Abstract 4588: Polylysine linker for successful manufacturing of hydrophobic peptides

Abstract

Abstract Hydrophobic peptides are common to be used in cancer vaccinations. Hydrophobic peptides, continue to present challenges for large scale production in peptide industry. Experimental Procedures: Full sequence prepared by Solid phase Synthesis:The peptide was synthesized with Fmoc-Lys(Boc)-Wang resin by using standard Fmoc/tBu chemistry. Protecting groups used for amino acids are: tBu for Thr, Boc for Trp and Lys and Boc-Ser- OH was used as a hydrolysis-labile ester provider. Fmoc-Leu-OH was coupled on the side chain of Ser by DIC/DMAP (Amino acid/DIC/DMAP, 3/1.5/0.3 eq). The other amino acids were assembled by repetitive removal of the Fmoc protecting group by treating with 2% DBU/2% piperidine in DMF 30 minutes and coupling of protected amino acid by using DIC/HOBt/NMM (4:4:4 eq) in 180 minutes to overnight. Ninhydrin test was performed after each coupling to check the coupling efficiency. After the last coupling,resin was washed and dried, then treated with reagent K for cleavage and removal of the side chain protecting groups. Crude peptide was precipitated from cold ether and collected by filtration. Table.1 Summary of 4 synthesize results Syn. NoLinker StructurePurity of CrudeDKP deletion: Product Hydrolysis YieldOverall Yield1No Linker61.4%12.3%2(O)-Ser56.5%1:252.8%12.5%3(O)-Ac-Ser40.8%1:370.6%28.2%4HMBA78.1%<1:2451.0%61.2% Conclusion: American Peptide demonstrated the utility of a hydrophilic peptide ‘tail’ in purification of a hydrophobic peptide on both small (20-30 mmol) research scale and large (800 mmol) cGMP scales to provide the peptide in hundreds-of-grams quantities. The approach described allowed for both time-and cost-competitive manufacturing of this difficult peptide for clinical trials. Citation Format: Firuz Shakoori, Archana Gangakhedkar. Polylysine linker for successful manufacturing of hydrophobic peptides. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4588. doi:10.1158/1538-7445.AM2014-4588