Improper refolding

Incubation in solutions of low denaturant concentration can sometimes cause inactivation. Formation of an incorrectly folded form of human carbonic anhydrase in 1.7M GuHCl which subsequently aggregates has been documented (77). Other proteins have also been observed to improperly refold at relatively low concentrations of denaturant These proteins include 6-galactosidase, tryptophanase, and elastase (6). 

For products located in inclusion bodies, product extraction and removal of extraction solutions must be validated. The extent to which the product can be refolded must be defined. Validation efforts are directed toward demonstrating consistency of refolding as well as removal downstream of any improperly folded product. 

Although aggregation is the predominant means by which proteins become inactivated during refolding, several other inactivation pathways have also been observed. Proteins can be inactivated by thiol-disulfide exchange or alteration of the primary structure by chemical modification of amino acid side chains. In addition, refolded proteins may be inactivate due to the absence of prosthetic groups and metals or because of improper association of the subunits in multimeric proteins (79). 

---