1. Lecture 9 Animal Cell Biotechnology Scaling up the production processpH• set point pH of 7.4 ± 0.1 common• without buffering the pH could fluctuate• for small scale operation, can maintain pH by using gaseous CO2 to control culture pH
2. Lecture 9 Animal Cell Biotechnology Scaling up the production process: Controlling the pH with CO2Butler, M. 2004. Animal cell culture and technology 2nd ed. London and New York:Garland Science/BIOS Scientific Publishers. P161.
3. Lecture 9 Animal Cell Biotechnology Scaling up the production process• for larger scale cultures, can directly add acid or base to maintain pH• insert probe into culture to detect changes in pH• acid or base pumped in accordingly, under automatic control → pH ↓, add base (concentrated sodium bicarbonate) → pH ↑, add acid (concentrated HCl) → not normally a problem due to lactic acid production
4. Lecture 9 Animal Cell Biotechnology Scaling up the production processOxygen requirements• supply of oxygen to satisfy cell metabolism is one of the major problems associated with culture scale-up• O2 consumption rate: 0.06-0.6 mM/hour for 106 cells/ml• for small volumes (< 1 litre) O2 diffusion from the headspace through the culture surface is sufficient to meet the oxygen demand• as the volume increases, the surface-volume ratio decreases
5. Dissolved oxygen polarographic electrodeDissolved oxygen polarographic electrode (InPro 6050, Metler-Toledo, 2006). The cathode, where the half-reaction with O2(O2+2H2O+4e-→4OH-) takes place, is in contact with amembrane, that allows the transport of the dissolved oxygenfrom the external medium. At the anode, the silver oxidation(Ag++Cl-→ AgCl + e-) takes place.
7. The limitation of O2 supply by diffusion through the head space Culture Head space O2 supply O2 demandvolume (L) area (cm2) (mmol/h) (mmol/h) 1 100 0.063 0.063 10 500 0.313 0.625 100 2500 1.56 6.25
8. Lecture 9 Animal Cell Biotechnology Scaling up the production processOTR = oxygen transfer rateOUR = oxygen uptake rateTo supply sufficient O2 to cells and to avoid O2 depletion: OTR > OUR
9. Lecture 9 Animal Cell Biotechnology Scaling up the production process• at > 1 L, the surface-volume ratio is too low to satisfy overall O2 demand• surface-volume ratio of a fermentor defined by aspect ratio: aspect ratio = diameter of culture/height of culture
10. Fig. 9.7 Aspect ratio = width/ height of culture
11. Lecture 9 Animal Cell BiotechnologyScaling up the production process: Bubble death !
12. Lecture 9 Animal Cell Biotechnology Scaling up the production processStrategies to prevent cell damage• use of chemical agents to reduce cell damage, such as 0.1% Pluronic F68 → synthetic copolymer of ethylene and propylene oxide, reduces cell:bubble interaction by preventing attachment of cells to bubbles• cover gas sparger with fine wire mesh to reduce the number of bubbles reaching cells• use of alternate fermentors (i.e. air lift fermentor)• sparge media in a secondary vessel• use gas permeable tubing (i.e. thin-walled silicone tubing) within bioreactor
13. Strategies for controlling dissolved oxygen Fig. 9.9 (b) Intermittent oxygen sparging (a) Change in air flow rate(c) Control of gas composition (d) Spin filter isolates cells from sparged gass
14. Dissolved oxygen control O2 flow rate controller Q1 QT = Q1 + Q2 PC N2 flow rate controller Q2 C (%)DO is controlled by the adjustment of the oxygen fraction in the sparged gas.Flow rate is kept constant and corresponds to the sum of the two controlled gases Q1 and Q2.
15. Fig. 9.11 Modulated feedback control +Set point _
16. A typical control loop controller actuator process + error electric PID bioreactorset-point resistance - sensor measured value thermometer A set-point is compared to the measured value by the sensor. An error measurement based on a signal to the electric resistance (actuator) is generated by the controller, that will heat up the bioreactor (process).
17. d erroractuation • P.error I. error.dt D. On-off controller, in which the action can only assume two states (on or dt off). • Controller modulated by pulse width (Pulse-Width Modulation or PWM). In this control type, the action is also on or off but the time that the actuator stays on within a certain cycle can be adjusted continuously. , allowing a final operation of different intensities. • Cascade controller, composed of one master and one slave loop. This is used when a more rigid control of a process variable is required, for instance, the temperature of the culture medium. • P-I-D controller, or Proportional-Integral-Derivative. Its based on the principle that the action is taken not only on how large is the error (difference between desired and measured values), but also on the sum of past errors (integral of the error) and to the rate that the error is changing (derivative of the error). where actuation is the controller output, error is the difference between the desired value (set-point) and the one measured by the sensor, t is time and P, I and D are constants that need to be adjusted for each system. The adjustment of the constants for a process, is called P-I-D controller tuning.
18. Proportional control.• The output of the controller is proportional to the error signal.• = 0 + k.E• where = output signal of the controller• where 0= output signal when the error is zero• where k = controller gain or proportional band• where E = error or deviation from the set point
19. Integral and derivative control.• Integral control. The output of the controller is a function of the integral of error and time. Here, the control action increases with time as long as the error is registered.• = 0 + tI E dt• where tI = integral time constant• Derivative time. The output of the controller is a function of the rate of change of the error. d E• = 0 + td . dt• where td = derivative time constant
20. Feeding flow rate control system based on glucose concentration measured in real time(adapted from Ozturk et al., 1997)
21. Other bioreactor types• Airlift fermenter• Packed bed bioreactor• Hollow fiber bioreactor• Single use bioreactor
22. Airlift fermenter• Gas mixture sparged into the reactor at the base.• The gas flow cause the culture medium to rise.• No mechanical agitators.
23. The fermenter is 200 high and 25 ft diam. (Chem. Eng. News, 10-Apr-78)
24. Lecture 10 Animal Cell Biotechnology Other fermentor Systems: Air lift bioreactorWaites et al. 2001. Industrial Microbiology: An Introduction. Oxford: Blackwell Science. P 98
26. Lecture 10 Animal Cell Biotechnology Other fermentor Systems: Air lift bioreactor• sparged gas agitates and aerates column• no mechanical parts, no shear stress• gas flow through inner tube lifts cells and medium,• cells and medium spill out over draft tube, circulate down side• 2-2000 litre reactors available
27. Hollow-fiber bioreactor• bioreactor consists of a cartridge containing bundles of synthetic, semi permeable hollow fibers which are similar to the matrix of the vascular system• good for anchorage-dependent or independent cells Cartwright, T. 1994. Animal cells as bioreactors. Cambridge:Cambridge University Press. p84
28. Hollow fiber bioreactor• In fibrous-bed bioreactor, the cells are immobilized on the fibers in the bioreactor.• Following is scanning electron microscope photos of human osteosarcoma cells in an artificial growth medium, a fibrous-bed bioreactor.• The cells cling to Dacron fibers
29. Fig. 9.13 Hollow fiber bioreactor
30. Electron micrograph of a cross section of hollow fibres showing a cell mass in the extracapillary spaceFig. 9.14
31. Fig. 9.15 Packed-bed bioreactor circulating media glass column gas out gas in glass beads circulation pump
32. Packed - Bed Bioreactor
33. Lecture 10 Animal Cell Biotechnology Other fermentor Systems: Packed-bed/fixed-bed bioreactor – glass bead column• good for anchorage-dependent cells• 1-100 litre volumes• cells attach to surface of beads (3-5 mm)• aerated medium is pumped in from a secondary vessel• inoculation could be a problem, uneven growth• heterogenous bioreactor – environment may not be the same throughout the column
35. Lecture 10 Animal Cell Biotechnology Other fermentor Systems: Packed-bed/fixed-bed bioreactor – ceramic bioreactor• ceramic cartridge (30 cm long, 4 cm wide) containing a series of parallel channels (1 mm2 square channels) → cells attach and grow on the walls of the channels• fresh medium is pumped in through the chambers, spent medium is returned to main reservoir• secreted products can be isolated from the spent medium
36. Fig. 9.17
37. Lecture 10 Animal Cell Biotechnology Other fermentor Systems: Packed-bed/fixed-bed bioreactor – the cell cubeButler, M. 2004. Animal cell culture and technology 2nd ed. London and New York:Garland Science/BIOS Scientific Publishers. P170-171.
38. Lecture 10 Animal Cell Biotechnology Other fermentor Systems: Packed-bed/fixed-bed bioreactor – the cell cube• stack of 20 cm2 polystyrene plates separated by 1 mm spacers• cells attach to either side of plate• culture medium is “sprayed” over the surface of the plates by multiple inlet ports
39. Lecture 10 Animal Cell Biotechnology Other fermentor Systems: Fluidized-bed bioreactor• similar to packed bed, but particles/ micro- carriers are separated by liquid media• immobilized cells are held in suspension by an upward flow of liquid medium M.Butler and M.Dawson. 1992. Cell Culture Labfax. Oxford:BIOS Scientific Publishers. p205
40. Lecture 10 Animal Cell Biotechnology Other fermentor Systems: Fluidized-bed bioreactorButler, M. 2004. Animal cell culture and technology 2nd ed. London and New York:Garland Science/BIOS Scientific Publishers. P172.
41. Fig. 9.19 Cytopilot Mini 2 litres
42. Cytopilot 100 litres
43. Single use bioreactorwww.applikon-bio.com Made of STEDIM 71 film
44. Summary• Types of bioreactors – Stirred tank – Airlift – Packed-bed – Fluidized-bed• Parameters to control – Stirring – Temperature – pH – Dissolved oxygen