Large pilot scale cultivation process study of adherent MDBK cells for porcine Influenza A virus propagation using a novel disposable stirred-tank bioreactor

Abstract

Influenza is one of the major viral diseases and has tremendous importance for human and animal health worldwide. The etiologic agent, Influenza A virus belongs to the family Orthomyxoviridae and is a single stranded (-) enveloped RNA virus with round shape and a diameter of 90-130 nm. Its genome consists of 8 segments which code for up to 11 proteins. The major surface proteins and antigenic determinants are hemagglutinin (HA) and neuraminidase (NA). Pigs are of special importance for Influenza. They are susceptible for a number of Influenza A viruses from different species (human, avian, porcine); in the case of multiple infections of swine new and dangerous virus strains can be assembled by reassortment of genes (antigenic shift). The strain which was used in this study is part of a trivalent inactivated vaccine against porcine Influenza (IDT Biologika GmbH). The cultivation of Influenza virus for the production of vaccines is often still done in embryonated hens eggs, although there are a number of cell lines available which propagate the virus, e. g. VERO or MDCK cells. Egg production processes are limited in terms of capacity whereas cell culture based processes are more flexible and easy to scale up when facing pandemic situations (H5N1 in 2005 and H1N1pan in 2009/10). Currently, adherent MDBK cells are used for the production of porcine Influenza A viruses at commercial scale (IDT Biologika GmbH). This process is performed in roller bottles so far using a biphasic process scheme (cell growth and virus propagation phase) and is thus limited in capacity, yields and process control. The main goal of this study was to explore options for direct transfer of this process into a novel disposable stirred-tank bioreactor (BIOSTAT CultiBag STR, Sartorius Stedim Biotech GmbH) using microcarriers for the attachment of MDBK cells. The advantage of this system is its scalability up to 1000 l, better process control and presumably higher virus yields. The use of disposables has positive effects on costs and efforts but also for cleaning validation in a multi-purpose facility. Based on the results of this study further optimization steps can be done and the implementation of such systems in GMP routine vaccine production can be evaluated.