Multivariate data analysis on multisensor measurement for inline process monitoring of adenovirus production in HEK293 cells.

In the era of Biopharma 4.0, process digitalization fundamentally requires accurate and timely monitoring of critical process parameters (CPPs) and quality attributes. Bioreactor systems are equipped with a variety of sensors to ensure process robustness and product quality. However, during the biphasic production of viral vectors or replication-competent viruses for gene and cell therapies and vaccination, current monitoring techniques relying on a single working sensor can be affected by the physiological state change of the cells due to infection/transduction/transfection step required to initiate production. To address this limitation, a multisensor (MS) monitoring system, which includes dual-wavelength fluorescence spectroscopy, dielectric signals, and a set of CPPs, such as oxygen uptake rate and pH control outputs, was employed to monitor the upstream process of adenovirus production in HEK293 cells in bioreactor. This system successfully identified characteristic responses to infection by comparing variations in these signals, and the correlation between signals and target critical variables was analyzed mechanistically and statistically. The predictive performance of several target CPPs using different multivariate data analysis (MVDA) methods on data from a single sensor/source or fused from multiple sensors were compared. An MS regression model can accurately predict viable cell density with a relative root mean squared error (rRMSE) as low as 8.3% regardless of the changes occurring over the infection phase. This is a significant improvement over the 12% rRMSE achieved with models based on a single source. The MS models also provide the best predictions for glucose, glutamine, lactate, and ammonium. These results demonstrate the potential of using MVDA on MS systems as a real-time monitoring approach for biphasic bioproduction processes. Yet, models based solely on the multiplicity and timing of infection outperformed both single-sensor and MS models, emphasizing the need for a deeper mechanistic understanding in virus production prediction.

Alloying Effect on Transformation Strain and Martensitic Transformation Temperature of Ti-Based Alloys from Ab Initio Calculations.

The accurate prediction of alloying effects on the martensitic transition temperature (Ms) is still a big challenge. To investigate the composition-dependent lattice deformation strain and the Ms upon the ß to α″ phase transition, we calculate the total energies and transformation strains for two selected Ti-Nb-Al and Ti-Nb-Ta ternaries employing a first-principles method. The adopted approach accurately estimates the alloying effect on lattice strain and the Ms by comparing it with the available measurements. The largest elongation and the largest compression due to the lattice strain occur along ±[011]ß and ±[100]ß, respectively. As compared to the overestimation of the Ms from existing empirical relationships, an improved Ms estimation can be realized using our proposed empirical relation by associating the measured Ms with the energy difference between the ß and α″ phases. There is a satisfactory agreement between the predicted and measured Ms, implying that the proposed empirical relation could accurately describe the coupling alloying effect on Ms. Both Al and Ta strongly decrease the Ms, which is in line with the available observations. A correlation between the Ms and elastic modulus, C44, is found, implying that elastic moduli may be regarded as a prefactor of composition-dependent Ms. This work sheds deep light on precisely and directly predicting the Ms of Ti-containing alloys from the first-principles method.

Production of adeno-associated viral vector serotype 6 by triple transfection of suspension HEK293 cells at higher cell densities.

In recent years, the use of adeno-associated viruses (AAVs) as vectors for gene and cell therapy has increased, leading to a rise in the amount of AAV vectors required during pre-clinical and clinical trials. AAV serotype 6 (AAV6) has been found to be efficient in transducing different cell types and has been successfully used in gene and cell therapy protocols. However, the number of vectors required to effectively deliver the transgene to one single cell has been estimated at 106 viral genomes (VG), making large-scale production of AAV6 necessary. Suspension cell-based platforms are currently limited to low cell density productions due to the widely reported cell density effect (CDE), which results in diminished production at high cell densities and decreased cell-specific productivity. This limitation hinders the potential of the suspension cell-based production process to increase yields. In this study, we investigated the improvement of the production of AAV6 at higher cell densities by transiently transfecting HEK293SF cells. The results showed that when the plasmid DNA was provided on a cell basis, the production could be carried out at medium cell density (MCD, 4 × 106  cells mL-1 ) resulting in titers above 1010  VG mL-1 . No detrimental effects on cell-specific virus yield or cell-specific functional titer were observed at MCD production. Furthermore, while medium supplementation alleviated the CDE in terms of VG/cell at high cell density (HCD, 10 × 106  cells mL-1 ) productions, the cell-specific functional titer was not maintained, and further studies are necessary to understand the observed limitations for AAV production in HCD processes. The MCD production method reported here lays the foundation for large-scale process operations, potentially solving the current vector shortage in AAV manufacturing.

Membrane Chromatography-Based Downstream Processing for Cell-Culture Produced Influenza 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.

A nanoparticle-based COVID-19 vaccine candidate elicits broad neutralizing antibodies and protects against SARS-CoV-2 infection.

A vaccine candidate to SARS-CoV-2 was constructed by coupling the viral receptor binding domain (RBD) to the surface of the papaya mosaic virus (PapMV) nanoparticle (nano) to generate the RBD-PapMV vaccine. Immunization of mice with the coupled RBD-PapMV vaccine enhanced the antibody titers and the T-cell mediated immune response directed to the RBD antigen as compared to immunization with the non-coupled vaccine formulation (RBD + PapMV nano). Anti-RBD antibodies, generated in vaccinated animals, neutralized SARS-CoV-2 infection in vitro against the ancestral, Delta and the Omicron variants. At last, immunization of mice susceptible to the infection by SARS-CoV-2 (K18-hACE2 transgenic mice) with the RBD-PapMV vaccine induced protection to the ancestral SARS-CoV-2 infectious challenge. The induction of the broad neutralization against SARS-CoV-2 variants induced by the RBD-PapMV vaccine demonstrate the potential of the PapMV vaccine platform in the development of efficient vaccines against viral respiratory infections.

Rapid High-Yield Production of Functional SARS-CoV-2 Receptor Binding Domain by Viral and Non-Viral Transient Expression for Pre-Clinical Evaluation.

Vaccine design strategies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are focused on the Spike protein or its subunits as the main antigen target of neutralizing antibodies. In this work, we propose rapid production methods of an extended segment of the Spike Receptor Binding Domain (RBD) in HEK293SF cells cultured in suspension, in serum-free media, as a major component of a COVID-19 subunit vaccine under development. The expression of RBD, engineered with a sortase-recognition motif for protein-based carrier coupling, was achieved at high yields by plasmid transient transfection or human type-5-adenoviral infection of the cells, in a period of only two and three weeks, respectively. Both production methods were evaluated in 3L-controlled bioreactors with upstream and downstream bioprocess improvements, resulting in a product recovery with over 95% purity. Adenoviral infection led to over 100 µg/mL of RBD in culture supernatants, which was around 7-fold higher than levels obtained in transfected cultures. The monosaccharide and sialic acid content was similar in the RBD protein from the two production approaches. It also exhibited a proper conformational structure as recognized by monoclonal antibodies directed against key native Spike epitopes. Efficient direct binding to ACE2 was also demonstrated at similar levels in RBD obtained from both methods and from different production lots. Overall, we provide bioprocess-related data for the rapid, scalable manufacturing of low cost RBD based vaccines against SARS-CoV-2, with the added value of making a functional antigen available to support further research on uncovering mechanisms of virus binding and entry as well as screening for potential COVID-19 therapeutics.