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 – – 1.0 Filtration/centrifugation (Step 1) 0.1 – – 2.0 Primary isolation (Step 2) 5.0 – – 10 Purification (Step 3) – 80 (30 – 90) Formulation/drying etc (Step 4) ____________________________________________________________

Removal Removal/separation of solids/particles/cells Cell harvesting in microbial fermentation Cell removal in animal cell culture

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. Methods

RIPP RemovalRemoval FiltrationCentrifugation

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

1.Direct filtration or dead-end filtration 2. Tangential flow filtration or cross-flow filtartion Filtration methods in Bioseparation

Factors affecting separation Size Vs. particle size Screens and strains Density Centrifuges Angstroms (Aº) Millicrons (nm) Microns (µm) Gravity sedimentation Ultracentrifuges Gel chromatography Ultrafiltration Microfilters Cloth and fiber filters Course range Fine range Micron range Macromolecular range Ionic range

Microfiltration 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 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: 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 and cross-flow filtration

ll the fluid passes through the membrane and all particles larger 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. Dead-end Filtration

Classical dead end filtration Feed stock Filtrate Filter cake Cake formation limits filtrate flow Only for big suspended material

Filtering improvements Filtering aids Filtering aids pH pH Temperature Temperature Duration of fermentation Duration of fermentation

The fluid (feed) stream runs tangential to the membrane, establishing The fluid (feed) stream runs tangential to the membrane, establishing a pressure differential across the membrane. a pressure differential across the membrane. This causes some of the particles to pass through the membrane. This causes some of the particles to pass through the membrane. Remaining particles continue to flow across the membrane, Remaining particles continue to flow across the membrane, "cleaning it". "cleaning it". The use of a tangential flow will prevent thicker particles from building The use of a tangential flow will prevent thicker particles from building up a "filter cake". up a "filter cake". Cross-flow filtration

Retentate Feed Permeate No cake formationNo cake formation Feed to retentate flow > > permeate flowFeed to retentate flow > > permeate flow

Cross-flow filtration 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

Permeate Filtrate Pressure Pressure Cross-flow Cross-flow filtration Dead-end filtration

Direct Flow Filtration Process Tangential Flow Filtration Process

Why use cross-flow filtration? Easy to set up and useEasy to set up and use No cake formationNo cake formation Fast and efficientFast and efficient Concentrate and diafilter in the same systemConcentrate and diafilter in the same system No scaling limitations- 10 ml to 1000 litres.No scaling limitations- 10 ml to 1000 litres. Economical, ReusableEconomical, Reusable

Applications of Tangential/Cross- Flow Filtration Cell harvestingCell harvesting Recover and removal of virusesRecover and removal of viruses Clarify cell lysates or tissue homogenatesClarify cell lysates or tissue homogenates Recover and purify plasmid DNA from cell lysates or chromosomalRecover and purify plasmid DNA from cell lysates or chromosomal DNA from whole blood DNA from whole blood Recover antibodies or recombinant proteins from cell cultureRecover antibodies or recombinant proteins from cell culture media media Concentrate and desalt, proteins, peptides, nucleic acidsConcentrate and desalt, proteins, peptides, nucleic acids

Basic Cross-flow filtration process (batch) CFF Retentate pump Feed Permeate Feed Concentrate Pressure control value

CFF Retentate Feed-pump FeedPermeate ConcentrateFeed Basic Cross-flow filtration process (continuous)

1845SchoenbeinNitrocellulose 1855 FickEsters of cellulose-nitrocellulose 1962 Gelman Instrument Co. Cellulose tri-acetate 1963 Sartorius Co. Regenerated cellulose 1963 Millipore, Gelman, Sartorius, S&S Polyvinyl chloride and polyamide 1963 General Electric Polycarbonate 1964 Selas Flotronics Silver membrane 1970 Celanese Co. Polypropylene 1970 Gore Corp. Polytetrafluoroethylene 1975 MembranelEnka Polypropylene 1979 Gelman Polysulfone 1980 MilliporePolyvinylidene fluoride 1981 NucleporePolyester 1984 Norton Co., CeraverAlumina 2000 Our researchCryomembranes/acrylamide/PVA Microfiltration - Membranes