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Magnetic bead separation

How can we specify a Magnetic Bead Separation Process?

When a new CLIA-IVD kit is transferred from R&D to production, all the manufacturing protocols should be adapted to the new throughput and volume. Biomarker specifications, buffers and coating protocols would benefit from the cumulated experience in non-magnetic kits. Coupling an antibody to magnetic beads is quite similar to doing it in colloidal gold or latex particles. But the washing protocols using Magnetic Bead Separation are something new. The use of classical (and dirty) centrifugation method makes not so much sense when we can use the magnetic properties of the beads. Similar reasoning applies to the use lateral flow filtration or other complex and time-consuming non-magnetic separation techniques.

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But, even if using Magnetic Bead Separation seems to be the obvious choice, they are some problems in practice. The first one is how to specify the process itself. When we discuss with IVD-kits manufacturers about their needs on magnetic bead separation, they usually refer to:

  • Separation time: how long it takes to separate the solid phase from the buffer.

  • Magnetic beads losses: what is the maximum amount of beads (and the coupled biomarkers) they accept to be lost during the process.

  • Batch volume: what is the required size of the lot and, sometimes, how to make compatible the different scales required during the process (development, validation lots, full production).

  • The need of avoiding irreversible aggregation: Having irreversible aggregation of the beads during the separation process force to expend a lot of resources to re-suspend them, and to check that the re-suspension is done properly. As each single kit (ml) should have the same characteristics, failure to do it correctly implies an increase in the variability.

However, all these parameters are ‘functional’: they describe the consequences of the Magnetic Bead Separation, but does not describe the process itself. What it is missed here is asking ourselves about the key parameter that defines a Magnetic Bead Separation process. What defines the behavior of the magnetic beads during the process?

The answer is quite simple, but usually overlooked. The key parameter in Magnetic Bead Separation processes is the magnetic force. The magnetic beads move at a definite speed, determined by the net force resultant of the competition between magnetic force and the drag force. This late is caused by the buffer’s viscosity.

It is important to notice that we are talking about the Magnetic Force, not the magnetic field. We should a basic concept: Applying a perfectly spatially-uniform Magnetic Field over a magnetic bead does not generate Magnetic Force. It will just generate a torque that would align the bead’s magnetic moment with the applied field. If we wish to generate a Magnetic Force (and consequently a movement of the magnetic beads), that we need to apply is a Magnetic Field that is not uniform in space.

When we use a simple permanent magnet for separating magnetic beads in a vial, we can do it because the applied magnetic field is not uniform. Then it make no sense to try to define the process by the value of the magnetic field (usually expressed in Tesla or Gauss), as what is key is how it changes with distance.

If we have a look on the formula expressing the value of the force, it would depend on the change of the scalar product of two factors: the bead’s magnetic moment and the magnetic field. The spatial variation of the magnetic field is defined by our magnetic separation rack and it would be fixed if permanent magnets are used as field source. The bead’s magnetic moment it would change, depending on the applied field. If the beads are superparamagnetic, the magnetic moment would vary linearly with the magnetic field when this is low (the materials would have constant magnetic susceptibility). At high values of the applied magnetic field, the bead’s magnetic moment becomes saturated, being approximately constant. On the bead’s linear magnetic response region, it is not easy to have a magnetic field profile leading to a well-defined homogenous force: we would need a constant gradient of the square of the applied field. However, if the magnetic beads are saturated, having a constant magnetic field gradient would lead to a well-defined homogenous magnetic force.

Then, if we fulfil both conditions – magnetic saturation of the field and constant magnetic field gradient-, we will have specified the magnetic force governing your Magnetic Bead Separation processes.

Figure 3 How to specify resized 600

Figure 3. Magnetic force expression for the different region of superparamagnetic beads magnetization curve

With this concepts in mind, it would be easy to understand how to avoid the most common and critical mistakes when using this technology in CLIA-IVD kits production. Remember that last week we published the first article in this series. You can check it in the following link:

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Lluis M. Martínez | SEPMAG Chief Scientific Officer

Founder of SEPMAG, Lluis holds a PhD in Magnetic Materials by the UAB. He has conducted research at German and Spanish academic institutions. Having worked in companies in Ireland, USA and Spain, he has more than 20 years of experience applying magnetic materials and sensors to industrial products and processes. He has filed several international patents on the field and co-authored more than 20 scientific papers, most of them on the subject of magnetic particle movement.

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