Cost-Effective NAb Assay Development: Techniques and Considerations
Many drug products, such as biosimilars, monoclonal antibodies, and growth factors, are large therapeutic molecules. These molecules have the risk of eliciting unwanted immune responses in the patient population. Factors affecting this immunogenicity drug profile include the manufacturing process, purity and size of the protein, health status of patients, and much more. Unwanted immunogenicity causes the formation of anti-drug antibodies and has a broad spectrum of clinical outcomes due to altered drug safety, efficacy, or pharmacokinetic profile. Neutralizing antibodies are a subset of anti-drug antibodies that bind to the drug product and alter its biological activity. Besides, neutralizing antibodies (NAb) may also react with endogenous analytes. Hence, bioanalytical laboratories such as LC-MS/MS service providers are increasingly focusing on immunogenicity assay development for identifying neutralizing anti-drug antibodies.
Several regulatory agencies have guidance documents for developing sensitive and valid immunogenicity assays as part of drug discovery and testing. The current article discusses NAb assay development and different techniques and considerations for cost-effective NAb assays.
Neutralizing antibody assays
Although standard radioimmunoprecipitation and ELISA assays are usually used to detect anti-drug antibodies, they fail to discriminate between antibody-drug interactions. Hence, detecting neutralizing antibodies needs specialized in vitro cell or non-cell-based assays. Both cell-based and non-cell-based ligand binding assays are vital in NAb assay development. However, cell-based assays are often recommended as they closely mimic in vivo biological mechanisms.
Each NAb assay has unique requirements that rely on the type of drug product, study population, and phase of study. Whether to use a direct or indirect NAb assay often depends on the mechanism of drug action. Although most NAb assays use a 96-well plate, they can be easily adapted for automated and high throughput experiments.
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The crucial steps of a cell-based neutralizing antibody assay include;
- Selecting an ideal cell line
- Choosing a suitable cellular response
- Identifying proper controls
- Optimizing assay parameters
- Validating assay method
The two main types of NAb assays, direct and indirect, are based on the mechanism of drug action and assay endpoints.
Direct NAb assays are generally for drug products that directly affect a cell, stimulating different responses, such as an increase in cytoplasmic ATP and receptor phosphorylation. In the absence of neutralizing antibodies, a drug product will bind to receptors on cell surfaces and induce a cellular response. On the other hand, in the presence of neutralizing antibodies, the cellular response is either ended or decreased.
Indirect assays are for drug products that block ligand binding to cell surface receptors, resulting in the absence or reduction of a cellular response. In the presence of neutralizing antibodies, they bind to the drug product and prevent it from attaching to cell receptors. Hence, the ligand binds to its promoter and induces specific cellular responses.
Today, a multi-tiered approach is often recommended for immunogenicity testing. Reactive study samples during NAb testing are further verioss-linfied through confirmatory assays. Removal of neutralizing antibodies using different depletion techniques, such as crking to bead agarose or chromatography, is recommended. However, as most biotherapeutics are monoclonal antibodies, they will bind to agarose beads and hence require a different strategy.