January 1, 2008 (Vol. 28, No. 1)

Novel Molecules Require Improvements in Synthesis, Scale-up, and Purification

Several presentations exploring issues related to the synthesis, scale-up, and purification of peptides designed for use as therapeutic agents were presented at the “EuroTIDES” meeting held in December in Berlin. The conference also featured speakers addressing the challenges inherent to synthetic peptide production, the changing face of the peptides industry, and the growing emphasis on optimization and quality control in process development.

Zelos Therapeutics’ (www.zelostherapeutics.com) presented 12-month Phase II trial data on Ostabolin-C™, a drug designed to stimulate bone formation, at the conference. Treatment with Ostabolin, a cyclic form of parathyroid hormone, was associated with clinically relevant increases in lumbar spine bone mineral density among postmenopausal women with low bone mass.

Ostabolin-C is a cyclized 31 amino acid peptide. Unlike the bis-phosphonate compounds commonly used to prevent osteoporosis such as Merck & Co.’s Fosamax, which inhibit bone resorption and slow bone loss, Ostabolin-C increases bone formation and is intended for use as a therapeutic agent in patients with moderate-to-severe osteoporosis, reported Paul Morley, Ph.D., CSO and cofounder of Zelos.

The lactam bridge that gives the compound its cyclic structure precludes synthesis of the peptide using recombinant technology. Zelos produces Ostabolin using solid-phase chemistry, which can be done cost effectively because of the high potency of the drug, noted Dr. Morely. It is active at doses ranging from 10 to 45 µg/day.

Peptide drugs in development are getting more complex, with more modifications and more unnatural amino acids. They are also increasingly being linked to carrier molecules to enhance their pharmacokinetic properties and drug-delivery options. Liquid- and solid-phase synthesis methods as well as hybrid processes, which exploit the beneficial properties of each method (peptide fragments are produced on a solid support and then combined into the full-length peptide in solution), all continue to be embraced by peptide producers.

Recombinant methods of peptide production continue to evolve as companies strive to improve titers, to explore multicopy gene constructs and novel expression systems, and to focus on optimizing and automating downstream purification.

The increasing complexity of the compounds is only one challenge for the peptide industry. Oleg Werbitzky, Ph.D, head of R&D, peptides, and oligonucleotides, at Lonza (www.lonza.com), identifies several others: increasing emphasis on improving product quality and process control combined with a need to move toward more robust, cost-effective, and environmentally friendly synthesis and purification strategies as well as demand for a greater scale of production as the range of therapeutic targets broadens.

All of these market pressures and internal demands are helping to drive the development and adoption of new synthetic, downstream purification, and analytical strategies and to push process design and optimization efforts to the forefront.

All of this activity, however, and the incremental changes in theory and practice are taking place in an uncertain regulatory environment. Unlike organic small molecule production, no clear regulatory guidance exists to help peptide manufacturers develop or modify chemical, analytical, and separation processes to improve peptide quality and simplify the regulatory review process.

Without defined regulatory guidance, “Peptides now fall somewhere in between small molecules and biologicals,” said Paul Little, Ph.D., senior chemist at 7TM Pharma (www.7tm.com). “There is no safety net for being able to point to a guidance document and determine what test to do at what step, yet there is still the possibility to take a peptide drug to market along a smooth regulatory path.”

This can be done by providing appropriate scientific justification and taking advantage of the opportunity to speak to the regulatory agencies throughout product and process development. “There is a rumor that regulatory guidance for peptides will be coming out soon,” added Dr. Little.

Top-Down Process Development

Each synthetic strategy for manufacturing peptides has its own set of advantages and limitations. Liquid-phase synthesis remains important for shorter peptides and for lower-volume and higher-value products. This approach suffers from the need to isolate and analyze a large number of intermediate products. Process development and maximizing operational efficiencies can be laborious. Solid-phase and recombinant processes are more easily scalable. Fermentation- and cell culture-based processes, in general, offer the least environmental impact.

Combination approaches that rely on both recombinant and chemical synthesis steps can help overcome one of the limitations of recombinant technology when it comes to producing peptides containing unnatural amino acid sequences.

The industry is making substantial strides in the area of analytics, applying emerging chromatographic techniques and mass spectrometry, observed Dr. Werbitzky. Also, a changing mindset is emerging within the regulatory bodies on both sides of the Atlantic. For example, the FDA is encouraging quality by design concepts in which “quality should be an intrinsic part of the process, not just demonstrated in the product,” said Dr. Werbitzky.

A good example of next-generation peptide synthesis and process optimization are the efforts under way at Trimeris (www.trimeris.com) to develop a fusion inhibitor with superior durability and pharmacokinetic properties compared to the company’s T-20 (enfuvirtide, Fuzeon) HIV fusion inhibitor now on the market. T-20 requires twice-a-day injections, but its acceptance by patients and physicians has been slowed by inconvenience and potential for injection-site reactions.

TRI-1144, a second-generation HIV fusion inhibitor, was engineered to form a stable helical structure, which gives it a longer half-life and stronger barrier against the development of resistance.

As with T-20, TRI-1144 is produced by solid-phase synthesis of peptide fragments, which are assembled into the full-length peptide in solution and then deprotected and purified. The synthetic process for TRI-1144 essentially mirrors that of T-20; however, during the global side chain deprotection step of T-20 synthesis, performed in a trifluoroacetic acid-based solution, Trimeris discovered two new impurities. These appeared in all batches synthesized, although their quantities varied depending on the deprotection conditions.

Trimeris attributes the impurities to side chain protecting groups and used chemical, enzymatic, and spectroscopic techniques to determine that they arise as a result of tryptophan alkylation by a derivative from the pentamethyl dihydrobenzofuran (pbf) protecting group.

“Impurity analysis is the essential part of process development and optimization, with the goal being to block the path to impurity formation and favor the path to product,” explained Huyi Zhang, Ph.D., a research investigator in process R&D at Trimeris.

Using a design of experiments approach, Trimeris assessed the various process conditions that might affect impurity levels such as the choice of reagents and the duration or temperature of a reaction and determined which combination of conditions was optimal for inhibiting impurities.

Dr. Zhang and colleagues observed that chemical rearrangements of the pbf protection group that take place during deprotection change the structure of some of the peptides formed. Initially, these impurities accounted for up to 15% of the peptides formed. After the implementation of DoE and process optimization, the two by-products comprised

Separation and Purification Issues

The length, modified amino acid composition, and 3-D conformational structures of some peptides in development present challenges for separation and purification downstream. Merck KGaA (www.merck.de) is working to improve separation efficiencies achieved with silica gel and polymer-based chromatography.

By experimenting with and refining the selectivity of separation conditions, companies can more reliably and efficiently separate impurities such as N-1 sequences, which tend to occur with greater frequency as the length of the peptide increases.

Designing separations based on selective properties other than hydrophobicity can introduce a new dimension that could help distinguish between closely related molecules. The presence of stereoisomers and the possibility of racemization during coupling also complicate production of longer peptides and require innovative purification strategies.

Michael Schulte, Ph.D., director of performance and life science chemicals/ R&D/life science solutions at Merck, used the multicolumn solvent gradient process (MCSGP) as an example of a recently developed separation technique that improves the cost effectiveness and efficiency of separating mixtures of complex drug molecules in a feedstream.

“Simulated moving bed chromatography has been scaled up within the last 10 years in the pharmaceutical industry to 1,500 tons of drug enantiomers a year produced by several systems,” says Dr. Schulte. “And the same success story might be possible for the MCSGP technology in the field of peptide and protein separation.

Toward Peptide Therapeutics

AplaGen (www.aplagen.com) employs several tools to synthesize its cytokine-mimetic peptide HemoMer®, a synthetic erythropoietin (EPO) mimetic that is the company’s flagship product in development. Natural EPO, which is produced in the kidney, stimulates red blood cell production; recombinant EPO is commercially available for the treatment of anemia.

AplaGen’s technologies include correctly folded peptide synthesis, which ensures that large peptides fold correctly and form the appropriate, biologically active, tertiary structure, as well as AGOX technology, which is used to form disulfide bridges that stabilize the peptide.

The company also uses microwave-assisted peptide synthesis to accelerate amino acid coupling and introduces structural elements called helical constraints.

AplaGen developed a carrier technology based on hydroxyethyl starch (HES) for its cytokine-mimetics.

HES, a semisynthetic polymer sometimes used as a plasma expander, increases the stability and half-life of the peptide in the bloodstream. Unlike PEG, HES is biodegradable and is broken down by amylases in the body.

By controlling the amount of hydroxyethylation, AplaGen can tailor the biodegradability and pharmacokinetic properties of the carrier. Multiple drug molecules can bind to a single HES carrier, yielding a supravalent compound.

“This supravalency results in increased efficacy,” said Marco Emgenbroich, Ph.D., head of supportive organic chemistry at AlphaGen. EPO receptors tend to be clustered on the cell surface in the bone marrow, and the ability of the HES carrier to deliver high concentrations of drug to these receptors may enhance drug binding and activity, noted Dr. Emgenbroich.

Synthetic peptide production, especially at industrial scale, continues to evolve as companies tackle the challenges presented by increasingly complex peptides in development. There is a clear focus on optimizing chemical synthesis and downstream separation processes that build in concepts and strategies drawn from the trend toward integration of quality by design and design of experiments methodologies early on in process development.

The emergence of innovative hybrid synthesis approaches, purification techniques, and drug delivery methods share the dual goal of reducing production costs and streamlining the path to regulatory approval and commercialization.

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