The baculovirus expression vector system (BEVS) has emerged as a powerful tool for the production of recombinant proteins used as therapeutics, reagents, and diagnostics. In order to maximize the system’s efficiency and thereby reduce costs, optimizing production parameters is imperative. A critical factor in optimization is the production of a high-quality baculovirus stock with a high-titer, pure clonal population of recombinant virus that is stable over time. Baculovirus stocks may contain alternate varieties of infectious virus due to cross-contamination, outgrowth of non-recombinant virus, and excision of inserts attributable to some recombinant virus production technologies. Since the advent of the BEVS, the “gold standard” for production of pure baculovirus stocks has been plaque purification. Briefly, plaque purification involves infecting a monolayer of cells with dilutions of virus before applying an agarose overlay to the monolayer. After a 5–7 day incubation, isolated plaques can be picked, virus eluted from the agarose plug, and amplified. The drawbacks of plaque purification are: (1) it is time and labor-intensive; (2) the results hinge greatly on the health of the cells and the cell density at infection; (3) identification and picking of isolated plaques is challenging; and (4) the integrity of the procedure is easily compromised by virus diffusion and mass flow of virus-containing liquid beneath the agarose overlay…
Tag: <span>baculovirus expression system</span>
The licensing of recombinant vaccines produced using the baculovirus expression vector system (BEVS) has cleared the way for the production of a variety of biopharmaceuticals produced using this technology. Obtaining accurate estimates of both total and infectious baculovirus titer in upstream and downstream bioprocess fluids is one of many process controls that will need to be addressed during the development phase of a product’s lifecycle. Traditional plaque-titer methods require 5–7 days of incubation in order to reveal plaques that may be enumerated, and is further complicated by plaques created by multiple viruses that may be scored as a single plaque, thereby lowering the titer estimate. Titer assays based on polymerase chain reaction (PCR) have been developed, but they measure the presence of baculovirus genes, not virus particles. This often results in titers one or two logs higher than the actual titer. Immunoassays correlate with host cell infection and virus replication, but they too can be time-consuming and difficult to interpret. Our goal was to identify a method that would provide estimates of both total and infectious virus particles in as close to real-time as possible. We have evaluated the ViroCyt Virus Counter over the course of three years and have found it to provide accurate and reproducible estimates of both titer types in as little as 30 minutes. We have created an algorithm that converts total virus particle counts into estimates of infectious titer and tested these values in virus amplifications. The Virus Counter method of titer determination has also been used to track the quantity of virus particles in the culture supernatant of stirred-tank bioreactors infected with standard baculovirus stocks and with baculovirus-infected insect cells (BIIC)…