1. Switch Between Cell Growth and Angiostain- Human IgG Fusion Protein Production of CHO Cells Using a Controllable Bottom Holding Time in a Novel Bioreactor Sing-Ying Hsieh, King-Ming Chang, Lewis Ho, Andy Chu, Shyh-Yu Shaw and Ing-Kae Wang Cesco Bioengineering, Co., Ltd

2. The Effect of Bottom Holding Time on Cell Growth and Protein Production in BelloCell Bioreactor

3. Mark AsRising rateTop holding timeDown rateBottom holding time 1101.5m1.0 mm/s0 sec1.0 mm/s1.5 mins 11009m1.0 mm/s0 sec1.0 mm/s9 mins 11030m1.0 mm/s0 sec1.0 mm/s30 mins 11060m1.0 mm/s0 sec1.0 mm/s60 mins 11090m1.0 mm/s0 sec1.0 mm/s90 mins Design HyQ PF-CHO MPS, 200 nM MTX Spinner Flask: 1/4 CM

4. GUR Comparison between Various Bottom Holding Time

5. Cell Growth Comparison between Various Bottom Holding Time

6. Time Point Protein Comparison between Various Bottom Holding Time

7. Accumulative Protein Comparison between Various Bottom Holding Time and Spinner Flask

8. Effect of Bottom Holding Time on Cell Growth and Productivity

9. Conclusion I Cell growth rate Protein production Cell growth and protein production were switched by control bottom holding time. The highest protein yield, 11060m, was 2.87 folds increasing compared with spinner flask in 459.5 hrs. In a same total yield (282.32 mg), spinner flask will spend around 50 days and consume 5.75 L medium.

10. Effect of Long-Term Culture on Cell Growth and Protein Production in BelloCell Bioreactor

11. object Nutrition effect (Glucose, Glutamine) in BelloCell-500 culture with CHO Cell The influence of seeding cell number (1x10 8, 2x10 8 ) in BelloCell-500 culture)

12. Design BC Serial No./ Spinner Gluc.(g/L)Gln(mM) Seeding Cell Number (x10 8 ) Mark As BC5004033.5223.5/2 BC5004047627/6 BC5004055425/4-2 BC5004065415/4-1 Spinner Flask 542Spinner Nutrition Test Seeding Cell Number Test Device Comparison HyQ PF-CHO MPS, 200 nM MTX, 11030 min Spinner Flask: 1/4 CM

13. The Reason of Setting 11030m of BelloCell for Long-Term Culture

14. GUR Comparison between 4 BelloCell and Spinner Flask 5/4-1 = 5/4-2 > 3.5/2 = 7/6 >Spinner Flask

15. Cell Number Comparison between 4 BelloCell and Spinner Flask Before 459.5 hrs: 3.5/2 > 5/4-1 = 5/4-2 > 7/6 > Spinner Flask After 459.5 hrs: 3.5/2 = 5/4-1 = 5/4-2 > 7/6 > Spinner Flask 3 BC underestimate after 363.5 hrs, 7/6:459.5 hrs Gluc, Gln cell growth ; Lactate, Ammonia, pH

16. Lactate Comparison between 4 BelloCell and Spinner Flask (7/6)>(5/4-1=5/4-2)>Spinner Flask>(3.5/2)

17. Ammonia Comparison between 4 BelloCell and Spinner Flask (7/6) >Spinner Flask >(5/4-1=5/4-2) >(3.5/2)

18. pH Comparison between 4 BelloCell and Spinner Flask (3.5/2)>(5/4-1=5/4-2)=Spinner Flask >(7/6) 6.8-7.8 6.8-7.8 6.8-7.3 6.7-7.9

19. L/G Comparison between 4 BelloCell and Spinner Flask Before 200 hr: aerobic(7/6, spinner flask) and anaerobic(3BC)

20. Time Point Protein Comparison between 4 BelloCell and Spinner Flask BelloCell(7/6) > BelloCell(3.5/2 、 5/4-1 、 5/4-2) = Spinner Flask

21. Accumulative Protein Comparison between 4 BelloCell and Spinner Flask 704.12 mg 573.55 mg 542.55 mg 528.46 mg 152.55 mg

22. Protein productivity (mg/L) Comparison between 4 BelloCell and Spinner Flask (7/6) > (5/4-2) = (3.5/2) = (5/4-1) = Spinner Flask 8L 15L 4.5L

23. Nutrition, Protein Production and Cell Growth between 4 BelloCell and Spinner Flask Nutrition Accumulative Protein=Protein Productivity Nutrition cell number ; cell growth protein production

24. Conclusion II The highest protein yield, 7/6, was 4.6 folds increasing compared with spinner flask in 35 days. In a same total yield (704.12 mg), spinner flask will spend around 145 days and consume 18 L medium. The highest protein productivity (mg/L), 7/6, was 2.6 folds increasing compared with spinner flask. The protein harvested could be prolonged from 24 hrs to 48 hrs and decreased consuming medium and increased protein concentration, when increased glucose to 7 g/L and glutamine to 6 mM.