Bioanalysis

The Bioanalysis of Antibody Drug Conjugates

Antibody Drug Conjugates (ADCs) are highly complex prodrugs, consisting of monoclonal antibodies (MAb) to which cytotoxic drugs, e.g. tubulin polymerization inhibitors or DNA damaging agents, are “loaded” using specific linkers. ADCs might also be defined as sophisticated delivery systems for payload drug delivery, intended to heighten both the safety and efficacy of chemotherapeutics.

Through targeted delivery to tumor cells via tumor-specific and/or over-expressed antigens, ADCs can improve the therapeutic window of a cytotoxic compound, increasing drug delivery to tumor cells and reducing normal-tissue drug exposure. In other words, even with a lower total dose, the local concentrations will be higher where needed, but the systemic side-effects will be lower.

To completely delineate the pharmacokinetics of an ADC, and hence its efficacy and safety, four assays should be developed, validated and applied to clinical plasma samples:

  • Freely circulating small molecule concentration (LC-MS/MS); the goal of this measurement is to assess whether concentrations stay under a certain maximum level, to assure the safety of the treatment (and/or to relate drug levels to side effects)
  • Small molecule concentrations bound to the ADCs (LC-MS/MS)
  • Total ADC concentration (ELISA)
  • Total free antibody (ELISA)
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In order to ensure the correct quantification of all of these different parameters, proper sample collection and processing (including freezing and storage) at the clinical site is essential. Inappropriate methodologies will invalidate the analytical results. Of course, the bioanalytical procedure must also be performed correctly.

For sample processing in the clinic and sample preparation in the laboratory, multiple options are available. QPS can propose a sampling and bioanalytical strategy based on your ADCs unique characteristics, e.g. applying immunoaffinity clean-up to separate the free fraction from the protein fraction, and subsequently using both fractions for further analyses and/or processing.

Quantifying the total ADC concentration separately from the total free antibody is more complicated; these need to be measured with different ELISAs, using antibodies that recognize different parts of the ADC-antibody complex, e.g. one to recognize the antibody, and one to recognize the payload/linker fraction. However, in practice, this may be even more complex than expected as different antibody batches may have different payload amounts, or even a mix of different payload amounts. Also, in vivo, the payload amounts may change due to detachment of fractions of the payload. Accurate estimations may be difficult to make, and data may be based on averages.

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