For all the screens, the data analysis and visualizations performed are used to determine the quality of the screen and to select hits for the next screen. At each phase, the information generated is used to determine which antibodies are to be moved to preclinical assays (Figure 2).
The serum-screening process begins with the extraction of serum samples before immunization. This first serum sample obtained is called pre-tap, which is used in every titer screening as a reference sample for that particular subject. After immunizations start, when subjects start to produce antibodies, scientists run the serum-screening assay every two weeks to collect serum samples referred to as tap.
The first step in selecting a hybridoma cell that produces antibodies with the desired characteristics involves plating out cells in different wells. Employing a highly automated and efficient process, Genmab uses Caliper Life Sciences’ Sciclone liquid-handling workstation to pipette the diluted samples into 384-well assay plates, which are filled up with a cell line expressing the target of interest and a control mock cell line.
These plates are then moved to Cello™ (The Automation Partnership), a high-throughput fully integrated imaging and culture system that can handle large hybridoma libraries. In a highly controlled manner, cells grow in wells and produce significant amounts of antibodies, which are then ready to be tested for their specificity and functional characteristics.
To determine potential antibody hits to progress to a primary screen, graphical information is compiled that visualizes serial dilutions of the tap and pre-tap on the target and the mock cell line, per individual subject and for the entire group. Trend analysis of the positive and negative controls within one plate and on all plates in one run is also performed.
For the immunization protocols performed subsequently to the serum screening, ActivityBase XE is used to collate similar graphical information, plus trend analysis of positive and negative controls over time. This analysis helps scientists to detect the subjects that are producing the antibodies with the desired characteristics.
When subjects are identified as producing potential hits, lymph and spleen cells are fused in a fusion protocol. Fused cells are placed in fusion plates in 96-well format and moved to the automated culture robot.
After fusion, several compound plates (harvest) are produced in the automated culture robot, which also outputs culture supernatants. Together with the output plates, a worksheet (fusion screening sheet) that contains all calculated assay data from the primary screening is produced, which is used as a basis for decisions for candidate selection in the next phase.
Scientists then perform primary screens on the supernatants, and the captured data is analyzed, which also allows further trend analyses on the positive and negative controls within one plate and on all plates in one run. Combined with the trend analyses of the on-plate effect, this information is used to help identify patterns of behavior within and across plates and to compare these trends in order to detect system errors.