Development & Validation of an Automated Workstation for HTS Flux Assays
Sikander Gill, Rajwant Gill, David Wicks, Joy Goswami and Dong Liang
Aurora Biomed Inc, Vancouver, BC, Canada
I. Abstract
III. Materials & Methods
IV. Results
E What values of the assay parameters (i.e., Z’,
Advances in genomics, proteomics, and combinatorial chemistries have window of detection, and IC
of standard blockers)
C. Was the liquid handling performance of VERSA 1000
dramatically increased the need for automation of biological assays and were achieved upon automating all the steps of the
verified before validation of the hERG assay?
microarrays. Automated liquid handling systems for carrying high-throughput HTS assay?
A. Which steps and conditions in the assay are being automated?
screening (HTS) assays have become an invaluable tool for the drug discovery and Table 1. Liquid handling performance of VERSA 1000
development industry. Therefore, the lack of such systems has been a major Versa Flux IC50 = 0.709 uM Hill slope = -0.76 Versa Flux IC50 = 104nM Hill slope = -1.0 bottleneck in the drug development process. In the recent years, the demand for Liquid Handling Specifications
higher throughput in the biotech and pharmaceutical sectors has initiated the Volume Range
development of versatile workstations from simple semi-automated bench-top liquid handlers to fully-automated integratable workstations.
With the objective of improving efficiency and to increase the level of automation CHO-hERG
and miniaturization, Aurora Biomed has developed a series of automated workstations, the VERSA Series This system can automate a range of applications 50,000 Cells/Well
in the fields of genomics, proteomics, drug discovery, and analytical applications. ReagentDrop
We describe the validation of this fully- automated workstation to run cell-based Na+/K+ Pump
Figure 3.
A window of detection showing activated and Inhibitor
assays using cells expressing an ion channel of interest. A panel of positive basal efflux of 69 and 19%, respectively was achieved while inhibitors of the ion flux were applied to determine their IC 5.4 mM RbCl
Syringe Pipette
performing all the steps of the assay with the VERSA 1000. values were then compared to those obtained This automated procedure resulted in a high Z’ factor value from the manually- performed assay. The SEM was used to measure the variability of 0.883 which suggests high robustness and low variability among the replicates. The Z’ factor values for both the automated system and D. Was the drug dilution and liquid handling performance
E-4031, carried out using the VERSA 1000 resulted an IC50 II. Introduction
VERSA 1000
of the VERSA 1000 in the presence of DMSO/water
values of 0.709 µM and 0.104 µM, respectively. These verified before validation of the hERG assay?
values were comparable to routine manual performances Need for automations of liquid handling processes in labs: As drug
discovery processes demand high productivity and low cost, companies are Automated Performance
Manual Performance
always looking for rapid, sensitive, robust and inexpensive technology to miniaturize assays and carry precise automated analyte delivery. To meet this Versa Flux IC50 = 0.907uM Hill slope = -.94 Manual Flux IC50 = 0.864 uM Hill slope = -1.3 V. Conclusion
demand, assay technologies have evolved rapidly over the past 5 to10 years1,2. Automation of hERG flux assay using VERSA 1000 Issues associated with automation of cell based assays: In order to
resulted in comparable parameters to that of an assay automate cell-based assays, specific technology and universal issues performed manually (i.e., Z’, window of detection, and IC associated with cell-based assays need to be considered3. Such issues of standard blockers. This suggests that the fully- include simplification of the assay parameters and steps, user friendly automated VERSA 1000 can increase the throughput of software controlling the automation systems, quality control and Versa Flux IC50 = 1.68 uM Hill slope = -1.1 Mannual Flux IC50 = 1.38 uM Hill slope = -1.1 standardization of procedures for automated large-scale screening, and the stability of the plated cells during wash steps etc. Solution to these issues: To provide solution to these demands of HTS and
to address these issues of automation, Aurora Biomed Inc, Vancouver, Canada VI. Acknowledgements
has developed the VERSA series of workstations. In the present validation Figure 1. Flow diagram of the HTS hERG assay protocol showing different studies, the VERSA 1000 was used for drug dilution and automating the cell- We are also thankful to Victor Navasero and Alicia Davis for steps, durations and conditions involving liquid handling. Validation of the hERG Figure 2. Liquid handling performance in the presence of DMSO
based nonradioactive rubidium flux assay for screening compounds against assay was carried out in 96 well-format using the VERSA 1000.
was verified using terfenadine, a DMSO soluble drug and a known blocker of hERG dissolved in 100% DMSO at 100X. Thioridazine, Why automate the cell-based assay? Automating cell-based assays brings
VII. References
a water soluble drug dissolved in water at 100X, was also used in the challenge of ensuring the integrity of the plated cell layer; therefore, a cell- B. What parameter of the HTS process were validated using the VERSA
these studies. Both the drugs were diluted to 8 different based assay (hERG assay) was chosen for the validation of the VERSA series. 1. Gill S et al.: Flux assays in high throughput screening of concentrations from the source plate, resulting 1X concentration of hERG channel activity is one of the most important factors in drug cardiac ion channels in drug discovery. ADDT 2003;1(5):709- terfenadine along with DMSO diluted to 1% and thioridazine to 1X toxicity4. Thus, in order to quickly eliminate cardiac-liable compounds, increase The following parameters were validated for hERG HTS assay: in assay buffers. Rest of the assay steps were performed chances of clinical success, and decrease development time, regulatory 2. Terstappen G: Nonradioactive rubidium ion efflux assay manually. The results indicated comparable IC agencies require Pharma companies to screen all compounds for activity and its applications in drug discovery and development. Signal-to-noise ratio (window of detection) µM (automated performance) and 0.864 µM (manual Standard error of the mean (SEM) among replicates performance) for terfenadine. Similarly, IC 3. Falconer M et al.: High-throughput screening for ion ¾IC determinations of some standard blockers However, manual performance of the hERG assay is labor-intensive and low (automated performance) and 1.38 µM (manual performance) for channel modulators. Biomol Screen 2002;7(5):460-465. throughput. As the throughput and precision of a robotic system is higher than thioridazine were obtained. The SEM values were very 4. Razvi E: HERG-technology and market analysis. Report the manual technique, we built a small, automated system to carry out this *Correspondence should be addressed to Aurora Biomed Inc., 1001 E. Pender St., Vancouver, BC, Canada, V6A 1W2, Tel: 1-604-215-8700, Fax: 1-604-215-9700, [email protected],


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