The body generates an immune response if it considers the administered protein to be foreign. Thus, proteins derived from animal sources such as bovine or porcine insulin are expected to be immunogenic. However, in reality, even recombinant human proteins are generally immunogenic, although to a lesser extent. This could be caused by various reasons. The protein may have product-related impurities such as oxidized forms or aggregates. Another major source of immunogenicity is the presence of impurities, such as those from the media or HCPs. The dose and route of administration also play a role, with subcutaneous routes generally more immunogenic than intramuscular or intravenous routes. The genetic makeup of the patient population also determines if the protein would be immunogenic115; thus, adverse effects may be race dependent. Thus, the immunogenicity of a protein cannot easily be predicted and is controlled by a complex multitude of factors and not a simple consideration of "self" versus "nonself."
Some proteins are intrinsically more immunogenic, and some do not seem to induce detectable immune responses. Administration of self-proteins to the body can break tolerance because of the presence of impurities or adjuvants or exposure to high levels of even "natural" sequences and may induce immunity, but sometimes the administration of even animal-derived proteins may not induce immunogenicity in most patients.70116 More structurally complex proteins are generally better antigens.117 However, sequence divergence from native proteins seems to play only a minor role.70 The degree and type of posttranslational modifications of the protein such as glycosy-lation can directly affect the immunogenicity of the protein.13 Thus, a protein produced in E. coli, which will not be glycosylated, is likely to induce a different immune response than the same protein produced in a mammalian cell line because it will be glycosylated in the latter case. Antibodies produced against an immunogenic protein may neutralize the efficacy of the protein, although this is not always the case. Generally, these antibodies do not cause any serious allergic or anaphylactic reactions, but if they neutralize the native protein, they can lead to devastating consequences.70118
Human proteins tested in animals are likely to be immunogenic to the animals, and this can be a major obstacle to preclinical studies, especially to long-term toxicology studies. Human proteins are expected to be immuno-genic to animals, but a high immune response in all tested species may be predictive of immunogenicity in humans, although this is by no means certain. The availability of immune-tolerant transgenic mice has been beneficial in this respect.70 Studies in nonhuman primates can more accurately predict immunogenicity prior to initiating clinical trials. Other predictive models such as major histocompatibility complex (MHC) binding of peptides, computer algorithms, or in vitro naive T-cell assays are also under development, but the accuracy of prediction would determine regulatory approval.119 Development of analytical methods to detect and characterize antibodies is an active area of research, and improvements in these capabilities will have a significant impact on our ability to monitor clinically relevant antibodies, measure low binding affinity antibodies, and distinguish between neutralizing or binding antibodies. Similarly, animal-derived proteins are likely to be immunogenic to humans. Examples of the last include the use of mouse-derived monoclonal antibodies or the use of bovine or porcine insulin in humans.
Even different animal strains may differ in their immunogenic response because of differing expressions of MHCs. For a discussion of the effect of antibody induction on pharmacokinetics of protein drugs, see Chapter 6. All new potential protein products need to be carefully evaluated for their immunogenic potential; once marketed, they should be reevaluated each time there is a significant manufacturing change or if the patient population changes.115
The need to monitor immunogenicity even after a product is marketed was highlighted by the development of pure red cell aplasia in patients treated with Eprex (e-poetin-a) in several countries where the product was marketed. The incidence was associated with subcutaneous administration, but the reasons for the sudden increase in immunogenicity after being on the market for several years was not clear. Manufacturing and formulation changes such as removal of human serum albumin from the formulation may be implicated but this is not proven. Other possible reasons that may be implicated could be a shift in the route of administration to subcutaneous or a change in product container to syringes.116118 Other factors that may be involved include changes in patient population such as those caused by marketing in different countries or changes in route of administration, formulation, storage, and handing.120 It has also been shown that a minute amount of epoetin may be associated with micelles of surfactant used in the formulation. It has been hypothesized that multiple epoetin molecules present in one micelle could lead to increased immunogenicity because of the presence of multiple epitopes exposed on the micellar surface.121 However, data presented at conferences suggest that the cause involved the generation of leachates from the interaction of polysorbate used in the formulation with the uncoated rubber syringe stoppers, and conversion to a Fluro Tec-coated stopper eliminated the problem.
Preparation of a pure protein that has identical structure to a body protein and does not have any aggregates or other product impurities and is free of process impurities will generally produce a protein that is likely to be nonimmunogenic. Aggregation is a major factor that may induce immu-nogenicity,122 and many existing analytical methods do not critically assess the presence of noncovalent aggregates. More novel approaches, such as identifying relevant epitopes and creating modified proteins, that can be tested in relevant assay systems are also under way.123 Impurities can come from the media, HCPs, or the columns used in purification or from the product itself, such as aggregates, fragments, or chemically modified forms such as oxidized forms. To detect these product or process-related impurities, specific and sensitive assays need to be developed.
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