The environmental impact of large-scale peptide production can be staggering. In his presentation at the “Avakado Media Peptide” conference held in April, Mimoun Ayoub, Ph.D., vp global business and strategic development at Peptisyntha (a Solvay company), noted that the production of 20 kilograms of an 18-residue peptide generates approximately 300 metric tons of used solvent and other waste.
The amount of waste is magnified for a company managing tens of commercial peptides at any given time, and the logistics for waste separation, storage, and disposal can present quite a burden.
Although solvent recycling may be useful for very large peptide products, “a good cost assessment needs to be made,” Dr. Ayoub said. “Solvents coming out of production are mixed together most of the time, and the separation by distillation requires a substantial investment that adds to energy consumption, analytical testing/release, and space utilization. These costs may exceed the price of fresh solvents.”
While purification of crude peptide is the driving force behind the cost of peptide manufacturing, according to Dr. Ayoub, the initial purity of the crude synthetic product is a critical factor in reducing processing costs.
To illustrate this point, Dr. Ayoub has analyzed manufacturing costs for the 18-mer linear peptide at different scales and using different synthetic strategies (solid-phase synthesis, solution-phase synthesis, and the hybrid approach).
In all cases, raw materials account for 20%–25% of the overall cost of production. “It is important to optimize the supply chain and look for cost-effective suppliers. However, the reliability in terms of quality and delivery time must remain the drivers in the choice of the supplier. The price is important, but should not drive this choice,” he said.
Even if alternative suppliers can offer raw materials at a 20%–30% savings, this may translate to only about a 5% reduction in the overall cost of API manufacturing, and this savings must be balanced against the risks associated with purchasing from a potentially unreliable supplier. These risks include the quality and purity of the materials and potential variability in delivery time. “Improving the crude purity by only 10 percent may result in a cost saving of >50 percent after purification and lyophilization,” added Dr. Ayoub.
Another decision peptide producers must grapple with, noted Dr. Ayoub, is whether to develop a solid-phase or liquid-phase synthesis scheme. Solid-phase peptide synthesis offers the benefits of shorter process development and manufacturing time and greater cost efficiency at smaller scale, but he contends that solution-phase synthesis may be more advantageous as product quantities increase.
Once again using the example of an 18-amino acid peptide, Dr. Ayoub explained that at small scale (1 to 3 kilograms), solid-phase synthesis is about half the cost of solution phase, whereas at 50 kilos, a solution-phase approach might be half the price of solid-phase synthesis, with substantially less solvent usage. Also important is that “the cost of regulatory filing is directly associated with the production strategy.”
Decisions should be made on a case-by-case basis, he suggested, based on a clear understanding of the advantages and limitations of each approach and the potential regulatory hurdles associated with changing a process mid-stream in development and scale-up.
Attention to Each Detail
For a CMO, both the evolution of synthesis and purification technology and changing customer demands drive innovation. Jan Pawlas, Ph.D., a member of the process development and support team at PolyPeptide Group (PPG), described two over-arching trends: an increased focus on the economic aspects of peptide manufacturing, and a demand for shorter delivery times.
In Dr. Pawlas’ view, a well-developed, efficient solid-phase synthetic process is suitable not only for small- to medium-scale production, but can also remain competitive for larger-scale processes and for producing longer sequences and more complex peptides with elaborate side chains.
The key is to optimize process development and achieve highly efficient conversion at every amino acid coupling to produce a high-purity crude synthetic peptide and minimize the demands on downstream processing. “If extensive purification is needed, then liquid-phase synthesis can become more competitive,” said Dr. Pawlas.
The path to more cost-efficient solid-phase peptide synthesis begins with a knowledge and experience base that allows a CMO to shorten the process development time and select an optimal synthetic route from the outset, according to Dr. Pawlas.
The other two main considerations in designing an efficient process are the quality and characteristics of the raw materials (amino acids, linkers, and coupling reagents), and selection of the resin. Quality of the amino acids is paramount, and the ability of manufacturers to purchase monomers in bulk for use in multiple projects provides economies of scale.
PolyPeptide Group has created a global sourcing program to coordinate the needs and purchasing activities across its global sites to benefit from better availability and lower prices for materials and equipment acquired in large quantities. Additionally, standardization of purchases simplifies the transfer of processes, technology, and projects across sites.
For resin selection, PPG screens batches of many different resins (acquired from commercial sources as well as novel materials developed in academia) with a variety of test sequences and under varying conditions. The goal is to identify a suitable polystyrene bead-based resin when possible to keep costs low.
Dr. Pawlas emphasized the importance of testing every new batch of polystyrene resin, as standard resin manufacturing can yield substantial batch-to-batch variation in quality due to uneven distribution of functional groups across the resin beads, for example.
Compared to traditional polystyrene bead manufacturing methods, copolymerization of the functionalized monomers “is the process of choice and could dramatically improve the yield of large-scale peptide synthesis and shorten production times,” he said.
Linker selection is an underappreciated aspect of solid-phase synthesis, according to Dr. Pawlas. For example, instability of linkers under standard Fmoc deprotection conditions may lead to the loss of valuable quantities of peptide product.
“This loss is not accounted for, as you will not see it in the crude peptide,” he explained. However, even a simple process change such as altering the composition from dimethyl formamide (DMF) to DMF/toluene can tremendously increase the stability of certain linkers during Fmoc deprotections,” he added.
Selection of coupling reagents also affects synthesis efficiency, as they play a critical role in mediating the formation of amino bonds during the coupling of each amino acid. Even small changes in temperature, solvent composition, concentration of the coupling reagent, or pH can all have a large impact on peptide yield and the occurrence of undesired side reactions.
“Not only the choice of the coupling reagent, but how you utilize it can make a big difference in process efficiency,” Dr. Pawlas concluded.