“Hybrid LC-MS/MS Technology Becoming Increasingly Valuable in the Bioanalysis of Antibody Drug Conjugates – Bioanalytical strategy Illustrated by Case Studies”

Dawn Dufield, Ph.D., Scientific Officer, KCAS Bio

Abstract

Recently, we have seen the role of hybrid LC-MS/MS in the analysis of antibody-drug conjugates (ADC) increase due to the complexity and number of bioanalytical assays needed to support this modality in the drug development process. Antibody drug conjugates tend to consist of a toxic small molecule payload attached via a linker to an antibody. The application of these drugs had mainly been in oncology where their mode of action is for the antibody to attach at the desired target site with subsequent release of the small molecule to combat tumor cells. Recently, we have seen a much wider application or use of “ADCs” to other modalities such as Antibody-peptide conjugates, Antibody-RNA-conjugates (ARCs) or even other more complicated multi-functional entities.  

Having a therapeutic entity with multiple distinct components means that we need to have analytical methodologies for various components. Traditionally, LC-MS/MS has mainly been applied to the quantitation of the small molecule payload or the linker moiety. Ligand-binding assays are highly successful for quantitation of the total antibody and sometimes may be suitable for quantification of intact antibody drug conjugate.

More recently, we have seen a trend towards all analyses being done by LC-MS/MS. Hybrid LC-MS/MS can easily be used to monitor both the total antibody as well as the ADC conjugate. Depending on the type of conjugation and linker used (site-specific vs Cys or Lys conjugation and cleavable vs non-cleavable,) and what reagents are available, hybrid LC-MS/MS offers several strategies or advantages to monitor the various constituent parts of the ADC.  

This gives the scientist flexibility for the quantitation of antibody drug conjugates. In many cases, ligand binding or hybrid LC-MS/MS can be applicable. However, poor reagents or limited availability of reagents can limit the immediate application of ligand-binding. The ability to approach the analysis from different angles to gain multiple insights into the analyte can increasingly nudge the decision towards the use of LC-MS/MS. In many preclinical cases, we can even use “generic” methods which make the method development and analysis much quicker and cost effective. At the very least, the availability of hybrid LC-MS/MS as an alternative technology greatly increases your chances of success in the bioanalysis of antibody drug conjugates.

The increasing use of a new technology for large molecule quantitation does present a challenge to industry. It is essential that your analysis partner has a thorough understanding of hybrid technology including potential immunoaffinity approaches, enzymatic digestion alternatives, chromatography and optimization of the mass spectrometer.

Bioanalysis is a continually evolving field. KCAS is focused on applying our LC-MS expertise to be at the forefront of the industry to help address our customers’ analytical challenges. Hybrid LC-MS/MS is proving to be an increasingly valuable tool for delivering answers about complex analytes such as antibody drug conjugates to enhance the drug development process. This presentation will give an overview of ADC bioanalysis as well as several case studies to illustrate the various strategic approaches.  

  1. Key Takeaways
    • Understand the bioanalytical challenges associated with antibody drug conjugates (ADCs)
    • Understand different types of modalities that are classified as antibody drug conjugates.
    • Understand the different strategies for developing "ADC assays" -  based on 
      • Information needed
      • Types of conjugation and linker chemistry
      • Reagents available - generic vs specific (pre-clinical and clinical) approaches
    • Understand minimally what approach is needed at various stages of MD in lifecycle
    • Highlight several strategies through various case study examples

Bio

Dawn Dufield is a Scientific Officer at KCAS Bio. She joined in 2018 and was previously with Pfizer for 20 years working in LC-MS/MS. She received her PhD from the University of Kansas in 1997.  She was an early pioneer of using immunoaffinity combined with LC-MS/MS to offer additional selectivity, now commonly referred to as Hybrid LCMS and is a co-author on a white paper “Recommendations for validation of LCMS based bioanalytical methods for protein biotherapeutics”. She has been working on ADCs for 15 years and has contributed to a book chapter on this topic. She has numerous publications and is an active member of AAPS and ASMS.