Improved endotoxin removal using ecofriendly detergents for intensified plasmid capture

Abstract

Increasing plasmid demand for both production of viral and gene therapies as well as nucleic acid based vaccines has highlighted bottlenecks in production. One bottleneck is traditional bead‐based chromatography as a capture step. To meet the needs of fast‐growing markets, new production solutions are needed. These solutions must enable efficient capture of a diverse range of plasmid types and excellent clearance of bacterial host impurities, such as endotoxin. Enhanced endotoxin clearance during chromatographic purification has previously been demonstrated with detergents such as Triton™ X‐100. However, degradation products of Triton™ X‐100 are known to have a negative environmental impact, and more sustainable, environmentally benign alternatives have been identified. This work establishes an efficient, intensified plasmid capture using convective anion exchange (AEX) chromatography. The feasibility of the intensified capture approach was assessed with different membrane and a monolith AEX supports. Various detergents from different physico‐chemical classes were evaluated with different AEX technologies. Purification efficiency evaluated endotoxin and host cell protein (HCP) clearance, plasmid yield, potential interference of the detergents with analytical in‐process control assays, and overall process compatibility. This comprehensive screening approach provides valuable insights to intensified plasmid production.

Engineering an Escherichia coli based in vivo mRNA manufacturing platform

“…Additionally, substantial increases in purity, as would be required for clinical contexts, could be achieved by (i) full optimization of chromatographic critical process parameters, such as buffer conditions and flow rates, and (ii) minimization of product degradation by producing mRNA under good manufacturing practice compliant conditions. Finally, for in vivo applications, it is essential that fully optimized two‐step chromatographic purification processes remove host cell DNA and endotoxin impurities, as is routinely achieved for protein and DNA products manufactured in E. coli (Kiesewetter et al, 2023). A simplified conceptual process flow diagram for large‐scale and small‐scale in vivo mRNA production processes is shown in Figure 5a.…”