Transcript Simulation Of Bioprocess ERT 315

Simulation Of
Bioprocess
ERT 315/4
Introduction
Stages of Biotech
• Ancient
• Classical
• Modern
Ancient Biotech
rd mill BC-Baking,
with early
•4th•/3Begins
civilization
brewing
(Egypt)
Use
•
Developments
in
microorganisms
•3rd mill. BC-Ethanol
agriculture and food
•17thproduction
century-Invention of
microscope
• Few records exist
Classical Biotech
• Follows ancient
• Makes wide spread use of
methods from ancient,
especially fermentation
• Methods adapted to
industrial production
Era of microorganism (19 century —1944)
18th
First vaccination in Europe (cowpox), heat sterilization of food
and organic chemistry
1857.
Pasteur :microorganism
1897.
Germany :Buchner :enzyme
A： primary metabolism product:ethanol, citric acid
B: anaerobic fermentation
1923
Commercial production of citric acid
1940s
Production of penicillin by fermentation
Most of amino acid isolated
1860-1890
Enzyme Engineering
2. Enzyme Engineering
1953. Grubhofer and Schleith
immobilization of enzyme
1969. Japan :application of immobilized
enzyme in industry
Amino acid production
1976 Genentech first specialist biotech
company
Modern Biotech
• Manipulation of genetic
material within organisms
• Based on genetics and the
use of microscopy,
biochemical methods,
related sciences and
technologies
3. Genetic Engineering
1974. US Boyer and Cohen recombinant
DNA
1976. first biotechnology company
Genentech was established
1977. Boyer hGH
1986 First rDNA vaccine approve
1995 First bacterial genome sequenced
2000 Human genome sequenced
Application of Biotechnology
Industry
Basic
chemicals
Fine
chemicals
Detergents
Health care/
cosmetics
Pharma
conventional
biopharma
Food/feed
Metal mining
Waste
treatment
Scale
Downstream Biocatalyst
Food
industry
complexity
Very large
Low
MO/enzymes
Medium
Medium
Medicine
MO/enzymes
Large
Smallmedium
Low
MO
MO/enzymes/
mammalian cells
Chemicals
Medium
Medium-high
Small
High
MO
Mammalian cells, MO
Very large
Very large
Very large
Medium
Low
Low
MO/enzymes
MO
MO
Environmental
Products
Biotech market
share
Organic small
molecules
Organic small
molecules
Enzymes
Proteins & small
molecules
Very low
Organic small
molecules
Proteins
Proteins
Metals
Purified water
Low
Medium
Medium
Low-medium
High
Medium
Very Low
high
Modeling
and Assessment
in
Process
Development
Why must modeling and simulation?
Process concept
• Close collaboration with the process Literature
Patents
design
Process design and
Modeling and
Expert
development
simulation
• Additional
information from
patents, knowledge
literature, and other external sources
• Simulation used to evaluate the process
Not ecoSustainability
Improvement
Stopaim
and
guide theassessment
R&D to the efficient
overall
needed
• Repeated iteratively
Ecoefficient
Industrial application
• To gain an understanding of the actual
future production
• To realize competitive industrial processes
and decision has to be made based on the
cost and potentials of a process
• To solve a problem that was previously
overlooked rises with the development
stage
• To give a complete picture of the expected
production-scale
Development
Of
Bioprocess
The Biotechnical Process
Cell
cultivation
Enzymatic
process
Reactor
Transgenetic Plant
And Animal
Fermenter
Agriculture
Enzymes Whole cells Extracellular Intracellular
Cell harvest
Product extraction
Extractive
technology
Raw material
Solid
Liquid
Homogenization
Biomass removalsolid/liquid separation
Concentration
Protein refolding
Product separation
Viral inactivation
Final formulation
Crystallization
Drying
Final filling
Unit Operations and
Unit Procedures
Unit Operations:
Basic step in production process
e.g. sterilization, fermentation, enzymatic reaction,
extraction, filtration, crystallization
Unit Procedures:
Set of operation that take place sequentially in
a piece of equipment
e.g. charging of substrate to a fermenter, addition of acid to
adjust pH, reaction, transfer of fermentation
broth to another vessel
Elements of bioprocess
• Upstream processing
• Bioreactor
• Downstream processing
Cell
cultivation
Enzymatic
process
Reactor
Transgenetic Plant
And Animal
Extractive
technology
Fermenter
Agriculture
Upstream
processing
Raw material
Enzymes Whole cells Extracellular Intracellular
Cell harvest
Product extraction
Solid
Liquid
Homogenization
Biomass removalsolid/liquid separation
Bioreactor
Concentration
Protein refolding
Product separation
Viral inactivation
Downstream
processing
Final formulation
Crystallization
Drying
Final filling
Upstream processing
P-3 / V-102
P-5 / V-103
1. Preparation and Storage of Solutions
-to provide and store that are needed at some point in the
process
e.g. preparation of the medium for the bioreactor/
buffers in the chromatography
-Liquid and solid mixture: filled in tank, mixed by agitation,
stored in the tank or transferred to a separate storage
tank until is needed in the process
-Raw material solutions: prepared with high concentrations
to keep the volume of the preparation tanks small
-Carbon and nitrogen sources: prepared in separate tanks
to avoid Maillard or
non-enzymatic browning reactions
Blending / Storage
Storage
Upstream processing
2. Sterilization of Input Materials
-to preclude contamination of the bioreactor
(i) Filtration
-to sterilize gaseous streams
-membrane filters: Pore size- 0.2-0.3µm
-prefilters used for dust and other particles
P-3 / AF-101
Air Filtration
(ii) Heat Sterilization
-heated by steam
-cooling water to bring the temperature back to normal
-Temperature: 121 °C (batch),140-45 °C (continuous),
holding time: 10-20 min
-For continuous: required heat exchanger
P-3 / ST-101
Heat Sterilization
Upstream processing
3. Inoculum preparation
-to provide a sufficient amount of active cell to inoculate
the production fermenter
P-3 / BBS-101
P-4 / RBR-101
P-5 / TFR-101
Dispos able Bioreac tor
Roller Bottle
T-Flask
P-6 / SFR-101
P-7 / TTR-101
Cleaning-in-Place (CIP)
-to prepare the equipment for the next cycle or batch
Shak e Flask
Tes t Tube
Bioreactor
1. Bioreactor Types
(i) Stirred tank bioreactor
-most commonly used in bioprocess
-depends on the complexity of the bioreaction
-air, supplied by a compressor, enter the vessel at the
bottom under pressure
-jacket and/or internal coils allow heating and cooling
Bioreactor
(ii) Airlift bioreactor
-mixing is achieved without mechanical agitation by the
convection caused by sparged air
-lower energy consumption
-used for plant and animal cell culture and for immobilized
biocatalysts
Bioreactor
(iii)Packed-bed and fluidized bed bioreactor
-The immobilized or particulate biocatalyst is filled in a
tube-shaped vessel
-medium flows through the column (upwards or
downwards)
-small particle attrition
-high velocity of the liquid phase promotes good mass
transfer
Bioreactor
2. Unit Procedures
(i) Filling and transfer of materials in vessels
P-3 / V-102
P-4 / V-103
-to bring materials (liquids, solids) into the
bioreactor
Vess el Procedure
Fermentation
-to transfer parts or the whole reactor volume to the next
operation at the end of the bioreaction
-the duration should be specified
-filled up to only 70-90% to keep some headspace for
foam build-up
(ii) Agitation
-to achieve and maintain homogeneity
-to enable efficient heat transfer
-energy consumption depends on the rotational speed of
bioreactor, fluid density and viscosity
Bioreactor
(iii) Aeration
-provides oxygen to meet the aerobic demand of the
cells during fermentation
-specified by the gas used (Air, pure O2, pure N2, or
air enriched with O2 or CO2) and the aeration rate
(0.1 and 2 vol. of gas per volume of solution per
minute (vvm)
(iv) Heat transfer
-to change and control the temperature of the
bioreactor
-to keep constant while exothermic reactions take
place in the fermenter
-for heating, heat is transferred from a heat-transfer
fluid via a heat- transfer surface to the reactor content
Bioreactor
-for cooling, heat transferred from the fermentor to the
cooling fluid
-used steam for heating
-heating rate depends on the bioreactor volume, typically
at 1.5-3.0 °C/min for a 10m3 reactor and 1-2 °C/min
for a 50 m3 reactor
-cooling agent : cooling water (20 °C), chilled water (5
°C), Freon, glycol, sodium chloride brine, calcium
chloride brine
(v) Foam control
-to control the foam formation from the combination of
agitation and aeration with the presence of foamproducing and foam-stabilizing substances
Bioreactor
(vi) pH control
-to control and reach the desired pH
-the medium is buffered- adjusting and
maintained the pH by adding acid or bases
(vii) Cleaning-in-place (CIP)
-to clean the equipment after every batch
Downstream processing
1. Biomass removal
-separate the biomass from the fermentation broth
-unit operations: centrifugation, microfiltration, rotary
vacuum filtration, decanting/sedimentation
-depends on a number of parameter (e.g. concentration,
particle size, density of biomass,scale operation etc)
2. Homogenization/Call Disruption
-to break open the cells to release the product into the
solution before purification
-unit operation: high pressure homogenization,
mechanical bead milling
P-3 / HG-101
P-4 / BM-101
Homogenization
Bead Milling
Downstream processing
3. Concentration
-to reduce the volume of the product stream that has to
be processed
-reducing equipment size and energy consumption
-three methods available:
(a)Partial evaporation of the solvent
-solution heated up to vaporize some of the solvent,
usually water
(b)Filtration
-semi-permeable membrane retains the product in the
retentate but transfers most of the solvent through the
membrane
(c)Precipitation
-adding a precipitation agent or by changing chemical or
physical conditions
Downstream processing
4. Phase Separation
(i) Centrifugation
-Used for biomass removal and solid separation
-based on density between solid particles and a solution
between two immiscible liquids
-sedimentation force is amplified by the particle or drop
size in centrifugal field in the centrifuge
-pretreatment is necessary to increase particle size
-maximum throughput defined by the sigma factor and
the settling velocity
P-3 / DS-101
Centrif ugation
P-5 / BCF-101
P-4 / BC-101
Bask et Centrif ugation Centrif ugation
Downstream processing
(ii) Filtration
-to separate particles or large molecules from a
suspension or solution
-semi-permeable membrane splits the components
according to their size
-microfiltration:
(a)Pore sizes of 0.1-10 µm
(b)Flux rate: 20 and 250 L/m2
-ultrafiltration(a)Pore sizes of 0.001-0.1 µm
(b)Flux rate: 20 and 200 L/m2
P-3 / MF-101
Microf iltration
P-4 / DF-101
D iaf iltration
P-5 / UF
U ltraf iltra
Downstream processing
-dead-end filtration:
(a)particles are retained as a cake through which solvent
must pass
(b)The pressure drop increases with solids accumulation
-cross-flow filtration:
(a)The feed is moved tangentially along the membrane
to reduce concentration polarization or filter-cake
thickness and associatedpressure drop
(b) Particles are obtained as concentrated slurry
-rotary vacuum filtration:
Used only for large-scale filtration with large particles
-diafiltration:
(a) used to change the buffer solution
P-7 / RVF-101
P-6 / DE-101
R otary Vacuum Filtration
Downstream processing
(iii) Sedimentation and decanting
-sedimentation:
-same as centrifugation, gravity is the
P-3 / V-102
P-4 / C
driving force
D ecanting
C larif i
-needs a longer settling time and large density difference
and particle size of the substances
-Applied for large-scale biomass removal mostly in
wastewater treatment
-decanting:
-for separation of liquid phases, e.g. water
-the layers are formed:
Solid or heavy liquid phase at the bottom, light liquid
phase on top and dispersion phase in between
-the parameters: density and viscosity of the two phases
Downstream processing
(iv) Condensation
-to liquefy the distillate in distillation (e.g. in product
separation or solvent recycling)
-to turn vaporized steam to liquid water after a
crystallization or concentration step
-use a typical shell-and-tube surface condenser-the
coolant flows in the tube while condensation of the vapor
occurs at the shell side
-parameter parameter: heat of vaporization, boiling
point, partition coefficient of the vapor component
P-3 / HX-101
C ondensation
Downstream processing
4. Product Separation and Purification
Extraction
-to separate a molecule from a solution by transferring to
another liquid phase
-based on the different solubilities of the product and the
impurities in the feed phase
-used when the product concentration is comparably low
or when distillation cannot be applied
-differential extraction column-top: the heavy phase
(aqueous solution), bottom: the light phase (organic
solvent) and moves upwards.
-key parameter: partition coefficient
P-3 / MSX-101
P-4 / DX-101
Downstream processing
(ii) Distillation
-for recovery of organic solvents
-based on the differences between the volatilities of
substances
-key parameter: Boiling point of the substances and the
linear velocity of the vapor
(iii) Electrodialysis
-an electromotive force is used to transport ions through
a semi-permeable, ion selective membrane by ion
diffusion
-the cations move through a cation membrane in acid
stream, the anions move through an anion membrane
into the supplied base stream
-key parameter: membrane flux (100-300 g/m2h)
P-3 / V-102
P-4 / V-103
Flas h
Batch D istillation
Downstream processing
(iv) Adsorption
-to retain either the product or impurities on a solid
matrix
-key parameters: binding capacity and selectivity of the
resin, binding yield of the target and non-target
molecules, volume of the eluent
(v) Chromatography
-to resolve and fractionate a mixture of compounds
based on differential migration
-basic principles are identical to purification by
adsorption
-Types of column:
Gel or exclusion chromatography (molecules), affinity
chromatography(ligand), Ion exchange chromatography
P-3 / GAC -101
GAC Adsorption
P-3 / C-
PBA Chrom a
Downstream processing
5. Viral Inactivation
-to preclude contamination of the bioreactor or impurities
in the product from bacteria, viruses and prions
-a combination methods is necessary because none of
the known methods inactivate all possible contamintants
(standard purification step + additional step)
-additional step : micro and ultrafiltration, heat, UV
radiotion, chemical substances
Protein Solubilization and Refolding
-to release the intracellular material and inclusion bodies
or water-insoluble pellets produced by heterologous
protein in bacteria and fungi
Downstream processing
6. Final Product Processing
(i) Crystallization
-converted the desired product from its soluble form crystallized (solid) form
-crystals are separated from the liquid solution, e.g. by filtration
-initiated by a volume reduction of the solution or by reducing the solubility
of the target molecules by addition of a crystallizing agents, or by changing
the physical or chemical conditions
-key parameter: crystallizaiton yield, crystallization heat, necessary residence time
P-3 / CR -101
C ry s tallization
(ii) Product stabilization
-to avoid premature degradation or denaturation
(iii) Drying
-removed water or another solvent from a solid product
-commonly used if the product is to be sold as powder
-contact dryer: the heat is provided via the drum wall form hot water, air,
or steam that flows outer side of the wall.
-convection dryer: preheated drying gas is mixed with the solid and the
solvent evaporates into the drying gas
(iv) Filling, labeling and packing
-to get the product ready for the customer or patient
P-3 / TD R-101
Tray D ry ing
Method
Separation principle
Typical
yield
(%)
Separated product
Centrifugation
Sedimentation
Microfiltation
Ultrafiltration
Chromatography
gel filtration
Ion exchange
Hydrophobic interaction
Reversed phase
Specific density
Specific density
Size/phase
Size
90-99
80-99
80-99
Cells, particles
Cells, particles
Cells, particles
cell debris, proteins &polymers
Affinity
Electrodialysis
Extraction
Distillaiton
Drying/evaporation
Crystallization
60-99
Size shape
Ionic charge
Hydrophobicity
Hydrophobicity/diffusity
specific binding
Molecular recognition
Ionic charge/diffusity
Solubility/phase affinity
Volatility
Volatility
Phase change
Large molecules
Ions
Hydrophilic or hydrophobic molecules
Hydrophilic or hydrophobic molecules
70-99
70-99
80-99
97-99
60-95
Molecules with specified epitopes
Ions
Hydrophilic or hydrophobic molecules
Volatiles
High-boiling molecules
Crystallized solids