On the way to greener SPPS: Replacing DMF

Bachem Baselland, Switzerland

At Bachem, we are committed to sustainability by taking responsibility towards the environment, society and our employees. We aim to redesign our processes to minimize the use and generation of hazardous substances and reduce the environmental impact. In the pharmaceutical and chemical industries, solvents consumption generates a significant environmental footprint.  Furthermore, regulatory agencies become more and more stringent with the usage of certain solvents common in peptide synthesis, such as dimethylformamide (DMF), dichloromethane (DCM) and N-methyl-2-pyrrolidone (NMP) because of their impact on health. For instance, DMF has recently been added to Annex XVII of the EU REACH regulation which means that use will be restricted in the EU.

Our goal is to develop efficient green and sustainable processes that provide high-quality peptides. Together with our partner Novo Nordisk, we made an important step towards greener solid-phase peptide synthesis (SPPS) in replacing DMF which we also published in peer-reviewed papers1Martin, V. et al. Green Chem., 23, 3295 (2021) 2Jadhav, S. et al. Green Chem., 23, 3312 (2021). Read our interview with Dr. Stefan Eissler, Vice President Peptide Manufacturing and watch our webinar to learn more on how we develop a toolbox for greener SPPS.

Can you please give us a brief introduction on SPPS and why this is a core technology for peptide synthesis?

When Bruce Merrifield developed the concept of an insoluble resin-bound peptide chain, a soluble activated amino acid and solvent to effect solid-phase peptide synthesis (SPPS), it was a new and revolutionary approach of organic synthesis. He was awarded of the Nobel prize in Chemistry in 1984 for the invention of Solid Phase Peptide Synthesis. 

SPPS allows to drive reactions to completion using a large excess in reagents and building blocks which makes SPPS an enabling technology for the synthesis of complex peptides. A high level of conversion is crucial for the synthesis of complex peptides via consecutive reactions without purification steps. With low levels of conversion, low yields and hardly separable mixtures with numerousness process-related impurities are obtained.  Today, most peptides are manufactured via SPPS because SPPS is an automatable, robust platform technology which enables the efficient manufacturing of complex peptides in high quality after preparative HPLC purification at all scales with limited process development effort. In addition, there is an established supply chain for common building blocks, starting resins and solvents.

With SPPS, synthesis of complex peptide APIs became more efficient and more scalable. At Bachem, we are a team of people who are highly specialized in this field and one of the few places in the world able to produce complex peptides that improve and save lives.

What are the reasons that trigger Bachem in looking for less impactful and greener SPPS?

Green chemistry has been in the focus of the chemical and pharmaceutical industry for quite a while now, and hence has been in the focus of Bachem as well. Furthermore, sustainability in general is a strategic pillar at Bachem. Unfortunately, SPPS goes against most of the 12 Principles of Green Chemistry. In particular, large quantities of waste are generated during peptide manufacturing via SPPS, which is typically quantified via process mass intensity, PMI. Furthermore, undesirable solvents are often used for SPPS. Please note that in contrast to that, peptides themselves have favorable properties, typically meeting the requirements of Designing Safer Chemicals and Design for Degradation.

Therefore, we investigate new solutions and alternative technologies to reduce the environmental footprint of our manufacturing processes, which includes measures to improve the SPPS technology in that regard. To that end, we also join forces with pharmaceutical companies and cooperate with our customers to reach sustainability goals.

What are the main challenges you encounter when developing a greener SPPS?

SPPS is a cutting-edge technology which at the current state of development meets high standards regarding conversion (ratio of how much reactant has reacted), purity (ratio between the desired product and impurities), yield (how much of desired product was formed), and process robustness. A greener alternative would need to meet the same standards, not only from a cost perspective, but also because a less efficient process is unlikely to be more environmentally friendly and might even fail to provide material in the required quality.

In addition to the environmental impact, regulatory agencies are more and more stringent regarding certain solvent usage. How does this impact Bachem?

The main SPPS solvent is dimethyl formamide (DMF). Unfortunately, DMF has reprotoxic properties and hence has recently been added to Annex XVII of REACH which means that use will be restricted in the European Union. Consequently, supply and use will be more difficult in the EU and such non-EU countries which are affected by or take over EU regulations. In summary, that is a strong motivation to investigate alternative SPPS solvents even though use of DMF for SPPS will still be possible in the future.

Being able to replace DMF with greener solvents is the future of SPPS. How does Bachem undertake such a change?

Fortunately, we benefit from the body of knowledge we have generated on the use of alternative solvents so far, which includes lab-scale work to establish an alternative toolbox as well as experience with manufacturing at scale. This allows us to speed up such activities. For a specific project, the first step is to establish a scalable manufacturing process based on initial process development on lab-scale. Using alternative solvents, proper process development is even more important than with DMF from my point of view.

What is your roadmap to develop such a toolbox?

As a first step we did a broad investigation of different alternative solvents and solvent mixtures considering several parameters in co-operation with Novo Nordisk. We focused our investigation on the physical properties of the solvents or solvent mixtures, resin swelling, Fmoc removal and coupling chemistry as well as solubilization properties. In the years to come, we will further develop and refine the tools in our toolbox and add new tools as appropriate – the aim is to improve continuously, such work is never done.

This would require extensive research before starting the SPPS. Did you come to an easy and quick way to assess new potential solvents?

We typically refer to our toolbox and our prior knowledge to define the initial screening conditions for a specific project, followed by more specific process development as appropriate for the purpose, mainly addressing process-related impurity formation to optimize purity, impurity profile and yield. For both tasks we benefit from the body of knowledge already generated, which streamlines process development and speeds up our process development activities. In principle, we prefer a single solvent or mixture of solvents that can be used throughout the complete synthesis, which may not be appropriate in specific cases.

In most of cases, DMF is still the solvent of choice for SPPS with properties supporting both Fmoc deprotection and coupling steps. So far, we have not seen an alternative, more environmentally friendly solvent that would equal or outperform DMF in that respect, though there are some solvent mixtures which come close. Hence, during process development we aim to get results as close as possible to those we would get using DMF with regards to purity, impurity profile and yield. That is not always easy because most alternative solvents have drawbacks that need to be dealt with by process development, which typically translates in somewhat more effort for process development to get a process with similar efficiency.

Have you tested these new solutions on a peptide API?

As a CDMO we have a strong interest to apply scientific results at scale to the benefit of our customers. Nevertheless, prior to use at production scale or scale-up, we demonstrate in small scale that scientific results are transferrable to relevant and representative examples. To this end, we have used a DMF-free set-up for the automated SPPS of bivalirudin and other peptides on 7.5 mmol scale with automated synthesizer and published the results. From that publication you may learn how we got results similar to DMF after a couple of iterative cycles of process development, which also gives a nice example of process development for a peptide. In that example, specific side-reactions had to be addressed, which of course also provides learnings that may be applied to the process development of other peptides. We also already did syntheses at scale for different customers using SPPS reactors with a capacity of up to 1000 L, and I am confident that there are more to come.

Paving the way to greener SPPS

In our studies in collaboration with Novo Nordisk, we set out to identify environmentally benign solvents suitable for large-scale SPPS. We assessed reagent solubility, resin swelling, amino acid coupling and Fmoc-removal efficiency in a collection of REACH-compatible neat solvents and selected binary solvent mixtures. To gain time in process development, we developed a convenient tool that predicts the usability of greener solvents and mixtures. These studies have shown that specific binary solvent mixtures are viable alternatives to DMF for the synthesis of therapeutic peptides.

Innovation in this field is key for the future and we are taking this challenge very seriously. We are proud of our advancements so far that will contribute to reduce our environmental impact and help our customers reach their sustainability goals. With these publications, our goal is to enable the entire peptide community to benefit from our results to advance the synthesis of more sustainable and environmentally friendly peptides.

At Bachem, we are a team of people who are highly specialized in this field and one of the few places in the world able to produce complex peptides that improve and save lives.

Stefan Eissler

Vice President Peptide Manufacturing, Bachem AG

About Bachem

Bachem is a leading, innovation-driven company specializing in the development and manufacture of peptides and oligonucleotides.
With over 50 years of experience and expertise Bachem provides products for research, clinical development and commercial application to pharmaceutical and biotechnology companies worldwide and offers a comprehensive range of services.
Bachem operates internationally with headquarters in Switzerland and locations in Europe, the US and Asia. The company is listed on the SIX Swiss Exchange.

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