Bioanalysis of Antibody Drug Conjugates

Considerations for biological analysis

• Heterogeneity of ADC

ADCs are actually mixtures, antibodies in ADCs are covalently linked to small molecule drugs by a linker, which can bind to the amino lysine side chain of the antibody, or to the sulfhydryl group resulting from the reduction of disulfide bonds between antibody chains, or to engineered cysteine residues introduced at specific sites on the antibody^1-3.

• Dynamic variation of DAR values in vivo

ADCs are theoretically stable in plasma, but the linker can still break down under special circumstances, allowing small molecule drugs to dissociate. The dissociation of small molecule drugs can change the DAR value or present a new DAR value. Studies have shown that the clearance rate of ADCs increases with DAR values⁴.

• Selection of drugs to be tested

For small molecule drugs and protein drugs, the exposure-response (ER) can be known by measuring the original drug. However, ADCs contain both antibody and small molecule drug components, and DAR values in vivo change dynamically, as well as limited clinical data, so it is difficult to determine which substance to be measured affects ER. In general, in the early stages of ADCs development, as many substances as possible are tested to understand the pharmacokinetic characteristics of each component of the ADCs. These tests generally include total antibodies,  ADCs and free small molecule drugs. In addition, anti-therapeutic antibodies (ATAs) may be produced when ADCs enter the body. ATAs may reduce the efficacy of ADCs, so it is necessary to evaluate ATAs during the development of ADCs.

Fig.2 Antibody–drug conjugate analytes.(Kaur, S.; et al. 2013)

Methods for pharmacokinetic analysis

• Determination of total antibody

Indirect-ELISA: The coated antigen was used to capture the total antibody, and the second antibody was used as the detection antibody according to the species property of the total antibody⁵. This method has the advantages of extensive application and mature technology, but sometimes it is difficult to obtain commercial antigen.

ECLA: ECLA uses electrochemiluminescence techniques with higher sensitivity and a wider dynamic range to obtain signals. One example is the highly sensitive MSD instrument developed by Meso Scale Diagnostics, inc.

• Determination of antibody conjugates

Conjugated antibodies are those that bind to small molecule drugs and have DAR≥1. The determination of conjugated antibody is mostly by ELISA.

Bridge-linked ELISA: Based on the high specificity and amplification effect of the binding between biotin and avidin or streptavidin (one molecule of avidin or streptavidin can bind four molecules of biotin), bridge-linked ELISA method can be designed to amplify the detection signal.

• Determination of binding small molecule drugs

For the determination of the toxin molecules linked to the antibody, the ADC is usually separated from the free small molecule toxin in some way, and then the small molecule toxin is released by chemical reaction or enzyme stimulation, and finally the accurate quantitative results are obtained by ELISA or LC-MS/MS.

ELISA: Anti-ID antibody, antigen ECD or anti-human IgG antibody were selected to capture the antibody part of an ADC, and anti-small molecule drug antibody was used as detection reagent.

LC-MS/MS: The conjugated antibody is first captured with reagents (such as protein A, anti-ID antibody, antigen, etc.), followed by the removal of the connecting arm, and finally, LC-MS /MS analysis is performed for the small molecule drugs.

• Determination of free small molecule drugs

The free small molecule drug is the part of the drug that has been dissociated from the antibody, and it can be determined by competitive-ELISA⁷ and LC-MS/MS⁸.

Competitive-ELISA: For the determination of free DM1, the sample was first precipitated by acetonitrile, the supernatant containing free DM1 was mixed with biotinylated mouse anti-DM1 antibody, and then transferred to a plate coated with BSA-DM1. DM1 in the sample will competitively bind anti-DM1 antibody with coated BSA-DM1. The higher the concentration of DM1 in the sample, the less DM1 antibody bound by BSA-DM1, and the lower the signal detected by streptavidin-HRP.

Quantitative LBA Assays for ADCs