(664g) Directed Evolution of Self-Cleaving Intein Purification Tags with Improved Cleavage Kinetics

Authors: 
Stimple, S. D., The Ohio State University
Coolbaugh, M. J., The Ohio State University
Wood, D. W., the Ohio State University

The lack of a purification platform technology for non-mAb (non-monoclonal antibody) proteins represents a primary impediment to product and process development in the biopharmaceutical industry. Advances in mAb-based technologies have been greatly bolstered by the development of protein A affinity chromatography into a generalizable platform for the purification of IgGs. Nevertheless, to date, no such platform exists for non-mAb products. Purification of non-mAb proteins has historically been problematic and costly, with each product purified using a uniquely optimized combination of chromatographic steps. The use of affinity purification tags represents a potential purification platform for non-mAb proteins, but the high costs associated with the tag removal step have greatly diminished the industrial relevance of such technologies. Self-cleaving intein purification tags circumvent the complications accompanying tag removal; however their use is currently limited to bacterial expression due to premature cleavage (leading to product loss) of the intein tag at mammalian cell culture conditions. This work aims to develop a CHO-suitable self-cleaving intein.

We have developed a yeast surface display-based platform for the directed evolution of inteins that have slower, more desirable, cleavage kinetics at the temperature and pH of CHO cell culture. Further screening experiments with the selected mutants have indicated that some of these mutant inteins also appear to retain full cleavage activity under purification conditions. This makes them ideal candidates for use in mammalian cell expression due to the enhanced pH-and-temperature sensitivity of the cleavage reaction. These mutants could also serve as the template/basis for future rounds of mutagenesis that may lead to the development of inteins possessing cleavage kinetics that are even more drastically pH-and-temperature dependent.