MPI Research and Perfinity Biosciences are working in partnership to streamline and validate bioanalysis of antibody drug conjugates. Vice President of Bioanalytical/Analytical Services Alan Breau and Senior Scientist Kevin Meyer come together to explain the process.

Antibody drug conjugates (ADCs) link precisely targeted antibodies to cytotoxic small-molecule drugs, with the goal of improving both the safety and efficacy of chemotherapeutics. Pivotal clinical trials of trastuzumab-DM1 for women with treatment-resistant HER2- positive breast cancer and of SGN-35 for relapsed/refractory Hodgkin’s lymphoma represent the vanguard of several dozen ADCs currently in various stages of clinical development. In ADCs, the antibody acts primarily as a carrier to deliver the potent drug to the target tissue, thus limiting total body exposure to the free and potent small molecule, a strategy that can mitigate toxic side effects. However, ADCs pose several challenges, including the tendency for the covalently-bound small molecules to detach and circulate freely in the body, the difficulty of manufacturing conjugates reproducibly and the metabolic fate of the conjugate and the antibody.

Currently the analysis of ADCs requires four distinct assays on each plasma sample, two small molecule assays and two ELISA assays. The small molecule assays detect freely circulating drug and bound drug that is chemically released from circulating ADCs.

Free or bound?

MPI Research and Perfinity Biosciences are currently collaborating to streamline and validate this process. One option is to measure the free and bound small-molecule levels by using immunosorbent technology to trap free drug for analysis, while the proteincontaining eluate is collected and hydrolysed to liberate bound drug for analysis. Thus, free and bound small molecules could be analysed from the same serum sample rather than from duplicates.

The proteins could be measured using two different ELISA assays: the small-molecule free antibody could be measured by conventional ELISA with an antibody that recognises the antibody/linker and the ADC, and the antibody-containing drug molecule could be measured by an ELISA that utilises an antibody that recognises the small molecule/linker portion of the ADC.

To further complicate analysis, ADCs are not homogenous products, but rather a mixture containing differing numbers of potent small molecules attached to each antibody. Therefore, the batch-to-batch distribution differences and the kinetics of the changing small-molecule distribution in vivo may impact the clinical effectiveness of the compounds. Currently only gross averaged data are obtained, which may confound an accurate pharmacokinetic/ pharmacodynamic prediction or batch potency determination.

Simultaneous quantification of total ADC, in vivo fragments of the ADC and drug conjugated at each site in the ADC can be achieved using MRM technology and carbon 13-labelled isopotomers of peptides and drug-bearing peptide conjugates. This approach quantifies small molecules linked at specific locations on the antibody and tracks their binding on the antibody to provide a more accurate indicator of the potency of each batch, as well as its potential in vivo performance.

High performance

Relative binding affinity with receptors also affects how well ADCs perform. For example, if antibody without any drug has a higher affinity for the receptor than antibody with drug, then the presence of drug-free antibody would block the desired binding of antibody-containing drug, potentially attenuating the ADC’s efficacy. We believe an assay that traps the ADC using an immobilised receptor protein or antibody, followed by deglycosylation and enzymatic digestion of the protein, would yield a series of peptides that could be analysed by liquid chromatography and mass spectrometry. The peptides that contain drug/linker would differ in retention and molecular weight from the analogous peptides that did not contain a linked small molecule. If tracked sites that were more labile to in vivo release could be identified, then product specifications could limit the amount of ADCs containing linked drug at these labile sites. The result would be a safer, more potent ADC mixture.

ELISA assay sensitivity need not be an issue. For example, the published assay of Herceptin – which has which has an average molecular weight of 145,531 daltons – has a validated range of range from 5-100 ng/ml. The samples in the published ELISA are diluted one to two-thousand-fold to correspond to an assay of 10-200 ìg/ml.

The published Cmax values for Herceptin are in the ìg/ml range, suggesting that the sensitivity of many ELISA assays is too low for protein therapeutics.

We believe that new bioanalytical and analytical processes will be necessary to support emerging ADC therapeutics. To that end, MPI Research and Perfinity Biosciences, in consultation with Professor Fred Regnier who founded Perfinity Biosciences, are actively investigating innovative bioanalytical/analytical methods for ADCs. We are proud to be leading the scientific advances necessary to allow ADCs to achieve commercialisation and widespread clinical application.