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Quantitative Analysis of ADC Components - Ligand Binding Assay (LBA) and LC-MS/MS

Ella Bette

There are both large and small ADC molecules. A variety of biological analysis methods and platforms are needed to analyze the diversity of ADCs. Conventional LC-MS/MS small-molecule assays are often used to analyze uncoupled payloads and DAR distributions over time, while ligand binding assays (LBA) and LC-MS/MS can also be used to analyze total antibodies (TAb) and total ADCs. The choice of analysis platform depends on the availability of key reagents, the required analytical sensitivity, and the different problems that need to be addressed at various stages of drug development. In this article, the classical LBA and LC-MS/MS methods for quantitative analysis of ADC components are briefly introduced.

1. Ligand binding assay (LBA)

LBA is the gold standard for macromolecular bioanalysis. LBA is typically used to measure TAb and total ADC, utilizing the mAb or payload component of the ADC, respectively.

1.1 LBA determination of total antibody (TAb)

Total Antibody (TAb) detection monitors antibody concentrations regardless of whether the payload is attached to the antibody, i.e., fully coupled, partially coupled, and completely uncoupled ADCs. This analysis is used for the overall PK evaluation of the ADC. In TAb analysis, specific reagents are used, such as recombinant target proteins, as well as general reagents, such as antibodies against the whole human IgG framework region or the (Fab')2 or Fc region, or against the light chain (LC) or the H+L chain region. These reagents bind to the antibody component of ADC regardless of its DAR value. Although these reagents do not directly bind to the payload, the payload can indirectly affect the binding of these reagents to the Ab component of the ADC due to steric hindrance. The interference is more pronounced in the species with higher DAR. Therefore, the analysis may not accurately calculate all of the expected DAR classes in the systemic circulation, thus affecting the overall PK characteristics of the ADC observed.

1.2 LBA determination of total ADC

LBA can monitor the concentration of antibodies carrying at least one payload molecule (i.e., ADC with DAR ≥ 1). The key reagents typically used for this LBA analysis can combine the payload component of the ADC with the antibody component. Similar to TAb assay, LBA for total ADC also shows sensitivity to the binding site and DAR heterogeneity of ADC. Solvent accessibility at the binding site may prevent the binding of anti-payload antibodies to the payload components of the ADC. In addition, proportional binding of the reagent to the Ab component of the ADC may not be possible due to the steric hindrance of multiple adjacent conjugate payload molecules. Therefore, LBA may not accurately measure ADCs with high and low DAR in systemic circulation.


In the last decade, LC-MS/MS analysis has become an alternative to LBA for TAb and total ADC bioanalytical measurements. LC-MS/MS combines the high selectivity of LBA with sensitivity. In addition, the LC-MS/MS approach overcomes the limitations of LBA in providing drug load or DAR information about the ADC. The three main steps of LC-MS/MS analysis are immune capture, release of alternative analytes, and detection.

2.1 Determination of total antibody by LC-MS/MS (TAb)

In recent years, LC-MS/MS has become an alternative platform for TAb measurement in non-clinical samples. In this form of analysis, ADCs are separated from the matrix using an immunoaffinity method. A generic capture reagent such as protein A or polyclonal anti-human antibody is used, and then broken down with an enzyme protein to produce a marker peptide from the Fc region of the antibody. Commercially stable labeled immunoglobulin antibodies, such as SILu™ monoclonal antibodies, are often used as the internal standard for this assay design.

Similar to LBA, general capture agents cannot be used for clinical LC-MS/MS analysis. This is due to the presence of excessive amounts of endogenous human immunoglobulin in clinical samples, which may interfere with the analysis. Therefore, specific capture reagents such as recombinant target antigens, and anti-idiotypic or CDR antibodies are used as immune capture agents in clinical LC-MS/MS analysis. It is often challenging to produce stable isotope labeled ADCs as internal standard. For this reason, stable isotope labeled (SIL) peptides are often used as internal standard and added to the test sample after the immunocapture step.

Soluble or shed target interference may also pose a challenge to LC-MS/MS based TAb analysis. Soluble or shed targets may prevent specific reagents (anti-idiotypic antibodies or recombinant target antigens) from binding to the ADC, thus interfering with immune capture. In vivo biotransformation of ADC antibody components is another challenge for TAb evaluation based on LC-MS/MS.

Another consideration in developing LC-MS/MS based TAb assays is that there should be no analytical bias due to changes in the ADC DAR in vivo. For example, higher DAR ADCs may be underestimated in analysis because the steric hindrance of the payload may prevent the ADC from binding to the capture reagent. Therefore, it may be necessary to test and analyze different DAR-enriched ADCs to ensure that there is no capture bias during the analysis.

2.2 Quantitative determination of total ADC (coupled antibody) by LC-MS/MS

The LC-MS/MS analysis platform can also be used to determine the total ADC. In this method, the anti-payload antibody is used to capture the ADC. Similar to LC-MS/MS based TAb analysis, peptides from the human Fc region or CDR region are used as marker peptides for non-clinical studies, whereas for clinical studies, marker peptides from the CDR region are used.

2.3 LC-MS/MS quantitative determination of total ADC (coupled payload)

The purpose of coupling payload analysis is to measure the concentration of payload molecules bound to the antibody. Coupling payloads involve direct measurement of payloads and thus can provide a more accurate assessment of the payload exposure at the target site and a correlation between exposure and efficacy.

LC-MS/MS is the main platform for coupling payload measurement. In LC-MS/MS coupled payload analysis, the capture agent is targeted at the Ab component of the ADC molecule. Therefore, the immune capture procedure is similar to TAb analysis based on LC-MS/MS. Once separated by an immunoaffinity method, the payload molecules are released from the ADC and the cutting payload is measured with LC-MS/MS, depending on linker chemistry, enzyme reactions, or chemical reactions that are used to cut the payload from the ADC. Although the alternative analyte in conjugated payload analysis is a small molecule drug, the use of immunocapture techniques guarantees validation guidelines for macromolecular bioanalytical methods for this analysis.

2.4 LC-MS/MS quantitative determination of uncoupled payload

Uncoupled payload analysis involves measuring the decoupled payload of the ADC in the plasma or target tissue after ADC administration, as well as the presence of uncoupled payload in the ADC formulation. LC-MS/MS is a preferred platform for uncoupled payload biological analysis. LBA is also used, but this depends on the availability of anti-payload antibodies. The stability of the ADC in the systemic circulation and target tumor microenvironment can be understood through uncoupled payload analysis.

The main challenge of uncoupled payload analysis is sensitivity. Because of the high cytotoxic activity of the payload, ADCs are usually administered at lower concentrations, making it difficult to quantify uncoupled payloads at lower levels. Stable ADCs release relatively little free payload in systemic circulation. In addition, to reduce metabolic risk, ADC payloads are typically more hydrophobic and have a higher molecular weight (>700 Da) compared to conventional small molecules, which adds to the analytical challenges.

Another difficulty in uncoupled payload quantification is that analytes sometimes need to be stabilized prior to LC-MS/MS analysis. For example, in the study described by Heudi et al., DM1 released from thioether-linked trastuzumab-DM1 contains a free mercaptan portion that needs to be reduced and alkylated prior to analysis. These steps prevent free DM1 from forming dimers on endogenous compounds such as cysteine or glutathione or reacting with free mercaptan groups.

The uncoupled payload of an ADC containing maleimide can form an adduct with an endogenous protein, resulting in an underestimation of the uncoupled payload concentration in the tested sample. Adduct formation can be evaluated by LC-MS/MS analysis. To evaluate the total uncoupled payload, additional steps can be added to the bioanalytical workflow to release the payload bound to the endogenous protein prior to the measurement.

BOC Sciences provides analytical characterization support for ADC drug development with rich experience and advanced technology, our services include total antibody assay, free antibody assay, binding payload assay, free payload assay, total ADC assay, DAR and drug distribution assay. If you are interested in our ADC analysis and characterization services, please click here to learn more!

Ella Bette
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