Step change of mobile phase flow rates to enhance protein folding in size exclusion chromatography

Minimal aggregation formation between injector and column inlet and enough residence time inside the column were identified to be the key for proper proteins refolding by size exclusion chromatography (SEC). Therefore, a step change of mobile phase flow rate strategy was developed. That is, during the injected denatured protein (sample) traveling from injector to column inlet, a higher rate of mobile phase should be applied to reduce aggregation. Then, a lower flow rate should be used to allow enough time for protein to refold inside the column. The refolding of denatured lysozyme in SEC by this method was shown to reduce the aggregation and thus resulting in good refolding yields, though not completely. Combining this method together with our previously proposed chaperon solvent plug strategy could obtain complete recovery of denatured protein, both mass and activity recoveries.