On-demand, cell-free biomanufacturing of conjugate vaccines at the point-of-care

Abstract

Conjugate vaccines are among the most effective methods for preventing bacterial infections, representing a promising strategy to combat drug-resistant pathogens. However, existing manufacturing approaches limit access to conjugate vaccines due to centralized production and cold chain distribution requirements. To address these limitations, we developed a modular technology for in vitro bioconjugate vaccine expression (iVAX) in portable, freeze-dried lysates from detoxified, nonpathogenic Escherichia coli. Upon rehydration, iVAX reactions synthesize clinically relevant doses of bioconjugate vaccines against diverse bacterial pathogens in one hour.We show that iVAX synthesized vaccines against the highly virulent pathogen Franciscella tularensis subsp. tularensis (type A) strain Schu S4 elicited pathogen-specific antibodies in mice at significantly higher levels compared to vaccines produced using engineered bacteria. The iVAX platform promises to accelerate development of new bioconjugate vaccines with increased access through refrigeration-independent distribution and point-of-care production.

Improving cell-free glycoprotein synthesis by characterizing and enriching native membrane vesicles

“…We To assess the impact of enriched vesicles on cell-free glycoprotein synthesis, we carried out reactions in two phases (Fig. 4A, Inset) (38). First, cell-free protein synthesis (CFPS) of the acceptor protein was run for a defined time, termed 'CFPS time'.…”

“…A body of work dedicated to optimization of extract preparation and reaction conditions has simplified, expedited, and improved the cost and performance of E. coli CFE systems (22,28,29). Optimized E. coli-based CFE reactions: (i) quickly synthesize grams of protein per liter in batch reactions (30)(31)(32), (ii) are scalable from the nL to 100 L scale (33,34), and (iii) can be freeze-dried for months of shelf-stability and distribution to the point of care (6,22,29,(35)(36)(37)(38)(39)(40). Freeze-dried CFE systems are poised to make disruptive impacts in biotechnology, having already been leveraged for point-of-use biosensing (41)(42)(43)(44)(45)(46), therapeutic and vaccine production (37,38,47), and educational kits (22,(48)(49)(50).…”

“…We recently described cellfree glycoprotein synthesis (CFGpS), a platform for one-pot biomanufacturing of defined glycoproteins in extracts enriched with heterologous, membrane-bound glycosylation machinery (47). To date, CFGpS has been used to produce model glycoproteins, human glycoproteins, and conjugate vaccines (38,47,(58)(59)(60). Importantly, CFGpS reactions can be freeze-dried for shelfstability and rehydrated at the point of care to make effective vaccines (38).…”

“…To date, CFGpS has been used to produce model glycoproteins, human glycoproteins, and conjugate vaccines (38,47,(58)(59)(60). Importantly, CFGpS reactions can be freeze-dried for shelfstability and rehydrated at the point of care to make effective vaccines (38). Because CFGpS activity relies on membrane-bound glycosylation components, methods to characterize and quality-control the membrane-bound components in extracts is paramount for moving the technology forward.…”

The Evolution of Cell Free Biomanufacturing

“…The ability to add membrane mimics such as surfactants or liposomes to the extract as stability agents has allowed the production of membrane proteins [48][49][50][51]. Vaccines [44,52], protein assemblies [53][54][55], and proteins incorporating non-natural amino acids [56][57][58] have also been synthesized using CFPS systems. Disulfide-bonded proteins and antibodies have also been synthesized by adding components that facilitate the formation of these bonds to the mixture such as glutathione reductase, thioredoxin reductase, iodoacetamide, and disulfide isomerase (DsbC) [26,29,59,60].…”