Virus-like particles (VLPs) are macromolecular structures with great potential as vehicles for targeted administration of functional molecules. Along with lipid nanoparticles and viral vector technologies, VLPs loaded with nucleic acids are a promising approach for nanocarriers needed for gene therapy.<br/>The VLP’s therapeutic efficacy is directly dependent on the loading and phase behaviour, and bound nucleic acids considerably affect both central downstream purification steps in the manufacturing process and analytical methods used in process development. The disassembly and subsequent reassembly reactions are frequently used for purification of these molecules being strongly dependent on the capsid stability, presumably affected by the presence of nucleic acids. To integrate the loading of VLPs with therapeutic nucleic acids into the downstream process, further general knowledge needs to be derived.<br/>The presented study investigates the phase behaviour and purification process of HBcAg VLPs in dependence of liquid phase conditions and presence of nucleic acid. Further, it demonstrates challenges associated with standard analytical methods used in process development for VLPs loaded with nucleic acids, proposing an optimized sample pretreatment method applicable for several analytical methods. HBcAg VLPs with different lengths of a nucleic acid binding site were produced in <i>E. coli</i>. The effects of both bound host cell nucleic acids and bound target nucleic acids were systematically investigated for different purification process steps including the capturing by precipitation, disassembly and reassembly. The purity and phase behavior of the VLPs were measured by capillary electrophoresis and size exclusion chromatography. For a reliable quantification of nucleic acids bound to VLPs by fluorescence dyes and qPCR, a column based nucleic acid extraction procedure was optimized and evaluated. In this study, effects of the presence of bound nucleic acids on the capsid stability were observed and a resulting correlation between this capsid stability and the purification process could be derived.

Influence of bound nucleic acids on the purification process and applied analytical methods of HBcAg virus-like particles for gene therapy

ACS Spring 2022

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