Since their introduction nearly a century ago, protein vaccines and therapeutics have revolutionized our ability to prevent and treat human disease. However, existing production processes for biopharmaceuticals are technically complex and rely on living cells, which necessitates highly centralized manufacturing in large-scale production facilities, specialized equipment, and cold-chain distribution. With increasing demands for medicines tailored to individuals or relatively small patient populations, there is growing interest in scaled down bioprocesses that can accommodate production of many different biologic molecules. In addition, the need for cold-chain refrigeration limits our ability to supply life-saving biologics to underdeveloped regions as well as in emergency situations, prompting efforts to develop on-demand protein production technologies that can be distributed without refrigeration. Overall, technologies for small-scale, decentralized biomanufacturing represent an emerging paradigm that promises to enable portable and personalized protein medicines. Still, existing technologies have been limited in their ability to produce glycosylated protein products, which represent over 70% of protein therapeutics and vaccines approved or in clinical development. This work seeks to address this limitation through the development of new technologies for on-demand biomanufacturing of glycosylated protein therapeutics and vaccines. We first developed cell-free glycoprotein synthesis (CFGpS) technology with the ability to produce glycosylated protein therapeutics at the point-of-care. The CFGpS platform uses crude Escherichia coli cell lysates containing the biological machinery for both protein synthesis and glycosylation to produce glycoproteins in simple, one-pot reactions. We show that CFGpS can produce glycoprotein medicines such as erythropoietin, as well as proteins bearing a range of bacterial and eukaryotic glycans. In parallel, we developed the in vitro bioconjugate vaccine expression (iVAX) platform that enables on-demand and portable biosynthesis of antibacterial vaccines via coordinated cell-free protein synthesis and glycosylation. iVAX reactions can synthesize single doses of vaccines against diverse bacterial pathogens in one hour, including the highly virulent Franciscella tularensis subsp. tularensis (type A) strain Schu S4 and pathogenic E. coli strains O78 and O7. In particular, we showed that anti-F. tularensis vaccines can be produced in iVAX for ~$6 per human dose and elicited pathogen-specific immune responses in mice. Together, the CFGpS and iVAX platforms represent key first steps toward modular, on-demand production of glycosylated protein therapeutics and vaccines, joining an emerging set of decentralized biomanufacturing platforms that promise to increase global access to costly drugs. Finally, we demonstrate that portable, cell-free protein production platforms can be adapted to enable educational kits for teaching molecular and synthetic biology, which we call BioBitsTM kits. The BioBitsTM kits alleviate many of the economic and logistical challenges associated with implementing hands-on molecular and synthetic biology activities in classrooms and other non-laboratory settings. As such, these kits have the potential to increase scientific literacy through the integration of cutting-edge molecular and synthetic biology topics into K-12 STEM education.

Last modified

• 02/12/2020

Creator

• Stark, Jessica C

DOI

• https://doi.org/10.21985/n2-xca6-cj67

Subject

• Chemical engineering

Language

• en

Alternate Identifier

• etdadmin_upload_686044
• http://dissertations.umi.com/northwestern:14826

Keyword

• glycoprotein therapeutics
• cell-free protein synthesis
• decentralized biomanufacturing
• synthetic biology
• STEM education
• conjugate vaccines

Date created

• 2019-01-01

Resource type

• Dissertation

Rights statement

• In Copyright