Protein Bioseparation - Classification 1. High-productivity, low resolution

1. Protein Bioseparation - Classification 1. High-productivity, low resolution 2. High resolution, low productivity 3. High resolution, high productivity

2. Downstream Processing Profile ____________________________________________ Product ---------------------------------------- Step concentration; g/L Quality, % ___________________________________________________________ Harvest broth 0.1 – 5 0.1 – 1.0 Filtration/centrifugation (Step1) 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) Formulation/drying etc (Step 4) 90 - 100 ____________________________________________________________

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

4. 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. Filtrationproduces moreconcentrated (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.

5. RIPP • Removal Filtration Centrifugation

6. Factors affecting separation/removal Broth Characteristicshigh 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 processbacterial, 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

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

8. Cloth and fiber filters Microfilters Ultrafiltration Gel chromatography Ultracentrifuges Gravity sedimentation Factors affecting separation Vs.particle size Size Screens and strains Density Centrifuges Angstroms (Aº) 1 10 102 103 104 105 106 107 Millicrons (nm) 10-1 1 10 102 103 104 105 106 Microns (µm) 10-4 10-3 10-2 10-1 1 10 102 103 Ionic range Micron range Fine range Course range Macromolecular range

9. 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

10. 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".

11. 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.

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

13. Filtering improvements • Filtering aids • pH • Temperature • Duration of fermentation

14. 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".

15. Retentate Feed Permeate Cross-flow filtration • No cake formation • Feed to retentate flow > > permeate flow

16. Water Proteins Cells • One stream in- two streams out technology • Tangential flow parallel to membrane for cell removal Cross-flow filtrationseparates 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

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

18. Direct Flow Filtration Process Tangential Flow Filtration Process

19. 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

20. 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

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

22. Feed Concentrate CFF Retentate Feed- pump Feed Permeate Basic Cross-flow filtration process (continuous)

23. Microfiltration - Membranes • 1845 Schoenbein Nitrocellulose • 1855 Fick Esters 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 Millipore Polyvinylidene fluoride • 1981 Nuclepore Polyester • 1984 Norton Co., CeraverAlumina • 2000 Our research Cryomembranes/acrylamide/PVA