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

Daniel Wohlfarth,Linh Minh Phuc Phan,Martin Vogel,Marcus Müller,Barbara Leuchs,Adrian Brunecker,Veronika Frehtman

doi:10.1007/s00253-021-11642-y

Daniel Wohlfarth, Linh Minh Phuc Phan + Show 5 more

Open Access

https://doi.org/10.1007/s00253-021-11642-y

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Abstract

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/cm2. Production also requires a 5% fetal bovine serum (FBS) supplementation and has a virus yield up to 3.1E7 plaque-forming units (PFU)/cm2. 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/cm2 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.

Highlights

According to the World Health Organization (WHO), cancer was the second leading cause of death worldwide in 2018, with an estimated 9.6 million deaths
For cell counting with trypan blue, the cell culture medium was removed, and the cell layer or carriers were washed with phosphate-buffered saline (PBS), trypsinated with approximately 0.017 ml/cm2 PBS/1 mM ethylenediaminetetraacetic acid (EDTA) 0.25% trypsin (Gibco, Grand Island, USA) at 37 °C until full cell detachment was observed by microscope
VP-SFMTM supplemented with 5% fetal bovine serum (FBS) or 0% FBS achieved an increased virus yield compared to MEM, at 2.7E7 plaque-forming units (PFU)/cm2 and 1.8E7 PFU/cm2, respectively

Summary

Introduction

According to the World Health Organization (WHO), cancer was the second leading cause of death worldwide in 2018, with an estimated 9.6 million deaths. The economic damage associated with cancer is significant and increasing, totaling approximately $1.16 trillion in 2010 alone (Stewart and Wild, 2014). Oncolytic virus (OV) therapy represents a promising approach to treating this disease. OVs are genetically engineered or naturally occurring viruses that selectively destroy cancer cells without harming healthy tissue (Fukuhara et al, 2016). In 2015, the first OV therapeutic (T-VEC or ImlygicTM) was approved by the Food and Drug Administration (FDA) (https://www.fda.gov/vaccines-blood-biologics/cellular-gene-therapy-products/imlygic-talimogene-laherparepvec) and followed by the European Medicines Agency (EMA) (https://www.ema.europa.eu/en/medicines/human/EPAR/imlygic). Other OV therapeutics based on different virus platforms (https://webs.iiitd.edu.in/raghava/ovirustdb/clinical.php) are in the development pipeline

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