DOWNSTREAM PROCESSING

The various procedure involved in the actual recovery of useful products after fermentation or any other process together constitute Downstream Processing. It is a very important step in the manufacture of different product in pharmaceutical industry (such as antibiotics, hormones, antibodies, vaccine, enzymes), Food industry etc. The product is either present in the cell, in the medium or both. The concentration of product is generally low, in either cases, and it is mixed with other molecules from which it has to be separated.

Industrial fermentations has both upstream processing (USP) and downstream processing (DSP) stages. USP involves all factors and processes leading to, and including, the fermentation, and consists of three main areas.

 1.      First aspect of USP deals with the producer microorganism. They include obtaining a suitable microorganism, industrial strain improvement to enhance productivity and yield, maintenance of strain purity, preparation of a suitable inoculum and the development of selected strains to increase the economic efficiency of the process.

2.      Second aspect of USP involves fermentation media, especially the selection of suitable cost-effective carbon and energy sources, along with other essential nutrients. Media optimization is a vital aspect of process development to ensure maximum yield and profit.

3.      The third component of USP relates to the fermentation, which is usually performed under controlled conditions. The ultimate aim is to optimize the growth of the organism or the production of a target microbial product.

 DSP includes all processes following the fermentation. It aims at efficient, safe and reproducible recovery of the target product with biological activity, purity, etc. The target product may be recovered by processing the cells or the spent medium depending upon whether it is an intracellular or extracellular product. Maximum recovery yield and minimum costs are therefore important to be ensured.

Two critical factors to be remembered during DSP

·         The level of purity of product which is usually determined by the specific use of the product.

·         The product’s biological activity is very important and must be retained.

 Fermentation factors affecting DSP include

·         The properties of microorganisms, particularly morphology, flocculation characteristics, size and cell wall rigidity. These factors have major influences on the filterability, sedimentation and homogenization efficiency.

·         The presence of fermentation by-products, media impurities and fermentation additives, such as antifoams, may interfere with DSP steps and accompanying product analysis.

·         Choice of fermentation substrate influences subsequent DSP. A cheap carbon and energy source containing many impurities may provide initial cost savings, but may require increased DSP costs. Overall cost savings may be achieved with a more expensive but purer substrate.

·         The physical and chemical properties of the product, along with its concentration and location, are key factors as they determine the initial separation steps and overall purification strategy. It may be the whole cells themselves that are the target product or an intracellular product, possibly located within an organelle or in the form of inclusion bodies. Alternatively, the target product may have been secreted into the periplasmic space of the producer cells or the fermentation medium.

·         Stability of the product also influences the requirement for any pretreatment necessary to prevent product inactivation and/or degradation. 

·         Adopting methods that use existing available equipment may be more cost-effective than introducing more efficient techniques which may need new facilities.

Steps in DSP 

DSP can be divided into a series of distinct unit processes linked together to achieve product purification.

The number of steps is kept to a minimum to reduce cost. Also, even though individual steps may obtain high yields, the overall losses of multistage purification processes may be huge. So, lesser the treatment steps, lesser the overall loss and cost.

The specific unit steps chosen will be influenced by the economics of the process, the required purity of the product, the yield attainable at each step and safety aspects.

In many cases, integration of fermentation and DSP is preferred to increase productivity, decrease the number of unit operations and reduce both the overall time and costs.

The fermentation broth at the time of harvesting is a complex heterogeneous mixture of cell debris, metabolic products, and unused portions of the medium. The required product usually forms a small proportion of the broth. The product required could be the cells themselves such as in yeast manufacture, or within the cells (such as in streptomycin or some enzymes) or free in the medium as with penicillin.

In a few cases no separation takes place such as in the acetone butanol fermentation, where the entire beer is used. In most cases, however, the separation methods used are filtration, centrifugation, decantation, and foam fractionation. Where the required fraction is in the cells then much of the impurities are removed with the filtrate after the cells have been isolated. 

The various methods used in solids removal are discussed below with the approximate level of purification obtained in each stage is shown below. The procedure followed within each stage depends on the material being extracted.

 

Step Process	Recovery/Purity (%)
1a. Cell harvesting	0.1-1.0 (if product is soluble)
	90-99 (if product is cell such as yeasts)
Filtration
Centrifugation
Decantation
1b. Disruption of cells
2. Primary isolation of the product	1-10
Adsorption and/or ion exchange
Solvent extraction
Precipitation
Ultracentrifugation
3. Purification	50-80
Fractional precipitation
Chromatography (adsorption, partition, ion exchange, affinity)
Chemical derivatization
Decolorization
4. Final product isolation	90-100
Crystallization
Drying
Solvent removal

DSP for the purification of products in the soluble portion of fermented broth

 

The various steps of Downstream Processing involve:

1. Separation
2. Cell disruption
3. Extraction
4. Isolation
5. Purification
6. Drying

 

1.     SEPARATION OF PARTICLES

·       It is first step of DSP and usually involve the separation of solids substances, from the liquid media.

·       Recovery of extracellular proteins is from the clarified medium, whereas disrupted cell preparations are used for both intracellular proteins and those within the periplasmic space.

·       In the case of some recombinant proteins expressed at high levels, they sometimes form inclusion bodies that are released by cell breakage.

·        Following cell disruption, soluble proteins are usually separated from cell debris by filtration

  

• v FILTRATION– it is used for filamentous fungi and bacteria.

 Different techniques of filtration are as follows:

1. Surface filtration
2. Depth filtration
3. Centrifugal filtration
4. Rotating drum vacuum filtration

  

Filtration

Conventional filtration of liquids containing suspended solids involves depth filters composed of porous media (cloth, glass wool or cellulose) that retain the solids and allow the clarified liquid filtrate to pass through. As filtration proceeds collected solids accumulate above the filter medium, resistance to filtration increases and flow through the filter decreases. These techniques are generally useful for harvesting filamentous fungi, but are less effective for collecting bacteria. The two main types of conventional filtration commonly used in industry are as follows.

 

1 Plate and frame filters or filter presses, which are industrial batch filtration systems. They are normally in the form of an alternating horizontal stack of porous plates and hollow frames. The stack is mounted in a support structure where it is held together with a hydraulic or screw ram. Filter cloths are held in place between the plates that contain flow channels for the feed and permeate streams. This essentially forms a series of cloth-lined chambers into which the cell suspension is forced under pressure. Following batch filtration, the apparatus must be dismantled to remove the collected filter cake. These systems are used for harvesting microorganisms from fermentations, including the preparation of blocks of baker’s yeast, the recovery of protein precipitates and the dewatering of sewage sludge. Similar horizontal and vertical pressure leaf filters are also available.

2 Rotary vacuum filters are simple continuous filtration systems that are used in several industrial processes, particularly for harvesting fungal mycelium during antibiotic manufacture, for baker’s yeast production and in dewatering sludge during waste-water treatment. The device comprises a hollow perforated drum that supports the filter medium. This drum slowly rotates in a continuously agitated tank containing the suspension to be filtered. Solids accumulate on the filter medium as liquid filtrate is drawn, under vacuum, through the filter medium into the hollow drum to a receiving vessel. As the drum rotates, collected solids held on the filter medium are removed by a knife that cuts/sloughs them off into a collection vessel.

Filter media may be precoated with a filter aid, e.g. Kieselguhr (diatomaceous earth), which can be continuously replenished. A filter aid improves filtering efficiency by building up a porous, permeable and rigid structure—the filter cake—that retains solid particles and helps flow control.

 The rate of filtration (flow of filtrate), is determined by the resistance of the cake and the filtration medium. In terms of biosafety, neither filtration system is suitable for processing toxic products, pathogens or certain recombinant DNA microorganisms and their products.

Membrane Filtration

Modern methods of filtration involve absolute filters rather than depth filters. These consist of supported membranes with specified pore sizes that can be divided into three main categories. They are, in decreasing order of pore size, microfiltration, ultrafiltration and reverse osmosis membranes. The suspension to be filtered is pumped across the membrane (cross-/tangential-flow) rather than at a right angle to it, as occurs with conventional filtration methods. This slows fouling of the membrane by particulate materials. Particles whose size is below the membrane ‘cut-off’ will pass through the membrane to become the ultrafiltrate or permeate, whereas the remainder is retained as the retentate.

Microfiltration is used to separate particles of 10-2μm to 10 μm, including removal of microbial cells from the fermentation medium. This method is relatively expensive due to the high cost of membranes, but it has several advantages such as quiet operation, lower energy requirements, the product can be easily washed, good temperature control is possible, containment is readily achieved and no bioaerosols are produced. Consequently, it is suitable for handling pathogens and recombinant microorganisms.

Ultrafiltration is similar to microfiltration except that the membranes have smaller pore sizes, and are used to fractionate solutions according to molecular weight, normally within the range 2000–500000Da. The membranes are composed of a thin membrane with pores of specified diameter providing selectivity, lying on top of a thick, highly porous, support structure. A membrane manufactured with an exclusion size of 100000Da, for example, should produce a retentate of proteins and other molecules over 100000 Da and an ultrafiltrate of all molecules below 100000 Da. Several of these ultrafiltration units can be linked together to produce a sophisticated purification system. These methods are extensively employed for the purification of proteins, and for separating and concentrating materials.

Ultrafiltration is also effective in removing pyrogens (bacterial cell wall lipopolysaccharides), cell debris and viruses from media, and for whey processing. Another variation on the ultrafiltration system is diafiltration, where water or other liquid is filtered to remove unwanted low molecular weight contaminants. This can be used as an alternative to gel filtration or dialysis for removing ammonium sulphate from a protein preparation precipitated by this salt, for changing a buffer or in water purification.

Reverse osmosis is used for dewatering or concentration steps and has been employed to desalinate sea water for drinking. In osmosis water will cross a semipermeable membrane if the concentration of osmotically active solutes, such as salt, is higher on the opposite side of the membrane. However, if pressure is applied to the ‘salt side’ then reverse osmosis will occur, and water will be driven across the membrane from the salt side. This reversal of osmosis requires a high pressure, consequently, a strong metal casing is required to house this equipment.

 

• Flocculation and flotation– It is used for small bacterial cells which are difficult to separate even by centrifugation.
• Flocculation: It involves the aggregation of cells which may be induced by inorganic salt, minerals, hydrocolloids and organic polyelectrolytes.
• Floatation: If flocculation isn’t effective then very minute gas bubbles is made by sparging. The gas bubble adsorbs and surround the cell, raising the gas bubbles to the surface of media in the form of foam.

·       The resultant supernatant, containing the proteins, is then processed by several different types of methods, such as precipitation/salting out of proteins and ultrafiltration.

 Precipitation is achieved by the addition of inorganic salts at high ionic strength, usually in the form of solid or saturated solutions of ammonium sulphate.

·       Ammonium sulphate is popular because of its high solubility, low toxicity and low cost. The solubility of the salt varies with temperature, so strict temperature control is required. Reduction of protein solubility can also be achieved by adding organic solvents, such as acetone, ethanol and isopropanol. This is performed at low temperature causing precipitation of the proteins.

 v CENTRIFUGATION

 Centrifugation may be used to separate particles and is also suitable for some liquid– liquid separations. Its effectiveness, depends on particle size, density difference between the cells and the medium, and medium viscosity. Instead of simply using gravitational force to separate suspended particles, if a centrifugal field is applied, the rate of solid–liquid separation is significantly increased and much smaller particles can be separated. The faster the operating speed and the greater the distance from the centre of rotation, the faster the sedimentation rate. Centrifuges can be compared using the relative centrifugal force (RCF) or g number (the ratio of the velocity in a centrifuge to the velocity under gravity). The choice of centrifuge depends on the particle size and density, and the viscosity of the medium.

Higher-speed centrifuges are required for the separation of smaller microorganisms, such as bacteria, compared with yeasts. For example, relatively slow centrifugation effectively recovers residual yeast cells remaining in beer after the bulk has sedimented out. But, an RCF of 20000g may be required to recover suspended bacterial cells, cell debris and protein precipitates from liquid media. Advantages of centrifugation include the availability of fully continuous systems that can rapidly process large volumes in small volume centrifuges. Centrifuges are steam sterilizable, allowing aseptic processing, and there are no consumable costs for membranes, chemicals or filter aids.

However, the disadvantages of centrifugation are the high initial capital costs, the noise generated during operation and the cost of electricity. Also, physical rupture of cells may occur due to high shear and the temperature may not be closely controllable, which can affect temperature-sensitive products. Bioaerosol generation is a further major disadvantage, particularly when centrifuges are used for certain recombinant DNA organisms or pathogens. Under these circumstances the equipment must be contained

 INDUSTRIAL CENTRIFUGES

Centrifuges can be divided into small-scale laboratory units and larger pilot- and industrial-scale centrifuges. Laboratory batch centrifuges include, bench-top, high-speed and ultracentrifuges, capable of applying RCFs of 5000– 500000g. Although industrial batch centrifuges are available, for most industrial purposes semicontinuous and continuous centrifuges are required to process the large volumes involved.

 Four main types of industrial centrifuge are commonly used.

1. Tubular centrifuges usually produce the highest centrifugal force of 13000–17000g. They have hollow tubular rotor bowls providing a long flow path for the suspension, which is pumped in at the bottom and flows up through the rotor. Particulate material is thrown to the side of the bowl, and clarified liquid passes out at the top for continuous collection. Since the particulate material accumulates on the inside of the bowl, there must be periodic removal of solids for efficient working

 2. Multichamber bowl centrifuges consist of a bowl that is divided by vertically mounted interconnecting cylinders and are capable of operating at 5000–10000g. The liquid feed passes from the centre through each chamber in turn, and the smaller particles collect in the outer chambers.

 3. Disc stack centrifuges can operate at 5000–13000g. The centrifuge bowl contains a stack of conical discs whose close packing aids separation. As liquid enters the centrifuge particulate material is thrown outwards, impinging on the underside of the cone discs. Particles then travel outwards to the bowl wall where they accumulate. These centrifuges usually have the facility to discharge the collected material periodically during operation.

 4 Screw-decanter centrifuges operate continuously at 1500–5000g and are suitable for dewatering coarse solid materials at high solids concentrations. They are used in sewage systems for the separation of sludge, and for harvesting yeasts and fungal mycelium.

 

2.     CELL DISRUPTION

• Disruption of microbial cell is usually difficult because of their small size, rigid cell walls and high osmotic pressure inside the cells. This is required in case of recovery of intracellular products.

Disruption of cell is generally achieved by mechanically, lysis or drying:

1. Mechanical cell disruption: this involves the uses of shear, E.g.-, colloid mill, ball mill grinder etc., homogenizer and ultrasound.
2. Drying: it involves the drying of cells by adding the cells into a huge amount of cold acetone and extracted using buffer or salt solution.
3. Lysis: lysis of microbial cells may be achieved by chemical means, e.g., salt or surfactants, osmotic shock, freezing, or Lytic enzymes, e.g., lysozyme, etc.

 

Some target products including many enzymes and recombinant proteins, form inclusion bodies and are intracellular. Therefore, methods are required to disrupt the microorganisms and release these products. The breaching of the cell wall/envelope and cytoplasmic membrane can pose problems, when cells possess strong cell walls. General problems associated with cell disruption include the liberation of DNA, which can increase the viscosity of the suspension. This may also affect further processing, such as pumping the suspension on to the next unit process and flow through chromatography columns. A nucleic acid precipitation step or the addition of DNase can help to prevent this problem. If mechanical disruption is used then heat is invariably generated, which denatures proteins unless appropriate cooling measures are implemented. Cell disruption can be achieved by both mechanical and non-mechanical methods. The disruption process is often quantified by monitoring changes in absorbance, particle size, total protein concentration or the activity of a specific intracellular enzyme released into the disrupted suspension.

 Mechanical cell disruption methods

Several mechanical methods are available for the disruption of cells. Those based on solid shear involve extrusion of frozen cell preparations through a narrow orifice at high pressure. This approach has been used at the laboratory scale, but not for large-scale operations.

 Methods utilizing liquid shear are generally more effective. The French press (pressure cell) is often used in the laboratory and the high-pressure homogenizers  may be used for bacterial and yeast cells, and fungal mycelium. In these devices the cell suspension is drawn through a check valve into a pump cylinder. It is forced under pressure and cell disruption is primarily achieved by high liquid shear in the orifice and the sudden pressure drop upon discharge causes explosion of the cells.

 The rate of protein release (efficiency of disruption) is independent of the cell concentration, but is a function of the pressure exerted, the number of cycles through the homogenizer and the temperature. A problem with this method of cell disruption is that all intracellular materials  are released. As a result, the product of interest must be separated from a complex mixture of proteins, nucleic acids and cell wall fragments. In addition, proteins may be denatured if the equipment is not sufficiently cooled and filamentous microorganisms may block the discharge valve. When used for certain categories of microorganisms, the homogenizers have to be contained to prevent the escape of aerosols.

 On a small scale, manual grinding of cells with abrasives, usually alumina, glass beads, kieselguhr or silica, can be an effective means of disruption, but results may not be reproducible. In industry, high-speed bead mills, equipped with cooling jackets, are often used to agitate a cell suspension with small beads (0.5–0.9μm diameter) of glass, zirconium oxide or titanium carbide. Cell breakage results from shear forces, grinding between beads and collisions with beads. The efficiency of cell breakage is a function of agitation speed, concentration of beads, bead density and diameter, broth density, flow rate and temperature. 

Ultrasonic disruption of cells involves cavitation, microscopic bubbles or cavities generated by pressure waves. It is performed by ultrasonic vibrators that produce a high-frequency sound. However, this technique also generates heat, which can denature thermolabile proteins. Rod-shaped bacteria are often easier to break than cocci, and Gram-negative organisms are more easily disrupted than Gram-positive cells. Sonication is effective on a small scale, but is not routinely used in large-scale operations, due to problems with the transmission of power and heat dissipation.

 Cell disruption is a somewhat neglected area of bioprocessing, as there has been relatively little innovation and progress. Newer disruption systems are being developed to give improved large-scale disruption, often with integral containment. They include a newly designed ball mill, the CoBall Mill, the Microfluidics impingement jet system and the Glass-col nebulizer.

 Non-mechanical cell disruption methods

An alternative to mechanical methods of cell disruption is by autolysis, osmotic shock, rupture with ice crystals (freezing/thawing) or heat shock. Autolysis, for example, has been used for the production of yeast extract and other yeast products. It has the advantages of lower cost and uses the microbes’ own enzymes, so that no foreign substances are introduced into the product.

 Osmotic shock is often useful for releasing products from the periplasmic space. This may be achieved by equilibrating the cells in 20% (w/v) buffered sucrose, then rapidly harvesting and resuspending in water at 4°C.

 A wide range of other techniques have been developed for small-scale microbial disruption using various chemicals and enzymes. However, some of these can lead to problems with subsequent purification steps. Organic solvents, usually acetone, butanol, chloroform or methanol, have been used to liberate enzymes and other substances from microorganisms by creating channels through the cell membrane. Simple treatment with alkali or detergents, such as sodium lauryl sulphate or Triton X-100, can also be effective.

 Several cell wall degrading enzymes have been successfully employed in cell disruption. For example, lysozyme, which disrupts the peptidoglycan of bacterial cell walls, is useful for lysing Gram-positive organisms. Addition of ethylene diamine tetraacetic acid (EDTA) to chelate metal ions also improves the effectiveness of lysozyme and other treatments on Gram-negative bacteria. EDTA has the ability to sequester the divalent cations that stabilize the structure of their outer membranes. Enzymic destruction of yeast cell walls can be achieved with snail gut enzymes. These enzyme preparations are also useful for producing living yeast spheroplasts or protoplasts. The antibiotics penicillin and cycloserine may be used to lyse actively growing bacterial cells, often in combination with an osmotic shock. Other permeabilization techniques include the use of basic proteins such as protamine; the cationic polysaccharide chitosan is effective for yeast cells; and streptolysin permeabiizes mammalian cells.

 3.     EXTRACTION

• It involves the recovery of a compound or a group of compounds from a mixture or from cells into a solvent phase.
• It usually achieved both separation of particles as well as concentration of the product.
• The process of extraction is frequently useful for the recovery of Antibiotics and most of the lipophilic substances.

 The process of extraction is achieved by:

1. Liquid-liquid extraction
2. Whole broth (medium + cells) extraction
3. Aqueous multiphase extraction

Aqueous two-phase separation involves partitioning the protein between the two phases, depending upon its molecular weight and charge. Commonly used systems include dextran and polyethylene glycol (PEG), or PEG and potassium phosphate. The two phases can then be separated by centrifugation. This method is cheap, gentle and versatile, and can be scaled up for industrial applications, including the purification of enzymes. Many alkaloids, antibiotics, steroids and vitamins are recovered by liquid–liquid extraction methods using organic solvents. Antibiotics, for example, are extracted from culture media into solvents such as amyl acetate.

Those solvents used should be non-toxic, selective, inexpensive, immiscible with broth and must have a high distribution coefficient for the product, i.e. the ratio of the product in the two phases.

Some of the product concentration may occur during the extraction step.

 4.     PURIFICATION

• It aims at recovery of the product in a highly purified state. Purification is achieved by

 Chromatographic methods: it is generally, used for the purification of low molecular mass compound from mixture of similar molecules, e.g., antibiotics(homologous) and macromolecules (enzymes).

 

The different chromatographic procedures are:

1. Adsorption
2. Ion exchange
3. Gel filtration
4. Hydrophobic
5. Affinity
6. Partition chromatography.

Chromatographic techniques are usually employed for higher-value products. These methods, normally involving columns of chromatographic media (stationary phase), are used for desalting, concentration and purification of protein preparations. In choosing a chromatographic technique for protein products, molecular weight, isoelectric point, hydrophobicity and biological affinity should be taken into consideration. Each of these properties can be exploited by specific chromatographic methods.

The order and choice of technique will depend on the particular product, but the following chromatographic parameters should be considered: capacity, recovery and resolving power (selectivity). The capacity refers to the sample size that can be applied to the system in terms of protein concentration and volume, and the recovery is the yield of product at each stage. Yield values should be as high as possible otherwise the overall process will be uneconomic. Resolving power and selectivity relate to the ability to separate two components, one being the product and the other the impurity. This is particularly important at the final purification stage. Care must be taken not to allow a protein to denature during purification, so columns are usually run at 4°C. Changes of pH, excessive dilution and addition of certain chemicals can also affect the stability of the protein.

1.               Adsorption chromatography separates according to the affinity of the protein, or other material, for the surfaces of the solid matrix. Alumina (Al2O3), hydroxyapatite (Ca10(PO4)6(OH)2) or silica (SiO2) are used for purifying non-polar molecules, whereas polystyrene based resins are effective matrices for polar molecules. This technique involves hydrogen bonding and/or van der Waals forces. The adsorbed proteins are usually eluted by increasing the ionic strength, often by using a gradient of increasing phosphate ion concentration.

2.               Affinity chromatography is a particularly powerful and highly selective purification technique. It can often give up to several thousand-fold purification in a singlestep. However, this chromatographic method is expensive on an industrial scale. The technique involves specific chemical interactions between solute molecules, such as proteins, and an immobilized ligand (functional molecule). Ligands are covalently linked to the matrix material, e.g. agarose. Some ligands interact with a group of proteins, notably nicotinamide adenine dinucleotide, adenosine monophosphate, and Procion and Cibacron dyes; other ligands are highly specific, especially substrates, substrate analogues and antibodies.

Since monoclonal antibodies have become more readily available, immunoaffinity chromatography methods have been developed for the purification of various antigens. The loading capacity of affinity columns is large, as the volume of the sample is unimportant and high resolution can be attained. This is also a high-speed technique and elution is achieved using specific cofactors or substrates; alternatively, non-specific elution may be performed with salt or pH change. For industrial-scale operations, it is often necessary to sterilize the chromatographic media in order to comply with various regulatory requirements. This can be problematical as some ligands are sensitive to sterilizing agents.

3.               Gel filtration chromatography involves separation on the basis of molecular size (molecular sieving), although molecular shape can also influence separation performance. Consequently, it is particularly useful for desalting protein preparations. The stationary phase consists of porous beads composed of acrylic polymers, agarose, cellulose, cross-linked dextran, etc., which have a defined pore size. These support materials should be sterilizable, chemically inert, stable, highly porous and hydrophilic, containing some ionic groups. Mechanical rigidity is particularly important in order to maintain good flow rates. The initial choice of stationary phase material is also a key factor, as some may interact with the target product, e.g. carbohydrate-based matrices interact with glycoproteins.

Solute molecules below the exclusion size of the support material pass in and out of the beads. Molecules above the exclusion size pass only around the outside of the beads through the interstitial spaces and the apparent volume of the column is smaller for these larger excluded molecules. As a result, they flow faster down the column, separating from smaller molecules and eluting first. Smaller molecules able to enter the pores are then eluted in decreasing order of size. Ion-exchange chromatography involves the selective adsorption of ions or electrically charged compounds onto ion-exchange resin particles by electrostatic forces. The matrix material is often based on cellulose substituted with various charged groups, either cations or anions.

A commonly used example is the anion-exchange resin diethyl aminoethyl (DEAE) cellulose. Proteins possess positive, negative or no charge depending on their isoelectric point (pI) and the pH of the surrounding buffer. If the pH of the buffer is below the pI, a protein has an overall positive charge, whereas a buffer at the pI results in the protein having no charge and pHs above its pI produce an overall negative charge. For example, if a protein has a pI of 4.2, at pH4.2 this protein is uncharged and will not bind to either positively or negatively charged resins. When the pH is raised to pH7.0 the protein is negatively charged and binds to positively charged resins. In conditions below pH4.0 the protein is positively charged and binds to negatively charged resins. If the protein is in an anionic state able to adsorb to DEAE cellulose, for example, any contaminants pass through the column. The product can then be desorbed as a purified fraction by altering the ionic strength of the buffer, often by using a gradient of increasing phosphate buffer concentration.

4.               High-performance liquid chromatography (HPLC) was originally developed for the separation of organic molecules in non-aqueous solvents, but is now used for proteins in aqueous solution. This method uses densely packed columns containing very small rigid particles, 5–50µm diameter, of silica or a cross-linked polymer. Consequently, high pressures are required. The method is fast and gives high resolution of solute molecules. Equipment for use in large-scale operations is now available.

5.                Hydrophobic chromatography relies on hydrophobic interaction between hydrophobic regions or domains of a solute protein and hydrophobic functional groups of the support particles. These supports are often agarose substituted with octyl or phenyl groups. Elution from the column is usually achieved by altering the ionic strength, changing the pH or increasing the concentration of chaotropic ions, e.g. thiocyanate. This technique provides good resolution and, like ion-exchange chromatography, has a very high capacity as it is not limited by sample volume.

6.               Metal chelate chromatography utilizes a matrix with attached metal ions, e.g. agarose containing calcium, copper or magnesium ions. The protein to be purified must have an affinity for this ion and binds to it by forming coordination complexes with groups such as the imidazole of histidine residues. Bound proteins are theneluted using solutions of free metal-binding ligands, e.g. amino acids.

 

5.               DIALYSIS & ELECTRODIALYSIS

These membrane separation techniques are primarily used for the removal of low molecular weight solutes and inorganic ions from a solution. The membranes involved are size selective with specific molecular weight cut-offs. Low molecular weight solutes move across the membrane by osmosis from a region of high concentration to one of low concentration. Electrodialysis methods separate charged molecules from a solution by the application of a direct electrical current carried by mobile counter-ions. Membranes used contain ionexchange groups and have a fixed charge; e.g. positively charged membranes allow the passage of anions and repel cations. They are essentially ion-exchange resins in sheet form and have also been used for the desalination of water.

 

6.                DISTILLATION

Distillation is used to recover fuel alcohol, acetone and other solvents from fermentation media, and for the preparation of potable spirits. Batch distillation in pot stills is used for the production of some whiskies, but with ethanol continuous distillation yields a product with 96.5% (v/v) ethanol concentration. This azeotropic mixture is the highest concentration that can be achieved from aqueous ethanol.

Some continuous stills may be in the form of four or five separate columns, but the Coffey-type still comprises just two columns, the ‘rectifier’ and ‘analyser’, each containing a stack of 30–32 perforated plates. Incoming fermentation broth is heated, as it passes down a coiled pipe within the rectifier column, by the ascending hot vapour produced by the analyser column. The now hot broth is released into a trough at the top of the analyser column and as it falls down the column it is heated by steam. Hot vapours generated are then conveyed from the top of the analyser column to the bottom of the rectifier column. As it passes upwards it is condensed on the coils carrying incoming broth. There is a temperature gradient in the rectifier column and each volatile compound condenses at its appropriate level, from where the fraction is collected.

 

7.    Finishing Steps

 CRYSTALLIZATION

Product crystallization may be achieved by evaporation, low-temperature treatment or the addition of a chemical reactive with the solute. The product’s solubility can be reduced by adding solvents, salts, polymers (e.g. nonionic PEG) and polyelectrolytes, or by altering the pH.

 DRYING

• Drying is the most important step in downstream processing which makes the product suitable for handling and storage.

The most frequent approaches of drying are:

1. Vacuum drying
2. Spray drying
3. Freeze drying.

 Drying involves the transfer of heat to the wet material and removal of the moisture as water vapour. Usually, this must be performed in such a way as to retain the biological activity of the product. Parameters affecting drying are the physical properties of the solid–liquid system, intrinsic properties of the solute, conditions of the drying environment and heat transfer parameters. Heat transfer may be by direct contact, convection or radiation.

·       Rotary drum driers remove water by heat conduction. A thin film of solution is applied to the steam heated surface of the drum, which is scraped with a knife to recover the dried product.

·       In vacuum tray driers the material to be dried is placed on heated shelves within a chamber to which a vacuum is applied. This allows lower temperatures to be used due to the lower boiling point of water at reduced pressure. The method is suitable for small batches of expensive materials, such as some pharmaceuticals.

·       Spray drying involves atomization and spraying of product solution into a heated chamber, and resultant dried particles are separated from gases using cyclones. Pneumatic conveyor driers use hot air that suspends and transports particles.

·         Freeze-drying (lyophilization) is often used where the final products are live cells, as in starter culture preparations, or for thermolabile products. This is especially useful for some enzymes, vaccines and other pharmaceuticals, where retention of biological activity is of major importance. In this method, frozen solutions of antibiotics, enzymes or microbial cell suspensions are prepared and the water is removed by sublimation under vacuum, directly from solid to vapour state. This method eliminates thermal and osmotic damage.