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

•HPLC detectors create photostress and cause protein oxidation.•Higher energy light source and more dilute fractions exacerbate the oxidation.•Fractionation process other than detectors also induces protein oxidation.•Fraction collection into scavenger buffers effectively prevented protein oxidation. Therapeutic monoclonal antibodies (mAbs) are inherently heterogeneous and hence generally studied and controlled by an array of orthogonal separation methods. During drug candidate development, fractionation by HPLC is regularly employed to assist peak identification and product understanding. One overlooked challenge is the protein oxidation introduced by the fractionation process. In this study, we report the extent of fractionation-induced protein oxidation, which tends to complicate data interpretation and peak assignments. Higher-energy detectors such as fluorescence detectors and lower fraction concentration were found to exacerbate the oxidation artifacts. Other contributing factors than the detector-induced photostress were also found to contribute significantly to protein oxidation. Furthermore, our study showed that collecting fractions into a solution with oxidation scavengers, such as histidine and methionine, was effective in eliminating the oxidation artifacts introduced by detector exposure and fraction processing steps. Through an example, we demonstrate that the modified fractionation workflow improves the accuracy of peak assignments.