Biology subjects

Aw, R.

Publications and source records attributed to Aw, R..

3 recordsLinked to original sources

bioRxiv2025

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 re-activated 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. Here, we present an optimized, 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 achieve >85% glycosylation efficiency and up to [~]450 mg/L of glycoprotein. We apply this platform to manufacturing 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 the 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.

synthetic biology↗

bioRxiv2025

Design-driven optimization of low-cost reagent formulations for reproducible and high-yielding cell-free gene expression

Access to recombinant proteins is vital in basic science and biotechnology research. Cell-free gene expression systems provide one approach to address this need, but widespread utilization remains limited by the cost, complexity, and inconsistency of current platforms. To address these limitations, we carry out a multi-dimensional definitive screening design to reduce the number of reagent components and remove costly secondary energy substrates. From more than 1,200 reagent formulations, we discover a simple and reproducible system based on 12 components. The optimized reagent formulation can produce 2.4 {+/-} 0.3 g/L of protein product at the 15-L scale ([~]$55/gprotein) and 3.7 {+/-} 0.2 g/L ([~]$36/gprotein) at the 4-mL scale with oxygen supplementation. This provides an 84 to 99% reduction in cost over previous cell-free reagent formulations. We further show that the optimized reagent formulation can produce nucleoside triphosphates from nucleotides and ribose and that it is robust to failure across batches of cell lysates, users/locations, and in the synthesis of different proteins. Specifically, we demonstrate the production of fifteen therapeutically relevant products, including full-length aglycosylated monoclonal antibodies. We anticipate that our optimized reagent formulation will further democratize the use of cell-free systems for protein manufacturing and synthetic biology applications.

synthetic biology↗

bioRxiv2024

Scalable cell-free production of active T7 RNA polymerase

The SARS-CoV-2 pandemic highlighted the urgent need for biomanufacturing paradigms that are robust and fast. Here, we demonstrate the rapid process development and scalable cell-free production of T7 RNA polymerase, a critical component in mRNA vaccine synthesis. We carry out a one-liter cell-free gene expression (CFE) reaction that achieves over 80% purity, low endotoxin levels, and enhanced activity relative to commercial T7 RNA polymerase. To achieve this demonstration, we implement rolling circle amplification to circumvent difficulties in DNA template generation, and tune cell-free reaction conditions, such as temperature, additives, purification tags and agitation to boost yields. We achieve production of a similar quality and titer of T7 RNA polymerase over more than 4 orders of magnitude reaction volume. This proof of principle positions CFE as a viable solution for decentralized biotherapeutic manufacturing, enhancing preparedness for future public health crises or emergent threats.

synthetic biology↗