Peptide Synthesis In Pharmaceutical Manufacturing
Mon, 06/22/2009 - 5:49am
With inherent abilities to block and/or enhance signal transfers in the human body, peptides, when harnessed as active pharmaceutical ingredients, can treat a host of metabolic diseases, cardiovascular and heart conditions, and neurodegenerative disorders.
Peptide-based drug targets are being identified at an increasingly rapid pace both in terms of recently introduced therapies and products in the development pipeline. In fact, a recent report by market and technology research firm Frost & Sullivan indicated that more than 40 approved peptide-based drugs are in use today and approximately 400 are being developed to treat allergies, cancer, Alzheimer's, Huntington's, and Parkinson's diseases.
Peptide-based therapies tap into the direct hard wiring of human physiology, yielding substantial and far-reaching benefits to drug treatments and therapies. Moreover, developments in peptide manufacturing and implementation have made these amino acid compounds more accessible to the market in terms of cost, flexibility, and effectiveness.
Compared to small molecule drugs, peptides offer lower toxicity, show higher specificity, and demonstrate fewer toxicology issues, and in some cases lead to the development of new compounds that are otherwise unavailable. For example, two biotechnology firms are working with Sunnyvale, California-based American Peptide Company, a manufacturer of these amino acid proteins, to develop peptide-based therapies for cardiovascular ailments, specifically heart failure; a condition affecting 5.3 million Americans.
One is a novel chimerical natriuretic peptide in clinical development for an initial indication of acute decompensated heart failure (ADHF). The other is a thrombin peptide that in preclinical studies has shown to minimize cardiovascular tissue damage by initiating a series of anti-apoptotic events.
The above examples are just two of the many life sciences companies pursuing the development of peptide-based therapies. However, manufacturing of peptides can be a complex process and requires careful considerations of manufacturing and process variables.
Chemical and Recombinant Synthesis
Chemical and recombinant peptide syntheses are the two basic amalgamations for these amino acids, each offering unique sets of advantages suited for different applications.
The recombinant method, for example, is a more natural process and can offer a price advantage at large production scales. It is also effective for longer sequences of more than 100 residues (residues are specific monomers within the polymeric chain of a polysaccharide, protein or nucleic acid). However, the development program for a recombinant peptide may be costly and can involve complex production steps.
Chemical synthesis, in contrast, is more flexible and easier to scale. It can modify unnatural amino acids and is not constrained to naturally occurring amino acids. Chemical synthesis can be cost effective from gram scale to multi-kilogram level depending on the synthesis route. Chemical synthesis is achieved by coupling the carboxyl group of an amino acid to the amino group of another amino acid.
For synthesis techniques, there are two distinct methods, solid-phase and solution-phase, each with unique applications. Liquid- or solution-based peptide synthesis is the older technique, though most labs today use solid-phase synthesis. Solution-phase is better for shorter peptide chains and is useful in large-scale production greater than 100 kg in scale. Solution-phase synthesis is still widely used in structure modification (peptide) synthesis, rare intermediates preparation and peptide/protein ligation and conjugation. In the peptide industry, solution-phase is more cost-efficient for large scale production of shorter chain peptides, such as luteinizing hormone-releasing hormone (LH-RH) analogues.
Solid-phase synthesis allows an for an innate mixing of natural peptides that are difficult to express in bacteria. It can incorporate amino acids that do not occur naturally and modify the peptide/protein backbone. In this method, amino acids attach to polymer beads suspended in a solution to build peptides, which remain attached to beads until cleaved by a reagent such as trifluoroacetic acid. This immobilizes the peptide during the synthesis so it can be captured during filtration, while liquid-phase reagents and by-products are simply flushed away. The benefits of solid-phase synthesis are that it greatly speeds production of peptides since it is a relatively simple process; it is easier to scale, and it is more suitable than solution-phase synthesis for longer sequences.
Within solid-phase there exist two different methods, (t)ert-(B)ut(o)xy(c)arbonyl, or t-Boc, and 9H-(f)luoren-9-yl(m)eth(o)xy(c)arbonyl, or Fmoc.
T-Boc is the original method used in solid-phase synthesis and uses acidic condition to remove Boc from a growing peptide chain. This requires the use of small quantities of hydrofluoric acid, which is generally regarded as safe, and requires specialized equipment. This method is preferred for complex syntheses and when synthesizing non-natural peptides.
Fmoc was pioneered later than t-Boc and makes cleaving peptides easier. It is also easier to hydrolyze the peptide from the resin with a weaker acid. This eliminates the need for specialized equipment. Again, both methods are valuable and each suit different applications. However, Fmoc is more widely used because it eliminates the need for hydrofluoric acid.
Process Variables to Increase Yields
There are a number of process variables that can enhance yield in the peptide synthesis process. First of all, sequence analysis and synthesis strategy design are crucial for the whole process. In this initial step, a chemist will determine automatic synthesis or manual synthesis, suitable resin type, coupling/deprotection/cleavage method that could eliminate potential side reaction and minimize by-product content; and whether or not to insert special building blocks and positions to prevent aggregation in sequence assembly.
As peptide chemistry and technology continue to advance, larger peptides made in greater quantities are possible. It is also possible to synthesize peptides that are more than 100 amino acids in length, although 10 to 50 amino acids long are more common for therapeutic peptides.
Purification is yet another important factor that must be carefully assessed among many methods to ensure desired results are achieved cost effectively.
ypically preparative high performance liquid chromatography (HPLC) is used to purify the peptide product. A common purification buffer is trifluoroacetic acid (TFA). However, when the final salt form is not TFA, an additional salt exchange step is required to convert the peptide to the desired salt form. Mass spectrometry data and amino acid analysis are obtained to confirm the identity of the target peptide.
Hydrophobic peptides can pose significant purification challenges because they are not readily soluble in typical purification buffers. Additionally, alternatives to freeze-drying of the purified peptides, such as large vessel precipitation and spray drying are under consideration, but spray drying can pose problems since peptides can be thermally unstable.
Another important purification technology is ion exchange, which is gaining importance in the purification of peptides. In addition, ultra performance liquid chromatography (UPLC) is becoming a useful analytical tool.
The promise of peptides as active pharmaceutical ingredients will not only reinvigorate drug innovation and discovery, it will also challenge the very ingenuity of pharmaceutical communities to develop novel delivery methods for present and future therapies. In-depth knowledge of peptide production methods to optimize yield and purity is critical to enabling cost-effective and faster commercialization of peptide-based therapies