(336g) Intein-Based Bioseparations: Development of a Disruptive Technology for a Conservative Industry

Over the past 20 years, my laboratory has worked on the development self-cleaving affinity tags for the capture and initial purification of recombinant proteins. The goal of this work is a truly general platform for the traceless and tagless affinity purification of virtually any target protein expressed in any recombinant host. Although tag-based affinity methods are common at laboratory scale, the expense and complexity of tag removal have prevented these methods from being adopted for the manufacture of therapeutic recombinant proteins. Thus, the development of a simple and inexpensive self-cleaving system constitutes a disruptive advance, with the potential to resolve tag removal issues and provide a new platform unit operation for the preparation of therapeutic products. Concurrent with our research, the biopharmaceutical industry has undergone major transformations. These include explosive increases in recombinant protein titer, the anticipation of biosimilars as patents expire, the increasing adoption of disposable single-use technology, and the growing interest in continuous processes in both upstream and downstream processing. In this presentation, I will discuss our development of intein-based self-cleaving tag methods in the context of the changing biopharmaceutical industry. This will include the basic approaches used to engineer new self-cleaving tags, and how those approaches relate to industry pressures in the highly regulated and conservative biopharmaceutical industry. In our most recent work, we have demonstrated the ability to purify fully processed glycoproteins from mammalian cell culture hosts, and we have incorporated this method into a portable biopharmaceutical factory platform. In both of these cases, we have completely eliminated premature cleavage of the intein tag through the adoption of a split-intein architecture, and we have introduced several additional mutations to impart highly pH-sensitive cleavage for tag removal. This platform is now capable of providing highly purified targets, in a traceless and tagless form using a single column step, from virtually any expression system capable of producing the folded protein. The simplicity of this approach suggest a strong future in the rapid development of new biosimilars, as well as additional applications in pure research and the production of novel non-mAb targets.