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Your magnetic beads separation process should be validated, and quality control should be implemented to ensure consistent results. This is simple when the magnetic force is constant and properly defined. If the magnetic force is constant, different batches of the same product should have the same separation curve. scientists can then check the separation process is running correctly by measuring the absorbance in real-time at different steps of the process.

In recent decades with the breakthrough of biotechnology, magnetic beads have been introduced as a useful, multifunctional tool in biomedical science. The utility of magnetic beads especially owes to their ability to selectively bind to and separate a huge variety of biomolecules of interest from biological samples. However, the outcome of protocols using magnetic beads highly relies on implementing strict quality control measures to ensure reliable yields.

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Validating the Magnetic Separation Process

Validation of a magnetic separation technique by the application of magnetic beads is an important component of the protocol to adhere to. To achieve this, the separation time and qualitative yields must be monitored. Based on the classical separation methods, the “transparency” of the supernatant marks the final phase of the separation procedure by eyesight (done by the operator) or by spectrophotometry (by sampling a small volume). Nevertheless, these methods don’t provide information about the process, just about the final state. For example, if beads aggregates are present or the supernatant should have the right properties at the end of the separation process.

Transparency Check

Checking the transparency of the supernatant has been one of the most widely accepted criteria for a separation procedure. Transparency, however, might not be reliable in terms of final yield as it is based on the operator’s subjective judgment and only offers the final outcome of the process with no information about the dynamic changes of the separation itself.

Spectrophotometry

Using spectrophotometry is another method to identify the accomplishment of the separation procedure which involves sampling a small volume of the supernatant and measuring its absorbance. Although spectrophotometry provides quantitative data on the final phase of the supernatant, it lacks the necessary information on the separation procedure itself.

Real-Time Monitoring for Quality Control

In order to optimize and elevate quality control of the magnetic separation procedure real-time monitoring of the changes that occur within the solution at each step is critical as it assures the desired quality criteria and consistency. Unlike the two aforementioned criteria, real-time monitoring provides detailed information on the ongoing process, detects early-phase issues, and enables troubleshooting them. With real-time monitoring different batches of the same product should have an easily measurable separation curve.

The Significance of Standardized Conditions

It should be borne in mind that optical changes of the separation procedure are established only when standardized magnetic separation conditions are met, otherwise it would be challenging to interpret them. This is because when using classical separators the magnetic beads at different regions within the sample may experience varying magnetic forces. This phenomenon can lead to a complex interplay between potential quality issues and spatial variations in magnetic force across the sample.

Nevertheless, a constant magnetic force system will ensure standardized conditions as the magnetic beads move at the same speed, regardless of their starting position.  This, in turn, leads to a more predictable and interpretable magnetic separation curve. When the magnetic force is constant, the real-time absorbance measure of monodisperse magnetic beads suspensions fits to a sigmoidal curve. From this information,  you can determine the critical parameters used to analyze your separation curve, t50 and the exponent p.

Parameters for Quality Assessment

The t50 parameter, representing the time required for half of the magnetic beads to complete separation, serves as a crucial metric for quality control. Changes in this parameter can indicate deviations from the expected behavior of the magnetic suspension. There are several factors that influence t50:

  • Presence of Aggregates: with aggregates and/or clumps in the sample, the t50 value will be shorter than expected, indicating incomplete separation.
  • Viscosity Changes: Alterations in the viscosity of the buffer solution can impact the separation speed. Higher viscosity leads to a slower separation process.
  • Concentration Effects: Changes in the concentration of magnetic beads in the suspension can also affect t50. According to theoretical models, t50 is inversely proportional to the fourth root of concentration. Higher concentrations slightly decrease the separation time, while lower concentrations slightly slow it down.
  • Temperature Variations: The temperature of the suspension can significantly alter the separation time. For instance, at room temperature, water viscosity decreases by approximately 2% per degree Celsius, which can have a substantial impact on the separation process.

Real-Time Monitoring for Early Detection and Cause Identification

The real-time measurement of the optical properties (absorbance) of the suspension during the separation when the magnetic separation conditions are properly standardized are increasingly important. Not only do they allow you to detect quality control problems in early stages of experimentation, but also help determine the cause of the problem.

Quality control in protocols involving magnetic beads is essential to guarantee reliable and reproducible results. Validating the magnetic separation process and implementing real-time monitoring under standardized conditions are pivotal steps in achieving this goal. These measures not only enhance the quality of the results but also offer insights into the underlying processes, enabling the identification of quality problems and their causes. Real-time monitoring, combined with standardized conditions, provides a comprehensive approach to quality control in the realm of magnetic beads technologies.

Quality Control of Protocols

Optical changes of magnetic bead suspension during the separation process. The initial state is an opaque homogenous liquid. When the solid magnetic phase is separated, the liquid has the optical properties of the buffer.

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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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