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Protein Bioseparation - Classification 1. High-productivity, low resolution 2. High resolution, low productivity 3. High resolution, high productivity Downstream Processing Profile ____________________________________________ Product ---------------------------------------Step concentration; g/L Quality, % ___________________________________________________________ Harvest broth 0.1 – 5 0.1 – 1.0 0.1 – 5 0.1 – 2.0 Primary isolation (Step 2) 5.0 – 10 1.0 – 10 Purification (Step 3) 50 - 200 50 – 80 (30 – 90) Filtration/centrifugation (Step 1) Formulation/drying etc (Step 4) 90 - 100 ____________________________________________________________ Removal Removal/separation of solids/particles/cells Cell harvesting in microbial fermentation Cell removal in animal cell culture Methods Settling/sedimentation used in large- scale waste treatment processes and traditional fermentation industry. The supernatant may still have some solid contents. Centrifugation produces a cell-concentrated stream- referred as cell cream with fluid behaviour. May have about 15% w/v solid content. Filtration produces more concentrated (dewatered) cell sludges. May have upto 40% w/v solid content. The accumulated biomass filter cake may provide filtration resistance. However, improved by new technologies. Selection of the method depends on starting broth, final desired cell density and scale of operation. RIPP •Removal Filtration Centrifugation Factors affecting separation/removal Broth Characteristics high viscosities, gelatinous broth materials, compressible filter cakes, particles with small density difference compared to water, high degree of initial dispersion, and diluteness of particulate suspension. Source and bioreactor process bacterial, viral, fungal, plant , animal etc., batch or continuous process. Separation improvements Broth pretreatments designed to increase ease of cell separation include: - cell aging, induce clumping of cells together - heat treatment - pH treatment - addition of chemicals to enhance flocculation like calcium chloride, clay, silica - addition of polymers like polyelectrolytes Filtration methods in Bioseparation 1. Direct filtration or dead-end filtration 2. Tangential flow filtration or cross-flow filtartion Factors affecting Vs. particle size separation Size Cloth and fiber filters Microfilters Screens and strains Ultrafiltration Gel chromatography Ultracentrifuges Density Centrifuges Gravity sedimentation Angstroms (Aº) 1 Millicrons (nm) 10-1 Microns (µm) 10-4 10 1 10-3 Ionic range 102 10 10-2 103 102 10-1 Macromolecular range 104 103 1 105 104 10 Micron range 106 105 102 Fine range 107 106 103 Course range Microfiltration Microfiltration is a way of removing contaminants in the size range of 0.1 to 10.0 µm from fluids or gases, by passage through a microporous medium such as a membrane. • Microfiltartion covers both: dead-end filtration and cross-flow filtration. • Microfiltration is used in both production and analytical applications, such as - Filtration of particles from liquid or gas streams for different industries, e.g.chemical or pharmaceutical - Production of pure water - Clarification and sterile filtration - Waste water treatment - Fermentations for bioseparations Dead end and cross-flow filtration Dead-end filtration: In the dead-end filtration technique all the fluid passes through the membrane, and all particles larger than the pore size of the membrane are retained at its surface. This means that the trapped particles start to build up a "filter cake" on the surface of the membrane, which has an impact on the efficiency of the filtration process. Cross-flow filtration: In cross flow filtration, a fluid (feed) stream runs tangential to a membrane, establishing a pressure differential across the membrane. This causes some of the particles to pass through the membrane. Remaining particles continue to flow across the membrane, "cleaning it". In contrast to the dead -end filtration technique, the use of a tangential flow will prevent thicker particles from building up a "filter cake". Dead-end Filtration All the fluid passes through the membrane and all particles larger than the pore sizes of the membrane are stopped at its surface. Their size prevents them from entering and passing through the filter medium. This means that the trapped particles start to build up a "filter cake" on the surface of the medium, which reduces the efficiency of the filtration process. Classical dead end filtration Feed stock Filter cake Filtrate •Cake formation limits filtrate flow •Only for big suspended material Filtering improvements • Filtering aids • pH • Temperature • Duration of fermentation Cross-flow filtration • The fluid (feed) stream runs tangential to the membrane, establishing a pressure differential across the membrane. • This causes some of the particles to pass through the membrane. Remaining particles continue to flow across the membrane, "cleaning it". • The use of a tangential flow will prevent thicker particles from building up a "filter cake". Cross-flow filtration Retentate Feed Permeate •No cake formation •Feed to retentate flow > > permeate flow Cross-flow filtration separates substances by passing them through semi-permeable membranes in hollow fibre or flat sheet formats. 1. Protein solutes are separated from insoluble material such as cells, insoluble particles, etc. 2. Can be used for micro-, ultra- filtration depending on the size of molecules to be separated. 3. Used for concentration or change of solvent 4. Also used for removal of viruses from biological solutions Water Proteins Cells • One stream in- two streams out technology • Tangential flow parallel to membrane for cell removal Cross-flow filtration Pressure Dead-end filtration Pressure Cross-flow Permeate Filtrate Direct Flow Filtration Process Tangential Flow Filtration Process Why use cross-flow filtration? •Easy to set up and use •No cake formation •Fast and efficient •Concentrate and diafilter in the same system •No scaling limitations- 10 ml to 1000 litres. •Economical, Reusable Applications of Tangential/Cross- Flow Filtration •Cell harvesting •Recover and removal of viruses •Clarify cell lysates or tissue homogenates •Recover and purify plasmid DNA from cell lysates or chromosomal DNA from whole blood •Recover antibodies or recombinant proteins from cell culture media •Concentrate and desalt, proteins, peptides, nucleic acids Basic Cross-flow filtration process (batch) Pressure control value Feed Retentate Concentrate Feed CFF pump Permeate Basic Cross-flow filtration process (continuous) Feed Concentrate CFF Feedpump Feed Permeate Retentate Microfiltration - Membranes 1845 1855 1962 1963 1963 1963 1964 1970 1970 1975 1979 1980 1981 1984 2000 Schoenbein Nitrocellulose Fick Esters of cellulose-nitrocellulose Gelman Instrument Co. Cellulose tri-acetate Sartorius Co. Regenerated cellulose Millipore, Gelman, Sartorius, S&S Polyvinyl chloride and polyamide General Electric Polycarbonate Selas Flotronics Silver membrane Celanese Co. Polypropylene Gore Corp. Polytetrafluoroethylene MembranelEnka Polypropylene Gelman Polysulfone Millipore Polyvinylidene fluoride Nuclepore Polyester Norton Co., CeraverAlumina Our research Cryomembranes/acrylamide/PVA