(W1030-02-07) Determination of the Concentration of Monoclonal Antibodies (Mab) Drug in Human Serum Using an Enzyme-Linked Immunosorbent Assay (ELISA)

Purpose: Monoclonal antibodies (Mab) are a type of targeted drug therapy. These drugs recognize and find specific proteins on cancer cells. There are many different Mab to treat cancer. They work in different ways to kill the cancer cell or stop it from growing. For this study, we developed an Enzyme-Linked Immunosorbent Assay (ELISA) method to quantitate Mab drug in human serum to support human clinical studies.

Methods: Mab drug was quantitated in human serum using an ELISA method. The method to quantitate Mab drug used the following steps. A 96‑well microplate was coated with human Mab capture protein. The plate was blocked plate by adding 300 μL of dilution buffer to each well and incubating for a minimum of 1 hour. 100 μL of each standard, QCs, and samples was added into duplicate wells on the plate and incubated for 2 hours. Anti- Mab biotinylated detection antibody to each well and incubated for 2 hours. Followed by Streptavidin-HRP for 20 minutes. 100 μL of 3,3’,5,5’-tetramethylbenzidine (TMB) reagent was incubated to each well, incubated for approximately 10~20 minutes and stopped with 50 μL of 2N Sulfuric acid. The plate was read at 450 nm with 540 nm as a reference wavelength. Data acquisition was be carried out using Gen5 software (Biotech Instruments, inc., version 2.06.10), and the raw data from Gen5 was be imported into Watson Laboratory Information Management System (LIMS) (Thermo Fisher Scientific, Inc., version 7.6.1), where regression was be performed.

Results: The ELISA method for Mab drug showed excellent linearity using a four parameter logistic (4PL) regression fit equation with weighting factor =1 at concentration range from 313 ng/mL to 20,000 ng/mL with coefficients of determination (r²) ≥ 0.9942. For Mab drug LLOQ samples in human serum, the intra‑assay CV ranged from 0.7% to 3.8% and the RE ranged from -18.8% to 2.6%; the inter‑assay CV was 7.7% and the RE was -6.4%. For Mab drug LQC, MQC, and HQC samples in human serum, the intra‑assay CV ranged from 0.7% to 11.0% and the RE ranged from -15.2% to 13.1%; The inter‑assay CV ranged from 3.5% to 10.8% and the RE ranged from -5.6% to -2.4%. For Mab drug ULOQ samples in human serum, the intra‑assay CV ranged from 1.2% to 6.1% and the RE ranged from -11.5% to -4.0%; the inter‑assay CV was 4.8% and the RE was -6.5%. Stability was established through 4 freeze-thaw cycles, ambient temperature (24 hours), as well as long term storage at ‑70°C and -20°C (33 days).

Conclusion: The ELISA method for the determination of the concentration of Mab drug in human serum has been established and fully validated for the quantitation of the concentration of Mab drug in human serum. The method validation parameters, including selectivity, sensitivity, precision, accuracy, and stability, were evaluated. The method has been successfully used in sample analysis to support human clinical studies with good reproducibility on incurred sample reanalysis.

References: Method Validation of a Ligand Binding Assay for the Quantitative Determination of Monoclonal Antibodies (Mab) drug in Human Serum, MBL Report No. RPT23222-MV1-1.0, Medpace Bioanalytical Laboratories, Cincinnati, OH, USA