INTRODUCTION
The industrial manufacturing of peptides via Solid-Phase Peptide Synthesis (SPPS) faces several sustainability challenges. First, low productivity, which results from the complex, multi-step deprotection, coupling, and washing, limits large-scale production (1).
SPPS also involves high consumption of organic solvents like DMF and DCM, increasing costs and waste generation. The hazardous nature of these solvents further poses environmental risks and health concerns, as many are toxic and difficult to manage (2).
Furthermore, SPPS suffers from low Process Mass Intensity (PMI) due to excessive solvent and reagent use, inefficient reactions, and high byproduct generation (3). Improving PMI is critical to making SPPS more resource-efficient and environmentally friendly.
Conventional SPPS, which involves side-chain protection to prevent undesired modifications, significantly reduces atom economy and necessitates large volumes of TFA for deprotection and peptide cleavage. This inefficiency is particularly pronounced in large-scale peptide manufacturing, where the volumes of TFA and associated anti-solvent requirements escalate costs and drastically reduce productivity. Overcoming these bottlenecks is a key area for innovation in large-scale peptide manufacturing (Figure 1).
The root cause of these challenges lies in certain unnecessary use of side-chain protecting groups. Essential protections include those for Lys, Cys, and terminal groups, while others, such as Arg, Asn, Gln, and His, are non-essential and can be omitted in an MP-SPPS approach. Previous efforts to minimize side-chain protection have been limited to small-scale or short peptides such as di- or tri-peptides, leaving a gap in large-scale API production (4).
The conventional solid-phase peptide synthesis (SPPS) process is also plagued by excessive organic solvent consumption, particularly for rinsing after amino acid coupling and Fmoc removal. It increases operational costs and raises environmental concerns due to the significant chemical waste generated. Minimal-rinsing SPPS (MR-SPPS) has been developed as a sustainable alternative to address these challenges. MR-SPPS aims to minimize DMF usage by optimizing the rinsing protocols, thus reducing solvent consumption and its associated environmental impact. This approach represents an important step towards more efficient and eco-friendly peptide synthesis practices, offering a potential solution to the excessive use of organic solvents in traditional SPPS.
Minimal-Protection SPPS (MP-SPPS)
Our study aims to address the above challenges by developing MP-SPPS using unprotected Arg, His, and Tyr. In addition to coupling these amino acids, we also focused on optimizing the peptide cleavage and isolation process to reduce TFA usage (5). The MP-SPPS process was tested on an angiotensin II analogue (Peptide Z) to demonstrate its broader applicability and advantages. The sequence of Peptide Z is H-Xaa1-Arg-Xaa3-Tyr-Xaa5-His-Xaa7-Xaa8-OH (Xaa1,3,5,7,8 are unreactive aliphatic or aromatic amino acids without nucleophilic side-chains).
Incorporating side-chain unprotected Arg into the minimal-protection SPPS (MP-SPPS) strategy is crucial due to the inherent challenges of removing the canonical Pbf-protecting group. The traditional Pbf group necessitates the use of highly concentrated TFA for its removal, which complicates the process and contradicts the green chemistry principles. By avoiding the need for such concentrated TFA, MP-SPPS can enhance efficiency and sustainability. Therefore, the successful integration of unprotected Arg is essential for realizing the full benefits of MP-SPPS, as it directly addresses the limitations associated with Pbf removal and aligns with the goals of greener, more efficient peptide synthesis.
The coupling of side-chain unprotected Fmoc-Arg-OH to a peptide immobilized on CTC resin was initially attempted using 1.5 equivalents of Fmoc-Arg-OH pre-activated with DIC/HOSu in DMF. However, the reaction, even after the addition of extra DIC, failed to yield the target product after 3 hours. Alternative coupling additives, including Oxyma and HOBt, were similarly ineffective. RP-HPLC and LC/MS analysis indicated extensive side reactions, including lactam formation and dimerization, attributed to the reactivity of the guanidino group.
Fmoc-Arg(HCl)-OH was tested to mitigate these issues, where the guanidino group is protonated, reducing its nucleophilicity and preventing lactam formation. Using DIC/Oxyma, this building block completed the coupling reaction within 1.5 hours, making it a suitable strategy for incorporating side-chain unprotected arginine in MP-SPPS.
The coupling of Fmoc-His-OH was completed in 90 minutes. However, RP-HPLC revealed 25% impurity due to an unwanted interaction between the unprotected imidazolyl group and DIC, resulting in a 126 Da mass increase (Figure 2). This impurity accumulated over subsequent DIC-mediated couplings.
Fmoc-His(Mtt)-OH, with its acid-labile protecting group, was incorporated using DIC/Oxyma to resolve this side reaction. This strategy completed all couplings efficiently, avoiding imidazolyl modification and reducing D-His formation to 0.09%. It reconciled the need for DIC/Oxyma for Arg while preserving His side-chain integrity, supporting the MP-SPPS approach. It has also been reported that TBEC (1-tert-Butyl-3-ethylcarbodiimide) in place of DIC as the coupling reagent could suppress the adduct formation and sustain the utility of Fmoc-His-OH as the building block for MP-SPPS (6).
In conventional SPPS, peptide Z was cleaved and deprotected using a concentrated TFA solution (175 equivalents of TFA, 2.7 equivalents of TIS, and 23.8 equivalents of H2O) with subsequent precipitation in MTBE. This process required 25.6 L/mol and 78.9 L/mol of TFA and MTBE, respectively, adversely affecting the manufacturing productivity.
To leverage the advantages of MP-SPPS, which aims to minimize TFA usage, we treated the Peptide Z resin derived from the MP-SPPS process by 10% TFA in the presence of 1.2 equivalents of TIS as a scavenger to Mtt cations.
Alternative solvents were explored to address the utility limitation of hazardous DCM. TFT (α,α,α-trifluorotoluene) performed comparably to DCM in peptide cleavage and Mtt removal, and it has a higher boiling point, improving safety.
The treated resin was rinsed with 10% HFIP (hexafluoroisopropanol)/TFT to improve the recovery. The peptide solution could be readily concentrated, and peptide Z was precipitated with cold MTBE, yielding a quantitative recovery. A 5.25-fold productivity enhancement was accomplished by the MP-SPPS process (Table 1). The comparison of the MP-SPPS and the conventional SPPS is demonstrated in Figure 3.
The MP-SPPS strategy demonstrates scalable, cost-effective peptide synthesis with high productivity and purity. While applicable to other acid-sensitive resins and peptides, the method’s effectiveness may vary based on peptide attributes. This strategy is recommended for peptides with favorable amino acid compositions, particularly for large-scale production.
Minimal-Rinsing SPPS (MR-SPPS)
In traditional SPPS, the excessive use of organic solvents is often required for resin rinsing, significantly contributing to the overall environmental and economic burden. This approach not only increases solvent waste but also lowers process efficiency and productivity, as larger amounts of solvent must be managed and disposed of. Hazardous solvents like dichloromethane (DCM) further exacerbate these concerns. The process mass intensity (PMI) remains high, indicating inefficiencies in material usage.
In a typical industrial-scale synthesis of a decapeptide using canonical SPPS, the process can require up to 1260 kg of DMF per mole of peptide produced just for the rinsing steps (7). This vast solvent consumption highlights the inefficiency of the method. The atom economy in such processes is extremely low, approximately 0.13%, even assuming a 100% yield in the synthesis. This figure underscores the environmental and material inefficiencies inherent in traditional SPPS, as most of the input materials do not end up in the final product but are consumed as waste.
Kumar et al. developed an in-situ Fmoc removal process that skipped the post-amino acid coupling rinsing by adding piperidine or 4-methylpiperidine directly to the coupling reaction solution to initiate the Fmoc removal. The resin was subsequently rinsed with DMF or 1% Oxyma/DMF solution. The process gave comparable product purity and reduced the solvent consumption by 75% (8).
To address these sustainability challenges, Minimal-Rinsing SPPS (MR-SPPS) offers a solution by reducing the volume of solvents needed for rinsing steps without compromising the quality of peptide synthesis. This innovation can greatly improve PMI, decrease hazardous solvent use, and enhance the overall productivity of industrial peptide manufacturing.
Minimal-Rinsing SPPS (MR-SPPS) can indeed be divided into two key categories based on the stage of the synthesis process:
Minimal-Rinsing Post Amino Acid Coupling: This approach minimizes the solvent after the amino acid coupling step by reducing rinsing volumes. The process aims to reduce rinsing volumes after the amino acid couplings while ensuring efficient coupling reactions. This category addresses solvent waste without compromising the completeness of peptide bond formation.
Minimal Rinsing Post Fmoc Removal: In this deployment, rinse is minimized following the Fmoc deprotection step. Fewer rinsing cycles are deployed after removing the Fmoc group, helping to reduce overall solvent consumption while maintaining effective removal of the base for Fmoc-deblocking and the formed by product.
Both strategies of MR-SPPS contribute to improving sustainability in peptide synthesis by reducing solvent use while maintaining process efficiency.
It is advisable to conduct a Failure Modes and Effects Analysis (FMEA) to assess the potential impacts and risks associated with implementing the MR-SPPS process. This risk management tool can systematically evaluate possible failure modes in MR-SPPS, including issues like inadequate resin rinsing, incomplete coupling reactions, and their potential effects on process efficiency, product yield, and purity. By performing FMEA, manufacturers can proactively identify critical areas for improvement, mitigate the risks, and ensure the scalability and reliability of MR-SPPS for sustainable peptide production.
As revealed in Table 2, insufficient rinsing after Fmoc-Xaa-OH coupling in MR-SPPS can result in the formation of endo-Xaa impurity, which has a low severity, whereas insufficient removal of piperidine after the Fmoc removal reaction may lead to a range of impurities, including Des-Xaa and DIC-endcapping species.
MR-SPPS was applied to synthesize an octapeptide. The baseline process used 8 ml DMF/g of initial MBHA resin, with three consecutive rinsing steps after each Fmoc-Xaa-OH coupling. In contrast, the MR-SPPS strategy eliminated DMF rinsing after the Fmoc-Xaa-OH coupling steps. RP-HPLC was performed at each Fmoc deblocking step and Fmoc-Xaa-OH coupling step. Additionally, colorimetric controls using ninhydrin and chloranil tests were implemented at each Fmoc-Xaa-OH coupling step.
The comparison between the baseline and MR-SPPS processes is outlined in Table 3.
The profiles of the two processes are comparable in terms of conversion rate, reaction kinetics, crude purity, and impurities. Only two impurities, endo-Gly and des-Leu, showed slight increases compared to the baseline process, which could also be attributed to natural process variation.
The MR-SPPS process significantly reduces the post-Fmoc removal rinse to a single rinse using 6.67% (w/v) Oxyma/DMF, amounting to 6.7 ml/g resin. In contrast, the baseline process requires 9 batch-wise DMF rinses, totaling approximately 61.2 ml/g MBHA resin. However, this led to many incomplete couplings, necessitating the recoupling of those amino acids to ensure proper sequence assembly.
In order to address the issue of incomplete couplings related to the MR-SPPS process, piperidine concentration was reduced from 25% (v/v, 15.5 equiv.) to 8% (v/v, 5 equiv.) with one rinse using 3 equiv. oxyma/DMF and a single neat DMF rinse post-Fmoc deblocking (5 ml/g resin). This adjustment achieved complete couplings for most amino acids with kinetics comparable to the baseline process, although incomplete couplings were still observed with AA2 and AA1. The retarded Fmoc-Xaa2-OH coupling favored the formation of an impurity DIC-(Xaa3-Xaa8)-NH2 (4.8%) due to the undesired reaction between DIC and the Nα functionality on the peptide chain (9).
A comparison between MR-SPPS and the baseline process is summarized in Table 4.
The fact that most Fmoc-Xaa-OH couplings were unaffected by the minimal rinsing strategy supports the applicability of the MR-SPPS process. However, Fmoc-Xaa2-OH and Boc-Xaa1-OH couplings were adversely impacted, with Fmoc-Xaa2-OH coupling resulting in significant DIC-(Xaa3-Xaa8)-NH2 impurity formation. While the MR-SPPS strategy is effective for common couplings, more challenging amino acid couplings may still benefit from more intensive rinsing after Fmoc removal to ensure product integrity. It also highlights the necessity of performing Failure Modes and Effects Analysis (FMEA) to map the sensitivity of each amino acid coupling to the MR-SPPS strategy. This approach would help identify which couplings are more prone to issues and guide adjustments to optimize the process for different amino acids.
SUMMARY
The MP-SPPS strategy has demonstrated its potential as a scalable and cost-effective method for peptide synthesis. It delivers high productivity and purity, particularly suited for peptides with favorable amino acid compositions and large-scale production. Meanwhile, the MR-SPPS approach effectively reduces organic solvent consumption, which addresses significant environmental and economic concerns associated with traditional peptide synthesis methods. However, while MR-SPPS has proven effective for most couplings, it presents challenges for certain amino acid sequences, which may require more intensive rinsing to ensure product integrity.
Looking ahead, both MP-SPPS and MR-SPPS could advance the field of sustainable peptide synthesis. The MP-SPPS strategy will likely be refined and applied to a broader range of peptides, optimizing performance across diverse amino acid compositions. MR-SPPS will continue to evolve, with ongoing efforts to improve rinsing protocols and overcome challenges associated with complex peptide sequences. Failure Modes and Effects Analysis (FMEA) will be instrumental in identifying and addressing specific issues in amino acid couplings, enabling targeted optimizations. As these strategies develop, they hold significant promise for reducing chemical waste and operational costs, leading to more environmentally friendly and efficient peptide synthesis practices.
ACKNOWLEDGMENT
I would like to thank Lena Hansen, Per Ryberg, Ileana Rodríguez León and Jörgen Kjellgren Sjögren from Ferring Pharmaceuticals for conducing and supporting the subject projects.
Figure 1. Root-cause tree of the low productivity/high cost analysis from the conventional SPPS.
Figure 2. Modification of Nim-His by DIC.
Figure 3. Comparison of peptide Z synthetic scheme from conventional SPPS and MP-SPPS.
Table 1. Comparison of the major indices between conventional SPPS and minimal-protection MPPS for peptide Z synthesis.
Table 2. FMEA of minimal rinsing
Table 3. Comparison between MR-SPPS (without post coupling rinsing) and Baseline process.
Table 4. Comparison between MR-SPPS (minimal rinsing post Fmoc removal) and Baseline process.
REFERENCES AND NOTES
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- Al Musaimi, O.; de la Torre, B. G.; Albericio, F. Greening Fmoc/tBu solid-phase peptide synthesis. Green Chem. 2020, 22, 996-1018.
- Ivy Kekessie, I.; Wegner, K.; Martinez, I. et al. Process Mass Intensity (PMI): A Holistic Analysis of Current Peptide Manufacturing Processes Informs Sustainability in Peptide Synthesis. J. Org. Chem. 2024, 89, 4261–4282.
- (a) Anuradha, M. V.; Ravindranath, B. Acylation of unprotected amino acids using ultrasound. Tetrahedron 1997, 53, 1123−1130; (b) Gagnon, P.; Huang, X.; Therrien, E.; Keillor, J. W. Peptide coupling of unprotected amino acids through in situ p-nitrophenyl ester formation. Tetrahedron Lett. 2002, 43, 7717−7719.
- Yang, Y.; Hansen, L.; Ryberg. P. Side-Chain Unprotected Fmoc-Arg/His/Tyr-OH Couplings and Their Application in Solid-Phase Peptide Synthesis through a Minimal-Protection/Green Chemistry Strategy. Org. Process Res. Dev. 2022, 26, 1520–1530.
- Fantoni, T.; Orlandin, A.; Di Stefano, I.; Macis, M.; Tolomelli, A.; Ricci, A.; Cabri, W.; Ferrazzano, L. Solid phase peptide synthesis using side-chain unprotected arginine and histidine with Oxyma Pure/TBEC in green solvents. Green Chem., 2024, 26, 10929-10939..
- Unpublished data from Ferring Pharmaceuticals.
- Kumar, A.; Sharma, A.; de la Torre B. G.; Albericio, F. In situ Fmoc removal – a sustainable solid-phase peptide synthesis approach. Green Chem. 2022, 24, 4887.
- Yang, Y. “Chapter 5. Side Reactions Upon Amino Acid/Peptide Carboxyl Activation” in Side Reactions in Peptide Synthesis. Academic Press, 2015, pp. 95-97.
