Two-peak elution behavior of therapeutic IgG4 in cation exchange chromatography (CEX) has been systemically investigated in this study. We report a case study in which IgG1 and IgG4 show significantly different chromatographic profiles in salt gradient and as well as isocratic elution in CEX. Process parameters and several other factors affecting the peak splitting phenomena have been evaluated in this study. Data suggests that, whereas pH, column loading, hold time, resin type, and residence time of elution are important, the hydrophobicity of the resin base matrix is likely a key contributing factor of the two-peak elution behaviors for IgG4. In-line fluorescence results indicate that different binding conformations for IgG4 exist during the two-peak elution process. The impact of the two-peak elution behaviors on CEX process development was also investigated. Finally, a hypothesis is proposed to explain the phenomenon observed. The study may provide insights in platform process development, analytical method development as well as therapeutic IgG4 molecular design in early discovery phase.

Two-peak elution behavior of therapeutic IgG4 on cation exchange chromatography

255th National Meeting (2018)

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