Introduction
Peptide synthesis has undergone remarkable transformations over the decades. From the early days of classical solution-phase peptide synthesis (CSPS) to the advent of solid-phase peptide synthesis (SPPS), researchers have sought more efficient and environmentally friendly ways to produce these vital therapeutic molecules. Peptides, known for their specificity and therapeutic potential, have gained considerable interest in treating a variety of conditions, including cancer, metabolic diseases, and infections.
Solid phase peptide synthesis has dominated peptide production due to its ease of use and automation. However, SPPS is inefficient, requiring excess reagents and huge volumes of solvent. Solid phase technology is also incompatible with traditional batch reactors, instead requiring the use of specialized reactors with limited availability, presenting significant supply-chain challenges for large volume peptides like the current GLP-1 agonists.
Faced with the challenge of producing large volumes of peptide therapeutics, LPPS has emerged as an alternative manufacturing technology able to dramatically improve scalability and environmental sustainability while also reducing costs. The newest liquid phase technologies are enabling more efficient manufacturing, improved quality control, and promise more robust supply chain for peptide therapeutics. This article reviews the progress and potential of LPPS, focusing on the advantages of LPPS for pharmaceutical manufacturing. For a deeper discussion of LPPS technology, the reader is referred to recently published reviews (1).
What is Liquid-Phase Peptide Synthesis?
Liquid-Phase Peptide Synthesis is a method for peptide production that occurs in solution rather than on a solid support. LPPS operates by using soluble support or tag, which allows the growing peptide chain to remain in solution during synthesis. Performing the synthetic steps in a homogeneous liquid phase eliminates the mass transport challenges typical of SPPS. At least some approaches to LPPS are fully compatible with standard batch processing equipment instead of the specialized equipment required of SPPS.
Some versions of LPPS, like the approach developed by Cambrex, remove of impurities, excess reagents, and byproducts from each step by aqueous workup, greatly reducing the demand for organic solvent used in washing these components from a solid phase support. Peptides are assembled via the sequential coupling of individual protected amino acids. The soluble support allows for more flexible purification strategies, less solvent waste and simplifies reaction monitoring, and is fully compatible with real-time PAT monitoring of reaction performance.
How does liquid phase peptide synthesis work?
The innovation behind LPPS lies in the ability to solubilize the growing peptide chains while still efficiently separating it from the excess reagents and byproducts of each reaction (2). A common approach to LPPS introduce C-terminal esters that impart solubility on the growing peptide (3). The esters are typically high molecular weight, hydrophobic benzyl ethers (4), medium sized polyethylene glycols, or fluorous alcohol esters installed on the first amino acid residue (5). Different esters offer tunable solubility or partition coefficient in different solvents, allowing for a reaction to be carried out in solution followed by selective precipitation of the peptide during purification steps. In other examples high molecular weight ester tags allow purification of the growing peptide by retention behind a nanofiltration membrane while permeating reactants and byproducts through the membrane (6). These methods seek to be a direct replacement for SPPS and thus applicable to linear construction of long peptides chains.
At Cambrex we have pursued an alternative LPPS technology designed for synthesis of the short chain fragments (10-12 mers) to support convergent approaches to long-chain peptides (7) for a fully liquid-phase synthesis of therapeutic peptides. Using a low molecular weight C-terminal esters peptide chains up to 12-mers can be solubilized in process friendly solvents such as 2-methyltetrahydrofuran with good volumetric efficiency.
Our technology is further differentiated from the approach using high molecular weight benzylic ester in that it is compatible with both Fmoc and Cbz protecting group strategies. The different protecting group strategies can even be used within the same fragment. Also, instead of relying on selective precipitation of the growing peptide, a simple aqueous workup is used between amino acid additions to remove reagents and byproducts to the aqueous phase while retaining the growing peptide in solution in the organic phase. Our LPPS method is performed in typical batch reactors, but has also been demonstrated in continuous flow, providing opportunities for further efficiency gains. The full-length peptide fragments are then isolated by precipitation or crystallization, to provide the fragments as solids with purity >98% typically observed.
Sustainability through LPPS
In conventional SPPS, reagents and reactants from the liquid phase react with the growing peptide attached to a solid polymer. Because of inefficiencies in transport of reactants from the solution phase to the reacting peptide, excess reagent is typically required. After each round of reaction, the byproducts and excess reagents are separated from the peptides by washing with a large volume of solvent. The solvents typically used in SPPS are classified as hazardous by regulatory agencies and are subject to strict regulations. For example, typical SPPS solvents N,N-dimethylformamide and dichloromethane are problematic under the European Chemicals Agency’s REACH regulation.
LPPS, by contrast, requires significantly less solvents and lower excess of reactants for each synthetic step. This not only minimizes environmental waste but also lowers production costs. A recent review of clinical and commercial peptide manufacturing processes found that LPPS can have more than 10x lower process mass intensity (PMI) per amino acid residue than SPPS (8). As noted above, the Cambrex LPPS technology is designed to use 2-methyltetrahydrofuran, a green and process friendly solvent, making it a more sustainable option for large-scale peptide production.
Applications of LPPS to Peptide Therapeutics
Peptides are essential therapeutics, however producing peptides on a commercial scale in a sustainable and cost-efficient manner is a significant challenge. With LPPS, and convergent synthetic approach, complex peptide sequences can be manufactured efficiently and sustainably. LPPS is compatible with standard batch reactors, significantly expanding the manufacturing capacity vs. specialized SPPS manufacturing facilities. LPPS typically operates at higher concentrations than SPPS, further increasing manufacturing efficiency. These characteristics of LPPS naturally lead to lower costs and greater supply chain options for these essential therapeutics.
Controlling product quality has typically been more of a challenge for peptides than for small molecule therapeutics. With solid phase synthesis of full-length peptides, impurities resulting from single amino acid deletion are significantly similar to the target making detection and removal of these impurities challenging. Opportunities to control purity in LPPS are greater than with SPPS. Application of convergent synthetic route allows for quality control of the fragments prior to assembly into the API, reducing the impurity burden on the API step and minimizing difficult to separate, high-homology impurities. With LPPS, PAT technologies such as on-line HPLC can be readily employed for in-process control.
Challenges and Limitations
LPPS is not without its challenges. SPPS is an automated platform technology that requires minimal process optimization to deliver the target peptide. For early phase clinical investigational peptides, SPPS provides rapid access to peptide API using automated commercial synthesizers. Coupled with preparative chromatography to purify the crude peptides, Linear synthesis of the full-length peptide by SPPS is typically the fastest route to those first few kilograms of cGMP peptides.
To realize the full benefits of LPPS technology requires process development, especially when applied in the context of a convergent synthetic strategy wherein two or more peptide fragments will be coupled to form the full-length target API. Identifying the appropriate disconnects back to fragments prepared by LPPS requires experimental data or prediction of sequence solubility and propensity for epimerization in tag cleavage and fragment coupling.
Future Directions for LPPS
The future of LPPS lies in continued innovation and the integration of green chemistry principles. At Cambrex and elsewhere researchers are actively exploring new solvent systems, coupling reagents, and protecting group strategies that are less toxic and more environmentally friendly, as well as developing new tags that can solubilize peptide sequences prone to aggregation and further simplify the purification process. We are also leveraging continuous manufacturing and industrial automation to further drive efficiency in LPPS, efforts that will streamline the production process and reduce human labor.
Conclusion
LPPS offers a sustainable and efficient manufacturing alternative to SPPS. By reducing solvent consumption, improving reaction efficiency, and enabling the use of standard batch and flow reactors for peptide manufacture, LPPS addresses many of the challenges faced in modern peptide production. As the demand for peptide therapeutics continues to rise, the ability to produce high-quality peptides efficiently, sustainably and at low cost will be critical to ensuring patient access.
REFERENCES AND NOTES
- Sharma A, Kumar A, de la Torre BG, Albericio F, Liquid-phase peptide synthesis (LPPS): a third wave for the preparation of peptides. Chem. Rev. 2022;122(16):13516-13546. Ferrazzano L, Catani M, Cavazzini A, Marelli G, Corbisiero D, et al. Sustainability in peptide chemistry: current synthesis and purification technologies and future challenges Green Chem. 2022;24:975-1020.
- Martin V, Egelund PHG, Johansson H, Quement STL, Wojcik F, Pedersen DS. Greening the synthesis of peptide therapeutics: an industrial perspective. RSC Adv. 2020;10:42457-42492.
- Less common is LPPS using a solubility tag on the N-terminus and growing the peptide from N-term to C-term. See for instance; Nakahara H, Sennari G, Noguchi Y, Hirose T, Sunazuka T. Development of a nitrogen-bound hydrophobic auxiliary: application to solid/hydrophobic-tag relay synthesis of calpinactam. Chem. Sci. 2023;14:6882-6889.
- Okada Y, Takasawa R, Kubo D, Iwanaga N, Fujita S, et. al. Improved tag-assisted liquid-phase peptide synthesis: application to the synthesis of the bradykinin receptor antagonist icatibant acetate. Org. Process. Res. Dev. 2019 ;23(11):2576-2581.
- Sharma A, Kumar A, de la Torre BG, Albericio F, Liquid-phase peptide synthesis (LPPS): a third wave for the preparation of peptides. Chem. Rev. 2022;122(16):13516-13546.
- So S, Peeva LG, Tate EW, Leatherbarrow RJ, Livingston AG. Organic solvent nanofiltration: a new paradigm in peptide synthesis. Org. Process Res. Dev. 2010:14(6)1313-1325.
- For an example of this convergent approach to peptide synthesis see; Frederick MO, Boyse RA, Braden TM, Calvin JR, Campbell BM, et. al. Kilogram-scale GMP manufacture of tirzepatide using a hybrid SPPS/LPPS approach with continuous manufacturing. Org. Process Res. Dev. 2021;25(7):1628-1636.
- Kekessie I, Wegner K, Martinez I, Kopach ME, White TD, et. al. Process mass intensity (PMI): a holistic analysis of current peptide manufacturing processes informs sustainability in peptide synthesis. J. Org. Chem. 2024:89(7): 4261-4282.
