Bioreactor Systems Ka-Yiu San A Fermenter / Bioreactor

Bioreactor Systems Ka-Yiu San A Fermenter / Bioreactor

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 1500L-Scale Bioreactors (courtesy of Tanox ) Bioreactor: Advantages

Controlled environment: 1. 2. 3. 4. Mixing pH Dissolved oxygen Temperature pH 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 O2 + H2O Pt+2e- 2OH- Anode (galvanic) Pb Pb2+ + 2eAnode (polargraphic) Ag + Cl- AgCl + e-

Mode Batch Fed-batch Continuous (e.g.. chemostat) Batch nutrient cell product Time Fed-Batch

Fresh nutrient Fresh nutrient nutrient volume cell product Time

Continuous Fresh nutrient cell Spent broth cell product volume nutrient Time

Recombinant processes - An engineering perspective a rm ti o Protein s fo an mRNA Transcription

n Tr on a ti Restriction sites io slat Lig Restriction

cleavage Recombined plasmid n Tra Restriction cleavage Gene of interest Cloning for rProtein production

n Cloning vector 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 (contd) 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 1. Spinner flasks 2. Continuous stirred bioreactors 3. Air (Liquid) lifted bioreactors 4. Hollow-fibers bioreactors 5. Microcarriers 6. Perfusion systems 7. Rotating wall bioreactors

Examples Nutrient considerations Two major classes serum supplemented serum-free (or low serum) Major functions of serum - basic nutrients - hormone and growth factors - binding proteins carrying hormone, vitamins, minerals, lipids, etc - non-specific protective functions - protease inhibitors - pH buffer

Environment considerations - nutrient supply - mixing - oxygen supply - pH - carbon dioxide - NaHCO3 or NaOH - temperature - waste accumulation - lactate - ammonia

Other considerations - inoculum - growth phase (late exponential phase) - density (varies, as a guide ~5x104 to 2x105 cells/ml) - mixing - shear 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 glycine glutamate alanine asparatate

-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 Oxygen supply (a challenging problem since oxygen is sparsely soluble in water) OTR = kla (C*-C) OTR: oxygen transfer rate kla: mass transfer coefficient C*:

saturated dissolved oxygen concentration C: dissolved oxygen concentration in the medium Methods for O2 supply - direct sparging - cell damage - pluronic F-68 supplement - surface aeration - limited surface area

- silicon tubing supplement - to increase surface area - perfusion Examples of performance of various aeration methods Methods of oxygenating a 40 liter Bioreactor (30 liter working volume with a 1.5: 1 aspect ratio) Oxygenating method Oxygen delivery (mg/l/h) No. cells x106/ml

supported AIR (10 ml/l/min at 40 r .p.m.) Surface aeration 0.5 Direct sparging 4.6 Spin filter sparging 3.0 Perfusion (1 vol/h) 12.6 Perfusion (1 vol/h) 15.9 + Spin filter sparging OXYGEN (10 ml/min at 80 r .p.m.)

Spring filter sparging 51.0 + Perfusion (1 vol/h) 92.0 (assuming oxygen utilization rate of 2-6 g/1 06 cells/h) 0.08 0.76 0.40 2.10 2.65 8.50 15.00 Cultivation methods for anchorage dependent cells

Commercially available spinner cultures. (A) LH Fermentation Biocul (1-20L); (B) Bellco and Wheaton Spinner Flasks (25 ml-2 liters); (C) Bellco and Cellon u spinner (25 ml-2 liters); (E) Techne (25 ml-5 liters); (E) Techne Cytostat (1 liter); (F) Techne BR-06 Bioreactor (3 liters). Hollow fiber reactors - consists of ultrafiltration capillary fibers - porous to macromolecules - thin wall - provide large surface area Flow diagram of a typical hollow fiber reactor c e ll c u lt u r e

w a s te fr e s h m e d iu m A ir ( o x y g e n ) o x y g e n a to r h I e

p p h Hollow fiber culture reactor and a diagrammatic representation of the pressure drop/nutrient gradient along the length of the cartridge. I, lumen of fibers; e, extracapillary space; h harvesting port; p, medium perfusion path fibre ri ro rc

[O2] oxygen conc [O2] [O2]c critical oxygen conc [O2]c ri ro rc Microcarriers Major Advantages: - possess high surface-to-volume ratio (as high as 2x107 cell/ml are achieved)

microcarriers can be settled easily facilitate cell and product harvesting cell propagation can be carried out in high productivity reactors enable control and monitoring of reactor environment possible to take representative sample for monitoring purposes Desired properties - functional attachment group -

buoyant density of the bead - for mixing consideration ( ~ 1.03 to 1.10 g/l) - size of the bead (100-200 m) - size distribution - smooth surface (allow cell spreading)

- transparency ( microscopic observation) - toxicity - rigidity A sample listing of commercially available microcarriers Trade Name

Manufacturer Material Acrobead Biosilon Bioglas Bioplas (Biospheres Biocarrier Cellfast Cytodex 1 Cytodex 2 Cytodex 3

Cytosphere Dormacell OE-53 Gelibead Mica Micarcel G Galil Nunc Solohill Eng. Solohill Eng. Microdex Superbeads Ventreglas

Ventregel Oextran Prod. Flow lab. Ventrex Ventrex Polyacrolein 1.04 Polystyrene. 1.05 Glass. 1.03 Polystyrene. 1.04

Collagen. 1.02 Polyacrylamide 1.04 Silica/Chitosan DEAE Sephadex 1.03 DEAE Sephadex 1.04 Collagen 1.04 Polystyrene 1.04 Dextran 1.05 Cellulose 1.03

Gelatin 1.04 Polyacy(amide 1.04 Polyacrylamide' 1.03 Collagen/glucoglycan DEAE Dextran 1.03 DEAE Sephadex 1.03 Glass 1.03 Gelatin 1.03

Biorad QDM lab. Pharmacia Pharmacia Pharmacia lux Pfeifer & Langen Whatman Hazelton lab. Muller-Ueheim Reactifs IBF SG Diam (m)

150 160-300 150-210. 150-210 150-210 120-180 160-230 115-200 130-210 160-230 140-240 Fibres 115-235 150

150-200 90-210 150-250 Area (cm2/g) 5000 255 350 350 350 5000 10000 6000 5500

4600 250 7000 4000 3800 350 5000 250 6000 300 4300 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?

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