Strategy for Selecting NAb Assay Formats:
NAb assay development is crucial in assessing the potential impact of biotherapeutics.
- 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 their uniqueness of function and biological effects. Thus, a neutralizing antibody test is essential for evaluating safety profiles comprehensively.
The preference for the cell-based format for NAb assay development is based on assessing the entirety of the biological action rather than a subset of neutralizing interactions. For example, Binding to the receptor may be necessary for the biological function, but it may not be sufficient. A structural change(s) may be required for downstream biology that other NAb analysis epitopes could affect.
The scientific basis for Neutralizing Antibody Assay format selection:
- Risk Based Assessment
- Mechanism of Action
- Assay performance
In NAb assay development, selecting the appropriate assay format is crucial for accurately assessing the presence of neutralizing antibodies (NAbs) against biotherapeutics. The Risk-based approach classifies biotherapeutics into high, medium, and low-risk categories; it may not be ideal for selecting the NAb analysis format. For instance, high-risk biotherapeutics like cytokines and growth factors may benefit from cell-based assays, but this approach may not suit all biotherapeutics.
The therapeutic Mechanism of Action (MoA)-driven approach is more effective in guiding the selection of the NAb analysis format for various biotherapeutics. For instance, high-risk biotherapeutics like cytokines and growth factors may benefit from cell based NAb assays, but this approach may not suit all biotherapeutics. Cell based neutralizing antibody assay development requires consideration of multiple factors, including process-related impurities and patient immune status. Similarly, neutralizing antibody assay validation is essential to ensure accuracy and reliability. Neutralizing antibody assay development should consider factors such as process-related impurities, patient immune status, genetic background, and route of therapeutic administration. The therapeutic MoA is a better determinant for choosing between cell based NAb assays and non-cell-based NAb assays, especially considering factors like process-related impurities, patient immune status, genetic background, and route of therapeutic administration. In the quest to accurately detect neutralizing antibody (NAb) activity, selecting an assay format is paramount to mirror the drug’s biological function in vivo. One commonly employed method is the neutralizing antibody titer assay, designed to gauge the potency of NAbs effectively. However, beyond mere selection, other crucial factors warrant consideration in neutralizing antibody testing. For instance, the NAb analysis’s sensitivity ensures reliable detection even at low NAb concentrations. Matrix interference, including specificity and selectivity against background components, is vital to maintain assay integrity. Moreover, assessing drug tolerance within the assay framework is crucial to avoid false positives or negatives. Additionally, the reactivity of cells to other sera components, including soluble targets, necessitates meticulous scrutiny to prevent erroneous interpretations. Ensuring the neutralizing antibody titer assay reproducibility is indispensable for consistent and reliable results across multiple tests. Lastly, the relevance of cell line receptor expression is pivotal, as it directly influences the assay’s ability to reflect in vivo interactions accurately.
These factors collectively contribute to developing robust and clinically relevant neutralizing antibody testing protocols.
Following is the guidance for the determination of the NAb analysis format based on 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 |
The cell based NAb assay is the 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 NAb 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 neutralizing antibody 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:
In NAb assay development, this format utilizes a cell line expressing the target receptor and measures the response induced by the drug’s direct interaction with its receptor. NAb interferes with this interaction, hindering drug function and diminishing the assay response. The neutralizing antibody test plays a critical role in evaluating the efficacy of therapeutic interventions, particularly in immunotherapy and biologics.
Agonist: Direct Cell Based Assay
DrugTarget
ReceptorNo NAb
Signal
DrugNAb PresentTarget
ReceptorNAb
No Signal
Indirect Cell-Based Assay:
This format requires a drug, a suitable cell line, and often an additional component like a ligand binding to the cellular receptor to generate 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 NAbNAb Present
No Signal
A Harmonized Future For Neutralizing Antibody Assay (NAb Assay):
NorthEast BioLab’s commitment to bioanalytical excellence manifests in our approach to NAb analysis. We specialize in neutralizing antibody assay development and neutralizing antibody assay validation processes of cell based neutralizing antibody assays, ensuring the accuracy and reliability of our tests. Harmonizing insights from regulatory guidance by agencies like EMEA and FDA, and white papers, we ensure our neutralizing antibody tests are developed and validated to seamlessly align with evolving regulatory expectations and industry standards. Our team is committed to the rigorous process of neutralizing antibody assay development and neutralizing antibody assay validation to provide our clients with high-quality data for therapeutic innovation. Join us in unraveling the complexities of NAb analysis 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