Simulation Of Bioprocess ERT 315/4

1. Simulation Of BioprocessERT 315/4

2. Introduction

3. Stages of Biotech • Ancient • Classical • Modern

4. 4th/3rd mill BC-Baking, brewing (Egypt) • 3rd mill. BC-Ethanol • 17th century-Invention of microscope Ancient Biotech • Begins with early civilization • Developments in agriculture and food production • Few records exist Use microorganisms

5. Classical Biotech • Follows ancient • Makes wide spread use of methods from ancient, especially fermentation • Methods adapted to industrial production

6. 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 • Commercial production of citric acid • 1940s Production of penicillin by fermentation Most of amino acid isolated 1860-1890 Enzyme Engineering

7. 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

8. Modern Biotech • Manipulation of genetic material within organisms • Based on genetics and the use of microscopy, biochemical methods, related sciences and technologies

9. 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

10. Application of Biotechnology • Food industry • Medicine • Chemicals • Environmental

12. Modeling and Assessment in Process Development

13. Why must modeling and simulation? Process concept • 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 Literature Patents Expert knowledge Process design and development Modeling and simulation Sustainability assessment Not eco- efficient Improvement needed Stop Eco- efficient Industrial application

14. 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

15. Development Of Bioprocess

16. The Biotechnical Process

17. Enzymatic process Cell cultivation Transgenetic Plant And Animal Extractive technology Agriculture Reactor Raw material Fermenter Enzymes Extracellular Intracellular Liquid Whole cells Cell harvest Homogenization Product extraction Biomass removal- solid/liquid separation Concentration Protein refolding Viral inactivation Product separation Final formulation Crystallization Drying Final filling Solid

18. Unit Operations & 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

19. Elements of bioprocess • Upstream processing • Bioreactor • Downstream processing

20. Enzymatic process Cell cultivation Transgenetic Plant And Animal Extractive technology Agriculture Reactor Raw material Fermenter Enzymes Extracellular Intracellular Liquid Whole cells Cell harvest Homogenization Product extraction Biomass removal- solid/liquid separation Concentration Protein refolding Viral inactivation Product separation Final formulation Crystallization Drying Final filling Upstream processing Solid Bioreactor Downstream processing

21. Upstream processing • 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

22. Upstream processing • 2. Sterilization of Input Materials • -to preclude contamination of the bioreactor • 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

23. Upstream processing 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

24. Bioreactor • Bioreactor Types • 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

25. 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

26. 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

27. Bioreactor 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

28. 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

29. 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/minfor 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

30. 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

31. Downstream processing • 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

32. 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: • 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

33. 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

34. 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: • Pore sizes of 0.1-10 µm • Flux rate: 20 and 250 L/m2 • -ultrafiltration- • Pore sizes of 0.001-0.1 µm • Flux rate: 20 and 200 L/m2

35. 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

36. Downstream processing (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

37. 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

38. 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

39. 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)

40. 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

41. 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

42. Downstream processing • 6. Final Product Processing • 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

43. Downstream processing (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

47. TYPES OF BIOPROCESSANDBIOPRODUCTS

48. Criteria to select appropriate biocatalyst • 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?

49. Characteristics of biocatalyst

50. Biocatalyst Enzyme 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