Question

Describe the different steps in down-stream processing required for purification of a functional recombinant protein produced in bacteria as an inclusion body.

Answer 1

The aim of downstream process should be to deliver the highest yield of the purest product at the shortest time/cost. Downstream processing contains three main stages, known as initial recovery including methods of extraction or isolation, purification involving removal of contaminants, and polishing by removal of specified contaminants formed during isolation and purification.

Initial recovery involves the separation between cell and supernatant or clarification of broth. Purification techniques such as centrifugation, filtration, sedimentation, and flotation can be employed based on the type of product. The recovery of proteins expressed as inclusion bodies or recombinants produced by E. coli, the process involves an extra step of protein refolding through buffer exchange. This may contribute to increase in production time and costs for intracellular biomolecules.

The purification process must be robust, reliable, and capable of removing the product-related impurities ensuring product safety. The Downstream process must consider parameters like purity, the speed of process, overall recovery and yield. Chromatography allows for high resolution and is traditional process protein purification and polishing.

Chromatography

Different strategies of chromatography can be employed for separation and purification of nucleic acids, peptides, and proteins. The chromatographic techniques frequently used include affinity, ion-exchange, hydrophobic interactions, size exclusion, and mixed-mode chromatography.

Affinity chromatography methods usually eliminate purification steps to increase yields. Traditional chromatographic techniques include particle-based resins, batch mode operation, and packed columns. The EBA chromatography is a 3-in-1 process in which the product is directly harvested from the cell suspension, combining clarification, concentration, and initial purification. It efficiently removes precipitates and captures target proteins from refold pools of E. coli-based production and it promotes enhanced recovery of Human Epidermal Growth Factor from E. coli.

Alternative separation techniques include affinity precipitation, high-performance tangential flow filtration. The filtration strategies are based on thiophilic and affinity interactions, two-phase aqueous systems, high-gradient magnetic fishing, preparative electrophoresis, and isoelectric focusing. Magnetic separation with immunocapture is employed by techniques used in purification kits, Filtration with ion-exchange membranes and flow-through chromatography for polishing steps. These techniques can remove contaminants such as host-cell proteins, nucleic acids, and viruses with increased flow rates, reduce buffer consumption and time, when compared to traditional polishing