Process Mass Intensity (PMI): A Holistic Analysis of Current Peptide Manufacturing Processes Informs Sustainability in Peptide Synthesis

Abstract

Small molecule therapeutics represent the majority of the FDA-approved drugs. Yet, many attractive targets are poorly tractable by small molecules, generating a need for new therapeutic modalities. Due to their biocompatibility profile and structural versatility, peptide-based therapeutics are a possible solution. Additionally, in the past two decades, advances in peptide design, delivery, formulation, and devices have occurred, making therapeutic peptides an attractive modality. However, peptide manufacturing is often limited to solid-phase peptide synthesis (SPPS), liquid phase peptide synthesis (LPPS), and to a lesser extent hybrid SPPS/LPPS, with SPPS emerging as a predominant platform technology for peptide synthesis. SPPS involves the use of excess solvents and reagents which negatively impact the environment, thus highlighting the need for newer technologies to reduce the environmental footprint. Herein, fourteen American Chemical Society Green Chemistry Institute Pharmaceutical Roundtable (ACS GCIPR) member companies with peptide-based therapeutics in their portfolio have compiled Process Mass Intensity (PMI) metrics to help inform the sustainability efforts in peptide synthesis. This includes PMI assessment on 40 synthetic peptide processes at various development stages in pharma, classified according to the development phase. This is the most comprehensive assessment of synthetic peptide environmental metrics to date. The synthetic peptide manufacturing process was divided into stages (synthesis, purification, isolation) to determine their respective PMI. On average, solid-phase peptide synthesis (SPPS) (PMI ≈ 13,000) does not compare favorably with other modalities such as small molecules (PMI median 168–308) and biopharmaceuticals (PMI ≈ 8300). Thus, the high PMI for peptide synthesis warrants more environmentally friendly processes in peptide manufacturing.

Peptides have recently received a revived focus in the pharmaceutical industry for reasons including their biocompatibility profile, structural versatility, and potential to modulate targets that are poorly tractable by small molecules.^1,2 Solid-phase peptide synthesis (SPPS) has been a reliable technology for the efficient synthesis of complex peptide targets, thus providing access to a broad chemical space in peptide therapeutics.³ However, SPPS and the vast majority of technologies currently used in peptide synthesis require a large excess of hazardous reagents and solvents. Consequently, improvements are needed to reduce their environmental impact.⁴⁻⁶ Problematic solvents include N,N-dimethylformamide (DMF) and its close derivatives N,N-dimethylacetamide (DMAc) and N-methyl-2-pyrrolidone (NMP). These solvents are globally classified as reprotoxic; their use may be restricted and potentially banned in the near future.^7,8 Other limitations of peptide synthesis include poor atom-efficiency of fluorenylmethyloxycarbonyl protected amino acids (Fmoc-AAs), potentially explosive and sensitizing coupling agents,^9,10 highly corrosive trifluoroacetic acid (TFA), and other toxic solvents such as dichloromethane (DCM), diethyl ether (DEE), and tert-butyl methyl ether (MTBE). These hazards are compounded by the large amount of solvent used for isolation and purification.

The Peptides Focus Team within the American Chemical Society Green Chemistry Institute Pharmaceutical Roundtable (ACS GCIPR) aims to facilitate a fast transition toward sustainable peptide synthesis. To achieve this goal, it would be pivotal to have accurate metrics for the current industrial processes of peptide synthesis to identify where the greatest impact can be made. This exercise has been extensively pursued for small molecules, and the Oligonucleotide Focus Team of the ACS GCIPR has recently conducted such exercise for oligonucleotides.¹¹ However, there is a lack of industry-wide studies for peptides. We aim to bridge this significant gap in this manuscript.

There are many established green chemistry metrics used to assess industrial processes.^12,13 One of the most comprehensive evaluations is a life cycle assessment (LCA) wherein the mass/energy balances and environmental impact of a product through all stages (e.g., raw materials, production, end-of-life treatment, disposal) is quantified. Since conducting a full LCA can be costly, time-consuming, and particularly challenging in early phase development, simple mass-based metrics are routinely used (Figure 1).

Figure 1.

Select mass based metrics.

Atom economy (AE) quantifies the efficiency of a reaction by measuring the number of reactant atoms that appear in the final product. It assumes both 100% yield and stoichiometric loading and therefore provides a measure of only the reaction design. Conventional metrics such as chemical yield, conversion, and selectivity quantify the actual conversion of the limiting reactant to the desired product. However, like AE, they exclude other significant resource requirements, such as solvent and other raw material inputs.

Complete environmental factor (cEF), which is another relevant metric to evaluate industrial processes, offers a measure of the complete waste stream and also factors in all process materials such as solvents and raw materials.^10,14

Furthermore, process mass intensity (PMI) provides a more holistic assessment of the mass requirements of a process, including synthesis, purification, and isolation. PMI is defined as the total mass of materials used (raw materials, reactants, and solvents) to produce a specified mass of product. It should, however, be noted that PMI does not account for the environmental impact incurred during the manufacture of starting building blocks and reagents. As PMI focuses on maximizing value and efficiency, the ACS GCIPR has identified it as the key mass-related green chemistry metric and as an indispensable indicator of the overall greenness of a process.¹⁵ A limitation to the PMI metric is that it does not take into account types of material and other factors such as energy usage, logistics, environmental impact, or starting material complexity.

Given the historical prominence of small molecules as medicines and their accessibility via well-controlled synthetic and analytical methods, it is not surprising that PMI was first developed for this modality. Typical small molecules have a reported PMI range of 168 to 308 kg material use per kg active pharmaceutical ingredient (API).¹⁶ In contrast, large molecules (or “biologics”), which are biotechnology-derived molecules—primarily monoclonal antibodies but also including fusion proteins, traditional vaccines, etc.—have been found to have an average PMI of ∼8,300 kg waste/kg API.¹⁷ Synthesized oligonucleotides, which are traditionally assembled in a conceptually similar manner to synthetic peptides with an excess of reagents and solvents (as well as energy) via solid-phase processes, challenging purifications, and burdensome isolations, have been appraised within a PMI range of 3,035 to 7,023 kg/kg with an average of 4,299 kg/kg.

In this article, cross-company peptide PMI data for synthetic peptides will be analyzed using a methodology similar to that in the oligonucleotides study. The unit operations are partitioned to better understand which stages are the most wasteful.¹¹ In addition, evaluation of PMI per unit amino acids (AAs) for peptides could help shed light on the sustainability impact of peptides of different chain lengths.

Current Peptide Manufacturing Processes

Synthesis

As of 2020, greater than 100 peptide APIs have been approved for pharmaceutical use with sequence lengths ranging up to ∼60 amino acids.¹⁸ These include lengths ranging from approximately 5AA residues (e.g., thymopentin, 5AA) to more than 30AA residues (e.g., enfuvirtide, 36AA; teriparatide, 34AA; aprotinin, 58AA). Chemical assembly is typically carried out via solid-phase peptide synthesis (SPPS) or liquid phase peptide synthesis (LPPS) (Figure 2). While a linear sequence is typically generated by SPPS (including branching side-chains, if applicable), LPPS allows for both linear and convergent syntheses. Additionally, some specific LPPS-related methods like soluble tag-assisted liquid phase peptide synthesis have been introduced.¹⁹⁻²¹ A hybrid approach, where SPPS synthesis of short fragments followed by joining of the fragments via LPPS has also been employed.²² Recombinant biotechnology or enzymatic assembly of deprotected fragments is a further orthogonal approach to chemical peptide synthesis.²³

Figure 2.

Illustration of solid vs liquid phase peptide synthesis cycles.

Regarding selection criteria for the synthesis strategy, aspects to be considered include material demand, required batch size, available manufacturing capability and capacity, complexity, scalability, potential for automation, availability and costs of starting materials, risk (process, timeline, costs), and regulatory control strategy. The chosen approach should fit the API life cycle as far as costs and timelines in development and manufacturing are concerned, which may be subject to change over time.

The LPPS strategy for the manufacture of shorter peptides, containing 5–10 AAs for example, offers some advantages. This approach allows for the utilization of different protecting groups such as Boc and Cbz protected AA for the synthesis of complex targets. The need for step-specific process optimization in LPPS provides the opportunity for limiting material and reagent usage, reducing impurity formation, and increasing impurity rejection during workup/isolation.²⁴ In contrast to SPPS, the reaction mixture may be analyzed by in-process analytical methods for reaction monitoring. The LPPS strategy may be used with conventional batch reactors; i.e., there are fewer restrictions with manufacturing sites. However, LPPS does present a few drawbacks. Typically, a greater effort is required to develop an LPPS-based manufacturing process compared to SPPS, resulting in higher initial costs and longer process development. Due to the limited extent of automation, LPPS requires more manual interactions during production, increasing risk due to human error. Additional issues may include an increased risk of racemization during coupling reactions and deprotections, particularly during the peptide elongation process, which often exhibits sluggish reaction kinetics. In prolonged reactions, chemical instability issues may surface during the reaction and work up of the growing peptide chain, especially in cases of larger peptides.

SPPS technology is a widely used and reliable platform. Established building blocks, reliable supply chains, and well-known chemistry conditions are available. Material generated early in the API life cycle has likely been produced via SPPS, demonstrating the general feasibility of the method, supported by the availability of automated lab scale synthesizers. As a consequence, less process development is required before scale up. These aspects make SPPS an attractive synthetic strategy for large-scale manufacturing of peptides. SPPS requires a filter reactor, which typically is a specifically designed fully or semiautomatic reactor (see examples in Figures 3, 4, 5, 6, and 7). At production scale, SPPS reactors with capacities from hundreds of liters such as the automatic 250 mL × 6 SPPS reactor (Figure 3) up to thousands of liters, as illustrated in the 6000 L SPPS reactor system (Figure 7), is employed.

Figure 3.

Automatic 250 mL × 6 SPPS reactor.

Figure 4.

Automatic 2 L × 4 SPPS reactor.

Figure 5.

Semiautomatic 100 L SPPS reactor.

Figure 6.

1000 L SPPS reactor.

Figure 7.

6000 L SPPS system.

Production-scale manufacturing of peptides with lengths in the range of 70 AA via SPPS has proven feasible in principle. However, even though the yield for each chemical transformation is typically high, it is usually less than 100%. Hence, despite the ambition to access ever larger peptides with varying chemical complexity by SPPS,²⁶ the length of peptides that can be manufactured efficiently by SPPS has a practical limit of approximately 100 amino acids. Furthermore, when synthesizing longer peptides, sometimes specific issues like inefficient coupling reactions, low yield, and excessive impurity formation may be observed, which are sometimes related to the formation of secondary structures of the protected peptide on the resin or specific properties of a building block.²⁷ In order to access more complex peptides or circumvent specific issues, alternative approaches can be applied. One is the hybrid approach,²⁸ which combines linear SPPS of smaller peptide fragments with fragment condensation in solution, i.e. LPPS. Please note that fragments may feasibly be manufactured by methods such as linear LPPS or soluble tag/anchor-assisted liquid phase peptide synthesis as well, which would result in a convergent LPPS process. An early example for production scale-manufacturing according to the hybrid approach is enfurvatide,²⁹ whereas a more recent example is tirzepatide.²² For the hybrid approach, the peptide is subdivided into several fragments, which are separately prepared by SPPS and conjugated to the complete peptide chain by LPPS in solution. The fragment approach adds complexity and additional manufacturing steps compared to linear SPPS, including the need for additional equipment. Nevertheless, the fragment approach may be more efficient than linear SPPS; e.g., if a higher initial resin substitution may be achieved, difficult coupling steps may be circumvented, resulting in a unique impurity profile that could make chromatography easier, a lower excess of AA derivatives may be sufficient, or a higher purity and yield could be achieved. Moreover, the hybrid strategy has potential to reduce overall manufacture duration, since the fragment production can be performed in parallel (convergent synthesis).³⁰

As both LPPS and SPPS have their specific limitations in terms of complexity and environmental impact, modified LPPS methods based on soluble tags/anchors have been proposed instead of an insoluble resin.^19,21 With that technology, peptide assembly takes place in a solution. The tag/anchor keeps the growing peptide chain in the organic layer, which allows the removal of side products, excess building blocks, and coupling reagents via aqueous extraction steps or precipitation. Consequently, less organic solvent than SPPS is employed. The technology is currently in use at multikilo scale for the manufacturing of short peptide sequences.²¹ As the tag is a relatively small molecule with a defined structure, both coupling and deprotection reactions could be monitored directly by TLC or HPLC, and the characterization of the tagged growing peptide is possible by NMR and MS analysis.

Chemical synthesis is the preferred method to produce peptide therapeutics under development. Alternatively, recombinant or semirecombinant production is a valuable and relatively sustainable strategy to produce peptides. The relatively limited use of recombinant strategy is mainly driven by two factors:³¹

• (1)Currently established recombinant platforms can only assemble peptides containing the natural L-AAs, limiting the applicability of recombinant strategy. Research activities are ongoing to extend the applicability of recombinant strategies.³²
• (2)In general, the development time is significantly shorter and the cost lower for the manufacture of peptides via a chemical synthesis route.

Consequently, the application of recombinant strategies is mainly focused on the large-scale manufacturing of long peptide sequences comprised of natural L-AAs.^23,33 A large-scale recombinant synthesis of peptides has the advantage of potentially driving cost down many folds. A key difference in product registrations is that peptides produced recombinantly are filed as Biologics License Applications (BLA), whereas synthetic peptides are filed as New Drug Applications (NDA) if their primary sequence is 40 amino acids or fewer. For synthetic peptides with a sequence greater than 40 amino acids, they will be filed as a BLA.

Purification

Purification of synthetic peptides is a major challenge in the production of peptide APIs. The crude peptide obtained by any of the aforementioned manufacturing strategies requires purification to achieve the specifications required for a pharmaceutical API.

The number of iterative steps in peptide synthesis potentially generates a large number of impurities (e.g., deletion and truncation), with a chemical structure close to that of the target peptide. The separation of these impurities is therefore complex based on the similarity of the chemical properties.

Crystallization can be a valuable option but with limited applicability to peptide manufacturing. It is mainly limited to short peptide sequences or cyclic peptides and requires a high initial crude purity as only a minimal increase in purity is observed with longer sequences.

Purification Technologies

Purification is an integral part of the synthetic peptide production process. Current processes for peptides are designed around chromatographic purification due to superior separation. Ion-exchange chromatography and reversed-phase high performance chromatography are the most established techniques using differences in molecular charge and hydrophobicity, respectively.³⁴

The purification process impacts the resource consumption in synthetic peptide production in two ways: First, the downstream processes account directly for approximately 50% of the Process Mass Intensity (PMI), due to the use of dilute product solutions and long chromatographic gradients. Although the created waste is rarely toxic and consists mainly of water, acetonitrile, and ethanol, organic solvents are not well-suited for recycling, as they are strongly diluted in the process. Second, the purification process yield has a direct impact on overall PMI as the fully synthesized product is handled, and losses in the downstream process need to be offset by increased amounts in the upstream process, thereby increasing overall PMI.

Reversed Phase, Ion Exchange, and Size Exclusion Chromatography

Preparative chromatography is the established technology for the purification of most peptide APIs³⁵ as it is known to be the most efficient approach to separate chemical substances with similar chemical structures. Large scale high-pressure and low-pressure chromatographic units are used for the manufacture of peptides and are scalable on both lab- and production-scale. The purification of peptide APIs is typically based on the use of reversed phase (RP) chromatography, ion exchange chromatography (IEC), and frequent size exclusion chromatography (SEC). Most of the manufacturing processes are based on an orthogonal strategy for purification (depending on the impurity profile of the crude and its batch-to-batch variability): 2 to 3 chromatographic steps are combined (RP and/or IEC). The proper strategy should target a complementarity between the purification steps to efficiently remove all impurities, based on the specific properties (e.g., pI, hydrophobicity).³⁶

The most common solvent mixture used for peptide purification is water/acetonitrile. Alternatively, alcohols can be used to replace acetonitrile. Ion pair additives/buffers can be included in the eluent to control pH, conductivity, and improve separation. Gradient elution is often required except for very short peptide sequences.

IEC can be used as an orthogonal method to RP, as it relies on different separation mechanisms. It can advantageously be used as a prepurification step to remove nonpeptide related hydrophobic impurities together with the residuals protecting groups and scavengers from cleavage. A final ion exchange step is often required after the last purification step to ensure the peptide is associated with the right counterion before going to the final isolation step.

Preparative chromatography is by nature a highly dilute process, and the purification step is an important contributor to the PMI for peptide manufacturing.

In the scope of greener purification processes, new developments as well as established techniques can be regarded as suitable to decrease downstream PMI and to increase process yield.

Multicolumn Counter Current Solvent Gradient Purification (MCSGP)

Multicolumn counter current solvent gradient purification (MCSGP) uses multiple columns to retain impure fractions containing product from a primary column for application on a secondary column and subsequent purification. Initially developed with up to 8 columns, now systems with 2 columns are established in laboratory and production scale. While the process step mass index is not reduced directly, increased yields compared to single column purification steps have been shown: Luca et al. showed for glucagon synthesized on a solid phase that a yield increase of +23% could be achieved on an optimized MCSGP process, compared to a single column separation, while maintaining comparable purity profiles,³⁷ thereby reducing the overall PMI.

Displacement Chromatography

Displacement chromatography uses the difference in binding strength of molecules to achieve separation on a chromatographic column during loading. Product elution can then be done with a displacement agent which binds stronger to the column.³⁸ Alternatively, in product displacement chromatography mode, multiple consecutive columns are used which are then eluted separately, thereby separating stronger binding impurities from the product.³⁹ This mode of chromatography has been established for a wide range of separation principles, including ion-exchange, reversed-phase, and hydrophobic interactions.⁴⁰

Displacement chromatography can be very efficient, as no organic modifier is needed to form an elution gradient, and the column can be loaded to maximum capacity. Additionally, the product elutes in a small volume from the column, reducing the need to concentrate and volume handling in subsequent process steps. The significantly reduced solvent consumption makes displacement chromatography a considerable green alternative for synthetic peptide purification.

Supercritical Fluid Chromatography

Supercritical fluid chromatography (SFC) uses liquified gases, often CO₂, as the liquid phase, which are enriched with additives to modulate peptide retention and peak shape. SFC is therein very efficient due to the lower viscosity of the liquid phase and better diffusion properties which is providing a better separation when compared to classic RP-HPLC systems. This improved separation allows for much shorter gradients, thereby increasing throughput and reducing solvent consumption.⁴¹ In pilot scale, SFC can be performed with CO₂ recycling,⁴² reducing the environmental impact even further. The product is furthermore eluted in higher concentration, compared to RP-HPLC, therefore reducing the need for subsequent solvent evaporation.⁴³

The shifting focus to greener processes has sparked an increasing interest in SFC; however, the high pressures and dedicated equipment needed for SFC are limiting the application in larger scale for preparative separations.

Process Design

Mechanistic process modeling is an important and well-established tool in the development of synthetic peptide purification process optimization strategies. Gétaz et al. showed that in silico models can be used to develop a multistep purification in production scale for a synthetic product and gain significant advancement in yield.⁴⁴

While the aim of optimization efforts is historically driven by process yield and cost, reduction of the PMI is often not yet an explicit optimization objective.

For a truly efficient process regarding PMI, upstream and downstream process steps need to be considered in the optimization efforts, given the impact of an increased yield in late process steps on overall PMI. Vice versa, the potential of process optimization in peptide synthesis might manifest in the overall process first in the reduction of purification steps or increased yield in downstream processing. This shows that when optimizing processes, it is essential to assess the impact across the whole process and not only on the subprocess level, especially in the synthesis-purification relationship.

Isolation

The peptide API is generally isolated as a dried powder. The most common strategy is lyophilization (freezing drying), which involves several steps. The dilute consolidated fractions contain the organic cosolvent and buffer used for the elution of the peptide API. For industrial scale isolation, the pooled fractions are typically concentrated by removing the cosolvent before a final bioburden reducing microfiltration and freeze-drying. The concentration and cosolvent removal are often performed using the following techniques:

• Tangential flow filtration with diafiltration of the solution

• Evaporation of the organic solvent

Large scale tray lyophilizers can be used for the isolation of API batches of less than 50 kg (Figure 8).

Figure 8.

Large scale tray lyophilizer (with associated isolator).

Alternatively, the spray drying isolation technique offers great advantages for large scale applications with higher throughput and lower energy consumption compared to freeze-drying. However, the peptide isolated product in this technique is exposed to high temperatures, which may negatively impact the purity and yield. This is often observed for the manufacturing of batches where small quantities are processed.⁴⁵

Assessing Sustainability of Current Processes

For this article, 14 ACS GCIPR member companies have compiled PMI metrics on 40 synthetic peptide processes at various development stages. These compounds included 34 SPPS processes, 4 LPPS processes, 1 hybrid SPPS/LPPS, and 1 chemo enzymatic peptide synthesis (CEPS) process. Peptides were classified according to phase of development, type of process, and number of AAs in sequence. The synthetic peptide manufacturing process was divided into three stages (synthesis, purification, and isolation). The inputs for the entire process were summed to determine the PMI for each individual synthesis. For this analysis, the route and PMI to Fmoc or Boc protected AAs were not factored into the final PMI. These are typically considered readily available raw materials, and vendor information on PMI is typically not available. It is acknowledged that the complexity of the AAs will affect the mass efficiency of the overall process, and future comparisons should strive to include this information to the extent that this is possible.

A summary of the output of this assessment is shown in Tables 1, 2, 3, and 4. The single hybrid SPPS/LPPS process was grouped with the 34 SPPS processes since 90% of the waste generating steps are from SPPS. Table 1 displays commercial and Phase 3 SPPS processes with PMI ranging from 1684 to 34,585 (average 13,063) for peptides ranging from 6 to 43 AAs in length (average 19 AAs). For better comparison between peptide processes, PMI per AA was calculated in order to factor sequence length into the PMI assessment. These values range from 163 to 2350 with an average of 874.5 for SPPS commercial and Phase 3 processes (Table 1). The overall PMIs for synthetic peptides from SPPS were significantly higher than those for synthetic oligonucleotides. Synthetic oligonucleotides had an average PMI of 4299 and average PMI per phosphoramidite building block 199.¹¹ The oligonucleotides process manufacturing is mostly linear synthesis using established platform methodology, whereas peptide manufacturing entails more elaborate downstream operations with highly variable yield depending on the target structure (linear vs cyclic structure; usage of natural amino acids vs. non-natural amino acids or small molecule building blocks) and its purity target. The SPPS PMI does not compare favorably either with other modalities such as small molecule (PMI median 168–308)¹³ and biopharmaceuticals (PMI ∼ 8300).¹⁷ Limited innovation and improvements in PMI are currently being observed at the later stages of development. This is depicted in the significant differences in the Phase 2 and 3/Commercial (Tables 1 and 2) vs Phase 1/preclinical processes (Table 3), with PMI per AA of 951/874.5 and 1464, respectively. A graphical representation of this assessment is also depicted in Figure 9. The lack of PMI improvement in early phase development may be related to lower volume process manufacturing (<10 kg), limited process development, and aggressive time to start first in human studies. In addition, the lack of a downward trend in PMI as development progresses is inconsistent with what is generally observed with small molecule API.¹⁸ This may be because SPPS peptide development and manufacturing platform technology have not yet advanced to present opportunities where PMI can be reduced during the development lifecycle. An interesting trend from the SPPS data sets is the increased peptide complexity in terms of manufacturing, often due to more rigorous quality standards per phase of development (average length commercial and Phase 3 = 19; Phase 2 = 23; Phase 1 = 28). However, the PMIs per AA for SPPS systems are static across the development lifecycle, most likely due to the small statistical differences in the number of AA on a manufacturing scale. The most promising data submitted were from the 4 LPPS processes, which account for 10% of the total processes submitted (Table 4). The PMI per AA ranged from 95 to 451 (average of 263 per AA) for sequences 10–20 AAs in length. The performance differential between LPPS and SPPS processes is highlighted in Figure 10. This underlines the need to continue to advance synthetic methodologies and technologies to improve peptide therapeutic PMI across the development phases. Interestingly, while the data set is limited, the decrease PMI across phase of development is consistent with PMI trends in small molecule commercialization.

Table 1. PMI Data for 14 Commercial and Phase 3 Peptides Produced by SPPS.

peptidea	phase of development	number of AAs	PMI	PMI per AA
X	Commercial	8	18798	2350
M	Commercial	10	19319	1932
L	Commercial	8	10655	1332
BB	Commercial	14	16671	1191
I	Commercial	39	34585	887
CC	Commercial	12	10370	864
E	Commercial	19	11419	601
H	Commercial	8	4414	552
A	Commercial	7	3274	468
W	Commercial	6	1683	281
R	Phase 3	43	7008	163
U	Phase 3	39	24742	634
V	Phase 3	15	12114	808
JJb	Phase 3	43	7836	182
Average		19	13063	874.5

^aPeptide sequences are blinded, and structures are not disclosed to comply with proprietary policies of participating companies (Tables 1–4).

^bHybrid SPPS/LPPS.

Table 2. PMI Data for 7 Phase 2 Peptides Produced by SPPS.

peptidea	Number of AAs	PMI	PMI per AA
F	30	15268	509
O	9	16299	1811
Q	9	19602	2178
S	43	7205	168
Z	38	6504	171
AA	12	10451	871
Average	23	12554	951

^aPeptide sequences are blinded, and structures are not disclosed to comply with proprietary policies of participating companies (Tables 1–4).

Table 3. PMI Data for 14 Phase 1 or Preclinical Peptides Produced by SPPS.

peptidea	phase of development	number of AAs	PMI	PMI per AA
B	Phase 1	34	79334	2333
C	Phase 1	15	27103	1807
D	Phase 1	16	16277	1017
G	Phase 1	38	15889	418
J	Phase 1	25	48306	1932
K	Phase 1	30	12617	421
N	Phase 1	9	38632	4293
P	Phase 1	9	22123	2458
T	Phase 1	42	10337	246
DD	Preclinical	17	14196	835
EE	Preclinical	36	74760	2077
FF	Preclinical	34	50309	1480
GG	Preclinical	46	21296	463
HH	Preclinical	46	32798	713
Average		28	33141	1464

^aPeptide sequences are blinded, and structures are not disclosed to comply with proprietary policies of participating companies (Tables 1–4).

Table 4. LPPS Processes.

peptidea	phase of development	number of AAs	PMI	PMI per AA
A	Phase 1	20	9019	451
B	Phase 2	12	4280	357
C	Phase 3	12	1139	95
D	Commercial	10	1497	150
Average		13.5	3984	263

^aPeptide sequences are blinded, and structures are not disclosed to comply with proprietary policies of participating companies (Tables 1–4).

Figure 9.

Average PMI per AA for 34 SPPS processes per phase.

Figure 10.

PMI for LPPS vs SPPS.

PMI was also subcategorized by unit operations (Synthesis, Purification, and Isolation) for SPPS processes. For preclinical and Phase 1, approximately double the waste is produced in the purification steps, whereas the waste profile flips to higher relative SPPS waste in the later stages. The trend may reflect the use of oversized chromatography columns in the early clinical phase and higher levels of washing required in a plant environment for SPPS and/or not fully developed synthesis conditions. Additionally, the low column loading, nonoptimized chromatographic conditions, and poor crude peptide purity at the initial process development contribute to high PMI at the early development stage. Increasing purification PMI contribution in Phase 3/commercial compared with Phase 2 may be related to a higher purity expectation of API for marketed production. Isolation accounted for very limited use of solvent primarily to just a single precipitation step for crude API and the near exclusive use of lyophilization to deliver final purified peptide API. While lyophilization is an energy intensive step (energy factor is not included in PMI calculation), it is a beneficial process for the isolation of the final peptide product contributing to the PMI due to high yield, compared to alternative procedures such as precipitation, crystallization, and spray drying. Lyophilization is very efficient from a PMI perspective as a standalone unit operation. Figures 11, 12, 13, and 14 show that approximately equal amounts of solvents and water (44%) are used in SPPS processes. There is a similar percentage of water used as in oligonucleotide processing (51%)¹¹ but higher compared with small molecule processing (28%)¹⁵ due to the chromatographic purification steps. The dominant organic solvent in SPPS is DMF (39%), which is the other main contributor to process waste due to extensive washing of peptide on resin intermediates. Interestingly, the next highest solvents used in synthetic steps, all at ∼1% individually, are MTBE, NMP, and 2-propanol. Acetonitrile (ACN ≈ 10%) is the other ubiquitous waste that is used extensively in the chromatographic purification step.

Figure 11.

Averages % of solvent usage of total PMI per process stage.

Figure 12.

Overall solvent composition SPPS process.

Figure 13.

Solvent composition (synthesis).

Figure 14.

Solvent composition (purification).

Safety Assessment vs Design Principles of Green Chemistry and Engineering

Safety is of paramount importance in any chemical enterprise and is at the heart of sustainability principles. As outlined in the 12 principles of green chemistry⁴⁶ and engineering,⁴⁷ several principles are grounded in safety (see Table 5, red). The peptide chemistry community continues to identify hazardous reagents that are in current use and offers guidance on their safe usage. Often significant research and development is pursued to furnish alternative, safer reagents of equal or superior performance.

Table 5. Principles of Green Chemistry.

Hydroxybenzotriazoles are known for their shock and friction sensitivity. Because dry 1-hydroxybentrotriazole is classified as a class 1 explosive. The other hydroxybenzotriazoles, such as HOAt, 6-Cl-HOBt, when dry also exhibit explosive properties. For this reason, HOBt hydrate form is exclusively used.⁴⁸ A comprehensive list of 45 peptide coupling reagents has been assessed for thermal stability, and a peptide reagent selection guide has been developed from a process safety standpoint that classified the reagents into “most preferred”, “use with caution”, or “least preferred” categories.⁴⁹ Ethyl cyanohydroxyiminoacetate (Oxyma) was deemed as a safer alternate to hydroxybenzotriazoles with lower risk of explosion based on calorimetry studies (DSC and ARC).⁵⁰ However, a recent study revealed that HCN gas was detected under normal reaction conditions during the use of Oxyma with DIC for AA activation.⁵¹ Since the publication of this article, further efforts to mitigate the safety risks of Oxyma have been reported. Usage of greener solvent options and introduction of dimethyl trisulfide (DMTS) as a HCN scavenger reduced the extent of HCN generation.⁵² A recent study further highlighted the relationship of the carbodiimide structure to the propensity to generate HCN, finally offering safer alternates to DIC for peptide coupling.⁵³ Peptide coupling reagents have been reported to be responsible for severe allergic anaphylactic reaction.^9,10 Several past reports in allergy and medical publications were not picked up by the synthetic chemistry community earlier. Since coupling reagents can all modify human protein, there are risks of potential sensitization, and therefore protocols for safe handling of potential sensitizers must be followed.⁹ Global peptide deprotection steps involved HF in the past when Boc- and Bn-protected AAs were used during synthesis. A switch to Fmoc synthesis was driven by safety concerns arising from the handling of HF in the case of Boc peptide chemistry. In Fmoc chemistry, TFA cocktails in organic solvents are used, typically containing scavengers to avoid reincorporation of highly reactive tertiary carbocations such as tBu often used for side chain protection. Moving forward, alternatives to TFA are recommended from a safety/sustainability standpoint.

In addition to safety considerations, many of the principles of green chemistry and green engineering (Table 5, blue) aim to maximize the mass and energy efficiency. Many of these principles correspond to challenges in the sustainability of the current approaches to peptide synthesis used today. The Fmoc SPPS makes use of AA starting materials bearing Fmoc and side-chain protecting groups. These starting materials are not aligned with the “maximize atom economy” and “reducing derivatives” principles. As noted above, most amide coupling conditions in peptide synthesis involve the use of benzotriazole derivatives as stoichiometric reagents since viable and industrially applicable catalytic protocols are lacking. Finally, the goal of “waste prevention”, which is linked to maximizing resource and mass efficiency, remains a significant challenge. Typically, peptide syntheses require large excesses of stoichiometric reagents (Fmoc AAs, coupling additives, piperidine base) to ensure both the coupling and Fmoc deblocking reaction proceed to completion. Organic solvents and water constitute the greatest proportion of the waste generated as a large excess of solvent is routinely used to rinse the resin after each coupling and deprotection operation.

Besides safety and mass energy efficiency, several other principles as outlined in Table 6 are relevant to the field of peptide synthesis. Use of analytical technology such as in situ monitoring of Fmoc removal and the various washing steps (following coupling, capping, and Fmoc removal steps) can help in tailoring the solvent usage and avoid the waste of solvents and reagents. Use of renewable feedstocks in the manufacture of key starting materials and reagents is a long-term sustainability goal. Design for separation in the context of peptides can refer to any advancement that can reduce the environmental impact of the peptide purification step. Technology that helps in furnishing cleaner crude peptide reduces the need for resource-intensive chromatography.⁵⁴ A comprehensive assessment of the sustainability challenges in peptide synthesis and purification has been published that details best practices and areas of improvement.

Table 6. Summary of Solvent Composition for Synthesis and Purification Steps.

synthesis step
Solvent	DMF	MTBE	NMP	IPA	DIPE	DCM	Others	ACN	DMAC	Heptane
% Norm	89.2	2.7	2.3	2.1	2	0.8	0.3	0.1	0.1	0.1

purification step
Solvent	Water	ACN	EtOH	MeOH	IPA	MTBE	Acetic Acid
% Norm	77.3	18.4	2.2	0.7	0.7	0.5	0.3

Improving Sustainability in Near Term

The synthesis, purification, and isolation processes of peptide APIs have been greatly improved in the past few years since the GCIPR team’s last publication.⁵ These key processes of synthesis, purification, and isolation, involving (i) solvent usage, (ii) coupling reagents, (iii) new synthesis platforms such as solid tag-assisted LPPS, and (iv) nanofiltration/membrane-based reactor systems, are discussed here as case studies on developments in the fields that are advancing short-term peptide sustainability goals. Short-term goals are defined as areas that can be advanced in 10 years or less.

Solvents

Solvents are the largest contributor to process mass intensity (PMI) and account for most of the waste generated and thereby the environmental impact of a drug substance. The PMI contribution of solvents is 80–90% for small molecules,⁵⁵ and a high(er) percentage is applicable for peptides, especially considering the totality of solvents required during (a) solid phase peptide synthesis, (b) solution phase chemistry such as cyclization, and (c) purification, usually by preparative reverse phase chromatography. DMF is the solvent of choice for SPPS coupling and Fmoc removal steps. Key reasons are favorable resin-swelling, ability to efficiently solubilize relevant starting materials and reagents as well as byproducts, familiarity, and good knowledge around potential side-reactions with these solvents. However, DMF is a highly hazardous solvent, that was restricted by EU REACH guidelines (December 2021) and was identified as SVHC (Substance of Very High Concern).^56,57

While water is the benchmark for green solvents, it is not fully compatible with the existing SPPS technology. Recently, however, promising progress has been made toward the development of water based SPPS where more water compatible resins, N^α protected amino acids and reagents have been investigated.⁵⁸ In the near term, finding a suitable replacement for DMF/NMP that works with most common AAs and reagents is critical to improve sustainability. Among the green solvents studied, N-butylpyrrolidinone (NBP) was favored in a study and was recently evaluated head-to-head against DMF.^8,57,59 NBP has been shown to exhibit superior performance compared to DMF in terms of reduced racemization and aspartimide formation during SPPS. The high viscosity of NBP is a trade-off that results in inferior resin swelling and starting material solubilization compared to DMF. Also, the very high BP (244 °C) would make it impractical from a solvent recyclability perspective. Alternate solvents exploration in SPPS spearheaded by the Albericio group includes ACN (Acetonitrile),⁶⁰ THF (Tetrahydrofuran),⁶⁰ MeTHF (2-Methyltetrahydrofuran),⁶¹ EtOAc (Ethyl acetate),⁶¹ CPME (Cyclopentyl methyl ether),⁶¹ GVL (γ-Valerolactone),^62,63 NFM (N-Formylmorpholine),⁶³ and PC (Propylene carbonate).⁶ Getting the right balance of properties to match the versatility of DMF has been challenging. GVL was found to react with AAs via ring opening.⁶⁴ While optimized conditions minimized this impurity during SPPS,⁶² there remains a continued need to investigate alternate organic solvents to afford chemists a broad solvent inventory to choose from based on their needs.

Ideally, having a single solvent will simplify the execution of the synthesis process. However, to strike a balance between process friendly implementation and feasibility, binary solvent mixtures have been considered as well. The advantage of having a binary solvent pair is that the polarity can be tuned by adjusting the solvent ratio according to solubility of reagents and byproducts, since the Fmoc removal step is favored by polar solvent mixtures, while the coupling steps are favored by a less polar environment.^2,65,66,57 Binary solvent mixtures were studied wherein DMSO combined with either dioxolane (DOL), MeTHF, or EtOAc was of suitable high polarity for the Fmoc removal step. The coupling steps were favored in DOL with NFM or NBP. Another study on the binary solvent mixture considered c oligonucleotides yrene or sulfolane with diethyl carbonate (DEC), and anisole with DCM (dichloromethane).⁶⁷ Swelling of various resins was conducted, and a moderate dependence on the ratio of the binary solvent mixture was observed. A subset of the best solvent combinations was chosen based on solubility of common Fmoc AAs and purity profile of peptides synthesized using the green solvent pair vs standard DMF. This approach is quite promising in the short term to afford successful SPPS. In general, a marked dependence of the overall purity profile is associated with the solvent polarity, and hence, a change of solvent should be accompanied by careful assessment of product purity and yield. Changing the solvent at a very late phase of a project in a pharmaceutical setting could be challenging since new impurities in the latter batches might necessitate additional toxicology and stability studies. It is recommended to consider sustainability aspects of the project early on, with early engagement by the project teams around implementing alternate solvents.

Additionally, piperidine is ubiquitously used to deprotect the Fmoc groups, which is problematic since piperidine falls under DEA list I of controlled chemicals (United States Drug Enforcement Administration). Few alternative bases such as methylpiperidines, DBU, piperazine, 4-methylpiperazine, and morpholine have been explored in DMF. Side reactions such as aspartimide formation must be monitored closely since this is a base-mediated side- reaction. Also, aspartimide formation is affected by polarity of the reaction mixture, as shown in the case of attenuated aspartimide formation when anisole was used as a solvent.⁶⁸ A recent report highlights the use of pyrrolidine to deprotect Fmoc group in conjunction with greener solvents.^65,69 The poor AA solubility in anisole is countered by including a cosolvent such as DMSO. There remains a need for a continued analysis of alternate bases as well as greener solvent combinations.

Alternatively, in 2004 Zinieris et al. published a study investigating if the piperidine concentration could be reduced for Fmoc-removal.⁷⁰ No significant difference in SPPS performance was observed between 5%, 10%, and 20% piperidine in DMF. After completed Fmoc-removal large amounts of solvent are used to remove byproducts and excess piperidine prior to initiating the next AA coupling. Likely a reduction in piperidine concentration would impact the required solvent amount used for washing considerably and consequently reduce the overall PMI of SPPS. Furthermore, Ravetti Duran et al. have demonstrated that applying a flow washing protocol after Fmoc-removal (rather than batch washing) can reduce solvent consumption by more than 50%.⁷¹ A combination of reduced piperidine concentration and flow washing provides a simple and ready-to-implement solution for reducing the PMI of SPPS considerably and deserves heightened attention by the industry.

Coupling Agents and Recycling Reagents

Typically, with the automation of SPPS, stoichiometric or excess reagents are added and then washed away from the solid phase, leading to large amounts of waste being generated. One way to reduce this waste burden is through a recycling strategy. Pawlas and Rasmussen have developed the concept of ReGreen SPPS, which not only focuses on optimizing the stoichiometry of the reagents in the various transformations but also on efficient recycling and reuse of reagents/solvents thus fulfilling the criteria of a circular economy.⁷² To mitigate the issue regarding the use of undesirable solvents in SPPS, the authors utilized mixed solvent systems employing the common industrial solvent EtOAc as the bulk component. Initial results reported on an SPPS-protocol using DMSO/EtOAc (1:9) on PS/DVB resin,⁷³ which demonstrated improved chemoselectivity compared to conventional solvent systems. This use of EtOAc as a cosolvent and its easy recyclability on industrial scale underpinned the development of ReGreen SPPS. Most notably, recycling of the waste stream enabled 86% of EtOAc, 70% of DMSO, and 38% of Oxyma to be recovered and reused in a new peptide synthesis with minimum impact in compliance with regulatory requirements. Benchmarking through life cycle analysis demonstrated that although the cEF for the DMF and DMSO/EtOAc processes are comparable, implementation of the recycling into the latter (∼84% of the total waste) led to an ∼4-fold reduction in cEF with cutting of the solvent cost in-half per amino-acid cycle (note further improvements of the latter are envisioned through optimization of volumes used in the washing steps).

An alternative paradigm is presented with the so-called “tea-bag” strategy, in which the polystyrene-resin located in a small propylene-sealed packet constitutes a microreactor, and this is the entity that is added to the reagents (for the reaction) or the solvents (for the washing steps). This concept was first developed by Houghten⁷⁴ in applying parallelization to SPPS and though initially demonstrated for the Boc/benzyl protecting scheme for peptide synthesis has subsequently been extended to the more common Fmoc/t-butyl based protecting groups. The advantage in terms of reducing solvent/reagent amounts is that one reagent solution can be utilized for multiple simultaneous reactions (for example, 20% piperidine in DMF for Fmoc removal) or on a number of discrete occasions for coupling of a specific AA. In addition, wash solutions can be employed in a similar manner. To illustrate the power of this approach, Guzmán and co-workers reported on the synthesis of 52 peptides with a wide amino-acid composition ranging between 7 to 20 amino-acid residues.⁷⁵ Though no correlation could be established between yield and physicochemical properties, the process showed excellent performance with a reduction in both DMF usage of 25% and a deprotection reagent of 50%. Recycling of reagents was integrated into the procedure with no adverse effect on the quality of the products generated.⁷

Alternatively, Mainkar and co-workers have developed benzoisothiazolone (BIT) as a fast, efficient, and fully recyclable redox reagent that can be utilized for SPPS (Figure 15).⁷⁷ Initial proof of concept demonstrated that BIT-derived thioesters could be formed through triethyl phosphite (P(OEt)₃)-mediated condensation of BIT with Fmoc-protected amino acids and could be successfully condensed with a solid-supported free amine in the presence of a Cu catalyst to recycle the BIT. The protocol was subsequently simplified with in situ formation of the thioester for coupling, which was demonstrated and benchmarked favorably against conventional hexafluorophosphate azabenzotriazole tetramethyl uronium (HATU) and (EDC) 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimid based SPPS reactions with near stoichiometric quantities of the Fmoc-protected acid being utilized. Although 3 equiv of BIT were shown to be optimal, this reagent was recovered in 87% yield after a high-yielding gram-scale reaction. To highlight the potential synthetic utility of this approach, SPPS of the teixobactin side chain was demonstrated using BIT as a recyclable acid activator.⁷⁸

Figure 15.

BIT recyclable reagent for SPP.

Greener Tag-Assisted LPPS (Tag LPPS)

Tag-assisted liquid phase peptide synthesis (Tag-LPPS) has been further developed by research groups, as there were practical challenges that remained for effective scale up. Soluble supports have been expanded to include diphenylphosphonyloxyl diphenyl ketone (DDK) tags, silylated tags, based on Chiba et al.’s lipophilic tags, and phosphonate tags, where both the silylated and phosphonate tags have a higher loading capacity than previously developed LPPS supports (DDK tag;⁷⁹ TBP tag;⁸⁰ Silyl tag⁸¹) (Figure 16). Another advantage of the small molecule DDK and phosphonate tags is that the tag can be regenerated and recycled for reuse. These have been demonstrated on smaller scale but are potential alternatives to advance tag-assisted LPPS. The lipophilic tags developed by Chiba and co-workers have substantially progressed with application to macrocycles and a larger scale demonstration in the last couple of years. Previous accounts still encountered long cycle times due to the required chromatography between each coupling step. To address the lengthy processing time, Okada et al.²¹ utilized the hydrophobic benzylic alcohol (HBA) tags previously developed by their group, for larger scale peptide production. Icatibant acetate, a Bradykinin receptor antagonist, was targeted and synthesized on 100 g scale. The larger scale was achieved by developing a one-pot coupling, deprotection, and precipitation process. Quenching excess activated AA with propylamine allowed the subsequent deprotection step to proceed without the need for precipitation. As a result, the number of precipitations and acetonitrile usage were reduced by half, while still using stoichiometric equivalents of reagents (1.05–1.5 equiv) and achieving >93% crude purity of the tagged peptide. The final peptide was still purified by RP-HPLC, but the overall reduction in chromatographic purifications and solvent usage is a large improvement over the previous demonstrations. This larger scale synthesis is a step in the right direction toward implementing this technology for larger scale production.

Figure 16.

Soluble tags for LPPS. (A) Phosphonate tag where R = Cl, OH, and N-OH for DDK-Cl, DDK-OH, and DDK-NOH respectively. (B) Phosphonate tag where R = Cl, OH, and N-OH for TBP-Cl, TBP-OH, and TBP-NOH respectively and R¹ = H or Cl. (C) Silylated tag B2STag. (D) Silylated tag B6-STag.

Yamagami et al.⁸² have also applied the tag-assisted technology to the synthesis of macrocycles, a modality with great therapeutic potential. Three different tagging positions were explored to form the macrocycle on the soluble tag, where the tag was either on the C-terminal, N-terminal, or in the middle of the peptide. When the tag was positioned in the center of the peptide, the highest yield was achieved with no major byproducts and without detectable epimerization. The authors mentioned how the physical properties of the precipitate are important for a successful process and that the tagging position may contribute to those properties, where the tag positioned in the center provided precipitate with the best filtration properties. To effectively scale up this technology, it will be important to better understand how to control the physical properties of the macrocycle precipitate and what variables can be optimized.

With these recent advances, the implementation of tag-assisted LPPS is being realized. Bachem is working with Jitsubo to use their Molecular Hiving technology, which employs these lipophilic tag anchors, for larger scale peptide production (15–20 kg).⁸³ Continued application of this technology to more peptide targets of different sequences and modifications will be important to progress the method for broader applicability and implementation on the manufacturing scale.

Organic Nanofiltration/Membrane-Based Reactor Systems

Organic nanofiltration and membrane-enhance peptide synthesis (MEPS) have been gaining traction in the field as a way to purify growing peptide chains, remove chromatographic isolations, and reduce solvent waste.²² Yeo et al.⁸⁴ published work on PEPSTAR, which stands for peptide synthesis via one-pot nanostar sieving. The technology uses organic solvent nanofiltration to purify growing peptides during solution phase synthesis. MEPS has been previously demonstrated but not scaled up due to lower loading capacity and lack of a monodisperse anchor. In this most recent report, a monodisperse anchor or “nanostar” was developed with an aromatic core, making it detectable by LC and amenable for in-process monitoring. Additionally, 3 peptide attachment sites are available to increase the peptide loading and improve mass efficiency due to the lower molecular weight of the nanostar. To facilitate a one-pot AA coupling, the activated ester was quenched, similar to the tag-assisted strategy, with excess piperidine used in the synthesis. This method was demonstrated where the synthesis and purification stages use the same equipment, resulting in a small footprint. Synthesis of di-, octa-, and decapeptides was successfully executed with this automated system and demonstrated the potential for larger scale automation and production. The PMI was calculated and used as a benchmark to optimize other LPPS technologies. The reported PMI is still close to that of solid phase synthesis, but the authors anticipate that the PMI will decrease with inclusion of downstream operations. The cost of goods for the PEPSTAR technology was determined to be about half of that needed for traditional solid phase synthesis, making a compelling case for this technology. With the enhancements described, PEPSTAR technology is a promising new technology for liquid phase synthesis that can be automated and potentially scaled up for larger production with continued progress.

Improving Sustainability in the Long Term

The Fmoc-Protecting Group

The N-α Fmoc-protecting group and the side chain protecting groups all add to the poor atom economy of SPPS. As an example, the molecular weight of glycine is increased 4-fold after Fmoc-protection. If the Fmoc-protecting group could be replaced by a simpler lower molecular weight protecting group, the atom economy and overall PMI for SPPS could be improved significantly. While considerable research has been devoted to identifying alternative side chain protecting groups,⁸⁵ largely driven by a desire to suppress side reactions, very little attention has been devoted to identifying an alternative to the Fmoc-protecting group. Imine based protecting groups based on dimethylbarbituric acid (DMB) and diethylthiobarbituric acid (DETB)⁸⁶ are two notable exceptions (Figure 17). The DMB group in particular has shown promise as a replacement for the Fmoc-group; however, switching from Fmoc to DMB only results in ca. 25% reduction in molecular weight. Furthermore, hydrazine is employed for deprotection, which is an undesirable reagent from a process chemistry perspective and has the potential to cause side reactions resulting in impurities.

Figure 17.

DMB and DETB protected amino acids are synthesized from enamine precursors 1. Deprotection is achieved using hydrazine to give the amino acid/peptide product and a pyrazole byproduct.

If an economically low molecular weight alternative to the Fmoc-protecting group could be identified, then a shift away from Fmoc could be envisaged for new processes. However, it is important that a holistic approach is taken to ensure that the problem is not merely removed from the SPPS stage, but rather a negative environmental impact is achieved in the manufacture of the required AA. Also, one has to ensure that the byproducts resulting from the deprotection of the N-α do not introduce new environmental hazards. Provided that the environmental and financial incentive is clear, a shift to an alternative protecting group strategy could be envisaged in the mid- to long-term.

Peptide Bond Formation

Coupling reagents are highly efficient at forming peptide bonds while ensuring that racemization and other side reactions are suppressed. However, excess reagents are required to drive reactions to completion. If a catalytic peptide bond forming reaction was developed, this could reduce both the PMI and the use of toxic chemicals. Recombinant and enzymatic technologies already exist that synthesize peptides catalytically.⁸⁷ The major drawback of these platforms is that the current technology does not allow routine incorporation of unnatural AAs. The development of a recombinant technology that allows the incorporation of selected unnatural AAs could be envisaged. However, the real landmark achievement would be the development of a promiscuous system that would allow for the incorporation of any unnatural AA.

Chemical catalytic peptide bond formation is another avenue of research that could significantly impact SPPS efficiency. Considerable attention has been devoted to the area of catalytic amide bond formation during the past 20 years,⁸⁸ but to date no such system that can replace standard SPPS coupling reagents has been identified, and only few examples on a scale beyond the research laboratory have been reported.⁸⁹

Arora and co-workers have reported progress toward this goal with the rational design of a biomimetic macrocyclic diselenide-based organocatalyst. The catalyst operates through activation of the carboxylic acid component in the coupling as a selenoester that undergoes the subsequent desired amidation reaction (Figure 18).⁹⁰ Critical to the success of the reaction is the addition of a phosphine to initiate the sequence through reduction of the diselenide and formation of a selenophosphonium derivative to react with the carboxylic acid as well as addition of a dehydrating reagent. However, both of these present barriers to sustainable implementation of this methodology as the optimal phosphine P(Bu)₃ are prone to oxidation, while separation of dehydrating reagents such as molecular sieves can prove challenging in solid-phase based protocols. The amidation reaction is successfully demonstrated for Fmoc AA derivatives using either DMF or ACN as the solvent and then impressively applied to the solid-phase synthesis of a pentapeptide. Removal of the sieves was achieved through the use of buoyant force with two coupling cycles required for extension of the chain beyond the third amino-acid residue. Purity of the final pentapeptide benchmarks well with that obtained using more conventional SPPS-based coupling approaches, while the hydrogen-bonded-based catalysis appears to be unaffected by the presence of multiple amide bonds in the molecule.

Figure 18.

Diselenide-based organocatalyzed amide bonds, where R = desired AA side chain and X = AA residue.

Most catalytic amide bond forming methodologies exhibit a poor substrate scope. There have been some reports on more general methodologies for the synthesis of amides⁹¹ and peptides,⁹² but unfortunately catalysis has been achieved at the expense of sustainability and does not offer a better alternative to current stoichiometric methods. Chiba and co-workers have recently reported an interesting approach using electrochemical peptide synthesis in a biphasic system.⁹³ Anodic oxidation of triphenylphosphine provides the coupling reagent in the form of the triphenylphosphine radical cation, producing triphenylphosphine oxide as a stoichiometric byproduct after coupling. The methodology is still in its infancy, and there are numerous challenges to overcome to make it of interest in an industrial setting, but it represents a step in the right direction by utilizing a simple, inexpensive, safe, and general coupling reagent (Figure 19).

Figure 19.

Electrochemical peptide bond formation using triphenylphosphine, where PG = protecting group.

To move this field forward, scientists must think about sustainability, simplicity, and robustness and demonstrate that the methodology is applicable on a multigram scale.

Mechanochemistry for peptide bond formation is another area with potential sustainability benefits, by dramatic solvent use reduction in the manufacture of small peptides.⁹⁴

The key next steps in the evolution of this field will be continued refinement of the approach, including description of scope and limitations and demonstration of this approach on scale-up.^95,96

A number of significant developments for peptide synthesis in water enabled by surfactants has positioned water to serve as a potential bulk medium in an iterative process and a real long-term alternative to reprotoxic polar aprotic solvents. Lipshutz for example demonstrated a simple two-stage unmasking-coupling process that could be rendered amenable to peptide synthesis. Cbz can be smoothly removed from an AA with minimal amount of palladium on charcoal as a catalyst and hydrogen gas as the reductant, and subsequently engaged into the required amide coupling step with very high efficiency, all in water with TPGS-750-M surfactant (Figure 18, see example 1 below).⁹⁷ The promises come from the encouraging preparation of a decapeptide in either a 5 + 5 or 8 + 2 strategy in 72% and 82% yield, respectively. Considering the plethora of options reported by both academia and from industry,⁹⁸ one could predict that this opportunity will soon be exploited, whether using this surfactant or others now in the literature.⁹⁹ A significant impact on kinetics is observed when using the surfactants such as PS-750-M enabling rapid reactions in the order of 10 to 30 min to reach completion (Figure 20, Example 2).¹⁰⁰

Figure 20.

Peptide synthesis in water using a surfactant.

Even more promising in terms of reaction acceleration has been the hydrophobic polymer effect reported by Braje at Abbvie (Figure 21).¹⁰⁰ A remarkable kinetic enhancement was observed when using HPMC (hydroxypropyl methylcellulose), which, by virtue of the hydrophobic effect, promoted amide bond formation within seconds with high selectivity and chemical compatibility. Preliminary data regarding epimerization were also promising.

Figure 21.

Amide bond formation in water with addition of HPMC.

In all these cases, a significant impact on the environmental footprint and even total carbon release can be envisioned once technical challenges associated with the contamination of the wastewater have been properly addressed.¹⁰¹

Conclusion

The ACS GCIPR has identified PMI as the key mass-related green chemistry metric, which is a key indicator of sustainability for the manufacturing process of APIs. Although LCA and AE are also alternative metrics in measuring the efficiency and thus greenness of the process, LCA remains a challenge in early phase development, and AE excludes other significant resource requirements such as solvent and other raw material inputs. Notwithstanding the noted limitation that PMI does not account for the environmental impact of starting materials (protected amino acid monomers), PMI still emerges as a preferred metric in this assessment.

The current manufacturing processes for peptide APIs consist of synthesis, purification, and isolation stages, with each stage contributing to PMI. Data presented and discussed in this perspective by 14 companies with peptide therapeutics in their portfolio have underscored the persistent issue of high PMI for the manufacturing of peptides as therapeutics in comparison to that of small molecules. Solvent usage during synthesis and purification contributes significantly to the PMI. From our findings, PMI values for synthesis and purification range from 30 to 70% of the overall PMI across development phases (Figure 10). Additionally, SPPS and LPPS current strategies often employed in peptide synthesis have average PMI values trending more favorably for LPPS (Figure 9), although SPPS is the most widely used synthetic process for peptide manufacturing. The rather high average PMI value of 13,603 for commercial and Phase 3 processes indicates the need for investigating and advancing synthetic methodology and technologies to improve peptide therapeutic PMI across the production phases.¹⁰²

Glossary

Abbreviations

ACN (MeCN)

methyl cyanide; acetonitrile

CPME

cyclopentyl methyl ether

DCM

dichloromethane

DEC

diethyl carbonate

DEE

diethyl ether

DIPE

diisopropylether

DMAC

dimethylacetamide

DOL

dioxolane

EtOAc

ethyl acetate

GVL

gamma-valerolactone

HPMC

hydroxypropyl methylcellulose

MeTHF

2-methyltetrahydrofuran

MCSGP

multicolumn counter current solvent gradient purification

NBP

N-butylpyrrolidinone

NFM

N-formylmorpholine

NMP

N-methylpyrrolidine

PC

propylene carbonate

SFC

supercritical fluid chromatography

THF

tetrahydrofuran

Biographies

Ivy A Kekessie obtained her PhD in Organic Chemistry from the University of California, Davis, CA under the supervision of Professor Gervay-Hague with research focused on the design and synthesis of HIV-1 entry inhibitors. Her postdoctoral studies with Professor Kit Lam at the University of California Davis Medical Center, Sacramento, CA, involved design, synthesis, scale up, and formulation of nanomicelles as delivery vehicles for chemotherapeutics targeting various cancer cells. Ivy joined Genentech (A Member of the Roche Group) in South San Francisco, CA in 2015 where she is currently a Scientist in the Peptide Therapeutics department working on peptide-derived therapeutics.

Katarzyna Wegner obtained her PhD. in Organic Chemistry (2007), Faculty of Chemistry, University of Gdańsk under the supervision of Professor Zbigniew Grzonka. In 2008, she joined IPSEN, Ireland where she is currently the Director of Active Pharmaceutical Ingredient Development group responsible for improvement and innovation of novel peptide and small molecule process manufacturing.

Isamir Martínez, Ph.D., is currently the Scientific Alliances and Business Engagement Sr. Portfolio Manager at the ACS Green Chemistry Institute/Office of Sustainability. Isamir is leading efforts to strategically accelerate and enable the implementation of green and sustainable chemistry and engineering throughout the global chemical enterprise. In her role, she leads global industrial collaborations, engages stakeholders, and other strategic collaborative programs that contribute to sustainability. She has a wide array of extensive scientific background in organic, medicinal/process/pharmaceutical chemistry, biocatalysis, and chemical sourcing. Prior to joining ACS, she worked in the pharmaceutical industry for over 12 years and taught chemistry at higher education institutions. Isamir’s passion is to develop green and sustainable chemistry methodologies that could be used to deliver efficient chemical processes and therefore contribute to a more sustainable future.

Michael Kopach earned his Ph.D. in Organic Chemistry from the University of Virginia in 1995. Following postdoctoral studies with Prof. A. I. Meyers at Colorado State University in natural product synthesis, he joined Roche in 1997. At Roche, he contributed to the Development group’s efforts in commercializing AIDS drugs such as Viracept and Fuzeon, the latter being the first synthetic peptide to reach commercialization on a large scale. In 2001, Dr. Kopach became a part of Eli Lilly and Company’s Synthetic Molecule Process Chemistry Division. Over the past decade, he has led Eli Lilly and Company’s initiatives in synthetic peptide commercialization and sustainability. Notably, he was instrumental in the route design and commercial implementation of Mounjaro, recognized as the first GLP-1 and GIP hormone dual agonist.

Tim is a Senior Director at Eli Lilly and Company with responsibilities for peptide and oligonucleotide process development activities. He has 25 years of experience as a process chemist with 9 years at Pfizer prior to joining Eli Lilly. Tim has worked across small molecules, antibody drug conjugates, peptides, and oligos and has a long-standing interest in improving sustainability and greening of chemical processes with large scale manufacturability in mind.

Janine Tom obtained her Ph.D. in Chemistry from UC Irvine in the lab of Professor Aaron Esser-Kahn, where she developed synthetic chemical tools to probe the innate immune response for vaccine development. She started at Amgen in 2017 and is currently a Process Development Senior Principal Scientist in the Drug Substance Technologies Synthetics group. At Amgen, she worked on several synthetic and hybrid modality programs, including traditional small molecule, antibody-drug conjugate, and siRNA modalities.

Martin Kenworthy studied Chemistry at University of Manchester (UK) and obtained his Ph.D. in 2003 with Prof. Jonathan Clayden. After postdoctoral studies with Prof. Richard Taylor at York University (UK), he joined AstraZeneca in 2006 (Macclesfield, UK), where he works in Chemical Development. Over the past decade, he has been responsible for AZ drug substance development for late clinical stage and commercial peptide projects and products.

Fabrice is a Distinguished Scientist at Novartis, Switzerland, responsible for scientific activities in Technical Research & Development. His research interests include the research and development of sustainable synthetic methodologies intended for large scale implementation.

John is a senior expert in science and technology at Novartis. His main responsibility is process development for the manufacture of peptides in large scale, and additionally he is very interested in sustainability and innovation towards data science applied to organic chemistry.

Stefan G. Koenig obtained his PhD in Organic Chemistry from Yale University in New Haven, CT (USA), under the supervision of Professor David J. Austin and then conducted postdoctoral work at the ETH in Zurich, Switzerland, in the laboratory of Professor Andrea T. Vasella. Stefan initiated his professional career at Sepracor in Marlboro, MA, and joined Genentech (A Member of the Roche Group) in South San Francisco, CA in 2010. He is currently a Distinguished Scientist in the External Science & Manufacturing group within Synthetic Molecule Pharmaceutical Sciences, leading various projects at partner companies.

Philippa (Pippa) Payne is an Associate Director in Global external manufacturing at Gilead Sciences. She completed her Ph.D. at the University of British Columbia, followed by postdoctoral studies at the University of North Carolina – Chapel Hill. At Gilead Sciences, she has worked on late-stage and commercial small molecule and peptide programs, advancing the application of green chemistry principles in process development and manufacturing. She was an elected cochair of the ACS Green Chemistry Institute Pharmaceutical Roundtable from 2021–2023.

Stefan Eissler studied biochemistry at Bielefeld University and completed these studies with a PhD thesis in organic chemistry under the supervision of Norbert Sewald. After a postdoc position at the laboratory for process research at the university of Zürich under the supervision of Thomas Bader, he joined Bachem AG in 2010 as a team leader. After various positions as team leader and director responsible for liquid-phase and solid-phase peptide synthesis as well as peptide purification, he currently holds the position of the Vice President Peptide Manufacturing Upstream.

Arumugam Balasubramanian (Bala) earned a Ph.D. in Chemistry from National Tsinghua University Taiwan, currently working as Director of research at Asymchem Tianjin for the process development and manufacturing of oligonucleotides, peptides, and small molecules for early to late phase molecules. He has experience in peptides and oligonucleotides process development and manufacturing with expertise in synthesis, purification, and isolation.

Changfeng Li obtained a Ph.D. in Organic Chemistry from Central China Normal University at Wuhan of China in 2009. He is currently working as a Senior Director of peptide team at CMMD, Asymchem. He is responsible for the CDMO business for SPPS and polypeptide laboratory management. Responsibilities also include customer management, process development, and production project management of early phase projects.

Subha Mukherjee earned his PhD in Chemistry from the University of Illinois at Urbana–Champaign in 2015. He worked under Professor Wilfred A. van der Donk’s supervision in peptide synthesis, bio-orthogonal chemistry, and chemical and enzymatic synthesis to investigate lanthipeptide and phosphonate natural products. In 2015, he joined the process chemistry department at Bristol Myers Squibb Company. Over the years at BMS, he has transitioned from enabling scalable process development to leading chemistry teams in the peptide, small molecule, and ADC portfolio as a modality-agnostic process chemist.

Albert Isidro-Llobet obtained his PhD in Organic Chemistry (2008) at the Institute for Research in Biomedicine, Barcelona Science Park, University of Barcelona under the supervision of Professors Fernando Albericio and Mercedes Alvarez. His PhD work involved the development of methodologies for peptide synthesis. In 2008, he received a Marie Curie Fellowship and joined the group of Prof. David Spring at the University of Cambridge where he worked on the diversity-oriented synthesis of macrocyclic peptidomimetics. He joined GSK, Stevenage, UK in 2012 as a peptide synthesis expert. He currently works at the Chemical Biology department in GSK where his role involves leading peptide, target identification, and data analysis projects.

Olivier Ludemann-Hombourger is the Global Director Innovation & Technology at PolyPeptide. He received his PhD in 2001 from the “Institut National PolyTechnique de Lorraine” in France, in collaboration with Novasep Process, for which he was leading the R&D activity over 10 years. He worked during three years for Lonza in Braine l’Alleud (Belgium) as head of Operations, before joining PolyPeptide as General Manager of the French entity for 7 years, until taking his current global position in 2019.

Paul Richardson earned his Ph.D. in Chemistry from the University of Sheffield (Synthetic Methodology/Total Synthesis, Professor Istvan Marko) and did postdoctoral studies at the University of Exeter (Biocatalysis, Professor Stan Roberts) and The Scripps Research Institute (Asymmetric Catalysis, Professor Barry Sharpless). He is currently the Director of Analytical and Synthesis Methodologies within Oncology Medicinal Chemistry at Pfizer in La Jolla.

Jörg Kittelmann is a Principal Scientist at Novo Nordisk specializing in purification process development for peptides and proteins. He has over 8 years of experience at the company and is very passionate about process sustainability and process analytical technologies. Jörg holds a PhD in Biotechnology from Delft University of Technology.

Daniel Sejer Pedersen obtained his PhD degree in synthetic organic chemistry from Cambridge University in 2005 and has held a variety of positions at universities and in industry. He is currently a Specialist in CMC Chemical Development at Novo Nordisk where he has been employed since 2018 working on synthetic phase 3 projects and the assessment and development of new manufacturing technologies.

Dr. Leendert van den Bos obtained his MSc and PhD degree in bio-organic chemistry from Leiden University. He joined Organon BioSciences as a Scientist in 2007 and worked subsequently for Schering-Plough, Merck Sharp & Dohme and Aspen Pharmacare mostly in the field of Process Research & Development. Leendert holds an Executive MBA from Nyenrode University. His research interests include process development, enzyme engineering, and continuous flow manufacturing.

Data Availability Statement

The data underlying this study are available in the published article.

Author Contributions

The manuscript was written through the contributions of all authors. All authors have given approval to the final version of the manuscript.

The authors declare the following competing financial interest(s): K.W. is an employee of IPSEN and owns stock or stock options. M.N.K. is an employee of AstraZeneca and owns stock or stock options. S.M. is an employee of Bristol Myers Squibb and owns stock or stock options. J.L. and F.G. are employees of Novartis and own stock or stock options. L.J.vdB. owns stock or stock options in EnzyTag. O.L.H. is an employee of PolyPeptide Laboratories. B.A. and C.F. Li are an employee of Asymchem Life Science own stock or stock options. A.I-L. is an employee of GSK and owns stock or stock options. I.A.K. and S.G.K. are employees of Genentech, a Member of the Roche Group, and own stock or stock options. M.E.K. is an employee of Eli Lilly and Company. S.E. is an employee of Bachem and owns stock or stock options. P.R. is an employee of Pfizer Inc. and owns stock or stock options. D.S.P. is an employee of Novo Nordisk and owns stock. J.K. is an employee of Novo Nordisk and owns stock. All other authors declare no competing financial interest.