Absolute Quantification of Hepatitis B Core Antigen (HBcAg) Virus-like Particles and Bound Nucleic Acids.

Effective process development towards intensified processing for gene delivery applications using Hepatitis B core Antigen (HBcAg) virus-like particles (VLPs) relies on analytical methods for the absolute quantification of HBcAg VLP proteins and bound nucleic acids. We investigated a silica spin column (SC)-based extraction procedure, including proteinase K lysis and silica chromatography, for the absolute quantification of different species of nucleic acids bound to HBcAg VLPs analyzed by dye-based fluorescence assays. This revealed load-dependent nucleic acid recoveries of the silica-SC-based extraction. We also developed a reversed-phase high-performance liquid chromatography (RP-HPLC) method to separate and quantify the HBcAg proteins and the bound nucleic acids simultaneously without prior sample treatment by dissociation reagents. The method demonstrated sufficient linearity, accuracy, and precision coefficients and is suited for determining absolute protein and nucleic acid concentrations and HBcAg protein purities at various purification stages. Both the silica-SC-based extraction and the RP-based extraction presented overcome the limitations of analytical techniques, which are restricted to relative or qualitative analyses for HBcAg VLPs with bound nucleic acids. In combination with existing analytics, the methods for an absolute quantification of HBcAg VLPs and bound nucleic acids presented here are required to evaluate downstream purification steps, such as the removal of host cell-derived nucleic acids, concurrent protein loss, and efficient loading with therapeutic nucleic acids. Hence, the methods are key for effective process development when using HBcAg VLP as potential gene delivery vehicles.

Production of acetoin from renewable resources under heterotrophic and mixotrophic conditions.

This study aimed to reveal whether Cupriavidus necator H16 is suited for the production of acetoin based on the carboxylic acids acetate, butyrate and propionate under heterotrophic and mixotrophic conditions. The chosen production strain, lacking the polyhydroxybutyrate synthases phaC1 and phaC2, was revealed to be beneficiary for autotrophic acetoin production. Proteomic analysis of the strain determined that the deletions do indeed have a significant impact on pyruvate formation and its subsequent direction towards the introduced acetoin-synthesis pathway. Moreover, the strain was tested for its ability to use typical dark fermentation products under hetero- and mixotrophic conditions. Growth with butyrate and acetate led to low efficiencies, while 46.54% ±0.78 of the added propionate was converted into acetoin. Interestingly, mixotrophic conditions led to simultaneous consumption of acetate and butyrate with the gaseous substrates and lowered efficiency. In contrast, mixotrophic propionate consumption led to diauxic behavior and high carbon efficiency of 71.2% ±0.64.