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)…
Tag: <span>cell counting</span>
Cell therapy products derived from adipose tissue have some unique processing issues with regard to obtaining accurate cell counts. This is because processing methods may not only show us the nucleated stromal vascular fraction (SVF) cells but also the micellular and microvesicle particles. This is true for both veterinary and human clinical products, and poses special concerns for in-clinic processing where the cell therapy dose is correlated with cell numbers and other QC data is not especially useful. In this study, multiple cell counting methods were compared for SVF cell preparation that were derived from canine adipose tissue using commercially-available processing kits. The data clearly showed that many non-nucleated particles appear cell-like by size and shape, and can lead to counting errors with automated counters. In addition, certain reagents important to processing can have properties wherein the reagents alone (e.g., lecithin) may be counted as cells. The most accurate cell numbers were from hemocytometer-counting of cells stained with 4´,6-diamidino-2-phenylindole (DAPI) which shows the nuclei in concert with a viability stain such as trypan blue. The data clearly showed that care must be taken when counting cells used as a therapeutic dose…