Dataset Information

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

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.

SUBMITTER: Wohlfarth D

PROVIDER: S-EPMC8648687 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Similar Datasets

Project description:BackgroundStemphylium blight incited by Stemphylium botryosum poses a significant threat to lentil crops worldwide, inducing severe defoliation and causing substantial yield losses in susceptible varieties under favorable conditions. While some moderate levels of resistance have been identified within lentil germplasm, a low number of resistant cultivars are available to farmers. Adding to the common constraints of resistance breeding, a notable challenge is generating a sufficient number of spores for large-scale screenings, which are essential for pinpointing additional sources of resistance for integration into breeding programs. Therefore, there is a pressing need to improve existing screening methods and tailor them for large-scale material selection. In pursuit of this objective, a protocol for the efficient production of fungal material has been adapted.ResultsOptimization of fungal material production was successfully achieved by comparing the use of fungal mycelia and spores. Spore production was found to be optimal when produced on solid V8-PDA(hi) medium, while liquid Richard's medium was identified as superior for mycelium yield. Furthermore, a refined screening method was developed by evaluating the resistance of six lentil accessions to stemphylium blight. This assessment included the use of either fungal mycelia (at densities ranging from 1 to 5 g L- 1) or spores (with densities ranging from 5 × 104 to 2 × 105 conidia mL- 1) under three different relative humidity levels (from 50 to 100%). Both humidity levels and inoculum dose significantly influenced the final disease rating (DR) and the relative Area Under the Disease Progress Curve (rAUDPC). Differences among genotypes in final symptom severity (DR) became more pronounced after inoculation with inoculum densities of 5 g L- 1 of mycelium or of 105 and 2 × 105 conidia mL- 1 of spore under 100% relative humidity. Given the challenges associated with the large-scale production of S. botryosum spores, inoculations with 5 g L- 1 of mycelium is highly recommended as a practical alternative for conducting mass-scale screenings.ConclusionsThe findings from this study underscore the critical importance of maintaining high level of humidity during inoculation and disease progression development for accurately assessing resistance to stemphylium blight. The optimization of mycelial production for suspension inoculation emerges as a more reliable and efficient approach for conducting large-scale screening to assess germplasm resistance against stemphylium blight in lentil crops.

Project description:Oncolytic viruses (OVs) represent a novel class of immunotherapeutics under development for the treatment of cancers. OVs that express a cognate or transgenic fusion protein is particularly promising as their enhanced intratumoral spread via syncytia formation can be a potent mechanism for tumor lysis and induction of antitumor immune responses. Rapid and efficient fusion of infected cells results in cell death before high titers are reached. Although this is an attractive safety feature, it also presents unique challenges for large-scale clinical-grade manufacture of OVs. Here we evaluate the use of four different suspension cell lines for the production of a novel fusogenic hybrid of vesicular stomatitis virus and Newcastle disease virus (rVSV-NDV). The candidate cell lines were screened for growth, metabolism, and virus productivity. Permissivity was evaluated based on extracellular infectious virus titers and cell-specific virus yields (CSVYs). For additional process optimizations, virus adaptation and multiplicity of infection (MOI) screenings were performed and confirmed in a 1 L bioreactor. BHK-21 and HEK293SF cells infected at concentrations of 2 × 106 cells/mL were identified as promising candidates for rVSV-NDV production, leading to infectious titers of 3.0 × 108 TCID50/mL and 7.5 × 107 TCID50/mL, and CSVYs of 153 and 9, respectively. Compared to the AGE1.CR.pIX reference produced in adherent cultures, oncolytic potency was not affected by production in suspension cultures and possibly even increased in cultures of HEK293SF and AGE1.CR.pIX. Our study describes promising suspension cell-based processes for efficient large-scale manufacturing of rVSV-NDV. KEY POINTS: • Cell contact-dependent oncolytic virus (OV) replicates in suspension cells. • Oncolytic potency is not encompassed during suspension cultivation. • Media composition, cell line, and MOI are critical process parameters for OV production. • The designed process is scalable and shows great promise for manufacturing clinical-grade material.

Dataset Information

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

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets