Critical aspects of anti-body conjugates: structure and analysis

By Dr. Robert Duff, Eurofins Lancaster Laboratories, USA

Targeted drug therapies for diseased cells (i.e. cancer) using monoclonal antibodies has been a topic of growing interest and an area of continued development. Antibody-drug conjugates (ADC) are an emerging class of targeted agents demonstrating tremendous potential both in vitro and in vivo. The mechanism of action is believed to be antigen recognition and binding followed by endocytosis during which the cell's lysosomal enzymes release the cytotoxin. These rationally designed conjugates, formed through the chemical linkage of a potent small molecule cytotoxin (drug) to a monoclonal antibody (mAb), have more complex and heterogeneous structures than the corresponding antibodies. Each part (the mAb, the drug, and the linker) must be carefully chosen in order to provide the best therapeutic index. The molecular diversity of the cytotoxins is remarkable. The commercial pipeline of antibody-based therapeutics continues to grow and now totals nearly 350 candidates.

The creation of linkers that are stable in circulation but labile upon binding of the ADC to its target has been an improvement  and has resulted in the current generation of ADCs having better stability and lower systemic toxicity.Regulatory agencies are primarily concerned with the selection of the most appropriate analytical methods for an ADC. Analytical methodologies need to discern primary structure (intact mass, peptide mapping (sequencing), NMR, FTIR, drug linkage discernment); secondary/tertiary structure (circular dichroism, SPR, X-ray), drug-antibody ratio (UV), fragments/ aggregates (AUC, SEC-MALS, SE-HPLC); and charge variants (CE, iCE, IEX, MS), glycosylation and other post translational modifications (LC-MS/MS). Antigen binding, biological activities and effector function are used as appropriate (ELISA, SPR (BiaCore)). Assays for free drug and (bio)process impurities such as synthetic impurities or host cell proteins should be included.

Overall, the ADC should be considered a new molecular entity  and not a combination product. ADCs show tremendous promise for the future, and now better methodologies are available to prove structure-function relationship through site-specific mAbs and non-natural amino acids.

For more information on this Eurofins Pharma service, contact: GMP_US@eurofins.com