July 1, 2008 (Vol. 28, No. 13)
Susan Aldridge, Ph.D.
Drugs Already on the Market and in Clinical Studies Drive Novel Method Development
Peptides are an emerging class of drugs, somewhere between a small molecule and a biologic. Their synthesis can be complex, which creates various challenges in large-scale production and purification. There are a number of companies involved in peptide manufacture and at least 150 peptide projects in the preclinical or Phase I stage.
Key issues in peptide synthesis and manufacture are the number of amino acids in the peptide chain and whether synthesis is done using solution-phase (conventional chemistry) methods, solid-phase (resin-based) techniques, or a combination of the two.
“Big pharma is now interested in peptides, which are being perceived as being more user friendly and more readily available,” remarks Satish Joshi, Ph.D., evp of Solvay Peptisyntha (www.peptisyntha.com). After their relative lack of success with small molecules, companies see peptides as being more natural, which could be a big plus.
Solvay Peptisyntha originally specialized in solution-phase peptide synthesis. By 2001, the company realized that solid phase was becoming more prominent so it decided to develop that too. Solvay Peptisyntha is now in the process of enhancing its QC/QA and will expand its manufacturing as more peptides reach Phase II and Phase III.
Mimotopes (www.mimotopes.com) is one of the original peptide companies. It specializes in the supply of peptides for research and preclinical work to both academic and pharma customers. The firm creates peptide libraries for mapping antigen-binding sites or protein-protein interactions using its SynPhase Lanterns synthesis platform.
This approach, says Mimotopes CEO, Nick Ede, Ph.D., allows peptide synthesis at a higher purity than with more traditional resins. “We have developed new surfaces specifically designed for peptide synthesis, especially longer peptides with more than 25 amino acid residues.”
Mimotopes has traditionally worked at the discovery end. About two years ago, the firm identified a need at the larger-scale, value-added end of the business. “We decided to form a global peptide alliance with Genzyme Pharmaceuticals (www.genzymepharmaceuticals.com), which is one of the top five GMP manufacturers, and so become a one-stop shop for researchers to access peptide experts from research to commercialization,” Dr. Ede explains. “This alliance brings together two players with core expertise at either end of the development spectrum, each sharing its sweet spot to give peptide researchers and developers support.”
Daniel Erne, Ph.D., svp and CTO at Bachem (www.bachem.com), says that growth in the peptide market is being driven by the need for highly specific drugs for unmet medical need. Thus, his company is constantly investing. “In the last two years we have spent more than 30 million Swiss Francs ($28 million) each year, and it looks as if we are going to continue at this pace,” notes Dr. Erne. The company supplies big pharma and biotech, making up to hundreds of kilograms of peptide if necessary.
“There is a real ground swell in preclinical and Phase I work using peptides,” Dr. Ede adds. “This is because peptides often have higher specific activity and lower toxicity compared with many small molecule drugs.” Dr. Joshi adds that there are now some interesting peptides in clinical trials with, for instance, cyclized disulfide bridges or carboxyls coupled to hydroxyls in their backbones. One such candidate, with four disulfide bridges, is in Phase II/III for brain glioma.
There are also complex peptide vaccine candidates, which are cocktails of peptides. “The most important peptide drugs today are the hormone-dependent treatments,” according to Dr. Erne. These include treatments for cancer, diabetes, obesity, and bone metabolism. Additionally, companies are developing various peptide mimetic drugs such as Vertex Pharmaceuticals’ Phase II molecule for hepatitis C.
“Efforts in GMP synthesis have brought down the cost of developing peptides,” points out Dr. Ede. “Now it is not outrageous to take a 30 mer this far. This has paved the way to experimental drugs as therapeutics such as antidiabetic peptides.”
Roche’s anti-HIV agent, Fuzeon, and Amylin Pharmaceuticals’ Exenatide for diabetes are leading market expansion and driving down the cost of large-scale peptide synthesis. Both have a chain length that would not have been possible five to ten years ago. “Costs have dropped significantly, with better availability for FMOC amino acids, resins, and the basic needs,” explains Dr. Ede.
“The development of Fuzeon means new players came into peptide supply. There is far more growth than there was a decade ago.” As peptides become more important as viable drugs, they become commodity items, and there is more pressure upon prices.
When it comes to the synthesis of peptides, whatever the scale, both solid-phase and solution-phase approaches are important. Many peptide companies are now adopting a hybrid technology, where peptide fragments are made by solid phase and then ligated in solution. Thus fragments of eight to fourteen amino acids might be synthesized on a solid-phase resin, removed and purified, and then these fragments will be coupled. Dr. Joshi reports that Solvay Peptisyntha is working on 120 mer peptides based upon 12 different 10 mer fragments, which is easier than making the peptide sequentially with 119 single steps.
Dr. Ede also notes that the hybrid approach is important now, making a protected fragment first, such as three or four amino acid fragments for synthesis of a >30 mer. In such a case, there may not be a requirement for formal purification of these fragments. “This is very much a trend in large-scale synthesis these days,” Dr. Ede comments. “Larger and larger peptides are becoming a reality with hybrid synthesis. The boundary where one shifts from chemical to biological synthesis has shifted. Soon we will be able to make cGMP peptides of more than 50 amino acids synthetically.”
There have been incremental improvements rather than any kind of quantum leap in the chemistries used to synthesize peptides, Dr. Erne explains. These include improved resins for solid-phase peptide synthesis, novel protecting groups for the amino acid components, and new coupling reagents. Novel peptides with unnatural amino acids and nonpeptide linkages have always been important because they can help prolong the otherwise short half-life of a natural peptide.
Mimotopes and Genzyme Pharmaceuticals are using pseudoproline reagents to obtain better crude peptides, which lowers development costs. Pseudoproline dipeptides are introduced into the synthesis of potentially difficult sequences that might otherwise aggregate, lowering yields and therefore increasing costs. “Pseudoprolines allow scale up to be developed in a way that will save money as you move into clinical trials,” says Dr. Ede. “It is a technique that is growing, and we are at the forefront.”
Large-scale peptide synthesis will remain within the realm of chemistry, at least for the foreseeable future. Starting production in cell culture, with all the issues around obtaining the right cells and vectors, is just too expensive and time consuming, and peptides with unnatural amino acids cannot be made with cells. Even relatively long peptides are made, successfully, by chemical synthesis.
When it comes to scaling up, the nature of the chemistry and the length of the peptide chain required are all important. “A solid-phase synthesis is a lot faster to develop for a new peptide structure,” says Dr Erne. “However, both are valid methods in their own right, and we carry out quite a few solution-phase syntheses at Bachem.”
Improvements in peptide chemistry and technology have pushed the limit of how big a peptide can be made in large quantities to as high as 80–100 amino acids. It is not easy, but it is possible to synthesize peptides of more than 100 amino acids in length. Most therapeutic peptides today, however, are 10–50 amino acids long.
When it comes to large-scale manufacture of peptides, a good understanding of the chemistry involved is a must. “If you design a process that cannot be scaled up, you are going to have tremendous problems,” says Dr. Erne. Scale-up applies to both synthesis and purification, and for the latter the target compound must be readily distinguishable and separable from its impurity.
When it comes to purification of peptides, ion exchange is becoming increasingly important and is taking over from reverse-phase HPLC. UPLC is also becoming important. Precipitation, done in a large vessel, and lyophilization are also key, although the latter is costly. Spray drying is a method that is being discussed although it can be problematic because peptides are known not to be particularly thermally stable. Sometimes it is difficult to separate the target compound from impurities that elute close to it. This is where UPLC can be useful. Mass spectrometry is used to see if the peptide peak is really pure.
A good synthesis with easy purification is the one with the lowest costs. As Dr. Erne points out, however, although peptides are expensive per unit weight owing to their complex structure, compared to drugs like aspirin, they are highly active, which reduces the actual cost per dose.