For biological therapeutics immunogenicity testing is an important aspect of clinical trials.  PBL supports clinical trials by performing immunogenicity testing and the Meso Scale Discovery (MSD) platform provides many advantages over the traditional ELISA analysis such as reducing matrix effects, providing high throughput options as well as exhibiting an increased dynamic range and lower sensitivity.

Immunogenicity occurs when patients produce antibodies to the drug which could render the drug ineffective and alter its pharmacokinetic profile by degrading the drug faster than would otherwise occur. Large molecules are complex three dimensional structures and are often recognized by the patient’s immune system as a foreign molecule creating an immune response.  Antibody production is a component of this immune response and could potentially bind to many different epitopes on the biological therapeutic.  Once an antibody-antigen complex is formed, the large molecule can be neutralized in a variety of ways. Antibodies binding the drug could cause the drug to become inactivated.  The antibody-drug complex can form aggregates effecting the solubility of the drug product. An antibody-drug complex could also stimulate internalization by phagocytes which would breakdown the drug product to its essential components. Unwanted side effects could also be attributed to antibody-drug complexes such as inflammation.

If a patient’s antibody binds a pharmacologically active portion of the drug, thereby inactivating it, that antibody is termed a neutralizing antibody (NAb). If a patient is shown to be immunogenic it is recommended to perform a NAb assay to determine if the antibodies created against the drug would inactivate the drug. MSD assays could be designed to investigate both immunogenicity and the presence of NAbs.

Performing MSD Immungenicity Assays

The MSD platform is GLP compliant and at PBL our QuickPlex SQ 120 instrument is fully validated and ready to use for regulated studies.  The general procedure for performing an immunogenicity study would be as follows:

1. Coat the MSD plate with the biological product at 0.05 to 5 pmole per well and incubate for 1 hour overnight.
2. Block with 150 µL of blocking agent for 1 hour.
3. Wash plate and add 25 µL of patient serum sample.
4. Add 25 µL of detection antibody.
5. Wash assay plate, add read buffer and read the plate.

For Antibody Drug Conjugates, a bridging assays would be the preferred method of analysis. Bridging assays typically bind the therapeutic antibody to the carbon electrode at the bottom of the well.  Any immunogenic antibodies would then bind the therapeutic antibody which is then detected by a second therapeutic antibody that has been linked to a Ru metal ion which would give off light when electrically stimulated.

A biotin streptavidin complex is used to bind the therapeutic antibody to the electrode which would allow the bridging complex to form before binding the therapeutic antibody to the plate which would result in greater sensitivity. Below is a the general procedure for performing this assay:

1. Combine biotin therapeutic antibody, detection therapeutic antibody and patient serum samples together. Incubate for one hour.
2. Transfer to a streptavidin blocked MSD plate and incubate for one hour.
3. Wash the plate, add read buffer and read the plate.