A great deal of work goes into characterizing and parameterizing the magnetic beads themselves. Almost every potential user knows how to specify them: their diameter, density, magnetic pigment content, surface activation (plain, covalent, bio-functionalized) and even the acceptable variations of these parameters.
However, magnetic bead separation involves more than just beads. A magnetic field from an external source is required to move them, which is usually a magnetic separation rack. Biomagnetic separation processes cannot be well defined by determining specifications for the magnetic beads alone.
This post is about magnetic bead separation and how to validate this process. If you are interested in this topic, and are willing to learn more about it, download our Free Guide The Starting Guide to Validate Biomagnetic Separation Processes:
Maintaining lot consistency in magnetic bead separation systems
How can lot-to-lot consistency be ensured without a well-specified magnetic rack? How can in-lot consistency be ensured if we do not know if the magnetic bead separation conditions are homogenous in the working volume?
The problem exists when working on a small scale, but can be masked by the variability of the magnetic beads themselves. It is when the magnetic bead separation process involves different working volumes (because validation is done at different scales or production volume is larger than final application), that the problem arises due to long separation times, significant increases of magnetic beads (and biomolecules) losses and irreversible aggregation problems (clumps).
These problems are often no more than the consequences of lack of specification in the magnetic field profile used for the magnetic bead separation because, in most cases, nobody is even aware of the need to take this factor into account.
To avoid these issues, we simply need to ask ourselves the key parameter to define a biomagnetic separation process.
The answer is not too complex. The key parameter to define the biomagnetic separation process is magnetic force. Magnetic force determines the magnetic bead separation speed by overcoming opposition to the drag force generated by the viscosity of the buffer.
Magnetic bead separation speed is defined by magnetic force
Let's look at some basic concepts of magnetic force. The first is that homogenous magnetic fields do not generate magnetic force. They only generate magnetic torque. Regardless of the strength of the magnetic field, if it is perfectly homogenous, the beads do not move. They merely spin to align their magnetic moments with the field.
To generate a magnetic force, a magnetic field is needed that changes with distance. In short, when a strong permanent magnet is used magnetic bead separation occurs because it generates a strong magnetic field variation, not because of the high value of the magnetic field.
A closer look at the formula shows that magnetic force depends on both the beads' magnetic moment and on the magnetic field profile. Depending on the magnetic behavior of the beads, magnetic moment changes linearly with the applied magnetic field (when susceptibility is constant) or, if the field is strong enough, their magnetic moment is constant (the bead is magnetically saturated).
By rewriting the magnetic force expression for each of the two magnetic behaviors, we found that the dynamics of the beads are different. For very low fields (when susceptibility can be considered constant), magnetic beads experience a force proportional to the gradient of the square of the magnetic field. However, when saturated (high fields), this depends on the gradient of the magnetic field intensity.
If we simultaneously fulfill the saturation of the magnetic material and a constant magnetic field gradient, we obtain a constant magnetic force (exactly what we achieve in our Sepmag Biomagnetic Separation Systems). Now, having defined the magnetic bead separation process, we can look more closely at the process dynamics.
The simplest assumption is that every magnetic bead moves independently. In this case, if we know the magnetic moment and the magnetic field gradient it is easy to calculate the magnetic bead separation speed. The separation time for the farthest bead is obtained by simply dividing distance by speed.
We can then test the formula by comparing experimental results. Using a cylindrical vessel and a Sepmag system that generates a radial magnetic field gradient, we can measure the light traversing the suspension. If we repeat the experiment with a second system with a different gradient value, we can determine whether the formula correctly describes the magnetic bead separation process. [Ref 1]
The results in the figure show very good agreement for the small diameter magnetic particles used in this experiment. However, the magnetic bead separation time lasts one day, and life science applications usually require separation times of a few seconds! This theory correctly predicted the separation time and the effect of doubling the value of the magnetic field gradient, but these small magnetic beads would be impractical for IVD applications such as CLIA immunoassays.
The same calculations can be done for suspensions with larger magnetic beads and using a similar magnetic field gradient. In this case, the predicted separation times are far longer than experimental values. The experiment showed that this magnetic bead separation takes less than three minutes, but the calculated value, assuming the beads move in the same way as isolated particles, gives a one-hour separation time.
What is happening? The explanation is quite simple. The magnetic beads used in most Life Science applications do not behave like ‘isolated’ particles.
Bead behavior in magnetic bead separation
When a magnetic field is applied, the magnetic beads become magnetized and each one behaves like a small magnet, aligning with their neighbors and forming chain-like structures. These clusters move like very large ‘beads’, much faster that isolated magnetic beads. Note that, as the video shows, the chains are formed in the direction of the magnetic field, but the movement is along the magnetic field gradient direction.
If magnetic beads are superparamagnetic, once the magnetic field is removed (i.e. the vessel moved away of the magnetic field) their magnetic moment becomes zero and the chains dissolve.
This collective behavior has important practical consequences. As chain-like structures are key to the magnetic bead separation process, the concentration of the beads in suspension has an important impact on the separation time. The higher the concentration, the faster the magnetic bead separation: the closer the neighbor, the easier and quicker it is to form chains. [Ref 2]
Then, having defined the magnetic bead separation conditions, we have found that our beads can work in isolation or cooperatively and that separation times can change by several order of magnitudes depending on this, regardless of the magnetic separation rack used.
How do we know when magnetic beads are acting cooperatively? Using homogenous biomagnetic separation system and monitoring the process, UAB and ICMAB researchers have developed models and tested them in cooperation with Sepmag R&D staff. [Ref 3]
They have defined an expression for the average length of the chains, N*. If N* is greater than 1, this denotes cooperative behavior. If N* is well below 1, the magnetic beads separate as isolated particles.
The value of N* is proportional to the square root of the concentration, but depends exponentially on, Γ, the ratio between the dipole-dipole magnetic energy and the thermal energy.
If we plot the value of N* according to the diameter for fixed magnetic pigment content, we see that by increasing the concentration we can collectively separate magnetic beads a little less bigger, but the key parameter is bead size (note the logarithmic scale for the Y-axis). Increasing magnetic pigment content (or its magnetization) may be also a good strategy, but would also usually increase bead density and therefore the sedimentation ratio.
To conclude, once we have correctly defined the magnetic bead separation conditions – or the biomagnetic separation process as we prefer to call it - it is easy to understand what is happening in the suspension when the magnetic field interacts with the beads. That is the key to developing products and processes that can easily be replicated and transferred to different working volumes. From microliters in the final analyzer to tens of liters in production facilities, if the full magnetic bead separation process, rather than just the beads themselves, is well defined, the result is a cost-efficient, smooth ramping production process.
If you found this article interesting and want to get a deeper insight in the topic of magnetic bead separation, make sure to check these articles from our blog:
- The key to consistency: validation of biomagnetic separation processes
- The weakest link in IVD production
- The 6 key factors affecting the behavior of magnetic beads
- M. Benelmekki & Ll. M. Martinez “Magnetophoresis of iron oxide nanoparticles: A tool for synthesis monitoring and biomagnetic applications” "Drug Delivery and Nanomedicine" vol 5. Editor J.N. Govil, Studium Press LLC, USA (2013).
- J. Faraudo & J. Camacho (2010). Colloid Polym. Sci., 288:207
- J. S. Andreu et al, PHYSICAL REVIEW E 84, 021402 (2011)