Production, purification, and process development of recombinant flagellin vaccine platform in Escherichia coli

Salmonella enterica ser.Typhimurium type Bflagellin monomer (FliC), when recombinantly expressed with an antigen, is an activator of cell types involved in innate and adaptive immunity by TLR5 stimulation. Large-scale production of recombinant full-length and fusion flagellin constructs has been achieved in E. coli. However, manufacturing outcomes vary with inserted antigen properties, and in vitro screening of fusion constructs is costly and time-consuming. In this study, scalable process manufacturing and high-throughput in silico screening of recombinant flagellin-based products were characterized. Full-length FliC was expressed in E. coli in both flask and bioreactor scale, subsequently purified, and compared to research-grade commercial FliC standard. To aid rapid in silico screening of recombinant FliC fusion constructs, a modified Synthetic FliC platform housing multiple restriction enzyme sites in the non-conserved hypervariable D3and terminal region was used to incorporate Respiratory Syncytial Virus (RSV) protein fragments G (aa 130 – 230) and conformation-sensitive F (Palivizumab-binding antigenic site II, aa 253 - 278). Fragments were inserted in multiple locations with varied length linker piece attachments and screened in silico for physiochemical properties, epitope conformation, and epitope exposure. Out of 22 FliC-RSV fusion candidates screened, five were selected, cloned into an inducible expression vector, transformed in E. coli BL21, and overexpressed in flasks or bioreactor. Expressed fusion protein was purified by PEG precipitation, 2-phase separation, and column chromatography, and assessed for bioactivity by indirect ELISA, specific TLR5 assay, and endotoxin levels using the LAL chromogenic kinetic assay. All FliC-RSVF fusion proteins demonstrated TLR5-activity, specific antibody affinity, \u3e95% purity, low manufacturing cost and time, and FDA acceptable endotoxin levels. These findings highlight the potential for a scalable, flexible, and cost-effective flagellin platform that is compatible with rational vaccine design, allowing rapid screening, production, and universal process standardization