Robust and resource-efficient production process suitable for large-scale production of baculovirus through high cell density seed train and optimized infection strategy.

The aim of this study was the development of a scalable production process for high titer (108 pfu/mL and above) recombinant baculovirus stocks with low cell line-derived impurities for the production of virus-like particles (VLP). To achieve this, we developed a high cell density (HCD) culture for low footprint cell proliferation, compared different infection strategies at multiplicity of infection (MOI) 0.05 and 0.005, different infection strategies and validated generally applicable harvest criteria of cell viability ≤ 80%. We also investigated online measurable parameters to observe the baculovirus production. The infection strategy employing a very low virus inoculum of MOI 0.005 and a 1:2 dilution with fresh medium one day after infection proved to be the most resource efficient. There, we achieved higher cell-specific titers and lower host cell protein concentrations at harvest than other tested infection strategies with the same MOI, while saving half of the virus stock for infecting the culture compared to other tested infection strategies. HCD culture by daily medium exchange was confirmed as suitable for seed train propagation, infection, and baculovirus production, equally efficient as the conventionally propagated seed train. Online measurable parameters for cell concentration and average cell diameter were found to be effective in monitoring the production process. The study concluded that a more efficient VLP production process in large scale can be achieved using this virus stock production strategy, which could also be extended to produce other proteins or extracellular vesicles with the baculovirus expression system.

A comprehensive antigen production and characterisation study for easy-to-implement, specific and quantitative SARS-CoV-2 serotests.

BACKGROUND: Antibody tests are essential tools to investigate humoral immunity following SARS-CoV-2 infection or vaccination. While first-generation antibody tests have primarily provided qualitative results, accurate seroprevalence studies and tracking of antibody levels over time require highly specific, sensitive and quantitative test setups. METHODS: We have developed two quantitative, easy-to-implement SARS-CoV-2 antibody tests, based on the spike receptor binding domain and the nucleocapsid protein. Comprehensive evaluation of antigens from several biotechnological platforms enabled the identification of superior antigen designs for reliable serodiagnostic. Cut-off modelling based on unprecedented large and heterogeneous multicentric validation cohorts allowed us to define optimal thresholds for the tests' broad applications in different aspects of clinical use, such as seroprevalence studies and convalescent plasma donor qualification. FINDINGS: Both developed serotests individually performed similarly-well as fully-automated CE-marked test systems. Our described sensitivity-improved orthogonal test approach assures highest specificity (99.8%); thereby enabling robust serodiagnosis in low-prevalence settings with simple test formats. The inclusion of a calibrator permits accurate quantitative monitoring of antibody concentrations in samples collected at different time points during the acute and convalescent phase of COVID-19 and disclosed antibody level thresholds that correlate well with robust neutralization of authentic SARS-CoV-2 virus. INTERPRETATION: We demonstrate that antigen source and purity strongly impact serotest performance. Comprehensive biotechnology-assisted selection of antigens and in-depth characterisation of the assays allowed us to overcome limitations of simple ELISA-based antibody test formats based on chromometric reporters, to yield comparable assay performance as fully-automated platforms. FUNDING: WWTF, Project No. COV20-016; BOKU, LBI/LBG.

Separation of virus-like particles and extracellular vesicles by flow-through and heparin affinity chromatography.

Separation of enveloped virus-like particles from other extracellular vesicles is a challenging separation problem due to the similarity of these bionanoparticles. Without simple and scalable methods for purification and analytics, it is difficult to gain deeper insight into their biological function. A two-step chromatographic purification method was developed. In the first step, virus-like particles and extracellular vesicles were collected and separated from smaller impurities in a flow-through mode. Benzonase® treated HEK 293 cell culture supernatant was directly loaded onto a column packed with core-shell beads. The collected flow-through was further purified using heparin affinity chromatography. In heparin affinity chromatography 54% of the total particle load were found in the flow-through, and 15% of the particles were eluted during the salt linear gradient. The particle characterization, especially particle size distribution and mass spectrometry data, suggests that extracellular vesicles dominate the flow-through fraction and HIV-1 gag VLPs are enriched in the elution peak. This is in part in contradiction to other protocols where the extracellular vesicles are recovered by binding to heparin affinity chromatography. The developed method is easily scalable to pilot and process scale and allows a fast accomplishment of this separation within one day.

Purification of HIV-1 gag virus-like particles and separation of other extracellular particles.

Enveloped virus-like particles (VLPs) are increasingly used as vaccines and immunotherapeutics. Frequently, very time consuming density gradient centrifugation techniques are used for purification of VLPs. However, the progress towards optimized large-scale VLP production increased the demand for fast, cost efficient and scale able purification processes. We developed a chromatographic procedure for purification of HIV-1 gag VLPs produced in CHO cells. The clarified and filtered cell culture supernatant was directly processed on an anion-exchange monolith. The majority of host cell impurities passed through the column, whereas the VLPs were eluted by a linear or step salt gradient; the major fraction of DNA was eluted prior to VLPs and particles in the range of 100-200nm in diameter could be separated into two fractions. The earlier eluted fraction was enriched with extracellular particles associated to exosomes or microvesicles, whereas the late eluting fractions contained the majority of most pure HIV-1 gag VLPs. DNA content in the exosome-containing fraction could not be reduced by Benzonase treatment which indicated that the DNA was encapsulated. Many exosome markers were identified by proteomic analysis in this fraction. We present a laboratory method that could serve as a basis for rapid downstream processing of enveloped VLPs. Up to 2000 doses, each containing 1×10(9) particles, could be processed with a 1mL monolith within 47min. The method compared to density gradient centrifugation has a 220-fold improvement in productivity.