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
DrugNAb PresentTarget
ReceptorNAb
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
DrugDrugNabTargetTarget ReceptorNo NAbNAb Present
A Harmonized future for Neutralization Antibody Assay (NAb 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