Collision-Induced Unfolding Reveals Stability Differences in Infliximab Therapeutics under Native and Heat Stress...

Area of Science:

• Biopharmaceutical analysis
• Analytical chemistry
• Structural biology

Background:

• Monoclonal antibody (mAb)-based biotherapeutics are critical therapeutics, necessitating robust analytical methods for quality control.
• Ion mobility-mass spectrometry (IM-MS) and collision-induced unfolding (CIU) show promise for characterizing mAb attributes like disulfide bridges, glycosylation, and conjugation.
• Existing studies highlight IM-MS and CIU's potential for biosimilar differentiation, but sensitivity to stress-induced changes, inter-platform reproducibility, and correlation with differential scanning calorimetry (DSC) require further investigation.

Purpose of the Study:

• To comprehensively evaluate the sensitivity and reproducibility of IM-MS and CIU for assessing heat-induced structural changes in Infliximab biosimilars.
• To compare the performance of CIU and DSC in detecting alterations in mAb higher-order structure (HOS) upon thermal stress.
• To assess the inter-platform reproducibility of IM-MS and CIU data for mAb analysis.

Main Methods:

• Acquisition of comprehensive IM-MS and CIU datasets for three Infliximab products (Remicade, Inflectra, Renflexis).
• Forced degradation of Infliximab samples via heat stress to induce structural changes.
• Comparative analysis of CIU and DSC data to assess sensitivity to structural alterations.
• Data acquisition across two distinct IM-MS instrument platforms (Waters G2 and Agilent 6560) to evaluate reproducibility.

Main Results:

• Both IM-MS and CIU demonstrated the ability to differentiate the three Infliximab products, revealing subtly different stability patterns upon heat stress.
• CIU proved more sensitive than DSC in detecting changes in mAb higher-order structure (HOS) induced by heat stress.
• Data acquired on two different instrument platforms showed similar capabilities for mAb differentiation, with minor variations noted.
• CIU-based heatmaps and classification enabled rapid assessment of differentiating charge states, estimating a 30-fold improvement in analysis time over DSC.

Conclusions:

• IM-MS and CIU are sensitive and reproducible techniques for monitoring stress-induced structural changes in mAb biotherapeutics.
• CIU offers a significant advantage over DSC in terms of sensitivity and speed for assessing mAb stability and HOS.
• These findings support the utility of IM-MS and CIU as powerful tools for biopharmaceutical characterization and quality control.