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Feature Articles : Jul 1, 2010 (Vol. 30, No. 13)

Biomagnetic Separations' Abilities Increase

Major Product Expansions Are Surfacing to Support an Assortment of Applications
  • Gail Dutton

After years of minor tweaks to biomagnetic separation technology, manufacturers are introducing major product additions that support molecular diagnostics, cell therapy, stem cell separations, and other new markets, as well as new separation devices that include microfluidics, modularity, and enhanced capabilities.

In May, Miltenyi Biotec announced a strategic collaboration with Stemgent to jointly develop and commercialize technologies focusing on embryonic and induced pluripotent stem cells including isolation products. Before that announcement, Miltenyi introduced a cancer stem cell isolation kit using the CD44 marker, which isolates a broader range of cancer stem cells than is possible using the standard CD133 marker.

Consequently, this kit will be useful in answering basic biological questions such as how tumor cells undergo epithelial mesenchymal transition following metastasis from the primary tumor site. Potential applications for the clinical market related to cancer stem cells may include diagnostics and the development of therapeutics, according to Kirt Braun, Ph.D., marketing manager at Miltenyi.

“Other new releases, anticipated this summer, include new products for our core customer base in immunology such as kits to isolate Langerhans cells, B cells, and memory B cells,” Dr. Braun says. “To further support Langerhans cell research, we will soon release a comprehensive solution that combines a mouse epidermal Langerhans cell isolation kit with a mouse epidermis dissociation kit to be used in conjunction with the gentleMACS™ dissociator. Together these replace home brew and mechanical methods of obtaining these cells, while providing high cell purity and recovery with greater than 90 percent viability rates.”

Miltenyi also developed a kit to isolate human mitochondria. “This is a true game changer,” remarks Dr. Braun. “Previously, mitochondria were separated through centrifugation, which isolates only a fraction of the total complement of mitochondria within a cell based solely on size.” Mitochondria isolation including sample preparation typically requires most of one day. “Miltenyi’s mitochondria isolation kit performed the isolation in less than one hour, isolating the full range of functional mitochondrial, often with higher purity.”

Microfluidics

Cynvenio Biosystems has developed an integrated biomagnetic separations system using microfluidics technology and is commercializing it with a major life science partner. According to H. Tom Soh, Ph.D., CTO, the micromagnetics technology used in the system improves the purity, rare-cell recovery, and throughput of the separation, and allows multiple process steps to be integrated in a closed, disposable system.

Previously, microfluidics technology was used for small sample volumes at relatively low throughputs. Cynvenio’s device, however, is scalable to throughputs exceeding 100 mL/hour in a single cartridge, which is ideal for clinical applications, Dr. Soh says. “We use miniaturization for features that give us large performance benefits. The throughput features, in contrast, are large to handle clinically relevant applications.”

Cynvenio is focused on developing the cartridge technology, wrapping an open platform instrument around it, and adding push-button automation. Cartridges range in size from that of a glass slide to about the size of an iPhone. The current device will integrate the sample-prep and separation processes, and work is under way to automate postseparation steps as well, says André de Fusco, CEO. Specific applications remain proprietary. “More work on the reagent side will come next year.”

A competing microfluidics biomagnetic separations device was just introduced by Life Technologies’ Invitrogen division. The MAGic™ Sample Processor processes up to 12 immunoprecipitations of His-tagged samples simultaneously using Dynabeads® technology. It uses a pipetting comb and cartridge format to reduce variability in manual sample and buffer preparation and handling, and features a heating and cooling mechanism to minimize sample degradation. Initial applications are in epigenetics and protein isolation, but the processor is designed to handle additional genomic and proteomic applications.

Paul Pickering, Ph.D., GM of Invitrogen’s cell therapy systems, says the MAGic Sample Processor is receiving great interest, but other applications are also being developed. His area, for example, is working closely with the California Institute for Regenerative Medicine in basic and translational research for embryonic and pluripotent stem cells.

“Translational work is not an easy process,” Dr. Pickering stresses. “The clinical research environment is more controlled than basic research.” Therefore, Life Technologies is upgrading its research products to accommodate those inherent variables.

“The inability to ensure the total separation of potentially teratoma-forming stem cells has slowed translation of stem cell therapies. We’re on a path to developing a scalable system that would be meaningful for human clinical purposes.” This device, the Gibco CTS MPC, can handle the liters of cell suspensions required for clinical utility.

For high-throughput molecular isolation, Miltenyi has introduced the MultiMACS M96 Thermo Separator. “It goes from cells to cDNA in 90 minutes in a 96-well format. There’s one workflow, one column, and no need for additional pipetting or precipitation,” Dr. Braun says, adding that the system increases sensitivity and decreases degradation and contamination. “It can be integrated with a robotic platform.”  

A system introduced last year, the MACS Quant flow cytometer, now features enrichment capabilities. “Pre-enrichment of rare cells reduces analysis time without compromising the analysis sensitivity and shortens workflow.”

PerkinElmer’s Janus® Automated Workstation offers full molecular biology capabilities, performing separations by biomagnetic, vacuum, or centrifugation technologies, according to Tim Cloutier, Ph.D., applications leader, automated solutions. There is a growing trend for nucleic acid separations to be done biomagnetically, he says, adding that nucleic acid separations are particularly important in forensics and that public health researchers and clinicians increasingly use DNA and RNA to identify strains of virus outbreaks and track mutations as they spread throughout a population. 

“We’re able to work with magnetic and microfluidics, using our flexible, modular Janus platform to actually perform the separations.”  That capability, perhaps, isn’t readily advertised, but people in the field understand that biomagnetic separations would be included in a total molecular biology liquid-handling workstation, he says. The Janus workstation includes versions for forensics, oil prep, and chitosan bead-based autoplexing, with modules for microplate handling, reagent dispensing, third-party instrument integration, and more.

At Ademtech the most recent advance replaces its Adem-MagMSV with the Modulo modular magnetic separators. The Modulo holds four microtubes of 1.5 mL or 2 mL and can incline those tubes to improve separation efficiency. The “brick” modules that hold the tubes can be added to the Modulo Classic to build an expandable  system while maintaining a homogenous magnetic force throughout the separator.

Spheres

Ademtech’s latest innovation is a ready-to-use bead for covalent coupling of proteins that has standardized the coupling procedure for immunoassays. Called the Activ-Adembeads, the preactivation step and coupling reagent are built in, according to Sandrine Godichaud, product manager. Monoclonal and polyclonal antibodies, high and low molecular weight proteins, and enzymes have been coupled to these beads, maintaining their activity for downstream applications. “Coupling is fast, flexible, and simple, and leads to a highly reproducible immobilization.” 

New superparamagnetic microspheres optimized for bioseparation of human cells or bacteria, biopurification of immunoglobulins or recombinant proteins, or biodetection for immunoassays or ELISA are being launched this month at the American Association for Clinical Chemistry conference by Merck Chimie and EMD. The line is introduced “in anticipation of the researcher’s expanding needs for biotools to meet precise assay requirements,” according to Fabrice Sultan, Ph.D., sales and marketing manager, noting that biocoated microsphere requirements for each of those applications differ.

The new line, called Bio-Estapor®, includes streptavidin, antimouse IgG, antihuman IgG, protein A and protein G coated microspheres, and 0.3 µm, 1.0 µm, and 2.6 µm sizes, with hydrophilic or hydrophobic properties and 30% to 50% ferrite content. The small beads separate slowly but have maximum binding capacity with a higher sensitivity and show “almost no sedimentation during the binding process,” Dr. Sultan says.

In contrast, “our large coated beads separate fast in a complex or viscous liquid  but will not remain long in solution without agitation or resuspension. The medium size combines efficient magnetophoretic mobility, high surface area, and a low sedimentation rate, making it the gold standard for chemiluminescent immunoassays.”

“Recently,” Dr. Sultan comments, “protein A and protein G Bio-Estapor microspheres have been used for antibody and Fc-fusion protein purification from cell culture supernatant. The binding activity of the purified proteins was totally preserved, with similar dose responses for proteins purified with protein A sepharose resin. Those coated microspheres have been reused more than 10 times with the same efficacy.”

Thermo Fisher Scientific recently launched a blocked magnetic particle to control nonspecific binding, according to Rick Galloway, director of particle technology. “Many companies spend a tremendous amount of money adding blockers to their test systems to reduce nonspecific interactions,” he says, increasing assay cost and complicating manufacturing. Instead, Thermo Fisher preblocks the surface of Sera-Mag SpeedBeads before creating an amino-modified surface or the streptavidin-coated version.

Polysciences’ Bangs Labs division recently introduced the ProMag™ Bind-IT™ and 1 µm ProMag microspheres. According to Kathy Turner, technical service manager, “ProMag Bind-IT are 3 µm polymer-based magnetic microspheres with a preactivated surface that allows ready binding of antibody without sacrificing stability.” The beads and proteins bind during a simple incubation, and Bind-IT’s chemistry immobilizes the antibody while “preserving its tertiary structure for optimal activity.”

Turner says a new size of ProMag magnetic microspheres is also available. Both the carboxyl and streptavidin versions are now available in a 1 µm size. “The smaller 1 µm size offers far greater surface area per unit weight, which can present a considerable advantage for the purification of biomolecules or capture of low-concentration targets. Smaller-diameter spheres additionally remain suspended for longer periods of time, simplifying assay incubation steps.”

Magnets

Stepmag Scale-up Systems, introduced last March by Sepmag, perform isolations in multiple tubes of varying sizes simultaneously. Bryan Rittenberry, responsible for Sepmag U.S. operations, says this technology is based on a circular magnetic system that creates homogeneous separation conditions, so the vast majority of beads experience the same magnetic force. “There are no stray magnetic fields, and up to 99 percent of the beads are recovered from the tubes.”

Tubes of 1.5 mL, 10 mL, and 50 mL can be placed in one holder within the ring for simultaneous separations, or 250 mL separations can be performed using the entire ring. “The Stepmag is a small version of a larger system, designed for R&D work.”  Scale-up to 30 liter batches can be performed on the same family of machines using the same technology.

Dexter Magnetic Technologies develops custom separations equipment and custom configurations of existing equipment for its customers. According to Doug Hartl, business manager, medical and LifeSep products, customers are interested in varying magnet placement and strength for its standard plate separators and bundling fixed-well plates with varying magnetics for high-throughput separations.