Around the world, pharmaceutical companies and their industrial and academic partners stand firm in their commitment to the application of green chemistry principles, tools, and metrics. However, new challenges continue to emerge as the industry seeks to compress development timelines and extend portfolios to newer and more complex modalities. In this work, we share perspectives from across the industry on establishing science-based targets and building laboratory culture that empowers sustainability-forward process development decisions. Effective direction of development decisions requires selecting appropriate metrics for the stage and scope of the process, and we explore solutions and current challenges to addressing this problem, including the extension of sustainability concepts beyond small molecules and into therapeutic peptides, oligonucleotides, and biologics.

Abstract Solid-phase peptide synthesis (SPPS) at commercial scale depends on three substances now subject to tightening regulatory pressure: dimethylformamide (DMF), classified as a reprotoxic substance of very high concern under EU REACH; dichloromethane (DCM), broadly prohibited for industrial use in the United States under the 2024 EPA TSCA rule; and piperidine, a DEA Schedule I precursor requiring licensed handling and disposal at manufacturing scale. We report a 50 mmol, DMF-free synthesis of the linear precursor of sunflower trypsin inhibitor-1 (SFTI-1) that simultaneously eliminates all three substances from a pharmaceutically relevant Fmoc/tBu SPPS process without compromising yield, purity, or stereochemical integrity. DCM-free resin loading and swelling were achieved using 1:1 EtOAc/ACN; coupling and Fmoc deprotection were conducted in 1:1 NBP/EtOAc throughout, with 2% DBU replacing 20% piperidine — reducing deprotection base consumption by 91%. Operating at 1.5 equivalents of amino acid and coupling reagents vs the conventional 3.0 equivalents delivered a 50% reduction in stoichiometric inputs. The principal synthetic challenge, arginine coupling, was addressed through high-throughput experimentation identifying 2:3 DMSO/1,3-dioxolane at 40 °C as a greener solvent system achieving 97% conversion. Head-to-head comparison against a conventional DMF benchmark confirmed equivalent potency-corrected peptide output, comparable crude purity, and no increase in epimerization across all nine chiral residues. Collectively, these improvements delivered an estimated 9% reduction in total process mass intensity (PMI) relative to the DMF process (847 vs 934 kg/kg crude peptide), with the dominant gains concentrated in deprotection base and reagent stoichiometry. Normalized per amino acid to enable comparison with reported industry benchmarks, both the conventional (66.7 kg/kg per amino acid) and green (60.5 kg/kg per amino acid) variants of this process already operate roughly an order of magnitude below the average PMI reported for commercial-scale SPPS in a recent multicompany benchmarking analysis (874.5 kg/kg per amino acid) (J. Org. Chem. 2024, 89, 4261−4282). These results provide a compliance-ready, industrially validated framework for commercial SPPS under current and forthcoming regulatory constraints.

The development of scalable and efficient manufacturing of high-volume complex synthetic peptides and proteins, like tirzepatide (TZP, 1), faces major hurdles due to the limitations of traditional Solid Phase Peptide Synthesis (SPPS) and Liquid Phase Peptide Synthesis (LPPS). To enable the commercial synthesis of tirzepatide, we pioneered an innovative four-fragment convergent hybrid SPPS/LPPS strategy combining their individual strengths. Integrating advanced techniques, like flow chemistry for fragment condensation and nanofiltration for intermediate purification, ensures high efficiency and scalability, setting a new standard for large-scale production. Given the broader reliance on inefficient linear SPPS in the field, our work underscores the potential of hybrid synthesis strategies to transform peptide manufacturing. The convergent approach allows for the simultaneous synthesis of high purity peptide fragments, significantly reducing overall manufacturing time and increasing API throughput. Concurrently, we established a two-fragment route leveraging Native Chemical Ligation (NCL) followed by tandem desulfurization. This method achieves the chemoselective coupling of unprotected peptide fragments in aqueous media, without epimerization. Crucially, we employed tangential flow filtration (TFF) to effectively purify intermediates between ligation and desulfurization, circumventing solvent-intensive steps. This NCL/TFF combination offers a powerful, orthogonal, and greener path to TZP, representing a meaningful innovation in synthetic peptide process chemistry.

Herein we describe a green-by-design approach to route selection and development, assisted by predictive analytics and historical data. In order to aid the selection of more efficient strategies, we created a user-friendly web application, the “PMI Prediction Calculator,” to foretell the probable efficiencies of proposed synthetic routes, prior to their evaluation in the laboratory. This tool can also be used to benchmark the outcome performance of a developed process. We expect that use of this app will bring greater awareness of sustainability during the ideation phase of route design and will contribute to a reduced environmental impact of pharmaceutical production. The app can be accessed following the link: https://acsgcipr-predictpmi.shinyapps.io/pmi_calculator/

Traditional solid-phase peptide synthesis (SPPS) is limited in its suitability for high-volume peptide/protein manufacturing applications due to its reliance on specialized equipment, high process mass intensity (PMI), and the use of environmentally concerning reagents. To address these limitations, a fully liquid-phase peptide synthesis (LPPS) has been developed and applied to the synthesis of a tirzepatide (30-39) fragment intermediate. This method leverages Fmoc- and Cbz-protecting group strategies to generate two high-purity crystalline pentamers entirely without solid-phase techniques. Subsequent solution-phase assembly of the pentamers yields the desired decapeptide. This green, efficient, and practical route offers a more economical and environmentally favorable alternative to SPPS, delivering a fragment intermediate of superior quality. Additionally, tirzepatide (30-39) decapeptide is a potential common intermediate for numerous other incretin peptides using hybrid SPPS/LPPS methodologies.

The peptide coupling reaction is one of the most critical steps in the solid phase synthesis of therapeutic peptides/proteins. Improper reaction conditions can result in several common impurities such as single amino acid deletions, additions, N-terminus modifications, and D-isomers, all while potentially impacting the active pharmaceutical ingredient critical quality attributes. In this work, we developed a first-principle mechanistic reaction kinetics model for the solid-phase peptide/protein coupling reaction based on well-established reaction mechanisms and experimental data from literature. Utilizing the reaction kinetics model, we present a systematic, quality by design approach for the coupling reaction control strategy. Critical process parameters are identified via univariate analysis and the design space is designated via multivariate risk assessment. The presented approach provides a novel solution for designing solid-phase peptide/protein synthesis control strategies and identifying normal operating ranges for each process parameter, as well as the associated design space.

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.

Phosphines and phosphites are critical tools for non-metal desulfurative methodologies due to the strength of the P=S bond. An overarching premise in these methods has been that stoichiometric (or excess) P(III) reagent is required for reactivity. Despite decades of research, a desulfurative process that is catalytic in phosphine/phosphite has not been reported. Here, we report the successful merging of two thermal radical processes: the desulfurization of unactivated and activated alkyl thiols and the reduction of P(V) = S to P(III) by reaction with a silyl radical species. We employ catalytic trimethyl phosphite, catalytic azobis(cyclohexyl)nitrile, and two equivalents of tris(trimethylsilyl)silane as the stoichiometric reductant and sulfur atom scavenger. This method is tolerant of common organic functional groups and affords products in good to excellent yields.

A ligation protocol has been developed in which a peptide containing an N-terminal serine is linked via its side-chain alcohol to the C-terminal carboxylate of a second peptide by Mitsunobu esterification. N-Deprotection of the serine and subsequent exposure to a weak amine base triggers O-to-N acyl transfer, delivering the desired ligation product. A unique aspect of this strategy is the use of the C-terminal carboxylate as a nucleophile rather than as an electrophile. As a result, the ligation occurs without evidence of epimerization. A broad scope of C-terminal nucleophiles is tolerated, including proline and hindered beta-branched residues.

Flow chemistry was initially used for speed to early-phase material delivery in the development labo-ratories, scaling-up chemical transformations that we would not or could not scale up batch for safety reasons. Some early examples included a Newman Kwart rearrangement, Claisen rearrangement, hydroformylation, and thermal imidazole cyclization. Next, flow chemistry was used to enable safe scale-up of hazardous chemistries to manufacturing plants. Examples included high-pressure hydrogenation, aerobic oxidation, and Grignard for-mation reactions. More recently, flow chemistry was used in Small Volume Continuous (SVC) processes, where highly potent oncolytic molecules were produced by fully continuous processes at about 10 kg/day including reaction, extraction, distillation, and crystallization, using disposable equipment contained in fume hoods.

Peptides are steadily gaining importance as pharmaceutical targets, and efficient, green methods for their preparation are critically needed. A key deficiency in the synthetic toolbox is the lack of an industrially viable peptide desulfurization method. Without this tool, the powerful native chemical ligation reaction typically used to assemble polypeptides and proteins remains out of reach for industrial preparation of drug targets. Current desulfurization methods require very large excesses of phosphine reagents and thiol additives or low-abundance metal catalysts. Here, we report a phosphine-only photodesulfurization (POP) using near-UV light that is clean, high-yielding, and requires as little as 1.2 equiv phosphine. The user-friendly reaction gives complete control to the chemist, allowing solvent and reagent selection based on starting material and phosphine solubility. It can be conducted in a range of solvents, including water or buffers, on protected or unprotected peptides, in low or high dilution and on gram scale. Oxidation-prone amino acids, π-bonds, aromatic rings, thio-aminal linkages, thioesters, and glycans are all stable to the POP reaction. We highlight the utility of this approach for desulfurization of industrially relevant targets including cyclic peptides and glucagon-like peptide 1 (GLP-1(7-36)). The method is also compatible with NCL buffer, and we highlight the robustness of the approach through the one-pot disulfide reduction/multidesulfurization of linaclotide, aprotinin, and wheat protein.

This review article highlights the need for the development of synthetic peptide and oligonucleotide manufacturing processes with improved sustainability credentials. These therapeutic classes are fast growing and have the potential to deliver multiple high volume products over the next decade. While there have been significant technical innovations in the past few years there is limited evidence for adoption of these new technologies in manufacturing, which still largely rely on traditional approaches that generate large quantities of waste and use many chemicals of concern. Examples of strategies that could improve the environmental footprint for peptides include Tag-Assisted Liquid-Phase Peptide Synthesis, Hybrid Solid/Liquid Phase Peptide Synthesis and the implementation of continuous processes. For oligonucleotides, convergent liquid-phase synthesis and enzyme-catalyzed synthesis strategies are highlighted which have potential for significant positive environmental improvement if incorporated into the development lifecycle. To guide development, the collection of baseline sustainability metrics such as Process Mass Intensity is recommended.

This study focused on investigating diketopiperazine (DKP) and the formation of associated double-amino-acid deletion impurities during linear solid-phase peptide synthesis (SPPS) of tirzepatide (TZP). We identified that the DKP formation primarily occurred during the Fmoc-deprotection reaction and post-coupling aging of the unstable Fmoc-Pro-Pro-Ser-resin active pharmaceutical ingredient (API) intermediate. Similar phenomena have also been observed for other TZP active pharmaceutical ingredient (API) intermediates that contain a penultimate proline amino acid, such as Fmoc-Ala-Pro-Pro-Pro-Ser-resin, Fmoc-Pro-Pro-Pro-Ser-resin, and Fmoc-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-resin, which are intermediates for both hybrid and linear synthesis approaches. During post-coupling aging, it is found that Fmoc deprotection can proceed in dimethylformamide (DMF), dimethyl sulfoxide (DMSO), N-methyl-2-pyrrolidone (NMP), and acetonitrile (ACN) solvents without any piperidine addition. Density functional theory (DFT) calculations showed that a peptide that has a penultimate proline stabilizes the transition state through the C-H···π interaction during Fmoc decomposition, which causes those peptides to be more prone to cascade-deprotection reactions. Pseudo-reaction pathways are then proposed, and a corresponding macrokinetics model is developed to allow accurate prediction of the TZP peptide intermediate self-deprotection and DKP formation rate. Based on those studies, control strategies for minimizing DKP formation were further investigated and an alternative to Fmoc protection was identified (Bsmoc-protected amino acids), which eliminated the formation of the DKP byproducts. In addition, the use of oxyma additives and lower storage temperature was demonstrated to markedly improve the peptide intermediate stability to DKP degradation pathways.

The large and steadily growing demand for medicines combined with their inherent resource-intensive manufacturingnecessitates a relentless push for their sustainable production.Pharmaceutical companies are constantly seeking to perform reliablelife cycle assessments of their medicinal products and assess the truevalue of their sustainable development achievements; however, theyfind themselves impeded by the lack of a universal metric systemthat allows for objective quantification of the underlying coredenominators. Guided by the unambivalent purpose of the UnitedNations Sustainable Development Goal 12, which aims atsubstantially reducing production waste by 2030, and driven by avision to catalyze greener active pharmaceutical ingredient (API)manufacturing around the globe, the authors set out to overcomecurrent obstacles by defining an improved model for the metric named innovation green aspiration level, iGAL 2.0. We propose yieldand convergence as new key sustainability indicators and include a new formula for convergence with potential applicability incomputer assisted synthesis planning (CASP) algorithms. The improved statistical model of iGAL 2.0 represents a valuableextension to the common API process waste metrics, process mass intensity (PMI) and complete E factor (cEF), by putting thosemeasures into perspective: iGAL 2.0 enables determination of relative process greenness (RPG) to identify potentiallyunderperforming and environmentally concerning processes early and thereby deliver environmental value. At the same time, iGAL2.0 generates economic value since reduced waste correlates to lower API production costs. The metric is complemented by itsscorecard companion to highlight the impact of innovation on reductions of API manufacturing waste, enabling scientists to readilycommunicate the value of their work to their peers, managers, and the general public. We believe that iGAL 2.0 can readily beadopted by pharmaceuticalfirms around the globe and thereby empower and inspire their scientists to make meaningful and significant contributions to global sustainability

Herein, a one-pot liquid phase peptide synthesis featuring iterative addition of amino acids to a “nanostar” support, with organic solvent nanofiltration (OSN) for isolation of the growing peptide after each synthesis cycle is reported. A cycle consists of coupling, Fmoc removal, then sieving out of the reaction by-products via nanofiltration in a reactor-separator, or synthesizer apparatus where no phase or material transfers are required between cycles. The three-armed and monodisperse nanostar facilitates both efficient nanofiltration and real-time reaction monitoring of each process cycle. This enabled the synthesis of peptides more efficiently while retaining the full benefits of liquid phase synthesis. PEPSTAR was validated initially with the synthesis of enkephalin-like model penta- and decapeptides, then octreotate amide and finally octreotate. The crude purities compared favorably to vendor produced samples from solid phase synthesis.

The large-scale manufacture of complex synthetic peptides is challenging due to many factors such as manufacturing risk (including failed product specifications) as well as processes that are often low in both yield and overall purity. To overcome these liabilities, a hybrid solid-phase peptide synthesis/liquid-phase peptide synthesis (SPPS/LPPS) approach was developed for the synthesis of tirzepatide. Continuous manufacturing and real-time analytical monitoring ensured the production of high-quality material, while nanofiltration provided intermediate purification without difficult precipitations. Implementation of the strategy worked very well, resulting in a robust process with high yields and purity.

A variety of high-pressure and low-pressure plug flow reactors (PFRs) are described in this chapter with manufacturing examples for each. Coiled tube PFRs and vertical pipes-in-series PFRs were used for two-phase gas–liquid reactions. A pulsating flow coiled tube PFR was used for gas–liquid reaction with solids precipitate. Superheated PFRs were used for reactions involving homogeneous solutions, heated above the boiling point of the solvent. Disposable coiled tube PFRs were used with highly potent compounds. Continuous stirred tank reactors (CSTRs) were needed for heterogeneous continuous processes. CSTRs, CSTRs-in-series, and intermittent flow CSTRs were used for reactions with long reaction times, positive order kinetics, and multiple reaction phases, either solid/liquid or liquid/liquid. This chapter also explains how to calculate the actual internal temperature profile along the length of a PFR, which is often not practical to measure.

With a renewed and growing interest in therapeutic oligonucleotides across the pharmaceutical industry, pressure is increasing on drug developers to take more seriously the sustainability ramifications of this modality. With 12 oligonucleotide drugs reaching the market to date and hundreds more in clinical trials and preclinical development, the current state of the art in oligonucleotide production poses a waste and cost burden to manufacturers. Legacy technologies make use of large volumes of hazardous reagents and solvents, as well as energy-intensive processes in synthesis, purification, and isolation. In 2016, the American Chemical Society (ACS) Green Chemistry Institute Pharmaceutical Roundtable (GCIPR) identified the development of greener processes for oligonucleotide Active Pharmaceutical Ingredients (APIs) as a critical unmet need. As a result, the Roundtable formed a focus team with the remit of identifying green chemistry and engineering improvements that would make oligonucleotide production more sustainable. In this Perspective, we summarize the present challenges in oligonucleotide synthesis, purification, and isolation; highlight potential solutions; and encourage synergies between academia; contract research, development and manufacturing organizations; and the pharmaceutical industry. A critical part of our assessment includes Process Mass Intensity (PMI) data from multiple companies to provide preliminary baseline metrics for current oligonucleotide manufacturing processes.