Described is a reproducible, effective and scalable process for parvovirus production including characterization strategies, preferably production of H-1PV.

The present invention relates to a separation of viruses of an essentially spherical shape from viruses with a rod-like shape that are comprised in a sample, wherein the sample comprising the viruses is subjected to filtration.

The present invention provides a method for purification of a virus or virus antigen comprising providing a virus preparation and centrifugation of said virus preparation in a gradient of a sugar established by the addition of two or more buffered sugar layers of different concentration. The method leads to higher yields and reduces unwanted aggregation of the virus or virus antigen by increasing the volume of the peak pool.

Described is an adenovirus derived helper virus which may comprise the adenoviral DNA sequences for E2a, S4 (orf6), the VA1 RNA gene, and the parvovirus VP capsid gene unit. Also described is a method of efficiently preparing a recombinant parvovirus (particle) which is based on the use of various adenoviral derived helper viruses/vectors.

The present invention provides a method for upstream optimization the large-scale parvovirus production, preferably the oncolytic protoparvovirus H-1 (H-1PV). It is based on microcarriers or macrocarriers and their respective use in suspension or fixed-bed, an optimized cell culture medium, and a medium exchange strategy. In summary, with the optimized cell culture medium and the new medium exchange strategy, the inventors established a reduction in seeded cell density and animal serum, leading to an animal serum-free harvest. The tested carriers are best suited for a high H-1PV yield, cell growth, and bead-to-bead transfer capability, wherein the inventors additionally scaled up the process from 24-well plates to Erlenmeyer, Spinner flask and iCellis nano. As a conclusion, the present invention provides a large-scale method for producing the oncolytic protoparvovirus H-1 with a high virus yield, while lowering production costs and avoiding undesired products of animal origin at the same time.

The purity and titer of virus stocks used for virus clearance studies have a significant influence on study outcome, and impact how well the scale-down model represents the production-scale process. Impurities in virus stocks are particularly important in the testing of small virus retentive filters because these impurities may cause a filter to foul prematurely, leading to underestimation of the true throughput capability of the filter and consequent inappropriate sizing of the production scale unit. In addition, impurities can also affect the levels of virus clearance observed by altering the fouling mechanisms and subsequent fluid passage through the virus filter. Described herein are methods for making, producing and using high purity virus stocks having high titer.

Described is an adenovirus derived helper virus which may comprise the adenoviral DNA sequences for E2a, S4 (orf6), the VA1 RNA gene, and the parvovirus VP capsid gene unit. Also described is a method of efficiently preparing a recombinant parvovirus (particle) which is based on the use of various adenoviral derived helper viruses/vectors.

The present invention provides a robust single clone Master Cell Bank (MCB) for an optimized production of H-1 parvovirus (H-1 PV) which is suitable to increase infectious parvovirus production compared to standard producer NB-324K mixed cells.