Development, optimisation and validation of a TNFα neutralising assay using a Quality by Design approach

Development and optimisation are essential steps in ensuring a bioassay consistently delivers reliable results during routine use. This application note describes the development of a TNFα neutralising assay for adalimumab potency testing using an Analytical Quality by Design (AQbD) approach.

By combining scientific understanding of the assay with statistical Design of Experiment tools, the method was systematically developed, optimised and validated. The approach supports the identification and control of critical method variables while demonstrating that the assay is fit for purpose.

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Key Highlights of the Method

• Development of a TNFα neutralising cell-based assay for adalimumab potency testing using an AQbD methodology
• Design of Experiment tools were used to identify, assess and optimise critical method parameters, including seeding rate, neutralisation time and contact time
• Final operating conditions selected for validation were 50,000 cells/well, 60 minutes neutralisation time and 23 hours contact time
• Robustness assessment demonstrated no statistically significant impact of reagent lot changes for TNFα, Actinomycin D, FBS or WST-1
• Validation criteria for accuracy, precision, linearity, range and specificity were met across the validated 50%–150% relative potency range.

How this supports your bioassay development programme?

A structured AQbD workflow provides a framework to understand analytical variability and define operating conditions that support consistent assay performance.
By integrating risk assessment, Design Space evaluation and validation activities, the approach strengthens confidence in method suitability for routine use.

• Supports the systematic identification of Critical Method Attributes (CMAs)
• Facilitates risk-based decision making through structured risk assessment and mitigation planning
• Enables optimisation of assay conditions using statistically designed experiments rather than trial-and-error approaches
• Establishes a Design Space to define acceptable operating ranges and reduce failure risk
• Demonstrates method performance through validation aligned with USP <1033> and ICH Q2 expectations
• Supports continued method verification through a defined control strategy based on control chart monitoring

By improving understanding of method variables, the AQbD approach helps reduce risks during validation and routine analytical use.

Typical Applications

• Potency testing of adalimumab through measurement of TNFα neutralising activity
• Development of cell-based bioassays using an Analytical Quality by Design framework
• Analytical method optimisation through Design of Experiment methodologies
• Validation and lifecycle management of potency assays intended for routine use