Membranes and beads combine to separate broths

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Ezine

  • Published: May 16, 2005
  • Author: Jon Evans
  • Channels: HPLC
thumbnail image: Membranes and beads combine to separate broths

Sometimes a fairly minor technical hitch can help to stymie a great idea. This is what almost happened to a group of researchers from the New Jersey Institute of Technology, Newark, US, who discovered that just such a hitch was ruining the efficiency of their newly-designed separation device.

The researchers, led by Kamalesh Sirkar, were investigating improved means of isolating specific bioproducts, such as proteins, from complex fermentation broths. Traditional methods tend to involve a number of time-consuming pre-treatment steps, such as centrifugation and flocculation, prior to standard column chromatography. The researchers wondered whether they could come up with a more efficient system by directly combining membrane filtration and chromatography.

To do this, the researchers customised a membrane technology known as hollow fibre modules, which consist of bundles of hollow fibre membranes within module shells. When the feed solution passes through the central core of a fibre, any particles able to pass through the fibre membrane collect in the outer casing of the module shell.

In their customised version of a hollow fibre module, the researchers inserted chromatographic resin beads in the space between the fibre membrane and the outer casing of the module shell. These beads are able to capture specific target proteins in the permeate, which are then released by washing the beads with an elution buffer.

Although this customised hollow fibre device did manage to isolate proteins, the researchers discovered that its performance was being severely affected by protein leakage. The problem was that proteins permeating through at the top of a fibre membrane could be captured by any number of beads as they made their way down with the permeate solution. However, those permeating through at the bottom of the fibre membrane, where the permeate flowed out of the module through an outlet port, could only be captured by a fairly narrow layer of beads, which soon became saturated with protein. Fairly quickly, therefore, target proteins began leaking out of the outlet port.

The researchers' first attempt at solving this problem involved adding an extended section to the end of the fibre, containing additional resin beads. But this caused the pressure in the system to drop too low, reducing the permeate flow rate. They then came up with a relatively simple, but highly effective, solution: stop any particles from passing through the membrane in the bottom section of the module.

They achieved this by coating the bottom quarter of the membrane with a layer of polyamide, which is completely impermeable to water. Because particles in the feed solution could now only pass through the top three-quarters of the membrane, this meant that the beads at the bottom didn't become saturated too quickly.

Testing their improved device on a mixture of the proteins myoglobin (Mb) and a-lactalbumin (a-LA), the researchers discovered that it didn't suffer from protein leakage to anywhere near the same extent as the original version of the device. It had a much higher absorption capacity for both proteins and was therefore able to handle much higher protein concentrations and loading times. The researchers also tested the device using both microfiltration (MF) and ultrafiltration (UF) membranes (MF membranes have much larger pores) and discovered that UF membranes provided an additional method of separation.

In the device, Mb and a-LA can be separated from each other during the eluting process, because Mb is released from the resin beads earlier than a-LA. However, the researchers found that changing the ionic strength of the feed solution had a major effect on this separation processes: at low ionic strengths, electrostatic repulsion prevented a-LA from passing through the UF membrane; at high ionic strengths, the beads were not very effective at capturing Mb. Changing the ionic strength of the field solution could thus offer a highly efficient and convenient way to isolate only a single type of protein from a fermentation broth.

Sirkar and his team are currently optimising certain aspects of the device and are looking for companies to help commercialise the technology.

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