Tech Resources - Application Notes
Validation of an Automated Cell-Based Bioluminescent TNFα Blocker Bioassay
31-Jan-12
Related Products: Precision, Synergy Mx
Authors: Brad Larson and Peter Banks, BioTek Instruments, Inc., Winooski, VT Tracy Worzella, Rich Moraver, Teresa Surowy, Promega Corporation, Madison, WI
Here we demonstrate the automation of a 96-well homogeneous bioluminescent TNFα blocker bioassay based on quantification of caspase 3 activity. The bioassay can be performed in a single day, and uses single-use, frozen U937 (human) cells which exhibit rapid response to TNFα. A simple, yet robust liquid handler was used to automate the assay steps of antibody titration and of cell and reagent dispensing.
Introduction
TNFα blocker biopharmaceuticals represent an important and successful class of protein drugs used in the treatment of several autoimmune diseases, including rheumatoid arthritis, psoriasis and Crohn’s disease. This success is driving the discovery of new versions of these protein drugs, new indications, and biosimilar development because some of these drugs come off patent protection soon.
The TNF signaling pathway leads to multiple endpoints, as is demonstrated in Figure 1. These include NFκB gene regulation, apoptosis, and cell death. A simple way to monitor pathway regulation by blocker antibodies in a bioassay is with a reagent that can quantify caspase 3 activity, as is the case here. Upon the addition of a proluminescent caspase-3/7 DEVD-aminoluciferin substrate, cells are lysed and caspase cleavage of the substrate takes place. The free aminoluciferin drives a luciferase reaction, creating a luminescent signal that is proportional to caspase activity in the well.
Figure 1. TNFα signaling pathway endpoints.
Bioassays are indispensible tools in biopharmaceutical drug development and commercialization. They are used to quantify biological activity (Ex. TNFα signaling activity) and stability of drugs or drug candidates. The automation of these assays can serve to create an accurate, robust process which can allow the researcher to perform other more important functions. Precision and accuracy of the automated bioassay are all-important in both drug discovery and development, and in manufactured biopharmaceutical lot release.
Part of bioassay development includes analysis of assay ruggedness, in which the influence of external factors on test results is measured. The study described here includes plate uniformity, as well as anti-TNFα blocker antibody titration tests. Variables included microplate used, run-to-run variability, as well as a comparison between manual and automated processing. Assessment of ruggedness was based on (a) variability around RLUs obtained in plate uniformity tests using a single dose of TNFα blocker antibody, and (b) variability of EC50 and Assay Window obtained between runs of full dose-response titrations of TNFα blocker antibody.
Material and Methods
Materials
Thaw-and-use U937 cells (a human cell line which rapidly responds to TNFα with caspase induction), TNFα blocker antibody, TNFα, and Caspase-Glo® 3/7 reagent (Catalog No. G8092) were provided by Promega Corporation.
Microtiter plates included in the ruggedness test included two flat, solid-bottom white 96-well plates; Corning catalog no. 3917 and Greiner Bio-One catalog no. 655083. Also included was a flat, clearbottom white 96-well plate; Corning catalog no. 3610.
Instrumentation
The Precision™ Microplate Pipetting System combines an 8-channel pipetting head and an 8-channel bulk reagent dispenser in one instrument. The instrument was used to dispense U937 cells, serially titrate blocker antibody across a 96-well PP plate, transfer samples from plate to plate, as well as for TNFα and detection reagent dispensing.
The Synergy™ Mx is a monochromator-based multi-mode microplate reader. A dedicated luminescence detection system is used to quantify the luminescent signal from each assay well.
Automated TNFα Assay Procedures
Plate Uniformity Test Method
- Manually dispense 200 μL/well of 2.5 ng/mL TNFα + 40 ng/mL anti-TNFα blocker antibody mix into the appropriate wells of a 96-well plate, and preincubate at 37° C / 5% CO2 for 60 minutes.
- Dispense 50 μL/well of U937 cells (15K cells/well) into a separate 96-well plate, and preincubate at 37° C / 5% CO2 for 30-40 minutes.
- Transfer 50 μL of the 2.5 ng/mL TNFα + 40 ng/mL anti-TNFα blocker antibody mix into each well of the cell plate, shake the plate for 30 seconds, and incubate at 37° C / 5% CO2 for 2.5 hours.
- Remove the plate from the incubator and allow to cool to room temperature (RT) for 30 minutes.
- Add 100 μL of Caspase-Glo® 3/7 reagent, shake the plate for 30 seconds, and incubate at RT for 60 minutes.
- Read the luminescent signal from the plate following the incubation period.
Automated TNFα Assay Procedures
Anti-TNFα Blocker Ab Titration Test Method
- Serially titrate the anti-TNFα blocker antibody in assay medium using a 100 μL total final volume per well.
- Dispense 100 μL/well of 10 ng/mL TNFα to each well of the titration series. Shake the 96-well plate for 30 seconds, and incubate at 37° C / 5% CO2 for 60 minutes.
- Dispense 50 μL/well of U937 cells (15K cells/well) into a separate 96-well plate, and preincubate at 37° C / 5% CO2 for 30-40 minutes.
- Transfer 50 μL of the TNFα/blocker antibody titration mix into the appropriate wells of the cell plate, shake the plate for 30 seconds, and incubate at 37° C / 5% CO2 for 2.5 hours.
- Remove the plate from the incubator and allow to cool to room temperature (RT) for 30 minutes.
- Add 100 μL of Caspase-Glo® 3/7 reagent, shake the plate for 30 seconds, and incubate at RT for 60 minutes.
- Read the luminescent signal from the plate following the incubation period.
All dispensing steps, except for step 1 of the Plate Uniformity Test, were performed manually, as well as robotically, using the Precision™.
Results and Discussion
Plate Uniformity Test
The goal of the plate uniformity study was to ensure that the cells, known ligand, blocker antibody, and detection reagent used in the assay could be consistently dispensed across a 96-well assay plate in an automated fashion. Manual dispensing was also performed as a control for comparison purposes. The test was independently performed a total of 6 times, using the three white 96-well plate types described previously. Average, standard deviation (SD), and % CV were computed for the 96 replicates tested on each microplate.
Figure 2. Manual and automated plate uniformity results. Average +/- SD data is shown for the three plates included with each individual performance of the test. Numbers at the bottom of each bar represent the % CV values computed from each average and standard deviation.
The low % CV values obtained through automated dispensing (from 3.4-5.2%) and the lack of any discernible negative dispensing pattern among all plate types tested demonstrate the ability of the Precision™ to consistently and evenly dispense the assay components in 96-well format. Also, compared to the % CV values obtained from manual pipetting (from 4.0-7.9%) by an experienced pipetter familiar with the assay, the % CVs of the automated system show a slight improvement, indicative of a more robust assay procedure. The lower luminescence values seen with the Corning 3610 can be attributed to the clear well bottom of this plate, while the other two plates have solid white well bottoms, which increases the luminescence signal from the wells.
Anti-TNFα Blocker Ab Titration Test
Anti-TNFα blocker antibody titration tests were” completed in order to ensure that the Precision could accurately and evenly dilute the blocker antibody across a 96-well plate. Serial 1:2 dilutions were performed to create a 12-point titration series with antibody concentrations ranging from 2000-0 ng/ mL. Inhibition curves, EC50 values, and assay window were compared between manual and automated processing. The test was once again performed a total of six times using the same three assay plate formats.
Figure 3. Representative curves showing increased blocking of the TNFα pathway with increasing antibody concentration.
Table 1. EC50 values generated from anti-TNFα blocker antibody inhibition curves. Individual values from each curve are shown, as well as averages from the six runs for robotic and manual dispensing with each plate type. SD and % CV are also shown with each average.
Figure 4. Average maximum and minimum signal, as well as assay window shown for the six manual and automated runs performed with each assay plate type. Error bars represent the SD for each average value.
The similarity between the curves generated from each antibody titration, as well as low variation among EC50 values, with % CVs ranging from 3.2-5.6%, demonstrate the capabilities of the Precision™ to consistently titrate the anti-TNFα blocker antibody in 96-well format, using each plate type. In addition, the close agreement in average EC50 values between all automated and manual runs completed, 40.73 and 41.37 ng/mL, respectively show that accurate dilutions are achieved with the Precision. Finally, matching average assay window values when compared to those generated using other microplate readers, which all range from 3.2-3.8 (experiment not shown), confirm that no loss in performance is seen when the Synergy™ Mx is used for signal detection.
Conclusions
Automation of the TNFα blocker bioassay can further simplify the assay process by freeing up time for the researcher to accomplish other, more important tasks. The data demonstrate that automation performs equivalent to, or better than, a manual user with this cell-based biologic assay. Automation provided lower % CV values in the plate uniformity test and equivalent EC50 values to manual in the TNFα blocker titration tests. These results confirm that the automated bioassay represents a complete, rugged solution to test biosimilars for their effectiveness in blocking TNFα activity.
AN013012_03
