(611a) Self-Removing Affinity Tags to Streamline Preclinical and Clinical Manufacturing Under a Single Purification Platform

The power and simplicity of Protein A affinity technology has made it ubiquitous in the biopharmaceutical industry for research and manufacturing of monoclonal antibodies (mAbs). Unfortunately, no corresponding method has been available for commercial scale manufacturing of non-mAbs (more generally referred to as just “recombinant proteins”). For biopharmaceutical applications, this method would allow any target protein to be purified in its unmodified native form via a single, universal capture method. In preclinical and basic science research, this goal is provided by cleavable affinity tags, which are used to quickly purify targets for initial characterization and preclinical toxicology studies. Full-scale clinical manufacturing processes for these recombinant proteins must then be developed later, and typically require complex multi-column approaches to be developed for each new product.

We propose that research and manufacturing of recombinant proteins could be streamlined under a single platform through the use of self-cleaving affinity tags. After years of work, we have finally developed and now commercialized the iCapTag^TM system. This system consists of an affinity capture resin, similar to Protein A, and a corresponding peptide tag. Strong and selective affinity between the ligand and tag facilitates purification of the target, but once purified, the target protein is released from the bound tag in a native form. The self-cleaving reaction is triggered by a small change in pH, allowing extended loading and washing times, followed by relatively repid release of the tagless target. This system thereby combines the universality of tag-based affinity methods with the ability to produce tagless products for clinical and full-scale manufacturing – all under a single, universal capture platform.

A key requirement for this technology has been the introduction of pH-sensitive cleaving to a split-intein tag. By optimizing key amino acids preceding our affinity ligand and within the tag, we were able to produce a self-cleaving reaction that is dependent purely on a mild change in pH, where no other salts, reducing agents, or enzymes are required. To increase reliability of the method, we have demonstrated that the cleaving reaction is predictably dependent on the first three amino acids of the target protein, allowing users to avoid unexpected failures of the technology while fine-tuning process parameters for their applications. Although this can require modifications of the target protein’s first or second N-terminal amino acids, these modifications are less invasive than those observed in many approved therapeutics. Finally, we have worked hard to develop highly robust and cost-effective manufacturing and regeneration processes for our affinity resin, which will allow its practical use at any scale from bench top to commercial manufacturing. We are now confident that this product will have a significant impact on the bioprocess industry as a truly next generation capture method.

In our most recent work, we have demonstrated this technology with a variety of proteins and prospective biosimilar products expressed in E. coli and mammalian cells. These include Filgrastim (Neupogen^®), Interferon alpha 2b (Viraferon^®), Streptokinase (Streptase^®), Covid-19 Receptor Binding Domain, as well as scFv molecules and other laboratory proteins – all delivered tagless and at high purity from a single capture column. Further, we have shown that the cleaving buffers used for target protein release can be modified with a wide variety of excipients, and allow cleaving directly into buffers that are optimized for subsequent polishing steps, or even directly into the drug’s formulation buffer. In this presentation, we will briefly explain the development of our self-cleaving tag technology and provide several case studies on how the resin performs with conventional mammalian and E. coli expression hosts, including impacts on expression and glycosylation, impurity clearance, recovery, yield and activity for different clinically relevant test proteins.

By providing a universal, scalable and process friendly solution for non-mAbs, this system is uniquely positioned to accelerate discovery and clinical development of many novel therapeutics and next-generation modalities in the coming decades, perhaps with a similar impact to that of Protein A technology on mAb development.