A Scalable Cell-Free Manufacturing Platform for Two-Step Bioproduction of Immunogenic Conjugate Vaccines.

Rapid and decentralized vaccine production is essential to ensure global preparedness against emerging and re-emerging infectious diseases. Cell-free gene expression systems, which can be freeze-dried for long-term storage and reactivated for point-of-use synthesis, offer a promising solution to address this need. However, scalable cell-free production of conjugate vaccines─highly effective tools against bacterial infections─has been hindered by low yields and inefficient glycosylation. To address these challenges, we developed a modular, cell-free platform for the synthesis and purification of conjugate vaccines. By decoupling cell-free protein expression from in vitro glycosylation in a two-step approach, we achieved >85% glycosylation efficiency and up to ∼450 mg/L of glycoprotein. We applied this platform to manufacture protein-polysaccharide conjugates composed of vaccine carrier proteins covalently modified with polysaccharide antigens from enterotoxigenic Escherichia coli O78 and Streptococcus pneumoniae serotype 4. Our workflow produced conjugate vaccine candidates in under 5 days with >87% product purity and low endotoxin levels suitable for preclinical evaluation. Immunization of mice with the pneumococcal conjugate vaccine induced a strong IgG response against S. pneumoniae serotype 4 capsular polysaccharide, confirming the immunogenicity of the conjugate. We anticipate that this cell-free platform will advance efforts in decentralized manufacturing and rapid response to bacterial disease threats.