Robert Dodge

Innovative Use of LC-MS/MS for Simultaneous Quantitation of Neutralizing Antibody, Residual Drug, and Human Immunoglobulin G in Immunogenicity Assay Development

Immunogenicity testing for antidrug antibodies (ADA) faces challenges when high levels of the drug are present in clinical patient samples. In addition, most functional cell-based assays designed to characterize the neutralizing ability of ADA are vulnerable to interference from endogenous serum components. Bead extraction and acid dissociation (BEAD) has been successfully applied to extract ADA from serum samples prior to conduction of cell-based assays. However, in the BEAD, certain amounts of the drug and endogenous serum components (so-called residual drug and serum components) from serum samples are carried over to final BEAD eluates due to formation of protein complexes with ADA or nonspecific binding with the beads. Using current enzyme-linked immunosorbent assay (ELISA)-based ligand-binding assays, it is difficult to evaluate the residual drug, which is complexed with excessive amounts of ADA and endogenous serum components in the BEAD eluates. Here, we describe an innovative application of LC-MS/MS for simultaneous detection of the residual human monoclonal antibody drug and endogenous human IgG and the neutralizing antibody positive-control (NAb-PC) in the BEAD eluates. In this study, the low levels of the residual drug and human IgG in the BEAD eluates indicate that the BEAD efficiently removed the high-concentration drug and serum components from the serum samples. Meanwhile, the NAb-PC recovery (∼42%) in the BEAD provided an acceptable detection limit for the cell-based assay. This novel application of LC-MS/MS to immunogenicity assay development demonstrates the advantages of LC-MS/MS in selectivity and multiplexing, which provides direct and fast measurements of multiple components for immunogenicity assay development.

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.

Development and characterization of a pre-treatment procedure to eliminate human monoclonal antibody therapeutic drug and matrix interference in cell-based functional neutralizing antibody assays

Biological therapeutics can induce an undesirable immune response resulting in the formation of anti-drug antibodies (ADA), including neutralizing antibodies (NAbs). Functional (usually cell-based) NAb assays are preferred to determine NAb presence in patient serum, but are often subject to interferences from numerous serum factors, such as growth factors and disease-related cytokines. Many functional cell-based NAb assays are essentially drug concentration assays that imply the presence of NAbs by the detection of small changes in functional drug concentration. Any drug contained in the test sample will increase the total amount of drug in the assay, thus reducing the sensitivity of NAb detection. Biotin-drug Extraction with Acid Dissociation (BEAD) has been successfully applied to extract ADA, thereby removing drug and other interfering factors from human serum samples. However, to date there has been no report to estimate the residual drug level after BEAD treatment when the drug itself is a human monoclonal antibody; mainly due to the limitation of traditional ligand-binding assays. Here we describe a universal BEAD optimization procedure for human monoclonal antibody (mAb) drugs by using a LC-MS/MS method to simultaneously measure drug (a mutant human IgG4), NAb positive control (a mouse IgG), and endogenous human IgGs as an indicator of nonspecific carry-over in the BEAD eluate. This is the first report demonstrating that residual human mAb drug level in clinical sample can be measured after BEAD pre-treatment, which is critical for further BEAD procedure optimization and downstream immunogenicity testing.

Challenges in developing antidrug antibody screening assays

Robert Dodge is the Laboratory Director for Immunochemistry and Cell Biology at Taylor Technology, a Pharmanet company in Princeton, NJ, USA. Taylor Technology is a contract research laboratory specializing in assay development and sample testing in a good laboratory practice environment. Robert oversees a staff of scientists responsible for the development of assays for use in protein drug quantitation, antidrug antibody screening and biomarker detection. Protein drugs may elicit an immune response in the form of production of antidrug antibodies (ADAs) by a subject. This ADA response may have serious safety implications for subjects or, at a minimum, may affect drug efficacy. Bioanalytical testing for antidrug antibodies has therefore become a required part of safety testing for subjects receiving protein drugs. Regulatory guidance and scientific white papers recommend a tiered approach for bioanalytical ADA testing. The first assay in the tiered testing scheme is a screening assay, which tests all subjects in the study for the presence of ADAs. The screening assay is quasiquantitative, typically lacks a specific positive control and must be designed to detect numerous isotypes and subclasses of antibodies. These characteristics of an ADA screening assay differ from a those of a standard immunoassay designed to quantitate a specific protein in matrix and thus present unique challenges. This article reviews the unique challenges encountered during the development of an ADA assay.

A systematic study of the effect of low pH acid treatment on anti-drug antibodies specific for a domain antibody therapeutic: Impact on drug tolerance, assay sensitivity and post-validation method assessment of ADA in clinical serum samples

We developed a homogeneous bridging anti-drug antibody (ADA) assay on an electro chemiluminescent immunoassay (ECLIA) platform to support the immunogenicity evaluation of a dimeric domain antibody (dAb) therapeutic in clinical studies. During method development we evaluated the impact of different types of acid at various pH levels on polyclonal and monoclonal ADA controls of differing affinities and on/off rates. The data shows for the first time that acids of different pH can have a differential effect on ADA of various affinities and this in turn impacts assay sensitivity and drug tolerance as defined by these surrogate controls. Acid treatment led to a reduction in signal of intermediate and low affinity ADA, but not high affinity or polyclonal ADA. We also found that acid pretreatment is a requisite for dissociation of drug bound high affinity ADA, but not for low affinity ADA-drug complexes. Although we were unable to identify an acid that would allow a 100% retrieval of ADA signal post-treatment, use of glycine pH3.0 enabled the detection of low, intermediate and high affinity antibodies (Abs) to various extents. Following optimization, the ADA assay method was validated for clinical sample analysis. Consistencies within various parameters of the clinical data such as dose dependent increases in ADA rates and titers were observed, indicating a reliable ADA method. Pre- and post-treatment ADA negative or positive clinical samples without detectable drug were reanalyzed in the absence of acid treatment or presence of added exogenous drug respectively to further assess the effectiveness of the final acid treatment procedure. The overall ADA results indicate that assay conditions developed and validated based on surrogate controls sufficed to provide a reliable clinical data set. The effect of low pH acid treatment on possible pre-existing ADA or soluble multimeric target in normal human serum was also evaluated, and preliminary data indicate that acid type and pH also affect drug-specific signal differentially in individual samples. The results presented here represent the most extensive analyses to date on acid treatment of a wide range of ADA affinities to explore sensitivity and drug tolerance issues. They have led to a refinement of our current best practices for ADA method development and provide a depth of data to interrogate low pH mediated immune complex dissociation.

Development and characterization of antibody reagents to assess anti-PEG IgG antibodies in clinical samples

BACKGROUND Polyethylene glycol (PEG) is a polymer that can be conjugated with therapeutic proteins. Monitoring anti-PEG antibodies in human subjects may be required as part of immunogenicity assessment. The lack of well-characterized anti-PEG reagents have limited our understanding of anti-PEG humoral response. RESULTS Antibodies reactive to PEG were engineered with a human IgG1 Fc. Surface plasmon resonance and plate-based methods demonstrated that their binding was dependent on molecular weight (MW) of PEG. Specificity experiments using chemical analogs identified their specificity. CONCLUSION Affinity, specificity and MW of PEG are critical characteristics that impact interactions of anti-PEG antibodies with PEG. These attributes especially MW of PEG and the assay formats may impact the ability to detect anti-PEG antibodies.

Challenges and opportunities in bioanalytical support for gene therapy medicinal product development

Gene and nucleic acid therapies have demonstrated patient benefits to address unmet medical needs. Beside considerations regarding the biological nature of the gene therapy, the quality of bioanalytical methods plays an important role in ensuring the success of these novel therapies. Inconsistent approaches among bioanalytical labs during preclinical and clinical phases have been observed. There are many underlying reasons for this inconsistency. Various platforms and reagents used in quantitative methods, lacking of detailed regulatory guidance on method validation and uncertainty of immunogenicity strategy in supporting gene therapy may all be influential. This review summarizes recent practices and considerations in bioanalytical support of pharmacokinetics/pharmacodynamics and immunogenicity evaluations in gene therapy development with insight into method design, development and validations.

Bead-extraction and heat-dissociation (BEHD): A novel way to overcome drug and matrix interference in immunogenicity testing

Biological therapeutics are foreign antigens and can potentially induce immune response resulting in the formation of anti-drug antibodies (ADA), which in turn may lead to a wide range of side effects. Neutralizing Ab (NAb) is a subset of ADA that can bind to the pharmacological activity regions of therapeutic to inhibit or complete neutralize its clinical efficacy. A cell-based functional NAb assay is preferred to characterize its neutralization activity. However, cell-based NAb assays are often vulnerable to drug interference, as well as interference from numerous serum factors, including but not limited to growth factors and disease-related cytokines. Bead Extraction with Acid Dissociation (BEAD) has been successfully applied to remove circulating drug and/or other interfering factors from human serum samples, thereby enriching for ADA/NAb. However, the harsh acid used in the extraction procedure can cause irreversible denaturing of NAb and lead to underestimated NAb measurement. Herein we describe a new approach when acid-dissociation is not optimal for a PEGylated domain antibody (Ab). We further demonstrate that heating at 62 °C can not only dissociate drug/ADA/NAb immune complex but also selectively and irreversibly denature domain Ab drug due to much lower thermal stability of the domain Ab, when compared to that of full antibodies. The irreversible denaturing of the drug favors the formation of an immune complex between ADA/NAb and the added biotinylated drug thus increasing the recovery of ADA/NAb from samples. We call this new procedure Bead Extraction with Heat Dissociation (BEHD), which can potentially be applied to other NAb assays that have poor compatibility with acid dissociation.

Development and validation of a functional cell-based neutralizing antibody assay for ipilimumab

Ipilimumab is the first US FDA-approved immune checkpoint-blocking antibody drug to harness the patients own immune cells. One of the postmarketing requirements is to develop a cell-based neutralizing antibody assay. Here, we share some of the most challenging aspects encountered during the assay development: new cell line construction; an unexpected inhibition of T-cell activation by low concentrations of ipilimumab; and two issues caused by sample pretreatment with acid dissociation to overcome drug interference: instability of neutralizing antibody positive control at low pH, and incompatibility of commonly used acid dissociation buffers in the cell assay. After troubleshooting and optimization, we successfully validated the assay and used the assay to test clinical samples to date.