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Jul 1, 2010 (Vol. 30, No. 13)

New Practices Simplify Peptide Synthesis

Burgeoning Demand Makes Rapid and Economical Production Strategies Essential

  • Sidebar: Peptide News & Trends

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    Polypeptide prides itself on its willingness to undertake difficult projects requiring large-scale manufacture, complex modifications, and high purity.

    Peptide synthesis is an expanding component of the biotechnology industry. With advances in the technology of linking amino acids together, the cost of synthetic peptides has dropped dramatically, while the ability to produce these complex molecules with sulfhydryl bonds and other ornate secondary modifications has continued to improve. The result is higher yields, greater purity, and a wider range of options for customers. Recent discussions with company representatives highlight the different ways that challenges are being met within the industry.  

    AnaSpec/Eurogentec was originally conceived as a peptide manufacturer and over the years has expanded its offerings. “While we started as a peptide company, we expanded into dyes, unusual amino acids, antibodies, and assay kits,” explains Anita Hong, founder of AnaSpec and now GM of AnaSpec/Eurogentec. 

    One product line in particular demand is zebrafish-specific antibodies, according to Hong. Prolific and far removed from the animal-rights debate, zebrafish are particularly suited to tracer experiments using fluorescently labeled antibodies. AnaSpec’s Z-Fish™ antibodies are raised from peptide antigens with 100% zebrafish sequence homology and are able to distinguish nonphosphorylated and phosphorylated peptides.

    AnaSpec offers a number of other research tools that are of interest to the biotech community such as the myelin oligodendrocyte glycoprotein (MOG), expressed on the surface of myelinating oligodendrocytes and the external lamellae of the myelin sheath. Because the MOG protein constitutes a crucial autoantigen for multiple sclerosis, AnaSpec has developed MOG peptides, recombinant protein forms, antibodies, and kits to characterize this substance and clarify its role in neurological disorders.

    The company also offers reagents for “click chemistry,” which is defined as an approach to the synthesis of drug-like molecules through a reaction that is wide in scope and easy to perform with readily available reagents, while being insensitive to oxygen and water. “We supply reagents for people who want to do their own click chemistry,” says Hong. One of the most widely used is TBTA, a stabilizing ligand required for many click chemistry reactions.

    Yet another widely used research tool is fluorescence resonance energy transfer (FRET), in which excited state energy is transferred from a donor to an acceptor molecule when the two are brought into close proximity, resulting in quenching of the signal. Using labeled peptides with appropriate cleavage sites, enzyme activities can be measured when the donor and acceptors are separated. Enzyme hydrolysis of the peptide results in spatial separation of the donor and acceptor, which leads to the recovery of the fluorescence of the donor. The company offers kits for measuring renin and various other proteases.  

    Peptide Synthesizers

    A manufacturer of peptide synthesizing instruments for 25 years, Protein Technologies just introduced the Overture™ Robotic Peptide Library Synthesizer. “When we conceived the initial plan of the Overture, we looked at the current market needs, identified the drawbacks of the existing devices, addressed these concerns, and produced a new offering,” explains CEO Mahendra Menakuru.

    Overture is built with six blocks, allowing six different protocols to be performed simultaneously. “Typically, robotic synthesizers use the same amino acid delivery technique, drawing their materials from bottles that have to be washed after every amino acid delivery, an expensive and time-consuming process. The Overture synthesizer, on the other hand, is built with dispensers that do not have to be cleaned, saving time and money. Other features of the Overture include redesigned software for an onboard computer that will accept a peptide sequence introduced through a USB port.” 

    Peptide Supply

    Peptisyntha bases its technology on work carried out at the University of Ghent in the 80s. The company was founded in 1987; its operation was enlarged in 2001 to focus on peptide synthesis by solid-phase technology.  

    Peptisyntha is experienced in solution-phase and solid-phase synthesis coupled with hybrid synthesis and has increasingly used ion-exchange chromatography for purification. Unprotected amino acids have been used over the years to improve economics of costs. “Our approach to small peptide production (<12 amino acids) uses a proprietary process that avoids HPLC, so we can generate these peptides at a reduced cost.”

    The company has also moved into the antimicrobial peptide arena. As more and more bacterial strains are developing resistance to conventional antibiotics, cationic peptides that lyse the cell wall are showing increasing appeal.

    “We are working with arginine-based peptides that we feel will be a new weapon in the battle against bacterial diseases. The strategy consists of manipulating side chain unprotected arginine residues in the form of their highly lipophilic tetraphenylborate salts. In this fashion, the peptide-coupling reaction can be run at high concentrations in solution  with straightforward recovery procedures.” 

    Polypeptide Group is a provider of custom and generic GMP-grade peptides, according to Rodney Lax, Ph.D., senior director of business development for North America. Worldwide, the privately held company has a staff of around 450 employees. “The fact that we are privately owned gives that additional degree of security that the company will be around for the long term.” This is a critical consideration, given the fact that the approval process for peptide drugs takes an average of 10–12 years.

    Dr. Lax emphasizes the willingness of his company to undertake difficult projects, requiring large-scale manufacture, complex modifications, and high purity. “We push analytical development from the outset. We prefer to solve problems early in development rather than have to redesign the manufacturing process later. Many peptide candidates nowadays are longer peptides, sometimes with 40 amino acids or more. Identification and separation of impurities represent challenges that cannot be addressed with the commonly used analytical systems.”  

    According to Lax, while there are probably no major changes in chemistry for large-scale manufacturing on the horizon, he foresees advances in equipment and technology associated with their increasing scale. “There will always be a demand for rapid progress to the clinic. For this purpose, the best approach is the adoption of solid-phase chemistry, which allows rapid production of large quantities of material.” 

    Lax believes that a universally applicable method for oral delivery of peptides would revolutionize peptide-based pharmaceuticals. This will require new encapsulation technologies to allow the peptides to reach the lower gut unscathed. The disadvantage of the oral route, or the use of other nonparenteral routes such as transdermal or inhalation strategies, is that the amount of peptide that finally reaches the circulation may be very low, requiring significantly larger doses of the peptide. “While this raises economic issues, many  peptide drugs are active in microgram quantities and using alternative delivery systems remains a very attractive option.”

    Cranking Up Production

    American Peptide Company produces research- and GMP-grade peptides at two separate production facilities in California.  The firm provides a variety of services related to peptide production, including process development, scale-up of production, and regulatory support. At this point, the bulk of its activities center around the expanded production of GMP-grade peptides for clinical studies and continued supply in the research-grade market, says Gary Hu, vp of sales and marketing. 

    An expansion was completed in late 2009, allowing the company to pursue large-scale purification under cGMP conditions. This included the installation of large 12-inch purification columns and expanded lyophilization capability to handle multikilogram single batch demands. Another expansion is under way to increase large-scale synthesis for solution-phase and solid-phase chemistries.

    PEGylation, a widely exploited approach to the delivery of protein-based therapeutics, has revolutionized the treatment of hepatitis C and other diseases due to the improved pharmacokinetics, decreased toxicity, and increased half-life in circulation.

    The process involves the chemical attachment of polyethylene glycol to the peptide or protein. Great care must be taken in chosing the appropriate attachment site if the maximum therapeutic benefit is to be realized. “We have substantial experience in the large-scale production of PEGylated peptides and proteins and feel this is one of our major strengths.”

    In addition to custom synthesis contracts, the company has a catalog of around 1,500 widely used peptides. Custom production runs from milligrams to kilograms. 

    “Peptide synthesis occupies a particular position in the world of biologics production. While it will not replace the use of recombinant DNA manipulation for very large proteins, for smaller peptides it represents a rapid and economical alternative.”

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