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.