FDA utilizes a risk assessment strategy to evaluate the potential immunogenicity of novel protein therapeutics and licensed products following manufacturing changes. The first two parts of this article discussed FDA's assessment of the critical factors influencing the immunogenicity of such products and the consequences of immune responses to treated patients. The first part focused on the clinical consequences of immune responses to protein therapeutics, while the second part examined product- and patient-related factors that influence immunogenicity. This third and final part focuses on the effects of changes in manufacturing and the utility of animal models in assessing a product's immunogenicity.

EFFECTS OF MANUFACTURING CHANGES ON IMMUNOGENICITY The following changes in the manufacture of protein products have a substantial potential to affect product attributes associated with immunogenicity:

• formulation
• virus or adventitious agent clearance and inactivation
• source material or cell line
• container closure.

Changes in source material or cell line may adversely affect proteins through changes in local concentration, as increased molecular collisions facilitate aggregation. Moreover, products made in bacterial systems are normally aggregated in inclusion bodies and require refolding and renaturation to make them soluble and functional.

UTILITY OF ANIMAL STUDIES Animal immunogenicity studies can be useful in assessing aspects of immune responses to therapeutic proteins. However, except in rare cases, animal models do not predict accurately the incidence of human immune responses to therapeutic-protein products. For proteins conserved in evolution, particularly those with a biologically unique function, animal models may illuminate the adverse effects of neutralizing antibody on the endogenous protein. This is particularly important where animal knockout (KO) models have not been generated or where the KO is lethal to the embryo or fetus. Since xenodeterminants certainly contribute to induction of immune responses to human proteins in animals, the incidence of immune responses to human proteins in animals reflects responses to a foreign protein, and thus will often overestimate the product's ability to induce such responses in humans. However, animal models do allow examination of the consequences of immune responses to therapeutic proteins.

Assessment of the inherent immunogenicity of a biological therapeutic requires treatment of animals with the species-specific recombinant animal homolog. This was indeed done in assessing the immunogenicity of thrombopoietin (TPO). When recombinant pegylated human megakaryocyte growth and development factor (PEG-MGDF) or TPO was tested in animal models, animals mounted immune responses that neutralized the product. Because TPO is highly conserved in evolution, such antibodies also cross-reactively neutralized the endogenous animal TPO, rendering animals thrombocytopenic.^74,75 This point illustrates the utility of animal models in assessing the consequence of an immune response. However, to test whether such responses were generated only because of responses to the xenodeterminants on the human TPO or PEG-MGDF, species-specific TPO was tested in several animal models. Stunningly, animals generated neutralizing-antibody responses to species-specific TPO which rendered them thrombocytopenic, reflecting the underlying lack of tolerance to this protein.⁷⁶ Therefore, the high sequence conservation of TPO in evolution, coupled with the findings of studies employing species-specific factor, markedly enhanced the predictive power of the animal studies for human immune responses. However, the species-specific protein product is likely to differ significantly from the recombinant human therapeutic in ways that affect immunogenicity (see Part 2). Further, species differences in major histocompatibility complex (MHC) proteins, which present peptides of the therapeutic protein product to CD4+ T cells, eliciting their help for antibody formation, also make it extremely difficult to model human immune responses in animals.