Traditionally, biomagnetic separation users have not monitored the separation process. The nature of classical separators, where the magnetic force changes with the distance, means you can determine when the separation is complete (the buffer becomes transparent), but it is difficult to interpret the optical changes during the process. This is because every location sampled will have a different bead concentration due to their different speeds. In addition, it is difficult to compare different batches as even a small difference in the vessel’s position within the separator will affect the beads’ behavior.
However, when the magnetic force is constant over the entire suspension, the separation speed is the same everywhere, making the optical changes easier to compare between batches and interpret (independent of changes in vessel position). As the magnetic force is simply the result of the competition between the magnetic force applied and the drag force generated by the buffer, when the magnetic gradient is constant, changes in separation speed are easy to link to differences in the magnetic bead suspension alone. Changes in the suspensions (e.g., size, magnetization distribution, concentration, buffer viscosity) will lead to different absorbance vs time curves.
Quality control of the separation process also becomes particularly important at larger scales. Ensuring that the process is the same for each batch can be achieved by monitoring and parameterizing your processes’ separation curves. Using optical monitoring software such as Sepmag’s ‘Qualitance’ allows users to gain greater control and insight into their process.
Figure 7.1 A vessel containing a suspension being placed in a Sepmag system.
Monitoring variation in the separation process
Using traditional methods to control the separation process, technicians often rely on the fact that the magnetic bead suspension is initially dark. During the separation process, the solid phase moves to the retention area and the buffer becomes transparent. The end time of the process is judged either by sight or, when more precision is required, by sampling at different time intervals and checking the absorbance using a spectrophotometer. This can then be compared against a reference value.
A key limitation of this approach is that it only allows analysis of the final result: whether the separation is complete after a given time. This doesn’t provide any information about what happened during the process. It is also not possible to detect a faster-than-expected process when aggregates are present if the absorbance is only measured at the separation time. So why might the process vary?
When using classical magnetic separators, any variations in magnetic behaviour due to changes in the suspension or beads will be different at different locations in the working volume, making the data difficult to interpret. It is therefore unusual for the separation process to be monitored when using classical separators. However, when using a constant magnetic force separation system, the information provided by real-time optical monitoring is far easier to analyze.
This is because the separation speed is the same throughout the working volume (resulting from the competition between the magnetic force and the drag force), so the dynamics of the magnetic bead depend directly on their magnetic moment, diameter, and concentration as well as the buffer viscosity. In other words, changes in the suspension are the sole source of variation in separation time when using a constant magnetic force. Any changes in the viscosity of the buffer caused by variations in temperature or substrate composition are therefore easily identifiable by monitoring the time absorbance variation during the separation process.
For production processes, monitoring the separation process can be highly valuable for users because while the magnetic force is constant, the initial state of the suspension can affect the process. For example, aggregates move faster than well-resuspended magnetic beads and so have a shorter separation time. Changes in temperature and any additive will also affect the viscosity of the buffer, which again will affect the separation time. For checking the batch-to batch consistency, users can simply check that the absorbance vs time curves are the same. Any errors in the re-suspension process, composition or even temperature, will lead to different behavior during the separation process.
The old saying ‘magnetic beads don’t work at large volumes’ is, simply, wrong. If you want to learn what leading IVD-manufacturers already know, this is the e-book for you!
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