Home / Back
Ultrafiltration
Ion Exchange Chromatography Hydrophobic Interaction Chrom. Chelate Chromatography Protein A/G Chromatography Heparin Chromatography Dye Chromatography Other Affinity Materials
Extraction
Membrane Chromatography
UltrafiltrationRunning an Ultrafiltration skid looks rather easy, but if you work with this unit operation you will see many screws you are able to turn like pressures and flows. The most important thing is getting the membranes clean afterwards so you will obtain consistent results. Spiral- Modules tend to be rather difficult to clean so try to cook them as high as possible (most of the cases between 45 and 80°C [nadir-filtration,I am getting a little annoyed of the former Hoechst sunbsidary, they are changing their name every few months, still they make reliable stuff]). Henkel makes the P-series where they tested most of the available Membranes for their resistance. Thinking of the module typ you will end up by decission between Plate and Frame, Cassette, Spiral or hollow fiber module. Hollow fiber will give you a very robust system with less membrane-surface per Volume but sometimes if you have fibres in your product they will clott the inlett of the fiber. The plate and Frame construction will give you a "medium" robust system which is mainly used in the dairy industry. The membrane prices per square meter (same in square feet for our anglo saxons) is nearly unbeatable, just equipping the skid is a little tricky but not impossible. The more luxe edition are the cassette systems which are only suitable for high price proteins (does anybody know why they are so expensive). The spiral modules give you the opportunity to put a lot of surface in a small room but be aware of the cleaning and don´t use too messy stuff.
Links Ultrafiltration
www.membrana.com
www.dksepsys.com
www.nadir-filtration.de
www.millipore.com
www.pall.com
www.sartorius.com
www.s-und-s.de
www.agtech.com
Planova/MicrozaIf somebody talks about chromatography in bio-process industry in 8 out of 10 cases he is using using Pharmacia media. They have a great support capability (qualification services) compared to others and the reason why Pharmacia media is used is because everybody uses it (you know the Henn and Egg Syndrom). From a developers viewpoint there are probably more advanced ideas on the market rigth now than the agarose based media from Pharmacia.
Merck and Tosouses mainly tentacle media which are supposed to have an increased capacity (at least for Lysozyme and BSA shown in experiments, in real live these media give somewhat surprising results, same for XL from Pharmacia).
A capacity increase is also claimed by Biosepra they fill their beads with some sort of gel which is heavily loaded with active sites and increase the capacity (Lysozyme and BSA again). The last interesting thing I saw on their website was a replacement for Protein A/G with a chemical group for Antibody purification! But I still ask myself why they put hydroxyapatite in an agarose shell. Because the advantage of hydroxyapatite is the rigid structure were you can bang on. After working with the material for a while I must admit that working with it is very tricky. The packing is normally not very stable and you always need some Ca or Phosphate in you buffer. We had the best results regenerating the column with 6M GuanidineHCl (watch the pH not going too low) and storing it in KOH pH 12. The most famous supplier for this stuff is Bio-Rad, the mechanism how it works is not 100% clear all the time, but a good interpretation was provided by Validated Inc. an inexpensive source for crude material is Trantor (Specifications and Prices).
For Sepragen you will better go to their Homepage, because I never used their stuff so far.
The Perseptive stuff is really nice and very robust. You can pump the hell out of it and no chemical will harm it (pretty much besides the Protein A/G media). The capacity is maybe not as high as other media but it is worth a try. Qiagen is not a real company for chromatography materials, but they have an exclusive license for the NiNTA material which is a chelating bead which keeps your metal much tighter bound than all the others available. This will make your people who are in charge of the waste water treatment much happier and also Qiagen which sells the stuff for a whole bunch of money. Upfront is offering a new material which they claim is a combined HIC with IEC and very usefull for MAB´s purification.
Talking about all the different chromatography modes and techniques would take too much time here, I still earn my money somewhere else. But Pharmacia will provide you with great booklets were all this is described very well (you don´t have to buy their stuff!)
Chromatography Columns are made by a lot of specialty companies and by Pharmacia (not great but OK), Upfront (experts for Expanded Bed Chromatography, maybe a little more advanced distributers than Pharmacia), Millipore (I think the Amicon things were not so bad so lucky Millipore), Kronlab (they build (do) everything for money), Bio-Rad ( Actually they are now distributing columns and process systems from the french Verdot company, which look very reliable), Sepragen (never seen) and Merck (Homemade stuff, made in a German garage).
There must be a great fractal distributer around to build your own column, but I am still looking for the company which produces it
Links Chromatography
Hard and Software
www.bioprocess.amershambiosciences.com
www.upfront-dk.com
www.bio-rad.com
www.dan-process.dk
www.merck.de
www.sepragen.com
www.kronlab.com
www.perseptive.com
www.millipore.com
Software
www.biosepra.com
www.dow.com
www.lewatit.bayer.de
www.tosohbiosep.com
www.qiagen.com
www.trantor.com.mx (crude Hydroxylapatite)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
http://www.bham.ac.uk/BRG/Brg.htm
Department of Biotechnology Royal Institute of Technology (KTH)This chromatography method is very useful for chromatography of large volumes with small product titers. Because the mechanism is not diffusion controlled, the targed protein will reach very rapidly the Linker attached to the membrane. The common Ion Exchange groups (Q,DEAE, S, SP) are available by the companies mentioned below.
Though you have to take into account the effective volume of a Membrane sheet is not much, and you need a lot of sheets (money) to get a satisfactory capacity. On the one site this will result into large flat sheets a few mm thick, with serious flow distribution problems or into spiral/hollowfiber-modules with poor breakthrough characteristics.
However, for analytical purposes membrane chromatography can very successfully be combined with spin columns (Vivascience), where speed, convenience, reproducibility and cost considerations are paramount to absolute binding capacity.Links Membrane-Chromatography
www.millipore.com
www.pall.com
www.sartorius.com
www.vivascience.com
www.merck.de (stopped the product)