The ongoing COVID-19 pandemic drew global attention to infectious diseases, attracting numerous resources for development of pandemic preparedness plans and vaccine platforms—technologies with robust manufacturing processes that can quickly be pivoted to target emerging diseases. Newcastle Disease Virus (NDV) has been studied as a viral vector for human and veterinary vaccines, but its production relies heavily on embryonated chicken eggs, with very few studies producing NDV in cell culture. Here, NDV is produced in suspension Vero cells, and analytical assays (TCID₅₀ and ddPCR) are developed to quantify infectious and total viral titer. NDV-GFP and NDV-FLS (SARS-CoV-2 full-length spike protein) constructs were adapted to replicate in Vero and HEK293 suspension cultures using serum-free media, while fine-tuning parameters such as MOI, temperature, and trypsin concentration. Shake flask productions with Vero cells resulted in infectious titers of 1.07 × 10⁸ TCID₅₀/mL for NDV-GFP and 1.33 × 10⁸ TCID₅₀/mL for NDV-FLS. Production in 1 L batch bioreactors also resulted in high titers in culture supernatants, reaching 2.37 × 10⁸ TCID₅₀/mL for NDV-GFP and 3.16 × 10⁷ TCID₅₀/mL for NDV-FLS. This shows effective NDV production in cell culture, building the basis for a scalable vectored-vaccine manufacturing process that can be applied to different targets.

Alcohol dehydrogenases (ADHs) together with the economical substrate-coupled cofactor regeneration system play a pivotal role in the asymmetric synthesis of chiral alcohols; however, severe challenges concerning the poor tolerance of enzymes to 2-propanol and the adverse effects of the by-product, acetone, limit its applications, causing this strategy to lapse. Herein, a novel ADH gene smadh2 was identified from Stenotrophomonas maltophilia by traditional genome mining technology. The gene was cloned into Escherichia coli cells and then expressed to yield SmADH2. SmADH2 has a broad substrate spectrum and exhibits excellent tolerance and superb activity to 2-propanol even at 10.5 M (80%, v/v) concentration. Moreover, a new thermostatic bubble column reactor (TBCR) system is successfully designed to alleviate the inhibition of the by-product acetone by gas flow and continuously supplement 2-propanol. The organic waste can be simultaneously recovered for the purpose of green synthesis. In the sustainable system, structurally diverse chiral alcohols are synthesised at a high substrate loading (>150 g L⁻¹) without adding external coenzymes. Among these, about 780 g L⁻¹ (6 M) ethyl acetoacetate is completely converted into ethyl (R)-3-hydroxybutyrate in only 2.5 h with 99.9% ee and 7488 g L⁻¹ d⁻¹ space–time yield. Molecular dynamics simulation results shed light on the high catalytic activity toward the substrate. Therefore, the high 2-propanol tolerance SmADH2 with the TBCR system proves to be a potent biocatalytic strategy for the synthesis of chiral alcohols on an industrial scale.

The administration of viral vectored vaccines remains one of the most effective ways to respond to the ongoing novel coronavirus disease 2019 (COVID-19) pandemic. However, pre-existing immunity to the viral vector hinders its potency, resulting in a limited choice of viral vectors. Moreover, the basic batch mode of manufacturing vectored vaccines does not allow one to cost-effectively meet the global demand for billions of doses per year. To date, the exposure of humans to VSV infection has been limited. Therefore, a recombinant vesicular stomatitis virus (rVSV), which expresses the spike protein of SARS-CoV-2, was selected as the vector. To determine the operating upstream process conditions for the most effective production of an rVSV-SARS-CoV-2 candidate vaccine, a set of critical process parameters was evaluated in an Ambr 250 modular system, whereas in the downstream process, a streamlined process that included DNase treatment, clarification, and a membrane-based anion exchange chromatography was developed. The design of the experiment was performed with the aim to obtain the optimal conditions for the chromatography step. Additionally, a continuous mode manufacturing process integrating upstream and downstream steps was evaluated. rVSV-SARS-CoV-2 was continuously harvested from the perfusion bioreactor and purified by membrane chromatography in three columns that were operated sequentially under a counter-current mode. Compared with the batch mode, the continuous mode of operation had a 2.55-fold increase in space–time yield and a reduction in the processing time by half. The integrated continuous manufacturing process provides a reference for the efficient production of other viral vectored vaccines.

New influenza strains are constantly emerging, causing seasonal epidemics and raising concerns to the risk of a new global pandemic. Since vaccination is an effective method to prevent the spread of the disease and reduce its severity, the development of robust bioprocesses for producing pandemic influenza vaccines is exceptionally important. Herein, a membrane chromatography-based downstream processing platform with a demonstrated industrial application potential was established. Cell culture-derived influenza virus H1N1/A/PR/8/34 was harvested from benchtop bioreactor cultures. For the clarification of the cell culture broth, a depth filtration was selected as an alternative to centrifugation. After inactivation, an anion exchange chromatography membrane was used for viral capture and further processing. Additionally, two pandemic influenza virus strains, the H7N9 subtype of the A/Anhui/1/2013 and H3N2/A/Hong Kong/8/64, were successfully processed through similar downstream process steps establishing optimized process parameters. Overall, 41.3–62.5% viral recovery was achieved, with the removal of 86.3–96.5% host cell DNA and 95.5–99.7% of proteins. The proposed membrane chromatography purification is a scalable and generic method for the processing of different influenza strains and is a promising alternative to the current industrial purification of influenza vaccines based on ultracentrifugation methodologies.

There have been considerable efforts on improving the lentiviral vector (LV) system and their production. However, there remains the persisting challenge of producing a sufficient quantity of LVs at manufacturing scale to support treatments beyond early clinical trials. Furthermore, their innately labile nature poses an equally important obstacle in LV production. As LVs lose function over time and they are sensitive to environmental factors in each unit operation in the bioprocess workflow, integrated continuous manufacturing is an attractive strategy for process intensification. This manuscript describes the implementation of nuclease treatment, clarification, and capture step in a semi-continuous mode. Combining the clarification and loading of the capture step as well as operating those steps in parallel to the purification of the capture step expedite the processing time, reducing it by 4-fold as compared to processing the same volume in batch mode using the same membrane size. This semi-continuous operation also improves the recoveries of functional vector particles and total vector particles by 26% and 18%, respectively, showing an added benefit in loading the capture membranes in series in continuous flow chromatography. Building on previously published upstream work using a scalable cell retention device in perfusion mode, this manuscript demonstrates the integration of upstream and downstream in a semi-continuous manner, reducing processing and hold times as well as showing improvements in LV product quality and recovery.