Understanding Biological Assays

Introduction to Biological Assays

• Biological assays, also known as bioassays, estimate drug potency by measuring the response a substance elicits in a biological test system compared to a standard preparation.
• Bioassays are applied to substances like vitamins, hormones, antibiotics, enzymes, and biotherapeutics, among others.
• The procedure is conducted on living entities such as animals, plants, tissues, cells, or micro-organisms.
• They are used for testing toxicity and pharmacological screening, supporting drug development, environmental monitoring, and pollutant detection.
• Bioassays help to test the biological activity of therapeutic agents for lead identification and optimization, stability, and batch release within the pharmaceutical industry.
• These assays can also identify contaminants or metabolites in relation to adverse drug reactions and may serve as limit tests for substances in active material preparations.
• Bioassays can assess water quality and characterize chemical or faecal pollution for environmental monitoring and pollutant detection.

Principles of Bioassays: Potency and Efficacy

• The fundamental principle involves correlating a test substance's concentration or potency with a standard preparation.
• Potency quantitatively assesses biological activity, reflecting the amount of a substance required to produce a specific effect.
• Efficacy indicates the maximum effect achievable by a drug.
• The maximum effect, Emax , in pharmacology is achieved when increasing drug concentration no longer increases the magnitude of the response.
• Equal efficacy means both drugs produce the same maximum effect.
• If Drug A achieves a maximum effect with less concentration than Drug B, Drug A is more potent.
• Drug A is more efficacious if it achieves a greater maximum effect than Drug B.

Relative Potency

• Absolute potency is difficult to measure due to inherent variability in biological systems, necessitating the use of relative potency, where biological activity is expressed as a sample's activity compared to a standard.
• For accuracy, bioassay methods must be precise, robust, accurate, and reproducible to reduce variability.
• Assayed sample preparations must elicit the same biological test system response as the standard.
• Standard and sample tests must occur simultaneously under identical conditions for reproducibility.

Standardisation of Bioassay Procedures

• Imprecision is inevitable in bioassays due to variabilities in the biological test system, instrumentation, sample preparation, inter-lot variation, and other factors.
• Precision is improved by using a well-chosen bioassay procedure, a verifiable standard, and suitable statistical analysis.
• Employing biological reference materials and standards ensures accurate comparison and calibration of bioassay results.
• Reference standards can come from the World Health Organisation (WHO), United States Pharmacopeia (USP), and European Pharmacopoeia (EP).

WHO Biological Reference Materials

• The WHO provides international standards to ensure the quality of biological medicines.
• These standards are used by laboratories and manufacturers to calibrate standards in biological activity.
• WHO standards are considered the ‘gold standard’ for regional, national, and international laboratories, which calibrate their standards against them.
• These standards are calibrated in biological activity units following extensive international studies.

Types of Biological Assays

• Bioassays can be categorized based on the living matter used: In vivo, in vitro, ex vivo, and microbiological assays.
• In vivo assays involve living organisms, usually laboratory animals, where the dose-response relationships are measured after administering dilutions of samples and standards.
• Ethical concerns and high costs have decreased animal use.
• In vitro assays are easier to perform, produce faster results (1-3 days), and are less expensive than animal models and can be automated.
• Bioassay performance is affected by the selection of cell type, the cell-handling process, and the bioassay design.
• Ex Vivo is a procedure utilizing a primary culture of rat hepatocyte cells.
• Microbiological assays are required by the EP and USP to test the activity or potency of antibiotics.

Pyrogen Test as an Example of an In Vivo Bioassay

• A pyrogen test detects substances causing fever and chills, which is a critical safety step for drugs administered parenterally.
• The process involves measuring temperature increases in rabbits after intravenous injection of a solution.
• Rabbits where the mean rise in temperature has exceeded 1.2 °C are permanently excluded.

Monocyte Activation Test (MAT)

• Regulatory agencies promote replacing in vivo assays with in vitro methods like the monocyte activation test (MAT).
• The MAT detects substances that activate human monocytic cells, releasing mediators involved in fever.

In Vitro (Cell-Based) Bioassay

• Cell culture-based bioassays offer information on the biological product effects, with potential imprecision due to variances in living cells but less risk of imprecision compared to animal models.
• One example is bioassays based on interferons’ inhibitory activity on viral cytopathic effects on human laryngeal cancer cell lines, measuring the potencies of different human interferon test preparations and dose-response relationships

Glucagon Bioassay as an example of Ex Vivo

• Glucagon bioassay involves culturing hepatocytes with a sample preparation and measuring glucose release.

Microbiological Assays

• Microbiological assays measure the activity of different antibiotics by demonstrating their inhibitory effect on microorganisms.
• Potency is determined through comparing growth inhibition of sensitive microorganisms by concentrations of the antibiotic with a reference substance.
• Assays include the cylinder-plate assay and the turbidimetric assay.
• A cylinder-plate assay involves antibiotic diffusion through an agar layer containing a target microorganism.
• The turbidimetric assay measures growth inhibition in a uniform antibiotic solution, assessed using a spectrophotometer.

In Vitro Testing

• In vitro testing should be used where possible instead of live animals.
• In vitro involves administering dilutions of a sample to clonal cell lines.
• Specific cells are isolated, enabling detailed analysis and stock generation for future use.

Ex Vivo Assays

• Include administering dilutions to cells from animal or human donors and calculating dose-response relationships.
• Cells in tissues are taken directly from a living organism and immediately tested in a lab environment.

Cylinder-Plate Assay

• Antibiotic diffuses from a vertical cylinder, producing an inhibition zone that can be measured
• Dishes are incubated for 16-18 hours

Turbidimetric Assay

• The method depends on the inhibition of microorganism growth in a solution
• Reference and sample preparations are distributed into test tubes.
• The turbidity of the medium is measured with a spectrophotometer.
• Tubidimetry is inversely proportional to the concentration of the antibiotic.

Graded and Quantal Assays

• Bioassays are graded when there is a proportional response to the concentration or dose.
• Graded responses reflect the nature of the expected effect, such as blood pressure response to adrenaline.
• Quantal responses are all-or-nothing events.
• The response varies with the dose, often reflecting a normal distribution.
• An example of quantal responses is digitalis causing cardiac arrest in guinea pigs.

Bioassay Design

• Bioassays involve variability, necessitating minimization at each design stage to prevent bias.
• Critical steps include blocking and randomization, replication, and optimized assay setup.
• Outliers must be taken into consideration.

Blocking and Randomisation for Mitigating Bias

• Blocking groups related experimental units, while randomization assigns samples and standards randomly to experimental groups.
• Grouping related experimental units and assigning samples randomly minimizes differences.

Examples of Poor and Good In Vivo Assay Designs

• A poor design does not randomize test samples and standards across the groups.
• A good design randomizes test samples and standards across the groups.

Replication Strategies

• Strategies involve repeating measures to avoid errors or variability, by selecting a number of replicates for each sample.
• At least four different sample concentrations are desirable to obtain reliable results

Assay Setup

• Most Bioassays include administering one of a series of concentrations of a test sample or standard to operational units.
• Key considerations is location-based error.

Analysis of Bioassay and Curve Models

• An effective model must approximate the true curve, average out random variation, and enable concentration predictions. and be compliant with current statistical requirements of the PE and the USP.
• Common evaluation models are the parallel line method and logistic models with four- and five-parameter fits.

Parallel Line Model

• A linear model with a linear relationship between the logarithm of the dose X and the biological response Y.
• It is useful when estimating the dose of a sample to achieve the same response as a known dose of standard.
• It is useful because it is easy to measure variability of the responses and parallelism of log dose-response plots.
• This model is represented by the equation Y = a + bX, where "a" is the intercept, and "b" is the slope.

Logistic Models

• Logistic models are non-linear, S-shaped functions considering the entire dose-response range constrained by asymptotes.
• This model could provide additional information and better precision.
• The 4-Parameter Logistic (4-PL) regression model is Y = D + (A-D)/ [1+ (X/C)B], where Y is theresponse, A is the response at no analyte, D at infinite analyte concentration, C is the inflection point, B is aslope factor, and X is the analyte concentration
• It is recommended a minimum of 8 concentration points to define a 4- PL Curve
• The 5PL model adds a parameter for asymmetry (G) better fitting non-symmetrical response curves: formula Y = D + (A-D)/ [1+ (X/C)B]G.

Conclusion

• Bioassays are effective for estimating sample concentration and biological activity.
• Assays are necessary for regulatory compliance with entities like the EP and USP, especially for antibiotics.
• Assays are useful for antibiotics, immunological products, hormones, blood and relatedproducts, enzymes, and more.
• These methods remain specialized but essential in drug evaluation.