Simulation Of BioprocessERT 315/4

1

Introduction

Stages of Biotech• Ancient

• Classical

• Modern

Ancient Biotech• Begins with early civilization• Developments in agriculture

and food production• Few records exist

•4th/3rd mill BC-Baking, brewing (Egypt)

•3rd mill. BC-Ethanol

•17th century-Invention of microscope

Use microorganisms

Classical Biotech• Follows ancient• Makes wide spread use of

methods from ancient, especially fermentation

• Methods adapted to industrial production

Era of microorganism Era of microorganism ((19 century —194419 century —1944))

1818thth First vaccination in Europe (cowpox), heat sterilization of First vaccination in Europe (cowpox), heat sterilization of food food and organic chemistryand organic chemistry

1857. 1857. Pasteur :microorganismPasteur :microorganism

1897. 1897. Germany :Buchner :enzymeGermany :Buchner :enzyme

AA ： ： primary metabolism product:ethanol, citric acidprimary metabolism product:ethanol, citric acidB: anaerobic fermentationB: anaerobic fermentation

19231923 Commercial production of citric acidCommercial production of citric acid

1940s Production of penicillin by fermentation1940s Production of penicillin by fermentation

Most of amino acid isolated Most of amino acid isolated 1860-18901860-1890

Enzyme Engineering

History of Bioprocess• Products of fermentation processes

Products type Example Products type Example

Alcohols Ethanol Butanol (+acetone)

Organic acids Citric acid Acetic acid

Amino acids Glutamic acid Lysine Polysaccharides Dectran Xanthan

Antibiotic Penicillins Tetracyclines

Purified waste water

BOD-removal Senitrification

Enzymes Proteases Amylases Single-cell protein

Fodder yeast

Hormones Insulin Human growth hormone

Starter cultures Baker’s yeast Lactic starters

Insecticides Thuringiensin Steroid transformation

Cortisone

Methane Biogasfrom sewage/water

Vaccines Tetanus

Nucleotides IMP GMP Vitamins Riboflavin

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

Mutation

random mutation, UV, NTG, rays

Cell fusion

protoplast fusion

hybridoma technique monoclonal anitibody Genetic engineering Extremphilic organisms:

pH, temperature, salt, pressure

Product Origin Producting organism

hGH Human Escherichia coli

IGF-I Human Escherichia coli

IFN-α-2a Human Escherichia coli

Protein G Streptococcus Escherichia coli

Insulin Human Saccharomyces cerevisiae

Hepatitis B vaccine Hepatitis virus Saccharomyces cerevisiae

Chymosin Calf Kluyveromyces lactis

Lipase Humicola lanuginosa Aspergillus oryzae

tPa Human Animal cells

Superoxide dismutase Human Animal cells

Some recombinant products that became available by the introduction of the genetic engineering technique

Application of Biotechnology

Food industry

Medicine

Chemicals

Environmental

Industry Scale Downstream complexity

Biocatalyst Products Biotech market share

Basic chemicals

Fine chemicals

Detergents

Health care/ cosmetics

Pharma

conventional

biopharma

Food/feed

Metal mining

Waste treatment

Very large

Medium

Large

Small-medium

Medium

Small

Very large

Very large

Very large

Low

Medium

Low

Medium-high

High

Medium

Low

Low

MO/enzymes

MO/enzymes

MO

MO/enzymes/

mammalian cells

MO

Mammalian cells, MO

MO/enzymes

MO

MO

Organic small molecules

Organic small molecules

Enzymes

Proteins & small molecules

Organic small molecules

Proteins

Proteins

Metals

Purified water

Very low

Low

Medium

Medium

Low-medium

High

Medium

Very Low

high

2

Development Of

Bioprocess

4. The Biotechnical Process

Enzymatic process

Cell cultivation

Transgenetic PlantAnd Animal

Extractive technology

Reactor Fermenter Agriculture Raw material

Enzymes Whole cells Extracellular Intracellular Solid Liquid

Cell harvest Homogenization

Product extractionBiomass removal-

solid/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 processe.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

Enzymatic process

Cell cultivation

Transgenetic PlantAnd Animal

Extractive technology

Reactor Fermenter Agriculture Raw material

Enzymes Whole cells Extracellular Intracellular Solid Liquid

Cell harvest Homogenization

Product extractionBiomass removal-

solid/liquid separation

Concentration

Protein refolding Product separation Viral inactivation

Final formulation

Crystallization Drying Final filling

Upstream processing

Bioreactor

Downstream processing

Upstream processing

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 is 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 are prepared with high concentrations to keep the the volume of the preparation tanks small

-Carbon and nitrogen sources are prepared in separate tanks to avoid Maillard or non-enzymatic browning reactions

P-3 / V-102

Blending / Storage

P-4 / SL-101

Solids Storage

P-5 / V-103

Storage

2. Sterilization of Input Materials-to preclude contamination of the bioreactor

(i) Filtration-to sterilize gaseous streams-membrane filters with pore size of 0.2-0.3µm-compressor creates the necessary pressure to assure air flow through membrane filters

that retain contaminants-prefilters used for dust and other particles

(ii) Heat SterilizationFor batch-heated by steam (in a jacket or sparged directly into the vessel)-cooling water to bring the temperature back to normal-Temperature: 121 °C, holding time: 10-20 minFor continuous-requires the necessary heat exchanger for heating and cooling-time to sterilize is much shorter and energy consumption is 80% lower-temperature: 140-45 °C

Upstream processing

P-3 / AF-101

Air Filtration

P-3 / ST-101

Heat Sterilization

P-3 / BBS-101

Disposable Bioreactor

P-4 / RBR-101

Roller Bottle

P-5 / TFR-101

T-FlaskP-6 / SFR-101

Shake Flask

P-7 / TTR-101

Test Tube

3. Inoculum preparation-to provide a sufficient amount of active cell to inoculate the production

fermenter-The volume factor:

(a)describes the increase of the volume from one inoculum preparation step for the next

(b) Defines the necessary number of inoculum preparation steps

Cleaning-in-Place (CIP)-to prepare the equipment for the next cycle or batch

Upstream processing

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-mixing and bubble dispersion are accomplished by mechanical agitation

-jacket and/or internal coils allow heating and cooling

(ii) Airlift bioreactorBioreactor

-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 -riser ( sparged gas) only in one part of vessel

-at the top, bubble disengage and the heavier medium moves to the downcomer (non-sparged part)

(iii)Packed-bed and fluidized bed bioreactor

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

P-4 / V-103

Fermentation

P-3 / V-102

Vessel Procedure

P-5 / V-104

Air Lift Fermentation

2. Unit Procedures(i) Filling and transfer of materials in vessels-to bring materials (liquids, solids) into the bioreactor -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 and the volume increase by aeration and subsequent substrate feeding

(ii) Agitation-to achieve and maintain homogeneity-to enable efficient heat transfer-in case of aerated fermentation, for the uniform distribution of the gas phase and gas-liquid mass transfer-energy consumption depends on the rotational speed of bioreactor, fluid density and viscosity, and baffling of the reactor

Bioreactor

(iii) Aeration-provides oxygen to meet the aerobic demand of the cells during fermentation-remove gaseous by-product, mainly carbon dioxide-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-for cooling, heat transferred form 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

Bioreactor

P-4 / PFAB-101

PF Aerobic BioOxidation

P-5 / HX-103

Heat Exchanging

P-4 / HX-102

Cooling

P-3 / HX-101

Heating

(v) Foam control-to control the foam formation from the combination of agitation and aeration with the presence of foam-producing and foam-stabilizing substances-addition of antifoam agents- deal with foaming cultures but reduce the oxygen transfer to the cells-disk rotating at high speed at the top of vessel can destroy the foam bubbles but required high energy consumption

(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

Bioreactor

Downstream processing1. 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-high pressure homogenization: (a) slurry pump through a narrow valve at a very high pressure (up to 1200)(b) Strong shear forces caused from the large pressure behind the valve lead to a disruption of the cell-mechanical bead milling:(a) The slurry is fed to a chamber with a rapidly rotating stirrer with steel, glass bead, other abrasives(b) High shear forces and impact during the grinding cause cell disruption

P-4 / BM-101

Bead Milling

P-3 / HG-101

Homogenization

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-requires a heat-stable product with a low vapor pressure to keep the product loss small and causes high energy costs.(b)Filtration-semi-permeable membrane retains the product in the retentate but transfers most of the solvent through the membrane-remove some impurities with a lower molecular size(c)Precipitation-adding a precipitation agent or by changing chemical or physical conditions-requires a product that can be easily and selectively precipitated without degradation

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-sigma factor:(a) Describes the centrifuge in terms of an equivalent area (m2) referenced to a settling tank and is the basis for scaling the centrifuge-settling velocity(a) Specific for the feed that has to be separated(b) Determined by the size and density of the particles, density and viscosity of

the solution

(

Downstream processing

P-5 / BCF-101

Basket Centrifugation

P-3 / DS-101

Centrifugation

P-4 / BC-101

Centrifugation

(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

-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 associated pressure drop(b) Particles are obtained as concentrated slurry-rotary vacuum filtration:(a)Used only for large-scale filtration with large particles-diafiltration:(a)used to change the buffer solution

Downstream processing

P-3 / MF-101

Microfiltration P-4 / DF-101

Diafiltration

P-5 / UF-101

UltrafiltrationP-6 / DE-101

Dead-End Filtration

P-7 / RVF-101

Rotary Vacuum Filtration

(iii) Sedimentation and decanting-sedimentation-same as centrifugation, gravity is the driving force-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-adding flocculating agents can enhance the sedimentation rate by increasing particle size-decanting-for separation of liquid phases, e.g. water and organic solvent-the layers are formed(a)Solid or heavy liquid phase at the bottom (b)Light liquid phase on top(c) Dispersion phase in between-the parameters: density and viscosity of the two phases

Downstream processing

P-4 / CL-101

Clarification

P-3 / V-102

Decanting

P-4 / DX-101

Differential Extraction

P-3 / MSX-101

Mixer-Settler Extraction

(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

4. Product Separation and Purification(i) 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-mixer/settler- two liquid phases are mixed in tank to enable the transfer across the phase boundaries of the product and then a sufficient time is allowed until the phase are separated-differential extraction column-top: the heavy phase (aqueous solution), bottom: the light phase (organic solvent) and moves upwards.-key parameter: partition coefficient- equilibrium concentration of a substance divided by its concentration in the feed phase

Downstream processing

P-3 / HX-101

Condensation

P-5 / C-101

Distillation

P-3 / V-102

Flash

P-4 / V-103

Batch Distillation

(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-the feed is preheated →continuous distillation column (several stages)

→volatile compounds evaporate →vapor moves upwards and leaves the column at the top as distillate →liquefied in the a condenser →high boiling compounds remain in the liquid phase, move downwards and the column at the bottom

(iii) Electrodialysis-an electromotive force is used to transport ions through a semi-permeable, ion

selective membrane by ion diffusion -separate the ion from aqueous solution- the cations move through a cation

membrane in the supplied acid stream, the anions move through an anion membrane into the supplied base stream

-key parameter: membrane flux (100-300 g/m2h) and the transport number (ratio of the flux of the desired ion and the flux of all ions through the membrane

Downstream processing

(iv) Adsorption-to retain either the product or impurities on a solid matrix-the solution is led though a column where the target bind to the resin. If the impurities

are retained → immediately eluted from the column with a buffer, if the product is retained → washing step is added in between

-key parameters: binding capacity and selectivity of the resin, bindingyield 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, i.e. the selective retardation of solutes during the passage through a chromatography column-basic principles are identical to purification by adsorption-Types of column:(a) Gel or exclusion chromatography-separate molecules to their size. (b) Affinity chromatography- based on the stereoselective binding of the solute to

immobilized molecules (ligand)(c) Ion exchange chromatography- electrostatic attraction between the target molecule

that is charged at the given pH and the charged resin.(d) Hydrophobic interaction chromatography (HIC)- separation of protein(e) Reversed-phase chromatography- uneven distribution of the solutes between two

immiscible liquid phases

Downstream processing

P-3 / GAC-101

GAC Adsorption

P-4 / C-102

EBA Chromatography

P-3 / C-101

PBA Chromatography

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- Centrifugation (to inactivate the cells and separate from the broth) →washed by mild detergent (to remove lipids, proteins, other impurities) →pellets are dissolve by adding high concentration of chaotropic reagents and detergents →reducing agent (to reduce disulfide bridges) →chelating agents (to prevent metal-catalyzed oxidation)

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

(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

Downstream processing

P-3 / CR-101

Crystallization

P-3 / TDR-101

Tray Drying

P-4 / BX-101

Packaging

Method Separation principle Typical yield (%)

Separated product

Centrifugation

Sedimentation

Microfiltation

Ultrafiltration

Chromatography

gel filtration

Ion exchange

Hydrophobic interaction

Reversed phase

Affinity

Electrodialysis

Extraction

Distillaiton

Drying/evaporation

Crystallization

Specific density

Specific density

Size/phase

Size

Size shape

Ionic charge

Hydrophobicity

Hydrophobicity/diffusity specific binding

Molecular recognition

Ionic charge/diffusity

Solubility/phase affinity

Volatility

Volatility

Phase change

90-99

80-99

80-99

60-99

70-99

70-99

80-99

97-99

60-95

Cells, particles

Cells, particles

Cells, particles

cell debris, proteins &polymers

Large molecules

Ions

Hydrophilic or hydrophobic molecules

Hydrophilic or hydrophobic molecules

Molecules with specified epitopes

Ions

Hydrophilic or hydrophobic molecules

Volatiles

High-boiling molecules

Crystallized solids

a