Upstream processing for viral vaccines–General aspects

Pelz, Lars; Göbel, Sven; Jaén, Karim E; Reichl, Udo; Genzel, Yvonne

doi:10.1201/9781003229797-5

Cited by 4 publications

(5 citation statements)

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“…General considerations on the appropriate choice for process parameters and intensification strategies for cell culture-based virus production were summarized previously by our group. 14 , 15 For influenza, temperature is typically maintained between 32°C and 37°C, pH between 7.2 and 7.6, and dissolved oxygen concentration above 30%. At the time of infection, seed virus is typically added along with a protease, such as trypsin, to enhance viral entry into the cell.…”

Section: Cell Culture-based Influenza Virus Productionmentioning

confidence: 99%

Innovations in cell culture-based influenza vaccine manufacturing – from static cultures to high cell density cultivations

Zinnecker,

Reichl,

Genzel

2024

Human Vaccines & Immunotherapeutics

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Section: Cell Culture-based Influenza Virus Productionmentioning

confidence: 99%

Innovations in cell culture-based influenza vaccine manufacturing – from static cultures to high cell density cultivations

Zinnecker,

Reichl,

Genzel

2024

Human Vaccines & Immunotherapeutics

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“…To address the unprecedented demand, as well as the high input doses required for many rVSV-based therapies, current batch-based manufacturing strategies need to be intensified. Usage of suspension cell lines in chemically defined media allows for the design of processes that are easier to scale-up, for higher cell concentrations and for smaller footprints compared to traditional adherent-based manufacturing (Pelz et al 2022 ). By the establishment of perfusion cultures, where depleted medium is continuously exchanged with fresh medium, while the cells are retained in the bioreactor by the use of a cell retention device, even higher cell concentrations can be achieved.…”

Section: Introductionmentioning

confidence: 99%

Production of recombinant vesicular stomatitis virus-based vectors by tangential flow depth filtration

Göbel,

Pelz,

Silva

et al. 2024

Appl Microbiol Biotechnol

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Cell culture-based production of vector-based vaccines and virotherapeutics is of increasing interest. The vectors used not only retain their ability to infect cells but also induce robust immune responses. Using two recombinant vesicular stomatitis virus (rVSV)-based constructs, we performed a proof-of-concept study regarding an integrated closed single-use perfusion system that allows continuous virus harvesting and clarification. Using suspension BHK-21 cells and a fusogenic oncolytic hybrid of vesicular stomatitis virus and Newcastle disease virus (rVSV-NDV), a modified alternating tangential flow device (mATF) or tangential flow depth filtration (TFDF) systems were used for cell retention. As the hollow fibers of the former are characterized by a large internal lumen (0.75 mm; pore size 0.65 μm), membrane blocking by the multi-nucleated syncytia formed during infection could be prevented. However, virus particles were completely retained. In contrast, the TFDF filter unit (lumen 3.15 mm, pore size 2–5 μm) allowed not only to achieve high viable cell concentrations (VCC, 16.4–20.6×106 cells/mL) but also continuous vector harvesting and clarification. Compared to an optimized batch process, 11-fold higher infectious virus titers were obtained in the clarified permeate (maximum 7.5×109 TCID50/mL). Using HEK293-SF cells and a rVSV vector expressing a green fluorescent protein, perfusion cultivations resulted in a maximum VCC of 11.3×106 cells/mL and infectious virus titers up to 7.1×1010 TCID50/mL in the permeate. Not only continuous harvesting but also clarification was possible. Although the cell-specific virus yield decreased relative to a batch process established as a control, an increased space-time yield was obtained. Key points • Viral vector production using a TFDF perfusion system resulted in a 460% increase in space-time yield • Use of a TFDF system allowed continuous virus harvesting and clarification • TFDF perfusion system has great potential towards the establishment of an intensified vector production

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“…Suspension cells allow for easy scale-up and passaging while maintaining rapid cell growth. Cultivation in stirred tank bioreactors (STR) can achieve high cell concentration in a small footprint, decreasing labor expenditures (Pelz et al, 2022). Moreover, suspension cultures allow the use of advanced process monitoring methods, enable process automation, and tight control of culture conditions.…”

Section: Introductionmentioning

confidence: 99%

“…Manufacturing methods for several oncolytic viruses (herpes simplex virus, vaccinia virus, reovirus, and adenovirus) for clinical trials, but also for rVSV‐NDV, are mainly performed in anchorage‐dependent cell lines (Abdullahi et al, 2018; Ungerechts et al, 2016). The use of adherent cells can be beneficial due to established and automated production facilities, years of experience with regulatory approval, and high cell‐specific virus yields (CSVY) (Pelz et al, 2022). However, for fusogenic OVs that cause formation of large multinucleated syncytia that die rapidly after induction only relatively low virus yields were achieved in several tested adherent cell lines (Abdullahi et al, 2018).…”

Section: Introductionmentioning

confidence: 99%

“…This property presents unique challenges for large‐scale clinical‐grade manufacturing, further compounded as scaling‐up of processes for adherent cells is mainly achieved by scaling‐out (e.g., increasing the surface area by multiplying the number of culture vessels) or the use of microcarrier systems. In static culture systems, there are also only limited options for monitoring and control of cultivation parameters such as pH values, dissolved oxygen concentration, feeding rates (Gallo–Ramírez et al, 2015; Pelz et al, 2022). Moreover, media for most adherent cells are supplemented with animal‐derived components such as fetal calf serum, which can lead to batch‐to‐batch variations, increased production and purification costs, and elevated contamination risks.…”

Section: Introductionmentioning

confidence: 99%

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Process intensification strategies toward cell culture‐based high‐yield production of a fusogenic oncolytic virus

Jaén

Dorn

Neumeyer

et al. 2023

Biotech & Bioengineering

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We present a proof‐of‐concept study for production of a recombinant vesicular stomatitis virus (rVSV)‐based fusogenic oncolytic virus (OV), rVSV‐Newcastle disease virus (NDV), at high cell densities (HCD). Based on comprehensive experiments in 1 L stirred tank reactors (STRs) in batch mode, first optimization studies at HCD were carried out in semi‐perfusion in small‐scale cultivations using shake flasks. Further, a perfusion process was established using an acoustic settler for cell retention. Growth, production yields, and process‐related impurities were evaluated for three candidate cell lines (AGE1.CR, BHK‐21, HEK293SF)infected at densities ranging from 15 to 30 × 106 cells/mL. The acoustic settler allowed continuous harvesting of rVSV‐NDV with high cell retention efficiencies (above 97%) and infectious virus titers (up to 2.4 × 109 TCID50/mL), more than 4–100 times higher than for optimized batch processes. No decrease in cell‐specific virus yield (CSVY) was observed at HCD, regardless of the cell substrate. Taking into account the accumulated number of virions both from the harvest and bioreactor, a 15–30 fold increased volumetric virus productivity for AGE1.CR and HEK293SF was obtained compared to batch processes performed at the same scale. In contrast to all previous findings, formation of syncytia was observed at HCD for the suspension cells BHK 21 and HEK293SF. Oncolytic potency was not affected compared to production in batch mode. Overall, our study describes promising options for the establishment of perfusion processes for efficient large‐scale manufacturing of fusogenic rVSV‐NDV at HCD for all three candidate cell lines.

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Upstream processing for viral vaccines–General aspects

Cited by 4 publications

References 0 publications

Innovations in cell culture-based influenza vaccine manufacturing – from static cultures to high cell density cultivations

Innovations in cell culture-based influenza vaccine manufacturing – from static cultures to high cell density cultivations

Production of recombinant vesicular stomatitis virus-based vectors by tangential flow depth filtration

Process intensification strategies toward cell culture‐based high‐yield production of a fusogenic oncolytic virus

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