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Capillary- pore and Tortuous- pore membranes • Capillary pore- straight through cylindrical capillaries • Tortuous pore-sponge with network of interconnecting tortuous pores Pore-size measurement techniques 1. Bubble –point method 2. Scanning elctron microscopy 3. Mercury porisimetry Organisms retained in various pore sizes Polytetra fluoroethylene (PTFE) membrane Polypropylene membrane CRYOGELS Doublet and triplet pores in capillary pore membranes Pore size distribution in 0.45 µm membranes (by mercury porisimetry) Plate (Sheet) and frame format membranes Hollofibre – Tubular format membranes Enhanced Cross-Flow Filtration Systems Hollow-fibre filtration system Manual benchtop tangential flow filtration (TFF) system. Typical Applications - Concentration and filtration - Desalting and buffer exchange - Cell harvesting/clarification - Virus harvesting/clarification Pellicon XL 50 Ultrafiltration Device Applications - Concentration, desalting, and buffer exchange of proteins, polysaccharides, lipid solutions, viruses, colloids, cell suspensions, and mammalian cells - Sample preparation - Preparation of material for clinical trials - Small volume manufacturing Labscale TFF System Centrifugation A centrifuge is a device for separating particles from a solution according to their size, shape, density, viscosity of the medium and rotor speed. In biology, the particles are usually cells, sub cellular organelles, viruses, large molecules such as proteins and nucleic acids. Analytical centrifugation Analytical centrifugation involves measuring the physical properties of the sedimenting particles such as sedimentation coefficient or molecular weight. Preparative centrifugation In preparative centrifugation objective is to isolate specific particles which can be reused. There are many type of preparative centrifugation such as rate zonal, differential, isopycnic centrifugation. Centrifugation classification based on speed Another system of classification is the rate or speed at which the centrifuge is turning. Ultracentrifugation is carried out at speed faster than 20,000 rpm. Super speed centrifugation is at speeds between 10,000 and 20,000 rpm. Low speed centrifugation is at speed below 10,000 rpm. Differential centrifugation A third method of defining centrifugation is by the way the samples are applied to the centrifuge tube. In moving boundary (or differential centrifugation), the entire tube is filled with sample and centrifuged. Through centrifugation, one obtains a separation of two particles but any particle in the mixture may end up in the supernatant or in the pellet or it may be distributed in both fractions, depending upon its size, shape, density, and conditions of centrifugation. Denisty gradient centrifugation It allows separation of many components in a mixture by creating density gradient during centrifugation There are two forms of density gradient centrifugation: rate zonal and isopycnic Rate Zonal Centrifugation (also termed sedimentation velocity, zone centrifugation) In rate zonal centrifugation, the sample is applied in a thin zone at the top of the centrifuge tube on a density gradient. Under centrifugal force, the particles will begin sedimenting through the gradient in separate zones according to their size shape and density. The run must be terminated before any of the separated particles reach the bottom of the tube. Swing bucket rotor Isopycnic centrifugation (also termed sedimentation equilibrium centrifugation) During the centrifugation, the CsCl generates a gradient (“self-generating gradient”), and the molecules move to the position in the gadient where their density is the same as the gradient material. Isopycnic means “same density,” so the molecules move to their “isopycnic position.” Fixed angle or swing bucket rotor Gradients Sucrose Cscl2 Glycerol Dextran Centrifugation The performance of a centrifuge is characterised by: Vc = dp2 . (ds - dl) . W2 r / 18 n where Vc = centrifugal sedimentation rate (gs-1), dp = particle diameter, ds = density of solid, dl = density of liquid, w= angular speed, r = distance of the particle to the axis of rotation and n = viscosity of medium. Major forces acting on solid particle during settlingGravitational force (FG) Drag force (FD) Buoyant force (FB) When the particles reach a terminal settling velocity, forces acting on a particle balance each other, resulting in zero net force. That is FG = FD + FB Nomogram for converting maximum relative centrifugal force (RCF, i.e., g-force) to RPM RCF to RPM: Determine centrifuge 's radius of rotation (in mm) by measuring distance from center of centrifuge spindle to bottom of device when inserted into rotor. Lay a ruler or draw a line from radius value in right-hand column value that corresponds to the device 's maximum rated g-force. Then read the maximum value from column at left. R I P P Isolation/Extraction Release of protein from biological host • To gain access to the product • Access to the product is simple and inexpensive when the protein is produced extracellularly • Microbial sources are preferred • Mammlian cell hosts are preferred when posttranslational modification is essential for the function of eukaryotic proteins • Bulk enzymes are invariably produced extracellularly by Bacillus species & fungi, as are the proteins produced by mammalian cell culture Cell envelops of bacteria and yeast Lipopolysaccharide membrane Mannan partially crosslinked by phosphodiester bridges Peptidoglycon layer Glucan layer with proteins Cytoplasmic membrane Gram-positive bacteria Gram-negative bacteria Animal cells: no cell wall, thus fragile in breaking Plant cells: composed of cellulose and other polysaccharides Yeasts Cell Disintegration Techniques Technique Example Principle Gentle Cell lysis Erythrocytes Enzyme digestion Lysozyme treatment of bacteria Chemical solubilization/ Toluene extraction autolysis of yeast Hand homogenizer Liver tissue Minicing (grinding) Muscle etc. Osmotic disruption of cell membrane Cell wall digested, leading to osmotic disruption Cell wall (membrane) partially solubilized chemically; lytic enzymes released complete the process Cells forced through narrow gap, disrupts cell membrane Cells disrupted during minicing process by shear force Cell Disintegration Techniques Technique Example Moderate Blade homogenizer (waring type) Muscle tissue, most animal tissues, plant tissues Grinding with abrasive Plant tissues, bacteria (sand, alumina) Vigorous French press Bacteria, plant cells Ultrasonication Cell suspensions Principle Chopping action breaks up large cells, shears apart smaller ones Microroughness rips off cell walls Cells forced through small orfice at very high pressure; shear forces disrupt cells Micro-scale high-pressure sound waves cause disruption by shear forces and cavitation Cell Disintegration Techniques Technique Bead mill Manton-Gaulin homogenizer Example Cell suspension Cell suspension Principle Rapid vibration with glass beads rips cell walls off As for French press, but on a larger scale Mechanical Methods • Mechanical methods can be applied to a liquid or solid medium • Most common mode, despite higher capital and operating costs • Disruption is based primarily on liquid or solid shear forces • Liquid shear cell disruption is associated with cavitation phenomenon that involves formation of vapor cavities in liquid due to local reduction in pressure that could be affected by ultrasonic vibrations, local increase in velocity, etc. Collapse and rebound of the cavities will occur until an incresae in pressure causes their destruction • On the collapse of the cavitation bubble, a large amount of energy is released as mechanical energy in the form of elastic waves that disintegarte into eddies which impart motions of diferent intensities to the cell, creating pressure difference across the cell. When the kinetic energy content of the cell exceeds the cell wall strength, the cell disintegrates. Mechanical Methods Ultrasonic vibrators (sonicators) are used to disrupt the cell wall and membrane of bacterial cells. An electronic generator is used to generate ultrasonic waves, and a transducer converts these waves into mechanical oscillations by a titanium probe immersed in a cell suspension. Wave density is usually around 20 kc/s. • Rods are broken more readily than cocci, and gram negative cells more easily than gram positive cells • The technique is not used at industrial scale primarily because the ultrasonic energy absorbed into suspension ultimately apears as heat, and good temperature control is necessary • In some cases results in denaturation of sensitive enzymes and fragmentation of cell debries Mechanical Methods High-Pressure homogenization: French Press: The french press is a hollow cylinder in a stainless-steel block that is filled with cell paste and subjected to high pressure. The cylinder has a needle value at the base, and the cells disrupt as they are extended through the value to atmospheric pressure. The flow restriction in the value assembly drives up pressure (in the range of 50 and 120 MPa) • Disruption follows first-order process at a given pressure in a highpressure homogenizer. The extent of protein release is represented by Rm = kNP ln Rm – R Where Rm and R are the maximal amount of protein available for release and the protein amount released at a certain time, respectively (kg protein/kg cells), k is the first-order rate constant, N the number of passages, P the operating pressure. Mechanical Methods High-Pressure homogenization: Manton-Gaulin homogenizer: Traditional form of high-pressure homogenizer, works as french press but on a larger scale. Bead Mill Disruption: Stirring a cell suspension with glass beads is an effective method of disruption of organisms. The process is normally performed in a bead mill, such as Dyno-Mill The principle of operation is to pump the cell suspension through a horizontal grinding chamber filled with about 80% beads. Within the grinding chamber is a shaft with specially designed discs. When rotated at high speeds, high shearing and impact forces from millions of beads break cell walls. • • • • Can be used effectively at large scale Available in sizes upto 275 l and can process 2000 kg/h of a cell suspension or about 340 kg dw/h of yeast Can work with algae, bacteria and fungi Better temperature control Mechanical Methods Limitations • High risk of damage to the product • Heat denaturation a major problem • The release of proteases from cellular compartments can lead to enzymatic degradation of the product • Bead mill have comparatively long residance times, products released early may be damaged • Products released encounter an oxidizing environment, that can cause denaturation and aggregation Non-Mechanical Methods Physical Rupture of Microbial Cells Desiccation: by slow drying in air, drum drying, etc followed by extraction of the microbial powder Osmotic shock: Changes in the osmotic pressure of the medium may result in the release of certain enzymes, particularly periplasmic proteins in gram negative cells. Suspending a cell suspension in a solution with high salt concentration High temperature: Exposure to high temperature can be an effective approach to cell disruption but is limited to heat-stable products. Heating to 50 – 55 ºC disrupts outer membrane, releases periplasmic proteins. Heating at 90 ˚C for 10 min can be used for releasing cytoplasmic proteins Non-Mechanical Methods Physical Rupture of Microbial Cells Freeze-thawing: Rupture with ice crystals is commonly used method. By slowly freezing and then thawing a cell paste, the cell wall and membrane may be broken, releasing enzymes into the media Nebulization: In nebulization gas is blown over a surface of liquid through a neck. Because of the differential flow within the neck, the cells are sheared Decompression: When pressurized, the microbial cells are gradually penetrated and filled with gas. After saturation by the gas, the applied pressure is suddenly released when the absorbed gas rapidly expands within the cells leading to rupture Note: Methods produce low protein yields and require long process time