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pH probe
1. Steam sterilizable2. Combination electrode
1. Two major typesa. Galvanic b. Polargraphic
Dissolved oxygen probe
Galvanic and Polargraphic Probes
Cathode 0.5 O2 + H2O +2e- 2OH-Pt
Anode (galvanic) Pb Pb2+ + 2e-
Anode (polargraphic) Ag + Cl- AgCl + e-
Cloning for rProtein productionR
est
rict
ion
site
s
Cloning vector
Ligation
Recombined plasmid
Restrictioncleavage
Restrictioncleavage
Ge
ne
of i
nte
rest
Transformation
Tra
nsc
rip
tion
Translation
mR
NA
Pro
tein
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 bias5. 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 aggregates7. Strain8. Process consideration - Medium - Temperature (growth vs. production) - Dissolved oxygen - Induction timing - Feeding profile (fed-batch) - waste product accumulation
Outline• Nutrient Considerations• Environment Considerations• Common Culturing Systems
1. Spinner flasks2. Continuous stirred bioreactors3. Air (Liquid) lifted bioreactors4. Hollow-fibers bioreactors5. Microcarriers6. Perfusion systems7. Rotating wall bioreactors
• Examples
Nutrient considerationsTwo 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- NaHCO or NaOH3
- 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
Kolmogorov length scale (microns)
Relative net growth rate versus Kolmogorov eddy length scale for FS-4 cultures with 0.2 g/l microcarriers
Rel
ativ
e sp
ecif
ic g
row
th r
ate
Nucleic acid synthesis
glutamine
glutamateglycine
alanine asparatate
TCA cycle
citrate malate
oxaloacetate
phosphoenolpyruvateglycolysis
glucose
pyruvate
lactate
-ketoglutarate
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 themedium
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/mlsupported
AIR (10 ml/l/min at 40 r .p.m.)Surface aeration 0.5 0.08Direct sparging 4.6 0.76Spin filter sparging 3.0 0.40Perfusion (1 vol/h) 12.6 2.10
Perfusion (1 vol/h) + Spin filter sparging
15.9 2.65
OXYGEN (10 ml/min at 80 r .p.m.)Spring filter sparging 51.0 8.50+ Perfusion (1 vol/h) 92.0 15.00
(assuming oxygen utilization rate of 2-6 g/1 06 cells/h)
Commercially available spinner cultures. (A) LH Fermentation Biocul (1-20L);(B) Bellco and Wheaton Spinner Flasks (25 ml-2 liters); (C) Bellco and Cellon uspinner (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
oxygenator
wastefreshm ed iu m
A ir(oxyg en )
c e ll c u ltu re
Flow diagram of a typical hollow fiber reactor
Hollow fiber culture reactor and a diagrammatic representation of the pressuredrop/nutrient gradient along the length of the cartridge. I, lumen of fibers;e, extracapillary space; h harvesting port; p, medium perfusion path
p p
hI e
h
MicrocarriersMajor 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 microcarriersTrade Name Manufacturer Material SG Diam (m) Area
(cm2/g)
Acrobead Galil Polyacrolein 1.04 150 5000Biosilon Nunc Polystyrene. 1.05 160-300 255Bioglas Solohill Eng. Glass. 1.03 150-210. 350Bioplas Solohill Eng. Polystyrene. 1.04 150-210 350(Biospheres Collagen. 1.02 150-210 350Biocarrier Biorad Polyacrylamide 1.04 120-180 5000Cellfast QDM lab. Silica/Chitosan 10000Cytodex 1 Pharmacia DEAE Sephadex 1.03 160-230 6000Cytodex 2 Pharmacia DEAE Sephadex 1.04 115-200 5500Cytodex 3 Pharmacia Collagen 1.04 130-210 4600Cytosphere lux Polystyrene 1.04 160-230 250Dormacell Pfeifer & Langen Dextran 1.05 140-240 7000OE-53 Whatman Cellulose 1.03 Fibres 4000Gelibead Hazelton lab. Gelatin 1.04 115-235 3800Mica Muller-Ueheim Polyacy(amide 1.04 350Micarcel G Reactifs IBF Polyacrylamide' 1.03 5000
Collagen/glucoglycanMicrodex Oextran Prod. DEAE Dextran 1.03 150 250Superbeads Flow lab. DEAE Sephadex 1.03 150-200 6000Ventreglas Ventrex Glass 1.03 90-210 300Ventregel Ventrex Gelatin 1.03 150-250 4300
Perfusion system- to provide fresh nutrient- to remove waste (especially toxic byproducts - mechanical signal
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