1. Bioreactor Systems
Ka-Yiu San

2. A Fermenter / Bioreactor And Its Parts
by Genentech, Corporate Communication

3. Single System for Anchorage-Dependent and Suspension Cultures
New Brunswick Scientific Company

4. 1500L-Scale Bioreactors (courtesy of Tanox )

5. Bioreactor: Advantages
Controlled environment:
Mixing pH
Dissolved oxygen
Temperature

6. Dissolved oxygen probe
pH probe
Steam sterilizable
Combination electrode
Dissolved oxygen probe
Two major types
Galvanic
Polargraphic

7. Galvanic and Polargraphic Probes
Cathode
0.5 O2 + H2O +2e-  2OH- Pt
Anode (galvanic)
Pb  Pb e-
Anode (polargraphic)
Ag + Cl-  AgCl + e-

8. Mode
Batch
Fed-batch
Continuous (e.g.. chemostat)

9. Batch
nutrient
cell
product
Time

10. Fed-Batch nutrient cell product Time nutrient cell product Time Fresh
volume

11. Continuous Time cell nutrient product Fresh nutrient cell Spent broth
volume
nutrient
cell
product
Time

12. Recombinant processes
- An engineering perspective

13. Cloning for rProtein production
Gene of interest
Ligation
Recombined
plasmid
Transformation
Transcription
Translation
mRNA
Protein
Host cell
Restriction sites
Restriction
cleavage
Restriction
cleavage
Cloning vector

14. Cloning vector properties
Origin
Size of backbone
Well characterized
Selective marker
Genetic marker
Unique restriction sites
Cloning vector
ori
marker

15. Some factors that affect gene expression
Gene dosage (copy number)
Plasmid stability (structural and segregational)
3. Transcription
- Promoter and terminator sequences
(promoter strength, inducible - leakage)
- Regulatory genes and sequences
4. Translation
- Ribosome binding site (Shine-Delgarno sequence) - Codon optimization to match host's codon bias
5. Final location of gene product
- Cytoplasmic or extracellular (secreted out of cell)

16. Some factors that affect gene expression (cont’d)
6. Protein stability
- Degradation by host proteases - Formation of insoluble aggregates
Strain
Process consideration
- Medium
- Temperature (growth vs. production)
- Dissolved oxygen
- Induction timing
- Feeding profile (fed-batch)
- waste product accumulation

17. Cell Culture
- An engineering perspective

18. Outline Nutrient Considerations Environment Considerations
Common Culturing Systems
Examples

19. Two major classes
serum supplemented
serum-free (or low serum)

20. Environment considerations
- nutrient supply
- mixing
- oxygen supply
- pH
- carbon dioxide
- NaHCO or NaOH 3
- temperature
- waste accumulation
- lactate
- ammonia

22. Kolmogorov length scale (microns)
Relative specific growth rate
Kolmogorov length scale (microns)
Relative net growth rate versus Kolmogorov eddy length scale for FS-4 cultures with 0.2 g/l microcarriers

23. Schematic representation of some of the interrelationships
Nucleic acid synthesis
glutamine
glutamate
glycine
alanine
asparatate
TCA
cycle
citrate
malate
oxaloacetate
phosphoenolpyruvate
glycolysis
glucose
pyruvate
lactate
-ketoglutarate a
NH4
Schematic representation of some of the interrelationships
of glucose an glutamine metabolism in mammalian cells

31. p h I e

32. fibre ri ro rc [O2] [O2]c [O2] – oxygen conc
[O2]c – critical oxygen conc

38. Typical cell growth on microcarriers

39. Typical cell growth on microcarriers

40. Perfusion system - to provide fresh nutrient
- to remove waste (especially toxic
byproducts
- mechanical signal

42. Questions?