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Overcoming Lot-to-Lot Variability in Protein Activity Using Epitope-Specific Calibration-Free Concentration Analysis.

Ian B Harvey1, Shannon D Chilewski1, Devyani Bhosale1

  • 1Translational Sciences and Diagnostics, Bristol-Myers Squibb, Princeton, New Jersey 08540, United States.

Analytical Chemistry
|April 11, 2024
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Summary
This summary is machine-generated.

Traditional protein concentration methods can misrepresent active protein levels, causing assay variability. A new calibration-free concentration analysis (CFCA) method quantifies active protein concentration, significantly improving assay consistency and reducing lot-to-lot variation.

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Area of Science:

  • Biochemistry
  • Protein characterization
  • Assay development

Background:

  • Accurate protein concentration determination is crucial for assay reproducibility.
  • Lot-to-lot variability in protein reagents persists despite traditional concentration measurements.
  • Inactive protein species can lead to inaccurate total protein concentration values.

Purpose of the Study:

  • To develop a standardized method for quantifying active protein concentration.
  • To address lot-to-lot variability in protein reagents.
  • To introduce calibration-free concentration analysis (CFCA) as a solution.

Main Methods:

  • Developed a standardized calibration-free concentration analysis (CFCA) method.
  • Quantified active protein concentration for recombinant soluble lymphocyte-activation gene 3 (sLAG3) batches.
  • Compared biophysical and immunoassay responses using total vs. active protein concentrations.

Main Results:

  • Defining sLAG3 reagents by assay-specific concentration improved kinetic binding parameter consistency.
  • Active concentration measurement decreased immunoassay lot-to-lot coefficients of variation (CVs) by over 600% compared to total protein concentration.
  • CFCA demonstrated improved consistency in assay results.

Conclusions:

  • Total protein concentration may not be the ideal metric for correlating in-assay signals between lots.
  • Quantifying assay-specific epitopes via CFCA can overcome lot-to-lot variability.
  • CFCA is a valuable tool for accurate protein reagent characterization.