Crossflow filtration, also called tangential flow filtration, is a method for the filtration of liquids. It is used especially in the food and pharmaceutical industries to filter or diafilter samples in volumes of several thousand liters. Unlike static filtration techniques, crossflow filtration systems are capable of clarifying liquids that contain relatively high quantities of colloids, haze or other suspended sediments. This is achieved by flux rates of 2.5 to 3 m/sec across the membrane, which prevents fouling, i.e., the buildup of a layer of these particles, which would otherwise result in the formation of a ‘filter cake’ causing rapid blockage of the membrane. Industrial crossflow systems must meet exceptionally high quality standards regarding cleaning, reusability and process control capability.
In laboratories, much smaller volumes are usually used. For sample volumes lower than 100 ml, centrifugation units, for example, are ideal for concentration and buffer exchange. These are available as single-use products in a wide choice of volume capacities and different pore sizes (Figure 1).
Figure 1. Shown is a Vivaflow 200 crossflow cassette for concentration of up to 5 l samples; Vivacell ultrafiltration units with a black pressurized cover for pressure filtration of samples of up to 250 ml; and Vivaspin ultrafiltration units (in the rack) for use in a centrifuge for concentration of sample of up to 20 ml.
For samples higher than 100 ml, stirred cells as well as pressurized cells are available, which are also dead-end filtration units in principle just as are the centrifuge units. Dead-end filtration means that a one-fold concentrated sample recovered is not recirculated as in crossflow filtration. Besides small samples, larger volumes from 100 ml to 5 l are also commonly used in laboratories and need to be concentrated or their buffers exchanged, such as cell culture supernatants prior to purification.
On the one hand, these volumes are too large to be concentrated in centrifuge or pressure-driven ultrafiltration units. On the other hand, they are too small to warrant the use of a process-scale crossflow system, apart from the high costs primarily associated with attributes not required by laboratories, such as GMP-compliant manufacture and exact monitoring of the concentration progress.
For such cases, the Vivaflow crossflow units are an ideal match for intermediate sample volumes. Made of polycarbonate or acryl, these units are a fast and simple solution for concentration of biological samples up to volumes of 5 l. On top of this, they look exceptionally attractive in any laboratory. The units are designed to operate according to the ‘plug & play’ principle. The pre-assembled tubing on the unit just needs to be threaded in a peristaltic pump, which continuously circulates the sample through the crossflow cassette until the desired volume has been attained.
A special feature of Vivaflow cassettes employed in water analysis is that they are easy to use. In Bergen, Norway, Vivaflow units are used to detect the infectious salmon anemia virus (ISAV) in salmon aquaculture ponds. For this purpose, two liters of water are concentrated using Vivaflow 200, with an incorporated 100 kDa PES membrane, down to 80 ml. This procedure is especially gentle and can be completed in less than half an hour. Afterwards, viral RNA is extracted from the concentrated sample and identified using RT-PCR (reverse transcription polymerase chain reaction). Virus quantities of only 5.5 virus particles/ml (TCID50 1,6 × 10−2 ml−1) have been successfully detected using this method.
Vivaflow can be used to concentrate bacteria and phytoplankton besides viruses, as was carried out by Marjolojn Tijdens et. al using a series of water samples taken from a shallow eutrophic lake in the Netherlands, Lake Loosdrecht. In this study, the population dynamics of water organisms and their related viral abundances in the lake depending on the season were to be determined. After 2 liters of lake water had been concentrated 40-fold as required using Vivaflow 200 with a 30 kDa PES membrane, the microorganisms were detected using epifluorescence microscopy and pulsed field gel electophoresis (PFGE). Depending on the season, 5.5 × 107 to 1.3 × 108 virus-like particles per ml−1 could be detected in this case. Vivaflow units are especially suitable for this application, as samples had to be prepared very quickly on site in order to ensure accurate analysis of the condition of the lake water.
A buffered solution
Vivaflow is also excellently suited for concentration of proteins from cell culture supernatants. Andrea Zocchi et al. report on the purification of a recombinant, glycolated avidin (recGAvi) from the methylotrophic yeast Pichia pastoris. Native avidin is a glycoprotein found in hen egg white, for instance, and is capable of binding biotin (vitamin H) and thus inhibiting cyboxylation of pyruvate.
Figure 2. This schematic diagram depicts the setup of a Vivaflow system if buffer exchange needs to be performed in addition to concentration. From the feed reservoir, new buffer flows into the sealed diafiltration reservoir containing the sample as filtrate (waste) is pumped off through the outlet tubing.
RecGAvi was expressed by P. pastori in the culture supernatant. Before purification of the protein, the bioreactor supernatant, which contained 330 mg/l RECGAvi, was removed by centrifugation and concentrated using Vivaflow 200 (30 kDa PES membrane) to 50 ml and, in the process, the medium was exchanged with a buffered solution, 0.05M NaHCO3 and 0.5M NaCl at a pH of 9.8. This is easily accomplished during a crossflow procedure by creating a closed system, which draws the same quantity of fresh buffer from a feed vessel into the concentrated sample as the quantity of filtrate that is pumped as waste out of the system (Figure 2). Thus, buffer exchange of up to 99% can be achieved. The rec-avidin sample concentrated and with the medium exchanged for a buffered solution could then be purified using affinity chromatography on a 2-iminobiotin agarose column.
In summary, crossflow filtration is excellent for use in many different laboratory applications entailing concentration and buffer exchange of biological samples. This technique facilitates and accelerates procedures by replacing lengthy centrifugation steps otherwise required for sample volumes of up to 5 l.
In addition, novel crossflow systems, such as Vivaflow, are specially designed to accommodate laboratory needs. They are optimally suited to perform reliable and fast concentration or diafiltration of up to 5-liter sample volumes and accomplish this at a very affordable price.
1 Microb Ecol. 2008 July; 56(1): 29–42. Population Dynamics and Diversity of Viruses, Bacteria and Phytoplankton in a Shallow Eutrophic Lake, Marjolijn Tijdens et al., Department of Microbial Wetland Ecology, Centre for Limnology, Netherlands Institute of Ecology.
2 Journal of Plankton Research 2008; 30(3): 261–273. Microbial dynamics during the decline of a spring diatom bloom in the Northeast Atlantic, Carole A. Llewellyn et al.1, Plymouth Marine Laboratory, Prospect Place, The Hoe, Plymouth PL1–3DH, UK.
3 Protein Expression and Purification 32 (2003) 167–174, Expression and purification of a recombinant avidin with a lowered isoelectric point in Pichia pastoris, Andrea Zocchi et al., Institut de Chimie, Universite de Neuchatel, Av. de Bellevaux 51, CH-2000 Neuchatel, Switzerland.