Downstream Processing

Downstream processing refers to the process steps taken following fermentation of microorganisms to the final product specification. 

The fermentation is sometimes referred to as a bioreactor. It will mean the production of:-

• biomass in the form of cells
• production of extracellular compounds and products i.e. products that are released by the cells into the fermentation medium 
• production of intracellular products i.e. compounds of interest that are retained within the cell wall of the microorganism.

Downstream processing can be as simple as is needed to produce an economically viable process. It can however involve many different steps in the process depending on the type of product to be generated.

Typical Examples of Fermentation Products For Further Processing

• The cell itself.

– Baker’s yeast and single cell protein  are typical large-scale fermentations which often produce biomass at a high concentration of the order of 30g/litre.

• Extracellular products.

-Compounds manufactured by this method include ethanol as in a brewery, organic acids such a citric acid, antibiotics, enzymes and amino acids. The food-grade organic acids and alcohols are produced in concentrations up to 100g/litre. Antibiotics and enzymes might be produced in concentrations up to 20 g/l.

• Intracellular products

-Compounds that are produced and retained within the cell usually have to be released before they can be used further. Typical molecules produced this way are recombinant DNA proteins, metabolic enzymes, pigments etc.

Ideally, extracellular products are preferred for purification because the medium into which they are secreted is simpler. The products, especially sophisticated ones such as proteins will have undergone post-translational modification.

Classification Of Bioproducts For Processing

The products of interest range from small molecules including gases through to macromolecules such as proteins, nucleic acids and nucleotides, polysaccharides including gums.

The Main Steps In Downstream Processing Affecting Clarification

The main step following fermentation is usually to separate the cells (biomass) from the fermentation medium, broth or supernatant. This requires some form of solid-liquid separation which separates the cells from the fermentation medium.

If the product is in the cell, then some type of cell disruption is needed to release the cell contents from which the cell. The cell debris is usually discarded.

When the product is extracellular or intracellular and is now present in some form of broth, there is a usually a recovery step which will involve concentration or modification of the broth so that the product of interest can be purified further.

The purification step can be highly sophisticated.  As a technology purification is the preferential concentration and isolation of a specific product from a mixture of other biomolecules.

The specification for the product will dictate the level of purification required. The extent and capability for purification depends on the source of the product, how it will be applied and its cost.

Following purification, product polishing is needed to make the product ready for sale or to meet particular product criteria.

The Criteria Or Driving Forces For Effective Primary Downstream Processing

In most cases, downstream processing is about recovering as much product as possible. Ideally any process developed must be consistent, repeatable and reliable. It should also involve as few steps as possible because an increasing number of steps implies rising capital and operating costs. 

Given that fermentations usually involve high levels of volume, the purification will rely on reducing the volume of liquid as much as possible. It is not unusual to have a 1000 fold reduction in volume with a corresponding increase in the concentration of the product.

Processing the product needs to occur as soon as the fermentation is finished with or when the product leaves the bioreactor. Any delay can spoil the product or involve expensive holding operations.

Type Of Unit Operations Involved

Products can be recovered by the following methods:

• centrifugation and decanting
• flocculation and flotation
• membrane separation (microfiltration, ultrafiltration)
• cell disruption
• precipitation using either organic solvents or salt
• evaporation
• organic solvent extraction
• spray and drum drying 

Scaling Up Issues

• the equipment size may be the limiting step
• process volumes are larger increase process times
• a reduction in yield
• increased material costs
• disappearance in performance efficiencies

Which Recovery Method To Use

One of the big issues in recovering a product from fermentation is whether it is extracellular or intracellular. It may also have a particular chemical state.

Usually the product of interest is in a fairly dilute system which will require its concentration. 

There are minimum purity requirements.  The presence of impurities too will also dictate what type of purification is needed.

The market price is often the determining factor.

Removing Particulates And Cells

The separation of the broth from the cells, cell debris and other particulates is often one of the most important steps of the process. The sort of unit operation needed to achieve this includes centrifugation, filtration, flocculation etc. (Pieracci et al., 2018).

The selection of equipment significantly affects subsequent operations. The choice of the primary clarification step is probably most significant because high quality, robust clarification will help later purification processes.

Sizing the equipment needed has to consider different types of filtration media, the quality and volume.

To account for process variability, scaling up must include a safety factor of between 20 and 60%. 

Undersized clarification processes  produce process deviations which can be extremely costly and lead to batch loss and delays in production. The impact is especially acute in GMP manufacturing. If you oversize the processing equipment it can produce unnecessary yield losses through incomplete product or biomass recovery.

 Early identification of issues is also a key consideration when preparing documentation for GMP purposes.

Centrifugation

A very effective separation method which is used to remove particulates from a liquid using gravitational force. The degree of separation depends on the particle size and density, and differences in viscosity between cells and the fermentation medium. The most commonly encountered types of centrifuge are the disc bowl, sedicanter, tubular bowl types.

Compared to filtration, a centrifuge is a substantially more expensive item but it makes up for the capital expenditure by being relative cheap to run because the cost of consumables is lower. A filtration unit often needs replacement membranes and the use of materials to clean the membranes

Product Purification

Following removal of cells and cleaning up the product stream, the next step is to purify the product. These are commonly proteins but they could be small molecules such as amino acids, carbohydrates etc.

In many cases the contaminants that need to be separated from the product of interest have similar properties from a physical and chemical perspective. Such separations are expensive to perform and if the similarities are especially close they are almost nigh impossible to achieve without some considerable expenditure. As we mentioned earlier, the specification of the final product is highly critical here so it often means extremely sophisticated and sensitive processing equipment is required to achieve the desired quality. Compared to fermentation and clarification, cost of subsequent purification is the most expensive part of the process.

Different types of chromatography can be employed to achieve near perfect separations. Consider:

• ion-exchange chromatography,
• affinity chromatography,
• hydroxyapatite chromatography
• size exclusion (gel filtration)
• reversed phase liquid chromatography.

Other methods including crystallization and fractional precipitation can also be considered. All these methods can be used sequentially to achieve the required level of purification. 

The process of chromatography is a significant process purification step. It relies on passing a relatively clean product called the mobile phase through a stationary phase which is a matrix of materials. The product is separated from the other molecules whilst the waste material passes through. 

Ion-exchange chromatography relies on charge differences in the matrix to bind to oppositely charged groups on the product. The stationary phase is usually resins and particulates carrying charged functional groups that retain the compound of interest. Altering the charge of the subsequent separation fluids dislodges the product of interest which is then processed further.

Crystallization is a process for producing solid crystals that precipitate out of solution. It is one of the oldest processing methods known and is a classic solid-liquid separation method.

Freeze-Drying and Lyophilization

 The product in solution is dried by freezing under vacuum. The frozen water sublimes to a gas without forming a liquid and leaving the product as a solid.

At this moment in time batch processing is still the most common industrial approach where downstream processing is concerned. In the last decade, continuous methods have been examined but they are not yet truly commercially ready although it is not that far off a process (Jungbauer, 2013) .

References

Jungbauer, A. (2013). Continuous downstream processing of biopharmaceuticals. Trends in Biotechnology, 31(8), pp. 479-492.

Pieracci, J.P., Armando, J.W., Westoby, M., Thommes, J. (2018) Industry Review of Cell Separation and Product Harvesting Methods. In: Biopharmaceutical Processing: Development, Design, And Implementation of Manufacturing Processes. Chapt. 9 Edt. G. Jagschies, E. Lindskog, K. Lacki, P. Galliher. Elsevier.