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Almost all biopharmaceutical products will cause a certain anti-drug antibody (ADA) response after entering the body, which refers to immune response. ADA may affect or change the pharmacodynamic effect between the therapeutic protein and its target. Its pharmacokinetic characteristics affect the efficacy of the drug, and in some cases may also cause serious adverse reactions and even endanger the life of the patient.

In this article, we summarize our drug tolerance method development process:

  • Pre-treatment of samples with acid
  • Solid phase extraction and acid dissociation
  • Magnetic bead Capturing and Acid Dissociation
  • Pre-treatment of samples using the principle of precipitation
  • ACE

1. Pre-treatment of samples with acid

Pre-treatment of samples with acid
There may be a large amount of free drug in the sample, and the presence of free drug can bind to the anti-drug antibody, thereby affecting the detection of ADA. The acidification treatment can separate the free drug from ADA to achieve the purpose of detecting ADA. The specific method is above.

2. Solid phase extraction and acid dissociation

Solid phase extraction and acid dissociation

This method ensures that all available ADA will be detected because it relies on a single binding site to extract ADA, while the bridging method relies on It is detected in an antibody with two free binding sites.

Acid dissociation dissociates ADA from the drug, but does not dissociate the very strong biotinstreptavidin bond.

3. Magnetic bead Capturing and Acid Dissociation

Magnetic beads have large surface area and high capture efficiency, and they can better bind to the target in a solution. The binding ability of magnetic beads is much stronger, so using magnetic beads instead of ELISA plates can greatly improve the binding of ADA.

In an increasing number of cases, magnetic beads are used to replace 96-well plates to improve the detection ability and drug tolerance of ADA.

4. Pre-treatment of samples using the principle of precipitation

The precipitation of PEG and target substance is based on the concentration of PEG and the size of the target substance (in terms of molecular weight, MW). The higher the PEG concentration, the lower the target MW for precipitation.

In the case of a large amount of drug in the sample, the ADA in the sample can be completely recovered and detected. This method can also be used for the detection of PK and Biomarker with interference factors.

Pre-treatment of samples using the principle of precipitation
ACE Method

5. ACE

The ACE method was developed to solve problems caused by that the free drug can affect the formation of a bridge reaction by only interfering with one end of the reaction.

It has been proven to be detectable even in the presence of more than 1000 times free drug (500 ng/ml anti-drug antibody can be detected in the presence of 500 μg/ml free drug).

In summary, all the above methods have impacts in improving drug tolerance. In the real-world method development process, methods will be adapted based on the particularity of each Assay and the characteristics of the drug.

However, when choosing the treatment method, it is necessary to consider the type of acid used, the pH of the acid, the acidification time, the concentration of PEG and so on.

Bioanalysis

References

Moxness, M., Tatarewicz, S., Weeraratne, D., et al., 2005]. Immunogenicity testing by electrochemiluminescent detection for antibodies directed against therapeutic human monoclonal antibodies. Clin. Chem. 51 (10), 1983.

Patton, A., Mullenix, MC, Swanson, SJ, Koren, E., 2005]. An acid dissociation bridging ELISA for detection of antibodies directed against therapeutic proteins in the presence of antigen. J. Immunol. Methods 304 (1–2 ), 189.

Smith, HW, Butterfield, A., Sun, D., 2007]. Detection of antibodies against therapeutic proteins in the presence of residual therapeutic protein using a solid-phase extraction with acid dissociation (SPEAD) sample treatment prior to ELISA. Regul. Toxicol. Pharmacol. 49 (3), 230.

Bourdage, JS, Cook, CA, Farrington, DL, Chain, JS, Konrad, RJ, 2007]. An Affinity Capture Elution (ACE) assay for detection of anti-drug antibody to monoclonal antibody therapeutics in the presence of high levels of drug . J. Immunol. Methods 327 (1–2), 10.

Zoghbi, J., et al.,] A breakthrough novel method to resolve the drug and target interference problem in immunogenicity assays, J. Immunol. Methods (2015).

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