Fully automated high-throughput immuno-µPlaque assay for live-attenuated tetravalent dengue vaccine development.

Dengue fever has remained a continuing global medical threat that impacts half of the world's population. Developing a highly effective dengue vaccine, with live-attenuated tetravalent vaccines as leading candidates, remains essential in preventing this disease. For the development of live virus vaccines (LVVs), potency measurements play a vital role in quantifying the active components of vaccine drug substance as well as drug product during various stages of research, development, and post-licensure evaluations. Traditional plaque-based assays are one of the most common potency test methods, but they generally take up to weeks to complete. Less labor and time-intensive potency assays are thus called for to aid in the acceleration of vaccine development, especially for multivalent LVVs. Here, we introduce a fully automated, 96-well format µPlaque assay that has been optimized as a high-throughput tool to evaluate process and formulation development of a live-attenuated tetravalent dengue vaccine. To the best of our knowledge, this is the first report of a miniaturized viral plaque method for dengue with full automation via an integrated robotic system. Compared to the traditional manual plaque assay, this newly developed method substantially reduces testing time by approximately half and allows for the evaluation of over ten times more samples per run. The fully automated workflow, from cell culture to plaque counting, significantly minimizes analyst hands-on time and improves assay repeatability. The study presents a pioneering solution for the rapid measurement of LVV viral titers, offering promising prospects for advancing vaccine development through high-throughput analytics.

Development and Characterization of an In Vitro Cell-Based Assay to Predict Potency of mRNA-LNP-Based Vaccines.

Messenger RNA (mRNA) vaccines have emerged as a flexible platform for vaccine development. The evolution of lipid nanoparticles as effective delivery vehicles for modified mRNA encoding vaccine antigens was demonstrated by the response to the COVID-19 pandemic. The ability to rapidly develop effective SARS-CoV-2 vaccines from the spike protein genome, and to then manufacture multibillions of doses per year was an extraordinary achievement and a vaccine milestone. Further development and application of this platform for additional pathogens is clearly of interest. This comes with the associated need for new analytical tools that can accurately predict the performance of these mRNA vaccine candidates and tie them to an immune response expected in humans. Described here is the development and characterization of an imaging based in vitro assay able to quantitate transgene protein expression efficiency, with utility to measure lipid nanoparticles (LNP)-encapsulated mRNA vaccine potency, efficacy, and stability. Multiple biologically relevant adherent cell lines were screened to identify a suitable cell substrate capable of providing a wide dose-response curve and dynamic range. Biologically relevant assay attributes were examined and optimized, including cell monolayer morphology, antigen expression kinetics, and assay sensitivity to LNP properties, such as polyethylene glycol-lipid (or PEG-lipid) composition, mRNA mass, and LNP size. Collectively, this study presents a strategy to quickly optimize and develop a robust cell-based potency assay for the development of future mRNA-based vaccines.

Glutathione affinity chromatography for the scalable purification of an oncolytic virus immunotherapy from microcarrier cell culture.

Therapeutic viral vectors are an emerging technology with several clinical applications in gene therapy, vaccines, and immunotherapy. Increased demand has required the redevelopment of conventional, low-throughput cell culture and purification manufacturing methods such as static cell stacks and ultracentrifugation. In this work, scalable methods were investigated for the manufacture of an oncolytic virus immunotherapy application consisting of a prototype strain of coxsackievirus A21 (CVA21) produced in adherent MRC-5 cells. Cell culture was established in stirred-tank microcarrier bioreactors, and an efficient affinity chromatography method was developed for the purification of harvested CVA21 through binding of the viral capsids to an immobilized glutathione (GSH) ligand. Bioreactor temperature during infection was investigated to maximize titer, and a decrease in temperature from 37°C to 34°C yielded a two-three-fold increase in infectivity. After purification of the 34°C harvests, the GSH affinity chromatography elution not only maintained a >two-fold increase in infectivity and viral genomes but also increased the proportion of empty capsids compared to 37°C harvests. Using material generated from both infection temperature setpoints, chromatographic parameters and mobile phase compositions were studied at the laboratory scale to maximize infectious particle yields and cell culture impurity clearance. Empty capsids that co-eluted with full capsids from 34°C infection temperature harvests were poorly resolved across the conditions tested, but subsequent polishing anion exchange and cation exchange chromatography steps were developed to clear residual empty capsids and other impurities. Oncolytic CVA21 production was scaled-up 75-fold from the laboratory scale and demonstrated across seven batches in 250 L single-use microcarrier bioreactors and purified with customized, prepacked, single-use 1.5 L GSH affinity chromatography columns. The large-scale bioreactors controlled at 34°C during infection maintained a three-fold increase in productivity in the GSH elution, and excellent clearance of host cell and media impurities was observed across all batches. This study presents a robust method for the manufacture of an oncolytic virus immunotherapy application that may be implemented for the scalable production of other viruses and viral vectors which interact with glutathione.

Investigation into the use of gamma irradiated Cytodex-1 microcarriers to produce a human cytomegalovirus (HCMV) vaccine candidate in epithelial cells.

V160 is a viral vaccine candidate against human cytomegalovirus (HCMV) that is manufactured using Adult Retinal Pigment Epithelial cells (ARPE-19) grown on Cytodex-1 microcarriers. The microcarriers are generally hydrated, washed, and autoclaved prior to use, which can be limiting at large production scales. To minimize microcarrier preparation and sterilization, the use of gamma irradiated Cytodex-1 was investigated. Similar ARPE-19 cell growth was observed on heat-sterilized and gamma irradiated Cytodex-1; however, significantly reduced virus production was observed in cultures exposed to gamma irradiated Cytodex-1. Additional experiments suggest that infection inhibition is not exclusive to ARPE-19 but is most directly linked to HCMV V160, as evidenced by similar inhibition of V160 with Vero cells and no inhibition of Measles virus with either cell type. These observations suggest a putative impact on HCMV infection from the presence of extractable(s)/leachable(s) in the gamma irradiated microcarriers. Thorough aseptic rinsing of gamma irradiated Cytodex-1 prior to use can mitigate this impact and enable comparable process performance to heat-sterilized Cytodex-1. Though not fully a "ready-to-use" product for the HCMV V160 production process, utilization of Cytodex-1 microcarriers was possible without requiring heat sterilization, suggesting a potential path forward for large scale production of V160.

Understanding the Spike Protein in COVID-19 Vaccine in Recombinant Vesicular Stomatitis Virus (rVSV) Using Automated Capillary Western Blots.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the viral agent that is responsible for the coronavirus disease-2019 (COVID-19) pandemic. One of the live virus vaccine candidates Merck and Co., Inc. was developing to help combat the pandemic was V590. V590 was a live-attenuated, replication-competent, recombinant vesicular stomatitis virus (rVSV) in which the envelope VSV glycoprotein (G protein) gene was replaced with the gene for the SARS-CoV-2 spike protein (S protein), the protein responsible for viral binding and fusion to the cell membrane. To assist with product and process development, a quantitative Simple Western (SW) assay was successfully developed and phase-appropriately qualified to quantitate the concentration of S protein expressed in V590 samples. A strong correlation was established between potency and S-protein concentration, which suggested that the S-protein SW assay could be used as a proxy for virus productivity optimization with faster data turnaround time (3 h vs 3 days). In addition, unlike potency, the SW assay was able to provide a qualitative profile assessment of the forms of S protein (S protein, S1 subunit, and S multimer) to ensure appropriate levels of S protein were maintained throughout process and product development. Finally, V590 stressed stability studies suggested that time and temperature contributed to the instability of S protein demonstrated by cleavage into its subunits, S1 and S2, and aggregation into S multimer. Both of which could potentially have a deleterious effect on the vaccine immunogenicity.

Development and scale-up of rVSV-SARS-CoV-2 vaccine process using single use bioreactor.

The outbreak of the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes the Coronavirus Disease 2019 (COVID-19) has spread through the globe at an alarming speed. The disease has become a global pandemic affecting millions of people and created public health crises worldwide. Among many efforts to urgently develop a vaccine against this disease, we developed an industrial-scale closed, single use manufacturing process for V590, a vaccine candidate for SARS-CoV-2. V590 is a recombinant vesicular stomatitis virus (rVSV) genetically engineered to express SARS-CoV-2 glycoprotein. In this work, we describe the development and optimization of serum-free microcarrier production of V590 in Vero cells in a closed system. To achieve the maximum virus productivity, we optimized pH and temperature during virus production in 3 liters (L) bioreactors. Virus productivity was improved (by ∼1 log) by using pH 7.0 and temperature at 34.0 °C. The optimal production condition was successfully scaled up to a 2000 L Single Use Bioreactor (SUB), producing a maximum virus titer of ∼1.0e+7 plaque forming units (PFU)/mL. Further process intensification and simplification, including growing Vero cells at 2 gs per liter (g/L) of Cytodex-1 Gamma microcarriers and eliminating the media exchange (MX) step prior to infection helped to increase virus productivity by ∼2-fold.

Monoclonal Antibody Reagent Stability and Expiry Recommendation Combining Experimental Data with Mathematical Modeling.

Monoclonal antibodies (mAbs) are widely used as critical reagents in analytical assays. While regulatory guidelines exist for stability monitoring of biopharmaceutical antibodies, they do not apply directly to the stability of mAbs used as assay reagent. We investigated alternative approaches to real-time stability monitoring of assay reagents. We compared functional (ELISA and cell-based) and biochemical (aggregation, deamidation) assay results using temperature-stressed mAb reagents. Data from both assay groups were compared for indications of antibody degradation. Arrhenius model kinetics was used to further extrapolate stability trends. Changes detected by traditionally monitored biochemical changes were not directly predictive of assay function. Instead, monitoring of reportable results was a closer indication of changes in assay performance related to mAb degradation. Using Arrhenius kinetic modeling, we combined forced degradation of individual reagents with reportable assay results to classify reagents into risk groups with associated re-evaluation and monitoring plans. This combined approach mitigates risk by monitoring each mAb reagent individually under stressed conditions while streamlining expiry assignment through simplified Arrhenius kinetics with only limited real-time stability data.