7.3.8 Confirmation of antibody positive samples

Biosimilarity: The FDA Perspective

282 While the assays described are useful for identifying antibody-positive samples, it is important to include an additional confirmatory step in the assessment strategy to ensure that the generated antibodies are specifically targeted to the therapeutic.

A confirmatory approach can include the use of different methods (ELISAs, competitive immunoassays, SPR, etc.), although an assay based on a different scientific principle from that used for the screening assay should usually be considered. It is also necessary to select a confirmatory assay taking into account the limitations and the characteristics of the screening assay. In most cases, assay specificity can be demonstrated by the addition of free antigen to a serum sample spiked with known amounts of antibody and by looking for inhibition of the expected signal. This approach itself can form the basis of a confirmatory assay. The use of the immunoblotting procedure, which provides information concerning the specificity of the antibodies detected, is valuable as the antibodies may have specificity for other components (e.g., contaminants) in the product and can cause data to be misinterpreted. For example, very small levels of expression system–derived bacterial proteins in rDNA products can cause significant antibody development, whereas the human sequence major protein present (the active principle) may be much less immunogenic. However, other procedures, e.g., analytical radioimmunoprecipitation assays, can also be used for specificity studies. The use of assays described earlier does not obviate the requirement for a functional cell-based neutralization assay. The latter should be incorporated into the strategy for immunogenicity assessment as it has been shown that results from bioassays can often be correlated with the effect of antibodies on clinical response. Some developers conduct neutralization assays at both preclinical and clinical levels as part of their product development program, while others implement these assays at the clinical stage after considering whether the therapeutic is low or high risk. Overall, the decision to put a biosimilar on the market is made if its effectiveness is similar, and its immunogenic profile is at least comparable or improved in comparison to the first licensed product. However, this decision is made on limited data. The similarity testing program may disclose substantial differences in terms of immunogenic profiles, but is probably unable to detect minor differences and rare events. For that reason, clinical trials complemented by a pharmacovigilance program are often required for evaluating a recombinant protein’s safety in patients. Undesirable effects of these drugs are very rare, yet require a follow-up during the life of the product. This includes testing all those attributes that can trigger a side effect such as immunogenicity; included here are aggregates, dimers, subvisible and

visible particles, HCP, host cell DNA, etc. The nonclinical studies are generally required to establish overall toxicology, such as in an animal model; however, in several situations, an animal model may not be readily available, such as in the case of mAbs. Several creative models have recently been proposed including transgenic mice, etc. Also included here are safety assessments at the toxicological level and in the clinical pharmacology of the product, such as PK and PD data. This is perhaps one of the most difficult areas of biosimilar product development. All protein therapeutic contain particles, and these can be immunogenic. Subvisible and submicron particles and aggregates of various sizes can be tested using methods such as HIAC, MFI, dynamic light scattering (DLS), FFF, AUC-sedimentation velocity, and SEC-HPLC-LS. It is most important to determine if a higher level of subvisible particles is nonproteinaceous. All protein therapeutics contain (higher or lower levels of) aggregates and particulate matter and most of these products are immunogenic. Aggregation results from fermentation/expression, purification, formulation, filling, transport, and storage administration. Some of the factors influencing protein aggregation include temperature, interfaces, freeze–thaw, container, pH, excipients, ionic strength, and concentration. Protein aggregates are assemblies of protein molecules that are highly heterogeneous regarding their size, reversibility, protein conformation, covalent modifications, and morphology. It is hard to predict which form will be more immunogenic. It is because even a simple dimer can adopt various shapes and characteristics based on pH, process stress, light, osmolality, etc. The micronsized IgG aggregates induced by shaking remain at subcutaneous injection site for longer than a month and how it affects the long-term immunogenicity remains unknown. The particles have different size ranges and the methods used to detect them are limited in their ability to detect them (Table 7.3). Most monomers are less than 10 nm and oligomers less than 100 nm.

Table 7.3 Methods Used to Analyze Different Particle Sizes

Methodology Range of Detection

HP-SEC 1 to 90 nm (does not differentiate other particles) SDS-PAGE 1 to 100 nm

DLS Nanoparticle tracking analysis FFF MFI Light obscuration Microscopy Visible

1 nm to 2 μm 30 nm to 2 μm 5 nm to 9 μm 0.8 to 200 μm 1 to 200 μm 5 to 200 μm 500 μm to 5 mm+ Safety similarity

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