Effect of sample loop dimension on lysozyme refolding in size-exclusion chromatography

The formation of misfolded protein aggregates, in particular inclusion bodies, has been widely considered as the major hindrance of good yield in refolding processes. To enhance the performance of protein refolding, extensive efforts were directed toward seeking out methods or means to reduce the aggregate production during the refolding process. Since simultaneous refolding and separation can be feasibly achieved within the packing matrices, size-exclusion chromatography (SEC) has been regarded as an efficient buffer exchange method to enhance protein refolding performance As of now, the effect of the process or operating parameters has yet to be thoroughly investigated. The present work is aimed at understanding how aggregate formation, as well as renaturation yield, varied with the diameter or length of sample loop in size-exclusion chromatography refolding process. Our results showed that not much difference was found in the patterns of aggregate formation for the contraction and the control cases. However, the formation of an additional peak was observed in the expansion cases. In addition, the amount of aggregates was not dependent on the sample loop diameter or length, but instead, influenced by injection volume and protein concentration. It was further concluded that a sample with large volume and low concentration was preferable for refolding process. We believe that the outcome from this work may shed light on the development of a more effective strategy for refolding processes.