Biotech And Drug Industry Interest Fueling The Centrifuge Revolution

Centrifuge Revolution (The Scientist, Vol:10, #6, p. 18-19, March 18, 1996) Increased interest by the biotechnology and biopharmaceutical industries in powerful instruments capable of separating the smallest of molecules has put a new spin on an old laboratory standard-the centrifuge. Small centrifuges (microfuges) and larger tabletop instruments critical for cell separations and DNA applications can be found on benches in practically every molecular biology laboratory. The larger ultracentrifuges are now used by scientists to perform analytical and highly quantitative analyses of biological molecules in solution. 'WHISPER QUIET': Savant's SFA13K miucrocentrifuge, left, and SFR13K refrigerated microcentrifuge respond to user's need for operation at lower noise levels. Along with these powerful machines, specially designed centrifuge tubes and containers have made their way to the laboratory products market. These supplies can withstand centrifugal forces hundreds of thousands of times the force of gravity. The theory that explains how particles move through a centrifugal field generated by a spinning rotor is peppered with analytical terms such as angular velocity, frictional coefficient, and solvent density. But even without an advanced degree in physics, life scientists can take full advantage of the current generation of user-friendly, microprocessor-controlled centrifuges to aid them in their research. "When a sample is spun in a centrifuge, a force is exerted on the particles in the sample, and they move toward the bottom of the tube," explains Suzanne Anderson, applications laboratory manager at DuPont Sorvall, a Newtown, Conn.-based company that specializes in centrifugation and centrifuge products. "How fast particles move through the centrifugal field depends on several factors, including the nature of the particle [size, shape, and charge, for instance] and the speed at which the sample spins." COMPACT UNIT: Fisher Scientific Compac Micro16 reportedly can achieve forces of 16,000 x g Other companies specializing in centrifugation products for molecular biology applications include Beckman Instruments Inc. in Fullerton, Calif.; Eppendorf (distributed in the United States by Eppendorf Division, Brinkmann Instruments Inc. in Madison, Wis.); International Equipment Co. (IEC) of Needham Heights, Mass.; and Savant Instruments of Holbrook, N.Y. Several models of centrifuges are available from large distributors, such as Pittsburgh-based Fisher Scientific Inc. Fisher carries Eppendorf and IEC centrifuges in addition to its own centrifuge product line. VWR Scientific of Buffalo Grove, Ill., and Krackler Scientific Inc. of Albany, N.Y., also market centrifuges for applications both large and small. The field of molecular biology essentially began with the discovery by Hamilton O. Smith at Johns Hopkins University of restriction enzymes capable of selectively cutting DNA (T.J. Kelley, H.O. Smith, Journal of Molecular Biology, 51:393-409, 1970; H.O. Smith, K.W. Wilcox, J. Molec. Biol., 51:379-91, 1970). The birth of this new discipline created a demand for centrifuges that could accommodate samples seldom exceeding a milliliter in volume. Molecular biologists, who often work with sample volumes in the microliter range, rely on microcentrifuges to generate powerful centrifugal forces, expressed as the number of times greater than the force of gravity (x g) or relative centrifugal force (rcf). Such great forces are required to separate exceedingly small and irregularly shaped molecules, such as DNA and protein, from a solution by driving the molecules toward the bottom of the tube. VACUUMLESS SEAL: VWR Scientific's screwcap microcentrifuge tubes are said to be leakproof. Eppendorf microcentrifuges such as the Model 5402 and Model 5415C-which are also available from Fisher Scientific-can be operated at a maximum speed of 14,000 revolutions per minute (rpm) and can achieve a force of 16,000 x g, or 16,000 times greater than the force of gravity. The Model 5416 microcentrifuge from Eppendorf, available through Krackler Scientific, can spin at up to 15,000 rpm with centrifugal forces in excess of 23,000 x g. The Micro-MB and MicroMax microcentrifuges from IEC generate g-forces in excess of 12,000 x g. The MicroMax features a microprocessor-controlled touch panel with several keys that regulate speed, time, acceleration, and braking. By fully utilizing the control features available with this microfuge, researchers can program the rotor to attain full speed in 7 seconds and come to a stop within 9 seconds. Refrigerated microcentrifuges counterbalance the heat generated during a centrifuge run, and allow researchers to spin delicate or heat-sensitive samples at maximum speed. In the RCM-14 refrigerated microcentrifuge from DuPont Sorvall, the conventional chlorofluorocarbon (CFC) refrigerants found in many types of cooled centrifuges prior to 1990 is replaced by Suva, a coolant developed by DuPont that reduces the output of ozone-depleting CFCs. The RCM-14 utilizes a new grade of Suva designated HP-134A, which offers CFC-free performance from the refrigerated microcentrifuge. Eppendorf's Model 5417R is a refrigerated microcentrifuge with a maximum speed of 14,000 rpm (which translates to 20,800 x g). This instrument is also CFC-free. The Model 5417R has a "soft run" feature that allows for gentle acceleration and deceleration, which can be selected for sensitive samples. In addition to heat energy, high-speed centrifuge runs generate noise. In a laboratory with one or more microcentrifuges in operation, the noise level can be quite distracting. Manufacturers are responding with instruments that lower the amount of noise to a tolerable level. Molecular biologists and visitors to their labs attest to the attractiveness of this feature. "The whine from a microcentrifuge in operation is annoying, and the quieter instruments are a definite improvement," declares microbiologist Denise McKeon of the department of biological sciences at the State University of New York, Albany. Eppendorf microcentrifuges are noted for their quiet operation, even at high speeds. Likewise, SpeedFuge microcentrifuges from Savant Instruments-which include the SFA13K and SFA13K Plus as well as the SFR13K refrigerated microcentrifuge-are described by the manufacturer as "whisper-quiet." These microcentrifuges are programmable in g-force up to 13,000 x g and can accommodate up to 40 sample tubes. Savant's SpeedVac Systems utilize vacuum pumps in addition to centrifugation for concentrating and drying down samples. In a SpeedVac system, for example, a researcher can dry ethanol-precipitated DNA or minute oligonucleotide preparations without losing any of the sample. For applications that require freeze-drying, SpeedVac units are equipped with deep vacuum rotary vane pumps and refrigerated vapor traps to achieve temperatures as low as -104° C. Savant offers several SpeedVac models, including a "personal" SpeedVac system designed for drying small samples such as DNA precipitations or drying oligonucleotides in ammonium hydroxide or other aqueous samples. For researchers with limited bench space or only a few samples to spin, Fisher Scientific produces the Fisher Compac line of "personal" centrifuges, capable of producing g-forces in the range of 5,300 to 16,000. These small units have digital displays and are equipped with rotor seals to contain potentially dangerous aerosols. IEC's MiniMax microcentrifuge is also a compact instrument that takes up only 8 inches of bench space. Fisher's Marathon Model 26KM centrifuge is a compact unit that performs microcentrifugations, but also has the capacity to handle larger samples. This Marathon model is practically the same size as a typical microcentrifuge, yet researchers can use this instrument for centrifugations of samples in 15-ml or 50-ml tubes. The Model 26KM has a maximum speed of 17,000 rpm (26,810 x g). It is equipped with safety features that ensure that each rotor is not operated in excess of its maximum speed, to protect both the centrifuge and the operator from damaging high-speed runs. The Marathon Model 26K/MR is Fisher's refrigerated version of this instrument. The g-forces generated by a spinning centrifuge can have profound effects-from a few cracks to complete annihilation-on the tubes in which a sample is spun. Several manufacturers market tubes and centrifuge "bottles," which hold larger volumes for separating large quantities of cells or solute. Some vendors feature tubes and other containers that are designed to withstand centrifugal forces that in some cases may exceed 500,000 x g. Fisher Scientific carries a line of tubes and bottles made of both plastics and glass from several suppliers, including Nalge Co. of Rochester, N.Y. Nalge's Nalgene product line of polypropylene, polycarbonate, and polyethylene centrifuge bottles are available from Fisher in several volume capacities. Nalgene polycarbonate centrifuge bottles, for example, can withstand g-forces up to 27,500 and can be obtained in sizes that range from 150 ml to 1 liter. Fisher also carries glass centrifuge tubes from Kimble Glass Co. in Vineland, N.J., which manufactures Kimax-brand tubes. Also available from Fisher are Pyrex-brand tubes from Corning Glass Co. of Corning, N.Y. Selected Suppliers Of Centrifuges And Related Equipment Amicon Inc. Beckman Instruments Inc. Composite Rotor Corning Glass Co. DuPont Sorvall Brinkmann Instruments Inc. Fisher Scientific Inc. International Equipment Company (IEC) Kimble Glass Co. Krackler Scientific Inc. Nalge Co. Pierce Chemical Co. Piramoon Technologies Savant Instruments VWR Scientific Krackler Scientific distributes Nalgene UltraPlus centrifuge ware. These products are designed to withstand the extreme g-forces generated in an ultracentrifuge, in which maximum revolutions per minute can range from 55,000 to 80,000 with a corresponding relative centrifugal force of up to 600,000, depending on the instrument. Nalgene thick-wall UltraTubes are available in polycarbonate or in polyallomer, to which proteins don't bind. Uncapped, Nalgene's UltraTubes can be used in spins up to a maximum of 100,000 rpm (in the range of 600,000 x g). For small spins, microcentrifuge tubes that hold a maximum of 1.5 to 2 ml can be purchased from a number of suppliers, and come in several sizes and volumes. FisherBrand microcentrifuge tubes from Fisher Scientific have attached "snap caps" to facilitate user handling, and can withstand forces to 13,000 x g. Microcentrifuge tubes are even available in a variety of colors, enabling researchers to color-code their samples or simply brighten up their labs with pastels or primary hues. Tubes made of colored plastic are available from Fisher as well as from Eppendorf, VWR Scientific, and Krackler Scientific. Eppendorf Biopur Safe-Lock microcentrifuge tubes have a positive sealing lid (held on by positive pressure as opposed to vacuum pressure), which allows for repeated opening and closing. These tubes are molded from dye-free polypropylene resins, and according to the company are irradiated to destroy exogenous pyrogens (bacterial toxins), DNA, RNase (enzymes that degrade RNA), and the energizing nucleotide ATP. VWR Scientific carries a line of VWRbrand screw-cap microcentrifuge tubes that can be both autoclaved (sterilized with superheated steam) and frozen. These polypropylene tubes are fitted with ethylene propylene O-rings, creating a leakproof seal without a vacuum. Screw-cap tubes such as these are valuable for storing samples at very low temperatures, or for researchers working with hazardous materials that may become airborne if they are flipped into the air from a snap cap lid that someone has opened too vigorously. CONCENTRATOR: Amicon's Microcon unit. For concentrating samples in a centrifuge, Amicon Inc. of Beverly, Mass., manufactures a variety of concentrators, which are essentially centrifuge tubes with filtration membranes. In a concentrator, centrifugation forces the liquid in a sample through an ultrafilter, trapping target molecules above. Concentrators are useful for concentrating samples of protein or nucleic acid, and to improve the effectiveness of the polymerase chain reaction (PCR) by removing impurities such as heme, heparin, or the anticoagulant EDTA, substances often found in blood-derived DNA. Investigators might also use a concentrator as a high-recovery alternative to dialysis for desalting and buffer-exchange applications. Amicon's concentrators are available in several sizes and with molecular cutoffs (the size of the pores in the filter) that range from 3,000 to 100,000 daltons. The Microcon and Micropure units, with a maximum process volume of less than half a milliliter, can remove primers from PCR-amplified DNA, recover oligonucleotides from acrylamide gels, or purify and concentrate antibodies produced in cell cultures of hybridoma (monoclonal antibody-secreting) cells. ULTRAFILTRATION UNITS: Pierce's KwikSpin units are available with a selection of molecular weight cutoffs. KwikSpin Ultrafiltration units from Pierce Chemical Co. of Rockford, Ill., are available with several ultrafiltration membranes, offering scientists a choice of molecular weight cutoffs. Kwikspin Micro Ultrafiltration Units have a maximum sample capacity of 400 ml. The Ultrafiltration Units accommodate samples up to 3 ml, while the KwikSpin Macro Ultrafiltration Units allow ultrafiltration of samples up to 25 ml in volume. Centrifuge applications range from pelleting whole cells or their nuclei in a low-speed centrifuge (accomplished at approximately 8,000 x g) to separating virus particles and subcellular bits such as proteins and plasmid DNA in an ultracentrifuge (at a force in the range of 600,000 x g). Ultracentrifugation is performed in floor-model centrifuges capable of achieving very high speeds and even higher g-forces. It has become the target of renewed interest by biotechnology and biopharmaceutical researchers studying macromolecules such as glycosylated proteins (proteins with attached sugar groups) with therapeutic potential. Via analytical ultracentrifugation (AU), investigators can determine absolute molecular weights of glycoproteins without using molecular weight standards or by inferring the information from an electrophoretic separation performed on a polyacrylamide gel. With this powerful technique, researchers can also observe how biological molecules self-associate and how they interact in solution. BROAD RANGE: Beckman's XL-1 series ultracentrifuge enables users to assess variables such as pH, ionic strength, concentration, and temperature. Beckman Instruments Inc. has developed the Optima XL-A and XL-1 Series Ultracentrifuges, which provide researchers with a broad range of AU capabilities. "With the XL-A and the XL-1, researchers can investigate the effects of pH, ionic strength, concentration, and temperature on the macromolecular association process," comments Ken Johnson, global product marketing manager for Beckman. The Optima XL-100 Ultracentrifuge is capable of generating 802,400 x g at speeds up to 100,000 rpm. The instrument has an air-cooled drive, which eliminates the use of CFCs, and its thermoelectric temperature control system runs without a compressor for less power usage. The Sorvall Ultraspeed line of ultracentrifuges from DuPont Sorvall began with the first oil turbine drive model in 1973. The Sorvall Ultra PRO 80 can be operated at speeds up to 80,000 rpm, or 605,500 x g. Like the Sorvall refrigerated microcentrifuge, the PRO 80 features CFC-free refrigeration and a new QUIKset operator interface system for simpler centrifuge run setup. The Sorvall Superspeed (26,000 rpm or 70,000 x g) models, such as the Super T 21 and the Superspeed RC-26, are supplied with a class of rotors designated SUPER-LITE. These are a family of aluminum rotors that weigh half as much as their predecessors. Because rotors occasionally must be removed from the centrifuge instrument, a rotor that weighs significantly less than the conventional units found in many of the older superspeeds (50 pounds or so) is a considerable improvement in the technology. "Changing a rotor in a superspeed is similar to lifting weights, only much harder on the back," comments SUNY-Albany's McKeon. Lighter rotors have an additional advantage beyond their back-saving properties in centrifugation. Because mass (weight) is one factor in determining the speed at which a rotor can be spun, lighter rotors will also translate into faster speeds and the generation of greater g-forces. However, a limiting factor in the development of lighter and faster rotors is the nature of the material from which both the centrifuge and the rotor are generally constructed-metal. "Metals are composed of inorganic crystals bonded by atomic forces along clearly defined lines," explains Mehmet Pamukcu, vice president of Piramoon Technologies Inc., a manufacturer of rotors based in Mountain View, Calif. "The problems of strength, structural integrity, corrosion resistance, and buildup of fatigue in metal rotors have become more apparent as the relative centrifugal force and the volumetric capacity increases." Some companies are investigating materials called composites with the goal of replacing metal as the structural basis for rotors. Composite materials are made from carbon filaments distributed in a controlled way into an organic resin, from which a rotor is formed. Composite rotors are lighter weight and do not corrode from the joint effects of high centrifugal forces and chemical reactions, as do rotors made of aluminum or other metals. "Rotors made of composites are very strong and lightweight, yet they won't corrode, as conventional metal rotors will," maintains Mitch Griffith, principal applications scientist for Composite Rotor, which has been manufacturing Compspin composite rotors in Mountain View, Calif., for six years. Composite Rotor markets 48 different rotors that can be selected according to the type of instrument and the speed of the centrifuge run. "Our Composite Rotor product line will fit centrifuges from several manufacturers, including [DuPont] Sorvall, IEC, and Beckman Instruments," reports Griffith. Piramoon Technologies, which also manufactures several types of composite rotors, has developed a prototype centrifuge based on the composite technology. The EMD Centrifuge prototype incorporates an electric motor embedded under the rotor, to spin the rotor directly. In a study conducted by Piramoon in which a conventional metal rotor was compared with an EMD composite rotor, the relative centrifugal force (g-force) achieved with the EMD was more than double that of the metal rotor. "In the future, it may be possible to build more efficient and less expensive centrifuges based on composite technology that will be safer to use and easier to maintain," says Griffith of Composite Rotor. Adds Piramoon's Pamukcu: "Centrifuges based on composites will address many well-known deficiencies imposed by conventional metal-based designs." Whether their rotors are made of metal or composite material, centrifuges are an essential piece of laboratory equipment, suitable for routine separations or for analytical studies of the interaction of biological molecules in a solution. Declares SUNY's McKeon: "Without a microcentrifuge or an ultracentrifuge, molecular biology would be next to impossible." Holly Ahern is a science writer and an assistant professor of biology at Adirondack Community College in Queensbury, N.Y.

Biotech And Drug Industry Interest Fueling The Centrifuge Revolution

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