Continuous influenza virus production in a tubular bioreactor system provides stable titers and avoids the "von Magnus effect".

Felipe Tapia,Udo Reichl,Volker Sandig,Daniel Wohlfarth,Yvonne Genzel,Ingo Jordan,Maria Serena Beato

doi:10.1371/journal.pone.0224317

Abstract

Continuous cell culture-based influenza vaccine production could significantly reduce footprint and manufacturing costs compared to current batch processing. However, yields of influenza virus in continuous mode can be affected by oscillations in virus titers caused by periodic accumulation of defective interfering particles. The generation of such particles has also been observed previously in cascades of continuous stirred tank reactors (CSTRs) and is known as the “von Magnus effect”. To improve virus yields and to avoid these oscillations, we have developed a novel continuous tubular bioreactor system for influenza A virus production. It was built using a 500 mL CSTR for cell growth linked to a 105 m long tubular plug-flow bioreactor (PFBR). Virus propagation took place only in the PFBR with a nominal residence time of 20 h and a production capacity of 0.2 mL/min. The bioreactor was first tested with suspension MDCK cells at different multiplicities of infection (MOI), and then with suspension avian AGE1.CR.pIX cells at a fixed nominal MOI of 0.02. Maximum hemagglutinin (HA) titers of 2.4 and 1.6 log10(HA units/100 μL) for suspension MDCK cells and AGE1.CR.pIX cells, respectively, were obtained. Flow cytometric analysis demonstrated that 100% infected cells with batch-like HA titers can be obtained at a MOI of at least 0.1. Stable HA and TCID50 titers over 18 days of production were confirmed using the AGE1.CR.pIX cell line, and PCR analysis demonstrated stable production of full-length genome. The contamination level of segments with deletions (potentially defective interfering particles), already present in the virus seed, was low and did not increase. Control experiments using batch and semi-continuous cultures confirmed these findings. A comparison showed that influenza virus production can be achieved with the tubular bioreactor system in about half the time with a space-time-yield up to two times higher than for typical batch cultures. In summary, a novel continuous tubular bioreactor system for cell culture-based influenza virus production was developed. One main advantage, an essentially single-passage amplification of viruses, should enable efficient production of vaccines as well as vectors for gene and cancer therapy.

Highlights

Influenza viruses are a major threat for human and animal health
A stable cell concentration of 5–6×106 cells/mL in the continuous stirred tank reactor (CSTR) was achieved after one week of culture
The nominal residence time (RT) of the plugflow bioreactor (PFBR) was set to h, the actual RT, determined by measuring the volume of harvest collected twice a day, was h

Summary

Introduction

Influenza viruses are a major threat for human and animal health. Influenza viruses have an approximate size of 100 nm and are characterized by an enveloped structure with a negativesense RNA genome. The genome is divided in 7–8 separated segments coding for more than 10 proteins depending on strains [1]. Hemagglutinin (HA) and neuraminidase (NA), the two main viral glycoprotein antigens, are located in the virus membrane. Infectious units are transmitted via air droplets and cause sudden fever and severe morbidity, sometimes leading to the death of the patients either directly or via bacterial sequelae. The most effective approach to control the disease is by vaccination [2]. Influenza vaccine production capacity increased to 6.4 billion doses in 2015, providing enough vaccines remains challenging especially in a pandemic situation [3]

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Conclusion