Enhanced Peptide Purification via Novel Orthogonal, Doped Reverse Phase Chromatography
ABSTRACT:Enhanced Peptide Purification via Novel
Orthogonal, Doped Reverse Phase Chromatography
Jürgen Machielse , †Prof. Dr. Kinkel , Andrea Wild , Tim O`Mara 
1. Zeochem AG, Uetikon am See, Switzerland
2. Former Prof. at TU Georg-Simon-Ohm, Nuremberg
3. Itochu Chemicals America Inc.
Peptides are important API’s for modern pharmaceuticals and h Moreave to be produced in industrial scale with increasing demand on separation costs.
RPC is the well-established chromatographic mode within the available tool-box of methods and procedures for the purification of this class of compounds in industrial scale production.
The paper will describe the beneficial use of Doped Reversed Phase packings in the repulsive-attractive mode compared to non-doped RP packings on crude peptides. The novel orthogonal Doped Reversed Phase materials combine the dual action of strong IEX groups (acidic or basic) and Reversed Phase ligands like octyl chains on the packing surface.
Usually, Mixed-Mode packings are applied under conditions which add the retention power of IEX groups by electrostatic attractive forces to the retention received from hydrophobic surface groups to the solutes. Both species, e. g. the peptide and the IEX group carry opposite charges.
In 2014, ETH-Zürich (Prof. Dr. M. Morbidelli et al) in cooperation of Zeochem AG have introduced the repulsive – attractive mode for peptide separation.
It can be shown that in the majority of all cases tested so far, improved selectivities and increased resolution at decreased retention time and solvent consumption can be obtained.
Poster ID# 2363
A Novel One-Pot Synthesis Strategy for Bicyclic Peptide Assembly
ABSTRACT:Bicyclic peptides are polypeptides forming two circular units. The cyclic structures often exhibit improved stability, higher potency and bioavailability. Therefore, they are considered as a novel therapeutic class, which lies between small molecules and monoclonal antibodies.
Bicyclic peptides can be prepared by both solution phase and solid phase More synthesis; however, their synthesis remains a challenge. The multiple steps of synthesis, cyclization, and purification often result in low overall yield. Therefore, the synthesis strategy plays a critical role.
We recently synthesized a 13-mer bicyclic peptide, containing one disulfide bridge and one triazole bridge. Multiple synthetic routes were tested, including both on-resin cyclization and cyclization in solution. The original protocol using step-wise cyclization in solution required multiple steps of cyclization and purification, which gave an overall yield of 18%. Using a novel, one-pot synthesis strategy, we were able to carry out two, sequential cyclizations in one reaction solution and purify the final product with only one final purification step to give an increased yield of 30%. For the production of 100mg of final, bicyclic peptide product, the entire synthesis time was shortened by one week using the one-pot reaction protocol. We believe scale up of the reaction process to produce gram quantities of final pure, bicyclic compound is feasible, and no further process development will be needed.
This novel strategy is applicable to facilitate the synthesis of a broad range of bicyclic peptides when the preparation of a disulfide bridge and triazole bridge within a single sequence is required.
Poster ID# 2408
Editing-deficient aminoacyl-tRNA synthetase mediated incorporation of backbone modified amino acids
ABSTRACT:Small molecules are only able to target the small population of human proteins containing a hydrophobic pocket which is a major problem in traditional small molecule drug development. Peptide based therapeutics offer the potential to target a much wider range of targets by inhibiting protein-protein interactions. The major drawbacks of peptide ther Moreapeutics include lack proteolytic stability and ill-defined secondary structures. These problems can be eloquently overcome by incorporation of unnatural amino acids into potential therapeutics to lower the rate of proteolytic cleavage and by creating cyclic peptides to lock the peptide into a conformation which lowers the entropic penalty of binding. Indeed, there are numerous examples of unnatural peptides exhibiting enhanced pharmacokinetics over their parent peptides. Taking in all these considerations, the ability to create libraries of random peptides containing unnatural amino acids is an appealing endeavor. Through a reconstituted cell-free translation system we demonstrate how an editing deficient valine tRNA synthetase (ValRS T222P) can be used for the incorporation of 13 unnatural amino acids into peptides including three cyclic beta amino acids and three alpha methyl amino acids. This system has the potential to be applied to the ligand discovery tool of mRNA display for identification of potent peptide inhibitors of disease relevant protein-protein interactions.
Poster ID# 2476
Functional Penetrating Phylomers (FPPs) for More Efficient Delivery of Biologics In Vivo
ABSTRACT:Structure-rich Phylomer peptide libraries, derived from biodiverse genomes, are a source of new cell penetrating peptides (CPPs) able to deliver therapeutic macromolecule cargoes to the intracellular space, thus opening up the druggable landscape. Employing a novel genetic screen known as the ‘endosome escape trap’ rare Phylomer CPPs were isolated Morethat were able to deliver 60nm phage nanoparticle cargoes to the cytoplasm. These new cell penetrating Phylomers known as ‘FPPs’ show no evidence of toxicity and can be targeted to particular cell types using receptor binding domains. A variety of functional assays have been used to determine the extent of delivery of peptide and protein cargoes to the cytoplasm or nucleus. One such assay, based on split GFP complementation, has shown Phylomer FPPs to be 37-160 times more efficient at cytoplasmic delivery than conventional CPPs such as TAT or R9. Phylomer FPPs have also been shown to effectively deliver a 90 amino acid MYC inhibitor in a mouse model of breast cancer. This new delivery approach is being applied in conjunction with Phylomer peptides targeting transcription factor oncoproteins such as cMyc, MycN and STAT5, for more efficient cell targeted intracellular delivery of protein conjugates of more than 50 kDa in size, and also for intracellular delivery of nucleic acids.
Poster ID# 2495
Group-Assisted Purification (GAP) Technology for Peptide Synthesis
ABSTRACT:Cole W. Seifert and Guigen Li
GAP Peptides LLC, Lubbock, TX 79415, United States; Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX 79409, United States; Institute of Chemistry and Biomedical Sciences, Nanjing University, Nanjing 210093, P. R. China.
The peptide therapeutics market has been growing rapidly for years, both More financially and as an increasing sector of the pharmaceutical industry.1 This market is projected to continue growing; therefore, new synthetic methodologies to reduce the cost of peptide synthesis and enable larger scale syntheses are highly desired.2 We have developed a solution-phase, Fmoc / tBu peptide synthesis method utilizing Group-Assisted Purification (GAP) chemistry, by design of a small-molecule (MW = 292 Da) protecting group. This protecting group, equipped with a phosphine oxide moiety, facilitates selective precipitation of the peptide from solution after each coupling step, with an average isolated yield per coupling of 97%. All fully protected peptides are white, crystalline solids that are easy to handle and can be stored for over 1 year without noticeable degradation. Reactions are run in homogeneous solution without the use of bulky polymers, leading to highly efficient couplings and high crude peptide purity. GAP peptide synthesis is amenable to a wide variety of coupling strategies, protecting groups, and deprotection reagents. Our process is also semi-automated, and we are working towards full automation. Examples of peptides synthesized using the GAP process are presented.
1. Craik, D. J.; Fairlie, D. P.; Liras, S.; Price, D., The Future of Peptide-based Drugs. Chem. Biol. Drug Des. 2013, 81 (1), 136-147.
2. Fosgerau, K.; Hoffmann, T., Peptide therapeutics: current status and future directions. Drug Discov. Today 2015, 20 (1), 122-128.
Poster ID# 2540
Bicyclic RGD-peptides with high affinity and selectivity for αvβ3, αvβ5, and α5β1 integrins
ABSTRACT:Bicyclic RGD-peptides with high affinity and selectivity for αvβ3, αvβ5, and α5β1 integrins
D. Bernhagen[a], L. De Laporte[b], P. Timmerman[a,c]
[a] Pepscan Therapeutics, Zuidersluisweg 2, 8243 RC Lelystad, Netherlands; [b] DWI – Leibniz Institute for Interactive Materials, Forckenbeckstr. 50, 52056 Aachen, Germany; [c] Van’t Hoff Institute for Mol Moreecular Sciences, University of Amsterdam, Sciencepark 904 XH Amsterdam, Netherlands
The ‘bicyclic peptide’ platform recently attracted considerable interest as a powerful platform for novel therapeutic drugs because of their high binding affinities and selectivities in combination with an appreciable proteolytic stability. So far, only few cellular proteins have been evaluated for their binding to bicyclic peptides. The integrins represent an interesting target class for novel therapeutic agents because of their significant role in key pathological processes. We therefore synthesized libraries of hundreds of different bicyclic peptides, one loop comprising the well-known “RGD”-motif and providing basic integrin-affinity, and second loop consisting of a random sequence (XXX) that allows for controlling the binding selectivity. In order to screen for affinity and selectivity to each integrin (αvβ3, αvβ5, and α5β1) we developed a robust and cost-effective screening assay using a knottin-RGD peptide as a reference binder.[4,5] After selection of the best binders for each integrin, we designed 2nd and 3rd generation diversity libraries and thus gradually improved the affinity and selectivity for each integrin. The best IC50 values that we obtained in this way were, for example, 30 nM for αvβ3 (GRGDS: 5 µM, knottin-RGD: 250 nM), and 90 nM for α5β1 (GRGDS: >30 µM, knottin-RGD: 100 nM). We also labeled the best bicyclic integrin-binders with a fluorescent dye and studied their integrin-binding on live cells. The most potent bicyclic integrin-binders showing highest were also immobilized on 2D hydrogels in order to investigate their effect on cell adhesion, cell proliferation and viability as compared to the conventional RGD-peptides.
 “Astrazeneca takes Bicycle to work in potential $1B multitarget collaboration”, Bioworld™ Today 2016, 27, 3.
 P. Li, P. P. Roller, Curr. Top. Med. Chem. 2002, 2, 325–341; V. Baeriswyl, C. Heinis, ChemMedChem 2013, 8, 377–384.
 M. Barczyk, S. Carracedo, D. Gullberg, Cell Tissue Res. 2010, 339, 269–280; Y. Takada, X. Ye, S. Simon, Genome Biol. 2007, 8, 215.
 D. Bernhagen, L. De Laporte, P. Timmerman, Anal. Chem. 2017, 89, 5991–5997.
 R. H. Kimura, A. M. Levin, F. V Cochran, J. R. Cochran, Proteins 2009, 77, 359–369.