Intensified Production of Vaccinia-Based Oncolytics in the High Density Cell Respirator (HDCR) Bioreactor Improves Vaccine Logistics and Economics

Abstract

Intensification of cell-based production processes is key to improving oncolytic vaccine logistics and economics by saving on GMP space, time, labor, and feedstock. Many promising vectors, including those based on vaccinia virus (e.g. CF33, JX-594/Pexa-Vec) are still produced using flask-based culture due to the cost, effort, and uncertainty involved in adapting to stirred-tank or perfusion processes. Based on prior success culturing adherent cells (e.g. HEK293, A549, CV-1) to high densities (107-8 cells/mL) using the scalable high-density cell respirator (HDCR) bioreactor, we hypothesized that the platform could support orders-of-magnitude-intensified production of replication competent viruses. Here we report on CF33 virus production as a proof-of-concept for oncolytic virotherapy. Bioreactor cartridges were produced based on a 4-stack of 600 cm² HDCR membranes, featuring a proprietary gas perfusable and permeable microarchitecture that optimally oxygenates cells even at high densities. Media perfusion was feedback-controlled based on glucose measurements in the waste stream. A549 cells were seeded into the bioreactor on microcarriers, expanded 10-fold to mid-10⁷ cells/mL densities, infected with CF33 virus (MOI of 0.1), and harvested 48 hours post-infection. CF33 virus was gradient purified and titered for functional virus by plaque forming assay. Multiple production runs using different strains of CF33 virus validated the reproducibility of the process, as summarized in Table 1. Importantly, cell specific titers (PFU/cell) remained comparable to conventional flask-based production, leading to significant intensification due to the higher cell densities supported in the HDCR bioreactor. Volumetric productivity is on the order of 100× that of cell factories. The efficient usage of media due to the gas-media decoupled operation of the HDCR bioreactor enables gradient-purified virus costs of around $500/1010 PFU. This is significantly below the $10,000/1010 PFU charged by CMOs and supports our mission of democratizing access to life saving medicines. The straightforward adaptation of CF33 virus production from a flask-based to a high yield, intensified process highlights the logistical and economical advantage of the HDCR platform for oncolytics.

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