Why Do Assays Matter?
After knowing what are assays, let us understand why they are important. The drug development pipeline initiates with the lead discovery phase, which involves identifying molecules that show desired biological activity at the drug target. The phase includes screening libraries of compounds to identify potential lead candidates. Assays help in this screening process, thus driving breakthroughs in drug discovery.
What Is Assay Development?
Since we know what assays are, let us focus on what is assay development. Since assays can assess the biological activity and safety of potential drug candidates, developing successful and efficient assays is crucial in drug discovery and development. These assays help determine the efficacy and safety of a drug candidate in the early stages of drug development, thus saving time and money. Assay development involves creating a platform for testing various parameters of the potential drug candidate. Assay validation establishes its accuracy and reliability, which builds confidence among assay developers and users.
Importance Of Assays In Drug Development
The previous section provided an overview of assay development steps and the need for assay development in drug development. This section will focus on the role of assays in identifying drug targets and high-throughput screening of libraries of compounds in drug discovery. Target identification is a crucial part of drug discovery and development. Most drug targets belong to protein families that are phylogenetically linked. Drug targets include protein kinases, hormone receptors, serine proteases, ion channels, and G-protein coupled receptors. Affinity-based pull-down and label-free methods can help identify protein targets. These methods use biological assays such as cellular thermal shift assay (CETSA). After drug target identification, several libraries of compounds are screened for the biological activities against the intended drug target, a process associated with “hit” discovery. This accelerates drug discovery by rapidly screening several libraries of compounds, equating to a few thousand compounds a day or a week. High-throughput (HTS) assays help in this screening process, and they can screen genomics, peptides, and protein libraries.
Types of Assays (Expertise, Services, and Solutions)
Beyond the domains of pharmaceuticals, assays find application in biotech, diagnostics, molecular biology, environmental biology, agricultural research, and mining to assess the presence of a substance or to quantify its amount and activity. In the biotech industry, biological and biochemical assays help to evaluate the performance of a genetically engineered microorganism, validate the production of recombinant proteins, and monitor various bioprocesses. In diagnostics, assays are critical in detecting disease and monitoring disease progression and response to therapies. For example, immunoassays can diagnose diseases such as HIV and COVID-19 by detecting the presence of disease-specific biomarkers or antibodies. In agricultural research, biological assays help to determine the safety of chemicals, pesticides, and insecticides commonly used in agriculture. Toxicity assays can assess the toxic effect of such substances on living organisms, thereby helping in risk assessment and regulatory decisions. Researchers also use bioassays to monitor the impact of environmental pollution on aquatic and terrestrial ecosystems.
4.1 Expertise: Core Assay Technologies
Mass Spectrometry (LC-MS) Assays
Due to its high sensitivity, liquid chromatography-mass spectrometry (LC-MS) assays find wide applications at different stages of drug development, including drug quantification and pharmacokinetics assessment.
ELISA & Immunoassays Assay
Enzyme-linked immunosorbent assay (ELISA) and other immunoassays are highly selective assays that detect and quantify the presence of antibodies, antigens, proteins, and hormones in biological samples. ELISA harnesses the catalytic properties of enzymes to assess the immunologic reactions and components.
qPCR and ddPCR Assay
Quantitative polymerase chain reaction (qPCR) and droplet digital (ddPCR) assays are used to quantify nucleic acids. ddPCR assays also find applications in microbial quantification and viral load testing.
Flow Cytometry Assay
Flow cytometry is a biological assay that is used for cell-based research such as cell sorting, cell cycle analysis, evaluating cell viability, apoptosis, immunophenotyping, and blood cell analysis.
Western Blot Assay
Western Blot assays find wide applications in biotech and clinical diagnosis. They are used to detect, separate, and quantify proteins in complex samples based on their weight. Thus, these assays help in the diagnosis of diseases such as HIV.
4.2 Services: Specialized Assay Offerings
Pharmacokinetics (PK) Assay
Pharmacokinetics assays help obtain drug concentration in biological fluids such as blood, serum, or plasma as a function of time. These assays find application in experiments exploring the mechanism of drug absorption, distribution, metabolism, and excretion (ADME).
ADA (Anti-Drug Antibody) & Neutralizing Antibody Assays
Drug administration can elicit an immune response, producing anti-drug antibodies (ADAs) and neutralizing antibodies (NAbs). These antibodies block and neutralize the pharmacological action of the drug. ADA and NAb assays are used to assess the immunogenicity of a drug during drug development.
Cytokine Assay
Cytokine assays can accurately quantify different cytokines produced in the body, thus enabling researchers to investigate immune responses to drugs and inflammation mechanisms.
Biomarker Assay
Biomarkers are molecular indicators that help detect diseases, predict disease prognosis of diseases, and monitor therapeutic responses. Biomarker assays help to identify and validate the presence of biomarkers in biological samples.
4.3 Solutions: Infrastructure And Collaboration
Clinical CRO & GLP Labs
Clinical contract research organizations (CROs) and GLP-certified laboratories offer product development services to pharmaceutical and biotech research organizations. These include the development and validation of different types of assays, adhering to the regulatory standards.
Preclinical CRO Assay
Preclinical CRO assays are CRO services that support assay-based preclinical studies in pharmaceutical and biotech companies before the drug enters the clinical trial. These assays are designed, developed, and validated by CROs following GLP practices and regulatory norms.
Method Development, Validation, and Transfer Assay
The key aspects of assay development are Method development, Method validation, and Method transfer. Assay development involves developing an assay that is acceptable for use. Validation helps to establish parameters such as accuracy, precision, specificity, robustness, range, limit of detection, and limit of quantification. Assay transfer allows the validated assay to be used consistently across different testing sites as intended, following stringent protocols and regulatory norms.
The Assay Development Process
The assay development process is complex and comprises several steps. The first step involves identifying and validating drug targets, e.g., receptors, enzymes, hormones, ion channels, or other biological molecules and pathways. Thereafter, the assay development steps include the design phase, where the assay is designed and optimized for the intended application involving a specific drug candidate. Applications include the high-throughput screening of compound libraries for hit identification, pharmacokinetics assessment, and others. Following assay development, multiple validation procedures are performed, such as pre-screen validation, in-screen validation, and cross-validation. Assay validation establishes its accuracy and reliability, and this, in turn, helps assay developers and users to use it confidently. If the assay fails at these validation steps, it will be redesigned and redeveloped for the intended application.
Optimization Tips
To improve the efficiency and reliability of assays, some of the best practices include the use of high-quality reagents, positive and negative controls, fine-tuning conditions such as incubation duration and temperature, and the use of automation to prevent human errors.
Key Factors In Assay Development
Different factors must be examined during assay development, such as relevance, reproducibility, quality, interference, cost, and regulatory requirements.
Relevance: This ensures that the assay can predict the specific disease state and identify compounds that display appropriate mechanisms of strength and action.
Reproducibility: Assays should produce consistent results across assay plates and screening sessions during the specific program.
Quality: The Z’-factor is estimated based on the variation in the signal, to determine the quality of an HTS assay. This is used as a measure to determine the acceptability of the assay for its intended application.
Interference: Assays must consider interference from solvents and/or reagents used.
Cost: Strategies should be reasonably adopted to minimize the assay cost.
Regulatory Considerations: Regulatory authorities have their guidance on assay development, which requires the use of reference standards and controls, processes, and documentation. Compliance and adherence to regulatory requirements are crucial in assay development.
Challenges In Assay Development
Despite multiple advantages of modern assays, such as high sensitivity and specificity, rapid detection, high throughput, multiplexing capabilities, and reproducibility, these testing platforms are not without challenges. These include managing false positives and negatives, variability, and non-specific interactions. Improving method design through strategic assay development and robust validation practices can help mitigate these challenges. In addition, secondary methods with different detection systems, such as NMR, UV spectrometry, mass spectrometry, and IR spectroscopy can be helpful.
Other limitations in assay development include additional research on molecular targets or modes of action involved in cell-based assays. Furthermore, biochemical and cell-based assays involve several steps and reagents, including culture media containing different compounds, buffers, antibiotics, etc. Thus, the procedure becomes time-consuming, with several experimental variables, such as pH and non-specific interactions that affect the assay results.
Integration of automation and bioinformatics has profoundly impacted assay development. Bioinformatics assists in virtual testing, optimizing assays, data analysis, and data management. Automation, on the other hand, can help to boost productivity, reduce human errors, enhance precision, and improve consistency.
Real-World Challenges
Different biochemical and cellular assays find application in drug discovery and development. These assays have strengths and limitations, which the researchers must know to avoid pitfalls. A review of case studies highlights the common pitfalls. One such example involves the determination of kinase inhibitor activity. Kinase inhibitors can be potential drug candidates for Duchenne muscular dystrophy. A commercial assay, using a KiNativ probe, was used to assess the activity of the CDK2 inhibitor against CDK2 and other kinases. The probe binds irreversibly to the conserved lysine residues at the outer pocket of most kinases, thereby interfering with the kinase inhibitor binding. Research efforts by another group using the HotSpot assay revealed a stark contrast in the findings. It highlights the need for secondary or orthogonal assays to confirm the findings.
Emerging Technologies In Assay Development
Existing assay and drug development technologies have become powerful by integrating emerging and advanced technologies. The use of modern detection technologies such as surface plasmon resonance (SPR), fluorescence, and other methods enable assays to detect low concentrations of analytes with ultrahigh sensitivity and selectively and makes them cost-effective.
Cell-based assays using 3D cell cultures mimic the biological complexity of tissues and organs and are thus well-suited for phenotypic drug discovery. Similarly, high content screening (HCS) is another advanced cell-based assay that allows functional analysis of live cells and can, therefore, contribute to cell-based screening in drug discovery.
Additionally, in silico assays offer virtual screening to predict characteristics and biological activities of drug molecules, such as binding mode, toxicity, carcinogenicity, and other risks. The results obtained from in silico assays help inform various drug design and developmental stages.
Future Innovations
Technologies such as AI and machine learning augment drug discovery and development in ways more than one. These technologies assist in assay optimization by predicting ideal experimental conditions and predicting activity in biological assays based on the molecular structure of the drug and the target. In the future, AI and machine learning will usher a paradigm shift towards improved efficiency, accuracy, precision, and scalability of assays. They will also pave the way for improving reliability and streamlining analyses.
Examples Of Assays In Research And Drug Development
Advanced assays are used in various drug discovery and development stages and molecular science research. Examples include ADP Hunter Assay, a fluorescence-based assay that measures kinase activity and is appropriate for HTS of kinase inhibitors. The simple assay protocol screened a library of 215,000 compounds to identify CKL1 inhibitors. CKL1 or CDC20like kinase 1 is a dual specificity kinase that plays a crucial role in alternative splicing. The simple and cost-effective ADP Hunter Assay identified 12 potential CLK1 inhibitors with good reliability and robustness.
Some other examples of assays in research are migration assays, ToxTracker Assays, and quenched fluorescence resonance energy transfer (FRET) assays. Migration assays help to assess the migratory behavior of cell populations, while the ToxTracker assay provides an informative screen for the genotoxic potential and associated mechanisms of compounds. FRET and quenched-FRET assays are fluorescence-based technologies with diverse applications in disease detection, drug discovery, and monitoring genetic variations between individuals.
Practical Insights
Assay development is a crucial part of drug development, which determines the success of drug candidates in later drug development stages. In addition, the integration of modern technologies and strategic designing of assays have improved the efficiency, accuracy, precision, and scalability of assays. Along with enhanced productivity, these assays promise to reduce the attrition rates in drug discovery and development, thus saving time, money, and effort.
Conclusion
Drug discovery and development is a complex and lengthy process. It usually takes several years for a drug to receive approval for clinical use. This process involves the identification of potential drug candidates, followed by the validation, optimization, efficacy, and safety testing through different preclinical and clinical trials. Only a few molecules successfully pass through the drug development pipeline to be launched in the market. Efficient and robust in vitro and in vivo assays of a drug candidate can successfully determine its efficacy and safety in the initial stages of drug development, thus saving time and money.
Compared to traditional testing methods, the advantages of assays are manifold. The superior sensitivity, specificity, precision, and reliability of validated assays make them an indispensable bioanalytical platform for drug discovery and development. In addition, integrating advanced technologies has improved reliability and consistency and reduced turnaround time in assay testing, thus reiterating the need for researchers to adopt advanced assay techniques.
