May 1, 2017 (Vol. 37, No. 9)

Method Serves as a Powerful Tool for Analyzing Cells and Secreted Proteins in Suspension

Flow cytometry, which uses light scattering and fluorescent technologies to analyze single cells, can serve as an appropriate and effective methodology for bioprocessing applications. GEN spoke to Thomas Duensing, Ph.D., chief technology officer, at IntelliCyt, to learn more about flow cytometry’s capabilities in a biomanufacturing environment.

GEN: Why should biomanufacturers seriously consider the use of flow technology for their operational activities?

Dr. Duensing: Flow cytometry is a powerful tool for analyzing cells and secreted proteins in suspension. In the majority of bioprocessing operations, genetically engineered production cells are cultured in suspension to secrete large quantities of the desired biologic molecule, which is in many cases a therapeutic antibody. Flow cytometry is thus a perfect tool for measuring quantities and attributes of secreted antibodies, and the number and health of the suspension cells that produce those antibodies.

GEN: Which technical capabilities of flow cytometry make it a useful, informational-gathering technique in biomanu-facturing?

Dr. Duensing: First, the ability to perform suspension assays is perfectly suited for evaluating suspension cell cultures and the proteins they produce. Second, flow cytometry is inherently a high-content technology. This means that multiple assay endpoints can be reported simultaneously. For bioprocessing, this means that secreted antibodies can be captured by beads, and the amount and characteristics of those antibodies can be measured.

The real power of multiplexing comes into play when these bead-based assays are combined with cell-based assays. Thus, in addition to measuring secreted antibodies, the number and health of the cells producing those antibodies can be simultaneously evaluated.

GEN: For which particular bioprocess operation is flow cytometry an ideal choice?

Dr. Duensing: Flow cytometry is ideal for testing and monitoring cell-productivity attributes throughout the bioprocessing workflow, including cell-line generation screening, media optimization, and production in bioreactors.

GEN: IntelliCyt recently introduced the Cy-Clone Plus assay system for ranking and selection of clones for cell-line development. This assay reportedly can accelerate cell-line development. How does it carry out this critical function?

Dr. Duensing: The first step in bioprocessing is identification of genetically engineered cell clones that can produce and secrete large amounts of the biologic product. This is most commonly done by arraying single cell clones in microtiter plates, culturing them for a number of days, and then testing the supernatants of those cultures for the amount of secreted protein present.

Clones are ranked based on the amount of product in the supernatant, and decisions regarding which clones to move forward are made based on those rankings.

The problem with making decisions at this stage, based solely on secreted protein levels, is that it’s possible that high levels of protein in supernatants results from highly productive cells, each of which secretes large amounts of product. However, it is also possible that each cell in the well is not very productive, and that high levels of secreted protein are simply due to the presence of large numbers of rapidly proliferating cells.

What’s needed in this field is the ability to perform high-throughput screens on large numbers of clones that simultaneously report both the amount of secreted proteins and information about the cells that are secreting those proteins.

The CyClone Plus kit, which runs on the iQue Screener Plus platform, enables high-throughput clone screens that report secreted protein levels on a per-cell basis. When this assay is performed on the iQue Screener Plus platform, 5000 clones can be tested in a few hours. Thus, in a high-throughput screening environment, rich information on how productive individual cells are, and on the health of individual cells is generated.

Having this information early in the process provides valuable information that enables scientists to make informed decisions about which clones to move forward, and ultimately increases their chance of success.

GEN: What impediments or barriers to expedited cell-line development does the Cy-Clone Plus assay overcome?

Dr. Duensing: Correlating IgG titers to individual cell productivity and health is usually done in the later stages of cell-line development, after screening for IgG titers. In that workflow, it is possible that highly productive, robust clones are missed and never taken forward into downstream cell-line development. Identifying productive clones directly in the initial high-throughput screening process could significantly decrease the chance of missing these valuable clones.