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Immunogenicity is a major challenge for protein therapeutics which can potentially reduce drug efficacy and safety and is often being monitored by anti-drug antibody (ADA) and neutralizing antibody (NAb) assays. Circulating targets and residual drugs in matrices can have significant impacts on accuracy of results from ADA and NAb assays, and sufficient drug and target tolerance for these assays are necessary. Here, we report the development of a competitive ligand binding (CLB) NAb assay for an anti-TFPI (tissue factor pathway inhibitor) monoclonal antibody (PF-06741086) with high drug and target tolerance to support ongoing clinical studies. A double acid affinity capture elution approach was used to mitigate drug interference, and a robust target removal strategy was employed to enhance target tolerance. The validated NAb assay has sensitivity of 313 ng/mL, drug tolerance of 50 μg/mL, and target tolerance of 1200 ng/mL. A step-by-step tutorial of assay development is described in this manuscript along with the rationale for using a high drug/target tolerant NAb assay. The NAb assay cut point factor obtained was 0.78. Other assay performance characteristics, e.g., precision and selectivity, are also discussed. This validated method demonstrated a superior drug and target tolerance to warrant specific and precise characterization of the NAb responses in support of ongoing clinical studies.
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Name: The AAPS journal
Discrimination of Anti-drug Antibodies With Neutralizing Capacity in Infliximab- and Adalimumab-Treated Patients: Comparison of the Homogeneous Mobility Shift Assay and the Affinity Capture and Elution Assay.
The measurement of anti-drug antibody (ADA) levels in adalimumab (ADAL)-treated and infliximab (IFX)-treated patients is critical for guiding therapeutic strategies. The homogeneous mobility shift ass...
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Immunochemical analysis which measures specific target antigen bound to antibody complex. Antibodies in assay form insoluble light scattering immune-aggregates which cause changes in the measured turbidity proportional to the concentration of the antigen.
A screening assay for circulating COMPLEMENT PROTEINS. Diluted SERUM samples are added to antibody-coated ERYTHROCYTES and the percentage of cell lysis is measured. The values are expressed by the so called CH50, in HEMOLYTIC COMPLEMENT units per milliliter, which is the dilution of serum required to lyse 50 percent of the erythrocytes in the assay.
The phenomenon of antibody-mediated target cell destruction by non-sensitized effector cells. The identity of the target cell varies, but it must possess surface IMMUNOGLOBULIN G whose Fc portion is intact. The effector cell is a "killer" cell possessing Fc receptors. It may be a lymphocyte lacking conventional B- or T-cell markers, or a monocyte, macrophage, or polynuclear leukocyte, depending on the identity of the target cell. The reaction is complement-independent.
Classic quantitative assay for detection of antigen-antibody reactions using a radioactively labeled substance (radioligand) either directly or indirectly to measure the binding of the unlabeled substance to a specific antibody or other receptor system. Non-immunogenic substances (e.g., haptens) can be measured if coupled to larger carrier proteins (e.g., bovine gamma-globulin or human serum albumin) capable of inducing antibody formation.
An immunoassay utilizing an antibody labeled with an enzyme marker such as horseradish peroxidase. While either the enzyme or the antibody is bound to an immunosorbent substrate, they both retain their biologic activity; the change in enzyme activity as a result of the enzyme-antibody-antigen reaction is proportional to the concentration of the antigen and can be measured spectrophotometrically or with the naked eye. Many variations of the method have been developed.
An antibody is a protein produced by the body's immune system when it detects harmful substances, called antigens. Examples of antigens include microorganisms (such as bacteria, fungi, parasites, and viruses) and chemicals. Antibodies may be produc...
An assay is an analytic procedure for qualitatively assessing or quantitatively measuring the presence or amount or the functional activity of a target entity. This can be a drug or biochemical substance or a cell in an organism or organic sample. ...
Clinical Approvals Clinical Trials Drug Approvals Drug Delivery Drug Discovery Generics Drugs Prescription Drugs In the fields of medicine, biotechnology and pharmacology, drug discovery is the process by which drugs are dis...