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An Algorithm for Building Multi-State Classifiers Based on Collision-Induced Unfolding Data.

Daniel A Polasky1, Sugyan M Dixit1, Daniel D Vallejo1

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Collision-induced unfolding (CIU) now uses multiple protein charge states for improved structural analysis. This enhanced method accurately distinguishes similar protein structures, aiding drug discovery and biotherapeutic characterization.

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Area of Science:

  • Biochemistry
  • Structural Biology
  • Analytical Chemistry

Background:

  • Collision-induced unfolding (CIU) distinguishes protein ions by their unfolding trajectories.
  • CIU is a promising high-throughput, structure-sensitive screening technique for drug discovery and biotherapeutic characterization.
  • Existing CIU workflows analyze single protein charge states, which can be insufficient for highly similar structures.

Purpose of the Study:

  • To expand CIU classification by incorporating data from multiple charge states and other structural perturbations.
  • To improve the accuracy of distinguishing protein subtypes and characterizing biotherapeutics.
  • To demonstrate the utility of multi-state CIU for differentiating highly similar innovator/biosimilar biotherapeutic pairs.

Main Methods:

  • Developed a combined CIU classification workflow integrating data from multiple protein charge states.
  • Applied the enhanced workflow to immunoglobulin (IgG) subtypes and Src kinase inhibitors.
  • Utilized multiple charge states and stress conditions to differentiate innovator/biosimilar biotherapeutics.

Main Results:

  • Improved accuracy of single-state CIU classifiers for IgG subtypes.
  • Developed an activation-state-sensitive classifier for Src kinase inhibitors where single-state data was insufficient.
  • Successfully distinguished a highly similar innovator/biosimilar biotherapeutic pair using combined multi-state and stress condition data.

Conclusions:

  • The expanded CIU classification method enhances the ability to distinguish complex and highly similar protein structures.
  • Multi-state CIU analysis significantly improves the accuracy and applicability of CIU for biotherapeutic characterization and drug discovery.
  • CIU, particularly with multi-state data integration, shows great potential as a rapid screening tool in pharmaceutical development.