Assignment-2: ChE 720 by Penny Dorka

1. 1 Assignment-2: ChE 720by Penny Dorka Improvement of mammalian cell culture process by adaptive, model-based dialysis fed-batch cultivation and suppression of apoptosis

2. 2 Objective To improve maximum cell-density and antibody production of the two NSO cell lines by culture mode and suppression of apoptosis

3. 3 System under analysis: Two NSO cell lines- 6A1 bcl-2 6A1 (100)3 producing chimeric cB72.3 IgG MAb for cancer therapy

4. 4 Culture Mode FED-BATCH Characterized by predetermined or controlled addition of nutrient medium in an otherwise batch culture (no withdrawal of culture) DIALYSIS FED-BATCH Fed-batch cultivation using dialysis

5. 5 Feed utilized FED-BATCH Feed containing 100mM glucose in an amino acid/vitamin concentrate employed BATCH

6. 6 Culture conditions Medium: serum-free ProCHO 4 CDM containing 23 mmol/L glucose supplemented with 25 �M L-Methionine suphoximine Temperature- 37 degrees centigrade DO at 50% air saturation Atmosphere of 5% CO2 pH maintained at 7 Feed different for BATCH and FED-BATCH culture

7. 7 Techniques adopted to improve cell culture Dialysis cultivation Adaptive fed-batch control bcl-2 overexpression

8. 8 Dialysis cultivation Dialysis: Separation of substances in solution by means of their unequal diffusion through semipermeable membranes Advantages: Facilitates removal of low molecular weight metabolites that can inhibit cell growth Larger monoclonal antibodies are retained

9. 9 Scheme of dialysis fed-batch process

10. 10 Adaptive model-based fed-batch control Use of an OLFO controller

11. 11 Adaptive model-based fed-batch control Advantages of an OLFO control: Universal character Permits adaptation, prediction of future course as well as its optimization

12. 12 bcl-2 overexpression Overexpression: Transfection of cell lines with vectors producing various proteins that regulate apoptosis Advantages: Suppression of apoptosis

13. 13 Schematic diagram for over-expression

14. 14 Procedures followed Bioreactor Cultivation: Process temperature maintained at 37 deg centigrade with DO at 50% air saturation and glucose concentration at 16 mmol/L. pH maintained at 7; stirring at 80-200 rpm. Cultures were routinely maintained in T25 flasks Analytical methods: 3 mL samples taken twice a day, analyzed for viable and total cell concentration; glucose; lactate; ammonia; antibody concentration; DNA content and osmolality

15. 15 Set-up for the membrane dialysis reactor http://scholarsportal.info/pdflinks/05092415570215774.pdf

16. 16 Procedures Model parameter identification and feed optimization algorithms Differential equations solved by fourth-order Runge-Kutta algorithm Model parameter identification and feed optimization done by Nelder-Mead algorithm Weighting factor of 1 was chosen by default for all parameters except, viable cell and glucose concentration for which weighting factor was 100.

17. 17 Model Equations Balances biophase Balances liquid phase inner reactor chamber Similar equations for Lactate; MAb and Limiting Substrate.

18. 18 Model Equations Balances liquid phase outer reactor chamber Similar equations for Lactate and Limiting substrate. Growth kinetics Death Kinetics [Monod-type equations]

19. 19 Performance of controller OLFO controller predicted viable cell concentration with an average accuracy of ~ 8% and glucose level with an average accuracy of ~ 5% accuracy =

20. 20 Results Dialysis fed-batch cultivation increases maximum cell density and product accumulation by a factor of ~ 5-7 6A1 bcl-2 cell line shows a prolonged growth phase and higher maximum cell density Viability of the cell line 6A1 bcl-2 ranges at 90-95% during growth phase and at 80-85%for the 6A1 (100)3 cell line

21. 21 Conclusions The fed-batch cultivation using dialysis increases maximum cell density and product accumulation in comparison to cultivation without dialysis The OLFO controller could control fed-batch and dialysis fed-batch cultivations of both cell lines The effect of the successful suppression of apoptosis by bcl-2 over-expression in one cell line gives positive results in contrast to the control cell line

22. 22 End of Presentation Questions?