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J Taris Dissertation (FINAL) 2021.pdf (7.57 MB)

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Development of a Novel Intein-Mediated Affinity Capture Platform for Production of Recombinant Proteins and Biopharmaceuticals

Taris, Joseph Edward
http://orcid.org/0000-0001-9704-9385
http://rave.ohiolink.edu/etdc/view?acc_num=osu163853540249581

Abstract Details

2021, Doctor of Philosophy, Ohio State University, Chemical Engineering.
The purification of recombinant proteins is critical to fundamental research and pharmaceutical development. In clinical manufacturing, biopharmaceuticals must be highly purified to meet stringent safety guidelines, and thus require a downstream purification process tailored specifically to each drug product. Paradoxically though, discovery-phase pharmaceutical research depends on screening diverse arrays of candidate molecules, making product-specific purification schemes impractical. This creates an inherent bottleneck in the transition from drug discovery to clinical evaluation to production-scale manufacturing. As a result, platform purification technologies like affinity chromatography – which enable highly specific isolation of proteins yet require minimal product-specific process optimization – have become a critical driver of pharmaceutical development. Affinity capture is performed using a stationary-phase substrate functionalized for direct affinity to a specific peptide or protein domain. In the absence of a known, convenient, naturally occurring affinity domain within a given protein of interest, an ‘affinity tag’ is recombinantly appended to the purification target to facilitate selective isolation of the desired product. Introducing an affinity tag is a fairly trivial modification with modern cloning and heterologous expression techniques, and this approach is widely practiced at bench scale due to its inherent versatility; affinity tag function is generally agnostic to the protein that the tag is fused with, thus, a single affinity tag platform can purify a variety of hypothetical products. Despite this utility, affinity tags have a few fundamental limitations that are particularly problematic for pharmaceutical production. Tags may alter structure or inhibit function of their fusion partners, and many tags are immunogenic and thus cannot be administered in fusion to a therapeutic. In response to these problems, several proteolytic techniques have been developed for post-purification affinity tag removal, however, these methods add processing complexity and generally are prohibitively expensive to scale up for biopharmaceutical manufacturing. This creates a dichotomy in the pharmaceutical development pipeline: affinity tags are used extensively in research and discovery to identify lead candidates, however, bespoke purification processes must then be developed and individually optimized to reproduce tagless analogs of each candidate for clinical evaluation. The work presented here aims to address these issues with the development of a novel split intein affinity capture platform. As demonstrated herein, split intein variants can be engineered to facilitate protein capture via natural affinity between their counterpart subunits, which upon assembly will catalyze a self-cleaving reaction, thereby removing the affinity tag while purifying the protein in a single-column capture process. Importantly, this platform leverages the advantages of traditional affinity tag capture, but produces a truly native, tagless and traceless product. By providing a versatile, unified platform – supporting all stages from discovery through manufacturing – this approach could significantly streamline the biopharmaceutical research and development. The work described covers the protein engineering used to develop intein activity amenable for purification; demonstrates techniques for high-throughput screening and characterization by means of automated parallel micro-scale purifications; describes predictive models for process scale up; and presents critical improvements to manufacturing of the intein capture media, which are imperative for the economic viability of the platform.
David Wood (Advisor)
Andre Palmer (Committee Member)
Jeffrey Chalmers (Committee Member)
277 p.
Biochemistry; Chemical Engineering; Molecular Biology; Pharmaceuticals
Biopharmaceuticals; Bioprocessing; Bioseparations; Downstream Process Development; Chromatography; Affinity Chromatography; Inteins; Split Inteins; Affinity Tag Removal; Protein Purification; Protein Engineering; Nostoc Punctiforme (Npu) DnaE;

Recommended Citations

Citations

  • Taris, J. E. (2021). Development of a Novel Intein-Mediated Affinity Capture Platform for Production of Recombinant Proteins and Biopharmaceuticals [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu163853540249581

    APA Style (7th edition)

  • Taris, Joseph. Development of a Novel Intein-Mediated Affinity Capture Platform for Production of Recombinant Proteins and Biopharmaceuticals. 2021. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu163853540249581.

    MLA Style (8th edition)

  • Taris, Joseph. "Development of a Novel Intein-Mediated Affinity Capture Platform for Production of Recombinant Proteins and Biopharmaceuticals." Doctoral dissertation, Ohio State University, 2021. http://rave.ohiolink.edu/etdc/view?acc_num=osu163853540249581

    Chicago Manual of Style (17th edition)

osu163853540249581
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© 2021, all rights reserved.
This open access ETD is published by The Ohio State University and OhioLINK.