The new tide in peptide purification and modification


by Dominik Sarma

Belyntic has developed a universally applicable linker molecule that allows the purification and modification of standard and difficult peptides in parallel using catch-and-release methodologies using our proprietary Peptide Easy Clean (PEC) technology. Our kits and services build on this molecule – the PEC-Linker RC+. 


Figure 1: The PEC-Linker RC+ for highly efficient, parallel peptide purification and modification

As a result of a trustful development collaboration with Bachem, an industry leader in peptide manufacturing, we celebrate the joint publication about developing the PEC-Linker RC+ and its application for peptide purification in the scientific journal Chemical Science.[1]  Check out the OPEN SOURCE article online (Link).

In this Blog article, we sum up the most important results in the context of their application in the development of novel peptide drugs and provide an outlook on a novel application of the linker technology: the highly efficient chemical modification of peptides after SPPS. 

We thank Bachem for supporting validation studies, material, and study design to create a comprehensive report. We look forward to a strong partnership to enable novel peptide therapeutics from development to production for our customers. 


Peptides have a high therapeutic potential due to their pivotal role in nature. Currently, more than 60 approved peptide drugs are on the market, and 157 active clinical trials are ongoing. [2] The trend to longer and chemically modified peptides imposes increased challenges for peptide synthesis as well as the subsequent purification using high-performance liquid chromatography (RP-HPLC). [3]

Catch-and-release (c&r) purification methods build on cleavable linker molecules and facilitate the purification process through chemo-selective isolation and high degrees of flexibility for peptide dissolution and handling. Yet, reported linker molecules could not provide side reaction-free or contamination-free c&r procedures due to the applied chemical routes, leaving this strategy experimental.[4]

With the PEC-Linker RC+ shown in Figure 1, we introduce a first-in-class approach for general application and provide the basis for the widespread use of c&r methods in industrial peptide development and manufacturing. The PEC-Linker RC+ is reductively cleavable for side reaction-free applications with all canonical amino acids. Moreover, the molecular design allows a safety-release to ensure the PEC-Linker's cleavage without traces of contamination from the cleavage reaction. The additional possibility for post-SPPS solid-phase modification emphasizes the game-changing potential of this new class of c&r technology. 

Faster peptide development 

Commercially available automated peptide synthesizers allow the parallel synthesis of dozens to several hundreds of peptides in a single run. In contrast, HPLC is a linear process, rendering purification the bottleneck in peptide manufacturing. Consequently, researchers mostly employ crude peptide libraries and, as such, accept a higher risk for false-positive responses, which hampers the speed and reliability of pharmaceutical development.[5] Moreover, the recent advancements in personalized cancer therapies require faster downstream processes to meet the demand for a short needle-to-needle time.

PEC is universally applicable. The absence of side reactions and contaminations in the final product using the PEC-Linker RC+ enables an increased throughput through parallel processing for any given set of peptides, including neoantigens. To demonstrate the strength and general applicability of the technology on a relevant example, we purified 20 neoantigens in a single day with a striking increase of purity from an average of 53% up to 89% (Figure 2). 


Figure 2: Purity gain of a neoantigen set of 20 peptides after PEC-purification.

A particular example is shown in Figure 3 that shows a challenging crude mixture for neoantigen peptide N14 out of which the target peptide could be isolated efficiently in a pure form. 


Figure 3: Purification of a neoantigen from a complex crude mixture

Difficult made easy

Peptide drugs become more and more complicated to purify due to increasingly challenging Physico-chemical profiles. This drawback manifests already during drug development: The addressable range from hydrophobic to hydrophilic peptides using chromatography limits the exploration of peptide candidate pools, leaving potential lead candidates undetected. 

PEC is an orthogonal approach. In contrast to chromatography, the PEC-Linker chemo-selectively isolates the target peptide out of its crude mixture. Therefore, it reflects an orthogonal strategy to HPLC and adds a vital option to tackle difficult peptides. The purification of the hydrophilic peptide Histone H3 (1-20), for example, is a formidable challenge due to the co-eluting impurities that form during synthesis. We showed how to achieve high purities using the PEC-Linker in a single purification step. The chromatogram shown in Figure 4 highlights how impurities can hide beneath an otherwise clean-looking main peak. Only mass analysis reveals the accumulation of truncations, which all represent a potential threat to your assay. PEC efficiently removes these impurities regardless of their similarity to the target peptide. 


Figure 4: Purification of a highly hydrophilic Histone fragment

New peptide API's – a future look

Another major trend is the chemical modification of peptide therapeutics to increase their stability and efficacy. The main modification types include lipidations, cyclizations, and disulfide formation. Unfortunately, purification difficulties typically accompany the modifications, rendering access to complex peptides difficult. 

PEC is a modification platform. The innovative safety-release enables conjugation chemistries in the purification-phase, which are highly efficient since they benefit from large possible excess and pseudo-high-dilution. To understand the tweak, we look at Figure 5: Halogenation at the aryl core renders the PEC-Linker stable after reducing the azide because it suppresses the spontaneous decomposition pathway. 


Figure 5: PEC-assisted solid-phase modification of peptides

At this stage, therefore, the unprotected peptide is bound to the purification material and ready for solid-phase modifications such as disulfide formations, or stapling reactions. Modifications like lipidations or PEGylations, that are unaffected by the DTT reductions can be performed before or after Linker-reduction. The modified product is then prone to decomposition after adding protons to the system, releasing the target complex peptide in pure form (Figure 6). 

Figure 6: Modification with the PEC-Linker RC+

Design for manufacturing

Ultimately, the challenging peptide API, modified or native, needs to be manufactured in high quality and at a large scale for the intended clinical applications. While many promising candidates in the peptide arena fail due to their impossible manufacturing in an economically and ecologically feasible way using standard chromatography, our PEC technology aims to ensure the manufacturability of even the most complex and difficult peptides at the larger scales, 

PEC serves the whole value chain. We obtain our PEC-Linker and related materials from renowned external manufacturers at the multi kg-scale. With this, we can ensure adequate material supply for clinical-stage projects and generic peptides, meeting the highest quality standards that our customers demand.

Together with our CMO partner Bachem, the leading independent supplier of peptide APIs, we can transfer customer projects from development to production seamlessly. Bachem has a proven track record in process development, large-scale cGMP manufacturing, in addition, to support market authorizations and approval processes. Furthermore, Bachem’s team is committed to continuously expanding their know-how in chemistries and technologies. 

Read also the Bachem Whitepaper about the PEC technology "A new turn in peptide purification" (Link)

This work for this publication was funded by the European Regional Development Fund. It lays the ground to enable new peptide therapeutics through access and scale-up using Belyntic's PEC technology and Bachem's longstanding manufacturing expertise. Please get in contact to discuss your challenge with us.


[1] R. Zitterbart, N. Berger, O. Reimann, G. Noble, S- Lüdtke, D. Sarma, and O. Seitz, Chemical Science, 2021

[2] A. C. Lee, J. L. Harris, K. K. Khanna and J. H. Hong, Int J Mol Sci, 2019, 20

[3] J. L. Lau and M. K. Dunn, Bioorg. Med. Chem., 2017, 2700-2707.

[4] O. Reimann, O. Seitz, D. Sarma and R. Zitterbart, J. Pept. Sci., 2019, 25, e3136.

[5] J. R. Currier, L. M. Galley, H. Wenschuh, V. Morafo, S. Ratto-Kim, et al., Clin. Vaccine Immunol., 2008, 15, 267-276; J. W. de Beukelaar, J. W. Gratama, P. A. Smitt, G. M. Verjans, J. Kraan, et al., Rapid Commun. Mass Spectrom., 2007, 21, 1282-1288.

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