Cell-based assays and analysis play a vital role in every stage of the drug development process from early discovery, pre-clinical, clinical and post market commitment phases. Cell-based assays are an analytical measurement defined by a set of reagents that produce a detectable signal for quantifying a biological process.
In this blog, we are going to cover cell-based assays in the various stages of drug development:
- Cell-Based Assays vs. Biochemical Assays
- Choosing an Appropriate Cell-Based Assay
- Cell-Based Assay Development Considerations
- Optimization of Cell-Based Assays
- The Future of Cell-Based Assays
Cell-Based Assays vs. Biochemical Assays
Cell based assays are regarded as more biologically relevant surrogates to predict the complexity of a therapeutic response in a biological system than non-cell-based, biochemical assays. Cell-based assays accelerate and enhance drug development to help therapeutics be brought to market in a quick and efficient manner.
Cell based assays aid in the following:
- Lead candidate selection,
- Provide invaluable information about therapeutic mechanism of action (MOA)
- Drug efficacy
- Off-target effects in a cellular environment
Furthermore, they can be used in the identification of patients that may respond to therapy, for monitoring pharmacodynamic biomarkers and for immunogenicity assessments. Cell-based assays are also vital for the evaluation of target engagement or saturation, determination of safety markers, and monitoring mechanisms of resistance.
Cell-based assays are also used to assess a variety of functional and biochemical effects, including cell viability, proliferation, cytotoxicity, apoptosis, signal transduction, enzyme activity, reporter gene activity, receptor occupancy, receptor binding, antibody (ADCC) and complement dependent cytotoxicity (CDC), among others.
The signal readout can be absorbance, fluorescence, luminescence, colorimetric, or radioactivity depending upon the variable to be measured and signals can be measured using microscopy, flow cytometers or plate readers.
As therapeutic assets evolve in complexity there is a need for more sophisticated bioanalytical methods especially in the cell-based assay arena.
The complexity of therapeutic modalities has evolved from monoclonal antibodies to antibody drug conjugates, bi/tri specifics, to cell and gene therapies with complex mechanisms of action. The effect of a drug on an organism is complex and involves interactions at multiple levels that cannot be predicted using biochemical assays alone.
Choosing an Appropriate Cell-based Assay
Cell-based assays are diverse in nature and a myriad of factors need to be considered when choosing a relevant cell-based assay at the appropriate time in the long road to biotherapeutic drug development and approval and will depend on the specific stage and goals of the program.
A clear understanding of the context of use for the assay and how the data will be used to support the program should be established, as this will drive the development of a biologically relevant assay that will yield high-quality data.
As the therapeutic progresses through its development lifecycle, the cell-based assays employed will evolve depending on the information needed to push the program forward. A cell-based assay utilized in discovery will look different from one used later in the clinical stage.
Cell Based Assay Development Considerations
Once the goal and context of use of the cell-based assay have been established then building the most relevant, reproducible, and robust assay ensues.
The cell-based assay must reflect some aspect of the drug’s MOA to ensure it is biologically relevant.
This requires the identification of a biologically representative cell line, either primary or immortalized, that will reflect at least one or more aspects of the therapeutics’ MOA and identifying an appropriate endpoint to measure.
Endpoints in Therapeutics’ MOA
Endpoints can be either early or late and each has advantages and disadvantages.
For example, early assay endpoints (receptor binding) that generate a measurable signal rapidly, have advantages over later endpoints (cell proliferation/cytotoxicity assays) that require several hours or days of incubation to develop a signal as they are convenient and reduce the chance of artifacts due to assay chemistry and matrix interference issues.
When choosing an assay, the time required for reagent preparation and the total length of time necessary to develop a signal from the assay chemistry and stability of the signal should be considered. A stable signal confers advantages such as convenience and flexibility in recording data and minimizes differences when processing large batches of plates.
Despite their challenges, cell-based assays can be streamlined and designed to provide valuable data efficiently using automation, multiplexing, and miniaturization.
Measuring one parameter may not be enough to accurately determine the functionality of a drug.
The ability to analyze several markers in multiplexed assays provides greater information on the drugs MOA, drug efficacy, toxicity, and immunogenicity. The ability to gather more than one set of data from the same sample in a multiplex format can save time and effort during drug development. To successfully multiplex, the detection signals of the different assays must be distinguishable from each other, and the assay chemistries must be compatible or separable in time and/or location to accurately interpret that data and avoid interferences.
Regardless of the model system chosen, establishing an assay with acceptable sensitivity, precision, selectivity, reproducibility, and robustness requires optimization of multiple parameters.
Since cell-based assays can be challenging due to inherent variability associated with biological systems, appropriate development and validation is required prior to implementation.
Optimization of Cell-Based Assays
Assay responsiveness to test compounds can be influenced by many subtle factors including culture medium, biological matrix, surface-to-volume ratio, gas exchange, evaporation of liquids, and edge effects.
These factors become even more important when attempting to scale up assay throughput by changing from 96- to 384- or 1536-well formats.
The use of multifactorial, statistical design of experiments (DOE) approach can be employed effectively in different stages of a bioassay’s life cycle to characterize, optimize, and validate the assay with the added benefit of resource efficiency.
Compared to traditional one-factor-at-a-time experiments, DOE has the potential to accelerate assay optimization and facilitate a more thorough evaluation of assay variables.
This method efficiently and systematically modulates the factors of interest to identify the key assay parameters, better understand the effects of each individual factor as well as estimate interactions between different factors to evaluate the effects on the system response and assists in the development and validation of a fit-for-purpose bioassay.
Optimization experiments depend upon the specifics of the bioassay such as:
- Assay Format
- Technology Being Used
An important aspect of optimization is to obtain a desirable assay window to interpret the results, improve the reproducibility and statistical performance of the assay by identifying conditions that increase the signal-to-noise ratio with respect to positive and negative controls, and to decrease the intra and inter assay variability of the assay by assessing coefficient of variations and Z’ values of the assay.
Furthermore, maintenance and handling of cell cultures at each step of the process should be standardized and validated for consistency.
Once the assay is developed, assay validation documents and confirms that the assay is acceptable for its intended purpose. Rigorous validation is critical considering that assays are expected to perform robustly over several years during the various phases of development and potential post market commitments.
The Future of Cell-Based Assays
Cell-based assays provide a complex and more biologically, physiologically relevant assay system than biochemical assays because they are conducted in a cellular environment that can mimic disease states, preserve signaling pathways, and model drug responses.
However, there are limitations, since most bioassays utilize a homogeneous population of cells grown from immortalized cell lines, that produce reproducible results but express target proteins or receptors in excessive, non-physiological amounts via transient or stable transfections that do not accurately reflect living systems in normal or diseased tissue.
Thus, the question remains as to how well they reflect real biology.
Although primary cells are more physiologically relevant, they are inherently variable, making it more difficult to deliver a robust cell-based assay.
Phenotypical consequences of a therapeutic of interest to the cell could reflect a combination of effects that a single cell-based assay would not be able to fully address.
Despite these challenges, cell-based assays play an integral part in the drug development process and several technologies are being utilized to overcome current limitations, such as genome-editing tools like CRISPR/Cas9 to allow easy engineering of mutations, knock-outs or knock-ins of a specific reporter or marker at precise location on the genome, 3D culture models, co-culture of cells, and artificial tissue techniques to mimic real biological environments and provide relevant in vivo cellular context.
Our cell-based team not only has extensive experience in developing and validating complex cell-based assays for a variety of therapeutic modalities, but also has a deep understanding of all regulatory recommendations and guidelines to help our clients in a science first manner to ensure success of their programs. Contact us today to learn more about our premier cell-based assay services.
Dr. Corinna Fiorotti brings almost two decades of drug development experience in large pharma/biotech which spans from research through clinical development, with expertise in building and executing on strategies for bioanalytical development. Dr. Fiorotti holds a Ph.D. in Immunology and Microbiology and completed a post-doctoral fellowship in Molecular Biology at Harvard Medical School where her work was featured as a cover story in the journal Nature. Dr. Fiorotti has deep knowledge in immunogenicity and bioanalytical assays which cover a wide range of disease areas and therapeutic modalities including protein therapeutics, and cell and gene therapy. She has extensive expertise in cell-based assays in addition to developing ADA and PK assays for both pre-clinical and clinical support. Dr. Fiorotti has an exceptional track record working with project teams to identify the critical questions/decision points and delivering strategies to address them, leading to efficient prosecution of their programs.