In general, therapeutic peptides currently in development across the industry tend to be a mix of long and short, simple and complex peptides. While agreeing with this assessment, Lax reports seeing a substantial increase in demand for “very complex” peptides moving through corporate pipelines. These include sequences of 40 amino acids or more, PEGylated peptides, and multicomponent peptide conjugates that require assembly using hybrid fragment technologies. The development of robust manufacturing processes for such molecules can take four to six months, far longer than for the typical peptide drug.
Another issue is the use of novel amino acid derivatives or coupling reagents. Access to such materials may prove challenging as projects move into larger-scale production. These reagents may not be readily available in bulk and may require time to produce; they may also introduce additional costs.
Aileron Therapeutics was established based on chemical cross-linking technology licensed from Harvard University and the Dana-Farber Cancer Institute. Aileron calls the synthetically locked, alpha-helical peptides it produces “stapled peptides.” Each “staple” is formed by cross-linking the olefin-tipped hydrocarbon side chains of two non-natural amino acids—merging two double bonds into one—in a process called olefin metathesis. An individual stapled peptide may contain one or more staples depending on the molecular conformation needed to achieve the desired activity, potency, and pharmacokinetic properties. Aileron has synthesized alpha-helical peptides that range in length from 10 to about 35 amino acids.
Early on, “we saw that the stapled peptides developed at Dana-Farber had a broader, more profound effect than small molecules,” says Nash.
The configuration of stapled peptides mimics the molecular structures typically found at the interface of protein-protein interactions. When locked into this stable configuration, stapled peptides are able to penetrate cells efficiently and to exert their effects on intracellular protein targets. The large surface area of the peptides gives them advantages over small molecules in their ability to disrupt specific signaling pathways by inhibiting targeted protein-protein interactions.
“Stapled peptides have a circulating half-life in rodents of more than 20 hours,” says Nash. If left unmodified, they would have a half-life of about five minutes.
Aileron is currently optimizing production of the one or more non-natural amino acids that are incorporated into each stapled peptide. The company has conducted pilot studies of its lead compounds in GMP manufacturing with CMO partners.
“We are about a year away from our first IND,” says Nash.
Aileron chose cancer as the first therapeutic target for its stapled peptides program with the aim of intervening in the BCL-mediated apoptotic pathway and, specifically, mimicking the activity of pro-apoptosis proteins such as BAX, a member of the BCL-2 protein family.
The natural apoptotic pathway is a multifaceted one, Nash explains. Recently in Nature, it was reported that a stapled alpha-helix of BIM BH3 domains directly activates BAX-mediated mitochondrial apoptosis. Because this mechanism directly reactivates the cell death pathway, Aileron is confident that these peptides will have efficacy against a broad range of cancers, and that the nature of the mechanism of action will minimize the ability of tumors to develop resistance to these drugs.
Looking to the future, the company is applying its technology to target transcription factors in the cell nucleus to modulate gene expression.
Frank Zhang, Ph.D., CEO of GenScript, describes a growing market for research-grade peptides, especially for epitope-mapping applications in antibody drug discovery and for the production of peptide arrays for screening. Custom peptide synthesis is one facet of GenScript’s CRO business, which also includes gene synthesis and cloning services, antibody development, and protein expression and purification.
In October, GenScript launched a series of GMP-grade peptides for use as anti-aging ingredients in cosmeceuticals and dermaceuticals. These peptides contain various modifications intended to enhance skin-permeating and antiwrinkle properties; examples include acetyl hexapeptide-3, palmitoyl hexapeptide, palmitoyl tetrapeptide-3, palmitoyl pentapeptide, GHK-Cu2+, and PAL-GHK, which have been incorporated into the products of companies such as Replexion, Bioque, Urban Nutration, Avon, Osmotics, Newspirit, and Neova.
GenScript’s FlexPeptide™ synthesis platform integrates solid-phase synthesis, liquid-phase synthesis, microwave technology, and the company’s ligation technology. The company uses a combination of these synthesis tools, basing the selection of which methods to use on the properties of the peptide to be synthesized. For difficult synthesis steps, Dr. Zhang notes that the increased coupling rates enabled by microwave energy offer particular advantages. Microwave technology is also useful for introducing site-specific modifications.
“We do not only use machines,” says Dr. Zhang, “we may also use manual synthesis,” which allows for greater control over the process.
GenScript can synthesize peptide fragments up to 50 amino acids in length, producing batches from a few milligrams up to two kilograms, according to Dr. Zhang. The company’s ligation technology allows for the production of peptides up to 200 amino acids in length.
The demand for synthetic peptides for use in proteomic research applications continues to increase, enjoying a growth spurt that began a few years ago, according to Heinrich Gausepohl, Ph.D., director of development at Intavis Bioanalytical Instruments, a manufacturer of automated peptide synthesizers. Demand is particularly strong for small-scale peptide synthesis of large numbers of different peptides, with orders in the tens of thousands not uncommon. Dr. Gausepohl attributes the rising interest in isotope-labeled peptides to a growing number of mass spectrometry-based research projects.
Intavis’ MultiPep RS employs solid-phase Fmoc peptide synthesis to generate milligram quantities of up to 384 peptides in four 96-well filter plates. For larger-scale syntheses, an optional column module allows for production of up to 72 peptides in volumes ranging from 10 to 100 µmol.
The MultiPep RS can also perform automated SPOT synthesis to produce peptide arrays on a derivatized cellulose membrane. Recently, Intavis added a new capability, enabling the instrument to produce peptide microarrays on glass slides, with up to 384 peptides arrayed in duplicate on each slide.