Solid vs. Liquid-Phase Synthesis
Conventional wisdom holds that for API process development and GMP manufacturing, solid-phase peptide synthesis (SPPS) is more cost effective for longer peptide sequences (>10 amino acids) and smaller volumes, and liquid-phase peptide synthesis (LPPS) is more suitable for producing short sequences and large volumes. Hybrid SPPS-LPPS strategies find their niche for long sequences at large volumes.
A cost analysis should take into account raw materials, synthesis, purification, and lyophilization costs. Volume and economies of scale, complexity of the peptide and chemistries, solvent use, and waste produced can all affect manufacturing costs, overall efficiency and productivity, as well as sustainability of the synthetic process and environmental factors.
For SPPS, at any given scale, purification costs drive manufacturing cost, according to Mimoun Ayoub, Ph.D., vp, global business, sales and strategic developments at Peptisyntha, a Solvay company. Critical factors that can be modified to reduce purification costs include time, optimization of the stationary and mobile phases, recycling of solvents, control of process parameters such as load and flow rate, and product yield.
A cost analysis performed by Peptisyntha demonstrates the importance of crude purity on cost. A 10% increase in crude purity will lead to higher purification yields and cost savings of >50% of the API.
“Having said that, it is important for a manufacturer to select the right synthesis approach (SPPS, LPPS, or hybrid) to achieve a robust and cost effective manufacturing process taking into account the volumes of the API, the crude delivered by the synthesis approach, and timelines,” says Dr. Ayoub.
He points to several strategies for improving the robustness of synthetic processes, including persilylation technology, phenyl-oxy-carbonyl (Phoc) chemistry, or tetraphenyl borate (TPB) technology used for synthesis of arginine-containing peptides, and urethane-protected N-carboxyanhydride building blocks.
Peptisyntha utilizes all of these technologies to enable cost-effective production by increasing yield and purity, reducing the number of chemical steps, avoiding racemization, and allowing for synthesis of difficult sequences.
Jon Holbech Rasmussen, director of global development, Polypeptide Group, considers the choice between classical solution phase vs. solid-phase synthesis for producing peptides 10 amino acids or shorter “a very interesting exercise.” Faced with the efficiencies of the solid phase, if we can understand and master solid-phase synthesis, and take some of the advantages intrinsic to solution-phase synthesis and transfer them to the solid phase, then we can push the limits of when the liquid and solid-phase synthesis methods reach the break-even point.
“You have to go into rather large volumes before solution phase becomes economically attractive,” notes Rasmussen, who dismisses the idea that solid-phase synthesis is only good for small scale and long peptides.
“It can be highly efficient for synthesizing shorter peptides at larger volumes. This change in thinking is related in part to better manufactured, more consistent solid phase resins and to a better understanding of what is actually going on in the resin during the synthesis reactions. By adopting this change in thinking we will be able to exploit the boundaries of this technology.”
The goal now is to determine which factors should guide technology selection for synthesizing a particular peptide. It is no longer a clear-cut, volume-dependent selection. One way of looking at it is to say that solid phase is merely one kind of protecting group.” From that perspective, it is only a matter of applying the available chemistries.
Rasmussen cites an example of a recent synthesis project in which “we were working with a peptide with a high number of unusual amino acids, and we realized that the classical solution phase was probably less efficient because it could only run dilute.
“Forcing the synthesis onto a diphasic solid phase system, we could increase the effective concentration of the synthesis, and that is really what drives cost. You can have a 5 cubic meter reactor, but if you can only fill it up with grams of material because the material is insoluble then it doesn’t really matter,” he says.
Furthermore, Rasmussen challenges the long-held belief that SPPS necessarily has heavy solvent consumption. Solvent consumption is linked to concentration, he explains, and optimizing wash cycles and exposure of amino acids to the growing peptide chain can reduce solvent use.
Polypeptide Group is increasingly using modeling to predict, design, and assess synthetic processes. The company has developed an in silico method that allows them to input a peptide sequence and generate information such as the potential for aggregation, a pH curve, or a molecular weight fingerprint, and to predict potential problems that might arise during synthesis.
Similarly, an impurity predictive program can predict 90% to 95% of impurities.
The biggest change in the peptide synthesis market in the past three to five years, according to Jason Chang of CS Bio, has been the increase in peptide modifications.
“It is rare that we get a project for a cGMP large peptide that is just a ‘straight’ peptide,” he says.
They more typically are pegylated or radiolabeled, have lipid additions or attached sugars, or are DNA/RNA-peptide combinations. Stapled peptides are becoming increasingly popular.
“Peptide science has come a long way,” he continues, for the purpose of making peptides more stable in the circulation and more viable as a drug candidates. Companies are even taking another look at “old” peptide drug candidates that may have been put on the shelf due to poor pharmacokinetics or bioavailability.
Manufacturing these modified peptides can introduce some challenges, particularly related to solubility when performing HPLC-based purification in aqueous solutions. But, “it is all within the realm of organic chemistry,” as is peptide synthesis is as well, “so it’s very compatible,” says Chang. “The chemistry is feasible, and because of that is has been a real boon for the industry.”