Bridging upstream and downstream for improved adenovirus 5 bioprocess.

Adenoviruses are well-known viral vectors that have been previously used in gene therapy and as a vaccine-delivery vehicle for humans and animals. During the COVID-19 pandemic, it gained renewed attention, but at the same time, it raised concerns due to side effects observed with some of the resulting vaccines administered to patients. It has been indicated that these side effects might be attributed to impurities present in the final product. Therefore, constant enhancement of the vaccine purity and further improvement of impurity detection methods are needed. In this work, we showcase an example of industry-relevant adenovirus bioprocess optimization. Our data show the effect of upstream parameters on the bioburden introduced to the downstream process. We provide an example of process optimization using a combination of the PATfix analytical method, ddPCR, infectivity, total DNA, and total protein analyses to optimize cell density, multiplicity of infection, and length of production. Additionally, we provide data illustrating the robustness of the convective interaction media quaternary amine monolithic chromatography step. This anion exchange strategy was shown to remove over 99% of protein and DNA impurities, including those unable to be addressed by tangential flow filtration, while maintaining high adenovirus recoveries.

Preferential exclusion chromatography as a capture step for extracellular AAV harvest from adherent and suspension productions.

Preferential exclusion chromatography (PXC) sometimes described as hydrophobic interaction chromatography is a well-known, but not widely used technique for purification of Adeno-associated viruses. It employs high molarity of preferentially excluded cosolvent (salt in our case). The downside of this method is that high molarity of salt can lead to aggregation and precipitation of different compounds from the sample. In the case of viruses that are excreted to medium, the concentration of impurities is much lower compared to cell lysates, and PXC can be used as a first chromatographic, serotype independent step to concentrate and purify adeno-associated virus (AAV). Here, we explored PXC for adherent and suspension harvests using monolithic chromatographic columns (CIMmultus). Suspension extracellular adeno-associated virus, serotype 9 (AAV9) harvest had more impurities compared to adherent harvest, therefore it required higher input regarding method development. Final conditions for suspension harvest included higher molarity of binding salt and using more open channel format of chromatographic column (6 µm channel size). Vector genome analysis with droplet digital polymerase chain reaction (ddPCR) revealed 84% and 97% recovery for suspension and adherent AAV9 harvest, respectively. After PXC capture step, adherent AAV9 was purified by already described ion exchange techniques. Overall process vector genome recovery, from clarified harvest to anion exchange elution fraction, was 54% measured by ddPCR. Residual host cell DNA was measured at 40 ng per 1E13 vector genome, and empty AAV was below 5% in final anion exchange chromatography fraction.