Here’s the situation: I had about 2 liters of conditioned medium from Sf9 cells that had been transformed to secrete my favorite recombinant protein. I verified the presence of the protein by immunoblot and estimated there to be about 5 mg of the protein present in the whole volume. However, the recombinant protein had to be in a purified state for my intended downstream applications. The easiest method of purification, affinity chromatography, was out because the recombinant protein has no affinity tag and an antibody column with a capacity anywhere close to what I needed would have been prohibitively expensive. Since that left conventional chromatography as the purification method, I needed to reduce the volume of the medium by 20-30 fold. Ammonium sulfate precipitation was an option but I didn’t know what percent saturation would effectively precipitate the recombinant protein. Luckily, my problem was solved with the Vivaflow 200 from VivaScience (Hannover, Germany).
The Vivaflow 200 is a ready-to-use tangential flow filtration device that can be used for concentration or buffer exchange (diafiltration). The 200 is recommended for volumes of 250 ml to 1-2 liters. For more than 2 liters, the devices may be combined in parallel. For smaller volumes, you can use the Vivaflow 50. Both the Vivaflow 200 and 50 work on the same principle. The solution to be concentrated or diafiltered is pumped across the face of a membrane filter of the desired molecular weight cut-off (MWCO). A flow restrictor on the exit port supplies sufficient backpressure to force some of the solution through the membrane and into the filtrate container; the remainder is recirculated across the membrane. Recirculation is allowed to continue until the desired degree of concentration has taken place. The Vivaflow unit can then be flushed, cleaned, and stored for several more uses.
The Vivaflow 200 is available with MWCOs from 5,000 to 100,000 Da with membranes made of polyethersulfone (PES) or 2 varieties of regenerated cellulose. The Vivaflow 50 is available with MWCOs from 3,000 to 100,000 Da with membranes of PES or regenerated cellulose. Accessory equipment and supplies are available from VivaScience. Both the 200 and the 50 are sold with pretty much everything you need except the pump.
For my sample, the starting volume was 1762 ml and the final volume of concentrate was 87 ml; a concentration factor of 20-fold. The figure shows a silver-stained gel of 10 µl each of the original solution (lane 2), the concentrate (lane 3), and the filtrate (lane 4). Lane 1 contains MW markers that range in size from 7 to 207 kDa. The gel clearly shows not only the effective concentration of the original solution, but also the absence of any protein leakage into the filtrate. For the current implementation, I used a PES membrane with a MWCO of 10,000 Da.
Overall, I have found the Vivaflow 200 to be an easy to use and effective means of concentrating large volumes of solution for a very reasonable price ($259). The device is very sturdily constructed of plexiglass with the filtration membrane and fluid flow in clear view at all times. Although the company recommends running the device at a pressure of 2.5 bar and a recirculation rate of 200-400 ml/min, I was not able to achieve either of these with the tubing supplied and my Masterflex peristaltic pump running at full speed. Hence, the 20-fold concentration of my sample took over 4 h. I assume this time would have been shortened markedly had I been able to run the device at the recommended pressure and flow rate. Using tubing more suited to a peristaltic pump may help.
Michael Campa, Ph.D.
Asst. Research Professor of Radiology
Duke University Medical Center