Adeno-associated virus (AAV) is being used successfully in gene therapy. Different serotypes of AAV target specific organs and tissues with high efficiency. There exists an increasing demand to manufacture various AAV serotypes in large quantities for pre-clinical and clinical trials.
View Article and Find Full Text PDFThe preparation of large amount of purified helper-dependent adenoviral vector material is hampered by the lack of development of downstream processes with proven records on separation and recovery efficiencies. In order to facilitate the use of clinical-grade helper-dependent virus material for large-scale in vivo studies, a three-step purification scheme consisting of (1) an anion-exchange chromatography for initial capturing of virus, (2) a shallow iodixanol density gradient ultracentrifugation for the removal of helper virus from helper-dependent virus, and (3) a size-exclusion chromatography for the removal of iodixanol and residual protein contaminants as a polishing step was developed. The use of a fast iodixanol density ultracentrifugation step was highly effective in separating infectious helper-dependent virus from contaminating helper virus.
View Article and Find Full Text PDFThe development of insect cells expressing recombinant proteins in a stable continuous manner is an attractive alternative to the BEV system for recombinant protein production. High cell density fed batch and continuous perfusion processes can be designed to maximize the productivity of stably transformed cells. A cell line (Sf-9SEAP) expressing high levels of the reporter protein SEAP stably was obtained by lipid-mediated transfection of Sf-9 insect cells and further selection and screening.
View Article and Find Full Text PDFIn a context of large-scale production of baculoviruses in serum-free media for use as gene delivery vectors, the stability of these viruses has become an important factor. The development of robust processes heavily relies on baculovirus stock stability. In the present work, we studied over a period of 300 days the stability of baculovirus vectors produced in serum-free media stored at 4, -20, or -80 degrees C or in liquid nitrogen.
View Article and Find Full Text PDFAggregation of viral particles represents a significant problem for baculoviral stock processing and storage. Aggregation may also affect the results of viral particle counting. A method using flow cytometry was previously developed in our lab to measure the concentration of baculovirus particles produced in insect cell cultures.
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