Field of Dreams

Magnetic Separation Systems Suppliers of Magnetic Microspheres for a Variety of Applications Labsystems' KingFisher Magnetic Particle Processor plunges into the magnetic separation scene. Since LabConsumer's last profile of magnetic bead technology, the use of paramagnetic particles in biological separations and detection has exploded.1 However, development of processing technologies has lagged behind refinements of the beads themselves. The instrumentation emerging within the last few years has for the most part been adapted from existing technologies; some, however, represent clear innovations. This profile will examine several of the new systems available for a variety of magnetic particle-based applications. Researchers doing magnetic separations are not limited to magnetic separation stands. Over the past few years, a variety of automated systems have emerged, increasing the speed, efficiency, and reproducibility of separations. Some of these systems are based on magnetic processing technology; others can be adapted for use with magnetic particles. The following sections discuss newer instrumentation as well as recent improvements in existing technologies. Labsystems of Franklin, Mass., offers the KingFisher(tm) instrument, a fully automatic system for DNA and RNA purification and processing.2 The KingFisher Magnetic Particle Processor eliminates the aspiration and other liquid handling steps that can lead to waste or contamination in systems that require vacuum extraction and manual liquid transfer methods. Purification is straightforward--samples, beads, and reagents are placed in a 96-well microplate that is contoured at the bottom to eliminate crevices in which materials and beads can "hide." A mechanical arm composed of a row of disposable plastic sleeves is lowered into the wells; the sleeves move like a plunger, mixing the liquids and beads. A second arm composed of a row of magnets then slides into the sleeves to gather the beads with the nucleic acids attached. The beads are washed, and purified nucleic acids are released into a small volume. Up to 24 samples can be processed in a run. According to marketing product manager David Jones, the KingFisher system offers many advantages over other separation techniques. "The advantages are ... efficiency, because you don't have carryover from any other previous processes; ... mechanical reliability, because all you're dealing with is permanent magnets and two motorized arms, and you don't have liquid handling functions or pumps, vacuums, and valves and so forth; ... and also the overall cost of the hardware is much less," says Jones. Labsystems offers purification kits for mRNA and single-stranded DNA designed specifically for the KingFisher system; kits for purifying genomic DNA and double-stranded DNA are in the works. A larger-volume version of the KingFisher instrument will be available within the year. Labsystems can also adapt other bead technologies and protocols to the KingFisher system. Roche Molecular Biochemicals of Indianapolis offers the MagNA Pure LC Instrument, automated for nucleic acid isolation and purification and postelution pipetting of reaction mixtures into glass capillaries, PCR tubes, or 96-well plates. The instrument is designed to purify PCR-quality DNA, RNA, and mRNA, but it also produces nucleic acids of sufficient volume, yield, and quality for applications such as Southern and northern blots. Reagents are automatically pipetted into the samples (blood or cultured cells); the samples are then mixed, incubated, and combined with a magnetic glass bead solution. Using an eight-nozzle piston-driven pipettor, the MagNA Pure LC draws the mixture into the pipet tips. A magnet then holds the beads/nucleic acids on the side of the tip while remaining material is ejected. The beads are washed three times before samples are heated in elution buffer to dissociate the nucleic acid from the beads; purified nucleic acids are transferred into a 4ºC storage block. According to Sharon Sheridan, marketing manager of pharmaceuticals and biotechnology at Roche, the MagNA Pure LC Instrument is the only totally walk-away instrument for purifying PCR-ready nucleic acids: "You can load your samples, your plates, and your master mixes, walk away, and come back and have a PCR reaction ready to go on your analytical instrument." As opposed to manual purification kits, MagNA Pure offers scalability. Sheridan notes, "Usually there's a sample size that gives you optimal yield with a manual kit. And if you want to purify higher or lower volumes of starting sample, you get a significant drop-off in the efficiency in terms of the recovery that you can get. [MagNA Pure] protocols and reagents are optimized so you get the same relative yield with a single protocol regardless of the starting sample you have." RNA and mRNA are free of DNA contamination after purification with the MagNA Pure LC instrument. Several companies offer components that can be combined with robotic systems for automated magnetic separations of nucleic acids and proteins. AGOWA GmbH of Berlin offers the AGOWA(r)sep 9600 for isolating and purifying nucleic acids. The separator is compatible with a variety of automated liquid handling systems, including TECAN, Hamilton, and QIAGEN robotic systems. It comprises a set of permanent magnets moved by a motor near the reaction tubes. Magnetic particles collect at either side of the reaction tubes; changing the position of the magnet pulls the particles through buffers during washing steps. In addition, the separator regulates temperature automatically. Dynal ASA of Oslo, Norway, offers the Dynal MPC(r)-auto96 automatic magnet station for 96-well microtiter plates, which is designed to be integrated into a robotic workstation or other automated system. The station comes with directions for using the Dynabeads(r) DNA DIRECT(tm) Auto96 DNA isolation kit with Beckman Coulter's Biomek(r) 2000 Workstation, handling 96 samples in 80 minutes. Other Dynal protocols can be implemented: Dynabeads mRNA DIRECT kits can directly isolate mRNA from blood, and DYNAPURE(tm) kits can purify sequencing products. Dynal also collaborates with other workstation suppliers. TECAN U.S. of Durham, N.C., offers the Te-MagS magnetic separator for use with its GENESIS Molecular Biology Workstation for automated purification and extraction of DNA and RNA as well as solid phase extraction. Pipetting, washing, and elution of purified product are all automatic with TECAN's robotic liquid handling arm and automation software. The magnetic separation capability, which involves a mobile magnetic bar that moves beads through the solution, is sold as an option package in the GENESIS system. QIAGEN Inc. of Valencia, Calif., offers the BioRobot(tm) 3000 series, which provides automated small-scale magnetic purification of up to 96 His-tagged proteins using Ni-NTA Magnetic Agarose Beads. BioRobot 3000 workstations can be custom designed with a variety of system components for any application; those employing magnetic separations include DNA-protein interaction assays, protein-protein interaction assays, and immunoassays. Beckman Coulter Inc. of Fullerton, Calif., offers the Biomek FX Solution for automated nucleic acid preparation, which is equipped with a magnetic Automated Labware Positioner (ALP) for magnetic bead separation applications. The MixSep biomagnetic separation system from Sigris Research Inc. of Brea, Calif., is a unique system that uses a rotating magnetic field to continuously levitate and move beads through rotating tubes.3 This occurs without agitating the solution, dramatically reducing fluid shear of nucleic acids or proteins or rupturing of cells. According to Iqbal Siddiqi, chief technology officer at Sigris, this feature allows researchers to extract very large fragments of genomic DNA, which are often difficult to purify with standard kits. The MixSep also offers interchangeable tube holders and magnet tracks, allowing use of different-size tubes according to the protocols. Because each tube mixes the beads in the same way, the system offers high reproducibility. The MixSep can be used in any procedure involving magnetic beads, and Siddiqi notes that Sigris has received requests to license the technology for high-throughput diagnostic and genomic applications. The company currently offers a 12-sample instrument. Beckman Coulter's Access Immunoassay System All Sorts of Cell Sorters While some of the systems discussed above can be used for cell selection, several companies offer systems--including specialized particles and instrumentation--specifically designed for cell separation. These have found use in cancer diagnostics and treatment. It should be noted that while many other companies offer magnetic particles that can be used in cell separations, the companies discussed below offer comprehensive systems. Miltenyi Biotec GmbH of Bergisch Gladbach, Germany, has a variety of tools for automated cell sorting and is a source of constant innovation in this field. The autoMACS is an automated benchtop magnetic cell sorter capable of sorting up to 10 million cells per second.4 The autoMACS targets cells for enrichment or depletion using 50 nm polysaccharide-coated iron oxide beads coupled with monoclonal antibodies. After presorting, cells are ready for further sorting by flow cytometry. Woody Woodward, general manager at Miltenyi Biotec Inc.-U.S.A., notes that although the autoMACS was initially targeted to the fluorescence-activated cell sorting (FACS) market, the system has found a niche in small and large laboratories in which large numbers of people with varied levels of training perform separations. "The autoMACS has really found a home there, because you don't need any training. You simply plug your sample in and press go, then go out and have a cup of coffee. Five minutes later you're done," says Woodward. The CliniMACS Cell Selection System, available for clinical use in Europe, offers larger-scale automated cell selection in a closed, sterile environment. According to Woodward, the instrument is currently being used under investigational device exemptions (IDEs) by clinical investigators in the United States for experiments in gene and cell therapy (for example, tumor cell purging). Along with other new products offered by Miltenyi Biotec, including its new line of dendritic cell markers and the recently introduced MACS Cytokine Secretion Assay, the CliniMACS is a powerful tool for cancer research. The Cytokine Secretion Assay allows detection and isolation of viable antigen-specific cells according to the cytokine they secrete. The specific cells can be further analyzed as expanded to generate, for example, tumor-specific T-cell clones; the assay is thus excellent for tumor or other vaccination studies. QIAGEN's BioRobot 3000 workstation Immunicon Corp. of Huntingdon Valley, Pa., offers an alternative cell selection and depletion technology based on ferrofluids, or highly magnetic nanoparticles. Like the beads used in Miltenyi Biotec's MACS system, these particles are small enough to limit interference with the visual observation of cells; however, they do not require addition of steel wool-packed columns to effect separations and can be used for whole blood and viscous solutions. Immunicon's technology thus combines high sensitivity with ease of use; as Dhanesh Gohel, principal scientist at Immunicon, notes, "We have the best of both worlds in a performance sense, because our technology is very user friendly and has a very high sensitivity as far as giving high-quality results." Because the particles behave as a true colloid, they do not settle due to gravity and do not need to be mixed constantly during use; they are thus ideal for automation. Immunicon has created specialized instrumentation, including magnetic gradient separators, for separations and isolations and has developed prototype sample preparation stations for clinical use of the company's cancer diagnostic tests. The technology developed by Immunicon has been used for applications such as cell diagnostics and immunodiagnostics. According to Gohel, the most recent application of the technology has been in drug discovery through high-throughput screening. Immunicon is known for its work in cancer diagnostics and has developed kits and protocols for isolation of tumor cells from whole blood. Gohel explains that Immunicon is also developing therapeutic applications for the particles, in particular for ex vivo cell selections or depletion. The particles can be used to isolate rare cells, or the technology can be scaled up to isolate more abundant cells. Oh Say Can You Assay Magnetic particle technology has recently been adapted for biomolecule labeling. Several instruments use magnetic carriers as probes for a variety of assays, replacing labeling techniques such as chemiluminescence, fluorescence, and radioactivity. Additionally, several instruments couple beads for magnetic separation with other labeling methods. Quantum Design of San Diego has developed the Magnetic Assay Reader (MAR III), a handheld instrument for quantification of biochemical analytes. The instrument uses magnetic particles as a reporter and operates via a mutual induction method in which the magnetic field expressed by the total mass of iron in the sample is detected. The value is then correlated to the number of molecules in the sample. Because it detects only the amount of magnetic materials bound to the analyte (for example, the iron oxides in superparamagnetic beads), it is not affected by iron from biological materials. The MAR III is highly sensitive, detecting as little as 20 pg of iron, and fast, with a reading time of 10-20 seconds. One of the primary applications of the MAR III is in the point-of-care market. According to Ron La Borde, director of biotechnology research and development, the instrument lends itself to lateral flow assays such as those used in medical applications (for example, over-the-counter pregnancy tests). In one scenario, the viral load of a patient with HIV could be monitored with only a pinprick of blood. Emergency rooms, where speed is key, could use the instrument to run blood analyses in seconds; current tests can take as long as 40 minutes. Other applications include detecting biohazards and biological contaminants for the military and environmental agencies and detecting contamination in the food handling industry. As La Borde explains, "In meat packing and the food handling industry, pathogens take a long time to be detected with existing tests ... the time to culture is longer [than an individual shift] ... and that's why you see all of these recalls." The MAR III, which detects Escherichia coli DNA directly, would eliminate these problems. Finally, the instrument can replace current biochemical assays in research laboratories, and a prototype model exists for array reading. According to La Borde, the instrument can detect a femtomole of DNA using a streptavidin-biotinylated magnetic particle as a probe. Another San Diego company leaping into magnetic labeling and detection is Ericomp Inc. Its Magnetic Messenger Labeling Technology detects and quantitates target molecules or cells using bound magnetic labels (carriers/beads). Many off-the-shelf magnetic beads can be used as a reporter in conjunction with Ericomp's technology. The system exploits a little-known property of paramagnetic beads: Such particles retain a small magnetic field after being placed in and then removed from a magnetizing field. This small field can be detected with highly sensitive instrumentation. Ericomp's prototype detection instrument currently has sensitivity of about 10 pg of 10 nanometer ferric oxide labels (~106 particles). As Lonnie Adelman, president of Ericomp, explains, "If you did measurements in the presence of a magnetizing field, any paramagnetic substance ... would give you some sort of an erroneous signal, but when you do the measurements the way we're doing them, without the magnetizing field ... there is no signal from those contaminants, which increases the signal-to-noise level dramatically." Quantum Design's Magnetic Assay Reader III Adelman explains that the system's low cost and low power requirements (the instrument can be run by a battery) combined with its ability to perform densely packed quantitative assays from a common analyte make it highly suitable for point-of-care applications. Adelman notes that in addition to the point-of-care market, the technology has a variety of potential applications, including nucleic acid analysis on microarrays. Ericomp is currently developing protocols in conjunction with several other companies and has tested a variety of laboratory membranes. Another unique use of magnetic bead technology in assay development is seen with the ORIGEN(r) M-SERIES(tm) M-8 Analyzer from IGEN International Inc. of Gaithersburg, Md.5 The system uses electrochemiluminescence technology to detect and quantitate the specific binding of two molecules. The process uses a ruthenium chelate as a label; labeled components are captured on the surface of paramagnetic beads, which are brought to the surface of an electrode via a magnet. After a low voltage is applied to the electrode, the ruthenium ion undergoes a redox reaction. The excited ion receives an electron from a second component in the assay buffer, tripropylamine (TPA), and relaxes to its original state, giving off light at 620 nm. The ruthenium chelate is recycled and the process continues, amplifying the electrochemiluminescent signal. Emitted light is measured and digitally stored. Because the light produced by the reaction occurs only 30 to 50 Å from the electrode, any unreacted components not removed by washing do not contribute to background signal. According to Irene Griff, application scientist manager at IGEN, this sensitivity sets the system apart from other technologies: "When it's compared head-to-head as far as assay performance goes with any other technology [for example, ELISA], we do much better in sensitivity and dynamic range, which allows the user ... to reduce the amount of rare reagent they are using." IGEN has licensed ORIGEN technology to Roche Diagnostics, Eisai Co. Ltd. of Tokyo, and Organon Teknika of Boxtel, The Netherlands. The M-SERIES M-8 Analyzer can be formatted for a variety of assays, including tyrosine kinase, protease, protein-protein binding, immunoassays, and nucleic acid quantitation. The technology is useful for high-throughput screening, assay development, secondary screening, ADME/Tox, and clinical trials. And, unlike many other automated systems, the M-8 Analyzer can go through the various stages of drug development. IGEN also has field scientists in the United States and Europe to support customers. The Access(r) Immunoassay System from Beckman Coulter couples magnetic separation with dioxetane-based chemiluminescent detection. The system is a random access analyzer that can perform a wide variety of sandwich assays and conduct up to 100 tests per hour. It also has a large loading capacity for up to three hours of continuous sample processing. The system is compact, offers 24 resident, refrigerated assays, and uses a unique ultrasonic primary pipetting probe for liquid handling steps to reduce sample carryover. The Access analyzer is designed to meet the needs of immunodiagnostic core laboratories and rapid response/STAT laboratories. The Future of the Field Magnetic bead technology appears to be following the high-throughput trend. Mary Meza, vice president of technology for Bangs Laboratories Inc. of Fishers, Ind., which manufactures custom magnetic microparticles for a wide variety of applications, notes that magnetic bead-based technologies lend themselves well to automation: "[Magnetic] separations can happen very quickly, depending on the physical configuration of the vessel and the magnetic separator. Some of the other separation methods, such as centrifugation or filtration, sometimes take a little bit longer. With high-throughput screening, speed's essential, and [magnetic separations] could be one avenue toward that." The new systems currently available and under development clearly point toward this and reveal the "attraction" of magnetics in diverse fields of research. S Aileen Constans can be contacted at aconstans@the-scientist.com. References 1. B. Sinclair, "To bead or not to bead," The Scientist, 12[13]:16-9, June 22, 1998. 2. M. Vettese-Dadey, "Gone fishin'," The Scientist, 13[15]:21, July 19, 1999. 3. L. de Francesco, "Let the field be with you," The Scientist, 12[2]:15, January 19, 1998. 4. G. Meisenholder, "Separation anxiety?" The Scientist, 13[16]:16, August 16, 1999. 5. B. Sinclair, "See the light," The Scientist, 14[10]:18, May 15, 2000. Magnetics in Drug Delivery Chemotherapy is notorious for causing unpleasant side effects such as hair loss and nausea in cancer patients. When cancer-fighting drugs are released throughout the body, they often damage healthy cells in addition to tumor cells. But what if a method existed that targeted oncological drugs directly to the tumor, preventing systemic circulation? FeRx Inc. of San Diego has designed 1-2 µm magnetic particles for the site-specific targeting, tissue retention, and sustained release of drugs. Termed MTCs, or Magnetic Targeted Carriers, these particles are composed of elemental iron and activated carbon. Elemental iron is more magnetic than the iron oxides normally used in magnetic beads, thus enabling the particles to be more easily manipulated in the body; activated carbon is highly absorptive, providing more efficient binding of the desired drug. FeRx Inc. uses magnetic carriers to deliver anticancer drugs to tumors. This method of drug delivery to tumors is relatively straightforward. A catheter is inserted into an arterial feed to the tumor, and a magnetic stand is positioned over the targeted site. Vialed MTCs are mixed with an anticancer drug already in solution; the mixture is then introduced into the catheter. The magnetic field pulls, or extravasates, the MTC-drug mixture through the artery to the tumor. The field is left in place for another 15 minutes; after removal of the magnet, the particles remain trapped in the tumor, where the drug is then released. According to FeRx president and CEO Jacqueline Johnson, this method offers significant advantages over traditional methods of solid-tumor chemotherapy: "The whole premise is to decrease significantly, if not eliminate, systemic circulation of the drugs, therefore eliminating the associated toxicities, while increasing the drug concentration at the desired site." Preclinical trials using MTCs to target tumors in swine models showed a high degree of localization and retention.1 Phase I/II clinical trials of MTCs coupled with the anticancer drug doxorubicin (MTC-DOX) to target liver tumors are currently taking place at a number of medical centers; these have also shown promise. "We have not seen any dose-limiting toxicities to date. We've seen some response in tumors, and in others the treated tumor has remained stable for the 28 days following treatment. Only a few patients have had their treated lesions grow. This is excellent in the course of liver cancer, which is a very aggressive cancer," says Johnson. Other applications of MTC technology that the company is preparing to investigate are the treatment of metastatic liver disease as well as a variety of other cancers, combination MTC-drug therapy, and delivery of therapeutic radioisotopes, or radionuclides, to tumors. For example, Urs Häfeli, staff scientist in the department of radiation oncology at the Cleveland Clinic Foundation Cancer Center, conducts research in collaboration with FeRx for treatment of liver and brain tumors using MTCs labeled with beta emitters such as Rhenium-188 and Yttrium-90.2 As Häfeli explains, by combining radiotherapy with drug therapy, tumors may be treated more effectively: "Many tumors are not sensitive to certain drugs; combination therapy might be what you need to treat the tumor." While the main area of current MTC research is cancer treatment, the technology is not limited to this--according to FeRx's literature, other drugs that are deliverable in this manner include antibiotics, thrombolytics, anti-inflammatories, peptides, and steroids. --Aileen Constans References 1. S. Goodwin et al., "Targeting and retention of magnetic targeted carriers (MTCs) enhancing intra-arterial chemotherapy," Journal of Magnetism and Magnetic Materials, 194:132-9, April 1999. 2. W. Schütt et al., "Biocompatible magnetic polymer carriers for in vivo radionuclide delivery," Artificial Organs, 23:98-103, January 1999.

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