Purification of Therapeutic Viruses Using Laterally-Fed Membrane Chromatography

Recent advances in the field of biotherapeutic viruses has created a demand for efficient and effective downstream purification processes to remove host-cell debris, proteins, nucleic acids, inactive virus particles, and virus aggregates from the final product. The most commonly used purification strategies for recombinant proteins such as chromatography face numerous obstacles due to the large size of virus particles. For example, Adenoviruses (which were used in this study) are approximately 90 nm in size and thus are restricted to the binding sites on the outer surface of conventional chromatography resin materials. Membrane chromatography (MC) is a promising alternative for the purification of biotherapeutic viruses due to the availability of materials with large pore sizes (typically greater than 1 μm), the dominance of convection-based mass transfer regime (enabling operation at higher flow rates), and the availability of single-use disposable systems. However, most commercial MC devices operate under a radial flow configuration, which results in non-uniform flow distribution along the membrane and ultimately the poor usage of binding sites and the dilution of samples. In contrast, the laterally-fed membrane chromatography (LFMC) device provides a homogeneous flow distribution which has been shown to be advantageous during protein purification, such as better resolution and lower product dilution^[1].

In this study, we have conducted the first ever evaluation of the LFMC process for the purification of therapeutic viruses. The purification of Adenoviruses was conducted using a BioRad NGC liquid chromatography system with the LFMC device and the radial-flow Sartobind Nano capsule (Sartorius) with both containing 1 mL of the strong anion-exchange Sartobind Q membrane. Based on elution times of NaCl pulses, it was determined that the total dead volume of the LFMC device is approximately three times lower than the Nano capsule. The feed sample of Adenovirus (containing 10⁹ infectious particles/mL) was obtained from a crude infected cell lysate that was treated with benzonase (to degrade DNA) and centrifuged (to remove cellular debris). At a flow rate of 5 mL/min, the elution peaks (based on UV absorbance) for the same gradient and step profiles were two times higher and twice as narrow for the LFMC compared to the Nano capsule – this indicates that the LFMC device gives a more homogeneous flow and lower degree of sample dilution. All of the fractions were collected in a biosafety cabinet and then analysed for the virus titer (via Hexon staining assay), protein concentration (via BCA assay), and DNA concentration (via PicoGreen assay). Initial results indicate up to 61% overall virus recovery with the LFMC and 54% with the Nano capsule. Our preliminary results indicate that the LFMC is an enhanced alternative for virus purification as it provides better resolution, lower sample dilution, smaller buffer volumes and shorter run time compared with conventional radial flow-based MC systems. Further studies are being run to optimize the virus recovery and purity in terms of solution conditions (e.g. pH, ionic strength) and gradient profiles.

[1] P. Madadkar, R. Ghosh, High-resolution protein separation using a laterally-fed membrane chromatography device, J. Membr. Sci. 499 (2016) 126–133.