Antibody drug conjugate (ADC) characterisation and release testing for structure, purity and impurities covering both conjugate drug and monoclonal antibody (mAb) components supporting development, registration and release

Antibody drug conjugates (ADCs) characterisation and analysis presents a range of complex challenges, all of which must be addressed to support successful product registration. ADCs are produced by covalently linking a monoclonal antibody via a chemical linker to small molecule potent cytotoxic drug. The inherent complexity of the mAb component, combined with the added variability introduced by the distribution of drug linked forms, presents significant complexity multiple analytical or bioanalytical obstacles to overcome in order to ensure a safe and effective drug product.

With over 20 years’ experience in large molecule bioanalysis and characterisation, our experts have considerable knowledge of the evolving regulatory framework across different regions and the scientific background to this product class. Bringing quality and safety to life, our scientists engage with clients from all around the world, helping them to meet all regulatory expectations and specifications for successful drug substance and drug product release. From laboratory testing in our GLP and GMP compliant laboratories to regulatory compliance and beyond, Intertek Total Quality Assurance expertise is delivered consistently with precision, pace and passion, enabling our customers to power ahead safely.

ADCs present unique bioanalytical challenges as they tend to be complex heterogeneous mixtures of multiple species with a range of drug-to-antibody ratios. ADCs typically incorporate both large and small molecule characteristics, and so our bioanalytical experts apply diverse bioanalytical methods, including ligand-binding assays and LC-MS methods, to quantify these species in serum/plasma for PK studies and strategies for assessing immunogenicity.  As part of the clinical phase of drug development, the immunogenicity of the ADC will need to be determined. Our experts can define the strategy for assessing immunogenicity and apply sensitive immunoassays.

Intertek provides complete bioanalytical capabilities for ADCs:

- Quantitative bioanalytical assays for preclinical and clinical studies
- Total antibody assays
- Total conjugated ADC assays
- Unconjugated (free drug) assays
Immunogenicity assessment – anti-drug antibody (ADA) assays

    Regardless of the regulatory pathway selected for an ADC product, characterization, comparability, release testing and stability assessments are required. Given the inherent complexity and heterogeneity of the mAb component, combined with the added variability introduced by the distribution of drug linked forms, a range of complex analytical approaches are deployed by our teams with an emphasis on mass spectrometry and chromatography methods as part of strategic packages pertinent to your actual need and phase of development supporting identity confirmation, characterization, comparability, purity, release testing and stability studies.

    To confirm ADC identity, our testing programs include a focus on binding assays, charge determination, peptide mapping and sequencing.

    The variability introduced by the numeric distribution of drug linked forms adds to the complexity of the overall product. Accordingly, our experts utilise advanced analytical methods to determine the drug load distribution or payload (the average number of drug molecules attached to the mAb or Drug-to-Antibody ratio (DAR)) and individual drug load variants. Our biopharma teams determine the conjugate attachment site through peptide mapping or sequencing studies. Typically, we then apply mass spectrometry (MS) (matrix-assisted laser desorption/ionisation time of flight and/or electrospray ionisation), chromatography, or ultraviolet (UV) spectroscopy methodologies to access a greater depth of detail regarding the ADC structure and confirm the DAR.

    We determine ADC product purity by applying a range analytical approaches which include SDS-PAGE/capillary gel electrophoresis, size-exclusion chromatography and other charge-variant assays. To build an impurity profile, a suite of chromatographic methods are deployed.

    Our biopharmaceutical stability study facilities perform ADC stability assessment via chromatographic and electrophoretic methods which evaluate the stability of the product while maintaining a particular focus on aggregation and fragmentation. Typically the analytical approaches that can be applied to the mAb can be applied or adapted to the ADC.  It is also important to determine that the conjugation process has not affected the conformation of the mAb during conjugation. Circular dichroism is perhaps the most accepted and reliable technique used to assess tertiary structure through evaluation of the near UV profile.

    Our experts conduct phase-appropriate comparability studies for both the mAb and the ADC product to assess comparability between the toxicology, clinical, and/or commercial batches. Our studies also incorporate comparability of the drug loading (distribution).
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