| Autor: |
Harvey IB; Translational Sciences and Diagnostics, Bristol-Myers Squibb, Princeton, New Jersey 08540, United States., Chilewski SD; Translational Sciences and Diagnostics, Bristol-Myers Squibb, Princeton, New Jersey 08540, United States., Bhosale D; Translational Sciences and Diagnostics, Bristol-Myers Squibb, Princeton, New Jersey 08540, United States., Tobia SE; Translational Sciences and Diagnostics, Bristol-Myers Squibb, Princeton, New Jersey 08540, United States., Gray C; Translational Sciences and Diagnostics, Bristol-Myers Squibb, Princeton, New Jersey 08540, United States., Gleason C; Global Biometrics and Data Sciences, Bristol-Myers Squibb, Princeton, New Jersey 08540, United States., Haulenbeek J; Translational Sciences and Diagnostics, Bristol-Myers Squibb, Princeton, New Jersey 08540, United States. |
| Abstrakt: |
Concentration determination is a fundamental hallmark of protein reagent characterization, providing a means to ensure reproducibility and unify measurements from various assays. However, lot-to-lot differences in protein activity often still occur, leading to uncertainty in the accuracy of downstream measurements. Here, we postulate that those differences are caused by a misrepresentation of the protein concentration as measured by traditional total protein techniques, which can include multiple types of inactive protein species. To overcome this, we developed a standardized method to quantify a protein's active concentration via calibration-free concentration analysis (CFCA). As a pilot study, we compare the biophysical and immunoassay responses from three batches of recombinant soluble lymphocyte-activation gene 3 (sLAG3), as defined by either their total or active concentrations. Defining the sLAG3 reagents by their assay-specific concentration improved consistency in reported kinetic binding parameters and decreased immunoassay lot-to-lot coefficients of variation (CVs) by over 600% compared to the total protein concentration. These findings suggest that the total concentration of a protein reagent may not be the ideal metric to correlate in-assay signals between lots, and by instead quantifying the concentrations of a reagent's assay-specific epitopes, CFCA may prove a useful tool in overcoming lot-to-lot variability. |
