Heather Myler

Bioanalytical Approaches to Quantify “Total” and “Free” Therapeutic Antibodies and Their Targets: Technical Challenges and PK/PD Applications Over the Course of Drug Development

The predominant driver of bioanalysis in supporting drug development is the intended use of the data. Ligand-binding assays (LBA) are widely used for the analysis of protein biotherapeutics and target ligands (L) to support pharmacokinetics/pharmacodynamics (PK/PD) and safety assessments. For monoclonal antibody drugs (mAb), in particular, which non-covalently bind to L, multiple forms of mAb and L can exist in vivo, including free mAb, free L, and mono- and/or bivalent complexes of mAb and L. Given the complexity of the dynamic binding equilibrium occurring in the body after dosing and multiple sources of perturbation of the equilibrium during bioanalysis, it is clear that ex vivo quantification of the forms of interest (free, bound, or total mAb and L) may differ from the actual ones in vivo. LBA reagents and assay formats can be designed in principle to measure the total or free forms of mAb and L. However, confirmation of the forms being measured under the specified conditions can be technically challenging. The assay forms and issues must be clearly communicated and understood appropriately by all stakeholders as the program proceeds through the development process. This paper focuses on monoclonal antibody biotherapeutics and their circulatory L that are either secreted as soluble forms or shed from membrane receptors. It presents an investigation into the theoretical and practical considerations for total/free analyte assessment to increase awareness in the scientific community and offer bioanalytical approaches to provide appropriate PK/PD information required at specific phases of drug development.

A White Paper—Consensus and Recommendations of a Global Harmonization Team on Assessing the Impact of Immunogenicity on Pharmacokinetic Measurements

The Global Bioanalysis Consortium (GBC) set up an international team to explore the impact of immunogenicity on pharmacokinetic (PK) assessments. The intent of this paper is to define the field and propose best practices when developing PK assays for biotherapeutics. We focus on the impact of anti-drug antibodies (ADA) on the performance of PK assay leading to the impact on the reported drug concentration and exposure. The manuscript describes strategies to assess whether the observed change in the drug concentration is due to the ADA impact on drug clearance rates or is a consequence of ADA interference in the bioanalytical method applied to measure drug concentration. This paper provides the bioanalytical scientist guidance for developing ADA-tolerant PK methods. It is essential that the data generated in the PK, ADA, pharmacodynamic and efficacy/toxicity evaluations are viewed together. Therefore, the extent for the investigation of the PK sensitivity to the presence of ADA should be driven by the project needs and risk based.

Quantitative bioanalysis of antibody-conjugated payload in monkey plasma using a hybrid immuno-capture LC-MS/MS approach: Assay development, validation, and a case study.

Antibody drug conjugates (ADCs) are complex molecules composed of two pharmacologically distinct components, the cytotoxic payload and the antibody. The measurement of the payload molecules that are attached to the antibody in vivo is important for the evaluation of the safety and efficacy of ADCs, and can also provide distinct information compared to the antibody-related analytes. However, analyzing the antibody-conjugated payload is challenging and in some cases may not be feasible. The in vivo change in drug antibody ratio (DAR), due to deconjugation, biotransformation or other clearance phenomena, generates unique and additional challenges for ADC analysis in biological samples. Here, we report a novel hybrid approach with immuno-capture of the ADC, payload cleavage by specific enzyme, and LC-MS/MS of the cleaved payload to quantitatively measure the concentration of payload molecules still attached to the antibody via linker in plasma. The ADC reference material used for the calibration curve is not likely to be identical to the ADC measured in study samples due to the change in DAR distribution over the PK time course. The assay clearly demonstrated that there was no bias in the measurement of antibody-conjugated payload for ADC with varying DAR, which thus allowed accurate quantification even when the DAR distribution dynamically changes in vivo. This hybrid assay was fully validated based on a combination of requirements for both chromatographic and ligand binding methods, and was successfully applied to support a GLP safety study in monkeys.

New FDA Draft Guidance on Immunogenicity

A “Late Breaking” session was held on May 20 at the 2013 American Association of Pharmaceutical Scientists-National Biotech Conference (AAPS-NBC) to discuss the US Food and Drug Administration’s (FDA) 2013 draft guidance on Immunogenicity Assessment for Therapeutic Protein Products. The session was initiated by a presentation from the FDA which highlighted several key aspects of the 2013 draft guidance pertaining to immunogenicity risk, the potential impact on patient safety and product efficacy, and risk mitigation. This was followed by an open discussion on the draft guidance which enabled delegates from biopharmaceutical companies to engage the FDA on topics that had emerged from their review of the draft guidance. The multidisciplinary audience fostered an environment that was conducive to scientific discussion on a broad range of topics such as clinical impact, immune mitigation strategies, immune prediction and the role of formulation, excipients, aggregates, and degradation products in immunogenicity. This meeting report highlights several key aspects of the 2013 draft guidance together with related dialog from the session.

2015 White Paper on recent issues in bioanalysis: focus on new technologies and biomarkers (Part 3 – LBA, biomarkers and immunogenicity)

The 2015 9th Workshop on Recent Issues in Bioanalysis (9th WRIB) took place in Miami, Florida with participation of 600 professionals from pharmaceutical and biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. WRIB was once again a 5 day, week-long event - A Full Immersion Bioanalytical Week - specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest in bioanalysis. The topics covered included both small and large molecules, and involved LCMS, hybrid LBA/LCMS and LBA approaches, including the focus on biomarkers and immunogenicity. This 2015 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2015 edition of this comprehensive White Paper has been divided into three parts. Part 3 discusses the recommendations for large molecule bioanalysis using LBA, biomarkers and immunogenicity. Part 1 (small molecule bioanalysis using LCMS) and Part 2 (hybrid LBA/LCMS and regulatory inputs from major global health authorities) have been published in volume 7, issues 22 and 23 of Bioanalysis, respectively.

An integrated multiplatform bioanalytical strategy for antibody–drug conjugates: a novel case study

BACKGROUND The bioanalytical strategy for antibody-drug conjugates (ADC) includes numerous measurements integrally designed to provide comprehensive characterization of PK, PD and immunogenicity. This manuscript describes the utilization of reagents specifically tailored to an ADC with a microtubule polymerization inhibitor payload and cathepsin B cleavable linker. METHODS The PK strategy includes the evaluation of physiological levels of total antibody, active ADC, total ADC, antibody-conjugated payload and unconjugated payload. These data are evaluated in the context of target and antidrug antibody levels to elucidate bioactive ADC. RESULTS & CONCLUSION Herein, we discuss how this strategy has been applied and present our preliminary observations. Continuously evolving to meet pipeline demands, the integrated bioanalytical data will provide critical insights into the exposure-response relationship.

Biotherapeutic bioanalysis: a multi-indication case study review

A thorough understanding of the structure and biology of a biotherapeutic is crucial to defining a suitable strategy for pharmacokinetic characterization in proof-of-concept disease models, toxicology species as well as the healthy and disease indication patient populations. This manuscript summarizes parameters that impact bioanalytical strategy for over 50 biotherapeutics indicated for the treatment of oncology, rheumatoid arthritis, allergy, multiple sclerosis, hematology, metabolism and infectious disease. We have addressed numerous therapeutic modalities including chimeric, humanized and fully human monoclonal antibodies, replacement proteins, peptides and fusion proteins, including polyethylene glycol and Fc fusions, as well as antibody-drug conjugates. With the rapid evolution of biotherapeutics over the last 20 years and the contraction of the pharmaceutical and biotechnology labor force, efficient workflow management becomes a crucial bioanalytical component. Thus, we have also addressed new technologies that have demonstrated either increased throughput or enhanced characterization, including Meso Scale Discovery, Gyrolab and affinity MS.

Pre-existing Antibody: Biotherapeutic Modality-Based Review

Pre-existing antibodies to biotherapeutic drugs have been detected in drug-naïve subjects for a variety of biotherapeutic modalities. Pre-existing antibodies are immunoglobulins that are either specific or cross-reacting with a protein or glycan epitopes on a biotherapeutic compound. Although the exact cause for pre-existing antibodies is often unknown, environmental exposures to non-human proteins, glycans, and structurally similar products are frequently proposed as factors. Clinical consequences of the pre-existing antibodies vary from an adverse effect on patient safety to no impact at all and remain highly dependent on the biotherapeutic drug modality and therapeutic indication. As such, pre-existing antibodies are viewed as an immunogenicity risk factor requiring a careful evaluation. Herein, the relationships between biotherapeutic modalities to the nature, prevalence, and clinical consequences of pre-existing antibodies are reviewed. Initial evidence for pre-existing antibody is often identified during anti-drug antibody (ADA) assay development. Other interfering factors known to cause false ADA positive signal, including circulating multimeric drug target, rheumatoid factors, and heterophilic antibodies, are discussed.

Large Molecule Specific Assay Operation: Recommendation for Best Practices and Harmonization from the Global Bioanalysis Consortium Harmonization Team

The L2 Global Harmonization Team on large molecule specific assay operation for protein bioanalysis in support of pharmacokinetics focused on the following topics: setting up a balanced validation design, specificity testing, selectivity testing, dilutional linearity, hook effect, parallelism, and testing of robustness and ruggedness. The team additionally considered the impact of lipemia, hemolysis, and the presence of endogenous analyte on selectivity assessments as well as the occurrence of hook effect in study samples when no hook effect had been observed during pre-study validation.

Anti-PEG antibody bioanalysis: a clinical case study with PEG-IFN-λ-1a and PEG-IFN-α2a in naive patients

BACKGROUND Extensive use of polyethylene glycol (PEG) in consumer products necessitates the assessment of anti-PEG antibodies (APAb). METHODS In clinical trials comparing PEG-IFN-λ to PEG-IFN-α, conventional bridge and direct assays were assessed. RESULTS & CONCLUSION The bridge assay detected IgM and IgG APAb reactive with common PEG sizes and derivatives at sufficient sensitivity, 15-500 ng/ml. Of subjects evaluated, 6% of PEG-IFN-λ and 9% of PEG-IFN-α subjects had persistent APAb while 60% of PEG-IFN-λ and 33% of PEG-IFN-α subjects had persistent anti-interferon antibodies (AIAb). Pre-existing APAb and AIAb prevalence was comparable (approximately 10% of subjects). APAb were earlier onset, less frequent, less persistent and lower titer than AIAb. No associated hypersensitivity events were reported.