Undestanding Neutralizing Antibody Assays

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Neutralizing Antibody Assay, NAb Assay Service

With the development of many biologics as therapeutics, immunogenicity assessment is becoming an important consideration for patient safety and product efficacy. Among the immunogenicity assessment, Neutralization Antibody Assays (NAb Assay) has emerged as an indispensable tool, offering nuanced insights into the impact of anti-drug neutralizing antibodies (NAbs). This comprehensive exploration delves into the intricacies of both cell-based and non-cell-based NAb Assays.

Biological therapeutics, including proteins, peptides, nucleic acids, and carbohydrates, can induce immune responses, leading to the formation of anti-drug antibodies (ADAs). While ADAs may not always result in adverse events, they can impact the pharmacological and pharmacokinetic properties of the therapeutic. This includes hypersensitivity reactions, immunologic cross-reactivity, reduced drug bioavailability, and neutralization of drug activity. The severity of the immune response influences the risk/benefit balance for the therapeutic.

Advancements in understanding immune responses to biologics highlight the importance of assessing ADA impact on drug safety, exposure, and pharmacological activity. Immunogenicity assays are designed to detect polyclonal antibody responses, employing a two-step strategy involving screening and confirmatory immunoassays (detecting binding ADAs) and subsequent assays for ADA characterization. NAb assays, conducted through cell-based or non-cell-based methods, assess the impact of detected ADAs on drug biological activity.

While, regulatory guidance with recommendations outlined by agencies like EMEA and FDA for validation of immunogenicity assays are limited, white papers have provided additional insights. Based on these recommendations, the approach by NorthEast Biolab for validating NAb assays, addressing assay performance characteristics and best practices for qualitative or quasi-quantitative assays are outlined below.

Strategy for Selecting NAb Assay Formats:

Neutralizing antibodies are associated with potential impact to the overall risk/benefit assessment of the biotherapeutic:

Potential efficacy impact in all biotherapeutics
Potential safety impact for molecules with endogenous counterparts, neutralizing both the biotherapeutic and the endogenous molecules
Neutralization of endogenous molecules can be life-threatening depending on the uniqueness of function and the biological effects.

The preference for the cell-based format is based on assessing the entirety of the biological action rather than a subset of interactions that are neutralizing. For example: Binding to the receptor may be necessary for the biological function, but it may not be sufficient. There may be a structural change(s) required for downstream biology that other NAb epitopes could affect.

Scientific basis for NAb assay format selection:

Risk Based Assessment
Mechanism of Action
Assay performance

The Risk-based approach classifies biotherapeutics into high, medium, and low-risk categories, it may not be ideal for selecting the NAb assay format. For instance, high-risk biotherapeutics like cytokines and growth factors may benefit from cell-based assays, but this approach may not be suitable for all types of biotherapeutics.

Therapeutic Mechanism of Action (MoA)-driven approach is more effective in guiding the selection of the NAb assay format for various biotherapeutics. For instance, high-risk biotherapeutics like cytokines and growth factors may benefit from cell-based assays, but this approach may not be suitable for all types of biotherapeutics. The therapeutic MoA is a better determinant for choosing between cell-based and non-cell-based NAb assays, especially considering factors like process-related impurities, patient immune status, genetic background, and route of therapeutic administration. The ultimate goal is to select an assay format that reflects the drug’s biological function in vivo to accurately detect NAb activity. Other additional things to consider are assay sensitivity, matrix interference (specificity and selectivity), drug tolerance, reactivity of cells to other sera components including soluble target, assay reproducibility and relevance of cell line receptor expression.

Following is the guidance for determination of NAb assay format based of Mechanism of action (MoA):

MoA	Drug Modality	Drug Target	Drug-target Interaction	Recommended Assay Format
Agonist	Recombinant protein or antibody	Cellular receptor	Drug binds and activates receptor	Cell-based assay as primary choice, non-cell-based assay as an alternative
Antagonist	Monoclonal Antibody	Humoral target	Drug binds and inhibits the target	Non-Cell based assay
	Monoclonal Antibody	Cellular receptor	Drug binds cellular receptor and competitively inhibits receptor-ligand interaction	Cell-based assay or non-cell-based assay
	Soluble receptor	Ligand	Soluble receptor binds ligand and blocks receptor-ligand interaction	Non-cell based Competitive LBA assay recommended; cell-based assay possible with a suitable cell line
Targeted intra-cellular delivery of a potent cytotoxin mediated by antibody	ADC	Cellular receptor	ADC binds the cellular receptor and mediates the internalization of payload	Cell-based assay
Target cell lysis through antibody effector function	Monoclonal antibody	Target cell receptor, FcγR or complement	Antibody binds to target cell receptor through variable region and FcγR or complement through Fc domain	Cell-based effector assay recommended, cell-based binding assay or non-cell based CLB assay acceptable with justification
Enzyme replacement	Enzyme	Replace deficient protein in circulation or in target cells; may need cellular receptor for enzyme uptake	Enzyme functions in circulation or through cellular uptake	Enzyme bioactivity assay and/or cell-based assay; two assays may be needed

As illustrated in the below figure, Direct and indirect formats for cell-based Neutralizing Antibody (NAb) assays are distinguished based on the biotherapeutic’s direct influence on cell response.

Direct Cell-Based Assay:

This format utilizes a cell line expressing the target receptor and measures the response induced by the direct interaction between the drug and its receptor. NAb interferes with this interaction, hindering drug function and thereby diminishing the assay response.

Agonist: Direct Cell Based Assay

DrugTarget
ReceptorNo Nab

Signal

DrugNo PresentTarget
ReceptorNab

Signal

Indirect Cell-Based Assay:

This format, requiring drug, a suitable cell line, and often an additional component like a ligand binding to the cellular receptor to generates a response. The ligand-receptor interaction creates the assay response, but the drug, neutralizing the ligand, decreases this response. NAb obstructs drug function, restoring the assay response.

Antagonist: Indirect Cell Based Assay

TargetTarget ReceptorNo Nab

Signal

DrugDrugNabTargetTarget ReceptorNo NabNo Present

Signal

A Harmonized future for Neutralization Antibody Assay (Nb Assay):

NorthEast Biolab’s commitment to bioanalytical excellence finds expression in our approach to NAb Assays. Harmonizing insights from regulatory guidance by agencies like EMEA and FDA, and white papers, NAb assays developed and validated should align seamlessly with evolving regulatory expectations and industry standards. Join us in unraveling the complexities of NAb Assays and shaping the future of therapeutic innovation.

References

Wu B, Chung S, Jiang XR, McNally J, Pedras-Vasconcelos J, Pillutla R, White JT, Xu Y, Gupta S. Strategies to Determine Assay Format for the Assessment of Neutralizing Antibody Responses to Biotherapeutics. AAPS J. 2016 Nov;18(6):1335-1350. doi: 10.1208/s12248-016-9954-6. Epub 2016 Aug 5. PMID: 27495119.

Gupta S, Devanarayan V, Finco D, Gunn GR 3rd, Kirshner S, Richards S, Rup B, Song A, Subramanyam M. Recommendations for the validation of cell-based assays used for the detection of neutralizing antibody immune responses elicited against biological therapeutics. J Pharm Biomed Anal. 2011 Jul 15;55(5):878-88. doi: 10.1016/j.jpba.2011.03.038. Epub 2011 Apr 6. PMID: 21531522.

United States Pharmacopeia (2023). General Chapter, 〈1106〉 Immunogenicity Assays—Design and Validation of Immunoassays to Detect Anti-Drug Antibodies. USP-NF. Rockville, MD: United States Pharmacopeia.

Immunogenicity Testing of Therapeutic Protein Products —Developing and Validating Assays for Anti-Drug Antibody Detection FEBRUARY 2019