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
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
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
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.)
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
INSCRIPCIONES Pilar González Serrano Planimetría del área del Lapis Niger Vista general de área arqueológica del Lapis Niger Propuesta de ubicación de los distintos elementos arquitectónicos que hubo en su día bajo el Lapis Niger Reconstrucción ideal de los vestigios...
White Desert - Egypt High Tide Bay of Fundy, New Brunswick Hopewell Rocks Provincial Park Canada Low Tide World famous El Arco - the rock arch of Los Cabos - Baja California Rocks on Cabo Pulmo beach are worn smooth...
"Pleasantly plump teenager Tracy Turnblad teaches 1962 Baltimore a thing or two about integration after landing a spot on a local TV dance show." -IMDb.com (Rating: 8.1/10) In the Hairspray musical, John Travolta danced, in heels, and a dress.
Attenzione: ricordarsi sempre che si è in un colloquio di selezione e alla fine c'è un giudizio! PROFESSIONALE Il conduttore pone domande in modo calmo che mirano ad ottenere informazioni sul soggetto senza però creargli ansia e cercando di instaurare...
"Regarded as the best of all the Grand Crus, Le Mesnil produces the most elegant champagnes the world has to offer."-Richard Juhlin. Le Mesnil-sur-Oger is the southernmost Grand Cru Village in the Côte des Blancs, the ridge of the Paris...
1-D STEFAN PROBLEM To see effects of concentrations at interface and at top of tube, let mass fraction of A in freestream be zero, while arbitrarily varying YA,i, interface mass fraction, from zero to unity In terms of an experiment,...
Ready to download the document? Go ahead and hit continue!