7.2.2.2 Structural factors

Biosimilarity: The FDA Perspective

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7.2.2.1.1 Intrinsic immunomodulatory effects The duration of treatment and the route of administration also influence the immune response; for example, if administration of a protein in a single dose results in the production of lowaffinity IgM antibodies, the repeated dosing might result in the production of highaffinity and hightiter IgG antibodies, which may be neutralizing. The conditions promoting an immune tolerance breakdown include • Chronic treatment with repeated doses for months, even years; • Absence of concomitant immunosuppressant treatment; and • Route of administration (the subcutaneous injection is more immunogenic than intramuscular, itself more immunogenic than an intravenous injection).

7.2.2.2 Structural factors Proteins are complex molecules with primary, secondary, and tertiary structures. Changes in the primary structure may be the cause of an immunogenic reaction. Several cases are well known and published in the literature: • Changing an insulin amino acid is enough to lead to a strong immunogenic response, whereas two amino acids inversion only leads to a PK change • The homology degree of a recombinant protein with the natural protein may explain an immunogenic reaction • Reactions of oxidation or deamidation of amino acids are known for triggering an immunogenic reaction by forming new epitopes. It is the example of human recombinant an interferon with one methionine, oxidized because of a modification of the purification process, that has led to nonneutralizing antibodies formation and which, returning to the initial process, has stopped being immunogenic • The modification of stability characteristics of a protein with aggregates formation may have significant consequences in terms of immunogenicity by tolerance breakdown of the immune system The significance of protein spatial conformation is well known for its biological activity as well as its stability. Partial modification of spatial conformation may occur after shear, by shaking, or by temperature modification (for example, temperature rise or freeze/thaw cycles).

7.2.2.2.1 Glycosylation Almost half of the therapeutic proteins that are approved or in clinical trials are glycosylated. Glycosylation is one of the most common and complex PTMs, which leads to the enzymatic addition of glycans on proteins. Glycans can influence the physicochemical (e.g., solubility, electrical charge, mass, size, folding, stability) as well as the biological (e.g., activity, half-life, cell surface receptor function) properties of proteins. The glycosylation profile of a protein is