Biological Chromatography: It's Not Just A Tool; It's A Discipline

SELECTIVITY: Supelco's SUPELCOSIL ABZ+Plus columns works like other reverse-phase columns yet provide selectivity for polar and charged compounds. For academic researchers and scientists who produce proteins on a large scale, separating substances by chromatography has never been easier. New and improved chromatographic support media, along with fully automated, computer-controlled instruments that are becoming increasingly user-friendly, have made it much simpler for even the most analytically challenged life scientist to perform perfect purifications. LOW-PRESSURE SYSTEM: Bio-Rad's BioLogic LP high-performance system for protein purification. "In many cases, chromatography is used by researchers simply as a tool to get at a particular protein" or other biological molecule, says Tom Pritchett, principal scientist for proteins at Beckman Instruments Inc., a major supplier of chromatography instruments based in Fullerton, Calif. "The growth of the biotechnology industry, however, has led to a large number of companies involved in the production of biopharmaceutical agents. For scientists involved in protein purification on a larger scale, chromatography is not just a tool, it's a discipline." Life scientists use chromatography to separate or purify many kinds of compounds, including carbohydrates, lipids, and nucleic acids. For biological molecules, liquid chromatography is the technology used most often. In liquid chromatography, a mixture of compounds in solution (the liquid phase) is passed through a column containing a support matrix (the solid phase). The compounds passing through the matrix behave differently-some may be attracted to the solid phase, others may be repelled, still others may be sieved according to their size as they pass through the column. The nature of the chromatography media used as the solid support determines the resolution capabilities for a chromatographic run. The newer generations of chromatography media consist of rigid particles, called beads, that are packed into columns that vary in size, depending on the application. The rigidity of the support matrix permits chromatographic separations to be carried out under pressure, which improves both the flow rate and the resolving power of the chromatographic process. PURIFIED PEPTIDES: Silica-based ZORBAX LP, manufactured by BTR-Separations, for liquid chromatography. The most common target molecule separated by liquid chromatography is a particular protein.A growing number of investigators are patenting newly discovered genes or their protein products with the hope that one eventually may have commercial value. But before the patenting stages comes purification and characterization of the target molecule, which is usually achieved by high-performance liquid chromatography (HPLC). "To achieve a fast, high-resolution separation of a protein from a mixture such as a crude cell extract, the best option is to use HPLC," comments William Hall, a research associate with the National Institute of Child Health and Human Development in Bethesda, Md. "New generations of chromatography media, along with columns made of stainless-steel or high-density glass that can withstand very high pressure, have significantly increased the resolution and speed of protein chromatography." For separating proteins from complex biological mixtures such as cell extracts prepared from bacterial cultures, "HPLC permits biologists to perform fast separations and achieve a highly purified product," explains Richard P. Cunningham, a professor of biology with the Center for Biochemistry and Biophysics at the State University of New York, Albany. Cunningham is in the process of patenting several enzymes that his laboratory has purified from E. coli cell extracts using fast protein liquid chromatography (FPLC), a trademark technology from Pharmacia Biotech Inc. in Piscataway, N.J. These enzymes function in the repair of DNA damaged by oxidizing agents and other potential mutagens. The repair enzymes, once patented and purified, will be marketed through Trevigen Inc., a Gaithersburg, Md. based biotech company. The "HP" in HPLC may stand for both "high performance" and "high pressure." Asserts Hall: "Without high pressure, chromatography could not be considered high-performance." Cellulose-based matrices, prevalent prior to the mid-1980s and still used by scientists performing low- or medium-pressure liquid chromatography, collapse under high pressure. "Chromatography media for HPLC must be rigid enough to withstand applied pressure, and still maintain the desired chromatographic qualities," says Pritchett of Beckman. The latest versions of chromatography media consist of synthetic beads, constructed from firm polymers that hold up under the intense pressure of HPLC. STREAMLINING THE PROCESS: PerSeptive Biosystems' BioCAD/SPRINT system. Perfusion Chromatography is one unique approach to bioseparation technology. This method, patented by PerSeptive Biosystems Inc. of Framingham, Mass., is based on the convective flow of the liquid phase through the interior of the rigid beads that make up the solid phase. Perfusion Chromatography requires PerSeptive's POROS media, a matrix of rigid particles engineered with two classes of pores. "The design of the POROS bead enhances the transport of molecules to particle binding sites by permitting convective flow through the beads as well as around them," explains Daryl Vanderburgh, marketing manager for laboratory-scale POROS media at PerSeptive. Perfusion Chromatography therefore occurs 10 to 100 times faster than conventional liquid chromatography, according to the company. To facilitate the execution of separations at such speeds, PerSeptive also offers the BioCAD/SPRINT system. It incorporates software, hardware, and data control to generate and manage complete experimental campaigns with the potential to yield answers from chromatography much more quickly. In addition, PerSeptive sells POROS as bulk media and in prepacked columns. POROS also is available in a Self Pack format, which allows investigators to pack their own high-performance Perfusion Chromatography columns. HyperDiffusion (HyperD) chromatography media, manufactured by BioSepra Inc. in Marlborough, Mass., and marketed by Beckman, also uses rigid beads with a porous structure. HyperD media, nicknamed "gel-in-a-shell," combine a high binding capacity characteristic of softer gel matrices with the high flow capability of a rigid bead constructed from synthetic polymers. Beckman has incorporated HyperD media into its ProScale program, a protein-purification system. Scaling-up production often poses problems for purifiers, since the separation scheme for small quantities of protein may not retain the desired chromatographic qualities when performed on a larger scale. The ProScale program allows investigators to explore separation strategies and design a purification scheme that works well in both environments. For most bioseparations-particularly those for which a highly purified product is sought-more than one chromatographic step is required. An investigator may pair size-exclusion chromatography, in which substances are separated according to their molecular size or shape, with one or more adsorption steps. In adsorption chromatography, either the target molecule or the compounds in the mixture are attracted to the support matrix. Media used for adsorption chromatography usually carry functional groups chemically bonded to the beads. Such groups may be charged to attract or repel various compounds, or they may carry ligands or antibodies that are highly specific for a particular target. Amicon Inc. in Beverly, Mass., carries an extensive line of chromatography media for commonly used bioseparation methods, including those for adsorption chromatography (ion exchange, hydrophobic interaction, and affinity chromatography), size exclusion (gel filtration and desalting), and partition chromatography. In partition chromatography, substances to be separated are sandwiched between two immiscible liquid phases (one stationary, one mobile). This technique is often used to resolve small organic molecules such as amino acids and small peptides, along with other acidic and neutral analytes such as cyclic amines and amides. For affinity chromatography, in which substances are targeted by antibodies or less specific ligands attached to the column material, Amicon's Dyematrix media are available in five different agarose dye ligand gels, each with a different binding characteristic. The dyes simulate specific ligands, such as coenzymes, that attract enzymes and other types of proteins. Also for affinity chromatography, Rainin Instrument Co. of Woburn, Mass., manufactures and markets several types of affinity media, including Hydropore-EP and Hydropore-Protein A. These are best used with Rainin's Dynamic Axial Compression (Dynamax) columns, which have an axial compression "nut" at one end that provides a dynamic high-pressure seal. The seal, which can slide along the inside surface of the HPLC column tube, allows the columns to be recompressed after they have been used several times. Axial compression of the column bed removes air spaces that form as the column is used; this extends the usable life of the column. Pharmacia Biotech's rProtein A Sepharose Fast Flow is an affinity medium specifically designed for rapid, high-capacity purification of immunoglobulins (antibodies) from the liquid surrounding cultured cells. The agarose particles carry a recombinant form of protein A, a ligand with a high affinity for antibody molecules. For researchers who prefer to prepare their own affinity media, Pharmacia offers CNBr-activated Sepharose 4 Fast Flow and NHS activated Sepharose 4 Fast Flow. These media permit researchers to attach ligands or antibodies specific for a target molecule to the chromatography media. The BAKERBOND family of liquid chromatography media from J.T. Baker Inc. in Phillipsburg, N.J., includes products for analytical and preparative liquid chromatography and biochromatography. They also are distributed by Albany, N.Y.-based Krackeler Scientific Inc. and VWR Scientific of West Chester, Pa. The BAKERBOND biochromatography product line consists of media for ion exchange, such as WP PEI and WP Sulfonic, affinity purification (BAKERBOND ABx), hydrophobic interaction (WP-HI-Propyl), and BAKERBOND standard analytical columns for partition chromatography. FROM TOSOHAAS: Toyopearl media are available for size=exclusion, ion-exchange, hyrdophobic interaction, and affinity chromatography. Chromatography media from TosoHaas of Montgomeryville, Pa., including TSK-Gel, Toyo-Pearl, and AmberChrom, are available in both bulk and prepacked form. Fisher Scientific of Pittsburgh distributes bulk chromatography media and columns from Whatman Inc. of Fairfield, N.J. These products include Whatman's line of Advanced Ion Exchange Cellulose (AIEC) media for resolving biologicals such as hormones and antibodies. Fisher also carries Whatman's Void Sealing (WVS) columns, which permit recompression of the column bed to lengthen the column's useful life. CAP columns from Alltech Associates Inc. of Deerfield, Ill., also feature an axial compression piston. When tightened, the piston compresses the packed column bed to repair damage from voids or channels that occur as columns age. Alltech also carries an extensive line of chromatography media and other products. Sigma Chemical Co. of St. Louis and Aldrich Chemical Co. in Milwaukee have aligned to form Sigma-Aldrich Chromatography, a company that sells several types of chromatography supports. SigmaChrom high-performance affinity columns and SuperChrom columns permit researchers to separate substances from mixed solutions with a minimum of chromatographic runs. Supelco Inc. of Bellefonte, Pa., manufactures the broad SUPELCOSIL line of HPLC columns. Supelco also distributes Hypersil, Nucleosil, Spherisorb, and Alpha Bond columns for Sigma-Aldrich Chromatography, which are best suited for separations involving small molecules. The chromatography product line of Bio-Rad Laboratories Inc. in Hercules, Calif., includes several chromatographic supports for separations performed by ion exchange, size exclusion, hydrophobic interaction, and affinity chromatography. These products are supported by Bio-Rad's chromatography instruments, including the BioLogic medium-pressure chromatography system and the BioLogic LP low-pressure chromatography system. Dionex Corp. of Sunnyvale, Calif., also carries a full line of products for chromatography, beginning with the DX500 HPLC system. This system is supported by a selection of pumps and detectors and a computer workstation, along with the company's BioPlus chromatography columns. Dionex is also the sole source of CarboPac columns for separating carbohydrates by chromatography. Advanced Separation Technologies Inc. Alltech Associates Inc. Amicon Inc. J.T. Baker Inc. Beckman Instruments Inc. Bio-Rad Laboratories Inc. BioSepra Inc. BTR-Separations Corp. Djonex Corp. EM Separations Technology Fisher Scientific Krackeler Scientific Inc. PerSeptive Biosystems Inc. Pharmacia Biotech Inc. Rainin Instrument Company Sigma-Aldrich Chromatography Supelco Inc. Thermo Separation Products Corporation TosoHaas Trevigen Inc. VWR Scientific Whatman Inc. For analytical and preparative HPLC, BTR-Separations Corp. in Wilmington, Del., provides a broad range of media available in bulk or prepacked into stainless-steel columns. Zorbax LP from BTR-Separations consists of silica-based particles specially processed to be quite uniform in size. Zorbax LP is available with a variety of chemical groups bonded to the solid phase for partition chromatography performed in both normal phase (in which the mobile phase is less polar than the stationary phase) and in reverse phase. Reverse-phase chromatography does not involve running a column backward, as the name might imply; rather, in reverse phase the chromatographic support is hydrophobic (nonpolar) and the mobile phase is polar. Thermo Separation Products Corp., an instrumentation company in San Jose, Calif., carries a line of columns that may be used with its SpectraSYSTEM HPLC unit. The SpectraSYSTEM is a complete HPLC unit that includes an autosampler with a built-in temperature control unit. This allows biochromatographers to perform heated (or cooled) separations. A full line of customizable preparative liquid chromatography products is available from EM Separations Technology in Wakefield, R.I. Annular Expansion (A/E) Columns feature EM Separations' patented annular expansion technology, which allows for rapid column packing and unpacking. With the A/E columns, hydraulic action on a piston drives a tapered shaft into the column, where it compresses the surrounding packing material outward toward the column wall. When proper pressure is achieved, the piston is locked in place. Many compounds with value to the biopharmaceutical industry exist as a mixture of the compound in different states, called isomers. Isomers of vitamin E (tocopherol), for example, include alpha-tocopherol, gamma-tocopherol, and sigma-tocopherol, which are only slightly different from one another. Therefore, separation of the isomers by chromatography has proved to be technically difficult. MODULAR SYSTEM: Thermo Separation Products' Spectra-SYSTEM provides complete LC analysis and automated sample preparation. To resolve optical isomers, chromatography beads or particles are bonded to a chiral selector that separates the different isomeric forms. Amicon carries chiral packings such as R-DNPG-100 chiral phase silica, which can be used with polar solvents, and BSA-300 for separating water-soluble compounds such as aromatic amino acids and their derivatives, benzodiazepines, and aromatic sulfoxides. Advanced Separation Technologies Inc. in Whippany, N.J., offers column packings that are bonded with a variety of chiral selectors such as human serum albumin (HSA) and cellobiohydrolase. CYCLOBOND from Advanced Separation is bonded with cyclodextrin, a chiral selector suitable for separations of small analytes often of interest to pharmaceutical companies. These include amino acids, peptides, and small chemical compounds such as antihistamines, anti-inflammatories, vitamins, and steroid hormones. CHIROBIOTIC V and CHIROBIOTIC T consist of macrocyclic antibiotics bonded to a silica gel support. The antibiotic vancomycin (in CHIROBIOTIC V) contains 18 chiral centers in a complex structure that favors the selection of particular isomers. Teicoplanin, the bonded phase in CHIROBIOTIC T, contains 23 chiral centers in an equally complex structure. Process scientists can perform chromatography with either normal-phase or reverse-phase solvents, with different chiral selectivities in each mode. "For chiral separations, using a macrocyclic antibiotic as a stationary phase in liquid chromatography is similar to using a protein-based stationary phase," comments George Reid, director of research at Advanced Separations. Because of their size and chemical structure, these antibiotics show greater stability and a higher sample capacity than other types of chiral selectors. "On a large scale, researchers who perform chromatography strive for high throughput with good resolution," concludes Hall of NIH. "Any product that offers these attributes will quickly gain the attention of people working on the process and production scale." Holly Ahern is a science writer and an assistant professor of biology at Adirondack Community College in Queensbury, N.Y.

Biological Chromatography: It's Not Just A Tool; It's A Discipline

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