Magnetic bead separation has become a very popular technique in life sciences. Magnetic bead separation is a quick, efficient, clean process that scientists use instead of filtration, centrifugation and separation techniques.
Magnetic beads and particles can be coated with specific affinity ligands for antigens, antibodies, catalyzers, proteins or nucleic acids. After being incubated with the suspension for a time, the vessel is introduced in a magnetic separation rack. The beads (and attached biomolecules) are attracted and retained by the magnetic force. Once separation is complete, the supernatant, which is not affected by the magnetic fields, can easily be removed. The biomolecule, attached to the magnetic beads, is already isolated from the original media and it can be eluted, if necessary.
This post is about biomagnetic separation with a magnetic separation rack, and how to scale-up this process. If you are interested in this topic, download our free ebook The Basic Guide to Scale-up Biomagnetic Separation Processes:
Our aim is to help you to understand how a life sciences magnetic separation device works. Once a few simple concepts have been grasped, it is easy to make optimal use of the magnetic field and to choose a suitable magnetic separation rack or 'biomagnetic separation system', as we prefer to call it.
Magnetic beads and the magnetic separation rack
There is copious reference material available on the subject of magnetic beads and how their characteristics (diameter, density, magnetic pigment content, surface activation) affect the efficiency of the process. It is because of this that when problems appear, i.e. high losses, slow separation, inconsistent results, clump formation, the focus tends to be on the magnetic beads themselves. Much time and money are spent analyzing bead size distribution and magnetic properties, testing alternative products/suppliers and reviewing coating and incubation protocols. This is often a waste of time and, as deadlines draw close and cumulated investments are put in jeopardy, relationships with providers, colleagues and management can become strained.
Before this happens, it is important to remember that the magnetic bead separation process involves more than magnetic beads alone. Magnetic carriers need to interact with a magnetic field in order to move. The magnetic field must generate a magnetic force which is b enough to overcome the drag force generated by buffer viscosity. If the magnetic separation rack that does not generate the appropriate magnetic force, which may not necessarily be a ber magnetic field or force, the entire process becomes inefficient, regardless of the characteristics of the magnetic beads. Despite the importance of the subject, detailed technical literature describing how magnetic fields interact with beads is very thin on the ground.
Knowing the importance of homogenous magnetic fields
The basic and oft-forgotten concept is that homogenous magnetic fields generate magnetic torque but not magnetic force. When beads are successfully separated with a simple neodymium-iron-boron magnet, this is not because of the strong magnetic field generated. It is because of the significant change in the magnetic field and distance. To generate a magnetic force, an inhomogeneous magnetic field is required.
The second important concept is that the strength of the magnetic force exerted over a bead depends on both magnetic moment and on the magnetic field profile. The magnetic moment of the bead depends on the magnetization curve. For superparamagnetic materials, when the magnetic field is very low, the magnetic moment is proportional to the intensity of the applied magnetic field (constant susceptibility). When the field applied is high enough, the magnetic properties become saturated and bead magnetic moment is constant. The exact ‘saturation field’ value depends on the specific material used for the ‘magnetic pigment’, but for a typical nanosized iron oxide used on commercial beads, the applied magnetic field should be over B>0.1 Tesla (80 kA/m).
A simple magnetic separation rack is a straightforward piece of permanent magnet. Calculating the value of a magnetic field in each point in space and then determining the gradient is not too complex. The graph shows the results as we move away from the center of the surface of the magnet. In the best case scenario (assuming the magnetic beads are always saturated) the magnetic force changes very quickly according to the distance from the surface. When different magnet sizes are compared, it may seem surprising that small magnets generate ber forces near the surface than larger ones, but this is because the larger the magnet, the more constant the magnetic field and, as we have just learned, a constant magnetic field does not generate force.
Some magnetic separation devices try to get around this by using two permanent magnets with opposite polarity. This means that the field is never constant near the center of the assembly and that there are greater forces near the permanent magnet surface where we want the magnetic beads to be at the end of the process.
However, the resulting magnetic force is extremely uneven, that is to say its value changes quickly with distance, which means that the force exerted on the beads closer to the magnet is very strong, while the force on the beads farther away from the surfaces of the magnet is very weak. This force profile has two undesired practical drawbacks on magnetic bead separation process:
- Very long separation time (or high sample losses): Because the force on the most distant beads is very weak, it takes a long time to recover all the magnetic beads and their attached biomolecules and in the case of vessels larger than a few milliliters, this can be up to several hours. There is an option to halt the process earlier but this would mean accepting significant losses.
- High risk of irreversible aggregation: Because the nearest beads are subjected to a greater force, they move very quickly. Once they reach the retention area, they experience an even greater force and build up against their neighboring beads, forming clumps. Given that the separation process of the farthest beads (with acceptable losses) involves an extensive separation time, those which quickly reach their final position are exposed to extremely high forces for a very long time.
Solving aggregation and distant bead loss
To make the process efficient, the magnetic separation rack must be modified to solve both problems simultaneously. By increasing the force, using, for example larger assemblies of magnet pieces, loss of the farthest beads is reduced but there is far more irreversible bead aggregation near the surface of the magnet. In contrast, reducing the magnetic force in the retention area palliates the irreversible aggregation problem but significantly increases loss of magnetic beads and their attached biomolecules.
To solve both problems simultaneously, more force must be exerted on the farthest beads while reducing the force in the retention area. It is difficult to find a solution with a magnetic force profile that changes with distance, as is the case with classical magnetic separation racks. The ideal solution is to have a magnetic force that does not change with distance. When the force is homogenous (as is the case with Sepmag’s Biomagnetic Separation Systems), it is possible to have a higher magnetic force farther from the retention area and a lower force near the magnet at the same time.
This means that the most distant beads travel faster and the entire magnetic bead separation process is faster without causing additional loss. The beads reach the retention area at a constant pace because all the beads move at the same speed and are subject to a gentle force – just strong enough to retain them when the supernatant is extracted. What's more, no clumps are formed and sonication is avoided, greatly simplifying the scaling up process.
Magnetic separation rack vs homogeneous systems
The difference between a magnetic separation rack (on the right in the video) and a homogeneous biomagnetic separation system (on the left in the video) is evident. Using the same number of permanent magnets, the separation time is much shorter even though the system on the left is far gentler on the beads throughout the process to avoid clumps.
The exact value of the force depends not only on magnetic gradient but also on the magnetic moment of the beads and buffer viscosity, therefore there is no universal magnetic field gradient value that can be recommended. Fortunately, the companies that develop and manufacture advanced biomagnetic separation systems have amassed a great deal of experience in the field and can help you to determine the final value.
The choice of the right gradient is important because too low a value makes it impossible to separate small beads or beads in high viscosity suspensions (such as, for example, whole blood). In contrast, too high a magnetic field gradient is counterproductive for large beads in water-based suspensions because the resultant magnetic force would be excessive and generate clumps. With most life science processes, there is no one system suitable for all magnetic bead separation processes.
Using devices with different magnetic forces at each point of the working volume makes it difficult to validate separation processes. Given that the magnetic separation conditions for each bead are different, it is difficult to parameterize the process and identify the key values of an efficient setup. However, working with a homogeneous magnetic force makes it possible to test the process in well-known conditions for the entire suspension, checking different magnetic forces as necessary and determining the right value for the magnetic bead separation process.
Don't forget to check these posts from our blog in order to get a deeper insight into the scaling-up of biomagnetic separation processes:
- Why do IVD companies scale up their biomagnetic separation processes?
- Why do different batch volumes require different magnetic fields?
- The 2 Questions to answer before scaling up your magnetic separation rack