Rapid Antibody Fragment Production and Binding Analysis Using Cell-Free Protein Synthesis Combined with Fluorescence Correlation Spectroscopy

ABSTRACT This study investigates the efficient development and production of single-chain variable fragments (scFvs) and antibody fragments (Fabs) using an E. coli-based cell-free protein synthesis system. Validation of the methodology was performed using a fluorescence correlation spectroscopy (FCS)-based assay to determine binding equilibrium constants (K D) between antibodies and the receptor binding domain (RBD) of SARS-CoV-2 Spike protein. An initial assessment employed two conventionally cell-produced anti-RBD antibodies. To optimize cell-free production, folding strategies were developed to enhance the solubility and yields of scFvs, including a two-stage refolding protocol that successfully recovered active proteins from misfolded precipitates. Fab fragments were also produced and characterized, with their binding properties analyzed to assess functionality. This study highlights the potential of cell-free systems for the rapid and efficient production of functional antibody fragments. The integration of advanced techniques, such as FCS-based kinetic measurements, underscores the versatility and applicability of cell-free platforms for antibody development and high-throughput screening. These findings offer a promising avenue for accelerating therapeutic antibody research and production. SIGNIFICANCE of WORK This study highlights the transformative potential of integrating cell-free protein synthesis (CFPS) with fluorescence correlation spectroscopy (FCS) for the rapid and scalable production and characterization of functional antibody fragments. By leveraging an E. coli-based CFPS platform, we successfully designed and optimized the production of single-chain variable fragments (scFvs) compared to Fab fragments, addressing some common challenges of low solubility and yield. The development of a cost-efficient two-stage refolding strategy further enhanced the scalability and functionality of scFvs, enabling higher recovery of active proteins from the unfolded state. Additionally, FCS provided a sensitive, rapid method for accurately quantifying antigen-antibody binding kinetics across a range of affinities. This work establishes CFPS and FCS as versatile and complementary tools, offering a robust framework for accelerating antibody fragment development, particularly in time-sensitive scenarios like infectious disease outbreaks.