Alternating tangential flow filtration

Alternating tangential flow filtration

Alternating tangential flow filtration (ATF) is a filtration method used in a variety of industrial applications to purify desired particles out of a sample. This technique is an optimized version of tangential flow filtration (TFF), making it useful to first develop an understanding of this original technology.

What is tangential flow filtration?

Tangential flow filtration (TFF) is a widely used separation method where particles are flowed tangential (parallel) to a filter rather than directly through a membrane. This method effectively separates out particles (the permeate) from the rest of the flow (the retentate) without quickly clogging the filter. This prevents particle build up and facilitates more complete removal of the permeate from the flow.

Diagram depicting cross flow in tangential flow filtration setup

Tangential flow filtration flow path

How does tangential flow filtration work?

TFF works by applying a pressure differential across a filter while continuously flowing a sample past it. As the particles pass the filter, the positive pressure on the permeate side causes particles to be pulled through. The retentate, on the other hand, continues to circulate through the system until all permeate is removed. If you are interested, this video shows a TFF overview.

What is alternating tangential flow filtration?

Alternating tangential flow filtration uses a similar technique as TFF. The primary difference is that ATF is a perfusion process, designed to minimize filter fouling.

In ATF, the permeate is removed to a collection while a diaphragm pump alternates between positive pressure and vacuum to move the retentate back and forth across the membrane. The retentate returns to the bioreactor between each cycle, mixing with the next fluid batch to be filtered. ATF is ideal for collecting the desired product within the permeate, as the retentate is continually pushed back into the bioreactor.

The alternating flow of the retentate cleans the filter, enabling it to be used much longer or for larger batches than equivalent TFF filters. For a better visual on how ATF works, watch this video.

Filter characterization in tangential flow filtration

Hollow fiber and cassette membranes are the two most common membranes for flow filtration. Hollow fiber membranes are cylindrical, with the membrane embedded as fibers in the cylinder. The fluid passes through the cylinder, with ports to separate permeate and retentate at either end of the cylinder. Cassette membrane layers are stacked and separated by screens. The fluid is flowed through the layers and the permeate is removed out of the side, while the retentate continues to flow.

Membranes are classified by their average pore size. Those used in bioprocessing are generally for either microfiltration or ultrafiltration. Microfiltration membranes have pores with diameters greater than 0.1 µm, and are generally used to separate proteins and other small molecules from cells (the larger cells are discarded in the retentate). Ultrafiltration membranes have pores with diameters smaller than 0.1 µm, and are often used to concentrate proteins. The proteins are in the retentate in this case, while water and various small solutes are discarded in the permeate.

Advantages of tangential flow filtration over dead-ended methods

Dead-ended filtration flows samples directly through a membrane, oftentimes resulting in a clogged filter. The tangential flow of TFF prevents clogging and is much faster and more efficient than traditional filtration methods.

In TFF, the flow stream with the desired product can be recycled and looped through the system as many times as necessary for complete separation of the permeate and retentate. This process can also be used to clean the filter, by flowing pure buffer through the system.

As with dead-ended filtration, the size of the membrane pores determine which particles are able to pass through the membrane. Pore size can be optimized to isolate the desired particles either in the permeate or the retentate.

Applications that use tangential flow filtration

  • When centrifugation isn’t available or practical, TFF is a convenient way to harvest cells. Microfiltration membranes will retain the whole cells while allowing for selective recovery of the supernatant.
  • After cells are lysed, TFF is useful for lysate clarification to remove debris such as unlysed cells and membrane fragments. This is often done as an intermediate purification step before chromatography.
  • Smaller and larger proteins may be separated using ultrafiltration membranes. The proteins must be of sufficiently different sizes for the membrane to adequately filter the components.
  • While chromatography often requires diluting protein samples, final products may require higher concentration. In this case, ultrafiltration may remove permeate while retaining the proteins at a higher concentration.

Mass flow for alternating & tangential flow filtration

Bidirectional mass flow controllers flowing air can alternate between positive pressure and vacuum to work with the diaphragm pump in the ATF setup. Vacuum can be used to pull the fluid through the filter for the initial permeate/retentate separation, and positive pressure to return the retentate to the reactor.