Recovery of therapeutic proteins from inclusion bodies: problems and process strategies.

The initial recovery of product in the form of inclusion bodies has many attractive process features which have been exploited by the biotechnology industry. The product, being sequestered in a near homogeneous state in dense granules of tight size distribution, may be recovered at high purities on a multi-kilogram scale by cell disruption followed by centrifugation [ 5-61. Soluble cellular components may be depleted by cycles of centrifugation and resuspension in buffer, leaving cell debris and associated membrane-bound components as the major Contaminants of inclusion body preparations. Some of these membrane proteins in the debris can be removed from the insoluble inclusion body product by selective extraction using buffers such as Triton X-l00/urea, originally developed as general methods for solubilizing membrane proteins. Alternatively, the density difference between cell debris and inclusion body granules can be exploited by differential centrifugation in sucrose buffers, inclusion body material being collected in the solids discharge of the centrifugal separator and cell debris rejected in the supernatant. Using these methods large quantities of highly enriched recombinant product have been processed routinely. The requirement for the removal of cell debris and the soluble constituents of the host is not necessarily obligatory, especially when accumulation levels of > 50% total cell protein have been achieved. Cell breakage by high pressure homogenization and the subsequent centrifugation operations are expensive in terms of fixed capital costs and energy consumption. The alternative strategy of recovering the inclusion body protein by directly solubilizing the cell paste in a strongly denaturing solvent, such as 8 M-guanidinium hydrochloride has therefore been advocated by some workers [7]. The choice may sometimes be determined more by constraints downstream than by the economics of recovery. For example, the recovery of renatured foot and mouth disease virus VPI coat protein, expressed in E. coli inclusion body fractions, is reduced by the presence of high molecular mass cellular components [ 8 ] and many process schemes employ an initial purification step before the removal of the solubilizing solvent [9, lo]. Most of the commonly used solubilizing agents, guanidine hydrochloride, acid or alkaline buffers and ionic detergent, reduce the choice of these initial pre-refolding purification steps to gel filtration. Urea solvents have found favour as solubilizing agents because their use permits consecutive subtractive ion-exchange chromatography, thereby removing nucleic acids, phospholipids and electronegative contaminant proteins from the process stream. The major contaminants found in inclusion body preparations are peptidoglycans, lipids, nucleic acids, lipopolysaccharide and membrane-associated proteins. Although most of these components will dissolve under the harsh conditions often required to solubilize inclusion bodies, these substances are often poorly soluble under renaturing conditions and the bulk will precipitate without influencing the refolding of the recombinant product. The process stream may then be enriched further by centrifugation and filtration.