Biological Drug ADA/PK Analysis

Techniques in ADA/PK Assays

• Bridging ELISA: This assay utilizes two antibodies to capture and detect the drug, offering high specificity and sensitivity for ADA detection. It is commonly used for evaluating therapeutic monoclonal antibodies and ADCs.
• Direct ELISA: In this method, the drug directly binds to immobilized antibodies. While it is simpler and faster, it often lacks the sensitivity required for detecting low levels of ADA.
• Competitive Binding Assays: This approach measures the ability of a labeled drug to compete with the sample for binding to an antibody. It is particularly useful for smaller molecules and certain peptide drugs.
• Cell-Based Assays: These assays assess the functional impact of ADA by examining how they affect cellular responses to the drug. This is important for determining neutralizing activity.
• Mass Spectrometry (MS): Liquid chromatography-tandem mass spectrometry (LC-MS/MS) offers high sensitivity and specificity, making it suitable for PK assay for small peptides and ADCs.

Antibody tools used in ADA/PK assays

Anti-payload antibodies

Typically, ADC drug PK assays focus on measuring conjugated antibodies, total antibodies, and the released payload. Creative diagnostics has developed anti-payload antibodies that exhibit high purity, specificity, and affinity, which are instrumental in analyzing the plasma/serum PK profiles, DAR values, and drug load distribution in ADC drugs. During the preclinical and clinical phases of ADC drug development, these payload antibodies are utilized to assess the safety and efficacy of ADCs, thereby expediting the development process.

Anti-idiotype Antibodies

An idiotype is the unique set of antigenic determinants (epitopes) of the variable portion of an antibody.  An anti-idiotypic (Anti-ID) antibody binds to the idiotype of another antibody, usually an antibody drug, which makes it a very powerful tool for antibody drug development, especially for immunogenicity and PK/PD analysis. Anti-idiotype antibodies could be categorized into three types: non-blocking, antigen-blocking and complex specific. Using different types of anti-idiotypes allows more flexibility in assay development. Type 1 anti-idiotypic antibodies are inhibitory for measuring free drug and are ideal for neutralizing assays and PK bridging ELISA. Type 1 anti-idiotypic antibodies could be used as a control or calibrator as a surrogate for human ADAs. Type 2 antibody is a non-inhibitory antibody that can be used to detect both free and bound drug in the sample. Type 3 antibodies are drug-target complex binders that can be used to quantify bound drug, as opposed to free drug levels.

Fig. 7 Three types of anti-idiotype antibodies

Common Applications of Anti-Idiotypic Antibodies

• Pharmacokinetic (PK) studies
• Immune Response (IR) immunogenicity assays
• Positive controls for anti-drug antibodies (ADA)
• Controls in ligand binding neutralizing assays
• Controls in antibody blocking assays

Fig. 8 Types of ELISA-based PK assays.

Anti-human IgG FC antibodies

Anti-human IgG Fc antibody can be used as a capture antibody to measure total ADA levels. It can also be used as detection antibody with anti-payload antibodies or anti-idiotype antibodies in the PK analysis of ADC drugs or antibody drug.