Northwestern University synthetic biologists have developed a new way to increase five-fold the production yields of protein-based vaccines, significantly broadening access to potentially lifesaving medicines.

The newly reported work builds on the team’s recently developed iVAX (in vitro conjugate vaccine expression) cell-free gene expression (CFE) platform for vaccine manufacture, which can quickly make shelf-stable vaccines at the point of care. The latest development has shown how enriching cell-free extracts with vesicles composed of cellular membrane— the components needed to made conjugate vaccines—improves glycoprotein synthesis, to vastly increase yields of the freeze-dried platform.

“Certainly, in the time of COVID-19, we have all realized how important it is to be able to make medicines when and where we need them,” said Northwestern’s Michael Jewett, PhD, who led the study. “This work will transform how vaccines are made, including for bio-readiness and pandemic response.” Jewett is a professor of chemical and biological engineering at Northwestern’s McCormick School of Engineering and director of Northwestern’s Center for Synthetic Biology.

The team’s work is published in Nature Communications, in a paper titled, “Improving cell-free glycoprotein synthesis by characterizing and enriching native membrane vesicles.” Jasmine Hershewe and Katherine Warfel, both graduate students in the Jewett laboratory, are co-first authors of the paper.

Cell-free gene expression systems activate transcription and translation using crude cellular extracts instead of living, intact cells, the authors explained. The technology essentially involves removing a cell’s outer wall (or membrane) and repurposing its internal machinery. “In recent years, these systems have matured from widely used tools in molecular biology to platforms for biomanufacturing and synthetic biology,” the authors continued. “Among CFE systems, Escherichia coli-based methods have been used the most.”

Optimized E. coli-based CFE systems can rapidly synthesize proteins, are scalable, and can be freeze dried, enabling months of shelf stability, the team continued. “The ability to readily store, distribute, and activate freeze-dried cell-free systems by simply adding water has opened new opportunities for point-of-use biosensing, portable therapeutic and vaccine production, and educational kits.”

The Northwestern University team recently developed a cell-free synthetic biology platform known as the iVAX manufacturing platform. The platform can be freeze dried, and adding water sets off a chemical reaction that activates the cell-free system, turning it into a catalyst for making usable medicines when and where needed. Remaining shelf stable for six months or longer, the platform eliminates the need for complicated supply chains and extreme refrigeration, making it a powerful tool for remote or low-resource settings, and potentially allowing on demand manufacturing of medicines, including protective vaccines.

In their previous study, Jewett’s team used the iVAX platform to produce conjugate vaccines to protect against bacterial infections. At the time, they repurposed molecular machinery from Escherichia coli to make one dose of vaccine in an hour, costing about $5 per dose. However, as Jewett noted, “It was still too expensive, and the yields were not high enough. We set a goal to reach $1 per dose and reached that goal here. By increasing yields and lowering costs, we thought we might be able to facilitate greater access to lifesaving medicines.”

Jewett and his team discovered that the key to reaching that goal lay within the cell’s membrane, which is typically discarded in cell-free synthetic biology. When broken apart, membranes naturally reassemble into vesicles, spherical structures that carry important molecular information.

The authors explained, “Despite the absence of intact cellular membranes, membrane structures are present in crude extract-based CFE systems.” Yet while there have been some examples of cell-derived membranes incorporated compounds to boost bacterial CFE systems. “… this area of research has remained understudied,” the investigators continued. “ … Yet, enriching native membrane-bound components in CFE systems, especially with heterologously expressed cargo, is poised to enable compelling applications.”

Protein glycosylation, for example, is mediated by membrane-bound components, and can have a profound impact on the folding, stability, and activity of proteins and therapeutics. “Introduction of cell-derived vesicles with machinery required for glycosylation could enable cell-free biomanufacturing of protein therapeutics and vaccines, especially for the point of care,” Jewett and colleagues noted.

Glycosylation may involve attaching to a carrier protein a sugar molecule that is unique to a pathogen. By learning to recognize that protein as a foreign substance, the body knows how to mount an immune response to attack it when encountered again. Attaching the sugar molecule to the carrier protein, however, is a difficult, complex process. Through their newly reported work, the researchers found that enriching vaccine extracts using membrane-bound machinery could significantly increase yields of usable vaccine doses.

They first characterized the vesicles and applied optimized methods to increase concentrations of vesicle-bound glycosylation machinery into the CFE system. “In this study, we aimed to characterize and engineer membrane vesicles (which form upon fragmentation of cell membranes during cell lysis) in E. coli CFE extracts,” they explained “Then, we used this knowledge to control enrichment of membrane-bound components for enhancing defined function, including improving glycoprotein synthesis.”

Their results showed how increasing vesicle concentration could be useful for enhancing the system’s glycosylation capacity for making the glycoprotein components for protein therapeutics such as conjugate vaccines. “By applying our optimized methods to increase concentrations of vesicle-bound glycosylation machinery, we shorten the time associated with extract preparation, increase glycosylation efficiencies, and enhance glycoprotein titers by up to ~170%,” the authors wrote. “… our work opens the door to engineering cell-free systems that rely on enriched membrane-bound components … Looking forward, we anticipate that our work will accelerate efforts to manufacture proteins that require membrane dependent modifications, such as glycoproteins.”

“For a variety of organisms, close to 30% of the genome is used to encode membrane proteins,” commented study co-author Neha Kamat, PhD, who is an assistant professor of biomedical engineering at McCormick and an expert on cell membranes. “Membrane proteins are a really important part of life. By learning how to use membrane proteins effectively, we can really advance cell-free systems.”

The work sets the stage to rapidly make medicines that address rising antibiotic-resistant bacteria as well as new viruses at 40,000 doses per liter per day, costing about $1 per dose. At that rate, the team could use a 1,000-liter reactor (about the size of a large garden waste bag) to generate 40 million doses per day, reaching 1 billion doses in less than a month. The authors concluded, “our results pave the way for efficient, accessible CFE systems that require membrane-bound activities for expanding system functionality and enabling a variety of synthetic biology applications.”