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

1. By Assit Prof Dr. P . Berciyal Golda VICAS

2. Downstream processing 🠶 Introduc tio n : - The various stages of processing that occur after co seperation , purification and packaging of products . The various stages of processing that occur after the completion of the fermentation or bioconversion stage, including separation, purification, and packaging of the product

3. Strategies to recovery and purify bio-products Fermentor Solid-liquid separation Recovery Purification Supernatant Cells Cell products Cell disruption or rupture Cell debris Crystallization and drying

4. Stages in Downstream Processing Removal of Insolubles Product Isolation Product Purification Product Polishing Product Packaging 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.

5. l. Removal of Insolubles Separation of cells, cell debris or other particulate matter Typical operations to achieve this: Filtration Centrifugation Sedimentation Flocculation a process where a solute comes out of solution in the form of floc or flakes. Gravity settling

6. a. Filtration :- 🠶 This i s a kin d of mechani c al ope r ation that used for s e p e r a tion of solids from fluids (liquids or gases )by interposing a medium to porous membrane through which the fluid can pass but solids in the fluuds are retained . 🠶 The sol i ds p a r t i c le s that d e posit e d o n the filter form of laye r w hi c h is known as filter cake. 🠶 All the solids particles are feeded and stopped by the cake and the cake grows at qhich particles are brought to its surface . 🠶 All the flu i ds goes through the ca k e a nd f lu i d medium . .

7. Continous Rotary Vaccum Filter :- 🠶 I t i s one of the m ost com m on l y used ty p e of filter fermentation.. 🠶 The d r um i s p r e c oated p rior to filtrat i o n. 🠶 A smal l ag e nt of c o agulating i s ad d ed to the b roth b e fore i t is pumped before it is pumped into the filter. 🠶 The drum rotates under vaccum and a thin layer of cells sticks to the drum. 🠶 The thi c k n ess of the laye r in c r e as e s i n the sectio n d e signed for forming the cakes.

8. Points to be considered while selecting the filter medium :- 🠶 Abi l it y to bui l d the soi l d. 🠶 Min i mu m r e sistanc e to flow the filtrate. 🠶 R e sistanc e to c h emi c al at t ac k . 🠶 Min i m u m c o st. 🠶 Lo n g l i f e .

9. b. Centrifugation :- 🠶 Centrifugation is used to separate particles of 100 – 0.1 micrometer from liquid by gravitational forces. 🠶 It depends on particles size , density difference between the cells and the broth and viscosity of broth. 🠶 Use of the centrifugal force for the seperation of mixtures. 🠶 More dense components migrate away from the axis of centrifuge . 🠶 Less dense components migrate towards the axis . Types of centrifuges used are 🠶 Tubular bowl centrifuge 🠶 Multichamber centrifuge 🠶 Disc bowl centrifuge etc………….

10. c. Sedimentation :- 🠶 It is applicable only for large particles greater than 100 micrometer flocs. 🠶 I t i s slo w p r o c ess and t akes ~ 3hrs. 🠶 It is used in process like activated sludge effluent treatment. 🠶 I t i s free set t l i ng p r o c ess dep e nds o n l y on gra v ity. 🠶 Particles settling is a high particle density suspension (hindered settling ). 🠶 Mostly applicable in every fermentors industries which are large scale or small scale……

11. d. Flocculation :- 🠶 Proces s whe r e a sol u te co m es out of soluti o n i n the form of flocs or flakes. 🠶 Par t i c le s finer than 0. 1 m i crometer i n wa t er r e main continoulsy in motion due to electrostatic charge which cause them to repel each other. 🠶 Once their electros t atic c h a r ge i n neut r ali z ed ( use o f coagulant ) the finer particles start to collide and combine together. 🠶 These larger and heavier flocs of particels are called flocs.

12. e. Gravity settings It is apllicable only for large particles greater than 100 micrometer flocs. It is a slow process and takes ~3 hours. It is used in process like activated sludge effluent treatment. It’s a free settling process depends only on gravity. Particles settling is a high particle density suspension(hindered settling).

13. II. Product Isolation Removal of those components whose properties vary markedly from that of the desired product. Water is the chief impurity Isolation steps are designed to remove it (i.e.dialysis) Reducing the volume Concentrating the product. Liquid –liquid extraction, adsorption, ultrafiltration, and precipitation are some of the unit operations involved.

14. Liquid -Liquid extraction It is a separation process that takes the advantage of the relative solubilities of solute in immiscible solvents. Solute is dissolved more readily and becomes more concentrated in the solvent in which it has a higher solubility. A partial separation occurs when a number of solutes have different relative solubilities in the two solvents used. Solvent should be non toxic, selective, inexpensive and immiscible with broth and should have a high distribution coefficent for the product.

15. Liquid–liquid extraction ( LLE ) :- 🠶 Liquid–liquid extraction ( LLE ), also known as solvent extraction and partitioning , is a method to separate compounds or metal complexes, based on their relative solubilities in two different immiscible liquids, usually water (polar) and an organic solvent (non-polar). 🠶 There is a net transfer of one or more species from one liquid into another liquid phase, generally from aqueous to organic. 🠶 The t r ans f er i s d r ive n b y chemi c al pot e ntial, i . e. on c e the transfer is complete, the overall system of protons and electrons that make up the solutes and the solvents are in a more stable configuration (lower free energy). The solvent that is enriched in solute(s) is called extract. 🠶 The f e ed solut i on that i s de p let ed i n solute(s ) i s ca l led the raffinate.

16. Ads o r p tion is a surface phenomenon It is the binding of molecules to the surface and different from absorption. The binding to the surface is weak and reversible. Compounds containing chromogenic group are usually strongly adsorbed on activated carbon. Common adsorbent used are activated carbon,silica gel,alumina becoz they present enormous surface areas per unit weight.

17. Ultrafiltration UF is basically a pressure-driven separation process. The operating pressure is usually between 0.1 and 1 MPa.

18. P r ecipi t a tion 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.

19. P r ecipi t a tion Salts such as ammonium & sodium sulphate are used for proteins to precipitate. Organic solvents methanol used to precipitate dextrans. Chilled ethanol and acetone used for protein precipitation. Non ionic polymer such as polyethylene glycol used in precipitation.

20. lll . Product Purification Done to separate those contaminants that resemble the product very closely in physical and chemical properties. Expensive to carry out Require sensitive and sophisticated equipment Significant fraction of the entire downstream processing expenditure. Examples of operations include affinity, size exclusion, reversed phase chromatography,crystallization and fractional precipitation.

21. Product purification :- Purification by 🠶 Ge l filtrat i o n 🠶 Io n exc h ange 🠶 Af f in i ty 🠶 Hyd r ophobic inter act i ons.

22. Chromatography Separation of mixtures Passing a mixture dissolved in a "mobile phase" through a stationary phase, which separates the analyte to be measured from other molecules in the mixture and allows it to be isolated.

23. Ion Exchange Chromatography Used charged stationary phase to separate charged compounds Resin that carries charged functional groups which interact with oppositely charged groups of thecompound to be retained. FPLC

24. Affinity chromatography: 🠶 This i s an elegant m ethod for the p urificat i on of p r oteins from a c o mplex mixture. 🠶 Af f in i ty c h romatog r a p hy i s b a se d on an inter act i on of a p r otein with an immobilized ligand. 🠶 The ligand can be a specific antibody, substrate, substrate analogue or an inhibitor. The immobilized ligand on a solid matrix can be effectively used to fish out complementary structures. 🠶 The p r otein bound to the l i gand can be eluted b y r edu c in g their interaction. 🠶 This can be ach i eved b y changi n g the pH o f the buf f e r , altering the i on i c strength or by using another free ligand molecule. 🠶 The fresh ligand used has to be removed in the subsequent steps

25. Definition: Ion Ion is an atom or molecule which has lost or gained one or more valence electrons, giving it a positive or negative electrical charge. Anions are negatively charged ions, formed when an atom gains electrons in a reaction. Anions are negatively charged because there are more electrons associated with them than there are protons in their nuclei. Cations are positively charged ions, formed when an atom loses electrons in a reaction, forming an 'electron hole'.

26. Affinity chromatography Affinity chromatography separates the protein of interest on the basis of a reversible interaction between it and its antibody coupled to a chromatography bead (here labeled antigen) . With high selectivity, high resolution, and high capacity for the protein of interest, purification levels in the order of several thousand-fold are achievable. The protein of interest is collected in a purified, concentrated form. Biological interactions between the antigen and the protein of interest can result from electrostatic interactions, van der Waals' forces and/or hydrogen bonding. To elute the protein of interest from the affinity beads, the interaction can be reversed by changing the pH or ionic strength. The concentrating effect enables large volumes to be processed. The protein of interest can be purified from high levels of contaminating substances. Making antibodies to the protein of interest is expensive, so affinity chromatography is the least economical choice for production chromatography.

27. Size exclusion chromatography Gel permeation/filtration chromatography (GPC) Separates molecules according to their size Low resolution"polishing" Tertiary/Quaternary structure(native)

28. Reversed phase chromatography Reversed-phase chromatography is an elution procedure used in liquid chromatography in which the mobile phase is significantly more polar than the stationary phase.

29. Definitions: Polarity The dipole-dipole intermolecular forces between the slightly positively-charged end of one molecule to the negative end of another or the same molecule. Molecular polarity is dependent on the difference in electronegativity between atoms in a compound and the asymmetry of the compound's structure.

30. Liquid Chromatography Mobile phase is a liquid. Carried out either in a column or a plane. HPLC In the HPLC technique, the sample is forced through a column that is packed with irregularly or spherically shaped particles or a porous monolithic layer (stationary phase) by a liquid (mobile phase) at high pressure.

31. HPLC Configuration

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

33. Cell disruption :- 🠶 Some product are intracellular including many enzymes and proteins required to disrupt the cell amd rrlease these products. E.g :- yeast 🠶 Cell disruption can be achieved by both mechanical and non mechanical methods. Mechanical :- 🠶 Sonication and liquid shear homogenization etc….. Non mechanical methods :- 🠶 Autolysis and osmosis shock etc…….

34. Solvent extraction :- 🠶 Reducing the volume of material to be handled and concentrating the product. 🠶 R e mova l of those co m ponents whose p r ope r ties va r y markedly from that of desired product … 🠶 Wat e r I s the Ch e i f impurit y . 🠶 L i quid l i quid ext r acti o n 🠶 Adsorp t i o n 🠶 Pr e c i pita t i o n 🠶 Are some of the unit operations involved in them….

35. Adsorption 🠶 A d s orp t i o n is the ad h esio n o f atoms, i o n s o r molecules f r om a gas, liquid or dissolved solid to a surface. 🠶 Thi s p r ocess c r eate s a film o f the adsorbate on the s u r fa c e o f the adsorbent. This process differs from absorption, in which a fluid (the absorbate) is dissolved by or permeates a liquid or solid (the absorbent), respectively. 🠶 Adsorption is a surface phenomenon, while absorption involves the whole volume of the ma terial. The term sorption encompasses both processes, while desorption is the reverse of it. 🠶 T he separatio n i s based on the interact i on o f the adsorbate w i th the adsorbent. The adsorbent is the surface and adsorbate is the molecules of interest which are getting adsorbed on the adsorbent.

36. Ultrafiltration 🠶 U ltr af i ltr at i on (UF) is a p r ess u r e -dr i ve n barr i e r t o s u spende d sol i ds, bacteria, viruses, endotoxins and other pathogens to produce water with very high purity and low silt density. 🠶 Ultrafiltration (UF) is a variety of membrane filtration in which hydrostatic pressure forces a liquid against a semi permeable membrane. 🠶 Th e variet y o f me m bran e fil t rati o n in which fo r ces like pressure or concentration gradients lead to a separation through a semipermeable membrane. 🠶 Suspended solids and solutes of high molecular weight are retained in them called retentate, while water and low molecular weight solutes pass through the membrane in the permeate (filtrate). 🠶 This separation process is used in industry and research for purifying and concentrating macromolecular (10 3 - 10 6 Da) solutions, especially protein solutions.

37. Precipitation :- 🠶 Precipitation is the most commonly used technique in industry for the concentration of macromolecules such as proteins and polysaccharides. 🠶 Further, precipitation technique can also be employed for the removal of certain unwanted byproducts e.g. nucleic acids, pigments. 🠶 Neutral salts, organic solvents, high molecular weight polymers (ionic or non-ionic), besides alteration in temperature and pH are used in precipitation. 🠶 In addition to these non-specific protein precipitation reactions (i.e. the nature of the protein is unimportant), there are some protein specific precipitations e.g., affinity precipitation, ligand precipitation. 🠶 The most commonly used salt is ammonium sulfate, since it is highly soluble, nontoxic to proteins and low-priced. 🠶 Ammonium sulfate increases hydrophobic interactions between protein molecules that result in their precipitation. 🠶 The precipitation of proteins is dependent on several factors such as protein concentration, pH and temperature.

38. Gel-filtration chromatography: 🠶 This is also referred to as size-exclusion chromatography. In this technique, the separation of molecules is based on the size, shape and molecular weight. 🠶 The sponge-like gel beads with pores serve as molecular sieves for separation of smaller and bigger molecules. 🠶 A solution mixture containing molecules of different sizes (e.g. different proteins) is applied to the column and eluted. 🠶 The smaller molecules enter the gel beads through their pores and get trapped. 🠶 On the other hand, the larger molecules cannot pass through the pores and therefore come out first with the mobile liquid (Fig. 20.7). 🠶 At the industrial scale, gel-filtration is particularly useful to remove salts and low molecular weight compounds from high molecular weight products.

39. Ion-exchange chromatography: 🠶 It involves the separation of molecules based on their surface charges. 🠶 Ion-exchangers are of two types (cation- exchangers which have negatively charged groups like carboxymethyl and sulfonate, and anion- exchangers with positively charged groups like diethylaminoethyl (DEAE). 🠶 The most commonly used cation-exchangers are Dowex HCR and Amberlite IR, the anion-exchangers are Dowex SAR and Amberlite IRA. 🠶 In ion-exchange chromatography, the pH of the medium is very crucial, since the net charge varies with pH. 🠶 I n other word s , the pH d e te r m i nes the e f fect i ve c h arge on both the target molecule and the ion-exchanger. 🠶 The i o nic bound mo l ecu l es can be eluted f r om the ma trix b y changi n g the pH of the eluant or by increasing the concentration of salt solution. 🠶 Ion-exchange chromatography is useful for the purification of antibiotics, besides the purification of proteins.

40. Product formulations :- 🠶 Formulation broadly refers to the maintenance of activity and stability of a biotechnological products during storage and distribution. 🠶 The formulation of low molecular weight products (solvents, organic acids) can be achieved by concentrating them with removal of most of the water. 🠶 For certain small molecules, (antibiotics, citric acid), formulation can be done by crystallization by adding salts. 🠶 Proteins are highly susceptible for loss of biological activity; hence their formulation requires special care. 🠶 Certain stabilizing additives are added to prolong the shelf life of protein. The stabilizers of protein formulation include sugars (sucrose, lactose), salts (sodium chloride, ammonium sulfate), polymers (polyethylene glycol) and polyhydric alcohols (glycerol). 🠶 Proteins may be formulated in the form of solutions, suspensions or dry powders.

41. lV. Product Polishing End with packaging of the product in a form that is stable, easily transportable and convenient. Crystallization Desiccation Lyophilization Spray drying May include: Sterilization of the product Remove or deactivate trace contaminants which might compromise product safety viruses or depyrogenation

42. lyophilization freezing the material reducing the surrounding pressure and adding enough heat to allow the frozen water in the material to sublime directly from the solid phase to gas.

43. V. Product packaging :- 🠶 Drying 🠶 F r e e z i ng 🠶 Spac e d rying

44. Thanking you ……