Bioreactor Systems KaYiu San A Fermenter Bioreactor And
Bioreactor Systems Ka-Yiu San
A Fermenter / Bioreactor And Its Parts by Genentech, Corporate Communication
Single System for Anchorage-Dependent and Suspension Cultures New Brunswick Scientific Company
1500 L-Scale Bioreactors (courtesy of Tanox )
Bioreactor: Advantages Controlled environment: 1. 2. 3. 4. Mixing p. H Dissolved oxygen Temperature
p. H probe 1. Steam sterilizable 2. Combination electrode Dissolved oxygen probe 1. Two major types a. Galvanic b. Polargraphic
Galvanic and Polargraphic Probes Cathode 0. 5 O 2 + H 2 O Pt+2 e- 2 OH- Anode (galvanic) Pb 2+ + 2 e. Anode (polargraphic) Ag + Cl- Ag. Cl + e-
Mode Batch Fed-batch Continuous (e. g. . chemostat)
Batch nutrient cell product Time
Fed-Batch Fresh nutrient volume cell product Time
Continuous Fresh nutrient cell Spent broth cell product volume nutrient Time
Recombinant processes - An engineering perspective
Protein m. RNA Transcription on Cloning vector ati Restriction cleavage m for ns Tra on ati Lig Restriction sites Recombined plasmid t nsla Tra Restriction cleavage Gene of interest Cloning for r. Protein production Host cell
Cloning vector properties - Origin - Size of backbone - Well characterized - Selective marker - Genetic marker - Unique restriction sites Cloning vector ori marker
Some factors that affect gene expression 1. Gene dosage (copy number) 2. 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)
Some factors that affect gene expression (cont’d) 6. Protein stability - Degradation by host proteases - Formation of insoluble aggregates 7. Strain 8. Process consideration - Medium - Temperature (growth vs. production) - Dissolved oxygen - Induction timing - Feeding profile (fed-batch) - waste product accumulation
Cell Culture - An engineering perspective
Outline • Nutrient Considerations • Environment Considerations • Common Culturing Systems • Examples
Two major classes • serum supplemented • serum-free (or low serum)
Environment considerations - nutrient supply - mixing - oxygen supply - p. H - carbon dioxide - Na. HCO 3 or Na. OH - temperature - waste accumulation - lactate - ammonia
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
Nucleic acid synthesis glutamine NH 4 glycine glutamate alanine asparatate NH 4 a -ketoglutarate TCA cycle citrate malate lactate pyruvate glucose glycolysis oxaloacetate phosphoenolpyruvate Schematic representation of some of the interrelationships of glucose an glutamine metabolism in mammalian cells
h I e p p h
fibre ri ro rc [O 2] – oxygen conc [O 2]c – critical oxygen conc [O 2]c ri ro rc
Typical cell growth on microcarriers
Typical cell growth on microcarriers
Perfusion system - to provide fresh nutrient - to remove waste (especially toxic byproducts - mechanical signal
Questions?
- Slides: 42