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The oncolytic virus H-1PV is a promising candidate for various cancer treatments. Therefore, production process needs to be optimized and scaled up for future market release. Currently, the virus is produced with minimum essential medium in 10-layer CellSTACK® chambers with limited scalability, requiring a minimum seeding density of 7.9E3 cells/cm 2 . Production also requires a 5% fetal bovine serum (FBS) supplementation and has a virus yield up to 3.1E7 plaque-forming units (PFU)/cm 2 . Using the animal-free cell culture medium VP-SFM™ and a new feeding strategy, we demonstrate a yield boost by a mean of 0.3 log while reducing seeding density to 5.0E3 cells/cm 2  and cutting FBS supplementation by up to 40% during the production process. Additionally, FBS is completely removed at the time of harvest. Eleven commercial micro- and macrocarriers were screened regarding cell growth, bead-to-bead transfer capability, and virus yield. We present a proof-of-concept study for producing H-1PV on a large scale with the microcarrier Cytodex® 1 in suspension and a macrocarrier for a fixed-bed iCELLis® bioreactor. A carrier-based H-1PV production process combined with an optimized cell culture medium and feeding strategy can facilitate future upscaling to industrial-scale production. Key points •  Virus yield increase and FBS-free harvest after switching to cell culture medium VP-SFM™ . •  We screened carriers for cell growth, bead-to-bead transfer capability, and H-1PV yield . •  High virus yield is achieved with Cytodex® 1 and macrocarrier for iCellis® in Erlenmeyer flasks .

Upstream process optimization and micro- and macrocarrier screening for large-scale production of the oncolytic H-1 protoparvovirus