Dietrich Annabelle, Heim Luca, Hubbuch Jürgen
Institute of Process Engineering in Life Sciences, Section IV: Biomolecular Separation Engineering, Karlsruhe Institute of Technology (KIT), Karlsruhe, Germany.
Biotechnol Bioeng. 2025 Apr;122(4):884-894. doi: 10.1002/bit.28914. Epub 2024 Dec 26.
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.
病毒样颗粒（VLPs）是一种多功能技术，可通过包装以及针对定向递送或免疫问题的潜在表面修饰来靶向递送遗传物质。尽管VLP的生产相对简单，因为它们可以使用诸如大肠杆菌等微生物进行重组生产，但其当前的下游加工通常依赖于单独开发的纯化策略。整合尺寸选择性分离技术可能允许在VLP纯化过程中采用标准化的平台工艺。本研究提出了一种创新的双级错流过滤（CFF）装置，用于VLPs的集成捕获和纯化，仅基于尺寸选择性分离技术（沉淀和过滤）进行处理。2μm/300 kDa截留分子量的膜配置允许选择性VLP沉淀的无缝集成，两个连续的渗滤步骤——首先，在第一膜阶段洗涤VLP沉淀，其次，通过在第二膜阶段连续去除沉淀剂和污染物来分离重新溶解的VLPs——以及用于设定目标VLP浓度的超滤。与单级CFF装置相比，这种具有集成自动化设计的双级CFF装置在保持高生产率的同时，展示了产品积累和污染物处理的能力。总体而言，本研究代表了通过沉淀和过滤对蛋白质纳米颗粒进行标准化平台加工的重大进展，并强调了将其应用扩展到各种生物分子、独特工艺条件、其他依赖相行为的过程以及连续加工的潜力。
