The continued strong demand for peptides as research tools and lead compounds in drug discovery is fueling technology development in high-throughput synthesis and purification.
Examples of peptide-based cancer therapeutics include luteinizing hormone-releasing hormone (LHRH) agonists to decrease testosterone production in prostate cancer, somatostatin analogues, and novel peptides that can specifically or preferentially bind to or penetrate tumor cells and offer opportunities for targeted therapies and for delivering radioactive or cytotoxic compounds to tumor tissue.
In addition to synthesizing anti-tumor peptides, American Peptide Company is developing cell penetrating peptides (CPPs) designed for cellular import of therapeutic molecules such as plasmids, DNA, siRNA, PNA, proteins, peptides, and nanoparticles. CPPs can form chemical linkages with their drug cargo, or they may form stable, non-covalent complexes with drugs. They are short peptides, composed of fewer than 40 amino acids, and share common features such as positively charged amino acids, hydrophobicity, and amphipathicity.
One approach the company is using to facilitate peptide synthesis is “click chemistry,” a modular strategy designed for rapidly combining small subunits.
“Click chemistry provides a number of avenues for peptide/protein modifications and could be combined with other techniques to make complex structures and multicomponent functionalized systems,” according to John McKinley, senior marketing manager. “Applications of click chemistry in peptide science include chemical ligation, cyclization, and bio-conjugation, imaging, synthesis of peptidomimetics based on traizole backbone, and conformational and backbone modifications.”
Optimizing for Speed
Automated peptide synthesizers can enable high-throughput, scalable production of high purity peptides for research or clinical applications.
“The speed of automated peptide synthesis will depend both on the optimization of reaction parameters and the throughput of the instrument used,” says James Cain, Ph.D., applications manager at Protein Technologies.
Variation of factors such as coupling and deprotection reagents, reagent excesses and concentrations, solvent selection, resin types, and resin loading make it possible to obtain high purity products with relatively short total reaction times, Dr. Cain explains.
Highly active coupling reagents—such as HATU and HCTU, for example—may not be well-suited for use on some robotic multiple peptide synthesizers due to long reagent dispensing times.
Dr. Cain points to human b-amyloid (1-42) as an example of a peptide that is difficult to synthesize and is of research and commercial interest. Its synthesis is difficult due to the high hydrophobicity of the C-terminal segment and tendency for on-resin aggregation.
Traditional approaches for making this peptide might require 50 to 60 hours of synthesis time, according to Dr. Cain. By optimizing a number of variables, it is possible to synthesize 24 human b-amyloid (1-42) peptides on the company’s Symphony X™ automated synthesizer in less than 14 hours using short reaction times and the instrument’s fluid delivery system.
In this example, the variables modified included use of low-loaded resin, a modified deprotection mixture containing 2% DBU added to the standard 20% piperidine in DMF, and HCTU as coupling reagent. Further efficiency is achieved through simultaneous addition of reagents to multiple vessels and washing of valve blocks and other components at the same time.
The Symphony’s IntelliSynth UV monitoring system, which captures UV readings every 10 seconds during the deprotection reaction, is particularly helpful for difficult sequences, such as the poly-alanine (Ala)10K, which commonly exhibits severe aggregation after the addition of the fifth residue, according to Dr. Cain.
Under standard deprotection conditions, longer deprotection times and more repetitions were required for full removal of the Fmoc protecting group after the onset of aggregation. More efficient removal could be achieved by adding 2% DBU to the deprotection mixture.
Infrared heating can also be incorporated into the design of the Symphony X. It has been shown to speed the synthesis of certain difficult sequences, such as an analog of ACP(65-74), in which the alanines have been replaced with sterically hindered aminoisobutyric acid (Aib) residues.
According to Dr. Cain, “While very pure product can be obtained using long coupling times at room temperature, the addition of IR heating allows for a ninefold reduction in the coupling time for the most difficult steps.”
CS Bio provides peptide manufacturing services, including custom and GMP capabilities, and offers medium- and large-scale automated synthesizers. For many years, the largest automated peptide synthesizer CS Bio offered was 50 L. Now demand for its large-scale 300 L synthesizer—which can produce kilograms of peptides and is validated for cGMP production—continues to grow, not only in the biotech industry but also more recently among pharma companies, which are developing internal peptide therapeutics groups, notes CS Bio’s director of operations, Jason Chang.
“It’s easy now to get good quality raw materials. People are trusting the chemistry, they are doing much larger syntheses, and they are getting much larger peptide batches in one shot as opposed to doing more small batches and combining them,” as they had in the past, Chang says.
Faster, High Purity Synthesis
A challenge for the typical peptide supplier that is set up to produce 1 mg to 5 mg quantities on automated synthesizers is the growing demand among researchers developing and running proteomic screens for very small quantities (as little as 20 nanomoles) of hundreds or thousands of peptides at low cost and fast turnaround, according to Mike Pennington, Ph.D., at Peptides International.
“We are seeing requests for increasingly complex peptides—multidisulfides, protein-peptide conjugates, and RNA-peptide conjugates, for example—longer peptides, especially for large-scale drug development; and small peptides with multiple disulfide bonds.”
In addition, he reports growth in the development of more complex combinatorial products such as vaccines having six, 12, even 18 peptide subcomponents and conjugates.
To improve product purity, Dr. Pennington describes greater reliance on specialized chemistries such as the use of pseudoproline derivatives and dipeptide building blocks that facilitate synthesis “through difficult stretches that tend to aggregate” and make it possible to produce peptides with 60 to 70 residues.
From a quality-control perspective, the addition of UPLC technology has “pushed the envelope of peptide technology to solve problems during synthesis,” he says. Errors in synthesis that would result in a deletion peptide that would have been difficult to resolve on conventional reverse-phase HPLC can be detected with UPLC, contributing to optimization of the synthetic chemistry and the use of specialized building blocks.
Peptides used for clinical applications and made under cGMP guidelines require low levels of endotoxin in the final product. Endotoxins are removed during HPLC purification, explains John McKinley of American Peptide, and subsequent downstream processing must be performed under controlled, clean room conditions.
“The use of tray lyophilizers will improve product handling since both the freezing and drying of purified peptide solution takes place inside the lyophilizer, compared to bottle lyophilizers where pre-freezing of peptide solution in several bottles is required,” he says.
The company installed a 294L-tray lyophilizer equipped with a clean-in-place system in a clean room at its Vista, CA-based GMP facility.
“The temperature of each step is controlled, and the conductivity of the water can be monitored,” McKinley explains. A control system will include full PLC automation with 21 CFR Part 11 compliant operation.