Recombinant proteins aggregated into IBs are usually inactive and denatured. The major challenge in processing IBs is being able to recover biologically active and/or soluble protein in high-yield. To accomplish this, the protein in these IBs must be isolated, solubilized, and refolded in vitro to its active state prior to downstream processing (Figure 3).
After isolation the IBs are resuspended and solubilized in buffer containing a strong denaturant (6-M Guanidinium Hydrochloride, G-HCl/Urea).
The solution at this stage will contain the solubilized IB along with the cellular debris and residual media and cellular proteins. The solubilized IB can then be separated from the cell debris using NFF, TFF, or centrifugation, where the IB will be contained in the filtrate, permeate, or the centrate. This solubilized IB then needs to be refolded.
To reduce the protein aggregation, the refolding process is usually carried out at low-protein concentrations (10100 g/mL). The process begins with the gradual removal of the denaturing agent (Urea/G-HCl) that can be achieved by methods including dialysis, rapid or slow dilution, or chromatography. Filtration steps to remove residual aggregates or contaminants are included at intervals appropriate to the particular process.
Clarification using TFF or NFF requires careful selection of filter or membrane type. Specific feed information, such as particle size and properties, must be considered along with the composition of the feed solution. Various vendors offer a portfolio of products that are suitable for these applications.