Why do biological drugs have to do immunogenicity experiments?

Immunogenicity refers to the ability to stimulate the body’s immune system to cause an immune response. Immunogenicity is one of the characteristics of antigens. It can act on the antigen recognition receptors of T lymphocytes and B lymphocytes to make them proliferate and differentiate, thereby producing immune effect substances, such as specific antibodies and sensitized lymphocytes. The strength of immunogenicity is usually related to molecular weight and chemical structure.

Substances with immunogenicity. Generally speaking, the larger the molecular weight, the stronger the immunogenicity. The molecular weight is less than 4000. Generally, it is not immunogenic. The molecular weight is between 4000 and 10,000. 10,000 people 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.
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Immunogenicity testing is an indispensable step in the development of antibody drugs and needs to be carried out both in the 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 patient’s life safety, while the latter can neutralize and inhibit the biological activity of biological drugs and weaken their efficacy.

Therefore, considering that immunogenicity will seriously affect the effectiveness and safety of drugs, the FDA and other drug regulatory agencies require that all biological drugs be tested for immunogenicity. However, the current forecasting methods are not good, and many drugs did not find ADA problems until clinical phase III. For enterprises, this undoubtedly increases R&D risk and capital investment.

Sources of immunogenicity of biopharmaceuticals

There are two main sources of immunogenicity of biological drugs: endogenous and exogenous. The endogenous immunogenicity comes from the amino acid sequence of non-human origin, the amino acid sequence of different people and some modifications (such as point mutation, glycosylation modification, trehalose removal, fatty acid addition, PEGylation, Fc Fusion, bispecific antibodies and multifunctional antibodies, etc.), which should be considered in the early stages of drug development.

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

How to detect the immunogenicity of biological drugs

The FDA recommends that immunogenicity risk testing is best carried out in the IND phase and clinical phase I. For biopharmaceuticals with high immunogenicity risk, pharmaceutical companies should conduct pre-verification at an early stage, and conduct real-time detection and analysis before samples are frozen. One of the conditions for passing the FDA drug certification is to have a complete protein immunogenicity test report. According to FDA guidelines, the immunogenicity test of biological drugs mainly includes the following 3 steps:

Immunogenicity screening: detection of ADA that recognizes antibody protein drugs. Common methods include ELISA, ECL and RIA.

ADA confirmation: Verify whether ADA is specific through protein drug competition, and eliminate false positive results.

ADA characterization: competitive ligand binding analysis, subtype analysis, binding stability analysis, antigen epitope specificity analysis, binding stability, neutralization ability analysis. Neutralization ability analysis is also called neutralization detection, and the commonly used method is competitive ligand binding (CBL). CBL is the preferred method of neutralization detection.

Commonly used immunogenicity testing methods

ELISA-bridge method

The immunogenicity test of antibody drugs is an important part of the application for clinical trials and registration of biotechnology drugs. Although existing animal tests have shown that immunogenicity does not necessarily produce the expected immune response in the human body, the evaluation of the drug immune response Still very important. The process of antigen or antibody immobilization is called coating. In other words, coating is the process of binding antigen or antibody to the surface of a solid carrier. The ELISA-bridge method coats drugs and detects them with labeled drugs. The advantage of this method is that it can detect various antibody subtypes, without species specificity, and can be tested with high throughput. The disadvantage is that it is not easy to detect low-affinity antibodies, and the epitope of the drug may be masked or changed during coating or labeling. , Susceptible to interference from the drug itself.

ELISA-direct method

Antibodies are generally coated by direct adsorption, and most protein antigens can also be coated by methods similar to antibodies. ELISA-direct method to coat the drug and detect it with a labeled antibody. The advantage of ELISA-direct method for immunogenicity detection is that it may increase the ability to detect low-affinity antibodies and has high throughput. However, when the antigenic determinant is present in or adjacent to the hydrophobic region, the direct adsorption of the antigen and the solid carrier can make the antigenic determinant not fully exposed. In this case, the direct coating effect is not good, and the indirect capture package can be used. By law. Moreover, the epitope of the drug may be masked or changed when directly coated, only a single subtype is detected, which is species-specific, and low-affinity antibodies are lost during multiple washings. The reagents may be different between the reference product and the sample.

ELISA-indirect method

ELISA-indirect method of coating monoclonal antibody or biotin, and then adding drugs, that is, the specific antibody against the antigen is pre-coated, and then the antigen is solid-phased through the antigen-antibody reaction. The antigen indirectly bound to the solid phase is far away from the surface of the carrier, and its antigenic determinants are also fully exposed. The indirectly coated antigen is subjected to the affinity chromatography of the solid phase antibody, and the purity of the antigen coated on the solid phase is greatly improved. Therefore, the antigen with more impurities can also be captured and coated to test the specificity and sensitivity of the test. This can be improved, and the repeatability is also good. Another advantage of indirect coating is that the amount of antigen is less, which is only 1/10 or even /100 of that of direct coating. Non-protein antigens that are not easily adsorbed on the polystyrene carrier can be coated in a special way.

Radioimmunoassay (RIA)

RIA is a microanalysis method that uses a radionuclide to label an antigen or antibody, and then combines with the tested antibody or antigen to form an antigen-antibody complex for analysis. It has both the sensitivity of radioisotopes and the specificity of antigen and antibody reactions. The method also has the advantages of high accuracy and precision, easy standardization, simple operation and economy. Because RIA has the advantages of high sensitivity, strong specificity, simple measurement and low cost, RIA has a certain vitality in application. However, because its most fatal weakness is the use of radionuclides, and the effective use time of markers is short, it is difficult to realize the automation of operation and measurement, and its further development is subject to some limitations. Now immunoassay technologies are developing towards the direction of non-isotopic labeling immunoassay.

Electrochemiluminescence (ECL)

Electrochemiluminescence (ECL) is derived from electrochemical methods and chemiluminescence methods, not only can be applied to all immunoassays, but also can be used for DNA/RNA probe detection. Its advantage is the liquid phase method, which can detect various antibody subtypes, has no species specificity, high throughput, can use high-concentration matrix, large detection surface area and stable signal. The disadvantage is that two kinds of markers (biotin and TAG) need to be prepared. The epitope of the marker molecule may change or the labeling process will change the molecule, and the reagents used are of poor versatility.

Surface plasmon resonance

The advantage of the surface plasmon resonance method is the liquid phase method, which does not require a conjugate (enzyme-labeled antibody), and can detect antibodies with different affinities and antibodies of various subtypes, without species specificity. The disadvantage is that chemical linkage may affect the molecule, and the linkage with dextran may affect the exposure of the epitope, and the renaturation may degrade the molecule. The reagents used are poor in versatility and low throughput. If the antibody produced is of the same subtype of the drug, It is difficult to confirm the anti-antibody response of a humanized antibody and the sensitivity is low.

Enzyme-linked immunospot method (ELISpot)

The principle of the enzyme-linked immunospot method (ELISpot) is similar to that of ELISA. It is also a method for detecting cytokines or other soluble proteins produced by cells. It can not only determine the amount of cytokines, but also count the frequency and sensitivity of cells secreting this cytokine. It is higher than ELISA, and the capture antibody used in the experiment will not affect the secretion of cytokines by activated cells. The disadvantage is that it is more complicated and time-consuming than the ELISA technology, and the experimental conditions need to be strictly controlled. The operator needs to be skilled and able to analyze the experimental results to reduce experimental deviation; it is a semi-quantitative method.

Immuno-PCR method (IPCR)

The immuno-PCR method (IPCR) is established on the basis of ELISA, which uses PCR amplification to replace the enzyme-catalyzed substrate of ELISA for color development. PCR has a strong amplification ability, can quantitatively detect DNA and RNA, and has very high sensitivity and specificity. Therefore, the specific antibody that binds to the antigen is combined with the DNA through the linking molecule, and then the antigen is quantitatively detected by PCR amplification. The sensitivity of IPCR is higher than that of ELISA. Compared with the corresponding ELISA, IPCR can increase the sensitivity of antibody detection by at least 1000 times, and only the dilution method in this method can eliminate the interference of the matrix in the biological sample.

The currently reported IPCRs all use the antigen to be tested to directly adsorb the solid phase, so the homogeneity of the solid phase has a great influence on the results; at the same time, other components in the tested sample can also be adsorbed to the solid phase, which is easy to cause excessive background or accuracy. Degree drops. Some antigens that are difficult to adsorb to the solid phase cannot be detected by immuno-PCR. The specificity and homogeneity of the linking molecule have a great impact on PCR, and the PCR amplification process is relatively simple. For example, a microplate is used as a solid phase and a matching PCR instrument is required. , Otherwise it needs to be moved into the reaction tube, which will inevitably lead to a large error, which can cause significant differences after amplification. IPCR has broad application prospects, but it is necessary to further improve the experimental process of IPCR and the development of supporting reagents.

The immunogenicity test of antibody drugs is not only a problem of drug treatment effects, but also a problem of drug safety. The side effects of people’s anti-antibodies are still unclear. However, the impact of anti-antibody production on the drug itself and the potential allergic reactions cannot be ignored. Therefore, the work of immunogenicity testing of antibody drugs needs continuous research.

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