Down Stream Processing

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.

1. Processing Basics of Fermentation By Chanakya Pachi

2. Basics of Fermentation and Its Technology

3. Fermentation Fermentation is the term used by microbiologists to describe any process for the production of a product by means of the mass culture of a microorganism. T he extraction and purification of a biotechnological product from fermentation.

4. Fermentation Basics The product can either be:

5. Batch Fermentation A batch fermentation can be considered to be a closed system. At time t=0 the sterilized nutrient solution in the fermenter is inoculated with microorganisms and incubation is allowed to proceed. In the course of the entire fermentation, nothing is added, except oxygen (in case of aerobic microorganisms), and acid or base to control the pH

6. Batch Fermentation The composition of the culture medium, the biomass concentration, and the metabolite concentration generally change constantly as a result of the metabolism of the cells. After the inoculation of a sterile nutrient solution with microorganisms and cultivation under physiological conditions, four typical phases of growth are observed

7. Growth Phases Lag phase Physicochemical equilibration between microorganism and the environment. Log phase Growth of the cell mass can now be described quantitatively as a doubling of cell number per unit time for bacteria.

8. Stationary phase As soon as the substrate is metabolized or toxic substances have been formed, growth slows down or is completely stopped. Death phase In this phase the energy reserves of the cells are exhausted.

9. Bio Reactors…

10. Down Stream Processing

11. General Steps in Downstream Purification

12. Downstream processing The various stages of processing that occur after the completion of the fermentation or bioconversion stage, including separation, purification, and packaging of the product.

13. Stages of Downstream Processing Removal of Insolubles Product Isolation Product Purification Product Polishing To markets

14. Stages in Downstream Processing A few product recovery methods may be considered to combine two or more stages. For example, expanded bed adsorption accomplishes removal of insolubles and product isolation in a single step. Affinity chromatography often isolates and purifies in a single step.

15. Unit operations in downstream processing

16. Separation of cells and medium Recovery of cells and/or medium (clarification) For intracellular enzyme, the cell fraction is required For extracellular enzymes, the culture medium is required On an industrial scale, cell/medium separation is almost always performed by centrifugation Industrial scale centrifuges may be batch, continuous, or continuous with desludging

17. Solid liquid separation Following steps are involved: Floatation- gas is introduced into the liquid it forms bubble. Cells and other solid particles are absorbed and removed. Flocculation -cells and debris from large aggregates and settles down. Addition of flocculating agents are often done. Filtration- most commonly used to separate biomass in culture medium.

18. Types of filtration used Depth filters: they composed of filamentous matrix. Particles are trapped in the matrix and fluid passes out. Absolute filters: the are of specific pore size than the particles to be removed. Mostly used to remove bacteria. Rotatory drum vacuum filters: consists of rotating drum immersed in tank of broth. As the d rum rotates it picks up the mass which forms a cake. Membrane filters:

19. C entrifugation Tubular bowl centrifuge: small, commonly used in pilot plants, can be run at high speed and can be run in both batch or continuous fermentation. Disc centrifuge: consists of several discs that separate bowl into settling zones. Slurry is fed through the centre. Multichamber centrifuge: modification of tubular bowl type. consists of several chambers which allows zigzag movement of feed.

20. Release of intracellular products Physical methods of cell disruption: Ultrasonication Osmotic shock: used to separate hydrolytic enzymes and binding proteins. Heat shock: High pressure homogenization: Grinding with glass:

21. Chemical methods Alkali treatment: Organic solvents: toulene is very oftenly used. It dissolves the membrane phospholipids. Detergents: triton x-100 or tweed is used

22. Enzymatic method Lysozymes: most frequently used for gram +ve bacteria, for –ve in combination with EDTA it is used.

23. Combination metods Concentration: the filtrate free from suspended particles contains 80% water. The water is removed to achieve concentration. Commonly used techniques are: Evaporation Liquid-liquid extraction Membrane filtration Precipitation adsorption

24. Evaporation The evaporators in general , have a heating device for supply of steam and unit for the seperation of concentrated product and vapour, a condenser, accessories and control equipment.

25. Dewatering Precipitation Salting out – addition of a high concentration of a soluble salt (typically ammonium sulphate) causes proteins to aggregate and precipitate. Addition of organic solvents Ultrafiltration The solution is forced under pressure through a membrane with micropores, which allows water, salts and small molecules to pass but retains large molecules (e.g., proteins) Spray drying Requires use of heat to evaporate water – unsuitable for most proteins

26. Protein purification Adsorption chromatography Ion exchange chromatography – binding and separation of proteins based on charge-charge interactions Proteins bind at low ionic strength, and are eluted at high ionic strength + + + + + + + + + + - - - - + + + + + + + + + + - - - + Positively charged (anionic) ion exchange matrix Net negatively charged (cationic) protein at selected pH Protein binds to matrix

27. Typical ion exchange protein separation Loading starts Loading ends, Low salt wash begins Protein absorbance Peak of unbound protein Salt gradient 1M Salt gradient begins Salt gradient ends Eluted peaks of weakly bound (I), moderately bound (II) and tightly bound (III) proteins II III I

28. Affinity chromatography Binding of a protein to a matrix via a protein-specific ligand Substrate or product analogue Antibody Inhibitor analogue Cofactor/coenzyme Specific protein is eluted by adding reagent which competes with binding

29. Affinity chromatography Matrix Spacer arm Affinity ligand + Active-site-bound enzyme 1. Substrate analogue affinity chromatography Matrix Spacer arm Antibody ligand + Antibody-bound enzyme 2. Immunoaffinity chromatography Protein epitope Enzyme

30. Gel permeation chromatography (GPC) Also known as ‘size exclusion chromatography’ and ‘gel filtration chromatography’ Separates molecules on the basis of molecular size Separation is based on the use of a porous matrix. Small molecules penetrate into the matrix more, and their path length of elution is longer. Large molecules appear first, smaller molecules later

31. Precipitation F ormation 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 .

32. Product Purification To separate contaminants that resemble the product very closely in physical and chemical properties . Expensive and require sensitive and sophisticated equipment .

33. Crystallization Process of formation of solid crystals precipitating from a solution, melt or more rarely deposited directly from a gas. C hemical solid-liquid separation technique, in which mass transfer of a solute from the liquid solution to a pure solid crystalline phase occurs.

34. Product Polishing F inal processing steps which end with packaging of the product in a form that is stable, easily transportable and convenient. Crystallization , desiccation, lyophilization and spray drying are typical unit operations

35. lyophilization Freezing the material R educing the surrounding pressure and adding enough heat to allow the frozen water in the material to sublime directly from the solid phase to gas.

36. Downstream processing should be modified based on target product Enzyme preparations for animal feed supplementation (e.g., phytase) are not purified Enzymes for industrial use may be partially purified (e.g., amylase for starch industry) Enzymes for analytical use (e.g., glucose oxidase) and pharmaceutical proteins (e.g., TPA) are very highly purified

37. Fermentation Culture supernatant Centrifugation to remove cells Liquid preparation to animal feed market Animal feed enzyme

38. Fermentation Culture supernatant Centrifugation to remove cells Protein precipitation Protein fraction 1 or 2 purification steps Semi-purified protein Lyophilisation Bottling To chemicals market Analytical enzyme

39. Fermentation Cell pellet Intracellular fraction Therapeutic Protein Centrifugation to remove medium Cell lysis Centrifugation Protein precipitation Protein fraction 3-4 purification steps Homogeneous protein Sterile bottling To pharmaceuticals market