FDA刚刚发布《治疗性蛋白质产品的免疫原性测试 - 开发和验证抗药物抗体检测方法行业指南》
This guidance provides recommendations to facilitate industry’s development and validation of assays for assessment of the immunogenicity of therapeutic protein products during clinical trials. Specifically, this document includes guidance regarding the development and validation of screening assays, confirmatory assays, titration assays, and neutralization assays.2,3 For the purposes of this guidance, immunogenicity is defined as the propensity of a therapeutic protein product to generate immune responses to itself and to related proteins or to induce immunologically related adverse clinical events. The recommendations for assay development and validation provided in this document apply to assays for the detection of one or more antidrug antibodies (ADAs).4 This guidance may also apply to some peptides, oligonucleotides, and combination products on a case-by-case basis.5
In general, this document does not discuss the rationale for ADA testing or the subject- and product-specific risk factors that may contribute to immunogenicity.6 Also, this guidance, including any discussions of terminology used in this guidance, does not apply to in vitro diagnostic products.7
In general, FDA’s guidance documents do not establish legally enforceable responsibilities. Instead, guidances describe the Agency’s current thinking on a topic and should be viewed only as recommendations, unless specific regulatory or statutory requirements are cited. The use of the word should in Agency guidances means that something is suggested or recommended, but not required.
Immune responses to therapeutic protein products have the potential to affect product pharmacokinetics, pharmacodynamics, safety, and efficacy.8 The clinical effects of immune responses in subjects are highly variable, ranging from no measurable effect to extremely harmful. Detection and analysis of ADA formation is a helpful tool in understanding potential immune responses. Information on immune responses observed during clinical trials, particularly the incidence of ADA induction or any implications of ADA responses affecting pharmacokinetics, pharmacodynamics, safety, or efficacy, is crucial for any therapeutic protein product development program. Accordingly, such information, if applicable, should be included in the prescribing information as a subsection of the ADVERSE REACTIONS section entitled Immunogenicity.9 Therefore, the development of valid, sensitive, specific, and selective assays to measure ADA responses is a key aspect of therapeutic protein product development.
III. GENERAL PRINCIPLES
The risk to subjects from mounting an ADA-generating immune response to a therapeutic protein product will vary with the product. FDA recommends adopting a risk-based approach to evaluating and managing immune responses to — or immunologically related adverse clinical events associated with — therapeutic protein products that affect their pharmacokinetics, pharmacodynamics, safety, and efficacy.10 Immunogenicity tests should be designed to detect ADA that could mediate unwanted biological or physiological consequences such as neutralizing activity or hypersensitivity responses.
A. Assays for ADA Detection
Screening assays, also known as binding antibody assays, are used to detect antibodies that bind to the therapeutic protein product. The specificity of ADA for the therapeutic protein product is usually established by competition with a therapeutic protein in a confirmatory assay. ADAs are characterized further using titration and neutralization assays. Titration assays characterize the magnitude of the ADA response. It is important to characterize this magnitude with titration assays because the impact of ADA on pharmacokinetics, pharmacodynamics, safety, and efficacy may correlate with ADA titer and persistence rather than incidence (Cohen and Rivera 2010). Neutralizing antibodies (NAbs) refer to those ADA with the ability to interfere with interactions between the therapeutic protein product and its target. Neutralization assays assess ADA for neutralizing activity. It is important to characterize neutralizing activity of ADA because the impact of ADA on pharmacokinetics, pharmacodynamics, safety, and efficacy may correlate with NAb activity rather than ADA incidence (Calabresi et al. 2007; Goodin et al. 2007; Cohen and Rivera 2010; Wang et al. 2016; Wu et al. 2016). Similarly, in some cases it may be useful to establish NAb titers in addition to NAb qualitative results (for example, positive or negative), depending on immunogenicity risk assessment. Additional characterization assays, including isotyping, epitope mapping, and assessing cross-reactivity (for example, to endogenous counterparts or to other products), may be useful.
The optimal time to design, develop, and validate ADA assays during therapeutic protein product development depends on the risk assessment of the product (Mire-Sluis et al. 2004; Gupta et al. 2007; Shankar et al. 2008; Gupta et al. 2011). The sponsor should provide an immunogenicity risk assessment as well as a rationale for the immunogenicity testing paradigm in the original investigational new drug application (IND). FDA encourages sponsors to test samples during phase 1 and phase 2 studies using suitable screening, confirmatory, and in some instances neutralization assays. Samples derived from pivotal clinical studies should be tested with fully validated assays.11 When immunogenicity poses a high clinical risk and real-time data concerning subject responses are needed (for example, when there is an endogenous counterpart with non-redundant function), FDA may request that assays suitable for their intended purpose be developed before initiating clinical studies and that testing be performed in real time. In such instances, timing and reporting of ADA assessment should be discussed with the Agency. In other situations, the sponsor may store subject samples so they can be tested when suitable assays are available. At the time of license application, the sponsor should provide data supporting full validation of the assays (see section VIII). Recommendations regarding the timing of ADA sample collection are provided in section VII.A.12
B. Limitations in Comparing ADA Incidence Across Products
Results from assays for detection of ADA facilitate understanding of the immunogenicity, pharmacokinetics, pharmacodynamics, safety, and efficacy of therapeutic protein products. However, detection of ADA is dependent on key operating parameters of the assays; for example, sensitivity, specificity.13 Although information on ADA incidence is typically included in the prescribing information under an Immunogenicity subsection of the ADVERSE REACTIONS section, FDA cautions that comparison of ADA incidence across products, even for products that share sequence or structural homology, can be misleading because detection of ADA formation is highly dependent on the sensitivity, specificity, and drug tolerance level of the assay. Additionally, the observed incidence of ADA is influenced by multiple factors including method, sample handling, timing of sample collection, concomitant medications, and disease condition. Therefore, comparing immunogenicity rates across therapeutic protein products with structural homology for the same indication is unsound, even though fully validated assays are employed. When a direct comparison of immunogenicity across different therapeutic protein products that have homology — or across similar therapeutic proteins from different sources — is needed, the comparison data should be obtained by conducting a head-to-head clinical study from which samples obtained are tested using an assay demonstrated to have equivalent sensitivity and specificity for antibodies against both therapeutic protein products.
The recommendations on assay development and validation provided in this guidance are based on common issues encountered by the Agency upon review of immunogenicity submissions. Sponsors should contact FDA for any product-specific advice, particularly for high-risk products; for example, products with endogenous counterparts that have non-redundant function.14 Assay designs for isotyping, epitope mapping, and cross-reactivity with endogenous counterparts should be discussed with FDA. Other publications may also be consulted for additional insight (Mire-Sluis et al. 2004; Gupta et al. 2007; Shankar et al. 2008; Gupta et al. 2011).15 In general, FDA recommends that sponsors develop assays that are optimized for sensitivity, specificity, selectivity, drug tolerance, precision, reproducibility, and robustness (see sections IV.C through H).
IV. ASSAY DESIGN ELEMENTS
This section applies to all types of assays for detection of ADA, unless specified otherwise. The bioanalytical scientist should evaluate the applicability of these factors and others based on emerging science. FDA’s thinking on this matter may change as the science evolves.
A. Testing Strategy
1. Multi-Tiered Testing Approach
FDA recommends a multi-tiered ADA testing approach (see Appendix). In this paradigm, a sensitive screening assay is initially used to assess clinical samples. To gain a more accurate understanding of the natural history of the ADA response, the screening assay should be sensitive and designed to detect low levels of low- and high-affinity ADA; for example, by minimizing wash steps. However, in most cases it is not necessary to empirically determine the affinity of antibodies that are detected by the initial screening assay. Samples testing positive in the screening assay are then subjected to a confirmatory assay to demonstrate that ADAs are specific for the therapeutic protein product. For example, a competition assay could confirm that an antibody is specifically binding to the therapeutic protein product and that the positive finding in the screening assay is not a result of non-specific interactions of the test serum or detection reagent with other materials in the assay milieu such as plastic or other proteins.
Samples identified as positive in the confirmatory assay should be further characterized in other assays, such as titration and neutralization assays. In some cases, assays to detect crossreactivity to other proteins, such as the corresponding endogenous protein, may be needed. For example, assessment of cross-reactivity may be needed when the therapeutic protein product belongs to a family of proteins with high homology and it is important to know whether other family members are affected by ADA. Further, in some cases tests to assess the isotype of the antibodies or their epitope specificity may also be recommended once samples containing antibodies are confirmed as positive. Epitope specificity determination of the ADA response is not frequently performed, although it is common to perform a more general assessment of domain specificity for multi-domain products such as pegylated proteins, antibody-drug conjugates, and bispecific antibodies (see section IV.A.3).
2. Immunoglobulin Isotypes or Subtypes
The initial screening assay should be able to detect all relevant immunoglobulin (Ig) isotypes. For non-mucosal routes of administration and in the absence of a risk of anaphylaxis, the relevant ADA isotypes are IgM and IgG. For mucosal routes of administration, IgA isotype ADAs are also relevant.16 Although FDA expects that all relevant isotypes be detected in screening assays, it is not necessary that the screening assay establishes which isotypes are being detected. For example, the bridging assay format can theoretically detect antibodies of most isotypes but does not provide information on which isotypes are being detected.17
In some circumstances the sponsor should develop assays that discriminate between antibody isotypes. For example, for therapeutic protein products where there is a high risk for anaphylaxis or where anaphylaxis has been observed, results from antigen-specific IgE assays may be informative.
Assessment of ADA subtype may be informative in some situations. For example, the generation of IgG4 antibodies has been associated with immune responses generated under conditions of chronic antigen exposure, such as factor VIII treatment, and in erythropoietintreated subjects with pure red cell aplasia (Matsumoto et al. 2001; Aalberse and Schuurman 2002). Consequently, depending on the clinical concern, assessing for specific isotypes or subtypes may be needed.
3. Domain Specificity
Some proteins possess multiple domains that function in different ways to mediate clinical efficacy. An immune response to one domain may inhibit a specific function while leaving others intact. FDA recommends that sponsors direct initial screening and confirmatory tests against the whole therapeutic protein product. For multi-domain therapeutic protein products, the sponsor may need to investigate whether the ADA binds to specific clinically relevant domains in the protein. For example, to adequately understand the risk of ADA to subjects for therapeutic protein products with modifications such as pegylation, sponsors should develop assays to determine the specificity of ADA for the protein component as well as the modification to the therapeutic protein product (Gorovits et al. 2014).
The domain specificity is generally assessed in ADA samples confirmed positive using the whole molecule. Examination of immune responses to therapeutic protein products with multiple functional domains such as bispecific antibodies may require development of multiple assays to measure immune responses to different domains of the molecules (see section IV.L.4).
B. Assay Cut-Point
The cut-point of the assay is the level of response of the assay that defines the sample response as positive or negative. Information specific to establishing the cut-point for the respective assay types is provided in sections V and VI. Establishing the appropriate cut-point is critical to minimizing the risk of false-negative results.
The cut-point of the assay can be influenced by a myriad of interfering product or matrix components.18 These components should be considered early on in assay development when defining the cut-point and are discussed in detail in section IV.K. Because samples from different target populations and disease states may have components that can cause the background signal from the assay to vary, different cut-points may be needed for discrete populations.
Where feasible, the cut-point should be statistically determined using samples from treatmentnaïve subjects.19 By performing replicate assay runs with these samples, the variability of the assay can be estimated. The statistical approach employed to determine the cut-point may entail various processes, such as removing statistical outliers from analyses, and using an approach to account for pre-existing antibodies. During assay development, a small number of samples may be used to estimate the cut-point.
The sponsor should consider the impact of statistically determined outlier values and truepositive samples when establishing the cut-point. The sponsor should provide justification for the removal of any data points, along with the respective method used to determine their status as outliers. Sponsors should consult with FDA if there is a concern regarding the exclusion of outliers.
Apparent positive values and samples may derive from the presence of pre-existing antibodies or other serum factors in subject samples (Ross et al. 1990; Turano et al. 1992; Coutinho et al. 1995; Caruso and Turano 1997; van der Meide and Schellekens 1997; Boes 2000). Although pre-existing antibodies to a variety of endogenous proteins are present in healthy individuals, these can be much higher in some disease states. The sponsor should identify those samples with pre-existing antibodies (for example, through competition with drug) and remove them from the cut-point analysis. If subjects in the study have pre-existing antibodies, it may be necessary to assign positive responses using a cut-point based on the difference between individual subject results before and after exposure to identify subjects in whom ADA increases following treatment, also known as treatment-boosted ADA. A common approach to evaluating treatmentboosted ADA responses is to assess changes in antibody titers. If it is not possible to use the methods described earlier in section IV.B for establishing the cut-point, sponsors should consult with the Agency to explore alternative methods.
1. Assay Sensitivity
Assay sensitivity is the lowest concentration at which the antibody preparation consistently produces either a positive result or a readout equal to the cut-point determined for that assay. The assays should have sufficient sensitivity to enable detection of ADA before they reach levels that can be associated with altered pharmacokinetic (PK), pharmacodynamic (PD), safety, or efficacy profiles. Assay sensitivity is assessed using positive control antibody preparations that may not represent the ADA response in a specific subject. For example, positive controls are frequently developed under conditions that enrich for high affinity antibodies. Such high affinity positive controls may overestimate the sensitivity of the assay. Because of this, the assay sensitivity determination contributes to the overall understanding of how the assay performs rather than setting an absolute mass of ADA that will be detected in any given subject. Because the measurement of assay sensitivity can be affected by onboard drug, it is also important to determine assay sensitivity in the presence of the expected concentration of onboard drug (see section IV.C.2).20 FDA recommends that screening and confirmatory IgG and IgM ADA assays achieve a sensitivity of at least 100 nanograms per milliliter (ng/mL) although a limit of sensitivity greater than 100 ng/mL may be acceptable depending on risk and prior knowledge. Traditionally, FDA has recommended sensitivity of at least 250 to 500 ng/mL. However, recent data suggest that concentrations as low as 100 ng/mL may be associated with clinical events (Plotkin 2010; Zhou et al. 2013). It is understood that neutralization assays may not achieve that level of sensitivity. Assays developed to assess IgE ADA should have sensitivity in the high picograms per milliliter (pg/mL) to low ng/mL range.
The sensitivity should be expressed as mass of antibody detectable/mL of undiluted matrix; for example, plasma, sera, saliva. Assay sensitivity should not be reported as titer. Assay sensitivity should be reported after factoring in the minimal required dilution (MRD). For example, an assay with 50 ng/mL sensitivity and an MRD of 20 would be reported as 1000 ng/mL. Testing of assay sensitivity should be performed with the relevant dilution of the same biological matrix as will be used to test the clinical samples. For example, assay sensitivity should be determined using the same anticoagulant as the diluent used with clinical samples.
During development, sensitivity may be assessed by testing serial dilutions of a positive control antibody of known concentration, using individual or pooled matrix from treatment-naïve subjects. The dilution series should be no greater than two- or threefold, and a minimum of five dilutions should be tested. The sensitivity can be calculated by interpolating the linear portion of the dilution curve to the assay cut-point.
A purified preparation of antibodies specific to the therapeutic protein product should be used as the positive control to determine the sensitivity of the assay so that assay sensitivity can be reported in mass units/mL of matrix. Positive control antibodies used to assess sensitivity can take the form of polyclonal preparations affinity purified against the therapeutic protein product or monoclonal antibodies (mAb).
During routine performance of the assay, a low positive system suitability control should be used to ensure that the sensitivity of the assay is acceptable across assay runs. Additionally, the low positive control should be consistently demonstrated as positive in both screening and confirmatory tiers (see section IV.J.1). Both positive and negative controls are discussed in detail in sections IV.J.1 and IV.J.2.
2. Drug Tolerance, Sensitivity, and Assay Suitability
The therapeutic protein product or its endogenous counterpart present in the serum may interfere with the sensitivity of the assay. The assessment of assay sensitivity in the presence of the expected levels of interfering therapeutic protein product, also known as the assay’s drug tolerance, is critical to understanding the sensitivity and suitability of the method for detecting ADA in dosed subjects.21 FDA recommends that sponsors examine assay drug tolerance early in assay development. The sponsor may examine drug tolerance by deliberately adding different known amounts of positive control antibody into ADA-negative control samples in the absence or presence of different quantities of the therapeutic protein product to determine whether the therapeutic protein product interferes with ADA detection. Results obtained in the absence and presence of different quantities of the therapeutic protein product under consideration should be compared. Drug tolerance may be improved using approaches such as acid dissociation that disrupt circulating ADA-drug complexes. The selectivity of the assay, the nature of the target, and the type of positive control should be taken into consideration when developing the assay because these factors impact the assessment of drug tolerance. For example, acid dissociation may not be appropriate when antibodies are acid labile or the drug target is soluble. Interference from the therapeutic protein product can be minimized by collecting subject samples at trough drug levels. See section VII.A for recommendations regarding the timing of ADA sample collection.
Specificity refers to the ability of a method to exclusively detect the target analyte, in this case the ADA.22 Lack of assay specificity can lead to false-positive results, which could obscure relationships between ADA generating immune response, pharmacokinetics, pharmacodynamics, and clinical safety and efficacy measures. Demonstrating the specificity of antibody responses to mAb, Fc-fusion proteins, and Ig-fusion proteins poses challenges because of the high concentration of Ig in human serum. The assay should specifically detect anti-mAb antibodies but not the mAb product itself, soluble drug target, non-specific endogenous antibodies, or antibody reagents used in the assay. Similarly, for subject populations with a high incidence of rheumatoid factor (RF), the sponsor should demonstrate that RF does not interfere with the detection method or that the assay can differentiate between RF and specific antibodies. RF is discussed in detail in section IV.L.2. In cases where ADA demonstrates cross-reactivity with host cell proteins and other product-related impurities, the specificity of these reactions may need further evaluation.
A straightforward approach to addressing specificity is to demonstrate that binding can be blocked by soluble or unlabeled purified therapeutic protein product. One approach is to incubate positive and negative control antibody samples with the purified therapeutic protein product or its components under consideration. Inhibition of signal in the presence of the relevant therapeutic protein product or its components indicates that the response is specific. Establishing the specificity of multimeric antibodies such as IgM by competitive inhibition may be difficult, so establishing assay capability for these circumstances requires careful development or additional approaches. For ADA to mAb products, inclusion of another mAb with the same Fc but different variable region can be informative. If the assay is specific and selective for ADA to the therapeutic protein product being studied, generally the addition of that therapeutic protein product or its components in solution will reduce the assay signal. Conversely, addition of the therapeutic protein product or its components should have little effect on antibodies of other specificities.
The selectivity of an ADA assay is its ability to identify ADAs specific to the therapeutic protein product in the presence of other components in the sample. Assay results may be affected by interference from the matrix or onboard therapeutic protein product. It is important to note that most assay matrices contain significant amounts of proteins of various sizes and charges. Failure to establish selectivity can contribute to non-specific signal, thereby obscuring positive results.
1. Matrix Interference
An important consideration is how the sample matrix (for example, plasma, serum, saliva) can affect assay performance. Some degree of signal suppression is expected when comparing assay performance in diluent versus matrix. Endogenous and exogenous components in a matrix may influence assay results, and it is usually necessary to dilute subject samples for testing to minimize such effects. The sponsor should define the matrix and dilution factor that will be used for preparation of subject samples before performing validation studies assessing potential interference of this matrix on assay results (see section IV.E.2 on MRD).
Various substances in the matrix, such as free hemoglobin (hemolysis), lipids (lipemia), bilirubin (interus), and presence of concomitant medications, can interfere with assay results. For example, the anticoagulants used during sample collection may have different effects on the assay, potentially affecting the assay sensitivity. The sponsor may examine matrix interference by spiking different known amounts of positive control antibodies in the presence or absence of matrix. Comparing the recovery of ADA in buffer alone with that in the matrix can provide input on the degree of interference from matrix components. Furthermore, such analysis may guide decisions on the MRD recommended for sample testing. This information may be useful to understanding assay sensitivity.
Buffer components that are chemically related to the therapeutic protein product may also cause interference in the assay. For example, polysorbate is chemically similar to polyethylene glycol(PEG) and therefore may interfere in the detection of anti-PEG antibodies. The chemical composition of the buffer should be carefully considered during assay development.
2. Minimal Required Dilution
Matrix components can contribute to non-specific signal, thereby obscuring positive results. Therefore, there is frequently a need to dilute subject samples to maintain a reasonable ability to detect ADA. Multiple definitions of MRD have been proposed, including the sample dilution that yields the highest signal-to-noise ratio; the sample dilution that results in a signal closest to assay diluent; and the sample dilution that results in the highest signal to variability ratio (MireSluis et al. 2004).23 Sponsors may use any of these definitions, but for the purposes of calculating assay sensitivity and titer, the MRD should take into consideration the final dilution of the sample in the assay, which typically ranges from 1:5 to 1:100 (that is, 1/5 to 1/100).
FDA recommends that sponsors determine the MRD from a panel of appropriate number of samples from treatment-naïve subjects. Determination of MRD usually involves serially diluting treatment-naïve ADA-negative samples, as well as testing known amounts of purified antibody at high, medium, and low concentrations in serially diluted matrix in comparison to the same amount of positive control antibody in diluent. This ensures a reasonable signal-to-noise ratio throughout the range of the assay. The MRD should be calculated using an appropriate number of individual serum samples. The appropriate number of samples will depend on various factors, including the variability of the individual samples; however, at least 10 samples are frequently recommended (Mire-Sluis et al. 2004).24
Although the MRD ultimately selected by the sponsor will depend on the assay design and subject population, FDA recommends that MRD not exceed 1:100. Higher MRD may result in false-negative responses. However, in some instances higher MRD may be required, and the overall effect of such MRD on assay sensitivity and immunogenicity risk assessment should be considered.
Precision is a measure of the variability in a series of measurements for the same material run in a method. Results should be reproducible within and between assay runs to assure adequate precision.25 Demonstrating assay precision is critical to the assessment of ADA because assay variability is the basis for determining the cut-points and ensuring that low positive samples are detected as positive. To provide reliable estimates, the sponsor should evaluate both intra-assay (repeatability) and inter-assay (intermediate precision) variability of assay responses. In cases where a floating cut-point is needed, inter-assay precision may be calculated using normalized values.
Reproducibility is an important consideration if an assay will be run by two or more independent laboratories during a study, and a sponsor should establish the comparability of the data produced by each laboratory.26 Comparable assay performance, including sensitivity, drug tolerance, and precision, should be established between laboratories.
H. Robustness and Sample Stability
Assay robustness is an indication of the assay’s reliability during normal usage27 and is assessed by the capacity of the assay to remain unaffected by small but deliberate variations in method and instrument performance that would be expected under relevant, real-life circumstances in routine laboratory practice. For example, changes in temperature, incubation times, or buffer characteristics such as pH and salt concentration can all impact assay results. The complexity of bioassays makes them particularly susceptible to variations in assay conditions, and it is essential to evaluate and optimize parameters such as cell passage number, incubation times, and culture media components. The sponsor should examine robustness during the development phase, and if small changes in specific steps in the assay affect results, precautions should be taken to control that step. Some aspects of robustness may be included in the assay validation exercise (see section VI.A). Because it is generally not feasible to establish the stability of subject samples, FDA recommends storing subject samples in a manner that preserves antibody reactivity at the time of testing. FDA recommends that sponsors minimize freeze-thaw cycles by appropriately aliquoting subjects’ samples because freezing and thawing such samples may also affect assay results. However, studies evaluating short-term stability, including, as relevant, freeze-thaw cycle and refrigerator- and room-temperature stability of positive control antibodies, may be useful.
I. Selection of Format
Different assay formats and instrumentation are available that can be used for detection of ADA. These include, but are not limited to, direct binding assays, bridging assays, and soluble-phase binding assays; for example, radioimmunoprecipitation assay. Each assay format has advantages and disadvantages, including throughput, sensitivity, selectivity, dynamic range, ability to detect various Ig isotypes, ability to detect rapidly dissociating antibodies, and availability of reagents. Bridging assay formats may be subject to false-negative results when the antigen (for example, PEG) has repetitive motifs. One of the major differences between these assay formats is the number and vigor of washes, which can influence assay sensitivity. Epitope exposure is also important to consider because binding to plastic or coupling to other agents (for example, fluorochrome, enzyme, or biotin reporters) can result in conformational changes of the antigen that can obscure, expose, modify, or destroy relevant antibody binding sites on the therapeutic protein product in question.
J. Selection of Reagents
Many components of the assays for ADA detection may be standard or obtained from commercial sources; for example, microtiter plates. Other components, however, including positive control antibodies, negative controls, and system suitability controls, may need to be generated specifically for the assay. Qualification and stability of critical reagents is important for ensuring consistent assay performance.
1. Development of Positive Control Antibodies
Sponsors may use the same or different positive control antibodies to develop, validate, and monitor system suitability during routine assessment of assay performance. For system suitability controls, a positive control antibody, either mono- or polyclonal, used at concentrations adjusted to ensure assay sensitivity and detect hook effects, should be included.28
Different approaches may be used to generate a positive control. Most frequently, positive control antibodies are generated by immunizing animals in the absence or presence of adjuvants. FDA recommends that positive control antibodies generated by immunizing animals be affinity purified using the therapeutic protein product. This approach enriches the polyclonal antibody preparation for ADA, which enables a better interpretation of sensitivity assessment results. The selection of animal species when generating positive control antibodies should be carefully considered. For example, if an anti-human Ig reagent will be used as a secondary reagent to detect antibodies in subjects, the positive control antibodies and quality control (QC) samples ideally should be detectable by that same reagent. When the positive control antibody is not detectable by that same reagent (for example, if the positive control is generated in a rabbit and a different secondary reagent is needed to detect the positive control antibody), a positive control antibody for the secondary reagent used to detect human antibodies in the subject samples also should be included in the assay to ensure that the reagent performs as expected. In some instances, the sponsor may be able to generate a positive control antibody from subjects’ samples.29 Such subject-derived positive controls can be very valuable but are generally not available in early trials. Alternatively, individual mAb or panels of mAb may be used as positive control antibodies. For therapeutic mAb, the sponsor should select a positive control antibody that binds to the variable region of the therapeutic mAb. Sponsors should discuss with FDA alternative approaches to assay development and validation in the rare event that a sponsor is not able to generate a positive control antibody.
Once a source of a positive control antibody has been identified, the sponsor should use that source to assess assay performance characteristics such as sensitivity, selectivity, specificity, drug tolerance, and reproducibility. FDA recommends that sponsors generate and reserve positive control antibody for use as a quality control or system suitability control during routine performance of the assay. For assay development and validation, dilutions should generate high, intermediate, and low assay signal values. The intermediate value is useful for assessing precision during assay validation. This is recommended even for development of qualitative assays to understand whether assay performance is acceptable across a broad range of antibody concentrations. Intermediate-value QC samples for detection of ADA are generally not needed for monitoring system suitability during routine assay performance.
2. Development of Negative Controls
FDA recommends that sponsors establish a negative control for validation studies and subject- sample testing. In this regard, a pool of sera from an appropriate number of treatment-naïve subjects can serve as a negative control. Importantly, the value obtained for the negative control should be below but close to the cut-point determined for the assay in the subject population being tested. Negative controls that yield values far below the mean value derived from individual serum samples used to establish the cut-point may not be useful in ensuring proper assay performance.
When possible, negative control samples should be collected from treatment-naïve subjects with the medical condition being studied and should include subjects with similar gender, age, and concomitant medications so that the sample matrix is representative of the study population. Control samples should be collected and handled in the same manner as study samples with respect to, for example, type of anticoagulant used, volume, and sample preparation and storage because these pre-analytical variables can impact the performance of control samples in the assay. It is frequently the case that such control samples are not available for use during development or pre-study validation exercises. In those situations, it is acceptable to use purchased samples or samples from healthy donors, but important parameters of assay performance such as cut-point, sensitivity, and selectivity should be confirmed when samples from treatment-naïve subjects from the appropriate target population become available. If cutpoint and selectivity differ when negative controls from different populations are used, reevaluating other assay parameters (for example, sensitivity) may be needed.
3. Controlling Non-Specific Binding
Every test component, from the plastic of the microtiter plates to the developing agent, can affect assay sensitivity and non-specific binding. One of the most critical elements is the selection of the proper assay buffer and blocking reagents used to prevent non-specific binding. The sponsor should carefully consider the number and timing of wash steps as well as the detergents added to the assay buffer (for example, blocking or wash buffer) to reduce background noise while maintaining sensitivity. A variety of proteins can be used as blocking reagents to provide acceptable signal-to-noise ratio. However, these proteins may not all perform equivalently in specific immunoassays. For example, they may not bind well to all types of solid phases or may show unexpected cross-reactivity with the detecting reagent. Therefore, the sponsor may need to test several blocking agents to optimize assay performance. Moreover, including uncoated wells is insufficient to assess non-specific binding. Rather, determining the capacity of ADAs to bind to an unrelated protein of similar size and charge that may be present in the sample may prove to be a better test of binding specificity.
K. Reporting Results for Qualitative and Quasi-Quantitative Assays
Several approaches may be used to report positive antibody responses, and the appropriateness of the approach used should be evaluated on a case-by-case basis. The most common approach is qualitative, with subjects reported as having a positive or negative antibody response.
For subjects who are confirmed to be ADA positive, determining antibody levels can be informative because it allows for stratified assessment of ADAs and their impact on safety and efficacy. Positive antibody levels may be evaluated using a titer. Reporting levels of antibodies in terms of titers is appropriate and generally understood by the medical community. Most frequently titer is determined from the reciprocal of the highest dilution that gives a value at or just above the cut-point of the assay. Alternatively, titer may be determined by extrapolating the dilution to the assay cut-point using the linear portion of the dose response curve. All sample dilutions, such as the MRD and acid dissociations, should be factored into the calculations of titers and provided when reporting titers.
When reporting results for neutralization assays, values may also be reported as amount of mass units of therapeutic protein product neutralized per volume serum with the caveat that these are arbitrary in vitro assay units and cannot be used to estimate in vivo availability of the therapeutic protein product.
Unless the assay method used allows for independent determination of mass per volume of undiluted matrix, antibody levels reported in mass units are generally not acceptable. This is because the mass unit estimations are based on interpolation of data from standard curves generated with a positive control antibody, and parallelism between the positive control and test article cannot be assumed. Furthermore, even if parallelism between the positive control and test article is demonstrated, the absolute mass units cannot accurately be calculated because the samples are likely to contain different populations of antibodies. Thus, FDA does not consider it necessary or desirable for the sponsor to report subject antibody results in terms of mass units unless (1) the results are determined by quantitative means or (2) a universally accepted and accessible source of validated antibody is available as a control and parallelism between the dilution curves of the control antibody and subject samples has been demonstrated.
L. Other Considerations for Assay Development
A myriad of factors can affect the assessment of ADA levels, such as subject-sample variability; therapeutic protein product-dose response of the cells used to generate the standard curve in a cell-based neutralization bioassay; affinity and avidity of the ADA; and concentration of competing product in confirmatory assays. Accounting for such factors is important to understand and analyze assay variability and avoid errors. Common factors that should be considered include the following:
、1. Pre-Existing Antibodies
Pre-existing antibodies may have clinical effects that affect the efficacy of the therapeutic protein product being tested. An alternative to the qualitative screening assay approach may be needed to assess the quantity and quality of ADA when pre-existing antibodies are present. For example, testing samples for an increase in ADA using a semi-quantitative assay such as a titration assay (see sections V.C and VI.D) can provide information on the impact of a therapeutic protein product on product immunogenicity that is not provided by a qualitative assay. When there are pre-existing antibodies and the titer of antibodies increases after exposure to the therapeutic protein product, they can be reported as treatment-boosted to differentiate them from treatment-induced antibody titers. For example, a boosted ADA response may be defined as a titer that is two dilution steps greater than the pre-treatment titer, when twofold dilutions are used to determine the titer.
2. Rheumatoid Factor
Measuring immune responses to therapeutic protein products that possess Fc regions, such as mAb and Fc-fusion proteins, may be particularly difficult when RF is present in the matrix. RF is generally an IgM antibody that recognizes IgG, although other RF Ig specificities have been noted. Consequently, RF will bind Fc regions, making it appear that specific antibody to the therapeutic protein product exists. Several approaches for minimizing interference from RF have proven useful, including treatment with aspartame (Ramsland et al. 1999) and careful optimization of reagent concentrations so as to reduce background binding. When examining immune responses to Fc-fusion proteins in clinical settings where RF generates false-positive results during development, FDA recommends developing an assay specific for the non-Fc region of the proteins rather than against the intact biotherapeutics.
3. Monoclonal Antibodies
Technologies reducing the presence of non-human sequences in mAb, such as chimerization and humanization, have reduced but not eliminated ADA. In these cases, the immune responses are directed largely against the variable regions of the mAb (Harding et al. 2010; van Schouwenburg et al. 2014). The assays that can detect the reactivity against variable regions are considered more appropriate to evaluate the potential impact of antibodies against mAb-based therapeutics in subjects. If the Fc region is engineered or bound to another molecule, an assay that characterizes this response may be needed.
4. Conjugated Proteins
Antibody-drug conjugates (ADCs) are antibodies conjugated with small molecule drugs, so they represent a classic hapten-carrier molecule. Therefore, the immunogenicity assays should measure the responses to all components of the ADC therapeutic protein product, including the antibody, linker-drug, and new epitopes that may result from conjugation. When ADCs need to be labeled for immunogenicity assays, the conjugation should consider the potential for increased hydrophobicity of the labeled molecules because they may cause aggregation. The stability and solubility of these capture reagents should be adequately characterized (see section IV.A.3).
V. ASSAY DEVELOPMENT
Information specific to the development of respective assay types is provided in sections A through D below. These sections supplement the information provided in section IV that is relevant to all assay types.
A. Development of Screening Assay
Based on the multi-tiered approach discussed previously in section IV.A, the first assay to be employed for detection of ADA should be a highly sensitive screening assay that detects low- and high-affinity ADA. Approximately 5 to 10 individual samples may be used to estimate the cut-point early in assay development; however, this may need to be adjusted when treatmentnaïve samples from the target population become available. A low but defined false-positive rate of approximately 5% is desirable for the initial screening assay because it maximizes detection of true positives. Subsequent assays can be employed to exclude false-positive results when determining the true incidence of immunogenicity.
B. Development of Confirmatory Assay
Because the screening assay is designed to broadly detect the presence of antibodies that bind product in serum samples with a defined false-positive rate of approximately 5%, FDA recommends that the sponsor develop assays to confirm the binding of antibodies that are specific to the therapeutic protein product. Implementation of a suitable confirmatory assay is important to prevent data on ADA false-positive subjects from confounding the analyses of the impact of ADA on safety and efficacy.