High-Throughput Screening (HTS) is an industry-standard automated process for rapidly testing thousands to millions of compounds for biological activity in cell-based or biochemical assays. HTS identifies hit molecules with significant biological activity, serving as starting points for medicinal chemistry optimization in collaboration with our partners. At Assay.Works, we leverage our extensive experience from a leading biopharmaceutical company's HTS Center, where we conducted over 250 HTS campaigns across major drug target classes during our 20-year tenure. We utilize small molecule diversity sets, client-provided libraries, and other substances like chemical mixtures, natural product extracts, oligonucleotides, and antibodies. Our success in industry-scale HTS campaigns relies on engineering robust, automation-compatible assays, robotics-assisted sample handling, and automated data processing.
Assay Format Examples
Read more on our Cell-based and Biochemical Assay Examples
Assay Quality
At Assay.Works, the quality of our assays is paramount, as it directly influences the success of drug discovery campaigns. We apply rigorous standards to ensure that our assays are fully "screening-ready," offering high biological relevance and robust performance. By adhering to the following criteria, our assays are designed to effectively identify novel chemical hits that can modulate therapeutic targets.
- Alignment with therapeutic modalities
- Target specificity and selectivity
- Consistent reliability & reproducibility across repeated experiments
- Optimization of assay parameters for peak performance
- Cost-efficiency without compromising quality
- Validation through known compounds or control samples
- Comprehensive data analysis and interpretation
By meeting these standards, our assays provide patho-physiologically meaningful results, maximizing their impact in drug discovery and development efforts.
Optimizing Assays for HTS
We design and refine robust, reliable, and scalable in vitro assays that precisely mirror the biology of therapeutic targets. Utilizing state-of-the-art automated, robotics-operated systems, our assays are geared to identify novel chemical compounds with potential as drug candidates.
Our approach to HTS prioritizes the use of unbiased statistical metrics to detect significant biological activity within extensive compound libraries. To ensure our assays meet industry standards, we emphasize meticulous optimization, enabling automated data analysis and clear differentiation between genuine biological activity and background noise. We focus on key quality parameters such as high signal-to-background ratios at minimal variance across the signal range, ensuring the accurate identification of true hits from large-scale screenings.
Robust Screening Assay Criteria
We implement stringent criteria to ensure the robustness of our screening assays. Critical success factors for HTS campaigns include:
- DMSO Tolerance: Ensuring resilience to varying DMSO concentrations.
- Calibration Controls: Maintaining accuracy with high and low signal control calibrations.
- Signal-to-Background Ratio: Optimizing for unambiguous detection of biological activity.
- Z’ / RZ’ Factor: Assessing assay quality and reliability.
- Signal Consistency: Minimizing variability across wells and experimental runs.
- Optimal Compound Concentration: Determining the ideal screening concentration.
- Outlier Detection: Monitoring for plate patterns and identifying anomalies.
- Quality Control: Upholding stringent intra- and inter-plate standards.
- Pilot Screening: Forecasting hit rates with preliminary pre-screens.
By adhering to robust screening criteria, Assay.Works guarantees the reliability and effectiveness of HTS processes, laying a strong foundation for successful drug discovery.
HTS Workflow
Our industrial HTS campaigns follow a scalable format that can be customized for any drug target across therapeutic areas. This standardized approach enables screening of compound libraries ranging from thousands to millions of molecules, identifying chemical entities that selectively modulate target activity in biochemical, biophysical, and cell-based assays.
Assay Development and Adaptation
- Design and refine robust, reliable, and scalable in vitro assays that precisely mirror target biology
- Adapt HTS assay to automated, robotics-operated procedures meeting all quality criteria ("bench-to-robot")
Primary Screening
- Screen the entire compound library once (n=1) at a predetermined concentration utilizing state-of-the-art automated systems
Hit Confirmation (Hit Selection)
- Automatically select primary hits using statistical cut-off mechanisms. Additional reading material for HIT selection in HTS (PDF).
- Confirm hits via re-testing in triplicates (n=3) at the same concentration
Concentration-response Screening (Hit Validation)
- Test confirmed hits at predefined ascending concentrations to establish concentration-dependency
- Conduct counter-screening in control settings lacking the drug target to identify artifacts and off-target effects
- Re-test compounds in alternative assay formats to eliminate compounds perturbing the HTS assay
Quality Control
- Assess purity and confirm integrity of hit compounds using liquid chromatography-coupled mass spectrometry (LC/MS)
Hit Package Delivery
- Compile available data for chemical matter passing the predefined triaging criteria and quality control standards
By adhering to this meticulous workflow, Assay.Works ensures our HTS assays are not only technically sound, segregate genuine biological activity from artificial compound behavior, and thereby successfully advance drug discovery efforts.
HTS Platform
- Versatile liquid handling
- hit.works integrated multi-device platform (Cybio robotics)
- Echo automated acoustic nanoliter dispensing
- Tecan Freedom EVO platforms
- Cybio-FeliX
- Thermo Fisher Multidrop Combi with high-speed plate stacker
- Biotek plate washers
- Complex assay procedures
- Different incubation temperatures with automated high capacity incubators
- Scheduled incubation and plate processing steps
- Flexible reader configuration
- 2x Operetta CLS – Confocal microscopy
- FLIPR Tetra – Fluorescence kinetics
- EnVision – Multimode
- Nanion SURFE2R 96SE
- Nanion PatchLiner
- BD Biosciences FACSCelesta
- Enterprise software
- HTS: Genedata Screener, Titian Mosaic Sample Management
- Imaging: Harmony and Cell Profiler
Data Analysis
Assay.Works uses industry grade data analysis softwares to standardize data across plates and runs, enabling accurate comparison of compound effects.
Genedata Screener
Genedata Screener is a plate-based screening data analysis software that offers modular architecture, seamless integration with various instruments and technologies, and capabilities for high-throughput screening and assay analysis in life sciences research.
Harmony
Harmony software used with the Operetta CLS system for image analysis along with PhenoLOGIC feature with machine learning component.
