The pharmaceutical industry is in need of a more efficient, sustainable and greener method to synthesize life changing therapeutics, of which many are long and complex peptides. As you will discover throughout the course of this article, the use of traditional Solid Phase Peptide Synthesis (SPPS) and recombinant expression methods, have many limitations in terms of product purity and lack of sustainability. One pioneering technology with the ability to manufacture therapeutic peptides economically, at a large scale and in an environmentally sustainable way is the Chemo Enzymatic Peptide Synthesis (CEPS) technology, available at Biosynth. From the successful synthesis of linear therapeutic peptides such as Exenatide and Thymosin-α1, to cyclic peptides like MCoTI-II, CEPS technology has already asserted itself as a ground-breaking technology in peptide therapeutics.

Developing Methods of Peptide Synthesis

Particularly throughout the 20th century, peptide synthesis techniques were forced to develop in order to meet the increasing demand for peptide therapeutics. Insulin is a prime example of this. Significantly insulin was first isolated from natural sources in 1921 and was the first peptide therapeutic to be developed for commercial use by diabetes mellitus patients. Since this pioneering discovery, many other bioactive peptides such as adrenocorticotrophic hormone were isolated, followed by the development of synthetic peptide drugs as peptide synthesis technologies become more advanced. Such that synthetic peptides such as recombinant human insulin and synthetic vasopressin were developed, as simply isolating insulin from natural sources became insufficient to meet demands.

SPPS

SPPS, originally developed by Robert Bruce Merrifield in the 1960s, is now a widely used method for the stepwise synthesis of precise peptide sequences. A solid support to which the c-terminal amino acid of the growing peptide chain is immobilized upon, acts as a scaffold for peptide synthesis and facilitates the removal of excess reagents and byproducts. Although this process allowed peptide and protein synthesis to be more accessible and efficient, the production of long and more complex peptides can be a challenge and lead to greater impurity levels as the chain length increases. Furthermore compared to biocatalytic methods, SPPS shows economic and environmental disadvantages in terms of the amount of CO2 emitted and materials used.

Recombinant Expression

One alternative is the recombinant expression of peptides, where genetically engineered microorganisms such as bacteria or yeast are used to produce desired peptides. This technique can produce long and complex peptides on a large scale. On the other hand this technique does not guarantee to give a soluble product, and can require significant development work, with changes required for each new sequence of interest. Moreover post translational modifications are also difficult to address for recombinant proteins. A solution to the majority of these problems is the use of the innovative technology CEPS.

CEPS

Chemo-Enzymatic Peptide Synthesis (CEPS) is a novel technology using an enzyme to generate longer peptides and small proteins through the ligation of shorter peptide sequences. Amazingly, it can synthesize cyclic peptides and peptide sequences with more than 100 amino acids, at a greater purity and is easily scalable. Not only that, but the unique CEPS enzyme has outstanding tolerance for a range of side chains, making the technology widely applicable for the majority of functionalized peptide sequences.

Overall CEPS technology provides a more sustainable, efficient and greener approach to peptide synthesis. Many of the advantages of this technology are only possible as a result of the outstanding properties of the enzyme. The enzyme itself is derived from an endotoxin-free process in Bacillus subtilis bacteria (a safe GRAS organism) and is active in aqueous conditions at a pH range of 7 to 8.5. As the enzyme remains active and stable where denaturing agents and organic co-solvents are present, the ligation of hydrophobic and folded peptides is also possible. Whats more, the enzyme can be described as having a broad substrate scope in that a unique recognition sequence does not need to be inserted, as it can recognize a large number of amino acid sequences. It is this that deems it a 'traceless technology'.

Advantages of CEPS Technology over Recombinant Expression

Key Features of CEPS technology at Biosynth

· CEPS generates long peptides well outside of the range of SPPS > 80 aa’s   

· Significantly higher yields than SPPS (>2x) in the synthesis of long or complex peptides

· High purity products- fragments used for ligation can be purified beforehand leading to much purer product compared to SPPS.   

· Capable of synthesizing >90% marketed pharmaceutical peptides

· Technology has significant applications in the discovery field by linking all sorts of payloads via a short peptide linker to proteins.

· Backbone cyclized peptides with large ring size (>12 aa’s). Alternatives require either cysteines (NCL) or fully protected linear precursor with poor solubility.   

· Cyclotides (cyclic peptides with 3 disulfide bridges, knot like structures) are possible

· Useful in connecting linker-systems i.e connecting non-natural fragments together to create bioconjugates and antibody drug conjugates

How does CEPS work?

At the heart of the CEPS technology is an engineered enzyme with an active site cysteine. As stated above this enzyme recognizes multiple sequences of amino acids, removing the need to insert specific recognition sequences.

When producing the required peptide at least two peptide fragments are needed. An ester fragment and an amine fragment. The active site cysteine reacts with the ester fragment (specifically the carbonyl of the ester), covalently attaching the ester fragment to the enzyme. At this point an organic alcohol leaves the active site. The amine fragment then comes in and the free amine reacts with the thioester. This is energetically favorable to form a product, with an amide bond. If necessary, the enzyme is then free to continue onto the next substrate, where the product becomes the new amine fragment.

Mechanism of CEPS Technology

The CEPS technology enzyme needs six amino acids for recognition and docking. Of these, four are in the ester fragment and two are supplied by the amine fragment. Again these amino acids do not need to be a fixed or unique sequence, offering a broad substrate scope. In cases where cyclic peptides are the end product, twelve amino acids are needed, six for recognition and six to close the loop.

The Success Stories of CEPS

Despite being such a novel technology CEPS has already asserted its dominance in the field of long peptide and protein synthesis. CEPS is perfect for the synthesis of peptides greater than 60 amino acids in length, chemokines, cytokines, nanobodies and non-antibody (small) protein scaffolds. Many of these proteins can be used as key peptide therapeutics, leading the way in medical advancement. To date CEPS has already successfully synthesized the likes of Liraglutide (CRB1001347), Semaglutide (FS171058), Exenatide (FE31731), MCoTI-II and Thymosin-α1 (FT109993).

Thymosin-α1 for example, can be made by ligating two fragments. Thymosin-α1 is a 28-mer therapeutic peptide, with immunoregulating activity. Currently it is used in medical applications such as hepatitis B and C and 'approved in more than 30 countries' (Schmidt, 2018). In response to a high demand for thymosin-α1, its synthesis was amplified, using solid and solution phase methods. However such methods provided low overall yields. Thymosin-α1 is difficult to synthesize due to the need for a large number of protecting groups and its ability to form β-sheets. When prokaryotic or eukaryotic expression systems were used low titers were given and there was a greater difficultly in isolating and purifying the product. The CEPS technology offered an efficient and cost effective solution to synthesizing thymosin-α1, in which two 14 mer peptides were ligated together. This was achievable on a gram scale, with a 94% coupling yield and a 55% increased yield. CEPS is notably a cutting-edge technology admirably serving the peptide synthesis and therapeutic industry (Schmidt, 2018).

Exenatide is another example of a CEPS technology success story. Exenatide, a 39-mer synthetic GLP-1 agonist, is an active pharmaceutical ingredient (API) in the antidiabetes therapeutics Bayetta® and Bydureon®. Using the CEPS technology exenatide, can be seamlessly manufactured on a scale of >50 grams, through the ligation of two fragments (Pawlas, 2019).

Not only does the CEPS technology successfully manufacture linear peptides but it can also manufacture disulfide-rich macrocyclic peptides (cyclotides) which show great potential as peptide therapeutics. This is due to them showing a higher metabolic stability compared to their linear analogues, and a high potency. These cyclotides can also be used as scaffold for bioactive epitopes to be fused onto, again demonstrating their pharmaceutical potential. Momordica cochinchinensis trypsin inhibitor-II (MCoTI-II) is such an example. MCoTI-II serves as a cyclic peptide scaffold to which peptides such as COG peptide, are fused to. COG is an antagonist of SET, which is overexpressed in cancer cells. This increases the stability of COG peptide so that the whole structure is cytotoxic to cancer cell lines (D'Souza, 2016). MCoTI-II, a cyclic 34-mer was successfully produced using CEPS, using a macrocyclization strategy (Schmidt, 2019).

Cytokines can also be manufactured using CEPS and as regulators of the immune system, offer themselves as potential candidates for therapeutics or targets for immune-related disorders. It is therefore important to understand the role that cytokines play in health and disease.

CEPS technology is truly a groundbreaking technology pushing forward scientific discovery and medical advancements. It has showcased its ability to produce new emerging and difficult to synthesize peptide therapeutics. As innovators, Biosynth strives to supply scientists with the most efficient and pioneering technologies. We have available to our customers, the CEPS technology, pushing the boundaries of long peptide and small protein synthesis. Visit our CEPS technology webpage or contact us to begin your peptide project today.

Liraglutide (CRB1001347), https://www.biosynth.com/p/CRB1001347/liraglutide

Semaglutide (FS171058), https://www.biosynth.com/p/FS171058/910463-68-2-semaglutide

Exenatide (FE31731), https://www.biosynth.com/p/FE31731/141758-74-9-exenatide

Thymosin-α1 (FT109993), https://www.biosynth.com/p/FT109993/1337515-90-8-thymosin-alpha1-trifluoroacetate

Chemo Enzymatic Peptide Synthesis (CEPS) technology https://www.biosynth.com/peptides/CEPS-technology

References

D'Souza, C., Henriques, S. T., Wang, C. K., Cheneval, O., Chan, L. Y., Bokil, N, J., Sweet, M. J., Craik, D. J. (2016) Using the MCoTI-II Cyclotide Scaffold To Design a Stable Cyclic Peptide Antagonist of SET, a Protein Overexpressed in Human Cancer. Biochemistry, 55(2): 396-405.

Pawlas, J., Nuijens, T., Persson, J., Svensson, T., Schmidt, M., Toplak, A., Nilsson, M., Rasmussen, J. H. (2019). Sustainable, cost-efficient manufacturing of therapeutic peptides using chemo-enzymatic peptide synthesis (CEPS). Green Chemistry, 21(23), 6451-6467.

Schmidt, M., Huang, Y-H., Rexeira de Oliveria, E. F., Toplak, A., Wijma, H. J., Janssen, D. B., van Maarseveen, H. J., Craik, D. J., Nuijens, T. (2019) Efficient Enzymatic Cyclization of Dislfide-Rich Peptides by Using Peptide Ligases. ChemBioChem, 20, 1524-1529.

Schmidt, M., Toplak, A., Rozeboom, H. J., Wijma, H. J., Quaedflieg, P. J. L. M., van Maarseveen, J. H., Janssen, D. B., Nuijen, T. (2018). Design of a substrate-tailored peptiligase variant for the efficient synthesis of thymosin-α1. Organic and Biomolecule Chemistry, 16(4), 609-618.

Wang, L., Wang, N., Zhang, W., Cheng, X., Yan, Z., Shao, G., Wang, X., Wang, R., Caiyun, F. (2022). Therapeutic peptides: current applications and future directions. Signal Transduction and Targeted Therapy, 7(48).