Development and validation of an antibody-drug conjugate bioassay

Antibody-drug conjugates (ADCs) are targeted biotherapeutics combining a monoclonal antibody with a cytotoxic payload. These complex molecules require robust analytical methods to demonstrate their quality, safety and efficacy. Among these, cytotoxicity bioassays are essential to evaluate both specific and non-specific effects on cells. This application note presents the development and validation of a bioassay using trastuzumab emtansine (T-DM1) as a model ADC, following USP <1033> requirements. The approach highlights key methodological choices and validation parameters.

KEY HIGHLIGHTS OF THE METHOD

• Cytotoxicity assay developed using SK-BR3 HER-2 expressing cells and trastuzumab emtansine (T-DM1)
• Optimised conditions: 20,000 cells/well, T-DM1 range from 200 to 5.2 ng/mL, 72-hour incubation
• Use of CellTox™ Green dye enabling direct measurement of cell death based on membrane integrity
• Validation according to USP <1033> covering specificity, precision, linearity and relative accuracy
• Demonstrated linearity (R² = 0.99; slope 1.02) and compliance with all predefined acceptance criteria

HOW THIS SUPPORTS YOUR ADC DEVELOPMENT PROGRAMME?

Reliable bioassays are critical for demonstrating the potency of ADCs throughout development of stability studies and quality control. This validated method provides a robust framework aligned with regulatory expectations.

• Supports regulatory compliance with validation aligned to USP <1033> guideline
• Enables accurate measurement of cytotoxicity with reduced variability compared to metabolic assays
• Improves data reliability through optimised assay conditions
• Allows detection of product degradation through demonstrated stability-indicating capability
• Ensures specificity by discriminating target-dependent and non-specific cytotoxic effects

This approach contributes to robust and interpretable potency data across development stages.