Princeton University Library Catalog

The Optimization of Streamlined Expressed Protein Ligation for Antibodies

Willis, John Alan [Browse]
Senior thesis
Muir, Thomas [Browse]
Princeton University. Department of Chemistry [Browse]
Class year:
92 pages
Restrictions note:
Walk-in Access. This thesis can only be viewed on computer terminals at the Mudd Manuscript Library.
Summary note:
The use of antibody-drug conjugates in medicine shows considerable therapeutic promise and the generation of these proteins is a hot topic in protein engineering. The recently developed Streamlined Expressed Protein Ligation(SEPL) greatly simplifies the generation of proteins with site-specific modifications in high yield, and is therefore an appealing tool for manufacturing antibody drug conjugates. However, the technique has a lower yield for antibodies than for many other proteins, due in part to significant antibody hydrolysis. I worked closely with Miquel Vila-Perelló to overcome this impediment to using SEPL to create antibody drug conjugates. Using a cysteine based hydrolysis assay, hydrolysis of the free antibody thioester was determined to be the source of much of the yield decreasing hydrolysis. An Npu-based thiolysis assay reveals that thiocholine, methylthioglycolate and thioglycolate may perform as well as the traditional MESNa in generating antibody Thioesters. Intein cognate pairing was shown to be unnecessary for high yield antibody SEPL using a synthetic AvaC peptide on a SEPL column. Overall, these results indicate that using concentrated, CHO expressed antibody supernatant with a high-speed thiol at a more acidic pH may show less hydrolysis than the previously investigated conditions.