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Feature Articles : Jul 1, 2009 (Vol. 29, No. 13)

European Overview: Firms Look for Niche in Peptide Synthesis

Unique Technology or Service Allows Companies to Stand Apart from Their Rivals
  • Sophia Fox

In May, TheraCode acquired JPT Peptide Technologies, a wholly owned subsidiary of Jerini. JPT offers peptide-based products and services for vaccine development, immune monitoring, and peptide biomarker discovery. “Our acquisition by TheraCode means that, in addition to continuing our work in the service arena, we will also develop immunodiagnostic and immunotherapeutic products in collaboration with our new parent,” explains Holger Wenschuh, Ph.D., managing director of JPT.

JPT’s high-throughput technology involves a step-wise solid-phase synthesis process, but instead of using traditional resin-based chemistry, flat membranes are used as a solid support.

“We dispense tiny droplets of activated amino acids directly onto the surface of these membranes, and it is these droplets that act as microreactors in which the chemical reactions between the amino acids and the peptides already on the membrane occur,” explains Dr. Wenschuh “The process involves a number of reaction rounds, with each spot on the membrane corresponding to an individual peptide construct and each membrane accommodating up to 2,000 spots.”

The main advantage of this approach is the speed with which large numbers of peptides can be created in parallel, says Dr. Wenschuh. “We can economically generate up to 50,000 peptides in a week as the need for consumables is low. Using our technology, peptide synthesis is several orders of magnitude cheaper than traditional solid-phase approaches,” he adds.

JPT’s core products include PepSpot™ and PepStar™ technologies for peptide arrays, PepMix™ peptide pools, as well as PepTrack™ and SpikeTide™ peptide libraries.

The company’s macro- and microscale peptide sets are complemented by preassembled tools for enzyme profiling such as kinase, phosphatase, and protease peptide microarrays,  and peptide microarrays for mapping immunodominant regions in antigens. JPT’s premium synthesis unit generates essential reagents for all phases of vaccine development.

While applications of the high-throughput technology are manifold, JPT says that the company’s main focus is on generating high-content peptide arrays, libraries, pools, and sets of proteotypic peptides for proteomic applications.

“Our high-throughput synthesis technology gives researchers the opportunity to monitor immune responses or quantify protein expression on a proteome-wide basis,” Dr. Wenschuh suggests. “As the application of such technologies in clinical research expands, we are confident that peptide arrays, pools, and libraries that cover, for example, the entire proteome of a pathogen, will have a significant impact on research into personalized medicines and diagnostics.”

Long-Chain Peptides

Almac offers a range of life science research and drug development services, including the development of both custom research peptides and GMP peptides as therapeutic candidates.

Specialist peptide expertise is a result of extensive investment in the production of long-chain peptides using a  tagging technology that caps the final peptide. The technology results in a high purity peptide even before chromatographic purification, and makes the production of peptides of over 100 amino acids long, including modified and labelled molecules, both cost-effective and efficient, claims Denis Geffroy, vp business development peptide and protein synthesis.

“One of the main stumbling blocks to the efficient generation of long-chain peptides is that the longer you make the molecule the less pure it becomes. That produces a huge purification challenge, requiring a lengthy and expensive HPLC process that inevitably leads to the loss of a lot of product. Our tagging technology means we can get 90–95 percent purity from the crude peptide and, in some cases, even bypass chromatographic purification completely.”

“We started our custom peptide synthesis business primarily for research applications, but over the last three years or so our GMP peptides business has grown to the point where it has overtaken the research peptides,” Geffroy points out. “To address the increased call for therapeutic peptides, we have established our Rapidd™ package as a complete set of solutions aimed at accelerating entry of APIs, including peptides, into early-stage clinical development. 

“The Rapidd package addresses not just peptide synthesis, but formulation, regulatory requirements, and preclinical safety and toxicology,” adds Geffroy. “It’s particularly tailored for start-up companies that may not have the financial security to spend two to three years moving a first product into the clinic.”

Fluorescent Dyes

Cambridge Research Biochemicals (CRB) works with small companies and academic groups to help commercialize new technologies relevant to the peptides market. CRB most recently signed a partnership deal with Cyanagen, giving it access to Cyanagen’s CHROMIS family of fluorescent dyes for labeling custom peptides.

CRB says that studies have shown the CHROMIS labels perform better than the most advanced commercial dyes, with increased brightness and performance across the 400–850 nm wavelength range. About 60 ready-to-use labels are already available, and the dyes can also be designed to display different charge values (anionic, cationic, zwitterionic, and neutral), and combine with over 40 crosslinkers and biotinylating reagents.

CRB will play an instrumental role in the EU-funded BIOSCENT project, a European collaborative program that aims to develop new bioactive polymeric scaffolds for use in stem cell-based tissue regeneration approaches to treating cardiovascular disorders and diseases. Announced in April, the project will leverage CRB’s peptide-synthesis expertise and the development of peptide-directed antibodies to identify cell signaling factors.

“We have established a core peptide technology team, which is working with peptide research scientists in the U.K. to look at new avenues for synthesizing complex molecules such as multiple-bridged and glycosylated peptides,” says Emily Humphrys, commercial director.

Tapping into the expertise of academically trained peptide chemists is not as easy as it sounds, however. While the custom-peptide market is growing steadily in terms of volume and peptide complexity to meet the increased demands of new proteomics applications and antibody technologies, the number of academically trained peptide chemists is dwindling, Humphrys points out.

“Not all peptide companies are created equal,” stresses Paul Sheppard, Ph.D., scientific development director for Enzo Life Sciences (ELS). “There are few formally trained peptide chemists left in either industry or academia today, and there is now an increasing reliance upon machines to make peptides, rather than on a human element that really understands peptide chemistry. 

“The upshot of this is that while the number of companies offering custom peptide-synthesis services is increasing, the knowledge and expertise residing within those companies has become greatly diluted; consequently the synthetic success rate and integrity of peptides obtained from some commercial providers may not rise to meet expectations.”

Collaborative research has proven most important to ELS. “Whereas the company’s deep involvement in an area such as the ubiquitin signaling pathways led to its inclusion in a five-year EU-funded Network of Excellence (RUBICON), its peptide-based capabilities allowed the company to share this expertise with all RUBICON  members as a core facility. Such involvement has undoubtedly proven to have been to the benefit of all involved,” adds Dr. Sheppard.

Broadening Focus

Intavis Bioanalytical Instruments was established nine years ago, primarily as a manufacturer of peptide synthesizers. Intavis used its expertise in the field to establish its own custom synthesis business three years ago, and this unit has experienced double-digit growth annually, says Heinrich Gausepohl, director of development.

“While the market for state-of-the-art automated peptide synthesizers has remained buoyant and is growing steadily, the proteomics revolution has also provided extensive opportunities for custom peptide services. Our experience in peptide chemistry meant it was an obvious step for us to add custom synthesis to our instrumentation business.”

Intavis offers custom peptides, peptide sets, and custom peptide sets for T-cell stimulation/epitope mapping. Products are centered on the company’s CelluSpots™ technology for custom peptide arrays and ready-to-use CelluSpots kinase substrate arrays.

CelluSpots technology was developed to address some of the drawbacks associated with SPOT technology for investigating protein-protein interactions, Dr. Gausepohl claims. “While SPOT synthesis technology is well recognized as a rapid and robust method to generate peptide libraries on membrane supports, it does have some shortcomings.

The reusability of traditional SPOT membranes is limited, the production of duplicate peptide SPOT arrays is time-consuming and expensive, and membranes are large compared with glass microarrays slides and require large sample volumes.”

In contrast, CelluSpots are arrays of peptide-cellulose conjugates spotted on planar surfaces as a 3-D matrix, rather than as a monomolecular layer. This essentially provides a high peptide loading and concentration. “The peptides are synthesized on modified cellulose disks that will dissolve after synthesis. The dissolved peptide-cellulose conjugates are then spotted onto coated microscope slides, and form a 3-D structure that is not dissolved in aqueous solutions,” Dr. Gausepohl explains.

“This three-dimensional structure holds up to 500 times more peptides per area in comparison with conventional monolayer deposition, and shifts the binding equilibrium to favor low-affinity protein-protein interactions. Pushing the reaction in the bound direction is particularly important as many key protein-protein interactions are believed to be weak, or occur only transiently, and would not necessarily be detected using other approaches.”

Intavis also believes that its CelluSpots technology has a number of other benefits compared with SPOT technology. “The CelluSpots approach displays comparable chemical properties to the original SPOT membranes and is compatible with standard microarray hybridization chambers and scanners. The method also allows numerous identical copies of the same quality to be prepared easily and cost-effectively, and requires far smaller sample volumes,” Dr. Gausepohl adds.

Spatially Defined Peptide Constructs

The addition of new technologies to custom peptide tools is allowing companies to carve their own niches in a market that is being flooded by me-too players. Like many custom peptide businesses, Pepscan’s peptides research business, Pepscan Presto, offers a range of peptide tools, including custom peptides and custom, kinase and protease PepChip microarrays. The company is also exploiting its CLIPS™ (chemically linked immunogenic peptides on scaffolds) technology, for the design and construction of peptides with spatially defined structures.

“Generally, when you take a peptide out of its large protein context it simply doesn’t fold into the correct 3-D structure,” explains Peter van Djiken, Ph.D., chief commercial officer. “This means the peptide doesn’t represent a true copy of the protein domain it’s derived from.

“Our CLIPS technology solves such problems by constructing conformationally correct peptides that mimic small parts of the native protein. As a result, CLIPS peptides are highly suitable for identifying and reconstructing complex protein interaction sites. The technology also improves the function of bioactive peptides 5- to 20-fold compared with ordinary peptides. Potential applications are manifold, including complex epitope mapping, raising monoclonal antibodies, and vaccine development.”

The CLIPS technology involves attaching small organic template molecules to anchor points incorporated at specific locations on the peptide sequences as it’s constructed. The templates induce the peptide to fold into desired loop, helix, or even sheet-like conformations.

“It’s a toolbox we can use to make all kinds of specially defined molecules,” Dr. van Djiken claims. “Advantages of the CLIPS technology include its simplicity—it is a one-step addition reaction carried out after coupling the last peptide, and its  high-yield, which can reach 100%. This makes it suitable for highly parallel synthesis of large combinatorial libraries of both soluble and solid-phase bound compounds.”

It’s also a useful tool for raising antibodies against target proteins that cannot be generated recombinantly, he suggests. “Membrane-spanning proteins such as GPCRs and ion channels are hard to reproduce because they don’t fold correctly out of the context of the lipid bilayer. With CLIPs, however, we can generate peptides that accurately mimic extracellular loops from transmembrane proteins, and use these to raise antibodies that fully cross-react with the native target.”

The CLIPS approach can be applied to any synthetic peptide, including modified or long peptides of 80 amino acids in length or greater, Dr. van Djiken says.