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Mobile Phase Optimization for the Analysis of an Antibody-Based Fusion Protein and Aggregates using a TSKgel Super SW3000 Size-Exclusion Column

T.J. Higley and Roy Eksteen, Tosoh Bioscience LLC, Montgomeryville, Pennsylvania and Scott Lauder, Lexigen Pharmaceuticals, Lexington, Massachusetts


Introduction:
During method development, many variables are examined to ensure method robustness. Factors such as elution profile, peak shape, and recovery are required to be consistent by GMP/GLP protocols. During a recent method re-qualification at Lexigen Pharmaceuticals, several variables were investigated to eliminate non-specific binding and increase the robustness of an established antibody separation method. Previous protocol required an unacceptable two-week "break-in" period to equilibrate the column with the mobile phase. Reduction of non-specific binding of proteins to SEC stationary phases can be achieved by exploring variables that affect either the ionic strength or hydrophobicity of the mobile phase. Typically, changes in mobile phase composition, such as buffer type and pH, salt concentration, the addition of organic modifiers, as well as the evaluation of competitive columns are the usual strategies to correcting non-specific adsorption. In this case, these options did not result in an improvement. Thus, an alternative approach to increase the chaotropic nature of the mobile phase was investigated. Concurrently, the buffer concentration was reduced to a minimum buffering capacity and the modifying salt was changed to NaClO4. A drastic improvement on the performance of the method was evident.


Experimental Conditions:
Gel filtration was performed with a 4.6mmID x 30cm TSKgel Super SW3000 column packed with 4m particles containing 250 pores. The original run
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