and NH 4 OH consumption for fed-batch culture of STM1 in CDM with different growth rate set point Growth rate Set point (h -1 ) Maximum dry cell weight (g/L) Maximum viable cell (cfu/mL) Feed time (h) NH 4 OH used for pH control (mL) 0.3 29.2  0.13 1.18  0.16  10 11 11.3 191  1.4 0.45 27.9  0.11 1.05  0.14  10 11 7.6 220  2.4 0.6 29.5  0.25 9.72  2.00  10 10 5.7 242  1.6

29. HCDC for Salmonella typhimurium Frozen seed vial (1 mL) Seed flask (100-500 mL) Seed fermenter (5-50 L) Production fermenter (100-1000 L) Blending with cryoprotectant and dispensing Freeze-drying Distribution Cell concentration (crossflow filtration) Storage Quality control

30. HCDC for Salmonella typhimurium Frozen seed vial (1 mL) Seed flask (100-500 mL) Seed fermenter (5-50 L) Production fermenter (100-1000 L) Blending with cryoprotectant and dispensing Freeze-drying Distribution Cell concentration (crossflow filtration) Storage Quality control

31. HCDC for Salmonella typhimurium Frozen seed vial (1 mL) Seed flask (100-500 mL) Seed fermenter (5-50 L) Blending with cryoprotectant and dispensing Freeze-drying Distribution Storage Quality control