Chromatography, Rooted In Chemistry, Is A Boon For Life Scientists

For Life Scientists Date: March 4, 1996 (The Scientist, Vol:10, #5, pg.17 & 19, March 4, 1996) (Copyright ©, The Scientist, Inc.) Sidebar:List of Vendors When two or more different biomolecules are present in a solution and a researcher wishes to study only one, the technique considered most often to achieve the separation is chromatography. Biochromatography in its various guises has been driven in part by the biopharmaceutical industry's demand for better and faster protein- purification technologies. It has evolved into a fully automated, computer-controlled process suitable for separating small amounts of material on the research scale, as well as to achieve process-scale quantities of therapeutic biochemicals. Life scientists use chromatography techniques to purify proteins or to separate nucleic acids as commonly as chemists use them to analyze organic and inorganic compounds. "As a science, chromatography has its roots in chemistry, where it has long been used to separate inorganic molecules and biochemicals such as amino acids," explains Richard Cunningham, a professor of biology with the center for biochemistry and biophysics at the State University of New York, Albany. "The development of specialized chromatography media that permits the rapid separation of biological molecules under high pressure enables biologists to perform separations on complex biological mixtures and achieve a highly purified product." "The biggest changes in chromatography have been associated with the types of chromatographic supports used in protein purification," notes Patrick McKay, senior research associate in the department of recovery sciences at Genentech Inc. in South San Francisco, Calif. McKay is a process scientist involved in the large-scale purification of recombinant DNA-derived proteins to be used as therapeutic biopharmaceuticals. Currently, chromatography supports and materials such as chromatography columns are available from large supply companies. These vendors include J.T. Baker Chemical Co. in Phillipsburg, N.J.; Sigma-Aldrich Chromatography of Milwaukee (an alliance of St. Louis-based Sigma Chemical Co. and Milwaukee-based Aldrich Chemical Co.); Fisher Scientific, located in Pittsburgh; and VWR Scientific of West Chester, Pa. Media can also be purchased from companies that specialize in aspects of protein chemistry, such as Pharmacia Biotech Inc. of Piscataway, N.J.; Pierce Chemical Co. of Rockford, Ill.; and PerSeptive Biosystems Inc., based in Framingham, Mass. In chromatography, chemical compounds enter a chromatographic medium in a mixture and emerge separately. In order for a separation to be achieved, the various components of the mixture must have differences, such as in size or charge, that affect the rate at which each will travel through the support matrix. At the heart of separation technology is the support matrix, which essentially determines the resolution capabilities of a chromatographic run. UNIVERSAL SYSTEM: PerSeptive Biosystems' BioCAD/SPRINT system for biomolecular purification and analysis. Before 1980, support matrices consisted mostly of cellulose particles, usually packed into a column, through which substances passed with differing speed. In general, such chromatographic separations proceeded quite slowly. The gravity-driven "flow rate," or the progress of the material as it passes through the chromatography medium, also kept the resolving power of the chromatographic process low. Chromatography media developed within the past decade use particles, or "beads," in place of cellulose as the solid support. "The current generation of chromatographic supports incorporates synthetic, polymeric beads with high flow rate capabilities, as well as new approaches in the design of the support particle, or bead," says McKay. "The key limiting factor for improving flow rates and resolution in chromatography has traditionally been the material used as the solid support," explains Tom Pritchett, principal scientist for proteins at Beckman Instruments Inc. of Fullerton, Calif., a major supplier of biochromatography instrumentation. "To achieve fast, high-resolution separations of biological molecules such as proteins, chromatography must be carried out under pressure. The media therefore must be rigid enough to withstand the applied pressure, and yet still maintain the desired chromatographic qualities." With the development of synthetic beads that could be used as chromatographic supports also came the potential to speed up the flow rate. The ability of the new bead matrices to remain rigid under pressure -- along with columns made of stainless steel, acrylic, or high-density glass -- enable researchers to perform chromatography under low or high pressure. "High-performance liquid chromatography [HPLC] was made possible by the development of steel columns and chromatographic media, which are able to handle pressures in excess of hundreds to thousands of psi," says McKay. Depending on the application and the type of molecules to be separated, researchers may choose from a wide array of chromatography media available from several sources. Fisher Scientific, for example, carries bulk chromatography media from Whatman Inc. of Fairfield, N.J., including Whatman's line of Advanced Ion Exchange Cellulose (AIEC) media for separating biopolymers such as hormones, peptides, and antibodies. Fisher also offers media pre-packed in columns for low-pressure or high-pressure chromatography applications. Sigma-Aldrich Chromatography distributes many different types of chromatography matrices for HPLC, including the SigmaChrom line of high-performance affinity columns. In affinity chromatography, molecules are separated based on their attraction (or lack of attraction) for the chromatography medium. Affinity chromatography is a powerful means of separating enzymes and other proteins from biological mixtures. With Sigma's SuperChrom affinity chromatography columns, for example, researchers can theoretically purify biologically active substances with a single chromatographic run. Matrex chromatography media from Amicon Inc. of Beverly, Mass., can also be applied to separations based on affinity chromatography. Amicon's Dyematrex product line consists of five dye ligand gels, each with differently colored dyes and different binding characteristics. Dyematrex media simulate a variety of specific ligands, such as coenzymes and substrates, allowing researchers to purify enzymes or proteins on the basis of specific chemical groups. J.T. Baker's line of BAKERBOND chromatography media includes bulk media and column-packed matrices suitable for a range of chromatography applications. For ion-exchange chromatography, in which proteins are separated on the basis of the attraction between a protein's charged side chain and the chromatography medium, J.T. Baker carries several types of resins for both anion and cation exchange. BAKERBOND chromatography products are also distributed by VWR Scientific. Pierce Chemical Co., which originated as a supplier of products for protein chemistry, offers chromatography media such as the Aquapore and Polypore product lines, packed in columns suitable for HPLC. Pierce's Aquapore media have a silica base that provides a chromatographic column with a wide pore size. This makes them especially effective for the separation of small peptides and antibiotics by ion-exchange chromatography or reverse-phase chromatography. Although the name may conjure up the image of a column running backward, reverse-phase chromatography is actually performed with a chromatographic support (the stationary phase), which is hydrophobic (nonpolar), and a polar moving solvent (the mobile phase). This is the "reverse" of conventional chromatography, in which the mobile phase is less polar than the stationary phase. Also amenable to reverse-phase chromatography, Pierce's Polypore columns are available in three forms. Polypore H is suitable for separating aqueous mixtures of organic substance. Polypore CA (with calcium) and PB (with lead) can be used by scientists separating carbohydrate molecules. Pure Proteins An advancement on the bioseparation front that has occurred within the last five years is the development of perfusion chromatography, a method patented by PerSeptive Biosystems Inc. In perfusion chromatography, molecules move through PerSeptive's POROS medium, a matrix consisting of rigid beads with pores. The use of perfusion chromatography is licensed through the purchase of POROS products. "In conventional chromatography, molecules move by convection around and between the beads that make up the solid support," says Daryl Vanderburgh, marketing manager for laboratory-scale POROS media at PerSeptive. With POROS media, convective flow occurs through the beads as well as around them. "The achievable flow rate with POROS is up to 100 times faster than with conventional chromatography media," he states. POROS media are available with a broad range of surface chemistries (chemical groups attached to the surface of the POROS matrix) and in several different pore sizes. Researchers can purchase the media in the POROS Self Pack format, which consists of media, empty columns, and a specially designed packing device. Alternatively, POROS can be purchased pre-packed in columns or as bulk media. Perfusion chromatography technology with POROS media is compatible with many types of chromatography instruments, including Pharmacia's FPLC (Fast Protein Liquid Chromatography) system and PerSeptive's BioCAD Workstation and BioCAD/SPRINT. PerSeptive's ConSep LC100, acquired from Millipore Corp. in Bedford, Mass., is a low-pressure chromatography system compatible with the POROS technology. PROTEIN PURIFICATION: Beckman Instruments has acuqired rights to market HyperD chromatography media. HyperD works with Beckman's new Biosys 2000 protein-purification workstation, shown at right. CHROMATOGRAPHY PRODUCTS: PerSeptive's POROS perfusion chromatography columns and media, left, and Scanalytics' AMBIS radioisotopic imager for radio-TLC Beckman Instruments plans to introduce a comprehensive program for protein purification this month. The ProScale program will consist of Beckman's new BioSys 2000 chromatography workstation with dedicated separation modeling software as well as HyperDiffusion (HyperD) media, for which Beckman has acquired exclusive marketing rights from BioSepra Inc. of Marlborough, Mass. BioSepra is a supplier of bioseparation systems, columns, and chromatographic media for process-development and production-scale protein purifications, which are critical to the biopharmaceutical industry. HyperD media, nicknamed the "gel-in-a-shell" by Beckman, combines the high flow of a rigid, porous structure with the high binding capacity of a soft gel matrix. "With the ProScale software on the BioSys 2000, researchers can perform simulations that allow them to explore separation strategies without having to actually perform a chromatography run," explains Prichett of Beckman. "The overall purpose of the ProScale program is to provide researchers with a rational way to approach protein purification." Owing to the open, porous structure of the HyperD beads, only one type of medium is required to purify proteins at both the research scale and the process scale, which means that researchers and production scientists don't have to reinvent the wheel at each stage as they move a protein from the research lab to the pilot scale and finally to the process scale. HPLC is probably the best-known of the various chromatographic techniques used to separate biological molecules. Fully automated HPLC systems make it possible for life scientists -- even those who lack extensive training in protein chemistry -- to analyze or purify biomolecules of their choice. Such systems are available from companies including Pharmacia, Beckman Instruments, PerSeptive Biosystems, and Bio-Rad Laboratories of Hercules, Calif. RESEARCH WITH TLC: Spectronics' Spectroline transilluminator with viewing and photodocumentation equipment. For research-scale applications, Pharmacia's FPLC is a high-pressure chromatography system that can be used with an assortment of chromatography columns. Pharmacia carries a broad line of FPLC columns, including Superdex and HiLoad Superdex, for gel-filtration chromatography. The company also offers other types of media, such as Mono Q and HiLoad Q Sepharose for ion-exchange chromatography. With this diversity of media types, researchers can perform all of the major liquid chromatography techniques on one system. Pharmacia's BioPilot system is a flexible, fully automated, benchtop liquid chromatography system for larger purification projects, such as process development and small-scale production. The system offers easy scaleup from the FPLC. The BioPilot system can be used with a variety of pre-packed BioPilot columns for gel filtration, ion exchange, and hydrophobic interaction chromatography. To meet the needs of researchers who prefer the modular approach to instrumentation, Bio-Rad Laboratories sells individual components for liquid chromatography as well as complete chromatography systems. Investigators can assemble a chromatography system from Bio-Rad's line of peristaltic pumps (low-pressure chromatography) and dual-piston pumps (HPLC), automatic sampling instruments, columns, and detection instruments. Rainin Instrument Co. Inc. of Emeryville, Calif., also offers HPLC components in its Dynamax hardware line. "Dynamax hardware utilizes dynamic axial compression, which maintains the integrity of the chromatography column," notes Eric Gerber, HPLC applications chemist at Rainin. "Because of the effects of high pressure on the silica matrix, gaps or void areas can occur, which essentially limits a column's useful life. Dynamax technology adds pressure to the column by means of a movable piston, which packs the matrix tightly to prevent shifting." When it comes to chromatography, some separations require TLC. The acronym does not stand for tender loving care, but rather for thin-layer chromatography, which is a technique used to separate small biological molecules such as amino acids and nucleotides. Because of its simplicity, TLC is often used by chemists and life scientists to analyze both biological and chemical compounds. In TLC, the stationary phase -- which is generally a matrix of silica, cellulose, or aluminum oxide -- is thinly layered onto the solid surface of a glass or plastic plate. The sample is deposited as a small spot on the stationary phase. When one edge of the coated plate is placed in a reservoir containing an appropriate organic solvent, capillary action draws the solvent through the matrix, carrying the sample along with it. Sample components are separated as they travel at different rates through the matrix based on their structural and chemical properties. TLC is rapidly gaining the status of a "high-performance" technology, which means that the chromatography media and the ability of researchers to automate some of the steps leads to fast, high-resolution separations. For some applications, TLC is preferred over HPLC because of its lower cost and higher sensitivity. Most investigators view TLC not as a competing technology for HPLC, but rather one that complements liquid chromatography in the laboratory. "While HPLC appears to provide a cleaner preparation, the output is generally a chromatogram showing peaks that correspond to various components in a mixture," comments Len Hook, product manager for gel imaging at Scanalytics in Billerica, Mass. "TLC allows the investigator to actually visualize the composition of the mixture by creating a convincing image that can be observed directly on the TLC plate." The two technologies can be used jointly for detecting substances such as pesticide or insecticide residues in environmental samples or in food. Rather than screen for hundreds of different compounds by HPLC, investigators can apply TLC as an initial screening step. Samples of food or water that test positive for a pesticide residue by TLC would then be subjected to HPLC for confirmation and identification. Scanalytics manufactures the AMBIS line of imagers for radio-TLC, in which test substances are radioactively labeled. The AMBIS radioisotopic imagers detect, quantify, and image radioactive "spots" directly on TLC plates. The AMBIS Model 4000 can be equipped with an optional 10-cassette sample auto-loader, which permits the instrument to operate virtually nonstop for overnight or weekend scans. Media for TLC can be purchased from most of the major chromatography companies. Investigators can also choose to buy TLC plates precoated, such as Sigma-Aldrich's silica and C18-bonded silica on glass plates for HPTLC (high performance TLC). To develop TLC plates following a chromatographic run, researchers have the option of purchasing spray reagents that react with substances in TLC gels to make them visible, along with developing tanks and other TLC accessories. The spots that constitute a TLC chromatogram can be detected with a Spectroline UV-viewing cabinet from Spectronics Corp. in Westbury, N.Y. Spectroline cabinets provide both long-wavelength and short-wavelength irradiation. CAMAG Scientific Inc., based in Wilmington, N.C., supplies a complete TLC system that consists of a sample application module and tanks for developing the TLC plates. These tanks include an automated developing chamber that develops, dries, and preserves the plate. CAMAG's system also includes a densitometric scanner for detecting chromatographic spots by absorbance or fluorescence. The TLC Scanner 3 from CAMAG, equipped with CATS software (which runs in a PC environment) gives investigators a densitometric evaluation of their TLC plates. VideoStore from CAMAG is a video documentation system particularly suitable for thin-layer chromatograms, providing researchers with image-analysis capabilities. By combining TLC and HPLC, researchers can improve upon the information gained by performing either one separately. "TLC is a technology that can be used alone, or it can be used to increase the power of other biochromatography techniques such as HPLC," says Hook of Scanalytics. "In a situation where it might take months to determine the composition of an unknown mixture by HPLC, the best place to start is with TLC to lay the groundwork." Holly Ahern is a science writer and an assistant professor of biology at Adirondack Community College in Queensbury, N.Y. Sidebar:List of Vendors

Chromatography, Rooted In Chemistry, Is A Boon For Life Scientists

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