In many fermentation processes, chromatographic techniques are used to isolate and purily relatively low concentrations of metabolic products. In this context, chromatography will be concerned with the passage and separation of different solutes as liquid is passed through a column, i.e. liquid chromatography. Depending on the mechanism by which the solutes may be differentially held in a column, the techniques can be grouped as follows:
(a) Adsorption chromatography.
(b) Ion-exchange chromatography.
(c) Gel permeation chromatography.
(d) Affinity chromatography.
(e) Reverse phase chromatography.
(f) High performance liquid chromatography.
Chromatographic techniques are also used in the final stages of purification of a number of products. The scale-up of chromatographic processes can prove difficult, and there is much current interest in the use of mathematical models and computer programmes to translate data obtained from small-scale processes into operating conditions for larger scale applications (Cowan et al., 1986, 1987).
Adsorption chromatography involves binding of the solute to the solid phase primarily by weak Van de Waals forces. The materials used for this purpose to pack columns include inorganic adsorbants (active carbon, aluminium oxide, aluminium hydroxide, magnesium oxide, silica gel) and organic macro-porous resins. Adsorption and affinity chromatography are mechanistically identical, but are strategically different. In affinity systems selectivity is designed rationally whilst in adsorption selectivity must be determined empirically.
Di-hydro-streptomycin can be extracted from filtrates using activated charcoal columns. It is then eluted with methanolic hydrochloric acid and purified in further stages (Nakazawa et al., 1960). Some other applications for small-scale antibiotic purification are quoted by Weinstein and Wagman (1978). Active carbon may be used to remove pigments to clarify broths. Penicillin-containing solvents may be treated with 0.25 to 0.5% active carbon to remove pigments and other impurities (Sylvester and Coghill, 1954).
Macro-porous adsorbants have also been tested. The first synthetic organic macro-porous adsorbants, the Amberlite XAD resins, were produced by Rohm and Haas in 1965. These resins have surface polarities which vary from non-polar to highly polar and do not possess any ionic functional groups. Voser (1982) considers their most interesting application to be in the isolation of hydrophilic fermentation products. He stated that these resins would be used at Ciba-Geigy in recovery of cephalosporin C (acidic amino acid), cefo-tiam (basic amino acid), desferrioxamine B (basic hy-droxamic acid) and paramethasone (neutral steroid).
Ion exchange can be defined as the reversible exchange of ions between a liquid phase and a solid phase (ion-exchange resin) which is not accompanied by any radical change in the solid structure. Cationic ion-exchange resins normally contain a sulphonic acid, carboxylic acid or phosphonic acid active group. Car-boxy-methyl cellulose is a common cation exchange resin. Positively charged solutes (e.g. certain proteins) will bind to the resin, the strength of attachment de pending on the net charge of the solute at the pH ,,f the column feed. After deposition solutes are sequentially washed off by the passage of buffers of increasing ionic strength or pH. Anionic ion-exchange resins normally contain a secondary amine, quaternary amine or quaternary ammonium active group. A common anion exchange resin, DEAE (diethylaminoethyl) cellulose k used in a similar manner to that described above for the separation of negatively charged solutes. Other functional groups may also be attached to the resin skeleton to provide more selective behaviour similar to that of affinity chromatography. The appropriate resin for a particular purpose will depend on various factors such as bead size, pore size, diffusion rate, resin capacity, range of reactive groups and the life of the resin before replacement is necessary. Weak-acid cation ion-exchange resins can be used in the isolation and purification of streptomycin, neomycin and similar antibiotics.
In the recovery of streptomycin, the harvested filtrate is fed on to a column of a weak-acid cationic resin such as Amberlite IRC 50 which is in the sodium form. The streptomycin is adsorbed on to the column and the sodium ions are displaced.
RCOO~NaJesin) + streptomycin
-» RCOO streptomycin Jesin) + NaOH
Flow rates of between 10 and 30 bed volumes per hour have been used. The resin bed is now rinsed with water and eluted with dilute hydrochloric acid to release the bound streptomycin.
RCOO~ streptomycin Jesin) + HC1
A slow flow is used to ensure the highest recovery of streptomycin using the smallest volume of eluant. In one step the antibiotic has been both purified and concentrated, maybe more than 100-fold. The resin column is regenerated to the sodium form by passing an adequate volume of NaOH slowly through the column and rinsing with distilled water to remove excess sodium ions.
The resin can have a capacity of 1 g of streptomycin g 1 resin. Commercially, it is not economic to regenerate the resin completely, therefore the capacity will be reduced. In practice, the filtered broth is taken through two columns in series while a third is being eluted and regenerated. When the first column is saturated, it is isolated for elution and regeneration while the third column is brought into operation.
Details for isolation of some other antibiotics are given in Weinstein and Wagman (1978). Ion-exchange chromatography may be combined with HPLC in, for example, the purification of somatotropin using DEAE cellulose columns and /3-urogastrone in multi-gram quantities using a cation exchange column (Brewer and Larsen, 1987).
This technique is also known as gel exclusion and gel filtration. Gel permeation separates molecules on the basis of their size. The smaller molecules diffuse into the gel more rapidly than the larger ones, and penetrate the pores of the gel to a greater degree. This means that once elution is started, the larger molecules which are still in the voids in the gel will be eluted first. A wide range of gels are available, including cross-linked dextrans (Sephadex and Sephacryl) and cross-linked agarose (Sepharose) with various pore sizes depending on the fractionation range required.
One early industrial application, although on a relatively small scale, was the purification of vaccines (Latham et al, 1967). Tetanus and diphtheria broths for batches of up to 100,000 human doses are passed through a 13 dm3 column of G 100 followed by a 13 dm3 column of G 200. This technique yields a fairly pure fraction which is then concentrated ten-fold by pressure dialysis to remove the eluant buffer (Na2HP04).
Affinity chromatography is a separation technique with many applications since it is possible to use it for separation and purification of most biological molecules on the basis of their function or chemical structure. This technique depends on the highly specific interactions between pairs of biological materials such as enzyme-substrate, enzyme-inhibitor, antigen-antibody, etc. The molecule to be purified is specifically adsorbed from, for example, a cell lysate applied to the affinity column by a binding substance (ligand) which is immobilized on an insoluble support (matrix). Eluent is then passed through the column to release the highly purified and concentrated molecule. The ligand is at tached to the matrix by physical absorption or chemically by a covalent bond. The pore size and ligand location must be carefully matched to the size of the product for effective separation. The latter method is preferred whenever possible. Porath (1974) and Yang and Tsao (1982) have reviewed methods and coupling procedures.
Coupling procedures have been developed using cyanogen bromide, bisoxiranes, disaziridines and perio-dates, for matrixes of gels and beads. Four polymers which are often used for matrix materials are agarose, cellulose, dextrose and polyacrylamide. Agarose activated with cyanogen bromide is one of the most commonly used supports for the coupling of amino ligands. Silica based solid phases have been shown to be an effective alternative to gel supports in affinity chromatography (Mohan and Lyddiatt, 1992).
Purification may be several thousand-fold with good recovery of active material. The method can however be quite costly and time consuming, and alternative affinity methods such as affinity cross-flow filtration, affinity precipitation and affinity partitioning may offer some advantages (Janson, 1984; Luong et al., 1987). Affinity chromatography was used initially in protein isolation and purification, particularly enzymes. Since then many other large-scale applications have been developed for enzyme inhibitors, antibodies, interferon and recombinant proteins (Janson and Hedman, 1982; Ostlund, 1986; Folena-Wasserman et al., 1987; Nach-man et al., 1992), and on a smaller scale for nucleic acids, cell organelles and whole cells (Yang and Tsao, 1982). In the scale-up of affinity chromatographic processes (Katoh, 1987) bed height limits the superficial velocity of the liquid, thus scale-up requires an increase in bed diameter or adsorption capacity.
Reverse phase chromatography (RFC)
This chromatographic method utilizes a solid phase (e.g. silica) which is modified so as to replace hy-drophilic groups with hydrophobic alkyl chains. This allows the separation of proteins according to their hydrophobicity. More-hydrophobic proteins bind most strongly to the stationary phase and are therefore eluted later than less-hydrophobic proteins. The alkyl groupings are normally eight or eighteen carbons in length (C8 and Clg). RPC can also be combined with affinity techniques in the separation of, for example, proteins and peptides (Davankov et al., 1990).
High performance liquid chromatography (HPLC)
HPLC is a high resolution column chromatographic technique. Improvements in the nature of column packing materials for a range of chromatographic techniques (e.g. gel permeation and ion-exchange) yield smaller, more rigid and more uniform beads. This allows packing in columns with minimum spaces between the beads, thus minimizing peak broadening of eluted species. It was originally known as high pressure liquid chromatography because of the high pressures required to drive solvents through silica based packed beds. Improvements in performance led to the name change and its widespread use in the separation and purification of a wide range of solute species, including bio-molecules. HPLC is distinguished from liquid chromatography by the use of improved media (in terms of their selectivity and physical properties) for the solid (stationary) phase through which the mobile (fluid) phase passes.
The stationary phase must have high surface area/unit volume, even size and shape and be resistant to mechanical and chemical damage. However, it is factors such as these which lead to high pressure requirements and cost. This may be acceptable for analytical work, but not for preparative separations. Thus, in preparative HPLC some resolution is often sacrificed (by the use of larger stationary-phase particles) to reduce operating and capital costs. For very high value products large-scale HPLC columns containing analytical media have been used.
Affinity techniques can be merged with HPLC to combine the selectivity of the former with the speed and resolving power of the latter (Forstecher et al., 1986; Shojaosadaty and Lyddiatt, 1987).
Although the concept of continuous enzyme isolation is well established (Dunnill and Lilly, 1972), the stage of least development is continuous chromatography. Fox et al. (1969) developed a continuous-fed column for this purpose (Fig. 10.30). It consisted of two concentric cylindrical sections clamped to a base plate. The space (1 cm wide) between the two sections was packed with the appropriate resin or gel giving a total column capacity of 2.58 dm3. A series of orifices in the circumference of the base plate below the column space led to collecting vessels. The column assembly was rotated in a slow-moving turntable (0.4-2.0 rpm). The mixture for separation was fed to the apparatus by
Fig. 10.30. The principle of continuous-partition chromatography.
---, faster-moving component; O O, slower-moving component
Fig. 10.30. The principle of continuous-partition chromatography.
---, faster-moving component; O O, slower-moving component
an applicator rotating at the same speed as the column, thus allowing application at a fixed point, while the eluent was fed evenly to the whole circumference of the column. The components of a mixture separated as a series of helical pathways, which varied with the retention properties of the constituent components. This method gave a satisfactory separation and recovery but the consumption of eluent and the unreliable throughput rate were not considered to be satisfactory for a large-scale method (Nicholas and Fox, 1969; Dunnill and Lilly, 1972). However, the development of such continuous separation equipment suitable for large-scale extraction would considerably simplify the use of chromatographic separation.
MEMBRANE PROCESSES Ultrafiltration and reverse osmosis
Both processes utilize semi-permeable membranes to separate molecules of different sizes and therefore act in a similar manner to conventional filters.
Ultrafiltration can be described as a process in which solutes of high molecular weight are retained when the solvent and low molecular weight solutes are forced under hydraulic pressure (around 7 atmospheres) through a membrane of a very fine pore size. It is therefore used for product concentration and purification. A range of membranes made from a variety of polymeric materials, with different molecular weight cut-offs (500 to 500,000), are available which makes possible the separation of macro-molecules such as proteins, enzymes, hormones and viruses. It is practical only to separate molecules whose molecular weights are a factor of ten different due to variability in pore size (Heath and Belfort, 1992). Because the flux through such a membrane is inversely proportional to its thickness, asymmetric membranes are used where the membrane (~ 0.3 /u.m thick) is supported by a mesh around 0.3 mm thick.
When considering the feasibility of ultrafiltration it is important to remember that factors other than the molecular weight of the solute affect the passage of molecules through the membranes (Melling and Westmacott, 1972). There may be concentration polarization caused by accumulation of solute at the membrane surface which can be reduced by increasing the shear forces at the membrane surface either by conventional agitation or by the use of a cross-flow system (see previous section). Secondly a slurry of protein may accumulate on the membrane surface forming a gel layer which is not easily removed by agitation. Formation of the gel layer may be partially controlled by careful choice of conditions such as pH (Bailey and Ollis, 1986). Finally, equipment and energy costs may be considerable because of the high pressures necessary; this also limits the life of ultrafiltration membranes.
There are numerous examples of the use of ultrafiltration for the recovery of bio-molecules: viruses (Weiss, 1980), enzymes (Atkinson and Mavituna, 1991), antibiotics (Pandey et al., 1985). Details of large scale applications are given by Lacey and Loeb (1972) and by Ricketts et al (1985). Affinity ultrafiltration (Luong et al, 1987; Luong and Nguyen, 1992) is a novel separation process developed to circumvent difficulties in affinity chromatography. It offers high selectivity, yield and concentration, but it is an expensive batch process and scale up is difficult.
Reverse osmosis is a separation process where the solvent molecules are forced by an applied pressure to flow through a semi-permeable membrane in the opposite direction to that dictated by osmotic forces, and hence is termed reverse osmosis. It is used for the concentration of smaller molecules than is possible by ultrafiltration. Concentration polarization is again a problem and must be controlled by increased turbulence at the membrane surface.
Liquid membranes are insoluble liquids (e.g. an organic solvent) which are selective for a given solute and separate two other liquid phases. Extraction takes place by the transport of solute from one liquid to the other. They are of great interest in the extraction and purification of biologicals for the following reasons:
(a) Large area for extraction.
(b) Separation and concentration are achieved in one step.
(c) Scale-up is relatively easy.
Their use has been reported in the extraction of lactic acid (Chaudhuri and Pyle, 1990) and citric acid using a supported liquid membrane (Sirman et al., 1990). The utilization of selective carriers to transport specific components across the liquid membrane at relatively high rates has increased interest in recent years (Strathmann, 1991). Liquid membranes may also be used in cell and enzyme immobilization, and thus provide the opportunity for combined production and isolation/extraction in a single unit (Mohan and Li, 1974, 1975). The potential use of liquid membranes has also been described for the production of alcohol reduced beer as having little effect on flavour or the physico-chemical properties of the product (Etuk and Murray, 1990).
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