Dual-Stage Cross-Flow Filtration: Integrated Capture and Purification of Virus-Like Particles.

Virus-like particles (VLPs) are a versatile technology for the targeted delivery of genetic material through packaging and potential surface modifications for directed delivery or immunological issues. Although VLP production is relatively simple as they can be recombinantly produced using microorganisms such as Escherichia coli, their current downstream processing often relies on individually developed purification strategies. Integrating size-selective separation techniques may allow standardized platform processing across VLP purification. This study presents an innovative dual-stage cross-flow filtration (CFF) set-up for integrated capture and purification of VLPs, enabling processing solely based on the size-selective separation techniques precipitation and filtration. The 2 μm/300 kDa MWCO membrane configuration allows the seamless integration of selective VLP precipitation, two consecutive diafiltration steps-first, for washing the VLP precipitates in the first membrane stage, and second, for isolating the re-dissolved VLPs by continuously removing precipitant and contaminants in the second membrane stage-and ultrafiltration for setting a target VLP concentration. Compared to a single-stage CFF set-up, this dual-stage CFF set-up with its integrative, automated design demonstrated the capabilities of product accumulation and contaminant handling while maintaining high productivity. Overall, this study represents a significant advancement toward standardized platform processing of protein nanoparticles through precipitation and filtration, and underscores the potential to expand its applicability to diverse biological molecules, unique process conditions, other phase behavior-dependent processes, and continuous processing.