Pierce™ Crossflow Filtration Cassette, 3 kDa MWCO, 1 cassette - FAQs

Pierce Crossflow Filtration Cassettes are not GMP grade.

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A pressure gauge is provided with every Pierce Crossflow Filtration Cassette. Pierce Crossflow Filtration Cassettes can be run without a pressure gauge; however, it is important to limit the flow rate to 200-300 mL/min to prevent tearing and potential collapse of the tubing. The flow rate can be determined by measuring the volume flow over 1 min. Alternatively, a pump speed of 300-350 rpm or a pressure of 1-1.5 bar will maintain a flow rate of 200-300 mL/min.

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High protein concentration and/or presence of particulate in the sample can slow down the rate of concentration/diafiltration. Adsorption of the sample to the membrane and lower temperature (4 degrees C vs room temperature) can also slow down concentration/diafiltration. Filtering your sample prior to processing with a crossflow filtration cassette will help speed up concentration/diafiltration.

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Crossflow ultrafiltration and stirred cell devices both effectively concentrate biomolecules with similar sample recoveries; however, a crossflow filtration device concentrates sample much faster than a stirred cell device.

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Crossflow filtration is much faster for concentration and diafiltration than dialysis and chromatography methods.

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Crossflow ultrafiltration in the biotech industry is typically for the following applications:
- Virus concentration
- Concentration of proteins in cell culture supernatant
- Diafiltration (desalting/buffer exchange) of protein or virus preparations

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Pierce Crossflow Filtration Cassettes are not endotoxin-free. The filtration cassettes can be flushed with 0.5 M NaOH to remove pyrogens (endotoxins).

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Pierce Crossflow Filtration Cassettes can be used to concentrate double-stranded and single-stranded nucleic acids.

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To reduce protein degradation during concentration, choose a buffer and pH that are compatible with your protein. If necessary, perform buffer exchange into a more compatible buffer using a diafiltration reservoir. If your protein is temperature sensitive, perform concentration at 4 degrees C. Lastly, add protease inhibitors to your sample if your protein is susceptible to degradation.

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To prevent the open end of the Feed Line from adhering to the wall of the sample reservoir, connect the Tube Reducer to the end of the Feed Line that will be submerged in the reservoir. Alternatively, perform an angled cut on the end of the Feed Line leaving an uneven opening.

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The Tube Reducer is used to connect size 16 tubing to size 15 tubing. This can be useful if you have a peristaltic pump with a size 15 pump head.

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We do not recommend leaving the Pierce Crossflow Filtration Cassette running, unsupervised, for an extended period of time, especially when nearing the desired concentration volume. We recommend being near the device and/or periodically checking that the feed line is submerged in the sample and that there is no loss of flow rate due to collapsed tubing or tubing adhered to the side of the reservoir.

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Pierce Crossflow Filtration Cassettes can be used for protein fractionation, although separation will not be 100%. We recommend selecting a filtration cassette with an appropriate molecular weight cut-off (MWCO) that will separate target proteins (e.g., to separate a 150 KDa protein from a 15 kDa protein, use a cassette with a 50 kDa MWCO). For efficient separation of two proteins, ensure that there is at least a 10-fold difference in molecular weight (MW).

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We do not recommend using Pierce Crossflow Filtration Cassettes for removal of cells and cell debris. Using the filtration cassette for this purpose will slow down the concentration time and potentially impact recovery. Instead, prior to concentration of the cell culture supernatant, we recommend centrifuging and filtering the supernatant to remove cells and cell debris.

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Pierce Crossflow Filtration Cassettes can be used to concentrate virus. We recommend using Pierce Crossflow Filtration Cassettes, 3-100 kDa MWCO (Cat. No. A57934) for this purpose.

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Concentrated antibody can be purified using protein A supports, e.g., Pierce High-Capacity Protein A MagBeads, alkali stable (Cat. No. A53035, A53036, A53037, A53038), or MabCapture C High-Capacity Protein A Resin (Cat. No. A53031, A53033, A53034). Alternatively, for IgG1 antibody species, we recommend using Pierce Protein A/G Magnetic Agarose Beads (Cat. No. 78609, 78610) or Pierce Protein A/G Plus Agarose (Cat. No. 20423, 20424). For more information regarding binding characteristics of antibody-binding proteins, please refer to Tech Tip #34 (https://assets.thermofisher.com/TFS-Assets/LSG/Application-Notes/TR0034-Ab-binding-proteins.pdf).

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Pierce Crossflow Filtration Cassettes can be reused up to 3 times. Successful reuse of Pierce Crossflow Filtration Cassettes is sample-dependent. We recommend using the same sample type for best results.

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The tubing supplied with the Pierce Crossflow Cassette can be reused. To reduce stress on the tubing and maintain it in good working condition, maintain the pressure at 1-1.5 bar using the pressure gauge provided. Replacement tubing can be obtained by purchasing the Crossflow Filtration Accessory Pack (Cat. No. A57937).

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In order to filter sample volumes larger than 3 liters (up to 6 liters), two Pierce Crossflow Filtration Cassettes need to be used. The Crossflow Y-Connector (Cat. No. A57936) is used to connect the sample feed line into the two Pierce Crossflow Filtration Cassettes.

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To perform buffer exchange manually, we recommend following this procedure:

1. Prepare Exchange Buffer that is 5X the volume of your sample.
2. Set up your Pierce Crossflow Filtration Cassette as stated in the user manual.
3. Turn on the pump and concentrate your sample until the volume is reduced ~90%.
4. Dilute the concentrated sample to the original volume with Exchange Buffer.
5. Repeat steps 3 and 4 until the desired solute removal has been achieved.

Note: Precipitation can occur at high concentration for some proteins. If this occurs, reduce sample volume by a lesser percentage in each step and increase the number of repeats.

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All of the required tubing and a pressure gauge are included with the Pierce Crossflow Filtration Cassette. Additional items required, but not provided, include a peristaltic pump and a pump head.

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Pierce Crossflow Filtration Cassettes work by Tangential Flow Filtration (TFF), which is also known as crossflow filtration. It involves the passage of fluid parallel to the filter rather than directly through it. The latter can cause the filter to clog and slow down the concentration process.

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We do not recommend autoclaving Pierce Crossflow Filtration Cassettes. High temperatures will significantly increase the membrane molecular weight cut-off (MWCO). To sterilize Pierce Crossflow Filtration Cassettes, use a 70% ethanol solution.

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More than 98% recovery of your target molecule can be achieved by performing a low-volume buffer rinse through the tubing and cassette following sample concentration.

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We recommend using a Masterflex pump and pump head that can be purchased through Fisher Scientific or from other major suppliers.

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Any pump and pump head that can deliver a flow rate of 200-300 mL/min can be used. The pump head must be compatible with size 16 tubing for use with 1 filtration cassette, and size 15 tubing for use with 2 filtration cassettes. We recommend using a Masterflex pump and pump head that can be purchased through Fisher Scientific or from other major suppliers.

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For maximum protein recovery, samples should have a molecular weight that is two-fold higher than the molecular weight cut-off (MWCO) of the filtration cassette.

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The Pierce Crossflow Filtration Cassettes contain a polyethersulfone (PES) membrane. Each cassette has a volume capacity of 0.5 to 2.5 L with 3 kDa, 10 kDa, 30 kDa, 50 kDa, and 100 kDa molecular weight cut-offs (MWCOs).

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