Addressing the Downstream Challenges of Viral Therapy for Cancer Treatment – a Study of Rhabdoviral Vector Purification

Oncolytic viruses (OVs) are a class of cancer therapies that are currently going through clinical trials on their way to full approval. For example, the world’s first clinical trial using a combination of two viruses to kill cancer cells and stimulate an immune response is currently being conducted. Existing downstream purification processes use a combination of chromatography and membrane-based processes to remove both process-related (e.g. cell debris, host cell proteins, and nucleic acids) and product-related impurities (e.g. aggregated and empty virus particles). One of the main challenges during viral vector manufacturing is the mandatory final sterile filtration step. The primary issue is the severe membrane fouling, as indicated by a dramatic and uncontrollable transmembrane pressure increase associated with the low recovery of the desired final product - recent studies reporting up to 80% losses in the total vector titer. We have evaluated the fouling behaviour of commercially available sterile filters with various membrane morphologies using a Rhabdoviral vector.

As a potential improvement in the downstream process, we have investigated the use of monolithic column chromatography. This technology features a continuous stationary phase with a network of interconnected pores that has been previously shown to be ideal for the production of ‘large’ biomolecules. The monolithic column (1 mL capacity) made of polymethacrylate copolymers functionalized with quaternary ammonium groups (BIA Separations) was evaluated using NGC Quest Plus chromatography system (BioRad). The flow rate and buffer composition conditions used for the monolith chromatography were optimized in terms of the recovery of the Rhabdoviral vector. At a relatively high flow rate of 4 ml/min, the purified Rhabdoviral vector sample was purified using a step-elution salt concentration gradient with the majority of impurities washed out at lower salt concentrations. To investigate the fouling propensity of the different biomolecules that were collected in each chromatographic fraction, a series of small-scale (13mm polycarbonate filter holder with the filter area of 0.5 cm²) sterile filtration studies were conducted. The resulting TMP profiles for each fraction (at constant flux) were analyzed using various pore plugging/blocking models. Our current hypothesis is that by increasing removal of the process- and product-related impurities using the monolithic column, we can improve the sterile filtration process performance in terms of throughput and product recovery that is needed for the large-scale production of oncolytic viruses.