Vincent Martin, Peter H. G. Egelund, Henrik Johansson, Sebastian Thordal Le Quement, Felix Wojcik and Daniel Sejer Pedersen Solid-phase peptide synthesis (SPPS) is generally the method of choice for the chemical synthesis of peptides, allowing routine synthesis of virtually any type of peptide sequence, including complex or cyclic peptide products. Importantly, SPPS can be automated and is scalable, which has led to its widespread adoption in the pharmaceutical industry, and a variety of marketed peptide-based drugs are now manufactured using this approach. However, SPPS-based synthetic strategies suffer from a negative environmental footprint mainly due to extensive solvent use. Moreover, most of the solvents used in peptide chemistry are classified as problematic by environmental agencies around the world and will soon need to be replaced, which in recent years has spurred a movement in academia and industry to make peptide synthesis greener. These efforts have been centred around solvent substitution, recycling and reduction, as well as exploring alternative synthetic methods. In this review, we focus on methods pertaining to solvent substitution and reduction with large-scale industrial production in mind, and further outline emerging technologies for peptide synthesis. Specifically, the technical requirements for large-scale manufacturing of peptide therapeutics are addressed.
The first synthetic peptide therapeutic oxytocin was introduced in 1962, and as of 2017 over 60 peptide drugs have been approved in the US, Europe and Japan, more than 150 drugs are currently in active clinical development, and >260 have been tested in human clinical trials. 1 An often vented concern about peptide therapeutics is their poor oral bioavailability, which seriously hampers oral administration. This drawback is usually circumvented by alternative routes of administration, such as subcutaneous injection or inhalation, 2 but advances in peptide formulation such as using permeation enhancers for increased oral absorption will undoubtedly accelerate the growth of this important class of therapeutic molecules. 3,4
Tellingly, the market for peptide therapeutics is currently valued at USD 23 billion, but is anticipated to increase to USD 57 billion by 2027. 5 Peptide and protein active pharmaceutical ingredients (APIs) can be prepared by either chemical or biological routes, where chemical synthesis is the current standard for preparation of peptides (especially those bearing unnatural amino acids or particular functional groups), while biological
Vincent Martin obtained his MSc and PhD degrees in organic chemistry from the University of Montpellier (France). After his PhD, he joined the laboratory of Prof. Krishnamurthy at the Scripps Research Institute as a Research Associate, working on origin of life and oligonucleotides chemistry from 2015 to 2017. Since 2018, he is a post-doctoral fellow at Novo Nordisk, focusing on the development of new methods for green solid-phase synthesis of peptides.
Peter H. G. Egelund obtained his BSc and MSc degrees in chemistry from the University of Southern Denmark (Denmark). He has performed several projects in organic synthesis, including potential antibiotics and anti-cancer medicines. Since 2020 he is a Development Scientist, focusing on finding green alternatives for DMF in solid-phase peptide synthesis. Novo Nordisk A/S, CMC API Development, Smørmosevej 17-19, DK-2880 Bagsværd, Denmark. E-mail: admin@frankenthalerfoundation.org; Tel: +45 4444 8888
Cite this: RSC Adv. , 2020, 10 , 42457 Received 21st August 2020 Accepted 3rd November 2020 DOI: 10.1039/d0ra07204d
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This journal is © The Royal Society of Chemistry 2020 RSC Adv. , 2020, 10 , 42457 –42492 | 42457 RSC Advances
REVIEW Open Access Article. Published on 24 November 2020. Downloaded on 3/11/2026 1:18:36 AM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online View Journal | View Issue routes such as recombinant expression and fermentation approaches, enzymatic or semisynthetic approaches are more advantageous for large peptides and proteins. 6 Chemical synthesis of peptides can be achieved either in solution- or by solid-phase, both strategies having their advantages and disadvantages. 7 For short peptides (#10 –15 amino acids), solution-phase synthesis is usually the strategy of choice. 6 On the other hand, the landmark invention of solid-phase peptide synthesis (SPPS) by Merrifield, i.e. the anchoring of a peptide to an insoluble solid support composed of a polymer ( e.g . polystyrene), 8 has enabled the synthesis of longer peptides that were previously unobtainable via traditional solution-phase chemistry. Steady improvements in protecting groups, coupling reagents, and peptide synthesis conditions have enabled routine access to peptides of high purity, in a scalable manner. 9–11 However, the SPPS synthetic cycle, comprised of a series of repetitive cycles of coupling, washing and deprotection steps with easy separation of reagents from the solid support by filtration, often employs super-stoichiometric amounts of reagents to push the reaction to completion, generally with poor atom economy (Fig. 1). Furthermore, owing to the major contribution of solvents to the mass balance of a typical SPPS process, 12,13 more environmentally benign alternatives to the most frequently used solvents are needed, as well as technologies that promote reduced solvent use and recycling. Today, most of the reagents and solvents applied in peptide chemistry are classified as environmentally problematic substances by the ECHA (European Chemicals Agency) under the REACH (Registration, Evaluation, Authorisation and Restriction of Chemicals) regulation. Current and impending regulation by REACH has the classic SPPS solvents dimethylformamide (DMF) and dichloromethane (CH 2 Cl 2) as well as
N-methyl-2-pyrrolidone (NMP) and dimethylacetamide (DMAc) heading for restriction (ECHA Annex XVII) and/or authorization for use (ECHA Annex XIV), 14 necessitating that alternative SPPS solvents are identified in the immediate future to avoid disruption of industrial production of therapeutic peptides. The greening of peptide synthesis has been reviewed from various perspectives in recent years. Albericio and co-workers have published two reviews primarily discussing the SPPS cycle, loading and cleavage and deprotection, water-based SPPS approaches as well as alternative synthetic approaches for
Henrik Johansson obtained his BSc and MSc degrees in organic chemistry from Lund University (Sweden), and his PhD in medicinal chemistry from The University of Copenhagen (Denmark). In 2015 he joined GSK and The Francis Crick Institute (UK) as a Postdoctoral Research Associate in chemical biology with the Crick-GSK Biomedical LinkLabs, working on Cys-reactive fragment screening and E3 ubiquitin ligases in the group of Katrin Rittinger. Since 2020 he is a Development Scientist at Novo Nordisk working with protein ligation.
Felix Wojcik studied chemistry at Heidelberg University (Germany) and obtained his doctor of natural sciences degree from the Free University of Berlin (Germany) for his work at the Max Planck Institute of Colloids and Interfaces (Biomolecular Systems Department). From 2014 –2019 he performed his postdoctoral studies in chemical biology at Princeton University (USA) with the group of Tom Muir. Since 2019 he holds a Development Scientist position at Novo Nordisk mainly focusing on early-stage chemical API processes including synthetic, semi-recombinant and enzymatic processes.
Sebastian Thordal Le Quement obtained his MSc and PhD degrees in synthetic organic chemistry from the Technical University of Denmark and the University of Copenhagen. After his PhD, he has held several postdoctoral and senior scientist positions in academia, including a stay at the Broad Institute of MIT and Harvard from 2012 – 2014. Since 2015, he has been employed at Novo Nordisk, and is now a Team Leader in chemical development mainly focusing on early-stage API development and manufacturing.
Daniel Sejer Pedersen obtained his MSc and PhD degrees in synthetic organic chemistry from the University of Copenhagen (Denmark) and Cambridge University (UK), respectively. Daniel has held several positions at Universities and in industry and was an Associate Professor at the University of Copenhagen from 2010 to 2017. Since 2017 he has been employed as a Senior Development Scientist in Chemical Development at Novo Nordisk focusing on GMP manufacturing for phase 3 clinical trials.
> 42458 |RSC Adv. , 2020, 10 , 42457 –42492 This journal is © The Royal Society of Chemistry 2020
RSC Advances Review Open Access Article. Published on 24 November 2020. Downloaded on 3/11/2026 1:18:36 AM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online peptide synthesis mostly of relevance to academic research. 15,16
Isidro-Llobet et al. have given a high-level overview on the status of green peptide synthesis and purification discussing all aspects of peptide synthesis and new technological platforms for peptide synthesis and purification from an industrial viewpoint. 17 Lax and Shah have reviewed the economic and environmental factors affecting the sustainability of peptide manufacturing from an industrial perspective, comparing solution and solid phase approaches with recombinant, semi-synthetic and ligation methods. 18 To date no reviews have delved on the specific technical requirements for a green peptide synthesis protocol from an industrial large-scale manufacturing perspective. The aim of the present review is therefore to outline and discuss the technical requirements that the pharmaceutical industry has for SPPS and a green peptide synthesis protocol, and to put the progress made to date into an API manufacturing context. In relation to SPPS this review will focus on greener improvements pertaining to the SPPS cycle, including swelling, coupling, Fmoc-removal and washing steps (Fig. 1). A detailed analysis of peptide cleavage and deprotection, precipitation, intermediate processing ( e.g. cyclisations, oxidation etc. ), downstream purification, isolation, and the synthesis of raw materials for SPPS ( e.g. amino acid building blocks and coupling reagents) is beyond the scope of the present review and is only briefly touched upon. In addition to SPPS, a selected number of emerging technologies for peptide synthesis ( e.g. water-based SPPS, protein ligation and peptide synthesis in continuous flow) with potential use for future large-scale manufacturing of peptide APIs are highlighted and discussed.
From the pharmaceutical industry perspective, the least disruptive short-term scenario for the continued manufacture of therapeutic peptides would be to adjust current SPPS protocols and transition to non-hazardous, green SPPS solvents. This would be greatly advantageous from a procurement point of view, as the necessary supply chain for a variety of raw materials (e.g. 9-fluorenylmethoxycarbonyl (Fmoc)-protected amino acids, activating reagents, resins or linkers) is fully established and cost-efficient building blocks can be easily procured from several independent suppliers in both non-GMP and GMP qualities. 18,19 In the longer term, the development of a completely new platform for the synthesis of peptides, which would utilize raw materials with lower environmental impact, is attractive. For example, Fmoc-protected amino acids and coupling reagents are used in excess during SPPS processes (poor atom economy) and are additionally manufactured by classical organic synthesis means, often involving the use of processes with a large negative environmental footprint. Thus, transitioning from the current Fmoc-based chemistry to emerging peptide synthesis technologies appears desirable from an environmental perspective and should be pursued by both academia and industry. For example, this can be done in consortiums such as the American Chemical Society Green Chemistry Institute, where pharmaceutical companies are working together with academia to promote greener chemistries, including SPPS. 20 Of crucial importance to this discussion, are considerations about the definition of a green solvent, especially in the context of organic synthesis. Several big pharmaceutical companies have published solvent selection guides which are very helpful when faced with the choice of process solvents. 21,22 However, these guides are generally not aligned, and it can sometimes be difficult to deduce which criteria the classification of the solvents have been based upon. Indeed, the classification can be influenced by the area of expertise of the company and what would be considered as a good solvent for substitution for one company, could prove less pertinent for another. As there is no such thing as a perfectly green solvent, it is often a balance between benefits and drawbacks for a specificapplication. The IMI-CHEM21, a European consortium which promotes sustainable biological and chemical methodologies has also published a guide which is based on a survey of publicly available solvent selection guides. 23 This consortium proposes a set of Safety, Health and Environment criteria aligned with the Global Harmonized System (GHS) and EU regulations. Finally, the volumes of solvents employed during manufacturing should also be kept in mind. For example, switching from a problematic solvent to a green solvent may have a larger negative environmental impact if the volumes are multiplied by several digits in the process, emphasizing the necessity for a holistic assessment of the process at hand. From a technical perspective, it may also not be possible to carry out the desired synthesis in a pilot plant beyond a certain volume (maximum capacity of reactors). At Novo Nordisk we have decided to focus on the ICH (International Council on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use) classification, 24 and REACH status of solvents when conducting solvent substitution, taking both environmental and safety factors into account. Currently, we operate with three solvent categories: (a) REACH compliant, (b) REACH affected, (c) Incomplete data (currently REACH compliant but a significant amount of data is not available). The classification of solvents is clearly much more complex than reflected by these three categories, but we have judged that it is better to
