Anti-drug Antibody Sample Testing and Reporting Harmonization

Statement of Compliance

While there is no formal regulatory guidance on the conduct of ADA testing, it is generally performed under the principles of GLP/GCP. It is recommended to include, at minimum, a statement regarding which guidelines were followed (e.g., GLP/GCP, sample analysis plan, relevant site SOPs) as well the extent of QC or Quality Assurance (QA) review. If QA review is conducted, a separate statement with specific details of the QA audit and associated signatures should be included.

Summary

The report summary provides a short review of the study information and ADA sample analysis. The summary includes information related to the drug name, study name and study number, study population and/or indication, as well as the method and its qualification or validation status, and study sample matrix. The number of samples received and analyzed, as well as the total number of samples that were positive in the screening and the confirmatory assays (if performed) for each clinical study, should be provided. The detection of any antibodies present prior to treatment (pre-existing antibodies) should be noted. The presence of pre-existing antibodies requires a careful assessment of post-treatment response (titer) to understand whether administration of the study drug led to a treatment-emergent (boosted) response. This interpretation is considered out of scope for the ADA BAR and should be included in the appropriate section of the CSR or ISI.

The format for reporting titer should clearly state the total final dilution applied to the sample, especially in the case that dilution beyond the assay minimum required dilution (MRD) is required. For example, if a dilution of 1:10 was applied during sample analysis (this being in addition to the assay MRD of 1:5), the final reported sample titer would be 1:50 (2019 FDA Guidance). If additional processing of samples is required, such as with acid dissociation, these dilutions should be accounted for in the MRD and final reported titer. Detailed examples of determination of MRD are provided in the Anti-Drug Antibody Validation Testing and Reporting Harmonization guidance (1).

The summary section may also provide details of sample characterization, such as isotype, domain specificity testing and any additional characterization(s) required due to specifics of the drug modality, such as bispecific molecules or antibody drug conjugate (ADC) drugs (1).

Bioanalytical Method

To properly evaluate the reported results, it is recommended that a brief summary of the study drug (biotherapeutic, gene therapy, ADC, monoclonal antibody therapy, etc.) and the method used for sample analysis be provided. The method summary can be presented in a tabular format and include information about assay platform (colorimetric vs. electrochemiluminescence) and assay format (sandwich, solution-based bridging, or direct/indirect assay). Any requirement for pre-treatment of samples, such as acid dissociation or immunoadsorption, added preservatives or enzyme inhibitors, and the associated data reduction required (raw response or normalized response) should be described. Additionally, method details and validation parameters should be summarized in a tabular format as noted in Section 5 of the example report template (Supplementary Material). This summary table should include a clear reference or link to the bioanalytical method and/or validation report for cross-referencing. The method summary table should also briefly list the study drug and sample matrix as well as a description of the positive control antibody recommended, including clonality, host and target species, and the final concentration for each positive control as determined in neat matrix.

System suitability controls such as negative, low, and high positive controls from each run are included to evaluate assay performance. During pre-study method development and validation, the low positive control should have been selected with an appropriate targeted failure rate to guarantee consistent sensitivity in-study (during the duration of sample analysis supporting the current study), (1, 5, 6). The MRD should be clearly defined as per industry-wide accepted guidance and be taken into consideration in the final reported dilution (titer or S/N) of the sample (1, 6, 7).

Critical validated method parameters, including screening and confirmatory cut point (and titration cut point, if applicable), assay sensitivity, drug tolerance, and target interference (in both screen and confirmatory assay tiers), matrix effect (or selectivity), and effect of hemolysis/lipemia on any of these parameters, should be summarized, if applicable (see Template Table 5-1). Multi-domain therapeutics that require assessment of more than one cut point or domain specificity assessment should be included. If any assay parameters (for example, cut point factor) changed in-study, these should be noted along with the original validated value.

Materials

The materials section should include a description of critical reagents and equipment and software used during sample analysis in tabular format as presented in Section 6 of the template.

Critical Reagents

Materials that are key to the performance of the method are considered critical reagents (8, 9). These include, but are not limited to, the study drug, positive controls, custom-labeled drug or detection reagent, and negative control matrix. All critical reagents should be listed in a table that includes available reagent description, reagent source, lot and catalog number, preparation date, concentration, expiration and/or retest date, and appropriate storage conditions. Documentation such as certificates of analysis for the drug substance or drug product or reagents unique to the assay, are not required, but may be included as an appendix to the report (10).

Positive Control Antibodies

A purified antibody with a defined concentration is preferable for the consistent evaluation of the surrogate positive control. In the case that the positive control antibody is not purified (such as with antibodies directed against toxin or anti-sera), any available information related to the positive control purity should be provided. A description of the positive control antibody clonality (monoclonal versus polyclonal), host and target species, and storage condition should be included.

Matrix and Controls

The negative control is a critical reagent as all sample results are evaluated against the assay cut point. Given the possible presence of interfering factors such as endogenous target, or the presence of a high false-positive individual within the negative control pool, the overall negative control results may be skewed by a non-representative sample. Therefore, the information from the individual lots that comprise the validated negative control pool should be summarized. This includes any assays needed for bridging the negative control pools used during sample analysis that are different from those used in method validation (e.g., when the original validated negative control pool is expended) (1). Therefore, it is recommended that a reference be provided to any documentation (e.g. notebook pages, report number) summarizing additional assays related to new negative control pools used during sample analysis.

The final qualified matrix pool used as the negative control should be included in Template Table 6.1. If the negative control pool is prepared from multiple individual matrix lots, the lot numbers of the individual matrix samples should be documented and retained in validation or study-related folders with the study records for cross reference, and therefore do not need to be individually listed in Table 6.1.

Equipment & Software

The equipment and software section should include the major equipment (e.g., plate reader, plate washer) and software only. Major equipment is critical to the performance of the assay, and is dependent on the assay being performed, and therefore does not include minor equipment such as freezers and incubators, used during sample analysis. If all equipment and software used are the same as those used in validation, this should be noted in the report for expedience and ease of reference. Ensure that specific equipment identification numbers are included to allow for study reconstruction. See Template, Section 6.2 (Supplementary Material).

Data Analysis

Describe the software used for the analysis of the results as well as a brief description of how the data were analyzed.

Data analysis should be performed and briefly described in accordance with the recommended tiered analysis including screening, confirmatory analysis, and characterization as described in Shankar (6) and Devanarayan (11) (Fig. 1). Any replicate outlier analysis, such as Grubb’s outlier analysis that was used in the assessment of positive or negative control samples should also be briefly described here. If an in-study cut point is required, provide a brief explanation in this section, including observed false-positive rate and data transformation. The details of in-study cut point calculation is discussed in the “Results” section.

Fig. 1figure 1

Tiered analysis for reporting of immunogenicity results (assumes false- positive rate is reflective of validation cut point population)

Sample results must be reported in a consistent format that reflects how they were analyzed. For example, in cases where data are reported as raw responses such as ECL units (relative luminescence units or RLU), all statistical evaluations (e.g., Std. Dev.) must be performed with raw data RLU numbers. In cases where data are reported as a signal to noise (S/N) ratio, all assay evaluations must be performed using the S/N ratio.

A statement that describes calculation of results taking place prior to rounding of numbers, as well as designating the total number of significant figures to be reported should be included. In addition, a brief description of how the data were transferred to the sponsor and/or clinical database (e.g., according to specifications listed in the Data Transfer Agreement) may be included, if applicable. This clarifies why manual calculations from displayed values may differ in the final significant figure from the original value.

Study Sample Receipt and Storage

Sample receipt and storage of samples is summarized in Table 8-1. Supportive documentation does not need to be included within the report, but should be referenced and stored with study records for sample tracking purposes. This may include additional documentation such as sample manifests, temperature data logger information, and sample reconciliation, as available.

A brief description of the condition of samples upon receipt as well as their storage and any temperature excursions is critical. For sample tracking, it is recommended to include sample identification number and/or accession number, and time point or visit information (as provided in the protocol, laboratory manual, or data transfer agreement documentation). Samples that were received in poor condition, had a temperature excursion, or an unreconciled discrepancy should be noted. If backup sample aliquots are provided, ensure the correct vial identification number and/or accession number is reported, especially in the case that the original and backup vials were required during analysis. If investigational sample discrepancies have been resolved, a statement should be included to reflect that no further investigation is required.

Sample laboratory origination locations, dates of receipt, and study center of origin are not necessary, assuming there are systems in place to ensure chain of custody through the assigned sample identification number.

As antibodies have proven to typically be stable for several years when stored frozen (12, 13), long-term stability is not always performed for anti-drug antibodies in study samples. Information on any excursions from established process stability (freeze-thaw cycles, benchtop, or refrigerated storage) should be described.

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