July 1, 2014 (Vol. 34, No. 13)

Bioavailability, Manufacturing, and Regulatory Issues Look Increasingly Tractable

Therapeutic peptides constitute an unusual drug category. Although most therapeutic peptides are synthetic, their molecular weights are well above those of small-molecule drugs, but well below those of proteins.

Like other peptides, therapeutic peptides use amino acid building blocks. Despite the nearly limitless combinatorial possibilities in constructing peptides of between 2 and 50 amino acid residues, peptides have not always been viewed favorably as drug candidates due to their poor oral bioavailability.

Nonetheless, prospects for development-stage peptide drugs are improving, thanks to advances in formulation, conjugation, and delivery methods, as well as the use of artificial amino acids. In fact, prospects have improved so much that this molecular class is experiencing a renaissance.

By 2010, 60 peptide drugs were approved by the FDA, 140 were in clinical trials, and 500 were in early development; by 2012, 6 peptide drugs had won approval—a record number—while 14 had entered late-stage clinical trials or had reached the review stage.

Such is the interest that U.S. Pharmacopeial Convention (USP) formed a Therapeutic Peptides Expert Panel in 2013 to evaluate quality attributes for synthetic peptides based on currently available regulatory guidance and expectations. The USP’s First Workshop on Synthetic Therapeutic Peptides, scheduled for October 1, 2014, will provide a forum where industry and regulators will examine the current status of regulation, manufacturing, and product characterization.

Moving in on Small Molecules

“Peptides are increasingly moving into domains traditionally held by small synthetic molecule drugs,” says Jonathan Collins, director of business development at CEM.

Peptide toxicity tends to be lower than for traditional small molecules because the human body breaks down peptide bonds rather easily, and has numerous ways to dispose of amino acids. Peptides are also a useful size, up to several thousand in molecular weight, so they can access larger, more complex biological targets.

Extending therapeutic peptides with cell-penetrating sequences has further broadened potential therapeutic possibilities. Approximately 10 residues long, cell-penetrating sequences are heavy in nitrogen-rich amino acids such as lysine and arginine, which readily penetrate cell membranes. Targets, notes Collins, are “no longer limited to cell surfaces.”

Over the years, researchers have devised dozens of ways to overcome bioavailability problems through aerosol sprays, delayed-release formulations, chemical conjugation (such as pegylation), and other strategies. “Patients no longer need to take peptides by injection,” Collins adds.

Another strategy for extending circulating half-life involves inserting one or more D-amino acids into the sequence. Most peptides in the body use L-amino acids exclusively, so digestive enzymes have a much harder time breaking down bonds to amino acids with the unnatural stereochemistry. Similarly, investigators have induced greater durability by chemically modifying amide bonds.

A technician operates CEM’s Liberty Blue, a microwave-based peptide synthesizer. Microwave energy is used for both the coupling and deprotection reactions during peptide synthesis.

Making Peptides

A significant contributor to the resurgence in interest in peptides has been the ready availability of peptide synthesis in the form of automated, standalone synthesizers, in addition to commercial synthesis services. Of the latter, synthesis specialists will supply quantities of up to several grams, while contract manufacturers can produce a hundred kilograms or more.

At small scale, the equipment used by researchers and suppliers is virtually identical. Resin-based systems prevail today, as they did when Robert Merrifield won a Nobel Prize for peptide synthesis work in 1984. Automated synthesizers construct peptides by means of repeated cycles of attachment and deprotection. The first amino acid, which is fixed to an appropriate resin, is attached to a second, protected second amino acid. Then the newly added residue is deprotected.

Additional attachment/deprotection cycles continue until the final step. At this point, all remaining protecting groups are cleaved and the peptide is freed from the resin.

Reagents, solvents, resins, and protecting groups have evolved over the years. And while the basic principle of resin-based peptide synthesis remains unchanged, vendors have introduced features that make synthesis faster, more reliable, and more environmentally friendly.

Most improvements have focused on greater automation and yield/purity per step. Yield restrictions have always plagued peptide synthesis. Constructing a 20-mer at 99% yield per step gives material of 82% purity, which most small-molecule chemists would take gladly. Unfortunately, the peptide impurities include many 19-mers, 18-mers, and so on, which are difficult if not impossible to separate from the product. A per-step yield of 99.9%, however, provides material of more than 98% purity, which is generally good enough for regulators.

CEM’s innovation, the use of microwave radiation as the energy source, overcomes all three drawbacks of traditional peptide synthesis. Microwaves are more efficient than heat and create none of the pressure problems. Whereas conventional synthesis takes one to two hours per residue, Collins claims CEM’s flagship synthesizer does the job in four minutes. In addition, CEM utilizes a safer deprotection base and approximately 90% less solvent.

Outsourcing Procurement

Rather than synthesizing their own peptides, many organizations employ custom synthesis service companies. Vendors provide anywhere from a few milligrams for research purposes to hundreds of kilos under cGMP. PolyPeptide Group, a worldwide network of cGMP peptide facilities, fufills gram-to-production scale requests, mostly from pharmaceutical and biotech companies. “Sixty percent of our business involves proprietary peptides, and the remainder generic,” says Trishul Shah, the company’s business development key account manager. The company’s low-scale limit, several grams, serves preclinical needs for early-stage programs.

PolyPeptide would not be price-competitive for customers interested in an array consisting of milligram quantities of a low molecular weight peptide with, say, a one amino acid substitution. Those labs are better served by a small-scale specialty synthesis firms, or by purchasing their own synthesizer. “We focus on products where quality and regulatory [compliance] are critical, where manufacturing is under GMP, and where interaction with regulatory bodies on CMC issues is common,” Shah notes.

While most peptide synthesis these days is automated, solution-phase chemistry is still around. In the past, common wisdom held that solution-phase synthesis was more efficient for very small peptides, especially at large scale. Those notions are now challenged by the high cost of protected amino acids, and the fact that solid phase does not require isolating the product after every step. “Solid phase is much more controllable as well,” Shah asserts.

Another option is to combine products of several smaller solution-phase processes, or of solution phase plus solid phase. These “convergent” synthetic strategies must make both chemical and economic sense.

PolyPeptide has experience with unusual building blocks, including D-amino acids and synthetic amino acid-like building blocks. These are not technologically problematical, according to Shah, but recent FDA guidances have placed extra burden on GMP manufacturers regarding ingredient sourcing.

Research Today, Cures Tomorrow

While peptide specialists that assist pharmaceutical companies and biotech firms often serve as research partners as well as suppliers, at least one peptide company, Peptides International, also works with academic partners to sustain long-term research projects. For example, the company’s senior chemist, Andrzej Czerwinski, Ph.D., has been working on the synthesis of numerous Arg-Gly-Asp (RGD) constructs with academics such as Shuang Liu, Ph.D., a professor of health sciences at Purdue University.

As indicated by an article published in Molecular Pharmacology in 2013, this collaboration resulted in a SPECT tumor imaging RGD peptide with improved pharmacokinetic properties due to the incorporation of carbohydrate, triazole, and polyethylene glycol moieties.

In addition, the company’s president and CEO, Michael Pennington, Ph.D., is part of a research effort that in 2011 received a five-year NIH R01 grant. This effort, which involves researchers from the Baylor College of Medicine, Monash University, and the University of California, Irvine, reflects Dr. Pennington’s long-standing interest in developing selective Kv 1.3 channel blocking peptides, which are derived from disulfide-rich peptides found in sea anemones and scorpions. In March 2014, Dr. Pennington and his colleagues published an article in Scientific Reports describing the potential of a scorpion toxin analogue as a therapeutic to fight autoimmune diseases.

Yet another research area of interest to Peptides International concerns the disulfide-rich peptide hormone hepcidin. The company notes that hepcidin has been of interest as a biomarker for disorders such as anemia of inflammation and myocardial infarction.

Progress on a hepcidin synthesis strategy developed by Peptides International in collaboration with researchers from the University of Queensland and the University of California, Los Angeles was reported February 2014 in Angewandte Chemie. The researchers introduced a safety-catch cysteine protecting group, which was designed and developed to expand the capabilities of synthetic strategies for the regioselective formation of disulfide bonds in cysteine-rich peptides.

According to Peptides International, such advances reflect a breadth of experience and expertise that may be relevant to the research programs of the company’s customers. Also of interest to potential collaborators are the company’s addition of new talent and upgrades in capacity.

For example, the company reports that it now has the capacity to handle kilograms of peptide production with 4-inch and 6-inch prep columns. In addition, several HPLCs are available to purify milligram-to-gram quantities of peptides.

To accommodate increased purification volumes, the company has acquired several lyophilizers to allow freeze-drying of multiple peptides and large volumes simultaneosly. Newly acquired instrumentation to accommodate small-scale peptide synthesis is also available, as are devices that can accomplish scale-up and medium-to-large scale synthesis.

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