Therapeutic viral vectors are an emerging technology with several clinical applications in gene therapy, vaccines, and immunotherapy. Increased demand has required the redevelopment of conventional, low-throughput cell culture and purification manufacturing methods such as static cell stacks and ultracentrifugation. In this work, scalable methods were investigated for the manufacture of an oncolytic virus immunotherapy application consisting of a prototype strain of coxsackievirus A21 (CVA21) produced in adherent MRC-5 cells. Cell culture was established in stirred-tank microcarrier bioreactors, and an efficient affinity chromatography method was developed for the purification of harvested CVA21 through binding of the viral capsids to an immobilized glutathione (GSH) ligand. Bioreactor temperature during infection was investigated to maximize titer, and a decrease in temperature from 37°C to 34°C yielded a two-three-fold increase in infectivity. After purification of the 34°C harvests, the GSH affinity chromatography elution not only maintained a >two-fold increase in infectivity and viral genomes but also increased the proportion of empty capsids compared to 37°C harvests. Using material generated from both infection temperature setpoints, chromatographic parameters and mobile phase compositions were studied at the laboratory scale to maximize infectious particle yields and cell culture impurity clearance. Empty capsids that co-eluted with full capsids from 34°C infection temperature harvests were poorly resolved across the conditions tested, but subsequent polishing anion exchange and cation exchange chromatography steps were developed to clear residual empty capsids and other impurities. Oncolytic CVA21 production was scaled-up 75-fold from the laboratory scale and demonstrated across seven batches in 250 L single-use microcarrier bioreactors and purified with customized, prepacked, single-use 1.5 L GSH affinity chromatography columns. The large-scale bioreactors controlled at 34°C during infection maintained a three-fold increase in productivity in the GSH elution, and excellent clearance of host cell and media impurities was observed across all batches. This study presents a robust method for the manufacture of an oncolytic virus immunotherapy application that may be implemented for the scalable production of other viruses and viral vectors which interact with glutathione.
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http://dx.doi.org/10.3389/fbioe.2023.1193454 | DOI Listing |
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December 2024
Guangxi Key Laboratory for Aquatic Genetic Breeding and Healthy Aquaculture, Guangxi Academy of Fishery Sciences, Nanning, Guangxi 530021, China. Electronic address:
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December 2024
Department of Chemistry, University of Nebraska-Lincoln, Lincoln, NE, USA. Electronic address:
Exp Cell Res
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Department of Maternal-Fetal Medicine Pregnancy Research Centre, Royal Women's Hospital, Parkville, VIC 3052, Australia; University of Melbourne Department of Obstetrics and Gynaecology and Newborn Health, Royal Women's Hospital, Parkville, VIC 3052, Australia. Electronic address:
Increasing evidence shows extracellular vesicles (EVs) are primarily responsible for the beneficial effects of cell-based therapies. EVs derived from mesenchymal stromal cells (MSCs) show promise as a source of EVs for cell-free therapies. The human placental fetal-maternal interface is a rich and abundant source of MSCs from which EVs can be isolated.
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December 2024
Department of Biotechnology, Faculty of Science and Humanities, SRM Institute of Science and Technology, Kattankulathur, Tamilnadu, 603203, India.
Mycobacterium tuberculosis is a human pathogen that causes Tuberculosis (TB) disease. Researchers have reported the activity of traditional medicinal plants against human pathogens. However, antimycobacterial studies of medicinal plants against M.
View Article and Find Full Text PDFZhongguo Zhong Yao Za Zhi
November 2024
Henan University of Chinese Medicine Zhengzhou 450046, China National Engineering Laboratory for Quality Control Technology of Chinese Herbal Medicines, Institute of Chinese Materia Medica,China Academy of Chinese Medical Sciences Beijing 100700, China.
Shaoyao Gancao Decoction(SGD) is a classic formula used in the clinical treatment of joint diseases, such as rheumatoid arthritis(RA), though its mechanism of action remains unclear. This study aimed to explore the mechanism of SGD in treating RA through chemical and network pharmacology analyses, combined with cellular experiments. UPLC-Orbitrap-MS~2 was used to qualitatively analyze SGD and drug-containing serum of rats after oral administration of SGD, thereby identifying the chemical composition and plasma components of SGD.
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