Examples

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EnzyPep’s chemo-enzymatic fragment condensation strategy proven on pharmaceutical products:

Examples, Exenatide and Thymosin-α-1 (using 2 segments)

To demonstrate the versatility of chemo-enzymatic peptide synthesis (CEPS), EnzyPep has developed manufacturing processes for a number of marketed pharmaceutical peptides using a combination of classical SPPS and enzymatic segment condensation. For instance exenatide, a 39-mer used for the treatment of diabetes type II, can only be manufactured with modest yield by straight-through SPPS. Exenatide has no Gly of Pro residues at strategic coupling positions and is therefore difficult to prepare via chemical fragment condensation (risk of racemization). EnzyPep embarked on the synthesisis of exenatide using two segments of similar length, i.e. an 21-mer and 18-mer (see graphical representation below).

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Both segments could be synthesized in good yield and purity (SPPS) and were obtained in a yield of 56% and 52%, respectively, and a purity >96% after one single preparative HPLC purification. Notably, using our selective ligases, no N- or C-terminal protecting groups are needed yielding the final exenatide product in a single ligation step. The coupling reaction was performed at gram scale with a reaction time of 90 minutes using only 1 mg of enzyme per gram of exentide product (pH 8.3, 20°C). The theoretical product concentration was 3.5 wt% and only 1.2 equivalents of amine (18-mer) segment were used. The figure on the right-hand side shows the kinetics for the enzymatic segment condensation reaction. The efficiency of the enzymatic ligation reaction (conversion of Cam-ester fragment to product) was 87%. Clearly, the product is very well separated from the two segments and could be purified by preparative HPLC (single run) to a purity of 99.6% and an overall yield of 43.5% based on the loading of the first amino acid of the Cam-ester. Compared to straight through SPPS of exenatide followed by (multiple) preparative HPLC purification(s), generally a yield of 20-25% based on the first amio acid, the enzymatic segment condensation strategy is almost twice as efficient. The purified exenatide product from the enzymatic condensation reaction was analyzed by several external companies and determined to be within specifications. The enantiomeric integrity of each amino acid was confirmed (CAT) as well as the peptide content. It was calculated (by EnzyPep, independent consultants and other companies) that the overall cost price reduction for the large scale production of exenatide could be around 50%.

Besides exenatide, EnzyPep has demonstrated the efficient synthesis of several other pharmaceutical peptides. A typical example is the synthesis of thymosin-α1, which is a particularly difficult peptide to synthesize using straight-through SPPS (28-mer). For the synthesis of thymosin-α1, two peptide segments of identical length were used, i.e. 14-mer + 14-mer. This time, the segments were not purified prior to ligation, but used in the crude form (precipitated after SPPS cleavage and deprotection), and with no lyophilization step. Again, the efficiency of the enzymatic ligation reaction was around 90% and the thymosin-α1 product was obtained in a crude HPLC purity of 60%. The enzymatic segment condensation approach was compared to the straight-through SPPS of thymosin-α1 using identical resins, reagents and protocols for both strategies, see below the crude thymosin-α1 product from the segment approach and of the straight through SPPS approach. Compared to the crude purity of 60%, the chemical synthesis of thymosin-α1 gave a crude HPLC purity of 45%, but more importantly, the impurities from the enzymatic segment condensation were much better separated from the product peak. The CEPS synthesized thymosin-α1 could be purified by one single preparative HPLC to a purity of 98.6% and an overall yield of 48%, whilst the SPPS version (using identical methods and peak collection protocols) was only 93.5% pure with a yield of 35%.

Extremely efficient head-to-tail cyclisation of peptides ranging from 12 to 50 amino acids:

Besides segment condensation, as discussed above, EnzyPep’s ligases can be used for very efficient head-to-tail cyclisation of peptides. Surprisingly, there seems to be no limit to the peptide length or sequence. Peptides ranging from 12 to 50 amino acids were cyclized in efficiencies >90% without any polymerization being observed. The enzymatic condensations could be performed at much higher concentrations than used for conventional chemical cyclisation reactions, which is an interesting for scale-up. Furthermore, the linear peptide precursor could be added incrementally to the enzyme catalyst resulting in final cyclic peptide product concentrations of ≈100 mg/mL. As an example, an exenatide Cam-ester was synthesized and enzymatically cyclized in an efficiency of 98%.

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Peptide-to-protein ligation, selective coupling of peptides to the N-terminus of proteins:

Peptides have also been ligated to proteins with similar efficiencies to those described above. When an excess of peptide (e.g. 5 equivalents of exenatide Cam-ester) was used, the quantitative ligation to an N-terminal extended human serum albumin (HSA, 585 amino acids) could be achieved (see figure on right). The excess peptide and ligase could be separated from the exenatide-HSA peptide-to-protein conjugate by one simple ultrafiltration step. Coupling of (pharmaceutical) peptides to many other proteins is ongoing. A model peptide has also been coupled successfully by CEPS to an XTEN protein with 864 amino acids. The upper sequence length limits of CEPS have not yet been reached and are still under investigation both in terms of peptide-to-peptide coupling and peptide-to-protein conjugates.

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This work is part of the
Project ENEF : New technologies and products
The world population continues to grow and that brings all sorts of challenges in terms of water, food, housing and energy with them. ENEF, an abbrevation for energy efficiency is concerned with the latter challenge. ENEF identifies possible improvements to existing materials, systems and processes and introduces them into the market so that they can serve as an example. It is not about small steps. The selected developments make a radical improvement of existing products and processes. They are game changers that drastically reduce CO2 emissions. SMEs are the driving force in this project.
More information on this Interreg project can be found at www.projectenef.eu

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