Peptide-Synthesis Services Lift A Load From Biological Research Labs

Among today's rapidly growing fields of biological study are those focusing on neurotransmitters, hormones, and various immunological and pharmacological agents. Central in many of these investigations is the analysis of biologically active peptides--chains of amino acids ranging from as few as two to more than 50 acids. Since these peptides can't be derived naturally in sufficient quantities--indeed, some cannot be naturally derived at all-- scientists increasingly are depending on synthetic development of peptides to meet their experimental needs. While many researchers have access to automated peptide-synthesis technology in their own laboratories or in core support facilities at their institutions, many are finding substantial advantages in contracting out the precise and time-consuming task of building a peptide to order. Thus, the growing need for peptides has spawned a robust young industry composed of commercial firms specializing in the synthesis of these amino acid chains. There are several virtues to using these outside suppliers. In- house resources may be overworked or technically limited, for instance. In some cases, it may simply be more cost-effective to turn to private support. In addition, recent advances in the catalytic chemistries used in peptide synthesis and improved instrumentation continue to extend the capabilities of these firms. "Many well-funded labs have gotten into doing peptide synthesis," says Martha Knight, president of Peptide Technologies Corp. in Gaithersburg, Md. "If they have money, they buy the synthesizer. But then they need to have a highly skilled technician or postdoc to do the work--and it's a lot of work. After a while they realize they're spending too much time in the synthesis and then characterization and purification of peptides. They'd rather be doing their more unique work. Researchers in biomedical labs everywhere now--medical schools, government labs, pharmaceutical companies--are coming to small companies like ours who make peptides." "With so many people asking for their services, the core facility of the university can get really loaded up," says Bosco Fong, an executive vice president of Peninsula Laboratories Inc. in Belmont, Calif. "When that happens, they go out and ask commercial companies like ours to do the peptide synthesis for them. We also get work from pharmaceutical companies for the same reasons. Most of the time, companies have their own facilities, but they are also getting too busy. They can't support all their R&D groups." Fong also points to another benefit of using commercial peptide- synthesis services: "When Scientist A asks Scientist B to do collaborative work [such as peptide synthesis], and there's a result, they have to coauthor on any papers. They don't have to do that with us. We provide them with the peptide, and they get all the credit." Automated peptide-synthesis technology has helped meet the needs of researchers for specific peptides since its invention in the 1960s by biochemist Robert Bruce Merrifield, now a professor, emeritus, at Rockefeller University in New York. In 1984, Merrifield was awarded the Nobel Prize in chemistry "for his development of methodology for chemical synthesis on a solid matrix." Improvements in the chemical reagent systems and the equipment used in the process of synthesizing peptides have led to better product yields. Still, the basic technique underlying the synthesis today remains the same as when Merrifield devised it. "It's possible to get purer long peptides with the methods used now than, say, 10 years ago, because there's been some improvement in the chemistry," says Knight, who trained in the labs of Merrifield and Rockefeller colleague John M. Stewart. "But the basic design of the chemistry still originates from Merrifield's work." "Our idea was to put the first amino acid of the peptide chain on a solid support, a polystyrene resin bead," says Merrifield. "You put it on by a rather stable bond. Then you carry out the addition of the second amino acid, so that you now have a dipeptide. You filter these polystyrene beads and wash them thoroughly to effect a partial purification. Then you're all ready to add the next amino acid, and you go through the same procedure." In this way, the peptide is built one amino acid at a time to the desired length. At the end of the synthesis, the peptide is removed from the solid support to be purified and characterized. "It's like stringing pearls," says Knight, "but you want to put them on in a certain sequence." In order for the synthesis to proceed in a controlled way, the several reactive parts of each amino acid must be activated and deactivated at appropriate times. Molecular "protecting groups" are added or removed to deactivate or activate the different parts, as needed. Commonly, the amino acid sequence to be synthesized is provided by the contracting researchers, perhaps implied by a cDNA sequence from a biological system they are currently studying. SUPPLIERS OF PEPTIDE-SYNTHESIS SERVICES AND EQUIPMENT Advanced Chemtech Louisville, Ky. Applied Biosystems Foster City, Calif. Bachem California Torrance, Calif. Biosearch Inc. Bedford, Mass. Clontech Laboratories Inc. Palo Alto, Calif. Coast Scientific San Diego Crescent Chemical Hauppauge, N.Y. Eppendorf North America Inc. Madison, Wis. Genosys Biotechnologies Inc. The Woodlands, Texas ICN Biomedicals Inc. Costa Mesa, Calif. INAMCO Chemicals & Lab Equipment Flushing, N.Y. Multiple Peptide Systems San Diego Peninsula Laboratories Inc. Belmont, Calif. Peptide Technologies Corp. Gaithersburg, Md. Peptides International Inc. Louisville, Ky. Perkin-Elmer Corp. Norwalk, Conn. Rent-A-Lab Inc. Vernon Hills, Ill. Research Genetics Huntsville, Ala. Research Organics Inc. Cleveland, Ohio Shimadzu Scientific Instruments Inc. Columbia, Md. Sigma Peptides & Amino Acids St. Louis Star Biochemicals Inc. Torrance, Calif. "There are three parts of an amino acid," says Freeman Stanfield, senior peptide scientist at Genosys Biotechnologies. "There's the carboxyl terminus, the side chain, and the amino terminus. "In the Merrifield method, 20 years ago and today, the carboxyl terminus is linked to the resin, the insoluble support, and there may or may not be a side chain protecting group, depending on the particular amino acid. And then there's an amine protecting group [at the amino terminus]. "At every cycle, the amine protecting group is removed, generating a free amino group. The amino terminus and, if necessary, the side chain of the incoming amino acid are also protected. The carboxyl terminus, then, of that second amino acid is coupled to the free amino [terminus] of the first amino acid. That process is repeated n number of times to make whatever size peptide that you want." Unfortunately, the chemistry used in this step-by-step synthesis process produces more side products as the peptide chain grows longer, resulting in smaller proportions of the desired peptide in the final product and a more difficult purification stage. It is in this area that new chemistries have been able to extend the technique's capabilities. The original Merrifield process uses a chemical called tBoc (tertiary butyloxycarbonyl) to protect the amino terminus and various benzyl groups to protect the side chains. An acid called TFA (trifluoroacetic acid) is the reagent used to remove the tBoc, in order to activate the terminus to receive the next amino acid. In the last step, a stronger acid, anhydrous hydrogen fluoride (HF), is used to cleave the peptide from the insoluble support. "The problem with that is that your peptide is exposed to [TFA] at every cycle," says Stanfield. "Over the [process of synthesizing], say, a 30- or 40-residue peptide, that's very deleterious. It's known that the benzylic side chain protecting groups in tBoc chemistry are not totally stable [during] the TFA treatment. So, you begin getting side products from that in longer synthesis." In a newer chemistry becoming more widely used, tBoc is replaced with a chemical called Fmoc (fluorenylmethyloxycarbonyl) to protect the amino terminus and tertiary butyl groups are used to protect the side chains. TFA is used only in the last cleavage step. As a result, the synthesis of longer peptide chains with fewer side reactions is now possible. The scientists at custom peptide-synthesis companies often engineer instruments according to their own specifications. At Genosys, for example, peptides are synthesized using a patented technique called segmented wafer synthesis, which is based on an adaptation of the company's proprietary DNA-synthesis machine. Over the years, however, a number of instrument manufacturers have identified peptide synthesis as a growing area and have developed automated machines for the task. Among these companies are Beckman Instruments Inc., Fullerton, Calif.; Biosearch Inc., Bedford, Mass.; Advanced Chemtech, Louisville, Ky.; Applied Biosystems, Foster City, Calif.; and Shimadzu Scientific Instruments, Columbia, Md. Often, these instrument makers will not seek to compete directly with the custom peptide-synthesis firms, but instead will target development of new chemistries and instrumentation that they then supply to the generally smaller service companies. At Biosearch, for example, chemical research and development is the company's focus, and it offers a number of proprietary reagents for peptide synthesis, according to Susan Hantman, a technical-applications specialist. For peptide synthesis, Biosearch markets the 9050 Plus PepSynthe- sizer for $59,500. The instrument is capable of synthesizing three peptides simultaneously and of using multiple activation chemistries within a single synthesis. With biologically active peptides, the purity of the final product is crucial. The omission or inadvertent inclusion of even one amino acid can dramatically alter the biological activity of a peptide. For this reason, a great deal of time and effort is expended after the synthesis in purifying and characterizing the peptide. Among the analytical methods commonly used are high- performance liquid chromatography (HPLC), mass spectrometry, electrophoresis, and amino acid analysis. "For analyzing a crude synthetic product to see how successful the synthesis was and for purifying that crude product to homogeneity, to whatever the desired purity level is, overwhelmingly HPLC is the method of choice," says Stanfield. "The other very useful analytical tools, in approximate order of importance, are mass spectrometry and amino acid analysis." Peptide-synthesis services range in cost from hundreds to thousands of dollars, depending on the length of the amino acid chain and other factors. While there are ways to reduce expenses in the synthesis process, Fong cautions that there are things customers may want to consider when trying to save money on peptide services. The analysis stage of peptide synthesis is an area in which some companies try to trim costs, he says. "One word of caution for people looking for cheap custom synthesis is to make sure that, if they're quoted a very cheap price, the peptides have gone through stringent quality-control tests," Fong advises. Inadequately purified and characterized peptide products put the reliability of whatever experiments a researcher might later conduct with that peptide at risk. "The cost of the peptide then becomes insignificant," says Fong, "when compared with the time wasted."

Peptide-Synthesis Services Lift A Load From Biological Research Labs

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