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When one scales up production using a classic magnetic separation system, one finds that the separation time increases quickly with an increase in production volume. An increase in separation time means that material losses are higher and aggregation problems become a serious problem. By using homogenous separation time, one finds that the magnetic separation process is shorter and the separation time can be easily estimated. In homogeneous systems material loss and bead aggregation is minimized. 

Magnetic beads need to be constantly mixed and homogenized to avoid sedimentation and clumping problems. Even when the sonication method is necessary to break up aggregates, mixing and homogenization is necessary.

In the Life Sciences, one of the most critical parameters for final IVD kit performance is magnetic bead concentration. The beads are functionalized before the magnetic separation process with antibodies or other biological molecules, so the concentration of magnetic beads also delivers a specific concentration of biologically active reagent. If you do not have the correct amount of beads/biological molecules in your preparation, the sensitivity of the kit changes significantly. Therefore volume control of the suspension is quite important.

In non-homogenous magnetic separators, monitoring the entire separation process is difficult to impossible. As a result, errors in the magnetic separation process, such as using the wrong magnetic beads or using buffers with the wrong properties are not detected until the final QC testing stage.

The sonication method is used to break up irreversible magnetic bead aggregates when they are generated during various steps of the production process. However, sonicating a large volume is not a straightforward process. To set up a largevolume sonication, you must choose the proper probe, the power, and the conditions, but choosing and determining these things can be tricky.

This post is about resuspension techniques, such as the sonication method, and how they can solve magnetic bead aggregation. If you are interested in this topic, download our free ebook The basic guide for resuspending magnetic beads:

One very important thing you must ask yourself is ‘why do I have aggregates in the first place?’. There are two possible causes of irreversible aggregation that you need to investigate:

1. Did you properly block the exposed hydrophilic bead surface after coating?

During the production process, you will coat the beads, but you will not always be able to coat their entire surface. If you have not blocked the parts of the beads not covered by your antibodies or antigen, these exposed surfaces can react with other proteins or with the protein on neighboring beads. If you are using the correct blocking reagent, you will reduce auto-aggregation, thereby reducing non-specific background reactions.

Since avoiding aggregate formation altogether greatly simplifies re-suspension, it is worth trying different blocking reagents to find the optimal re-suspension conditions (e.g. BSA, Tween 20, Triton-X 100, etc.).

2. Are you applying excessive magnetic force when separating the beads?

The magnetic force in classical separation systems decreases quickly with distance from the magnets. Because of this, technicians need to generate extremely high forces in the magnetic bead retention areas in order to have enough force to capture beads far from the magnets. In addition, since the forces are weak far from the magnets, separation times are long, causing the beads in the retention area to experience high magnetic forces over long periods of time. High magnetic forces can cause irreversible bead aggregation in spite of optimal blocking techniques.

Modern homogeneous biomagnetic separation systems, however, generate the same force in the entire working volume, thus decreasing the time for bead recovery, increasing the amount of beads recovered and decreasing the forces the beads experience in the retention area over time. Gentler retention forces, larger retention areas and the shorter exposure time to magnetic forces can result in an avoidance of irreversible aggregation altogether.

If you can avoid irreversible aggregation, mixing via homogenization is the only technique necessary to resuspend the beads allowing for a simpler, more predictable process that is easy to scale up.

Don't forget to check these posts from our blog in order to get a deeper insight into magnetic bead resuspension:

• Three Questions to Help Decide What Technique to Use for Magnetic Bead Mixing and Homogenizing
• The 2 Most Popular Magnetic Bead Mixing and Homogenizing Technologies
• Mixing and Homogenizing Magnetic Beads without Touching the Suspension

When magnetic bead reagents are produced in quantity, often you cannot know if you have the correct properties of the beads until the final quality control step. But if these properties are wrong, finding out the properties at the end of the magnetic separation process for production does not allow you to salvage the lot. Knowing magnetic bead properties, such as size, magnetic charge and surface charge, is critical in order to have excellent reproducibility in the final product (e.g. IVD kits).

If scientists and technicians link their production results solely to the separation time on one specific piece of classic biomagnetic separation equipment, they will not be able to translate that success. This is applied to both different batch sizes or even the same batch size on a different piece of equipment, unless they optimize the separation time for the new conditions.

Resuspension of the magnetic beads is the key to guaranteeing both in lot and lot to lot consistency of the resulting in vitro diagnostic (IVD) magnetic bead reagents. In order to achieve resuspension of solids with liquids, scientists and technicians in the lab typically use five different general techniques:

When a lab has finally optimized their production process, they often link their process to a very specific piece of equipment and, by extension, have locked themselves into a constant volume. Often a lab develops its magnetic separation process for production with a specific magnetic separation device – this is normal. Usually the only parameter that needs to be adjusted during production is the separation time.

The problems with magnetic bead suspensions

The major problem with non-colloidal solids suspended in liquids is that they will eventually sediment and clump over time. Magnetic bead suspensions are no exception. Even though magnetic separation is generally gentler than separation by centrifugation, clumps and aggregates can still form. Aggregate formation happens at a much greater frequency if you do not use homogenous biomagnetic separation conditions.