Immunological Applications Top List Of Peptide-Synthesis Services

Peptide-Synthesis Services Peptides-linear chains of amino acids-are the building blocks of nature. Understanding their action figures prominently in recent advances in many fields, particularly immunology. Such insight, for example, allows investigators to elucidate the details of antigen-antibody interactions of the human immune system. VERSATILE: Services at Richmond, VA.-based Commonwealth Biotechnologies include peptide and DNA synthesis. Because scientists aren't able to extract sufficient quantities of peptides from natural sources for their experiments, a growing number of companies are custom-manufacturing peptides for research. Synthetic peptides are used for a variety of molecular biological applications, including monoclonal or polyclonal antibodies for immunological research, peptidomemetics, and the testing of prospective drugs in clinical trials. Researchers generally target a specific amino acid sequence of the protein they're working on and order that sequence in quantities ranging from a few milligrams to many grams, depending on the project. "We have customers throughout the research community," notes Mark Peterson, manager of commercial operations at Advanced ChemTech Inc., a Louisville, Ky.-based company that performs custom synthesis in addition to manufacturing and selling synthesizers. "In industry, our clientele ranges from small biotechs who may not have the capabilities to do synthesis themselves to large companies who may need the service to bolster their own capacity. We also have clients in government and academia." SIMPLICITY: Advanced ChemTech's tabletop peptide synthesizers. The company also performs custom synthesis. Saving time is the main reason more and more researchers are farming out their peptide synthesis to companies like Advanced ChemTech (F. Hoke, The Scientist, Aug. 22, 1994, page 17). "Probably the No. 1 reason for researchers going to a private company for synthesis is speed," says Peterson. "There are two types of people who come to us for peptide services. One is the scientist who just needs help in getting everything done. The second is the researcher who calls, saying: 'This is a troublesome sequence, and I need help. My time is now better spent not doing this.'" In the last two years, pharmaceutical companies' rush to embrace combinatorial chemistry as a new way to discover drugs has also changed the face of the peptide-synthesis industry (K.S. Brown, The Scientist, May 13, 1996, page 1). Some companies are now offering combinatorial libraries of peptides, which are collections of chemical variations on different classes of these amino acid chains. "Combinatorial peptide libraries and multiple-peptide synthesis are slowly but surely replacing much of the demand for single peptides," observes biochemist Robert Harris, president of Commonwealth Biotechnologies Inc. in Richmond, Va. "Researchers want lots of different sequences that may vary by a small amount. They also want sequences that incorporate unusual amino acids or organic synthons that replace amino acids." About 35 commercial companies offer custom-made peptides and a variety of premade peptides through catalogs. For example, Research Organics Inc. (http://www.resorg.com), located in Cleveland, sells a large variety of blocked amino acid derivatives to academic labs so that faculty researchers can conduct their own syntheses, according to Dan Flowers, director of research and development. The firm also custom-synthesizes small peptides. Other firms, though not as many, produce the equipment to perform the synthesis. In the United States, there are about five major manufacturers of synthesizers (S. Veggeberg, The Scientist, Jan. 23, 1995, page 17). They are Advanced ChemTech; Applied Biosystems Inc. in Foster City, Calif.; Gilson Medical Electronics Inc. in Middleton, Wis.; PerSeptive Biosystems Inc. in Bedford, Mass.; and Rainin Instrument Co. Inc. in Woburn, Mass. Solid Support Peptide-synthesis companies-as well as academic and government-based synthesis facilities-use two basic manufacturing methods. The most common is the solid-phase Merrifield method, named for Robert Bruce Merrifield, a professor, emeritus, at Rockefeller University who won the 1984 Nobel Prize in chemistry for developing the procedure. In this process, the first amino acid in the peptide string is attached to a resin support bead. A series of mechanical filtering and washing steps between each addition of a new amino acid follows. When the peptide reaches the target length, it is cleaved off the bead with harsh chemicals. Genosys Biotechnologies Inc., located in The Woodlands, Texas, is working on ways to improve the efficiency of the Merrifield method. "What's different about us is that we use the proprietary Abacus technology," says Alan Bergold, the company's director of peptide operations. "The synthesis is done in individual wafers in the Abacus system. Ultimately, this means that we generally have higher-purity products off the instrument. About 63 percent of all peptides that are 15 amino acids or less come off at greater than 75 percent purity. Other people are limited to synthesizing a single peptide at a time, whereas we can do 48 peptides simultaneously in the wafers." Genosys just received a Small Business Innovation Research grant from the National Institutes of Health to expand its multiple-synthesis technology. "Our ultimate goal is to have a synthesizer that will make 100 peptides within one day," declares Bergold. CUSTOMIZED: Peptides made to order at Peptides International in Louisville, KY The second, far less common procedure is called solution-phase synthesis, in which small peptides, sometimes with unusual chemistries, are linked together in solution to make longer ones. "With the Merrifield method, you put the amino acids on by machine one at a time, so the peptide grows but isn't usually purified until the end of the synthesis," explains Arno F. Spatola, president of Peptides International Inc. (http://www.pepnet.com) in Louisville, Ky., and a professor of chemistry at the University of Louisville. "Solution-phase gives a purer product," he says. "The down side is that it's more expensive." However, he notes, researchers doing specialized R&D projects at pharmaceutical companies are interested in peptides derived in this way. Peptides International uses the Merrifield method for its operations, but it also distributes solution-phase-prepared, rare toxins. These molecules and a variety of bioactive peptides for specialized research are sold through the firm's cooperative agreement with the Peptide Institute, a peptide-synthesis company in Osaka, Japan. One peptide that Peptides International sells-121-amino-acid-long Midkine-has the distinction of being the longest synthetically made peptide that is commercially available. Midkine regulates growth and cell differentiation. Midkine aside, researchers and company representatives agree that a major technological limitation is making peptides above a certain length quickly and inexpensively. "We usually won't tackle making any peptide above 50 or so amino acids," says Peterson. For longer peptides, the choice of custom-synthesizing vs. using some other method for researchers comes down to cost-effectiveness. Recombinant technology might be a more efficient way to go, Peterson adds. Peptides that are 100 amino acids or more in length have been synthesized mechanically, but not on a cost-effective, industrial scale. "The newest thing in peptides right now is ligation, which allows you to make longer sequences," says Mark Sanders, marketing director at Bio-Synthesis Inc.(http://www.biosyn.com) in Lewisville, Texas. "But that's still in its early stages. We can go up to the mid-40s right now. We make a series of, say, 20 amino-acid-long peptides and stick them together in the proper order." But ligation is still being refined, especially the step that involves tacking the shorter amino acid strings together in the proper order. "A lot of people are working on this, though, because many researchers need 80- to 90-amino-acid-long peptides for neuroscience and cell biology projects," notes Sanders. On the other hand, remarks Advanced ChemTech's Peterson, "With more large biomolecules-such as cytokines and chemokines-being isolated, researchers are trying to determine the molecules' active sites or modify those sites. Because of this, I also see the need arising for smaller, more easily handled peptides." PRECISE PURITY: Peptides are synthesized and purified for both clinical and nonclinical applications at American Peptide Co. based in Sunnyvale, Calif. Among the myriad applications for which scientists use peptides, immunological projects use far more than any other type of study. "Most of the demand that we're seeing is for immunological-grade peptides to make antibodies for various purposes-for example, diagnostics or identifying active amino acid sequences within a protein," relates Peterson. "The antibody itself will recognize the sequence, and researchers use that antibody to determine what molecule they are recognizing." POWERFUL PRODUCTS: Vials of endothelium produced by American Peptide for hypertension research. "The most common application of our peptides is for polyclonal antibodies and epitope mapping," says Bio-Synthesis' Sanders. An epitope is a region on the surface of an antigen capable of triggering an immune response and combining with the particular antibody produced by that response. "If researchers are trying to find an epitope, they'll send me an order for 10 or so discovery-scale peptides with the exact same sequence, except that in one position an amino acid will be changed," comments Sanders. "One of those will work on the epitope and nine won't. Then they'll come back and order a larger, more pure peptide from the one peptide sequence that works." Immunologists use San Diego-based Chiron Mimotope's Multipin synthesis technology for screening antigens from already characterized proteins. The Multipin is based on the design of the familiar, 96-well microtiter plate. By synthesizing an individual peptide on each of 96 pins that correspond to each microtiter well, one can produce large numbers of custom epitopes inexpensively, according to technical consultant Michael Routhe. Immunological diagnostic kits also figure prominently in researchers' peptide needs. Martha Knight, president of Peptide Technologies Corp. in Gaithersburg, Md., comments that "in the last year, we've been synthesizing more peptides with unusual chemical groups attached. We've been putting fluorescent dyes on peptides for developing diagnostic antibody kits for all kinds of diseases." Her company will offer peptides for determining enzyme activity in apoptosis in the near future. Selected Peptide Synthesis Companies and Suppliers of Peptide Synthesis Equipment: Advanced ChemTech Inc. American Peptide Company Applied Biosystems Inc. Bachem Bioscience Inc. Bio-Synthesis Inc. Chiron Mimotope Coast Scientific Inc. Commonwealth Biotechnologies Inc. Genosys Biotechnologies Inc. Gilson Medical Electronics Inc. Peninsula Laboratories Inc. Peptide Technologies Corp. Peptides International Inc. PerSeptive Biosystems Inc. Rainin Instrument Co. Inc. Research Organics Inc. While immunologists rely heavily on synthetic peptides, they are used in a variety of nonimmunological research projects. Shawn Lee, vice president of operations at American Peptide Co. in Sunnyvale, Calif., says his company custom-synthesizes peptides for both clinical and nonclinical applications. "We are the largest producer of endothelins, a family of hormones used in hypertension research," notes Lee. "We also produce beta-amyloids for studying Alzheimer's disease." Bosco Fong, executive vice president of Peninsula Laboratories Inc., located in Belmont, Calif., states that one of his company's specializations is peptidomemetics. This is a drug-development technique in which a natural protein sequence is modified so that it will perform such desired functions as crossing the blood-brain barrier and cell membranes, as well as prolonging its residence time in the human body. This is achieved by putting a special amino acid into the sequence to make it more soluble, for example. Bachem Bioscience Inc. in King of Prussia, Pa., is also working extensively on peptidomemetics, according to executive vice president Jos} deChastonay. "Researchers try to find out the three-dimensional structure of a peptide and what part of that structure makes it bioactive," he says. HANDY KITS: Making peptides for immunological diagnostic kits is one of the services offered by Gaithersburg, Md.-based Peptide Technologies. Many peptide-synthesis companies either have just added or have plans to add production of combinatorial peptide libraries to their services. For example, Coast Scientific Inc. of San Diego is aiming to be able to produce combinatorial arrays of peptides and antibodies within the next six months. The big change for peptide synthesis, notes Fong, is on the drug-discovery end via combinatorial chemistry. "In the old days, when we wanted to modify a peptide we had to do hundreds of sequences blindly, but with combinatorial chemistry techniques, it's a more targeted approach." "Adding combinatorial chemistry as a service is certainly something that you have to be able to do," says Peterson. "We have done that in the last two years. One of our instruments specifically makes combinatorial libraries." Automated synthesizers were first made commercially available in the mid-1980s. Now, various machines are capable of synthesizing amino acids into peptides en masse for combinatorial screening. Harris predicts that there will be an "explosion" in clients screening libraries for a variety of activities, but that combinatorial approaches to peptide synthesis will continue to be primarily for immunological applications. Some companies offer such related services as DNA synthesis in addition to making peptides. "For instance, a scientist will deliver us an idea," says Commonwealth's Harris. "They might have an antigen or an antibody that has some biological properties they're interested in pursuing. They'll give us a source of that molecule and we'll purify, fragment, and sequence the protein. Then we'll do the cloning to eventually get the DNA. From this, we'll make peptides based on the primary amino acid sequence laid out in the genetic instructions." Most peptide-synthesis companies do just the synthesis, with other companies performing the DNA work. Bio-Synthesis is another company that works with both DNA and peptides. "We're one of the only companies that does both custom DNA and peptide synthesis," notes marketing director Mark Sanders. He says that the advantage to this approach is that both types of sequences are related. "A lot of researchers will find the amino acid sequence of the protein they're working on, and then they'll need peptides made out of that sequence. They need both the peptides and oligonucleotides [short chains of DNA or RNA bases] to be made synthetically so they can perform PCR [the polymerase chain reaction], for example. From a researcher's standpoint, DNA and peptide sequences are not mutually exclusive. You have to work with both at some point, although lots of projects will have different sets of people working on each phase." FDA-APPROVED: A GMP-grade facility at Penninsula Labs in Belmont, Calif. Not all peptides are synthesized for basic research projects. Some are headed for clinical trials and must be extremely pure. These peptides are made in Good Manufacturing Procedure (GMP)-approved labs. GMP-approved procedures (a Food and Drug Administration standard) for making peptides ensure these molecules won't compromise the clinical-trial data that will ultimately have to pass muster with FDA reviewers. A few companies, such as American Peptide, Coast Scientific, Peninsula, Bachem, and Advanced ChemTech, do GMP-level synthesis. However, the expense and special expertise involved can be prohibitive. "If customers have a certain peptide and they want to bring it through clinical trials, that has to be performed under GMP methods," says Peninsula's Fong. "We are facilitators for that: We have the facility, the personnel, and the quality-control-approved labs." Peninsula focuses on contract GMP peptide synthesis, but also takes on smaller, more specialized jobs. Keeping a meticulously accurate paper trail is essential for GMP peptide production, notes Fong. "If something goes wrong with the peptide made in a GMP environment, you can trace step-by-step through the records to see where the mistake occurred." Bachem's deChastonay points out that his company, in addition to selling more than 6,000 premade peptides, custom-synthesizes GMP and non-GMP-grade peptides. "We're actually the only peptide company that manufactures, on a contractual basis, drugs that are found on the pharmacy shelf in the U.S., Japan, and most European countries. I would say a good half of our turnover is achieved by synthesis of bulk drugs for large pharmaceutical firms and biotech firms." Because of rapid technological advances in combinatorial chemistry and immunology, company representatives agree, the range of peptide-synthesis options for investigators is better than ever.

Immunological Applications Top List Of Peptide-Synthesis Services

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