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Ruggedness Testing Including an Evaluation of Automation of a Cell- Based Bioluminescent TNFα Blocker Bioassay下載
September 23, 2011
Authors: Tracy Worzella, Rich Moravec, Neal Cosby, Frank Fan, and Teresa Surowy, Promega Corporation, Madison WI; Brad Larson, Peter Banks, BioTek Instruments, Winooski VT
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.
Bioassays are indispensible tools in biopharmaceutical drug development and commercialization. They are used to quantify biological activity and stability of drugs or drug candidates. Precision and accuracy of the bioassay are allimportant in both drug discovery and development, and in manufactured biopharmaceutical lot release, yet many bioassays suffer from drawbacks such as complexity and variability of results.
We developed a simple, homogeneous and robust bioluminescent TNFα blocker bioassay based on quantification of caspase 3 activity. The bioassay has good precision and accuracy and can be performed in one day. It uses U937 (human) cells which exhibit rapid response to TNFα. By developing and using U937 cells in single-use frozen, thaw-and-use format, we were able to remove variability arising from continuous cell culture.
Part of bioassay development also includes analysis of bioassay ruggedness, in which the influence of external factors on bioassay test results is measured. Our study here describes such an analysis of our TNFα blocker bioassay using a 96-well plate format. We included evaluation of automation in the analysis, as some bioassay laboratories use automation for liquid handling. For this, the assay steps of antibody titration and of cell and reagent dispensing were automated using a simple, yet robust liquid handler. Validation of the use of instrumentation with the assay chemistry was thus part of the study. Ruggedness variables evaluated were (i) manual and automated pipetting, (ii) bioassay plate used, (iii) luminometer / microplate reader used, and (iv) bioassay run (day). 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 assay EC50 and Hill Slope obtained between assay runs of full dose-response titrations of TNFα blocker antibody.