Large scale tangential-flow filtration of water samples DO NOT PUBLISH WITHOUT PERMISSION OF AUTHORS K. Eric Wommack
 
Materials:
1.    Large 20 l and 50 l polypropylene carboys with caps (Nalgene)
2.    9.5mm diameter PharMed peristaltic pump tubing (Cole-Parmer)
3.    9.5mm diameter Tygon tubing for plumbing of water sample between filters, pumps and reservoirs (Nalgene)
4.    Cartridge sediment filter made of string wound polypropylene fiber (Kenmore, Sears, Roebuck & Co. Hoffman Estates, Il) capable of removing particles > 25µm.
5.    Filter housing for cartridge sediment filter.
6.    Peristaltic pump capable of providing sufficient cross-flow for a 0.85 m2 ultrafiltration membrane (eg. M12, Millipore, Corp. Bedford, MA).
7.    0.5 m2 0.22 µm Pellicon, microporous tangential-flow membrane (Millipore, Corp. Bedford, MA)
                        0.85m2 Helicon S10 30 kD molecular weight cut-off (MWCO) ultrafiltration cartridge (Millipore, Corp.
                        Bedford, MA)
 
 
Methods:
1.    Pre-filter 20 to 50 L water sample through a sediment cartridge filter of string wound polypropylene fiber capable of removing particles >25 µm. These filters are commercially available as home water filtration devices. The water sample should be pumped through the filter using a diaphragm or peristaltic pump and collected into an appropriately sized carboy. Pre-filtration is necessary as subsequent filtration of particulates > 100 µm can result in clogging of passages within the tangential flow filtration cartridges.
2.    Prior to filling, rinse the carboy 3 X with a few liters of the 25 µm pre-filtered water. Rinse all carboys 3 X with appropriate filtrate prior to filling.
3.    Plumb the 0.22 µm 0.5 m2 Pellicon TFF filter by placing Tygon tubing lines to the retentate and permeate ports. The tube from the carboy to the peristaltic pump head and one of the retentate ports is the feed line, while the tube from the other retentate port back to the carboy is the return line. The section of the feed line within the peristaltic pump head should be replaced by equally sized PharMed tubing, which can withstand long term peristaltic use. The retentate ports on the Pellicon housing can serve as either the feed or return as there is no set direction for the tangential flow in the Pellicon cartridge. The permeate line is a section of tubing from the permeate port of the Pellicon filter housing to a different carboy that will collect the 0.22µm filtered water. A schematic diagram of the plumbing for the large large-scale TFF system is shown in Fig. 1.
4.    Because TFF is a pressure filtration, the direction of the peristaltic flow should be from the water sample towards the filter
5.    Turn the permeate control valve off and start pumping the 25 µm filtered water through the Pellicon at a low 10% pump speed and an occlusion setting of 5 on the pump head. Continue pumping at low speed until the lines and filter is completely full and all air has been completely expelled. It may be necessary to elevate the feed and return lines to assist in air expulsion, if necessary. Once the system is fully primed, adjust the occlusion setting back to 3 to minimize wear on the PharMed tubing.
6.    Slowly raise the pump speed to 45% and open the permeate valve so that the ratio of retentate to permeate flow is 2:1. Relative flow rates can be determined by collecting permeate and retentate for a given amount of time with a graduated cylinder. A 2:1 flow ratio is achieved when the volume of water collected from the retentate is 2 X the volume collected from the permeate for a given same amount of time.
7.    As tangential flow filtration proceeds, bacterial cells and particles between 0.22 and 25 µm in size will be concentrated in the retentate of the Pellicon filter. The Pellicon filter permeate will include viruses and dissolved solutes < 0.22 µm. Do not allow the volume of the >0.22 µm retentate to fall below 1/20 of the original volume (eg. 50 L to 2.5 L). Excessive concentration of bacteria and particles between 0.22 and 25 µm makes cleaning difficult and may result in filter clogging.
8.    Once at least 20 L of 0.22 µm-filtered water has been obtained, TFF to concentrate virus particles can begin. Use of two peristaltic pump heads on one pump allows for simultaneous operation of the Pellicon filter and 0.85m2 Helicon S10 30 kD MWCO ultrafiltration cartridge.
9.    After reducing the pump speed to 10% and stopping the pump, plumb the feed and return lines of the Helicon filter cartridge in a similar fashion to that of the Pellicon in step #3. Use the <0.22 µm permeate from the Pellicon filter as the feed water for the Helicon cartridge. Note, the direction of tangential flow is set for the Helicon cartridge and should not be reversed.
10.    Fully open the permeate control valve and set the occlusion knob on the pump head to 5 to allow for priming of the lines and Helicon cartridge. Start the pump at a low setting of 10%, then raise to 20% to fill the cartridge and expel air. Once the cartridge is full and all air is expelled adjust the occlusion knob back to 3.
11.    Slowly raise the pump speed to 45%, then slowly close the permeate control valve until a retentate : permeate flow of 3:1 is achieved.
12.    Viruses and dissolved solutes between 30 kD and 0.22 µm will be concentrated within the retentate of the Helicon filter. The permeate of the Helicon filter is effectively free of viruses.
13.    Collect 1 L of the virus-free permeate in a 2 L plastic bottle for subsequent rinsing of the Helicon.
14.    Collect the rest of the virus-free permeate in a 50 L carboy. This water will be used for washing the TFF filters.
15.    Continue TFF until the level of the retentate of the Helicon is near the minimum hold-up volume of the filtration system, ~ 1 L. Do not allow the filer to run dry or bubble excessively. Open the permeate valve, reduce the pump speed to 10% and then stop the pump. Release the feed line from the peristaltic pump head and allow the Helicon filter and line to drain completely. Store the 1 L viral concentrate at 4°C.
16.    Place the feed, return, and permeate lines into a 1 L volume of previously collected virus-free permeate. Place the feed line back into the peristaltic pump head, then prime the lines and filter at an occlusion setting of 5 and a pump speed of 10% until all air is expelled.
17.    Recirculate the virus-free permeate through the Helicon filter at 30% pump speed for about 5 minutes. Avoid excessive bubbling of air during this step.
18.    Reduce the pump speed to 10% stop the pump, open the pump head and drain all lines into the 2 L virus-free permeate rinse bottle.  
19.    Add the 1 L rinse water to the 1 L viral concentrate. Store the viral concentrate at 4°C until further TFF concentration.
20.    Wash the Pellicon filter by first flushing with 25 L of virus-free permeate at a pump setting of at least 45%. Follow this initial wash with ~ 25 L of RO or distilled water. Flushing is achieved by not recirculating, but rather directing the return and permeate lines to waste. Reverse the direction of tangential flow 2X during washing to assist in rinsing. Do not drain the filter. Instead, clamp the lines to keep the cartridge filled with ultrapure water.
21.    Wash the Helicon in a similar fashion; however, the direction of flow cannot be reversed in this filter. Keep the cartridge filled with ultrapure water.
22. If the cartridges are not to be used for 2 days or longer, filter membranes should be cleaned following manufacturer’s instructions. Care must be taken to ensure that cleaning and storage solutions are compatible with the filtration membrane.  Integrity test filter according to manufacturer’s directions after every 5-10 uses.