Introduction Stages of Biotech Ancient Classical Modern.

IntroductionIntroduction

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

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

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


Enzymes

Proteins & small molecules


Proteins

Proteins

Metals

Purified water

Very low

Low

Medium

Medium

Low-medium

High

Medium

Very Low

high

Modeling Modeling and Assessment and Assessment

ininProcess Process

DevelopmentDevelopment

Process design and development

Modeling and simulation

Sustainabilityassessment

Stop

Process concept

Industrial application

LiteraturePatentsExpertknowledge

Eco-efficient

Not eco-efficient

Improvementneeded

Why must modeling and simulation?

Close collaboration with the process design Additional information from patents,

literature, and other external sources Simulation used to evaluate the process

and guide the R&D to the overall aim Repeated iteratively

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 Development Of Of

BioprocessBioprocess

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:

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

Upstream processing

Bioreactor

Downstream processing

Enzymatic process

Cell cultivation

Transgenetic PlantAnd Animal


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


1. Preparation and Storage of Solutions-to provide and store that are needed at some point in the

processe.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

P-3 / V-102

Blending / 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: Pore size- 0.2-0.3µm-prefilters used for dust and other particles

(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

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

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

Upstream processing

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

(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

(iii)Packed-bed and fluidized bed bioreactorBioreactor

-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(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 thecells during fermentation-specified by the gas used (Air, pure O2, pure N2, orair 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 foam-producing 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

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

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(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

without degradation


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-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


P-3 / MF-101

Microfiltration P-4 / DF-101

Diafiltration

P-5 / UF-101

Ultrafiltration

-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-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-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


P-4 / CL-101

Clarification

P-3 / V-102

Decanting

(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

Condensation

Downstream processing4. 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-4 / DX-101

Differential Extraction

P-3 / MSX-101

Mixer-Settler Extraction

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(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)


(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 compoundsbased 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

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 substancesProtein Solubilization and Refolding-to release the intracellular material and inclusion bodies or water-insoluble pellets produced by heterologous protein in bacteria and fungi


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

Crystallization

(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 / TDR-101

Tray Drying

P-4 / BX-101

Packaging

Method Separation principle 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


Molecules with specified epitopes

Ions


Volatiles

High-boiling molecules

Crystallized solids

TYPES OF BIOPROCESSAND

BIOPRODUCTS

What yield, product concentration, and productivity can be reached?

What substrate can be utilized, what additional media components are required, and how does it all effect downstream processing?

What by-products are formed and how do they affect yield and downstream processing?

What are the challenges in biocatalyst preparation, storage, propagation, security, and safety?

What are the optimal reaction conditions, e.g. temperature, oxygen supply, shear sensitivity, foam formation, etc?

How well do we understand the reaction mechanisms, are they robust and genetically stable?

If the product is expressed intracellularly, how is it extracted? How do we purify the desired product form the many

impurities in the process?

Biocatalyst

Production device

Raw

material

Time-scale Purification

Complex protein structure

Viral/prion

risk

Process

examples

Enzymes

Bacteria and yeast

Fungi

Mammalian cells

Plant cells

Transgenic plants

Transgenic animals


Bioreactor

Bioreactor

Bioreactor

Bioreactor

Bioreactor

Bioreactor

Whole plant

Whole animal

Pure substrates

Simple media

Simple media

Complex media

Simple media

Fertilizer, CO2, various others

Various plant & animal materials

Certain parts of plants, animals and humans

Short

Short

Medium

Medium

Medium

Long

Long

Long

Simple

Medium

Medium

Medium

Medium

Complex

Complex

Complex

No

No

No

Yes

Possible

Possible

Yes

Possible

No

Small

Small

Medium

Small

Small

High

High

Cyclodextrin,

Acrlyamide, L-dopa

Lysine,Vitamin B2,

Insulin

Citric acid, antibiotics

Monoclonal

Antibodies,

Interferons

Taxol, shikonin,

Methyldigoxin

Antibodies, antibody

Fragments, HAS,

PHB

Α1-antirypsin, HAS,

Lactoferrin

Plasma,components,taxol

Enzyme BiotransformationEnzyme Biotransformation

-enzymes: protein with a unique three-dimensional structure able to bind a substrate, usually but not always a small molecule, and catalyze a specific reaction, similar to chemical catalysis but under mild conditions of temperature and pressure

-classified in six groups according to the chemical reaction: oxido-reductase, transferases, hydrolases, lyases, isomerases, ligases

-highly selective and specific in the reaction: regio-, stereo- and enantioselective

-can be in solution or immobilized

Metabolic Bioconversion using cell cultivationMetabolic Bioconversion using cell cultivation- Classified into five groups: Bacteria, Fungi, Mammalian

cells, insect cells, plant cells(i) Bacteria-unicellar prokaryotes with a rigid cell wall-key determinants for their growth: media composition, temp., gaseous environment, pH-show a range responses to oxygen: aerobic bacteria- require oxygen for their growth; anaerobic bacteria- grow only at the absence of oxygen; facultatively-anaerobic bacteria- able to grow under both conditions-temperature optimal for growth: psychrophiles (20-30 °C), mesophiles (30-40 °C), thermophiles (45-60 °C), extreme thermophiles (extremophiles) (80-105°C)-optimum pH: 6.5-7.5

(ii) Fungi-divide into two subgroups: yeasts, molds-yeast: small, single cells that can grow as individual cells or clumps-molds: multicellular, vegetative structure call mycelium, as usually highly-branched systems of tubules(iii) Mammalian cells-produce correctly folded proteins and secrete them to the culture environment-grow quite slowly, with typical doubling times of 12-20 h-temperature : 37 °C, pH: 7.3-mammalian cell product: monoclonal antibodies, interferons, vaccines, erythropoietin

(iv) Insect cells-produce recombinant proteins less expensively and more quickly than mammalian cells and at high expression levels -typically grow at around 28 °C, and pH 6.2-used for veterinary vaccines for the swine fever virus(v) Plant cells-10 to 100 times larger than microbial cells and more sensitive to shear-slow metabolism, with doubling times of 20-100h -cultivate as a callus or a lump of undifferentiated plant tissue growing on a solid nutrient medium or as aggregated plant cells in suspension-used to produce secondary metabolites, anticancer drug paclitexel (taxol), recombinant proteins of high value

Transgenic PlantsTransgenic Plants-genetically modified plants to produce a wide variety of

products-the expression can take place in the whole plant or only in a certain part as in the seeds-commonly used plants: tobacco, potato, rice, wheat-inexpensive, easy to scale-up, free of human pathogens

Transgenic AnimalsTransgenic Animals-reduce the dependency on the seasonal and geographical conditions, post-translational modifications are more likely to mimic the native structure-usually done by injecting exogenous DNA into egg cells to produce a vital embryo that is later able to express the Desired product

Extractive TechnologiesExtractive Technologies-comprise all processes where a product is extracted from natural material-Used in the extraction of pharmaceuticals from human or animal blood and from plant material-the products usually chemically complex non-protein

materials

Product Classifications/Characteristics-according to size, bioproducts can be divided into:Small molecules, Large molecules and Solid particles-Small moleculesi. sugars, amino acids, organic acids, vitaminsii. Molecular weight of 30-600 Da and a radius smaller than 1 nmIii divided into primary and secondary metabolites:a. Primary metabolites: sugar, organic alcohols, acids-produced in the primary growth face of the organismb. Secondary metabolites: formed at or near the beginning of the stationary phase, e.g. antibiotics and steroids-large moleculesi. proteins, nucleic acids, polysaccharidesii. Molecular weight of 103-106 Da -solid particlei. Whole cell like yeast, animal cell, ribosomes, virusesii. A radius of up to several µm

-by the scale of production, bioproducts can be classified into:Bulk or commodity chemicals made at large scale, Fine

chemicals and Pharmaceuticals made a smaller scale-bulk chemicalsi. Produced in very large amounts ii. Simple downstream processingiii. Sold at a relatively low price-pharmaceuticalsi. produced in a small amounts ii. high priceiii. used expensive media and complex equipment with low

productivities-fine chemicalsi. Used as intermediates and have application in a variety of

industriesii. Annual production, price, and required purity lie between bulk

chemicals and pharmaceuticals

Product classes-describe by its function (proteins, organic acids, lipid) or application (food and feed additives, pharmaceuticals, detergents, chemical intermediates, agriculturally used products)i. organic alcohol and ketones-produced in anaerobic fermentationsii. organic acids:-used as intermediates or as food additives-major organic acids produced are citric, lactic, gluconic acid.iii. amino acids:-the building blocks of protein and are connected via peptide bonds-used as food additives, feed additives, and in pharmaceuticalsiv. nucleic acids: -used as therapeutics, e.g. DNA vaccines, gene therapyv. antibiotics:-frequent use in human and animal health-produced on fungal fermentation

vi. vitamins:-produced in bioprocesses, e.g. vitamin A, C, E, and the B vitaminsvii. biodegradable biopolymers:-plastics derived from renewable material-common biopolymer are polyhydroxyalkanoates (PHA)viii. dextran and xanthan:-industrially produced microbial polysaccharides-used as thickening, gelatinizing, suspending agentsix. carotenoids:-natural pigments (yellow or red color)-produced by microorganismsx. pesticidesxi. lipids:-including fats, oils, waxes, phospholipids, steroids-commercially produced lipid: prostaglandins, leukotrienes, xii. proteins

-Water:-dominant raw material-other component of the reaction medium: macronutrients and micronutrients-macronutrient:-needed in concentrations larger than 10-4 M-including carbon–energy source, oxygen, nitrogen, phosphate, sulfur, and some minerals such as magnesium and potassium ions-carbon-energy source:-provides the carbon for biosynthesis as well as energy derived by its oxidation-Typically used carbons sources: glucose, starch, corn syrup, molasses. soybean oil, palm oil, ethanol, methanol-50% is incorporated in the biomass, and remaining 50% is used to derived energy for biosynthesis-nitrogen:-accounts for 10-14% of the dry cell mass-most widely used are ammonia and ammonia salts, proteins,

-oxygen and hydrogen:-20% of the cell mass (O2), 8% (H2)-phosphorus: 3% of cell dry weight and is provided by phosphate salts -sulfate: 0.5% of cell mass is added as sulfate salts or with amino acids contained in complex media-magnesium and potassium ions: provided as inorganic potassium and magnesium sulfate-micronutrients:-required in low concentrations:-including iron, zinc, manganese,copper, sodium, calcium, boron-added as inorganic salts-also can be classified into defined or synthetic media and complex or natural media-defined media:-contain specific amounts of pure chemicals with a known composition-complex media:-include one or more natural materials whose chemical composition is not exactly known and which may vary with source of time

-natural media:-cheaper-cause less reproducible fermentation and more complex downstram processing-bacteria and fungi: need only a relative simple media and very low cost-mammalian cells: more complex medium is necessary, need serum as required ingredient(complex media) or not (synthetic media)-plant cell: require a carbohydrate cell source, inorganic macronutrients and micronutrients.

Introduction Stages of Biotech Ancient Classical Modern.

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Transcript of Introduction Stages of Biotech Ancient Classical Modern.