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From single-cell cloning to high-yield influenza virus production - implementing advanced technologies in vaccine process development.

ABSTRACT: Innovations in viral vaccine manufacturing are crucial for pandemic preparedness and to meet ever-rising global demands. For influenza, however, production still mainly relies on technologies established decades ago. Although modern production shifts from egg-based towards cell culture technologies, the full potential has not yet been fully exploited. Here, we evaluate whether implementation of state-of-the-art technologies for cell culture-based recombinant protein production are capable to challenge outdated approaches in viral vaccine process development. For this, a fully automated single-cell cloning strategy was established to generate monoclonal suspension Madin-Darby canine kidney (MDCK) cells. Among selected cell clones, we could observe distinct metabolic and growth characteristics, with C59 reaching a maximum viable cell concentration of 17.3 × 10⁶ cells/mL and low doubling times in batch mode. Screening for virus production using a panel of human vaccine-relevant influenza A and B viruses in an ambr15 system revealed high titers with yields competing or even outperforming available MDCK cell lines. With C113, we achieved cell-specific virus yields of up to 25,000 virions/cell, making this cell clone highly attractive for vaccine production. Finally, we confirmed process performance at a 50-fold higher working volume. In summary, we present a scalable and powerful approach for accelerated development of high-yield influenza virus production in chemically defined medium starting from a single cell.

SUBMITTER: Zinnecker T

PROVIDER: S-EPMC10991716 | biostudies-literature | 2024 Apr

REPOSITORIES: biostudies-literature

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From single-cell cloning to high-yield influenza virus production - implementing advanced technologies in vaccine process development.

Zinnecker Tilia T Badri Najd N Araujo Diogo D Thiele Kristin K Reichl Udo U Genzel Yvonne Y

Engineering in life sciences 20240218 4

Innovations in viral vaccine manufacturing are crucial for pandemic preparedness and to meet ever-rising global demands. For influenza, however, production still mainly relies on technologies established decades ago. Although modern production shifts from egg-based towards cell culture technologies, the full potential has not yet been fully exploited. Here, we evaluate whether implementation of state-of-the-art technologies for cell culture-based recombinant protein production are capable to cha ...[more]

PMID: 38584687

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Project description:Similar to the recent COVID-19 pandemic, influenza A virus poses a constant threat to the global community. For the treatment of flu disease, both antivirals and vaccines are available with vaccines the most effective and safest approach. In order to overcome limitations in egg-based vaccine manufacturing, cell culture-based processes have been established. While this production method avoids egg-associated risks in face of pandemics, process intensification using animal suspension cells in high cell density perfusion cultures should allow to further increase manufacturing capacities worldwide. In this work, we demonstrate the development of a perfusion process using Madin-Darby canine kidney (MDCK) suspension cells for influenza A (H1N1) virus production from scale-down shake flask cultivations to laboratory scale stirred tank bioreactors. Shake flask cultivations using semi-perfusion mode enabled high-yield virus harvests (4.25 log10(HAU/100 μL)) from MDCK cells grown up to 41 × 106 cells/mL. Scale-up to bioreactors with an alternating tangential flow (ATF) perfusion system required optimization of pH control and implementation of a temperature shift during the infection phase. Use of a capacitance probe for on-line perfusion control allowed to minimize medium consumption. This contributed to a better process control and a more economical performance while maintaining a maximum virus titer of 4.37 log10(HAU/100 μL) and an infectious virus titer of 1.83 × 1010 virions/mL. Overall, this study clearly demonstrates recent advances in cell culture-based perfusion processes for next-generation high-yield influenza vaccine manufacturing for pandemic preparedness. KEY POINTS: • First MDCK suspension cell-based perfusion process for IAV produciton was established. • "Cell density effect" was overcome and process was intensified by reduction of medium use and automated process control. • The process achieved cell density over 40 × 106 cells/mL and virus yield over 4.37 log10(HAU/100 μL).

Project description:Ageing societies and a rising prevalence of dementia are associated with increasing demand for care home places. Monitoring technologies (e.g. bed-monitoring systems; wearable location-tracking devices) are appealing to care homes as they may enhance safety, increase resident freedom, and reduce staff burden. However, there are ethical concerns about the use of such technologies, and it is unclear how they might be implemented to deliver their full range of potential benefits.This study explored facilitators and barriers to the implementation of monitoring technologies in care homes.Embedded multiple-case study with qualitative methods.Three dementia-specialist care homes in North-West England.Purposive sample of 24 staff (including registered nurses, clinical specialists, senior managers and care workers), 9 relatives and 9 residents.36 semi-structured interviews with staff, relatives and residents; 175h of observation; resident care record review. Data collection informed by Normalization Process Theory, which seeks to account for how novel interventions become routine practice. Data analysed using Framework Analysis.Findings are presented under three main themes: 1. Reasons for using technologies: The primary reason for using monitoring technologies was to enhance safety. This often seemed to override consideration of other potential benefits (e.g. increased resident freedom) or ethical concerns (e.g. resident privacy); 2. Ways in which technologies were implemented: Some staff, relatives and residents were not involved in discussions and decision-making, which seemed to limit understandings of the potential benefits and challenges from the technologies. Involvement of residents appeared particularly challenging. Staff highlighted the importance of training, but staff training appeared mainly informal which did not seem sufficient to ensure that staff fully understood the technologies; 3. Use of technologies in practice: Technologies generated frequent alarms that placed a burden upon staff, but staff were able to use their contextual knowledge to help to counter some of this burden. Some technologies offered a range of data-gathering capabilities, but were not always perceived as useful complements to practice.Implementation of monitoring technologies may be facilitated by the extent to which the technologies are perceived to enhance safety. Implementation may be further facilitated through greater involvement of all stakeholders in discussions and decision-making in order to deepen understandings about the range of potential benefits and challenges from the use of monitoring technologies. Staff training might need to move beyond functional instruction to include deeper exploration of anticipated benefits and the underlying rationale for using monitoring technologies.

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From single-cell cloning to high-yield influenza virus production - implementing advanced technologies in vaccine process development.

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From single-cell cloning to high-yield influenza virus production - implementing advanced technologies in vaccine process development.

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