Bioreactor Systems

Ka-Yiu San

by Genentech, Corporate Communication

A Fermenter / Bioreactor And Its Parts

Single System for Anchorage-Dependent and Suspension CulturesNew Brunswick Scientific Company

1500L-Scale Bioreactors(courtesy of Tanox )

Bioreactor: Advantages

Controlled environment:1. Mixing2. pH3. Dissolved oxygen4. Temperature

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-

Mode

BatchFed-batchContinuous (e.g.. chemostat)

Batch

nutrient

cell product

Time

Fed-BatchFresh nutrient

Fresh nutrient

volume

nutrient

cellproduct

Time

nutrient

cellproduct

Time

ContinuousFresh nutrient

cellSpent broth

volume

nutrient

cell

product

Time

Recombinant processes

- An engineering perspective

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

Cell Culture

- An engineering perspective

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)

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 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

ri

ro

rc

[O2]

[O2]c

ri ro rc

fibre

[O2] – oxygen conc

[O2]c – critical oxygen conc

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

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?