Boosting Accuracy and Efficiency: Assay Development and Validation Process for Drug Discovery

The development of a new drug is a complex, time-consuming, and costly process that takes an average of 10-15 years and costs anywhere from $1-2.5 billion USD^1,2. A significant component of the drug discovery process involves assessing various drug properties through robust assays. Such assays include binding affinity assays, such as the enzyme-linked immunosorbent assay (ELISA), which are crucial for screening compound libraries to identify candidates with the desired effect^3,4.

Then, enzyme activity assays are employed during candidate characterization during candidate characterization, involving the quantification of enzyme-substrate binding, usually measured by colorimetric indicators^5,6.

Finally, in the compound optimization phase, cell viability assays are used to monitor cell health in response to incubation with compounds by quantifying specific cellular or metabolic changes associated with viability or lack thereof^7,8. 

Well-designed assays are instrumental in helping researchers identify molecules with the desired therapeutic effect while filtering out ineffective ones. Thus, accurate and efficient assay development and validation pipelines are crucial for drug discovery success. Here, we explore key considerations, challenges, and the role of emerging technologies in assay development and validation for drug discovery. 

Key Considerations for Assay Development and Validation

Design of experiments (DoE) for assay development is an approach that enables researchers to strategically and methodically refine the experimental parameters and conditions of a drug discovery assay⁹. It is a systematic approach that helps researchers understand the relationship between variables and their effect on assay outcomes, informing assay format, reagent and control selection, and condition optimization for sensitivity and specificity. Employing DoE techniques enables scientists to diminish experimental variation, lower expenses, and expedite the introduction of novel therapeutics. Its integral role in fine-tuning assay conditions increases the probability of success in drug discovery¹⁰. 

Following assay development, researchers must validate assays in terms of their robustness, reproducibility, and ability to measure what they are designed to measure accurately¹¹. This involves extensive quality control of measurement performance following comprehensive and predefined method validation requirements. Method validation requirements will be specific to the assay but will likely include tests to validate the assay's specificity, linearity, range, accuracy, precision, detection and quantitation limits, robustness, and system compatibility¹². 

Researchers often face considerable challenges in drug discovery assays, which they must overcome through strategic assay development and validation. Some of the most common challenges include:

1. False positives/negatives

False positives occur when an assay incorrectly identifies an inactive compound as active, leading to wasted resources¹³. Conversely, false negatives miss potential therapeutic compounds due to assay insensitivity or interference¹⁴. Strategies to mitigate these include improved assay design, controls, and analytical pipelines.

2. Variable results

Assay variability arises from biological differences, reagent inconsistency, instrument variability, and human error. Addressing this requires standardized protocols, rigorous quality control, and automation to enhance consistency and reliability^15,16.

3. Interference from non-specific interactions

Non-specific interactions can falsely indicate compound activity by interacting with components other than the target molecule¹⁷. Counter-screens and careful assay design aimed at minimizing these interactions help improve the specificity and accuracy of results¹⁸.

The Role of Emerging Technologies in Supporting Assay Development and Validation

Emerging technologies play a pivotal role in supporting assay development and validation. They provide innovative solutions that enhance the efficiency and reproducibility of drug discovery assays, contributing significantly to the overall drug development process.

Microfluidic devices enable drugs to be tested on cells under controlled environments. The environments created by microfluidic devices mimic physiological conditions, allowing the cells to be monitored for long periods of time¹⁹. Moreover, microfluidic technologies facilitate assay miniaturization, increasing assay throughput and reducing sample volume requirements, which is advantageous for quickly testing various conditions in assay development and validation workflows²⁰. 

Similarly, biosensors, devices that use biological or chemical receptors to detect specific analytes, can streamline assay development and validation processes by monitoring chemical or biological parameters with high sensitivity and specificity, helping researchers fine-tune assays^21,22.

Artificial intelligence is another central technology in streamlining assay development and validation; it accelerates hit identification and drug design through its predictive and modeling capabilities and streamlines experimental design, risk assessments, and data analysis during the assay development phases^23,24. However, AI models used in drug discovery must be thoroughly tested and robustly validated. 

Another technology that can enhance assay development and validation pipelines is automated liquid handling²⁵. Not only does it enable researchers to increase their productivity and output by increasing assay throughput, freeing up skilled scientists for other work, and enabling miniaturization, but it also enhances the accuracy and precision of assays and minimizes the risk of human error often introduced during manual pipetting steps^26,27. 

With the I.DOT Liquid Handler (Fig. 1), researchers can easily create gradients of concentrations and volumes of assay components, allowing them to systematically explore the impact of different variables on assay outcomes. 

Figure 1. The I.DOT HT enables automated liquid handling with up to 384 source wells for streamlined drug discovery assay development and validation processes. 

This automated dispenser streamlines the DoE process by providing reliable and consistent dispensing, facilitating the optimization of experimental conditions, and enhancing the efficiency and reproducibility of drug discovery assays. Book your personalized demo with a DISPENDIX representative today!

References

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