Establishing Suspension Cell Cultures for Improved Manufacturing of Oncolytic Adenovirus

Abstract

Recent clinical trials have shown the potential of oncolytic adenoviruses as a cancer immunotherapy. A successful transition of oncolytic adenovirus to clinical applications requires efficient and good manufacturing practice compatible production and purification bioprocesses. Suspension cultures are preferable for virus production as they can reduce process costs and increase product quality and consistency. This work describes the adaptation of the A549 cell line to suspension culture in serum‐reduced medium validated by oncolytic adenovirus production in stirred tank bioreactor. Cell concentrations up to 3 × 106 cells mL−1 are obtained during the production process. At harvest 1.4 × 1010 infectious particles mL−1 and 6.9 ± 1.1 × 1010 viral genome mL−1 are obtained corresponding to a viral genome: infectious particles ratio of 5.2 (± 1.9): 1 confirming the virus quality. Overall, the suspension characteristics of these A549 cells support an easily scalable, less time‐consuming, and more cost‐effective process for expanded success in the use of oncolytic viruses for cancer therapy.

Animal Cell Lines as Expression Platforms in Viral Vaccine Production: A Post Covid-19 Perspective

“…However, it has been shown that it can also be used in the production of adenovirus, , which has become popular again in recent years . A-549, which is also human origin, is a cell line with a very high potential for use after its reliability in viral vaccine production has been demonstrated in more detail. , However, since only approved and commercially used cellular platforms are covered in this review, all these other potential vaccine expression platforms are not elaborated.…”

“… , This situation creates additional costs in production and prolongs the preprocessing time in preparation for production. Additionally, it is difficult to obtain large amounts of cells because cell proliferation is limited by the available surface area. ,,, Since it is known that primary cultures and diploid cell lines retain their adherent character and have limited adaptability to suspended conditions, they are produced adherently in viral vaccine production, either using microcarriers or in packed-bed bioreactors (such as iCellis) where other suitable support materials are used. , Cells with suspension character have advantages such as ease of scale-up since they are not dependent on any surface, less labor required, growth is limited only by the cell concentration in the nutrient medium, and the cellular microenvironment in the production environment is more homogeneous. , However, as mentioned, since almost all of the expressional platforms used in vaccine production show adherent character, if the use of suspension cultures is aimed in production, the cellular substrates must be adapted to the suspension growth condition. , This adaptation is possible by culturing adherent cells in dynamic systems, using serum-free nutrient media specifically developed for the cell line, or applying both methods simultaneously. ,,, In this way, very high success can be achieved in continuous cell lines. ,, However, since the cellular character changes greatly during adaptation, it must be constantly checked that this suspended culture developed by cell engineering synthesizes the vaccine material in sufficient quantities and in a suitable form for industrial use. Additionally, the safety properties of adapted cells, such as tumorigenicity, should also be re-evaluated and checked. ,, The main disadvantage of suspension cultures is the need for cell-retention devices in case of any nutrient media change or washing process during production …”

Bridging upstream and downstream for improved adenovirus 5 bioprocess

“…Adenovirus harvests from adherent and suspension cell lines differ in multiple aspects, including not only the method of cultivation but also the raw materials used and necessary production equipment. Considering the process scalability, costs, product quality, and its consistency, the choice of upstream platform plays an important role [26]. In terms of robustness, the chromatographic step capacity was checked, and H4 was included as additional material from an adherent cell line.…”

Incubation Temperature and Period During Denarase Treatment and Microfiltration Affect the Yield of Recombinant Adenoviral Vectors During Downstream Processing

“…Cell lysis using detergent is easy to perform in the laboratory and at the production scale. In this step, most commonly, non-ionic detergents such as Tween-20 and Tween-80 and ionic detergents such as sodium deoxycholate can be used to solubilize cell membranes and release viruses [ 21 , 22 ]. Tween-20 detergent, also known as Polysorbate-20, is frequently used for protein solubility, protein–lipid, lipid–lipid aggregation, and enveloped virus clearance in the pharma industry [ 23 , 24 ].…”

“…In downstream processes of adenoviral vectors, incubation times vary from half an hour to 4 h during the release of viruses from the cell [ 22 , 28 ]. The incubation temperature varies between room temperature and 37 °C [ 21 , 22 , 28 ]. However, the effects of incubation periods and temperature on vector yield and stabilization are not well known.…”