Incubation Temperature and Period During Denarase Treatment and Microfiltration Affect the Yield of Recombinant Adenoviral Vectors During Downstream Processing.

Adenoviral vectors (AV) are commonly used as vaccine and gene therapy vehicles because of their ease of construction, ability to grow to high titers in the large-scale production process, and safety for human applications. However, the efficiency rate of downstream processes for adenoviral vectors still varies greatly. In the current study, we aimed to investigate the effect of the downstream treatment protocol and microfiltration of the harvested upstream material on viral vector yield. We compared the performance of the repeated freeze-thaw (RFT) and the Tween-20 detergent lysis (DLT) methods. In addition, the effects of the cell lysis method, incubation temperature, and time on viral yield were investigated. The samples were incubated at either room temperature or 37 °C for 1-, 2-, and 4-h periods. Samples were filtered with PES and SFCA membrane. Virus yield and infectivity were assayed by qPCR and immuno-titration. In conclusion, our results suggest that 2-h incubation gives the best results when incubated at 37 °C for denarase activity when Tween-20 is used for virus recovery. If the room temperature is preferred, 4-h incubation could be preferred. A phase 1 clinical trial (NCT05526183, January 21, 2022) was started with the recombinant adenovirus used in the study.

Recent Progress on Vaccines Produced in Transgenic Plants.

The development of vaccines from plants has been going on for over two decades now. Vaccine production in plants requires time and a lot of effort. Despite global efforts in plant-made vaccine development, there are still challenges that hinder the realization of the final objective of manufacturing approved and safe products. Despite delays in the commercialization of plant-made vaccines, there are some human vaccines that are in clinical trials. The novel coronavirus (SARS-CoV-2) and its resultant disease, coronavirus disease 2019 (COVID-19), have reminded the global scientific community of the importance of vaccines. Plant-made vaccines could not be more important in tackling such unexpected pandemics as COVID-19. In this review, we explore current progress in the development of vaccines manufactured in transgenic plants for different human diseases over the past 5 years. However, we first explore the different host species and plant expression systems during recombinant protein production, including their shortcomings and benefits. Lastly, we address the optimization of existing plant-dependent vaccine production protocols that are aimed at improving the recovery and purification of these recombinant proteins.

Production of full-length SARS-CoV-2 nucleocapsid protein from Escherichia coli optimized by native hydrophobic interaction chromatography hyphenated to multi-angle light scattering detection.

The nucleocapsid protein (NP) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical for several steps of the viral life cycle, and is abundantly expressed during infection, making it an ideal diagnostic target protein. This protein has a strong tendency for dimerization and interaction with nucleic acids. For the first time, high titers of NP were expressed in E. coli with a CASPON tag, using a growth-decoupled protein expression system. Purification was accomplished by nuclease treatment of the cell homogenate and a sequence of downstream processing (DSP) steps. An analytical method consisting of native hydrophobic interaction chromatography hyphenated to multi-angle light scattering detection (HIC-MALS) was established for in-process control, in particular, to monitor product fragmentation and multimerization throughout the purification process. 730 mg purified NP per liter of fermentation could be produced by the optimized process, corresponding to a yield of 77% after cell lysis. The HIC-MALS method was used to demonstrate that the NP product can be produced with a purity of 95%. The molecular mass of the main NP fraction is consistent with dimerized protein as was verified by a complementary native size-exclusion separation (SEC)-MALS analysis. Peptide mapping mass spectrometry and host cell specific enzyme-linked immunosorbent assay confirmed the high product purity, and the presence of a minor endogenous chaperone explained the residual impurities. The optimized HIC-MALS method enables monitoring of the product purity, and simultaneously access its molecular mass, providing orthogonal information complementary to established SEC-MALS methods. Enhanced resolving power can be achieved over SEC, attributed to the extended variables to tune selectivity in HIC mode.

Towards integrated production of an influenza A vaccine candidate with MDCK suspension cells.

Seasonal influenza epidemics occur both in northern and southern hemispheres every year. Despite the differences in influenza virus surface antigens and virulence of seasonal subtypes, manufacturers are well-adapted to respond to this periodical vaccine demand. Due to decades of influenza virus research, the development of new influenza vaccines is relatively straight forward. In similarity with the ongoing coronavirus disease 2019 pandemic, vaccine manufacturing is a major bottleneck for a rapid supply of the billions of doses required worldwide. In particular, egg-based vaccine production would be difficult to schedule and shortages of other egg-based vaccines with high demands also have to be anticipated. Cell culture-based production systems enable the manufacturing of large amounts of vaccines within a short time frame and expand significantly our options to respond to pandemics and emerging viral diseases. In this study, we present an integrated process for the production of inactivated influenza A virus vaccines based on a Madin-Darby Canine Kidney (MDCK) suspension cell line cultivated in a chemically defined medium. Very high titers of 3.6 log10 (HAU/100 µl) were achieved using fast-growing MDCK cells at concentrations up to 9.5 × 106 cells/ml infected with influenza A/PR/8/34 H1N1 virus in 1 L stirred tank bioreactors. A combination of membrane-based steric-exclusion chromatography followed by pseudo-affinity chromatography with a sulfated cellulose membrane adsorber enabled full recovery for the virus capture step and up to 80% recovery for the virus polishing step. Purified virus particles showed a homogenous size distribution with a mean diameter of 80 nm. Based on a monovalent dose of 15 µg hemagglutinin (single-radial immunodiffusion assay), the level of total protein and host cell DNA was 58 µg and 10 ng, respectively. Furthermore, all process steps can be fully scaled up to industrial quantities for commercial manufacturing of either seasonal or pandemic influenza virus vaccines. Fast production of up to 300 vaccine doses per liter within 4-5 days makes this process competitive not only to other cell-based processes but to egg-based processes as well.