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Agarose Resin Chromatography

Introduction to Chemiluminescence immunoassays

Using Agarose Resin chromatography allows for a versatile, prepacked column that enables small-scale, high-resolution size exclusion chromatography for preparation, characterization, and analysis of proteins and other biomolecules. Size exclusion chromatography, also known as gel filtration, is a common technique used to separate compounds of small molecules, such as proteins, polysaccharides, and nucleic acids when in an aqueous solution. This can be extremely useful in numerous commercial applications, such as analysis and determination of an unknown sample, removal of large proteins for purification of a sample, for removing small molecules such as dyes and primers, and buffer exchanges. Such chromatography is purchased as a prepacked column that allows for increased resolution and analysis, quick runtime, with a high pH tolerance to allow applications to a variety of molecules and substances. One example of a commercially available separation is Superose 6 chromatography, found here.

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Monitoring Biomagnetic Separation Processes: Sepmag® launches QCRv2

The traditional way to check whether a biomagnetic separation process is complete is by sight. The technician/researcher looks at the suspension: at the beginning of the process, the suspension is homogenous and opaque, but when the separation process is complete, the magnetic beads are left on the walls of the vessel and the supernatant is transparent. When the suspension is ‘transparent’, the technician stops the process by extracting the supernatant, leaving the magnetic beads in the bottle.

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Perfecting the magnetic separation process in magnetic bead IVD kit production

Magnetic bead kits are widely used in bioscience laboratories nowadays, as their development and perfecting during recent years has been significant. Both scientists in research labs, for immunoassays and magnetic separation processes, and lab technicians in hospitals, using IVD kits for molecular diagnostics, have experienced an improvement in their separation processes due to these magnetic bead kits. As using a biomagnetic separation process has proven useful for these applications, the increasing demand in magnetic beads has lead providers to develop cost-effective ways for their production.

Download our FREE guide about Biomagnetic Separation for Production HERE
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Will IKERLAT superparamagnetic latex microspheres work with Sepmag® Systems?

When new magnetic beads reach the market, one of the questions users have is, how well will it separate?

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Resuspension problems during the biomagnetic separation process

End product variability is caused in large part by resuspension problems in your process. During a magnetic separation process, the retention forces in the system need to be high enough that losses of the beads will be avoided when the buffer is pumped out. But the forces should not be too great because if they are excessive, beads will aggregate.

Download our FREE guide about Biomagnetic Separation for Production HERE
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The Safe Use of Biomagnetic Separation Systems in Production Facilities

Large magnetic systems create potentially high risk environments in terms of safety during a magnetic separation process. These systems generate large magnetic fields which result in the generation of large stray fields. The stray fields affect the surrounding environment and can attract ferromagnetic objects, potentially injuring system operators. The larger the system, the higher the risk, especially if workers do not follow Health and Safety protocols.

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The Effect of Magnetic Bead Concentration in Biomagnetic Separation

The concentration of magnetic beads is an important step in a magnetic separation process. Separation time is dependent on magnetic bead concentration, and final kit performance is also very dependent on accurate concentrating techniques, but liquid handling inaccuracies can lead to serious errors. If these concentration errors are not detected early in the process, excessive time, money and effort will need to be spent to either correct or redo the batch.

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New paper about the behavior of magnetic beads separation

Learning more about the behavior of magnetic beads separation when strong magnetic fields are applied

 

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Determining the Right Separation Time during Biomagnetic Separation Processes

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. 

Download our FREE guide about Biomagnetic Separation for Production HERE
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How do concentration changes affect Biomagnetic Separation Processes?

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.

Download our FREE guide about Biomagnetic Separation for Production HERE
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Monitoring with Homogenous Biomagnetic Separation to detect QC Issues

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.

Download our FREE guide about Biomagnetic Separation for Production HERE
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Issues with lot-to-lot inconsistencies in magnetic bead processing

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).

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3 Key Parameters for Defining a Production Process in Biomagnetic Separation

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.

Download our FREE guide about Biomagnetic Separation for Production HERE
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Problems from linking your biomagnetic separation process to a specific type of equipment

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.

Download our FREE guide about Biomagnetic Separation for Production HERE
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How to save space in the clean room during a magnetic separation process

Classic magnetic separation equipment requires a large amount of space in order to comply with health and safety regulations. While the magnetic separation process has numerous advantages, the magnetic fields surrounding the devices may be so large that they fall within the ‘danger’ and/or ‘caution’ areas.

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Objectively Quantifying the Separation Time of your Biomagnetic Separation Process

The separation time in standard magnetic separation devices is usually determined by analyzing aliquots of solution taken at different times. The problem is that each aliquot gives the technician information about one spatial point in time. Therefore, the design of validation experiments becomes a very complex endeavor.

Download our FREE guide about Biomagnetic Separation for Production HERE
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Safety Risks using Magnetic Systems during Magnetic Separation Processes

When using biomagnetic separation systems, customers are always curious about how to comply with the various health and safety regulations that are in effect. When customers use small systems for a small scale magnetic separation process, there is very little risk from the magnets. The only risk would be if the technician has a pacemaker and in that case, they would be extremely careful around even the smallest system. There is also a small risk of pinching one’s fingers between two magnets.

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Keep Magnetic Bead and Biomolecule Losses near Zero during Production

When scaling up a process using a traditional magnetic separation rack, the percentage of bead and biomolecule losses significantly increases with an increase in volume. One way of dealing with this problem is by applying a higher force at longer distances. But for this to work, you must apply this greater force without increasing the forces in the retention area during the magnetic separation process, in order to avoid irreversible aggregation.

Download our FREE guide about Biomagnetic Separation for Production HERE
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How to Guarantee Lot-to-Lot Consistency in Biomagnetic Separation

If one wants to scale up production from small lab lots to full-scale large lots, a non-homogenous magnetic separation process will result in lot-to-lot inconsistencies. Homogenous biomagnetic separation conditions, however, guarantee consistent results regardless of production scale.

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Centrifugation and Filtering with Biomagnetic Separation

Biomagnetic separation techniques are faster, cheaper and easier to use than non-magnetic techniques. In addition, when a magnetic separation process is performed under homogenous conditions, these techniques are also scalable and easily validated.

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Detect Resuspension Problems with Biomagnetic Separation Processes

Due to the inherent properties of classic non-homogenous biomagnetic separators, beads can aggregate during the magnetic separation process. When this happens, technicians try to resolve the magnetic beads separation problem by using special resuspension techniques like the sonication method. But problems with resuspension can ultimately lead to end-product variability, especially if aggregation is not detected early.

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The Use of Biomagnetic Separation in Production Processes

Magnetic separation is a breakthrough technique for in vitro diagnostics (IVD). Scientists, hospitals and companies have taken advantage of the magnetic separation process for immunoassays, molecular diagnostic and genetic testing systems and kits. However, this type of technology is typically utilized by the end-user in very small quantities.

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Avoiding Irreversible Aggregation Problems during Production

In-lot consistency is the key to reproducibility at the level of a kit. Unfortunately, in non-homogenous systems irreversible aggregation is one of the main sources of in-lot variability. If all of the beads are exposed to the same force as they are in homogenous magnetic systems, the risk of aggregation is greatly reduced. Because of this, it is important to know how to avoid irreversible aggregation problems during a magnetic separation process.

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Why does separation time increase more quickly than the production volume?

A recognized problem in the biomagnetic separation industry is that when one increases the batch size to scale up production of magnetic beads, the magnetic separation process time increases unproportionally to the increase in volume if one is working with standard magnetic separation devices.

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Why do bead losses increase during production?

One of the biggest problems of producing magnetic beads when scaling up the production is that compared with smaller lot production, larger lot production seems to result in a much larger disproportionate loss of beads. This seems to happen even when the beads are produced in conditions that are similar to the small lot production in a magnetic separation process. The assumption is that when you scale up a process, you will have greater efficiency, but this does not happen when scaling up production of magnetic beads using classical separators.

Download our FREE guide about Biomagnetic Separation for Production HERE
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How do non-homogeneous magnetic separators jeopardize lot consistency?

It is understandably important to end users that every kit within a particular lot have the same properties. In other words, when one is producing lots of material to be used in an IVD kit, one necessarily strives for maximum reproducibility and minimal variability. With standard magnetic separators, it is very difficult to achieve this goal in a magnetic separation process.

Download our FREE guide about Biomagnetic Separation for Production HERE
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Common mistakes that lead to inconsistency

Often when a lab produces a product that becomes popular, the impetus is to move forward and scale up production of that product. The problem is that moving from the production of small lots to full scale production usually produces surprising results. Scaling up is not trivial, and the magnetic separation process is no exception. When one scales up production, results become very inconsistent.

Download our FREE guide about Biomagnetic Separation for Production HERE
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Production of magnetic bead kits: To be or not to be magnetic

What are the problems of the classic magnetic separation process? Typically the classic ways to produce magnetic bead reagents and kits are slow, very high maintenance and costly to run. The three classic techniques, centrifugation, filtration and tangential filtration, are not straightforward techniques.

Download our FREE guide about Biomagnetic Separation for Production HERE
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Set up of Biomagnetic Separation Production Processes

Scientists in academic research labs and pharmaceutical labs perform magnetic separation process with magnetic bead kits for immunoassays and separation science. Doctors, lab technicians and scientists use magnetic beads in IVD kits as molecular diagnostics devices.

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