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Different fluorescence-based techniques are used to study the protein dynamics in living cells. These techniques include FRAP, FRET, and PET.
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In-Vial Detection of Protein Denaturation Using Intrinsic Fluorescence Anisotropy.

Krishnakumar Chullipalliyalil1, Khaled Elkassas2, Michael A P McAuliffe1

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This study introduces an in-vial fluorescence method to monitor biopharmaceutical stability, avoiding destructive testing. This technique offers a non-destructive way to assess protein denaturation without compromising sterility.

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Area of Science:

  • Biopharmaceutical analysis
  • Protein stability assessment
  • Analytical chemistry

Background:

  • Conventional biopharmaceutical quality control methods for denaturation (fragmentation, deamidation, oxidation, aggregation) are destructive and require sample removal, risking sterility.
  • Existing techniques like SDS-PAGE, IEX, IF, RP-HPLC, and SEC-HPLC are lab-based and necessitate breaching the vial's seal.

Purpose of the Study:

  • To investigate heat- and surfactant-induced denaturation of bovine serum albumin (BSA) using in-vial intrinsic fluorescence.
  • To develop a non-destructive method for monitoring biopharmaceutical stability within sealed vials.

Main Methods:

  • Analysis of intrinsic fluorescence polarization of in-vial stored BSA under induced denaturation conditions.
  • Utilized a bespoke lab-based setup for in-vial measurements.
  • Compared fluorescence data with circular dichroism and size-exclusion HPLC.

Main Results:

  • Demonstrated that in-vial fluorescence measurements can effectively detect and quantify protein denaturation.
  • Observed changes in fluorescence polarization correlated with denaturation levels.
  • Results were consistent with established methods like circular dichroism and size-exclusion HPLC.

Conclusions:

  • In-vial fluorescence measurement is a viable non-destructive technique for monitoring biopharmaceutical product stability.
  • This method avoids sample removal, preserving sterility and reducing product loss.
  • Potential for developing a cost-effective, portable solution for real-time stability monitoring from manufacturing to patient administration.