Steric exclusion chromatography for purification of cell culture-derived influenza A virus using regenerated cellulose membranes and polyethylene glycol

- Cellulose membranes are a suitable matrix for steric exclusion chromatography.
- Virus particles were eluted in Tris-HCl and PBS buffers with >95% recovery.
- Maximum depletion of host cell DNA and protein was 99.7% and 92.4%, respectively.
- Operation is single-use and fully scalable to industrial manufacture.
- The DBC5 of the hemagglutinin antigen was 3.4 μgHA cm−2 (906 μgHA mL−1).

Steric exclusion chromatography has been used for the purification of proteins and bacteriophages using monoliths. The operation is carried out by mixing a crude sample containing the target species with a predetermined concentration and molecular weight of polyethylene glycol (PEG) and loading it onto a non-reactive hydrophilic surface. Product capture occurs by the mutual steric exclusion of PEG between the product and the matrix. Selectivity is significantly influenced by target product size. Product elution is achieved by decreasing the PEG concentration. In this study, a 75 cm2 cellulose membrane adsorber was used for the purification of a clarified and inactivated influenza A virus broth produced in a 5 L bioreactor using suspension Madin Darby canine kidney cells. Product recovery was above 95% based on hemagglutination activity and single radial immunodiffusion assays. Maximum depletion of double stranded host cell DNA and total protein was 99.7% and 92.4%, respectively. Purified virus particles showed no aggregation with a monodisperse peak around 84 nm. 250 mL of the clarified inactivated virus broth was purified within 40 min. The surface area productivity based on the recovery of the viral hemagglutinin antigen was 28-50 mg m−2 h−1 depending on the feed and loading conditions.