Diese Präsentation wurde erfolgreich gemeldet.
Wir verwenden Ihre LinkedIn Profilangaben und Informationen zu Ihren Aktivitäten, um Anzeigen zu personalisieren und Ihnen relevantere Inhalte anzuzeigen. Sie können Ihre Anzeigeneinstellungen jederzeit ändern.

Downstream processing group ppt

Different processes involved in Ds processing.

  • Loggen Sie sich ein, um Kommentare anzuzeigen.

Downstream processing group ppt

  2. 2. Downstream processing refers to the recovery and purification of biosynthetic products, particularly pharmaceuticals, from natural sources such as animal or plant tissue or fermentation broth, including the recycling of salvageable components and the proper treatment and disposal of waste. It is an essential step in the manufacture of pharmaceuticals such as antibiotics, hormones (e.g. insulin and humans growth hormone), antibodies (e.g. infliximab and abciximab) and vaccines; antibodies and enzymes used in diagnostics; industrial enzymes; and natural fragrance and flavor compounds.
  3. 3. Filtration is the most commonly used technique for separating the biomass and culture filtrate. The efficiency of filtration depends on many factors— the size of the organism, presence of other organisms, viscosity of the medium, and temperature.
  4. 4. These filters are with specific pore sizes that are smaller than the particles to be removed. Bacteria from culture medium can be removed by absolute filters.
  5. 5. These filters are frequently used for separation of broth containing 10-40% solids (by volume) and particles in the size of 0.5- 10µm. Rotary drum vacuum filters have been successfully used for filtration of yeast cells and filamentous fungi
  6. 6. In this type of filtration, membranes with specific pore sizes can be used. However, clogging of filters is a major limitation. There are two types of membrane filtrations—static filtration and cross-flow filtration
  7. 7.  Filtration is used at various stages of the downstream processing in the bioreactor harvest as well as processing of purified products.  Several filtration process are used. Most common ones are- 1. Microfiltration - used at the start of the downstream process to clarify the feed 2. Ultrafiltration – used between chromatography steps to concentrate the product and change the buffer conditions 3. Reverse Osmosis- use of pressure for osmosis
  8. 8.  Cell disruption is an essential part of biotechnology and the downstream processes related to the manufacturing of biological products.  The disruption of cells is necessary for the extraction and retrieval of the desired products, as cell disruption significantly enhances the recovery of biological products.
  9. 9.  Mechanical  Non-mechanical
  10. 10.  Bead mill- The main principle requires a jacketed grinding chamber with a rotating shaft, running in its center.  Agitators are fitted with the shaft, and provide kinetic energy to the small beads that are present in the chamber. That makes the beads collide with each other causing disruption
  11. 11.  Ultrasound- Ultrasonic disruption is caused by ultrasonic vibrators that produce a high frequency sound with a wave density of about 20 kHz/s.  A transducer then converts the waves into mechanical oscillations through a titanium probe, which is immersed into the cell suspension. Such a method is used for both bacterial and fungal cell disruption.
  12. 12.  French press and high pressure homogenizer- In a French press, or high pressure homogenization, the cell suspension is drawn through a valve into a pump cylinder  Then it is forced under pressure of up to 1500 bar, through a narrow annular gap and discharge valve, where the pressure drops to atmospheric. Cell disruption is achieved due to the sudden drop in pressure upon the discharge, causing the cells to explode.
  13. 13.  Thermolysis- use of heat to disrupt the cell membrane.  Periplasmic proteins in G(-) bacteria are released when the cells are heated up to 50ºC.  Cytoplasmic proteins can be released from E.coli within 10min at 90 ºC.  Freezing and thawing of a cell slurry can cause the cells to burst due to the formation and melting of ice crystals.
  14. 14.  Decompression- During explosive decompression, the cell suspension is mixed with pressurized subcritical gas for a specified time, depending on the cell type.  The gas enters the cell and expends on release, causing the cell to burst. Gases like carbon dioxide can be used
  15. 15.  Osmotic shocks- here cells are first exposed to either high or low salt concentration.Then the conditions are quickly changed to opposite conditions which leads to osmotic pressure and cell lysis.
  16. 16.  In addition to physical and mechanical methods, several chemical methods for cell disruption exist. These methods rely on utilization of chemical substances or enzymes in disruption process.  The mechanisms of actions are multiple, but the most widely used methods act by destroying the cell wall by enzymes, osmotic pressure, or by interfering or precipitating cell wall proteins.
  17. 17.  Detergents- Detergents that are used for disrupting cells are divided into anionic, cationic and non- ionic detergents.  The common thing for all detergents is that they directly damage the cell wall or membrane, and this will lead to release of intracellular content.
  18. 18.  Solvents- Solvents which can be used for cell lysis include for example some alcohols, dimethyl sulfoxide, methyl ethyl ketone or toluene.  These solvents extract cell wall’s lipid components which leads to release of intracellular components.
  19. 19.  Enzymes- Use of enzymes depending on the cell wall composition  For example, lysozyme is commonly used enzyme to digest cell wall of gram positive bacteria. Lysozyme hydrolyzes β-1-4- glucosidic bonds in the peptidoglycan
  20. 20.  Liquid -liquid extraction (LLE) is the process of separation of a liquid mixture of components where liquid solvents are used followed by dilution of one or more components of the initial mixture.  This downstream process is significantly useful in Bioprocess technology.  This is a unit process which requires the knowledge of phase behavior and physicochemical characterization of different compounds.
  21. 21.  In liquid-liquid extraction, components in the fed material, consisting of liquid phases are separated when third liquid also known as solvent is added to the process.  By adding this new component which is insoluble in the feed, a new phase is formed.  The component which is more important during the extraction or which is the desired component to be extracted during the process is transferred to extract.
  22. 22.  The extraction is carried out in two ways of mixing; countercurrent and co-current mixing. The co-current flow is limited to one stage per extraction, whereas, counter current is controlled as multi stages per unit.  Depending on the density of the solvent to the carrier liquid the counter current extraction can be carried out on two ways  If the solvent is less dense than carrier liquid, solvent is fed from the bottom.  The reverse phenomenon happens if the solvent is denser than the carrier liquid.
  24. 24. PRECIPITATION ■ Formation of a solid in a solution during a chemical reaction. ■ Solid formed is called the precipitate and the liquid remaining above the solid is called the supernate. ■ It is the most commonly used technique in industry for the concentration of macromolecules. ■ It can also be employed for the removal of certain unwanted by-products. ■ Neutral salts, organic salts, alteration in temperature and pH are used in precipitation.
  25. 25. ■ Precipitation of protein is widely used in downstream processing in order to concentrate proteins and purify them from various contaminants. ■ Protein precipitation can be – non-specific protein precipitation - protein specific precipitation Protein specific precipitation – e.g.- affinity precipitation - ligand precipitation
  26. 26. AFFINITY PRECIPITATION ■ In affinity precipitation, the protein is free in solution, rather than bound to an insoluble support. ■ Ligand binding gives rise to the precipitation of the protein from solution, which is then followed by centrifugation. ■ The pellet contains the protein of interest and the ligand, whereas the other components of the mixture remain in the supernatant, allowing easy separation.
  27. 27. METHODS OF PRECIPITATION ■ Salting out ■ Isoelectric precipitation ■ Precipitation with miscible solvents ■ Non-ionic hydrophilic polymers Polymers such as dextrans and polyethylene glycol ■ Flocculation by pyroelectrolytes Alginate, carboxymethylcellulose, tannic acid polyacrylic acid and phosphatases are used ■ Polyvalent metallic ions Ca2+, Mg2+, Mn2+ are used ■ Increase in temperature ■ Change in pH
  28. 28. SOLID-LIQUID SEPARATION ■ the separation of cells from the culture broth, removal of cell debris, collection of protein precipitate, etc. ■ The term harvesting of microbial cells are used for the separation of cells from the culture medium. ■ Several methods used for solid-liquid separation are – flotation flocculation filtration centrifugation
  29. 29. FLOTATION ■ When gas is introduced into the liquid broth, it forms bubbles. ■ The cells and other solid particles get absorbed on gas bubbles. ■ These bubbles rise to the foam layer which can be collected and removed. ■ Certain substances called as collector substances are used to facilitate stable foam formation. ■ Collector substances used are like – long chain fatty acids - amines
  30. 30. FLOCCULATION ■ In flocculation, the cells or cell debris form large aggregates to settle down for easy removal. ■ The process of flocculation depends on the nature of cells and the ionic constituents of the medium. ■ Sometimes flocculating agents are also used to achieve appropriate flocculation. ■ Some flocculating agents are – inorganic salts, organic polyelectrolyte, mineral hydrocolloid.
  31. 31. FILTRATION ■ Filtration is the most commonly used technique for separating the biomass and culture filtrate. ■ The mixture goes through a filter which retains the particles according to size while allows the passage of fluid through the filter. ■ The efficiency of filtration depends on many factors – size of the organism, viscosity of the medium, temperature. ■ Several filters are in use like – depth filter absolute filter rotary drum vacuum filter membrane filter
  33. 33. CENTRIFUGATION ■ Separation by means of the accelerated gravitational force achieved by a rapid rotation. ■ Relies on the density difference between the particles and the surrounding medium. ■ Most effective when the particles to be separated are large, the liquid viscosity is low and the density difference between particles and fluid is great. ■ Batch centrifuge is common in the labs but the low processing capacity limits its use in large scale. ■ Continuous centrifuges are common in large-scale processing in which the deposited solids are removed continuously or intermittently.
  34. 34. TUBULAR BOWL CENTRIFUGE ■ High speed ■ Length diameter ratio 4.8 ■ 15000r.p.m. ■ Used widely in emulsion ■ Used in solid with small amount ■ Can be run in both batch or continuous mode
  35. 35. DISC CENTRIFUGE ■ Contain conical sheets of metal (discs) which are stacked with clearances. ■ Disc size – 0.3mm ■ The discs rotate with the bowl to split the liquid into thin layers. ■ The slurry is fed through a central tube. ■ The clarified fluid moves upward while the solids settle at the lower surface.
  36. 36. MULTICHAMBER CENTRIFUGE ■ Modification of tubular bowl centrifuge. ■ Consist of several chambers in such a way that feed flows in a zigzag fashion. ■ Particle size – 0.1 to 200 micrometre diameter. ■ Variation in centrifugal force in different chambers. ■ Force is higher in the periphery chambers. ■ Smallest particle settle down in the outermost chamber.
  37. 37. SCROLL CENTRIFUGE OR DECANTER ■ Composed of a rotating horizontal bowl tapered at one end. ■ Used to concentrate fluid with high solid concentration. ■ Solids are deposited on the wall of the bowl.
  38. 38. CHROMATOGRAPHY  ‘Chromatography’ is an analytical technique commonly used for separating a mixture of chemical substances into its individual components, so that the individual components can be thoroughly analyzed.  The mixture is dissolved in a fluid called the mobile phase, which carries it through a structure holding another material called the stationary phase.  Chromatography is used in downstream processing to effectively purify the biological products (proteins, pharmaceuticals, diagnostic compounds and research materials)  There are many types of chromatography e.g., liquid chromatography, gas chromatography, ion-exchange chromatography, affinity chromatography, but all of these employ the same basic principles.
  39. 39. PRINCIPLE  Chromatography is based on the principle of separation of compounds into different bands (color graphs) and then identification of those bands.  The preferential separation is done due to differential affinities of compounds towards stationary and mobile phase. After separation of the compounds, they are identified by suitable detection methods.  The differences in affinities arise due to relative adsorption or partition coefficient in between components towards both the phases.
  41. 41. APPLICATIONS OF CHROMATOGRAPHY  The chromatographic technique is used for the separation of amino acids, proteins & carbohydrates.  It is also used for the analysis of drugs,hormones,vitamins .  Helpful for the qualitative & quantitative analysis of complex mixtures.  The technique is also useful for the determination of molecular weight of proteins
  42. 42. DRYING DEVICES LYOPHILIZATION  A stabilizing process in which a substance is first frozen and then the quantity of the solvent is reduced, first by sublimation (primary drying stage) and then desorption (secondary drying stage) to values that will no longer support biological activity or chemical reactions.  It is a drying process applicable to manufacture of certain pharmaceuticals and biologicals that are thermolabile or otherwise unstable in aqueous solutions for prolonged storage periods, but that are stable in the dry state.
  43. 43. PRINCIPLE:-  Lyophilization is based on a simple principle of physics called “SUBLIMATION”. Sublimation is the process of transition of a substance from solid to the vapor state without passing through an intermediate liquid phase.  Lyophilization is performed at temperature and pressure conditions below the triple point, to enable sublimation of ice.  The material to be dried is first frozen and then subjected under a high vacuum to heat (by conduction or radiation or by both) so that frozen liquid sublimes leaving only solid ,dried components of the original liquid.
  44. 44. Equipment used for Lyophilization – LYOPHILIZER A lyophilizer consists of a vacuum chamber containing product shelves which are capable of cooling and heating containers and their contents. A vacuum pump, a refrigeration unit, which is associated controls are connected to the vacuum chamber.
  45. 45. How does it work  Fundamental process steps are: 1.Freezing: the product is frozen. This provides a necessary condition for low temperature 2.Vacuum: after freezing, the product is placed under vacuum. This enables the frozen solvent in the product to vaporize without passing through liquid phase, a process known as SUBLIMATION. 3.Heat: Heat is applied to the frozen product to accelerate sublimation. 4.Condensation: Low-temperature condenser plates remove the vaporized solvent from the vacuum chamber by converting it back to a solid. This completes the separation process. Resulting product has a very large surface area thus promoting rapid dissolution of dried product.
  47. 47. FREEZING PRIMARY DRYING STAGE SECONDARY DRYING STAGE PACKING • The product must be frozen to a low enough temperature to be completely solidify and be adequately pre- frozen. • Decrease the shelf temperature to -50⁰c. • Low temperature and low atmospheric pressure are maintained • Formation of ice crystals occurs. • Heat is introduced from shelf to the product under graded control by electrical resistance coils or circulating silicone. • The temperature and pressure should be below the triple point of water i.e., 0.0098°C and 4.58mmHg. • Easily removes moisture up to 98% to 99%. • The temperature is raised to 50°C – 60°C and vacuum is lowered about 50mmHg. • Bound water is removed. • Rate of drying is low. • It takes about 10-20 hrs • This process is called ‘Isothermal Desorption’ as the bound water is desorbed from the product. • After drying the vacuum is replaced by filtered dry air or nitrogen to establish atmospheric pressure • Vials and bottles are sealed with rubber closures and aluminum caps • e.g., penicillin can be freeze dried directly in ampules.
  48. 48. APPLICATIONS  PHARMACEUTICAL AND BIOTECHNOLOGY 1. Pharmaceutical companies often use freeze-drying to increase the shelf life of products, such as vaccines and other injectables. 2. By removing the water from the material and sealing the material in a vial, the material can be easily stored, shipped, and later reconstituted to its original form for injection.  FOOD INDUSTRY 1. Freeze-drying is used to preserve food and make it very lightweight. 2. The process has been popularized in the forms of freeze-dried ice cream, an example of astronaut food.  TECHNOLOGICAL INDUSTRY 1. In chemical synthesis, products are often freeze- dried to make them more stable, or easier to dissolve in water for subsequent use. 2. In bio- separations, freeze-drying can be used also as a late-stage purification procedure, because it can effectively remove solvents.
  49. 49. SPRAY DRY TECHNOLOGY  Spray drying is used for drying large volumes of liquids. In spray drying, small droplets of liquid containing the product are passed through a nozzle directing it over a stream of hot gas. The water evaporates and the solid particles are left behind.  It is a kind of continuous atmospheric dryer which can be used to dry materials such as fuel, intermediates, soap powder, or inorganic salts, etc.
  50. 50. HOW DOES IT WORK? A spray dryer uses the spray method to transform the material into fog droplets in order to be dispersed into the hot gas stream.  The material connects with the hot air in a co-current, countercurrent, or mixed flow manner so that the water can evaporate quickly to achieve the drying effect.  The spray dryer provides a large surface area for heat and mass transfer by atomizing the liquid to small droplets. These are sprayed into a stream of hot air, so that each droplet dries to a solid particle. The drying chamber resembles the cyclone ensuring good circulation of air, to facilitate heat and mass transfer, and that dried particles are separated by the centrifugal
  51. 51. APPLICATIONS  Spray dryers are used for the drying of liquid materials like emulsion, suspension, solution, slurries, thin pastes, etc.  Spray drying can be used to dry materials that are sensitive to heat or oxidation without degrading them, even when high temperature air is employed.  The liquid feed is dispersed into droplets, which are dried in seconds because of their high surface area and intimate contact with the drying gas.  The product is kept cool by the vaporization of the enveloping liquid, and the dried product is kept from overheating by rapid removal from the drying zone.  The improvement in flow and reduction of air entrapment make the spray-dried material suitable for use in the manufacturing of tablets and capsules.
  52. 52. BIOSENSORS Biological components + Physiochemical Detectors
  53. 53. DEFINITION ▪ Self-contained integrated device that is capable of providing specific qualitative or semi-quantitative analytical information using a biological recognition element which is in direct-spatial contact with a transduction element. (IUPAC,1998) ▪ In simple words, Biosensors detect analytes of interest by combining a biological component with a physiochemical detector via electronic signals. ▪ They can be Nano Biosensors, Amperometric Biosensors, Blood Glucose Biosensors, Quantum Mechanical- Based Biosensors etc. ▪ NOTE- Biosensors ≠ Bioanalytical system 56
  54. 54. COMMON EXAMPLES ▪ Glucometer Pregnancy Tests ▪ Fitbit Amit Gothe 57
  56. 56. WORKING PRINCIPLE ▪ Analytes diffuse from the solution to the surface of the Biosensor. ▪ Analytes react specifically & efficiently with the Biological Component of the Biosensor. ▪ This reaction changes the physiochemical properties of the Transducer surface. ▪ This leads to a change in the optical/electronic properties of the Transducer Surface. ▪ The change in the optical/electronic properties is measured/converted into electrical signal, which is detected. 59
  57. 57. FLOW CHART Amit Gothe 60
  58. 58. … 61
  59. 59. CONSTRUCTION Amit Gothe 62
  60. 60. Bioreceptor ▪ In a biosensor, the bioreceptor is designed to interact with the specific analyte of interest to produce an effect measurable by the transducer. ▪ High selectivity for the analyte among a matrix of other chemical or biological components is a key requirement of the Bioreceptor. ▪ While the type of biomolecule used can vary widely, biosensors can be classified according to common types Bioreceptor interactions involving: antibody/antigen, enzymes/ligands, nucleic acids/DNA, cellular structures/cells, or biomimetic materials. 63
  61. 61. Surface attachment of biological elements ▪ An important part in a biosensor is to attach the biological elements (small molecules/protein/cells) to the surface of the sensor. ▪ The simplest way is to functionalize the surface in order to coat it with the biological elements. ▪ This can be done by polylysine, aminosilane, epoxysilane or nitrocellulose in the case of silicon chips/silica glass. Subsequently, the bound biological agent may be for example fixed by Layer by layer deposition of alternatively charged polymer coatings. 64
  62. 62. Biotransducer ▪ A Biotransducer is the recognition-transduction component of a biosensor system. It consists of two intimately coupled parts; a bio- recognition layer and a physicochemical transducer, which acting together converts a biochemical signal to an electronic or optical signal. ▪ As a result of the presence and biochemical action of the analyte (target of interest), a physico-chemical change is produced within the biorecognition layer that is measured by the physicochemical transducer producing a signal that is proportionate to the concentration of the analyte. 65
  63. 63. TYPES OF BIOSENSORS ▪ Biosensors can be classified by their Biotransducer type. The most common types of Biotransducer used in biosensors are discussed in the next slides : 66
  64. 64. … Calorimetric/Thermal Detection Biosensors. Optical Biosensors. Resonant Biosensors. Piezoelectric Biosensors. Electrochemical Biosensors. • Conductimetric Sensors. • Amperometric Sensors (Blood Glucose Biosensor). • Potentiometric Sensors. 67
  65. 65. 1. Calorimetric / Thermal Detection Biosensors. ▪ Uses Absorption / Production of Heat. ▪ Many enzyme catalyzed reactions are exothermic, generating heat which may be used as a basis for measuring the rate of reaction and, hence, the analyte concentration. This represents the most generally applicable type of biosensor. ▪ Temp. measured by Enzyme Thermistors - The temperature changes are usually determined by means of thermistors at the entrance and exit of small packed bed columns containing immobilized enzymes within a constant temperature environment ▪ Uses: Detection of: (1) Pesticides . (2) Pathogenic Bacteria 68
  66. 66. 2. Optical Biosensors ▪ Colorimetric for color - Measures change in Light Adsorption. ▪ Photometric for Light Intensity - Detects the Photon output. ▪ Raman effect 69
  67. 67. 3. Resonant Biosensors ▪ An Acoustic Wave Transducer is coupled with Bioelement. ▪ Measures the change in Resonant Frequency. 70
  68. 68. 4. Piezoelectric Biosensor ▪ The principle of Piezoelectric Biosensor is used in sound vibrations, hence it is called acoustic Biosensors. The basics of the Biosensors are formed by the piezoelectric crystals and the characteristic frequencies are trembling with the crystals of positive and negative charge. By using the electronic devices we can measure the certain molecules on the crystal surface and alters the response frequencies using these crystals we can attaché the inhibitors. 71
  69. 69. 5. Electrochemical Biosensors ▪ Electrochemical Biosensor is a simple device. It measures the measurement of electronic current, ionic or by conductance changes carried by bio-electrodes. 72
  70. 70. i. Conductimetric Biosensors ▪ Measures Electrical Conductance/Resistance of the solution. ▪ Conductance Measurements have relatively Low Sensitivity. ▪ Electrical Field is generated using sinusoidal(ac) voltage, which helps in minimizing undesirable effects like: i. Faradaic processes. ii. Double layer charging & iii. Concentration polarization 73
  71. 71. ii. Blood Glucose Biosensor ▪ The Blood glucose Biosensors are used widely throughout the world for diabetic patients. It has a single use disposable electrodes with glucose oxide and derivatives of a mediator (Ferrocene) and the shape of the blood glucose Biosensor looks like a watch pen. With the help of hydrophilic mesh electrodes are converted. 74
  72. 72. iii. Potentiometric Sensors. ▪ Working Principle – When ramp voltage is applied to an electrode in solution, a current flow occurs because of electrochemical reactions. ▪ Measured Parameter – Oxidation / reduction Potential of an Electrochemical run. Amit Gothe 75
  73. 73. APPLICATIONS Amit Gothe 76
  74. 74. CRYSTALLIZATION ▪ Crystallization is a separation and purification method widely used for final purification of components. ▪ Crystallization consists of two stages: formation of nuclei and growth of crystals. ▪ For crystallization to occur the solution should be first supersaturated. Amit Gothe 77
  75. 75. NUCLEATION ▪ The first step of crystallization is formation of nucleation where crystals are formed when solute molecules dispersed in the solvent start to gather into clusters. And became stable under the current operating condition . ▪ These stable structures together form a nuclei . It is at the stage of nucleation that atoms arrange in periodic manner to form crystal structure. ▪ There are two different nucleation formations – primary and secondary. Amit Gothe 78
  76. 76. Crystal growth ▪ The second step of crystallization is crystal growth where nucleus size increases after the critical cluster size is achieved. ▪ It is the Growth of nuclei to the next stage . ▪ Crystal growth rate is affected by various physical factors, such as surface tension of solution, pressure, temperature, relative crystal velocity in the solution, Reynolds number and other factors ▪ Polymorphism : ability to crystallize with different crystal structures Amit Gothe 79
  77. 77. Crystallization processes ▪ Influencing factors : temperature and concentration A) cooling crystallization B) evaporative crystallization - Generating crystals by evaporating a solution at constant temperature.Most of the industrial crystallizers are evaporative. ▪ Example: sodium chloride and sucrose Amit Gothe 80
  78. 78. Applications ▪ Crystallization is an important downstream processing method in bioprocess technology and in all chemical industry. ▪ Downstream processing can contribute to a large portion of end product price. Therefore crystallization has an advantage compared to other solid liquid separation operations such as distillation since crystallization is a rather energy efficient unit operation. ▪ In addition the yielded product has very high purity level and therefore wide scale of end use opportunities. Amit Gothe 81
  79. 79. Amit Gothe 82