20 May / June 2017
Accelerated Development of Quantitative Assays for Antibody Drug Conjugates
by Khatereh Motamedchaboki, PhD, Scientist, Remco Van Soest, MSc, Senior Applications Specialist, Kelli Jonakin, PhD, Global Marketing Manager, Pharma/BioPharma, and Ian Moore, PhD, Global Technical Marketing Manager, Pharma Quant and MetID Sciex, 71 Four Valley Drive, Concord, Ontario, L4K 4V8, Canada
Antibody drug conjugates (ADCs) represent an exciting and rapidly growing class of biotherapeutics. Because of their unique molecular structure, pharmacokinetic studies of ADCs can be particularly challenging. Ligand binding assays (LBA) have typically been used but can suffer from poor reproducibility, limited dynamic range, and cross reactivity. Liquid chromatography tandem mass spectrometry (LC-MS/MS) assays generally have high selectivity, dynamic range, and reproducibility but can lack in sensitivity when applied to protein therapeutics. In this article, we describe a hybrid LBA/LC-MS/MS technique that uses a universal immunoenrichment strategy and microflow LC to create a novel quantitative analysis solution that accelerates method development and improves performance of ADC pharmacokinetic assays.
Introduction
Within the last several decades, monoclonal antibody (mAb) based drugs have become an established class of biotherapeutics and represent the fastest growing segment of the global biopharmaceutical market [1-3]. Because of their inherent advantages in specificity, efficacy, and safety, pharmaceutical companies have dedicated large portions of their research and development efforts to mAb-based therapies with the list of approved and investigational mAb based drugs now reaching into the hundreds [4].
MAb based therapies represent a way to specifically target a disease by using an immune molecule which, by design only targets the diseased cells of interest. Antibody drug conjugates (ADCs) are a class of biotherapeutics that combine a mAb with a cytotoxic small molecule drug attached to the mAb through a chemical linker group. The idea behind an ADC is that the antibody provides the specificity while the small molecule drug provides the cytotoxic payload to treat the disease. This targeted delivery of the cytotoxic drug improves the drug’s overall efficacy and minimises systemic toxicity.
The unique chemical structure of ADCs, as both an antibody and a small molecule drug, can present challenges for scientists
Figure 1. Binding Kinetics of BioBA Beads. 583 µg of biotinylated anti-human IgG was added to 4.25 mg of beads. After 120 min, 255 µg remained in solution resulting in a binding capacity 77µg/mg.
tasked with their bioanalysis. Most ADCs are heterogeneous molecules. One or more drugs may attach to the antibody at various sites resulting in varying drug to antibody ratios (DAR). To add to the complexity, the chemical lability of the linker group as well as the chemistry used for conjugation can affect the in vivo stability and heterogeneity of the ADC. Thus, it can be a challenge to determine which form or forms of the ADC molecule should be monitored.
Traditionally, ligand binding assays (LBA) such as enzyme linked immunosorbent assays (ELISA) have been used for the bioanalysis of mAbs and mAb based biotherapeutics. LBAs typically have high sensitivity and high throughput, are relatively
inexpensive, and require limited sample preparation. However, LBAs can suffer from high variability, narrow dynamic range, and problems with selectivity. Additionally, internal standards cannot be added which help to correct for anomalies and ensure accurate quantification.
Mass spectrometry, and in particular liquid chromatography tandem mass spectrometry (LC-MS/MS) has found widespread use for the quantitative analysis of small molecule drugs. Assays based on LC-MS/MS are exceedingly sensitive and selective with excellent dynamic range. Additionally, assays can be multiplexed and internal standards are easily added to any experiment. In more recent years LC-MS/MS has been applied to
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