Eliminating protein oxidation artifacts during High Performance Liquid Chromatography peak fractionation processes

Area of Science:

• Biochemistry
• Analytical Chemistry
• Pharmaceutical Science

Background:

• Therapeutic monoclonal antibodies (mAbs) are complex biologics requiring rigorous characterization.
• High-performance liquid chromatography (HPLC) is crucial for mAb analysis and product understanding during development.
• Protein oxidation during fractionation is an underappreciated challenge impacting data interpretation.

Purpose of the Study:

• To quantify protein oxidation artifacts induced by HPLC fractionation of therapeutic monoclonal antibodies (mAbs).
• To identify factors contributing to fractionation-induced oxidation.
• To develop and validate a method to mitigate oxidation during mAb fractionation.

Main Methods:

• HPLC fractionation of therapeutic monoclonal antibodies (mAbs).
• Analysis of oxidation using various detectors and fraction concentrations.
• Assessment of oxidation mitigation strategies using oxidation scavengers (histidine, methionine).

Main Results:

• Higher-energy detectors and lower fraction concentrations significantly increased oxidation artifacts.
• Non-detector-related factors also contributed to protein oxidation.
• Collecting fractions in solutions containing histidine and methionine effectively eliminated oxidation.

Conclusions:

• Fractionation-induced protein oxidation complicates mAb analysis.
• Oxidation scavengers are effective in preventing artifacts during HPLC fractionation.
• A modified workflow using oxidation scavengers enhances the accuracy of mAb peak assignments.