7.3.5 Assays for detection of antibodies

Biosimilarity: The FDA Perspective

278 when project-specific immunological reagents are not available. To run a bridging ELISA, a labeled drug is needed, however. The label may be detected directly (e.g., fluorescein) or indirectly via an amplification system.

Common methods available include binding assays based on immunochemical procedures such as solid or liquid phase immunoassays, RIPA, and biophysical methods such as SPR. These assays determine the presence (or the absence) of antibodies based on the ability of the antibodies to recognize the relevant antigenic determinants in the therapeutic protein. These assays have their own advantages and disadvantages as described in the following: • Direct-format binding assays (coating with antigen and detecting with labeled anti-Ig) are rapid and relatively easy to use but prone to spurious binding or matrix effects and antigen mobilization may mask or alter epitopes. • Indirect-format binding assays (coating with a specific mAb or biotin, etc. followed by antigen) have high throughput, excellent sensitivity, and coating with a specific mAb keeping the antigen in oriented position and provide consistent coating and maintains antigen conformation, species specificity, and isotype detection determined by secondary reagent; however, the detection reagent may differ between control and sample and extensive studies are required to demonstrate that the coating mAb does not mask or alter epitopes. The test may also fail to detect low-affinity antibodies and has species specificity, and its isotype detection requires determination by secondary reagents. • Bridging-format electrochemiluminescence offers high throughput and use of high concentrations of matrix but requires two antigen conjugates (biotin and TAG) and antigen labeling may alter/denature antigen, or mask/alter epitopes. • RIPA (SPR) is tolerant to interference from antigen, provides signal detection consistent during life of TAG conjugate, has moderate to high throughput, good sensitivity, and can be concrete and automated; it contains information on specificity, isotypes, relative binding affinity, and relative concentration and enables detection of both low-affinity and high-affinity antibodies, and detection reagents are not required. However, it requires dedicated equipment; reagents are costly and vendor specific. It can be isotype specific, may fail to detect rapidly dissociating antibodies, and requires radiolabeled antigens that may alter or denature the antigen; decay of radiolabel may affect antigen stability. Also, it may require immobilization of the antigen, which may alter the conformation of the native protein and the regeneration step may degrade the antigen; the sensitivity is less than that found in binding assays.