Downstream Processing :- An Overview and Process steps

Definition

Downstream processing is concerned with the extraction of desired products from the upstream biomass. It refers to the purification of biosynthetic products from their crude preparation like tissue fluids or fermentation broth. In downstream processing, the products which are desired are purified and separated through fermentation or enzyme reactions. The desired products are temperature sensitive, have multiple items to separate, in aqueous medium they are diluted, may have properties similar to contaminants.

Downstream Processing means recovery of desired products from its crude extract. For eg. Recovery of antibiotics from fermentation broth to make it as a tablet form.

Aim

• Downstream processing involves the purification of a specific molecule such as a protein or nucleic acid from the wet biomass. The main aim is to purify the active pharmaceutical ingredient (API) to make it in a tablet form.

Downstream Methods

Downstream processing embraces wide range of fractionation and purification methods. They are enlisted below:-

-Filtration

-Chromatography

-Distillation

-Centrifugation and precipitation

Principles of Downstream Processing

Most important principle of downstream processing is that one size definitely does not fit all(no universally applicable arrangement of operational units which define ideal downstream processing). A combination of two to three purification techniques need to be employed for successful separation of desired product. The simplest and cheapest separations should be carried out early in the process. Where expensive and complex separations are necessary, these should be run towards the end of process.

Downstream Process Steps

The steps of downstream process are:-

-Cell disruption and harvesting

-Cell lysis

-Clarification

-Capture

-Purification and Polishing

-Formulation, fill and finish

Harvesting, Cell Disruption & Clarification

The first downstream processing steps are cell harvesting and cell disruption followed by clarification to remove insoluble and particulate matter. The first processing step is to harvest the cells by separating the cellular biomass from the fermentation broth. Generally achieved by Centrifugation with separator’s or tangential flow filtration on microporous membranes.

Cell lysis

Cell lysis is achieved by mechanical disruption under pressure, releasing the desired product into the lysate. This is followed by another round of tangential flow filtration using a finer microporous membrane to remove the subcellular debris. Next step depends on whether the product is soluble or accumulates as inclusion bodies.

Clarification

If product is soluble, it ends up in the clarified permeate and can be sent to next processing step for product capture. If the product accumulates a inclusion body, continuous Centrifugation can separate the dense inclusion bodies from other particulate matter. Additional solubilizing step is required involving the use of strong, denaturing buffers, prior to protein refolding and recovery by high resolution chromatography.

Capture

The starting point for product capture is a clean filtrate which contains desired product plus many different contaminants. Capturing is usually achieved by first chromatography step which is designed as rough cut to eliminate as many impurities as possible. Capture or polish model approach is currently used where the capturing step achieves most of the purification in one module and significantly reduces the feed volume by simultaneously concentrating the product in the flow-through.

Purificaion And Polishing

Purificaion refers to the elimination of all contaminating molecules and the presentation of the desired product in a pure and homogeneous form. It generally involves a series of chromatography and filtration procedures, polishing being the final stages of purification. It is the most challenging stage because it involves separating the desired product from contaminants.

Formulation, Fill And Finish

The operation that occur subsequent to protein purification, such as freeing of the purified protein bulk, thawing of the bulk, formulation(excipient addition), sterile filtration, filling, freeze dying and inspection are termed as Formulation and fill-finish operations.