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Feature Articles : Jul 1, 2010 ( )
New Practices Simplify Peptide Synthesis
Burgeoning Demand Makes Rapid and Economical Production Strategies Essential!--h2>
The molecular properties of peptides are of keen interest to the biotech industry. Midway between the classic, low molecular weight drugs and the large protein-based biologics, peptides, ranging in size from a few to a few hundred amino acids, perform a variety of vital physiological functions. As such, they represent appealing targets for innovative pharmaceuticals, yet their synthesis has proved daunting. As presentations at two recent symposia demonstrate, new technologies are paving the way for rapid and economical methods of peptide production.
Ron Raines, Ph.D., professor of biochemistry of the University of Wisconsin in Madison, talked about the effects of stereoelectronics on peptide and protein conformation at the Gordon Research Conference on the “Chemistry and Biology of Peptides.”
“Collagen comprises one-third of total protein and is the most prevalent component of the extracellular matrix. Twenty-eight different types of collagen composed of at least 46 distinct polypeptide chains have been identified in vertebrates and many other proteins contain collagenous domains.”
Indeed, collagen is the major component of skin and other fibrous tissues, and an understanding of the modifications that take place in the course of the aging process are vital for a thorough understanding of how these events affect the longevity of the individual.
This ubiquitous molecule is defined by a structural motif in which three parallel polypeptide strands coil about each other to form a right-handed triple helix. Every third residue of the amino acid sequence of collagen is glycine, so the repeating order is XaaYaaGly. The most common residents of the X and Y positions are proline and hydroxyproline. This sequence of amino acids gives rise to the 3-D conformation of the molecule and endows it with its characteristics of insolubility and durability.
Whole collagen molecules are difficult to study at atomic resolution. As a result, Dr. Raines and his team have focused on peptides to obtain a detailed picture of collagen’s 3-D structure. Their investigations have elucidated the role of stereoelectronic effects on peptide and protein conformation, that is, the chemical consequences of orbital overlap, a phenomenon that can play a significant role in the structure and function of organic molecules.
Dr. Raines’ studies have also revealed simple mechanisms for the synthesis of long collagen triple helices and fibrils, which in years to come could play an essential role in biomedicine and nanotechnology. Indeed, early animal studies have revealed that synthetic, stronger collagen fibrils can expedite wound healing, anchoring growth factors more effectively and speeding the growth of new skin.
Other investigations by Dr. Raines’ lab have revealed that collagen-related molecules can be coated with gold and other conductive metal to form nanowires. In the future these might form constituents of nanodevices that could be used to repair damaged tissues and monitor internal physiological conditions.
Andrei Yudin, Ph.D., professor at the University of Toronto, discussed the properties of natural and synthetic cyclic peptides, molecules that have found utility in a variety of pharmaceutical and industrial applications. Because these structures can act as nanomaterials, imaging agents, and drug candidates, there is great interest in their properties.
Dr. Yudin explained that peptides have a zwitterionic nature, that is, they are both positively and negatively charged, with the amino end of the molecule positive and the C-terminal being negative. So even though cyclization of peptides is not thermodynamically favored, enthalpic contributions from electrostatic and polar interaction can drive the formation of ring structures. In the case of small peptides (<7 amino acids), amphoteric molecules that can behave as both an electrophile and a nucleophile can be used to promote peptide cyclization.
It is well known that conventional activation reagents remove the zwitterionic character of the peptide. To achieve the preferred reaction in a workable timeframe, Dr. Yudin’s group employed the “Ugi Reaction,” which favors the thermodynamic equilibrium of amide bond formation. Using an amphoteric amino aldehyde, the product is a cyclic peptide molecule that can facilitate the synthesis of peptides with a range of amino acids.
“Amphoteric amino aldehydes have led to the development of a novel peptide macrocyclization process. The resulting molecules possess useful structural features that allow specific modifications such that fluorescent tags, solubilizing groups, and conformation tuning. Given the prevalence of cyclic peptides in chemistry and biology, this operationally simple method should find utility in many areas.
“Furthermore, the method should be adaptable to other linear molecule substrates, leading to a wide range of novel macrocyclic architectures, for instance cyclic oligonucleotides.”
Purity and reproducibility for pharmaceutical manufacture is a major challenge in peptide synthesis, according to Jeffrey Bode, Ph.D., associate professor at the University of Pennsylvania. To deal with these complex problems, he has developed a new reaction for amide formation in which two unprotected peptides, of virtually any sequence, can be ligated together to build up long peptides of high purity.
“Ketoacid-hydroxylamine amide formation, can be employed as a general method for peptide ligation.”
Dr. Bode noted that peptide targets include large 4,000–8,000 molecular weight therapeutic peptides, foldamers, and cyclic and hydrophobic peptides. A cogent example is the therapeutic peptide Fuzeon (Roche), which currently requires 106 chemical steps to synthesize, generates a massive quantity of waste byproduct, and can run up a bill of $20,000 per patient year.
Chemical ligation, on the other hand, has the potential to shorten the process and avoid the formation of difficult to remove side products commonly formed in chemical peptide synthesis. To achieve this result, the team developed a new reaction for peptide bond formation, specifically the combination of an alpha-ketoacid and a hydroxylamine ligation.
This unusual reaction makes possible peptide fragment couplings that do not need reagents or catalysts, do not generate byproducts, perform with multiple substrates, and can handle the unprotected functional groups found on peptide side chains.
At this time the most significant challenge in the application of the process is the development of a practical synthetic method for C-terminal peptide ketoacids and N-terminal hydroxylamines, Dr. Bode explained. A pivotal step in this strategy is the development of a chemoselective conversion of cyanosulfurylides to alpha-ketoacids and the implementation of solid-phase synthesis of N-terminal hydroxylamines. Using this approach, a high purity final product can be obtained with chemistry that should be amenable to upscaling to kilogram amounts.
Looking forward, Dr. Bode feels that the technology of peptide synthesis by ligation can fill a significant gap in the production of therapeutic molecules. “For large proteins, cloning and genetic engineering is still the way to go,” he stated, but for molecules in the range of 30 to 50 amino acids, peptide synthesis is faster and more cost effective.”
This may not be the first option that directors of biotechnology programs would favor, but given the dramatic advances reported at these conferences, it may be the most efficacious.
A presentation at Cambridge Chemistry’s “Peptide Conference” highlighted a new approach to the design of solid supports for peptide synthesis, concentrating on the industrial-scale production of peptides.
Don Wellings, Ph.D., CEO of SpheriTech, outlined his company’s technology for the manufacturing of solid supports for peptide synthesis. The concept of a polymer support contained within a column was put together in the 1980s as a means of minimizing solvent usage in a closed system that lends itself to automation.
“Our polymer-encapsulation technology employs hollow glass beads, low cost and of high quality, they favor encapsulation of any polymer.”
Dr. Wellings has developed a technology based on an idea that complements continuous flow in peptide synthesis. “Imagine if the polymer particles floated; you could do solid-phase synthesis in a standard solution-phase reactor or even a separating funnel, no filter plate, no sinter, no porous disc.
Preformed hollow glass microspheres form a seed for preparation of polymer particles at a low cost per cubic meter of glass beads. From a theoretical point of view, virtually any polymer, such as polydimethyl acrylamide and polystyrene, can be coated on the outside of a hollow sphere. The advantage here lies not only in the hollow glass seed but in the ease of handling a buoyant polymeric particle.
Peptide synthetic chemistry is not a new endeavor, but it has undergone dramatic changes in recent years. As new peptides are discovered the demand for their production in bulk grows stronger. The improvements and optimizations in solid-phase peptide synthesis mean that the use of large quantities of noxious organic reagents can be avoided. This means a more environmentally friendly and economical means of production of peptides is now practical and readily available.
Sidebar: Peptide News & Trends
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.
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.”
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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