Production of canine adenovirus type 2 in serum-free suspension cultures of MDCK cells

Abstract

The potential of adherent Madin Darby Canine Kidney (MDCK) cells for the production of influenza viruses and canine adenovirus type 2 (CAV-2) for vaccines or gene therapy approaches has been shown. Recently, a new MDCK cell line (MDCK.SUS2) that was able to grow in suspension in a fully defined system was established. In this work, we investigated whether the new MDCK.SUS2 suspension cell line is suitable for the amplification of CAV-2 under serum-free culture conditions. Cell growth performance and CAV-2 production were evaluated in three serum-free media: AEM, SMIF8, and EXCELL MDCK. CAV-2 production in shake flasks was maximal when AEM medium was used, resulting in an amplification ratio of infectious particles (IP) of 142 IP out/IP in and volumetric and cell-specific productivities of 2.1 × 10(8) IP/mL and 482 IP/cell, respectively. CAV-2 production was further improved when cells were cultivated in a 0.5-L stirred tank bioreactor. To monitor infection and virus production, cells were analyzed by flow cytometry. A correlation between the side scatter measurement and CAV-2 productivity was found, which represents a key feature to determine the best harvesting time during process development of gene therapy vectors that do not express reporter genes. This work demonstrates that MDCK.SUS2 is a suitable cell substrate for CAV-2 production, constituting a step forward in developing a production process transferable to industrial scales. This could allow for the production of high CAV-2 titers either for vaccination or for gene therapy purposes.

Cell substrates for the production of viral vaccines

“…Safety concerns, fluctuating cost levels and risk of serum shortage are additional constraints which do not support the use of FBS. This encourages vaccine manufacturers to move toward animal-component-free cell cultures and the development of cell-specific media [68][69][70]. Media development and production are often outsourced to media suppliers (e.g.…”

Cell Culture-based Viral Vaccines: Current Status and Future Prospects

“…Furthermore, the characteristics of cell substrate including cell growth and cytogenetic patterns, tests for infectious agents and oncogenic ability are observed. Finally, the compatibility between the viral vaccine and cell substrate needs to be evaluated as the genotype or phenotype of the virus may be altered due to selective pressure during passaging, therefore, it is essential to confirm the stability of vaccine virus [12,[114][115][116][117].…”

Advancements in the design and scalable production of viral gene transfer vectors

“…CAd2 for instance is produced in the Madin-Darby Canine Kidney Epithelial (MDCK) cells engineered to express the CAd2 E1 protein (Szelechowski, Bergeron, Gonzalez-Dunia, & Klonjkowski, 2013). Anchorage-dependent cells are fundamentally more difficult to scale, requiring the use of microcarriers or culture in fixed bed bioreactors, though there has been some recent progress bringing MDCK cells into suspension culture (Castro et al, 2015).…”

“…serotype's endogenous E4-ORF6 (Figure 3a) (Alonso-Padilla et al, 2016).The production of adenovirus vectors from non-human serotypes is more problematic, often requiring the use of species-specific producer cell lines that may lack the scalability of HEK-293 or PER.C6 cells(Figure 3a). Anchorage-dependent cells are fundamentally more difficult to scale, requiring the use of microcarriers or culture in fixed bed bioreactors, though there has been some recent progress bringing MDCK cells into suspension culture(Castro et al, 2015). Anchorage-dependent cells are fundamentally more difficult to scale, requiring the use of microcarriers or culture in fixed bed bioreactors, though there has been some recent progress bringing MDCK cells into suspension culture(Castro et al, 2015).…”