Why must immunogenicity experiments be done for biopharmaceutic

  • Immunogenicity refers to the characteristics that can stimulate the body's immune system to elicit an immune response. Immunogenicity is one of the characteristics of antigens, which can act on the antigen recognition receptors of T lymphocytes and B lymphocytes to proliferate and differentiate them, thereby producing immune effector substances, such as specific antibodies and sensitized lymphocytes. The strength of immunogenicity is generally related to the size and chemical structure of molecular weight.

    Substances with immunogenicity, generally speaking, the larger the molecular weight, the stronger the immunogenicity. The molecular weight is less than 4000 and is generally not immunogenic. The molecular weight is between 4000 and 10000, showing weak immunogenicity. Those with a molecular weight higher than 10,000 have strong immunogenicity. But there are exceptions, such as gelatin, which has a molecular weight of up to 100,000, but is weakly immunogenic because it is a linear amino acid structure that is easily degraded.

    Immunogenicity testing is an essential step in the development of antibody drugs and needs to be carried out in both preclinical and clinical stages. Immunogenic drugs may induce harmful immune responses in the body and can form anti-drug antibodies (ADAs) and neutralizing antibodies (NAbs). The former will cause a strong immune response in patients and even endanger the life safety of patients, and the latter can neutralize the ability and inhibit the biological activity of biological drugs and weaken their efficacy.

    Therefore, considering that immunogenicity can seriously affect the efficacy and safety of drugs, FDA and other regulatory authorities require that immunogenicity testing should be performed for all biopharmaceuticals. However, the current predictive tools are not good, and many drugs do not identify ADA problems until clinical phase III. For companies, this undoubtedly increases R & D risk and capital investment.


    Source of biopharmaceuticals immunogenicity

    There are two main sources of immunogenicity of biopharmaceuticals: endogenous and exogenous. Endogenous immunogenicity comes from amino acid sequences of non-human origin, amino acid sequences of different people, and some modifications made to enhance efficacy or prolong half-life (such as point mutation, glycosylation modification, trehalose removal, fatty acid addition, PEGylation, Fc fusion, bispecific antibody and multifunctional antibody, etc.), which should be considered in the early stage of drug research and development.

    Exogenous immunogenicity mainly comes from CMC (CMC mainly refers to production process, impurity study and quality study), materials and transportation. For example, increasing antibody expression while glycosylation does not keep up leads to the production of polymers, which are one of the important causes of immunogenicity. For another example, common surfactants can cause a strong immune response. In addition, the route, frequency, and timing of administration may also affect the immunogenicity of the protein.


    How to Detect Immunogenicity of Biopharmaceuticals


    FDA recommends that immunogenicity risk testing should preferably be performed at the IND stage and in clinical phase I. For biopharmaceuticals with high immunogenicity risk, pharmaceutical companies should conduct pre-validation at an early stage and conduct real-time detection and analysis before sample cryopreservation. One of the conditions for passing FDA drug certification is that there is a complete report of protein immunogenicity testing. According to FDA guidelines, immunogenicity testing of biopharmaceuticals mainly includes the following three steps:

    1. Immunogenicity screening: detection of ADAs that recognize antibody protein drugs. Commonly used methods include ELISA, ECL, and RIA.


    1. ADA confirmation: verify whether ADA is specific by protein-drug competition and exclude false positive results.

      3. ADA characterization: competitive ligand binding analysis, isoform analysis, binding stability analysis, antigen epitope specificity analysis, binding stability, and neutralizing capacity analysis. Neutralization capacity analysis, also known as neutralization assay, is commonly performed by competitive ligand binding (CBL), whichis the method of choice for neutralization testing.