Two AEX resins were evaluated for the capture step. For downstream purification, normal flow filtration (NFF) was used for clarification and tangential flow filtration (TFF) was used for concentration and buffer exchange. In the harvest step, 0.5% Tween 20 was used for cell lysis to release adenovirus from the host cell instead of the commonly used Triton™ X-100, now on the authorization list (Annex XIV) of registration, evaluation, authorization, and restriction of chemicals (REACH) (5). In this work, performed by iBET, Oeiras, PT, anchorage-dependent A549 cells were grown in serumcontaining medium using HYPERFlask™ cell culture vessels, and thereafter in suspension for production of oncolytic adenovirus in serum-free medium using the ReadyToProcess WAVE 25 bioreactor system. Previous work has demonstrated adenovirus production in HEK293 suspension cells using the ReadyToProcess WAVE 25 bioreactor system (1–4). One attractive alternative for scale-up using adherent cells is the use of microcarriers. However, adaptation of suspension cells can be time-consuming and difficult, and might affect virus titer negatively, making alternative solutions more feasible for laboratory- and clinical-scale production. As scaling up of anchorage-dependent cells can be a challenge, suspension cell cultures are therefore preferred as these are more easily scaled. A549 cells are traditionally propagated in adherent culture with serum-containing medium. Human lung carcinoma cells (A549) are commonly used for production of recombinant adenovirus for human gene therapy, including oncolytic adenovirus. As one of the most studied vectors for experimental and clinical use, adenovirus serotype 5 (AdV5) is a suitable system for development of a process for oncolytic adenovirus production. Recently, oncolytic adenovirus has successfully been applied as cancer immunotherapies or tumor vaccines. Also, generally causing a mild nature of disease, adenoviruses are considered as safe delivery vectors for gene therapy applications. Selectively engineered, the virus does not only destruct the target tumor cell, but also stimulate the host’s anti-tumor immune response.Īdenovirus is an extensively characterized and well-studied viral vector that infect both dividing and non-dividing cells without the risk of integration into the host genome. These viruses selectively replicate in tumor cells and effectively kill these cells without harming normal cells. Oncolytic viruses constitute a new promising therapeutic approach for treatment of cancer. Well-established analytical methods were used to ensure accurate monitoring of the processed material. The downstream process was optimized to meet regulatory demands on product purity and quality. Anion exchange (AEX) chromatography was used for virus capture and size exclusion chromatography (SEC) for polishing. Virus was released from the host cells by treatment with Tween™ 20 and clarified using ULTA™ filter capsules followed by concentration and buffer exchange using hollow fiber filters. Upstream virus production was performed in A549 cells using the single-use ReadyToProcess WAVE™ 25 rocking bioreactor system. This application note describes a process for oncolytic adenovirus production, from upstream cell culture to downstream purification, using modern tools and technologies.
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