Peptide, Oligonucleotide Synthesis: Key In Molecular Studies

HIGH THROUGHPUT: PE Applied Biosystems’ ABI 394 DNA/RNA Synthesizer is controlled by a Macintosh computer. Polypeptides (proteins) and polynucleotides (DNA/RNA) are the two essentials of organic life. Just as detailed blueprints and clay bricks are essential to construction workers, shorter synthetic versions of these two molecular workhorses-peptides and oligonucleotides-are crucial to experimental biologists. Biologists put oligonucleotides to nearly as many uses as Mother Nature does the genes that are constructed from them. Most common, perhaps, is the use of short stretches of DNA or RNA as primers for the polymerase chain reaction (PCR), in which often very dilute genetic material of a specific organism is amplified to usable or detectable levels. Labeled oligos can "probe" samples by linking to an opposing strand through the Watson-Crick base pairing rules: Guanine will find its cytosine counterpart (G-C), while adenine and thymine converge (A-T). The presence of the target genetic material may be signaled by light emitted by the label, or the label may itself serve as the target for antibodies or other small molecules, which themselves provide a signal. Oligonucleotides are also wending their way into therapeutic use. Varying cancer, viral, and even genetic disease therapies rely on oligonucleotides or closely related derivatives to intercept the messenger RNA (mRNA) that carries the message from the DNA in the nucleus to the protein-manufacturing apparatus in the cytoplasm of the cell; this is dubbed the antisense approach. The antisense oligos are the opposing strand of the target mRNA, binding tightly to it. Once gagged, the mRNA falls out of circulation, and the protein it encodes either is reduced or is not produced at all. As they gear up for research production, clinical trials, and (they hope) mass production, companies like Isis Pharmaceuticals Inc. of Carlsbad, Calif., are in need of oligonucleotide synthesizers that can meet big production needs while simultaneously keeping the cost of materials down. With a need to manufacture kilograms of individual oligos, Isis turned to Pharmacia Biotech Inc. of Piscataway, N.J. The partnership has proved fruitful, reports Anthony Scozzari, assistant director of drug substance manufacturing at Isis. Pharmacia's OligoProcess DNA/RNA Synthesizer can handle runs of up to 100 mmol, which is up to 20 times greater than the capacity of competing synthesizers, says Scozzari. The linear flow-through design saves reagents and solvent by flushing reagents through. "Other instruments I've seen work through a serial dilution mechanism-so you add a little bit [of reagent], mix it, then drain it, and then you add a little bit, mix it, and drain it, so you're constantly diluting the next step with materials from the prior step," he notes. By contrast, the OligoProcess flushes reagents and solvents through linearly and then sequentially adds new reagents for the next synthesis step. DESIGNED FOR SCALEUP: Pharmacia's OligoPilot Ii DNA/RNA Synthesizer shares software with the company's OligoProcess unit. Pharmacia's OligoPilot II DNA/RNA Synthesizer is a complementary instrument, designed for scaling up synthesis processes for production. It can run batches up to 5 mmol-also larger than any comparable pilot synthesizer, according to Jack Ball, vice president for sales and marketing in the specialty bioproducts group at Pharmacia. The OligoPilot and the OligoProcess share software, simplifying the scaleup procedure, says Ball. "In addition, if someone wants to purify the crude oligo after synthesis, Pharmacia offers a full range of purification systems which utilize the same software platform as OligoPilot and OligoProcess." For users in need of smaller quantities of many different oligos, PE Applied Biosystems, a division of Perkin-Elmer in Foster City, Calif., sells the high-throughput ABI 394 DNA/RNA Synthesizer. Simultaneous synthesis of four oligonucleotides takes a few minutes more than other instruments need to produce two, the company claims, and system upgrades are available to enable handling of larger bottles for phosphoramidites and reagents, doubling capacity. The eight positions on the ABI 394 allow the user to keep two full sets of bases on the instrument, making it possible to switch from one set to the other to extend unattended runs. The instrument can make up to four primers or probes simultaneously, each with a different reporting group, according to the company. The instrument is controlled by a Macintosh computer, and an unlimited number of instruments can reside on an AppleTalk network. Password protection gives control over access privileges. Operators can set up the instrument and monitor synthesis without leaving their desks. HIGH-VOLUME SYNTHESIZER: Beckman’s multicolumn Oligo 1000M DNA Synthesizer is designed to be flexible. The multicolumn Oligo 1000M DNA Synthesizer, sold by Beckman Instruments Inc. of Fullerton, Calif., is a high-volume synthesizer with a similarly flexible design. Its two-minute cycle times and 10-minute cleavage and deprotection reactions produce oligos faster and more economically than other DNA synthesizers do, the company says. Each of its eight columns can be programmed to synthesize different oligos. The random sequential access lets the user change, insert, delete, and reorder previously queued sequences, even while the instrument is in the middle of a run. Beckman's Oligo 1000 DNA Synthesizer is designed for low-volume oligo production. It can produce two research-ready 20-mer primers in less than two hours, while consuming up to 50 percent less of the phosphoramidite synthesis reagent than other synthesizers do, according to the company. The optional Binary-Pak saves additional operator time, reportedly ensuring error-free dilution of the phosphoramidite. The diluent and phosphoramidite are premeasured into separate sections of the bottle. Peptide fragments of an enzyme or a receptor are commonly used to probe its structure, on the assumption that the individual fragments will have a three-dimensional structure similar to the one they have when embedded in the larger protein. They also are used to produce site-specific antibodies, to identify the regions (epitopes) of a protein antigen that antibodies recognize and bind specifically to, and as tabletop sweeteners (the dipeptide aspartame, or NutraSweet), among other applications. The vast role that proteins play in regulating biochemical processes, signaling, DNA transcription, and a variety of other biological processes has drawn intense interest in using proteins and peptides therapeutically. Early attempts were set back in part by the overabundance of proteases in the bloodstream that can rapidly degrade would-be peptide drugs. As those challenges are gradually overcome, peptides will likely stake a growing place in the future pharmaceutical marketplace. FOR LOW-VOLUME USERS: Advanced Chemtech’s ACT Model 90 tabletop peptide synthesizer is suitable for low-budget academic labs. With such needs in mind, Advanced Chemtech Inc. of Louisville, Ky., sells its Model 400 production-scale peptide synthesizer. The instrument is targeted to the manufacturer that follows the Food and Drug Administration's Good Manufacturing Practices (GMP) regulations for producing drug products. "We have built in a number of safeguards to prevent users from doing something that would compromise a regulated process. . . . It's possible to have $50,000 worth of materials in the reaction mixture. You certainly don't want to lose something [to contamination] at that point," says Mark L Peterson, vice president of technology at Advanced Chemtech. Advanced Chemtech also produces peptides, and it looks no farther than its own instrument line for its manufacturing needs. "We use all of the instruments internally for our own production. That's something no other company in the peptide field can claim," maintains Peterson. The company's Model 348 multiple peptide synthesizer can make up to 48 peptides in quantities of up to 150 mg of crude peptide per reactor well. It can also handle larger-scale synthesis beginning with up to 1 gram of starting resin, all without requiring the user to purchase additional hardware, the company claims. The vortex agitation system employed by the instrument increases reaction rates while ensuring homogeneous mixing for high yields, according to the company. It also grants operator control of reaction mix times and speeds, allowing the user to adjust mix protocols for material viscosity and quantities. The 348 comes with a standard 48-well reactor, but 8- and 16-well reactor options are also available. BUILT-IN SAFEGUARDS: Advanced Chemtech’s Model 400 production-scale peptide synthesizer is designed for labs performing FDA-regulated processes. For the low-volume user, Advanced Chemtech offers its ACT Model 90 tabletop peptide synthesizer. The unit is targeted primarily to low-budget academic labs, according to Peterson, though some companies use it for process development and scaleup. It can make two peptides concurrently at scales from 0.1 to 10 mmol, and it can be standardized to one preferred chemistry and scale so that little peptide synthesis experience is necessary. "It's a very low-cost entry into peptide synthesis," says Peterson. Of course, even low-cost devices can be prohibitive for some. But for labs whose budgets are tight, custom synthesis houses can be a sensible alternative. At least for oligonucleotides, cost shouldn't deter anyone, observes Asim Esen, a geneticist at Virginia Polytechnic Institute and State University (Virginia Tech). "It started [in the 1980s] at $3 to $4 per residue . . . [the cost of oligos is] down to 84 cents a residue . . . making them yourself [via manual lab methods] is not worth fooling with," he says. At least one custom oligo supplier fears that the falling prices may have produced an unexpected backlash. The chemistry of oligonucleotide synthesis-like any chemistry-is imperfect. Side reactions on the nucleotide bases, failed couplings, and misfired capping steps can occasionally result in lots that must be remade. "Unfortunately, the user who is ignorant of the limitations of oligo synthesis is also unaware that there are going to be 'bad' oligos that are defective-not because any of the suppliers are incompetent or lazy, but [because] this is an imperfect world and errors do occur," states Michael T. MacDonnell, chief scientific officer at Ransom Hill Bioscience Inc., a Ramona, Calif.,-based supplier of custom oligos. The number of ill-fated lots is about seven per 1,000, says MacDonnell. Noting the rapidly decreasing prices, some customers perceive these occasional do-overs as resulting from cutting corners to reduce costs, he notes. In fact, such errors have decreased slightly in the last decade as the techniques and instrumentation improve. "There is probably no sector of the biotechnology service industry with more conscientious effort made to deliver a good product than can be found in the synthetic oligo industry," he maintains. LINEAR FLOW-THROUGH: Pharmacia’s OligoProcess DNA/RNA Synthesizer saves reagents and solvent by flushing reagents through. Madison, Wis.-based Eppendorf Scientific Inc. provides custom oligonucleotides with an eye toward meeting specific labeling needs of researchers. The company can tag the oligos at the 5« end with its multihydroxy linker, which acts as a branch that can support multiple tags to help increase the signal level. But the company will also meet the customer's requirements. "If a customer requires something that is not a common label, we'll work to accommodate their needs," notes Julie Robinson, product manager at Eppendorf. Selected Suppliers of Peptide and Oligonucleotide Synthesis Equipment And Services: Advanced Chemtech Inc. BACHEM Bioscience Inc. Beckman Instruments Inc. Chiron Technologies Eppendorf Scientific Inc. Isis Pharmaceuticals Inc. PE Applied Biosystems Pharmacia Biotech Inc. Ransom Hill Bioscience Inc. Quality Controlled Biochemicals Inc. Wako BioProducts Some will shy away from custom synthesis, taking to heart the old adage: "If you want something done right, do it yourself." "A lot of times we don't have a rigorous way of checking the quality of the [peptides] we receive, other than the data sheets that are provided" by the custom-synthesis company, says Esen. A few years ago, even the cost of custom synthesis-at $15 to $20 per residue for 20 mg to 50 mg of peptide-was out of his reach, so he made them himself, using the TEA-BAG procedure developed by Richard Houghton at Scripps Clinic and Research Foundation in La Jolla, Calif. (R. Houghton et al., Proceedings of the National Academy of Sciences, 82:5131-5, 1985). The procedure allows synthesis of up to 100 peptides simultaneously, but the process is time-consuming and labor-intensive. If you are concerned about the quality of custom-synthesized peptides, go to a company with a long history of providing quality materials, advises Lora Hartzell, product manager at BACHEM Bioscience Inc. of King of Prussia, Pa. "Because we've been in business for 26 years now, we have a lot of experience that we've built upon. . . . In most cases, our peptides are greater than 95 percent pure," as compared with the more typical 80 percent to 95 percent, Hartzell claims. Different suppliers provide peptides in varying forms. San Diego-based Chiron Technologies, formerly Chiron Mimotopes, synthesizes the peptides onto specially prepared polyethylene pins mounted on blocks. (The resulting set is called a Pepset.) The company then will either cleave the peptides from the pin or provide them to the researcher still attached in a defined format that is compatible with the standard 96-well microtiter plate formatted for many biological assays. If supplied still attached to the pin, the 96-pin block can be inverted into the assay and removed at the end to be used again. "Simply disrupt the molecular interactions [antigen-antibody affinity] with sonication or some other method, and then reuse the peptide and the pin in a new assay," says Michael Routhe, technical consultant at Chiron. Quality Controlled Biochemicals Inc. (QCB) of Hopkinton, Mass., routinely makes peptides of three to 65 amino acids in length, with purities to 98 percent and in quantities from 5 mg to 50 grams, says Julie Szabo, technical support manager at QCB. The company also carries more than 225 bioactive peptides, including kinase subscrates, inhibitors, apoptosis peptides, neuropeptides, and more than 35 different fragments of the b-amyloid protein, which takes part in formation of the fibrillar plaques that may be involved in the pathogenesis of Alzheimer's disease. Wako BioProducts of Richmond, Va.-a division of Wako Chemicals USA Inc.-aims to tailor its custom-built peptides to a researcher's needs. For what the company describes as a "modest" charge, it will provide sequence analysis or mass spectroscopy in addition to the amino acid compositional analysis and a high-performance liquid chromatogram that it provides free of charge. "The majority of price-per-amino acid suppliers simply provide a peptide without assessing [what happens after] the researcher opens the peptide's vial. Wako previews each request and keeps the ultimate application [of the peptide] in mind for each custom request," maintains Renee A. Davis, technical representative at Wako. As in any pursuit, it is of course the final result of biological research that gets noticed. But no research project will get off the ground without reliable starting materials. Custom synthesis houses and instrument providers will continue to offer varying services and devices that should meet just about everyone's need-the trick, perhaps, is finding the company or instrument whose capabilities best match your project's requirements. James Kling, a science writer based in Bellingham, Wash., can be reached online at jkling@nasw.org.

Peptide, Oligonucleotide Synthesis: Key In Molecular Studies

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