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mRNA Extraction

Overview of mRNA extraction

The “central dogma” states that DNA gets transcribed into mRNA which is then translated into a protein. The mRNA strands for messenger ribonucleic acid as it is the messenger between the directions of DNA and the creation of proteins by the ribosome. Researchers and industry laboratories extract mRNA from cells to study processes occurring in the cell. mRNA only accounts for 5% of the RNA in the cell so it is important to have a technique which will specifically purify this type of RNA. RNA is also very sensitive to Rnase contamination, which is found all over your skin as an antimicrobial. To avoid contamination it is helpful to have an efficient and simple method for mRNA extraction. A common method for mRNA extraction is the use of magnetic beads

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A glimpse of CACLP 2018, the major Chinese IVD trade show

The China Association for Chemical Laboratory Practice (CACLP) Expo is the biggest and most influential IVD-exhibition in the Asian giant. We attended the 2018 edition, held in the city of Chongqing from March 17th to March 19th. Here are the impressions about the event of some colleagues and our CSO.

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Scale-up Challenges in Biomagnetic Separation Processes

The last two decades have seen an explosive growth in the use of magnetic beads in Life Science, with sustained double figure sales increase all across the industry. The main driver of this success has been the use of magnetic beads as a solid phase on Chemiluminescence Immunoassays (CLIA) kits. Thanks to its easy automation, this technique has become the preferred choice for high throughput In Vitro Diagnostic.

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SEPMAG announces the renewed VERSION of its classical e-books.

Renewed version of ‘The Basic Guide to Scale-Up Biomagnetic Separation Processes’ e-book is already available (FREE)!

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Looking at Operational Safety when Scaling-up Biomagnetic Separation Processes

Scaling up biomagnetic separation process is not just about quantity and quality of the production. One of the main concerns is the operational safety of using ‘big magnets’. As most of the classical magnetic separators are assemblies of permanent magnets in an open configuration, the use of large versions of these devices raises legitimate concerns about the risk for the operators and other laboratory/production equipment. As we will discussed later, the problem does not longer exist using advanced biomagnetic separation systems, but understanding the risks is also key for successfully implement in production environment.

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How to Monitor Scaling-up Process

 

Successfully scaling-up biomagnetic separation processes relies on determining the right working conditions. Having a constant magnetic force in the whole working volume guarantees the in-lot consistency, but manufacturing also needs to guarantee the lot-to-lot consistency.

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How to Scale-up the conditions to a new volume

Almost all life science magnetic beads projects start at small volume. The high cost of the biomolecules (antibodies, protein, nucleic acids….) and the uncertainties involved –what would be the right surface and protocol to coat the beads- make sound to work initially at scales of few milliliters.

As previously discussed, the problem is not working at small scale, but that we don’t pay careful attention when defining the biomagnetic separation conditions. If we leave this task for later stages of the development, we may find important bottlenecks for the scaling-up and, many times, jeopardize the whole project, as the initial conditions may not be scalable at a reasonable cost.

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How to Select the Right Biomagnetic Separation Conditions

To scale-up a Biomagnetic Separation process, selecting the correct working conditions is beyond paramount. Almost any set of conditions may appear to work well enough at very small volume. Classical magnetic separators generate inhomogeneous magnetic force, having some beads magnetically saturated on regions near the retention areas and non-saturated beads in the rest of the working volume. For tubes of one milliliter or less the separation may apparently seems working fine, as the irreversible aggregation problem would not be noticed, the separation time is short and the magnetic beads losses not appreciable.

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Know How Your Particle Magnetic Behaves

 To successfully scale-up a biomagnetic separation process it is necessary to understand how the magnetic beads behave. The separation speed depends on the balance between the magnetic force (generated by the field pattern and the moment of the beads) and the drag force (caused by the buffer viscosity). Thus, it is important to understand how this two forces act on a real magnetic bead suspension.

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Understanding the Magnetic Force for Scaling-up Biomagnetic Separation Processes

To successfully scale up a biomagnetic separation process is necessary to understand the key parameter governing it. To move a magnetic bead we need to apply a magnetic force over it. This force would make the bead move in a direction and be in equilibrium with the drag force generated by the viscosity of the buffer. The result would be a constant velocity (if the magnetic force is constant).

Free guide: 7 Keys to Successfully Scaling-up Biomagnetic Separation Processes

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Magnetophoretic Separation of Microalgae under Homogeneous Magnetic Field Gradient

Microalgae become the exclusive focus in research of biofuel production to meet global energy demand. Photosynthetic microalgae use the sunlight to form biomass from the supplement of carbon dioxide and water. One of the main constituents of microalgal biomass is the natural oil stored within the cells. This natural oil can be further transformed into biodiesel through a transesterification process. The biofuel is renewable with huge potential to replace the fossil fuel. The International Energy Agency has reported that the total final oil consumption of the world in 2010 has reached 3575 Mtoe.1

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Why Do Different Batch Volumes Require Different Magnetic Fields?

Do you want to learn how to take the most of your magnetic separation rack? There are lots of common mistakes related to the scale-up of biomagnetic separation processes, and lots of them imply the use of non-homogeneous magnetic racks.

Download our Free Guide on Biomagnetic Separation Scale-up HERE.
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Why do IVD Companies Scale Up Their Biomagnetic Separation Processes?

When biomagnetic particle kits are initially developed, R&D companies work with small volumes in a magnetic separation rack in order to test and optimize a number of variables. When the kit is deemed successful, the company obviously wants to take the kit to market and consequently ramp up production.

Download our Free Guide on Biomagnetic Separation Scale-up HERE.

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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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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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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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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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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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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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