FERMENTATION-DIFFERENT STAGES

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THE SLIDE INCLUDES DIFFERENT STAGES INVOLVED IN FERMENTATION PROCESS.............HAVE A LOOK...

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FERMENTATION (Different Stages Of Operation) :

FERMENTATION (Different Stages Of Operation) Do what you can, with what you have, where you are. Theodore Roosevelt 1 JEEJO N.U M.PHARM PHARMACEUTICS jeejonu9@gmail.com

DISCUSSION ON::

DISCUSSION ON: MAINTENANCE OF CULTURE INOCULUM PRODUCTION DOWNSTREAM PROCESSING FERMENTATION SCALE-UP 2

MAINTENANCE OF CULTURE:

MAINTENANCE OF CULTURE To maintain the physiological properties of high yielding strains. Agar Slope Under Paraffin Deep Freezing Lyophilization 3

INOCULUM PRODUCTION:

INOCULUM PRODUCTION Most critical stage in any fermentation process. The size of the inoculum is between 5-10% of fermentation process. In each stage rigorous control is necessary to avoid both mutation and contamination. 4

DOWNSTREAM PROCESSING-MULTISTAGE OPERATION :

DOWNSTREAM PROCESSING-MULTISTAGE OPERATION ‘The isolation and purification of a biotechnological product to a form suitable for its intended use’. Depends on the nature of end product, its concentration, stability and degree of purification required. Product recovery yield is higher if the number of steps in DSP is lower. 5

STAGES IN DSP:

STAGES IN DSP Solid-liquid separation/clarification Release of intracellular components Extraction Concentration Purification Drying Formulation. 6

(I) SOLID-LIQUID SEPARATION/CLARIFICATION:

(I) SOLID-LIQUID SEPARATION/CLARIFICATION FLOTATION FLOCCULATION FILTRATION CENTRIFUGATION 7

FLOTATION::

FLOTATION: Particles are adsorbed on gas bubbles, get trapped in a foam layer & collected. 8

FLOCCULATION::

FLOCCULATION: The cells form large aggregates to settle down for easy removal. Flocculating agents : Inorganic salts, Organic polyelectrolyte's, Mineral hydrocolloid’s. 9

FILTRATION:

FILTRATION For separating the biomass and culture filtrate. Efficiency of filtration depends on: Size of organism Presence of other organisms Viscosity of the medium Temperature 10

FILTRATION EQUIPMENTS::

FILTRATION EQUIPMENTS: ROTARY DRUM VACUUM FILTER 11

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12 FILTER PRESS

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MEMBRANE FILTER PRESS 13

CENTRIFUGATION: :

CENTRIFUGATION: Removal of microbial cells and other discrete large particles. Batch centrifugation Continuous flow centrifugation Separation of particulate debris is inefficient by centrifugation. 14

CENTRIFUGES: :

CENTRIFUGES: TUBULAR BOWL TYPE CENTRIFUGE 15

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MULTICHAMBER BOWL TYPE CENTRIFUGE 16

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DISC STACK CENTRIFUGE 17

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THE DECANTER OR SCROLL CENTRIFUGE 18

PROPERTIES OF INDUSTRIAL CENTRIFUGES:

PROPERTIES OF INDUSTRIAL CENTRIFUGES Tube High centrifugal force Good dewatering Easy to clean Chamber Large solids capacity Good dewatering Bowl cooling possible Disc type Solids discharge No foaming Bowl cooling possible Limited solids capacity Foams Difficult to recover protein No solids discharge Cleaning difficult Solids recovery difficult Poor dewatering Difficult to clean

Centrifugation properties of different cell types:

Centrifugation properties of different cell types Bacteria Small cell size Resilient Yeast cells Large cells Resilient Filamentous fungi Mycelial Resilient Cultured animal cells Large cells Very fragile High speed required Low cell damage Lower speed required Low cell damage Lower speed required High water retention in pellet Very susceptible to damage

(II) RELEASE OF INTRACELLULAR COMPONENTS :

21 (II) RELEASE OF INTRACELLULAR COMPONENTS PHYSICAL METHODS CHEMICAL METHODS ENZYMATIC METHODS Ultrasonication Alkalies Lysozyme Osmotic shock Organic solvents Glucanase , mannanase & protease. Thermolysis Detergents High-pressure homogenization Impingement Grinding with glass beads CELL DISRUPTION

(III)EXTRACTION::

(III) EXTRACTION: The process of removing a compound or a group of compounds from a mixture or from cells into a solvent phase is called extraction. Liquid – liquid extraction Whole broth (medium + cells) extraction Aqueous multiphase extraction 22

(IV)CONCENTRATION:

(IV) CONCENTRATION Evaporation Liquid-liquid extraction Membrane filtration Precipitation Adsorption 23

EVAPORATORS:

24 EVAPORATORS PLATE EVAPORATOR

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25 FALLING FILM EVAPORATOR

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26 THIN FILM EVAPORATOR

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27 CENTRIFUGAL TYPE THIN-FILM EVAPORATOR

LIQUID-LIQUID EXTRACTION:

LIQUID-LIQUID EXTRACTION “ Transferring the solute from one liquid phase to another liquid phase” Extraction of low MW products. Extraction of high MW products. 28

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29 EXTRACTION OF LOW MW PRODUCTS. EXTRACTION OF HIGH MW PRODUCTS. PHYSICAL EXTRACTION AQUEOUS TWO-PHASE SYSTEMS (ATPS) DISSOCIATIVE EXTRACTION REVERSE MICELLER SYSTEMS REACTIVE EXTRACTION SUPERCRITICAL FLUID EXTRACTION (SCF)

MEMBRANE FILTRATION:

MEMBRANE FILTRATION Ion exchange resins Adsorption resins-styrene, sulfoxide,acrylic ester. Pervaporation - membrane filtration coupled with evaporation. Perstraction - membrane filtration coupled with solvent extraction. 30

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TYPE PARTICLE SIZE COMPOUND OR PARTICLE SEPARATED MICROFILTRATION 0.1 – 10 μ m Cells or cell fractions, Viruses. ULTRAFILTRATION 0.001- 0.1 μ m Compounds with MW > 1000 (e.g. enzymes). HYPERFILTRATION 0.0001- 0.001 μ m Compounds with MW < 1000 (e.g. lactose). Major Types Of Filtration Processes 31

PRECIPITATION:

PRECIPITATION Separation technique used for concentration of proteins and polysaccharides. Constitutes the only unit operation in the recovery of bulk proteins. 32

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33 MODE EXAMPLE Addition of neutral salts (NH 4 ) 2 SO 4 Addition of organic solvents Ethanol, acetone , Propanol Addition of non-ionic polymer PEG Addition of charged polymer Polyacrylic acid , Polyethyleneimine Increase in temperature change in pH MODES OF PROTEIN PRECIPITATION

ADSORPTION:

ADSORPTION Activated charcoal. Cellulose based adsorbents- for protein precipitation. Polystyrene,methacrylate and acrylate based adsorbents- for low molecular weight compounds . 34

(V) PURIFICATION:

(V) PURIFICATION CRYSTALLIZATION CHROMATOGRAPHIC METHODS 1. Adsorption chromatography 2.Ion exchange chromatography 3.Hydrophobic interaction chromatography 4. Affinity chromatography 5.Size exclusion chromatography 6.Radial flow chromatography 7.Perfusion chromatography 8.HPLC 35

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36 Chromatography Separation principle Size-exclusion (gel filtration) Size & charge Ion-exchange chromatography Net charge Chromatofocusing Net charge Hydrophobic interaction chromatography Hydrophobicity Affinity chromatography Molecular recognition Immobilized metal-ion affinity chromatography Metal ion binding Covalent chromatography Content of free –SH groups. VARIOUS CHROMATOGRAPHIC TECHNIQUES FOR PROTEIN SEPARATION

CHROMATOGRAPHY SYSTEM:

CHROMATOGRAPHY SYSTEM Lab Scale Chromatography System Large Scale Chromatography System

(VI) DRYING::

(VI) DRYING: Makes the products suitable for handling and storage. (i) Vacuum drying (ii) Freeze drying (iii) Spray drying 38

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39 SOME OF THE FREQUENTLY USED DRYING EQUIPMENTS Type of dryer Mode of heat transfer Movement of the product Belt dryer Convection Intensive due to gas flow Fluidized bed dryer Convection Intensive due to gas flow Spray- dryer Convection Intensive due to gas flow Freeze- dryer Contact and radiation None or mechanical Drum- dryer Contact Slight mechanical Chamber dryer Convection and contact None

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40 FREEZE-DRYER SPRAY-DRYER VACUUM -DRYER

(VII) FORMULATION:

(VII) FORMULATION The commercial viability of a biotechnological product is dependent on the maintenance of its activity and stability during distribution and storage. Stabilizing agents are included in the formulations to prolong the product shelf life . 41

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Media Prep Working Cell Bank Sub- Culture Inoculum Sub- Culture Sub- Culture Sub- Culture Sub- Culture Large Scale Bioreactor Wave Bag Seed Bioreactors Fermentation 150L Bioreactor 750L Bioreactor 5,000L Bioreactor 26,000L Bioreactor Depth Filtration Collection Centrifuge Harvest/Recovery Harvest Collection Tank 1,500L Filter Chromatography Skid Anion Exchange Chromatography (QXL) Column Eluate Hold Tank 8,000L Eluate Hold Tank 6,000L Filter Chromatography Skid Protein A Chromatography Column Chromatography Skid Column Eluate Hold Tank 20,000L Hydrophobic Interaction Chromatography (HIC) Eluate Hold Tank 20,000L Viral Inactivation Eluate Hold Tank 5,000L Filter Chromatography Skid Anion Exchange Chromatography (QFF - Fast Flow) Column Post-viral Hold Vessel 3,000L Viral Filtering Ultra Filtration Diafiltration Bulk Fill Purification 24 days 31 days 8 days 1 day Upstream Downstream Process Overview

FERMENTATION SCALE-UP:

FERMENTATION SCALE-UP ‘ The phenomenon of developing industrial fermentation process in stages ’. To develop optimal environmental and operating conditions at different levels. 43

REFERENCES:

REFERENCES Colin Ratledge & Bjorn Kristiansen, Basic Biotechnology, Cambridge University Press, 151-211. Vinita Kale & Kishore Busari, Applied Microbiology, Himalaya Publishing House, First edition-2001, 163-202,303-330. Biotechnology by B D Singh, Kalyani publishers, First edition- 1998. Giriraj Kulkarni T, Biotechnology And its applications in pharmacy, jaypee publications, first edition 2002; page no:73-99. S S Kori & M A Halki, Pharmaceutical Biotechnology-Fundamentals and applications, page no: 135-156. Dr. U. Satyanarayana, Biotechnology, First edition-2005, Books and Allied pvt.ltd, page no:268-280. 44

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