ADCs represent a promising class of targeted therapies, and understanding the intricacies of their analysis is crucial for their successful development. In this blog, we address common questions to guide you through the various considerations involved in ADC research and testing. We will cover key topics related to the analysis of ADCs, including payloads and conjugations, the influence of cleavable or non-cleavable conjugation on assay setups, recommended PK methods, critical reagents platforms for PK ADC methods, and immunogenicity testing methods.
Question: What PK methods do you recommend for ADC analysis?
For ADC development, key PK methods include:
- *Total Antibody Concentration (TAb): Measured by LBA (Ligand Binding Assay) or hybrid LC-MS/MS
- *Conjugated Drug (ADC): Quantified using LBA or hybrid LC-MS/MS
- *Free Payload: Assessed via LC-MS/MS.
- Metabolite Profiling or biotransformation: Identifies breakdown products, typically using LC-MS/MS.
- PK Modeling: Population modeling to optimize dosing.
- Tissue Distribution: Tracks distribution in tissues for deeper insights.
These methods provide a comprehensive PK profile, essential for optimizing ADC dosing and efficacy.
*Indicates required/recommended PK assays needed for GLP Tox studies and clinical studies.
Question: What assays are needed at what point in the Drug Discovery Process?
We believe in delivering a fit-for-purpose analysis. Early in drug discovery, when you might be screening several ADC’s or payload/linker combinations, we believe it is more important to look at ADC concentrations as well as ADC stability. The stability can be assessed by measuring ADC only. This can be done by several approaches depending on whether the ADC has a cleavable linker or not. We have a suite of commercially available reagents for both LBA and Hybrid LC-MS/MS that can be used for ‘generic assays’ to help quantify ADC. Additionally, we can screen various ADC stability assuming there are chemistries we can leverage such as screening various DAR/linker chemistries with similar payloads and can use this same approach for early pre-clinical PK analyses. Later in the drug development process, typically it is required/recommended to measure 3 parts for PK (ADC, Tab and Free payload) plus immunogenicity (ADA with domain specificity and NAb).
Question: What is the recommended platform for PK Antibody-Drug Conjugates (ADCs) quantification?
There is no one-size-fits-all platform for PK ADC assays. There are advantages to using both LBA and Hybrid LC-MS/MS. LBA for detecting both unconjugated antibody and ADC, offers good sensitivity, precision, and high throughput for sample analysis. However, recent advances in LC-MS have made it an increasingly important complementary technique for ADC analysis. For comprehensive ADC bioanalysis, investigating a hybrid approach using both LBA and LC-MS/MS methodologies can be beneficial. Typically, LBA can be a more cost-effective approach (especially for large clinical studies) for either Total Ab or ADC, however there is a recent trend toward the use of Hybrid LC-MS/MS for several parts of the analysis for many ADCs due to increased specificity as well as less reliance on critical reagents. This combination covers different dimensions of ADC bioanalysis, including:
- ADC PK assay (Ab-conjugated payload)
- Total antibody PK assay
- Free payload PK assay
Question: What technology should you use to analyze ADCs?
We believe ADC analysis should use several technologies depending on the questions and stage of drug discovery including: LBA or ligand binding analysis as well as LC/MS – particularly Hybrid LC-MS/MS as well as “traditional” LC-MS/MS for the free payload part of the analysis. The technologies recommended will depend on the stage of the project, the reagents that are available, and the type of conjugation chemistry. We would love to share our experience and help you navigate the drug development of ADCs for your organization
Question: Why should I consider Hybrid LC-MS/MS as a viable alternative to LBA for ADC Bioanalysis?
LBA had traditionally been the main bioanalytical approach for Total Ab and ADC, but more recently there has been a trend toward using Hybrid LC-MS/MS for many of the PK analyses. The recent 2024 WRIB paper suggests that “The choice of method type for TAb and ADC is debatable” 1. LBA has been viewed as more cost-effective and higher throughput. Hybrid assays (IA-MS) have been viewed as enabling more accurate measurement of potential deconjugation and thus changes in the drug-to-antibody ratio (DAR) due to the direct measurement of payload released from the ADC, while not requiring specific anti-payload reagents like LBA” We agree and think that both techniques have their potential advantages and disadvantages and would love to discuss your particular projects to help you navigate this complex field.
Question: What type of payloads and conjugations do you work with for the ADCs?
We have worked with both cleavable and non-cleavable conjugations and a variety of payloads including tubulin inhibitors (like MMAE, DM1), Topoisomerase I Inhibitors (like SN-38, Camptothecin, Dxd, exatecan)
Question: How does the type of conjugation (Cleavable or non-Cleavable) influence the ADC assay setup?
We let science and biology dictate the platform. If custom critical reagents can be robustly produced the LBA option for PK ADC is a good starting point. An advantage of Hybrid-LC-MS/MS, the analysis of Antibody-Drug Conjugates (ADCs) can be approached differently depending on the type of linker:
Cleavable linker ADCs:
- Use a generic Immunoprecipitation (IP) or specific capture (receptor) IP to isolate the antibody.
- Cleave off the payload and monitor the released payload. This will also give some information about Avg Drug-to-Ab ratio or DAR
Non-cleavable linker ADCs:
- Perform IP using an Anti-Payload Antibody and quantitate a surrogate peptide from the antibody.
- Alternatively, use a generic or specific capture (receptor) IP and quantitate both the linker and the payload. This works best for site specific conjugation
Question: What are common challenges in the key PK methods for ADC analysis?
PK methods for free payload, ADC, and total antibody analysis are essential for monitoring the behavior of ADCs in the body.
For free payload, LC-MS/MS is commonly used to quantify the unbound drug, though challenges like ion suppression and sample matrix effects can affect accuracy, as well as needing to follow various metabolites or stability concerns of the ADC itself.
PK ADC methods focus on measuring the intact ADC, using LBA or hybrid LC-MS/MS techniques, but issues such as cross-reactivity or variability in capture reagents may arise as well as understanding ADC stability or the need for Avg DAR assessment
Total antibody methods assess the overall concentration of the antibody, typically using LBA, though issues like non-specific binding or interference from other proteins can impact results. Addressing these challenges ensures more accurate PK profiles, supporting optimized dosing and efficacy evaluation. Hybrid LC/MS/MS can address some of these issues. The potential value of Hybrid LC/MS/MS is having the ability to multiplex Total Antibody with the ADC analysis. ,
Question: What do I need for critical reagents for PK assays for ADCs?
For PK assays of antibody-drug conjugates (ADCs), you need several critical reagents:
- Anti-idiotypic antibodies: These are used as capture and detection antibodies in LBAs or Hybrid LC-MS/MS to measure total antibody and ADC concentrations. For preclinical applications an anti-human IgG or anti-human FC can also be used.
- Polyclonal Antibodies: Partially purified polyclonal antibodies may be used as capture and detection antibodies in LBA or Hybrid LC-MS/MS to measure total antibody and ADC concentrations.
- Anti-payload antibodies: These are used to capture ADC molecules containing the payload for conjugated payload assays and can be used in both LBA and LC-MS/MS approaches.
- Reference standards: These include the ADC drug product and assay reference standards with different drug-to-antibody ratios (DARs), including a DAR = 0 fraction for specificity testing.
- Internal STD’s (although maybe not considered technically “critical”: These are useful in LC-MS/MS assays and can be full protein stable labeled internal standards or surrogate peptide internal standards (flanked or “short”).
Critical reagents for PK assays should be well-documented, characterized, and produced in sufficient quantities for the product’s lifecycle. For ADC assays, it’s important to consider ADC characteristics, like DAR and distribution, as they affect binding and target recognition.
Question: What are the required methods for Immunogenicity of ADCs?
Immunogenicity testing for antibody-drug conjugates (ADCs) typically follows a tiered approach similar to that used for conventional biotherapeutics, with some additional characterization specific to ADCs. The required methods include:
- Screening Assay: Used to determine if a sample is positive or negative for anti-drug antibodies (ADAs).
- Confirmatory Assay: Confirms screening-positive responses, typically using competitive binding assays with the ADC.
- Characterization Assays (dependent on the drug and mode of action):
- Domain Specificity: Determines ADA reactivity to different components of the ADC (antibody, linker, and small-molecule drug).
- Titer Determination: Assesses relative levels of ADAs.
- Neutralizing Antibody (NAb) Assays: Evaluates the neutralizing activity of ADAs, using either cell-based bioassays or non-cell-based competitive ligand-binding assays.
Question: What do I need for critical reagents for ADA assays for ADCs?
Critical reagents for Anti-Drug Antibody (ADA) assays for Antibody-Drug Conjugates (ADCs) typically include:
- Labeled drug conjugates: These are often used in bridging assay formats.
- Positive control (polyclonal antibody).
- Domain-specific confirmatory reagents: These are particularly important for multi-specific drugs like ADCs.
To ensure quality, reagents should be characterized for purity, affinity, and specificity, produced in large batches to minimize variability, and managed through proper storage and stability monitoring. Validation studies should include drug tolerance and sensitivity testing.
In conclusion, understanding ADC analysis is crucial for their successful development. From selecting the right assays to ensuring proper immunogenicity testing, every step plays a vital role. Reach out to our experts for guidance on optimizing ADC analysis, and let us help you navigate these complex processes to achieve the best possible outcomes.