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Virus Filtration/Removal
Endotoxin Removal
Sterile Filtration
Gel-Chromatography
RP-Chromatography
Drying
If you are lucky your protein is heat or pH resistant, if not don´t give up easily fight for your succes! A lot of viruses are very easy to inactivate but if you have to deal with the Parvo guys then you are in trouble. Commonly pH values between 2 and 4 or above 11 are used for virus inactivation steps. As you can imagine most of the proteins are not very happy at these conditions. Another way of getting the bad guys inactivated is the use of heat. While dry heat kills a lot of virus species you will be happy to have a Log reduction of 2 after 72h of 80°C which is just enough to be significant. Heating with microwaves in aqueous media is promoted by the German godfather of Cell Culture production (Prof. Walter at Boehringer Ingelheim) and sold by Charm. Here the mechanism relies on the very short heating times, unfortunately the capacity is limited to 80 L/h (there working on more) but I think it is worth a try. A Virus can also be inactivated by putting a lot of junk in your Protein mixture like Ethyleneimine, Urea, iodine or whatever you can think of. But you never know before how it will affect your protein. Different Buffers will give different resistance to denaturation. In the Case of Parvo, you always have to keep in mind, that it has a protein shell and if you want to hurt it you will hurt your product.
Virus removal can be achieved either by adsorption or filtration. Most of the Viruses will stick to Q or DEAE exchangers at moderate pH´s so you can either bind your protein to a cation exchanger at let the bad guys run through or take a anion exchanger and elute very carefully so you separate the bad from the good guys. Filtration is pretty tough if you have to deal with Parvo (again, small nasty and tough) in many cases this Virus is as big as your protein and filtration is only possible because of rigid structure of the virus (at least that is what the companies tell you). Here you need very narrow distributed pores in your UF-Membrane like AGT. Or custom cutoffs which are delivered by the Millipore and Pall for their Virus filters (always keep in mind that there is no real standard procedure to measure the cutt off so you can´t really compare the numbers of different companies). SeitzSchenk makes dead end filters which releases Iodine and inactivate the virus.www.agtech.com
Planova/Microza
Charm
www.seitzschenk.de
www.millipore.com
www.pall.com
www.argonide.com
Endotoxin Removal
besides using packed bed or batch chromatography the adsorption of endotoxins to Membranes seem to be very promising. PALL offers a whole range of possibilities with different sorts of surfaces like modified Nylon-66 called Posidyne membranes or the Mustang series E(ndotoxin), Q and DEAE whereas Q and D are real IEX mebranes like MERCK and Sartorius (Sartobind) sell. Pall´s E series is a mixture of IEX and HIC which apparently is very suitable for endotoxin removal . Because elution of endotoxins is far besides trivial, the filter may have to be disposed. Especially for all products besides Posidyne this will be very expensive.www.pall.com
www.sartorius.com
www.merck.de
Sterile Filtration
Every month at least 10 people would like to sell you filter cartridges and they all claim that they have the best ones in the world. If you look behind the box and the plastic you will often find the same membrane. Because there are just not so many companies around which make membranes or they make some and buy the rest somewhere else. The same applies to ultrafiltration membranes by the way. While it was common to use 0.2 µm membranes (I always wonder how Millipore exactly knows 0.22) the tendence goes smaller to 0.1 or less. There were some microbioligists who found some nanobacteria (so we better call them nanobiologists) and they scared everybody a lot.To be continued !!!
www.millipore.com
www.pall.com
www.sartorius.com
www.seitzschenk.de
www.s-und-s.de
www.cuno.com
Links Chromatography
Hard (and Soft) ware
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.tosohbiosep.com