Preparative Protein Purification by Magnetic Beads

Isolating and purifying biological products by magnetic microsorbents, so-called magnetic beads, is an interesting alternative to the classical treatment of bioproducts, e.g., by precipitation or chromatography. The basic principle of protein isolation by magnetic beads that is shown in the figure below has been used on an analytical scale routinely and very successfully for a long time already.

In a first step, magnetic beads with a specific functionalization are added to the biosuspension in order to bind the target protein. Because of the small size of the particles, thorough mixing in the next step achieves very fast binding of the product. Subsequently, the loaded magnetic beads are retained selectively by magnetic separation and separated from the supernatant. Final washing and elution steps deliver the target product free from solids in a purified, often also concentrated form.

Functioning

While the process described represents the state of the art on an analytical scale, the appropriate equipment technology has so far been lacking for implementation on a preparative or technical scale. Our new separation system now constitutes an automated combination, capable of being scaled up, of liquid handling and magnet technology. To carry out protein sorption, stock the wash and elution buffers, and collect the supernatant, the wash solutions and the product solutions a total of six receiving vessels are available (see figure below). The magnetic beads are separated by means of a magnetic separator which, as a result of its patented separation matrix designed in accordance with a rotor/stator principle, guarantees not only effective separation, but also, and just as importantly, effective resuspension of the magnetic beads.

For product isolation, the magnetic beads are first added to the initial solution externally, and the suspension produced in this way is mixed for approx. five minutes. Then the receiving vessel is connected to the instrument and the desired program run is started. The suspension is fed through the magnetic separator by means of a peristaltic pump. In the magnetic separator, the magnetic beads are retained in the separation matrix while the supernatant, which has been cleared of the target protein, inclusive of any solids, such as cells or cell debris, is discharged as waste. After the end of the product feed phase, the remaining supernatant in the magnetic separator is displaced by wash buffer, with the magnetic field switched on, and a closed circuit is achieved by switching the valves.

After turning off the magnetic field, the magnetic beads can now be resuspended quickly and efficiently within the wash buffer by using the rotor/stator system (1st  washing step). Under the impact of the magnetic field, the magnetic beads are separated again after approx. two minutes. The following washing and elution steps are carried out with fresh wash and elution buffers, respectively, in accordance with the pattern outlined above. This is followed by optional cleaning and equilibration steps for the magnetic beads, which makes them available again for the next cycles. The program sequences can be programmed and stored freely for different applications.

Applications

This new instrument can be used to handle up to 10 g of magnetic beads. As a function of their binding capacity and the product concentration in the starting medium, this is able to bind approx. 0,5 - 1 g of target protein. Protein generation on this scale is an important precondition in numerous scientific studies, such as

  • extendet protein analysis by X-ray diffractometry or solid state NMR,
  • tests of enzyme activity and stability,
  • production and characterization of protein immobilisation products.

Advantages

  • Direct protein isolation from starting media containing solids.
  • Simple, automated execution.
  • Very short process time of less than one hour.
  • Flexible as a result of the choice of type and quantity of magnetic beads used.