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"Capture" Step and Cell Separation

During the first step after fermentation or grinding of a natural product a main part of your yield can be lost instantly.

• Centrifugation

 
• Microfiltration

 
• Extraction

 
• Expanded Bed Chromatography

 
• Batch adsorption

Centrifugation is a common method for the separation of cells from broth or the removal of debris. Although the fluid phase is cleared the water content of the solid phase is still around 60%. This means that for fermentations of yeasts like Pichia or Hansenula where a dry weigth of 150 g/L is common an additional 275 g/L of broth is lost in the cells, which is around 1/3 of the possible yield. An additional washing step will increase the yield but also increase the volume which has to be processed in the next step. For the removal of cell debris the density gradients are often not sufficient enough to result into a separation of the phases. The viscosity increase during cell rupture can be decreased by the addition of DNAses.
Links Centrifugation:
http://www.westfalia-separator.com/
http://www.sorvall.com/
http://www.heraeus-instruments.de/
http://www.cinc-co.com/
http://www.auxill.com/
http://www.alfalaval.com/
http://www.jouan.com/
http://www.hettich-zentrifugen.de/

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Microfiltration gives somewhat the same problems. If the yield has to be in acceptable limits  high dilution factors are necessary for high density cultivations. Viscous fluids are also difficult to handle. If the target protein is extracellular the cells should be intact during separation. Shear forces acros the membrane can rupture the cells and release unwanted protein into the broth.

Links Microfiltration
www.membrana.com
www.dksepsys.com
www.nadir-filtration.de (I gave up finding the new sites of the former Hoechst membranes every few months)
www.millipore.com
www.pall.com
www.sartorius.com
www.s-und-s.de
www.cuno.com
www.seitzschenk.de
http://filtteck.com.tw

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Extraction is a very suitable method as a capture step. Not only cells or debris are removed but if the conditions are right a purification and a volume reduction can be achieved. Here aqueous two phase systems like PolyEthyleneGlycol/Salt, Poly- Ethylene Glycol/Carbohydrate, Water/Tensid or PolyEthyleneGlycol/Copolymer Systems are commonly used. But also systems containing Water/Salt/Ethanol or Propanol result into a suitable system for some proteins. After phase separation the target phase can be directly applied to a chromatographic step (ion exchange, HIC or affinity) or diafiltrated or the protein can be precipitated.

Links Extraction
http://bama.ua.edu/~rdrogers/aq2phase/index.html
http://www.iet.uni-duesseldorf.de/
Department of Biochemistry, Lund University
Department of Biotechnology Royal Institute of Technology (KTH)

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Expanded Bed Chromatography is a relative new method for the purification of cell broth. The Media is not tightly packed in the column but is fluidised during the run. This will enable cells to flow through the column and proteins to bind to the media. Many functional groups are available from Amersham-Pharmacia-Biotech and Biosepra, hydroxylappatite is available from BioRad on request. The conductivity has to be sufficient for the media but high enough to prevent cells from binding.

Links EBA
http://www.iet.uni-duesseldorf.de/
http://www.cheng.cam.ac.uk/groups/biochem/
http://www.bioprocess.amershambiosciences.com
http://www.upfront-dk.com/
http://www.biosepra.com/
 

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Normally inexpensive ionexchange (5-10$/L for bulk amounts) media (Lewatit, Bayer or DOWex, DOW) are used for Batch adsorption. If the conductivity is sufficient low these method will obtain a great volume reduction for the further process. Many different matrixes, shapes and functional groups are available. The media can also be used to decolorise the solution.

Links Batch adsorption
www.lewatit.bayer.de
www.dow.com

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