Large-scale Suspension Cultures Of Mammalian Cells To Produce Adenovirus Technology Experimental Research

Adenovirus vectors are one of the most promising gene transfer systems. Adenoviral vectors have a number of advantages such as their ability to infect post-mitotic tissues. They are produced at high titers and are currently used in 26% of clinical protocols through different strategies.They are of great value for gene therapy because these vectors achieve temporal high-level transgene expression and high gene transfer efficiency. To meet increasing needs of adenovirus vectors for gene therapy programs, parallel development of efficient, scalable and reproducible production processes is required. Perfusion cultivation of 293 cells is one of the most commonly used methods to produce adenovirus vectors and it is suitable for industrialized production specially. Experimental studies had been carried out by perfusion cultivation of HEK293 N3S cells in a 5L stirring bioreactors to produce recombinant adenovirus containing the green fluorescent protein gene (Ad-GFP), Adenovirus vector encoding hepatocyte growth factor gene(Ad-HGF), Adenovirus vector encoding p53, GM-CSF, B7-1 gene (BB-102) and so on. Perfusion rate was 1~2 volume/day. To infect the 293 N3S cells with Ad-GFP at the density of 2~4×106 cells/ml. The time of collecting cells was 48 hours post infection. By perfusion cultivation of 293 N3S cells in a 5L stirring bioreactors, we established the production process for adenovirus vectors, which paves a way to produce recombinant adenovirus for gene therapy.Purification of recombinant adenovirus vector was achieved using two rounds of cesium chloride (CsCl) density gradient ultracentrifugation. This technique was useful for generation of small scale clinical-grade lots. Some quality testings were carried out according to the production process and the national standards of Chinese Requirements for Biological Products (CRBP), such as physic examination, asepsis testing, viral activity examination, viral purity determination, replicative competent adenovirus testing, bacteria endotoxin determination, residues testing, characteristic gene identification and so on. The products were qualified basically.Although the CsCl density gradient method was useful for generation of small scale clinical-grade lots, it was not scalable. With the advent of gene therapy protocols, large quantities of AdV are required. Other purification procedures were evaluated, for example, ion exchange, hydrophobic interaction, metal chelate and size-exclusion chromatography. These methods were evaluated for capture and purification of type-5 adenovirus vector. The anion-exchange chromatography was applied to purifying Adenovirus vectors in the experiments. The Q Sepharose XL was used in the purification, the results demonstrated that the hybridproteins and cellular nucleic acids were removed from the cell lysate. The final recovery of adenovirus vector was about 70% through the chromatography. The purity of the products was determined by the A260/A280 ratio, the high performance liquid chromatography (HPLC) assay, SDS-PAGE and so on. The products coming from the chromatography was similar to which came from CsCl gradient centrifugation. This time- and labor-saving process could be flexibly enlarged and is suitable to large-scale production.