Staff Scientist III Labcorp Drug Development Madison, Wisconsin
Biotherapeutic drugs have a propensity to generate an immune response (immunogenicity), producing anti-drug antibodies (ADA) with significant implications to pharmacokinetics, pharmacodynamics, safety, and efficacy. The Food and Drug Administration (FDA) published guidance for method development and validation of assays for anti-drug antibody detection/immunogenicity testing in 2019. Bridging ligand binding assay (LBA) is the gold standard for immunogenicity testing but other technologies are also implemented, such as electrochemiluminescence, surface plasmon resonance, etc. These technologies are used to address the tier-based approach to ADA testing including: screening, confirmatory, titration, neutralization, cross-reactivity, and isotype/subtype assays.
Hybrid LBA LC-MS has been widely adopted as an alternative to traditional LBA for bioanalysis of biotherapeutics. However, very few cases of LC-MS for ADA detection have been reported. Hybrid LBA LC-MS offers many advantages over the existing technologies including plug-and-play methodology, multiplexing of ADA isotypes/subtypes, semi-quantitation, and reduced biological interference. A single plug-and-play hybrid LBA LC-MS methodology can be directly applied to different biotherapeutics with limited redevelopment or need for specialized reagents. ADAs against any biotherapeutic drug can be specifically affinity enriched by charging streptavidin magnetic beads with the biotinylated drug substance. Affinity enrichment is required to differentiate the drug-specific immunoglobulins (Igs) from the high concentrations of endogenous Igs. During method development, a purified positive control ADA, typically generated in non-human species, is used to optimize affinity enrichment performance. Affinity enriched ADAs are subsequently digested by trypsin to produce LC-MS compatible peptides for quantitation. Multiplexing is achieved by monitoring conserved, highly selective surrogate peptides from each Ig isotype/subclass in a single LC-MS/MS analysis. A stable isotopically labeled peptide for each surrogate peptide is used to control for LC-MS variability. LC-MS offers sufficient sensitivity (down to 100 ng/mL) and semi-quantitative ADA concentrations are back calculated against calibration curves prepared from a purified Ig isotype/subclass in surrogate matrix. Absolute quantitation cannot be achieved due to the biological variability in ADA affinities (and hence recoveries) and lack of authentic reference materials. The combination of high binding capacity and high LC-MS specificity makes the assay less sensitive to biological interferences relative to LBA.
We will discuss the development of a hybrid LBA LC-MS assay for the semi-quantitation of IgG subclasses (IgG1 (500 to 50,000 ng/mL), IgG2 (150 to 15,000 ng/mL), and IgG3/4 (100 to 10,000 ng/mL)) to support the development of a ‘tolerogenic vaccine’ protein therapeutic where a shift in the relative IgG subclass concentrations is clinically relevant. We will highlight the challenges of developing the affinity enrichment procedure including the importance of drug substance integrity, biotinylation, bead capacity/recovery, magnetic bead quality, wash buffer composition, and non-specific binding of endogenous Igs. We also highlight the challenges of identifying surrogate peptides, optimizing trypsin digestion conditions, and LC-MS/MS parameters. Finally, we will discuss the challenges of developing a robust assay using surrogate reference materials (purified human Igs), surrogate matrix (TBS-T), and synthetic peptide internal standards.
Learning Objectives:
List challenges and advantages associated with ADA assays by LC-MS
Describe a typical workflow for hybrid LBA LC-MS assays
Implement a similar workflow for ADA assays for novel protein therapeutics