Peptide- and protein-based drugs and the technologies used for their assembly are becoming increasingly important. In spite of considerable improvements in both solid phase chemistry and recombinant procedures, the commercial manufacture of many long peptides and post-translationally modified proteins remain beyond the bounds of available methodology. Any route open to achieving the assembly of this class of molecule will most probably involve a hybrid fragment approach using either solution chemistry, native chemical ligation (NCL) or enzymatic ligation. However, while NCL has been used to manufacture a plethora of peptides and proteins, it has yet to be used in large-scale commercial manufacture due to scalability issues. A number of natural and engineered enzymes have been used to ligate polypeptides. Most of these are thermodynamically driven and do not achieve ample conversion to support commercial manufacture. Sortases, butelase and trypsiniligase - like NCL - leave foreign footprints in the final product. In the last four years, we have produced over 250 novel ligases offering a wide range of selectivity, such that 90% of all peptide fragments are recognized and can be coupled - free of footprints - to each other. These enzymes have been used to manufacture long peptides and protein conjugates at a range of scales. They can also be used to site selectively address the A- and B-chains of insulins and the VH and VL chains of Fab fragments.
Timo Nuijens graduated in Drug Innovation at the University of Utrecht. He performed his Ph.D. project on enzymatic peptide synthesis within DSM Innovative Synthesis B.V. (Geleen) under the supervision of Dr. Peter Quaedflieg (DSM Innovative Synthesis B.V.) and Prof. Rob Liskamp (Univ. Utrecht). His Ph.D. was obtained in May 2012. Since mid 2012 he is working as a lead scientist at Enzypep and has the responsibility of the scientific development and co-ordinating the R&D team in Geleen.