Systematic evaluation of suspension MDCK cells, adherent MDCK cells, and LLC-MK2 cells for preparing influenza vaccine seed virus

Nakamura, Kenzo; Harada, Yu; Takahashi, Hitoshi; Trusheim, Heidi; Roth, Bernhard; Hamamoto, Itsuki; Hirata-Saito, Asumi; Ogane, Teruko; Mizuta, Katsumi; Konomi, Nami; Konomi, Yasushi; Asanuma, Hideki; Odagiri, Takato; Tashiro, Masato; Yamamoto, Norio

doi:10.1016/j.vaccine.2019.08.064

Cited by 9 publications

(8 citation statements)

References 35 publications

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“…The optimization of the pH control resulted in a better process performance and a slight increase in HA acc , but the CSVY was significantly lower compared to the batch process (10,476 virions/cell), which seemed to be a demonstration of a "cell density effect" as described above. As an important process parameter, the temperature may influence the cell behavior and virus replication (Nakamura et al 2019). Thus, we compared the effect of 37°C and 33°C applied in the virus infection phase in HCD cultivations on the cell growth, virus replication, and virus retention in a subsequent run (ATF3).…”

Section: Process Optimization In Bioreactors At Hcdmentioning

confidence: 99%

High cell density perfusion process for high yield of influenza A virus production using MDCK suspension cells

Bissinger

Genzel

et al. 2021

Appl Microbiol Biotechnol

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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 log 10 (HAU/100 μL)) from MDCK cells grown up to 41 × 10 6 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 log 10 (HAU/100 μL) and an infectious virus titer of 1.83 × 10 10 virions/mL. Overall, this study clearly demonstrates recent advances in cell culture-based perfusion processes for nextgeneration 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 × 10 6 cells/mL and virus yield over 4.37 log 10 (HAU/100 μL).

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Section: Process Optimization In Bioreactors At Hcdmentioning

confidence: 99%

High cell density perfusion process for high yield of influenza A virus production using MDCK suspension cells

Bissinger

Genzel

et al. 2021

Appl Microbiol Biotechnol

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“…In the future, it could be an alternative to generate high-growth CVVs for emerging influenza H7N9 virus via introducing HA-I120T or HA-A151T substitutions, because it is acceptable to introduce special amino acid into the HA protein of influenza CVVs without affecting its antigenicity and pathogenicity [36]. Based on genetic and antigenic stability of influenza viruses, a suspension MDCK cell line, MDCK33016PF, has been considered as a suitable platform for the isolation and preparation of influenza vaccine seed viruses, especially H3N2 and B/Yamagata viruses [37]. The sequencing results of eight segments from NHRI-RG5 and -RG6 was quite stable in sMDCK cells after at least serial 7 passages, but several adaptive mutations, I120T, K163R, A292T, I421V, and I421M, were found in NHRI-RG3 and -RG4 after serial 3 passages and required for improving growth efficiency.…”

Section: Discussionmentioning

confidence: 99%

Development of high-growth influenza H7N9 prepandemic candidate vaccine viruses in suspension MDCK cells

et al. 2020

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Background: Influenza vaccine manufacturers traditionally use egg-derived candidate vaccine viruses (CVVs) to produce high-yield influenza viruses for seasonal or pandemic vaccines; however, these egg-derived CVVs need an adaptation process for the virus to grow in mammalian cells. The low yields of cell-based manufacturing systems using egg-derived CVVs remain an unsolved issue. This study aimed to develop high-growth cell-derived CVVs for MDCK cell-based vaccine manufacturing platforms. Methods: Four H7N9 CVVs were generated in characterized Vero and adherent MDCK (aMDCK) cells. Furthermore, reassortant viruses were amplified in adherent MDCK (aMDCK) cells with certification, and their growth characteristics were detected in aMDCK cells and new suspension MDCK (sMDCK) cells. Finally, the plaque-forming ability, biosafety, and immunogenicity of H7N9 reassortant viruses were evaluated.Results: The HA titers of these CVVs produced in proprietary suspension MDCK (sMDCK) cells and chicken embryos were 2-to 8-fold higher than those in aMDCK cells. All H7N9 CVVs showed attenuated characteristics by trypsindependent plaque assay and chicken embryo lethality test. The alum-adjuvanted NHRI-RG5 (derived from the fifth wave H7N9 virus A/Guangdong/SP440/2017) vaccine had the highest immunogenicity and cross-reactivity among the four H7N9 CVVs. Finally, we found that AddaVax adjuvant improved the cross-reactivity of low pathogenic H7N9 virus against highly pathogenic H7N9 viruses. Conclusions:Our study indicates that cell-derived H7N9 CVVs possessed high growth rate in new sMDCK cells and low pathogenicity in chicken embryo, and that CVVs generated by this platform are also suitable for both cell-and egg-based prepandemic vaccine production.

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“…Two similar cell lines, 293T (human kidney cell, similar to Vero) and Hep G2 (human hepatoma cell, similar to Huh7) were also included for the target cell investigation. In addition, another two mammal cell lines were also employed for target cell selection, including CHO (Chinese hamster ovary cell) and MDCK (dog kidney cell), which were widely employed in recombinant protein [23] or vaccine [24] production. Taken together, six types of cells were tested ( Figure 2).…”

Section: Optimization Of Sars-cov-2 Pbnamentioning

confidence: 99%

Establishment and validation of a pseudovirus neutralization assay for SARS-CoV-2

Nie

et al. 2020

Emerging Microbes & Infections

682

748

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Pseudoviruses are useful virological tools because of their safety and versatility, especially for emerging and re-emerging viruses. Due to its high pathogenicity and infectivity and the lack of effective vaccines and therapeutics, live SARS-CoV-2 has to be handled under biosafety level 3 conditions, which has hindered the development of vaccines and therapeutics. Based on a VSV pseudovirus production system, a pseudovirus-based neutralization assay has been developed for evaluating neutralizing antibodies against SARS-CoV-2 in biosafety level 2 facilities. The key parameters for this assay were optimized, including cell types, cell numbers, virus inoculum. When tested against the SARS-CoV-2 pseudovirus, SARS-CoV-2 convalescent patient sera showed high neutralizing potency, which underscore its potential as therapeutics. The limit of detection for this assay was determined as 22.1 and 43.2 for human and mouse serum samples respectively using a panel of 120 negative samples. The cutoff values were set as 30 and 50 for human and mouse serum samples, respectively. This assay showed relatively low coefficient of variations with 15.9% and 16.2% for the intra-and inter-assay analyses respectively. Taken together, we established a robust pseudovirus-based neutralization assay for SARS-CoV-2 and are glad to share pseudoviruses and related protocols with the developers of vaccines or therapeutics to fight against this lethal virus.

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Systematic evaluation of suspension MDCK cells, adherent MDCK cells, and LLC-MK2 cells for preparing influenza vaccine seed virus

Cited by 9 publications

References 35 publications

High cell density perfusion process for high yield of influenza A virus production using MDCK suspension cells

High cell density perfusion process for high yield of influenza A virus production using MDCK suspension cells

Development of high-growth influenza H7N9 prepandemic candidate vaccine viruses in suspension MDCK cells

Establishment and validation of a pseudovirus neutralization assay for SARS-CoV-2

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