《食品和生物分离过程》（英文版） Chapter 7 Innovative separation methods in bioprocessing.pdf_大学文库

Chapter 7 Innovative separation methods in bioprocessing J. A. ASENJO, Biochemical Engineering Laboratory, Department of Food Science and CHAUDHURI, School of Chemical Engineering, University of Bath, Bath BA2 7AY UK 7.1 INTRODUCTION Discoveries and achievements in modern biology and recombinant dna technology in the last few years have resulted in the development of a number of new therapeutics for human use such as insulin, human growth hormone(hGH), tissue plasminogen activator (tPA)for cardiac disease, erythropoietin(EPO)and hepatitis B vaccine and thus the possibility of their industrial large-scale production. This poses a tremendous challenge for the chemical and biochemical engineer in terms of developing efficient separation processes for these new proteins. As they are intended for human use the levels of purity required are of the order of 99.9% or 99.98% or even higher(depending on dosage)and they have to be separated from a very large number of contaminants, other proteins nucleic acids, polysaccharides and many other components present in the cell culture or cell lysate used to manufacture these proteins. Competitive advantage in production depends not only on innovations in molecular biology and other areas of basic biological sciences but also on innovation and optimisation of separation and downstream proc esses The main issues important for the development of novel separation techniques to give mproved resolution, simplicity, speed, ease of scale-up and possibly continuou operation are presented and discussed. The assessment of the state of the art as well as promising future developments concentrate on the separation and purification of proteins from complex mixtures. The present trend to develop techniques that exploit fundamental physicochemical principles more efficiently is emphasised. This includes the analysis of the physicochemical properties of proteins such as pl, charge as a function of pH, biological affinity (including metal ion and dye affinity), hydrophobicity and size and its

Chapter 7 Innovative separation methods in bioprocessing J. A. ASENJO, Biochemical Engineering Laboratory, Department of Food Science and Technology, The University of Reading, Reading RG6 6AP, UK and J. B. CHAUDHURI, School of Chemical Engineering, University of Bath, Bath BA2 7AY, UK 7.1 INTRODUCTION Discoveries and achievements in modern biology and recombinant DNA technology in the last few years have resulted in the development of a number of new therapeutics for human use such as insulin, human growth hormone (hGH), tissue plasminogen activator (tPA) for cardiac disease, erythropoietin (EPO) and hepatitis B vaccine and thus the possibility of their industrial large-scale production. This poses a tremendous challenge for the chemical and biochemical engineer in terms of developing efficient separation processes for these new proteins. As they are intended for human use the levels of purity required are of the order of 99.9% or 99.98% or even higher (depending on dosage) and they have to be separated from a very large number of contaminants, other proteins, nucleic acids, polysaccharides and many other components present in the cell culture or cell lysate used to manufacture these proteins. Competitive advantage in production depends not only on innovations in molecular biology and other areas of basic biological sciences but also on innovation and optimisation of separation and downstream processes. The main issues important for the development of novel separation techniques to give improved resolution, simplicity, speed, ease of scale-up and possibly continuous operation are presented and discussed. The assessment of the state of the art as well as promising future developments concentrate on the separation and purification of proteins from complex mixtures. The present trend to develop techniques that exploit fundamental physicochemical principles more efficiently is emphasised. This includes the analysis of the physicochemical properties of proteins such as PI, charge as a function of pH, biological affinity (including metal ion and dye affinity), hydrophobicity and size and its

Innovative separation methods in bioprocessing 181 7. 2. 2 Kinetics and mass transfer The physical behaviour of the system has an effect on the development of novel separation processes. Processes can be divided into equilibrium and rate processes. In equilibrium processes selective separation depends on the attainment of a favourable equilibrium state. This, for example, includes liquid-liquid extraction and ion exchange chromatography. Rate processes, on the other hand, separate different proteins on basis of their response to an imposed field(such as an electric field). Mobility and similar properties determine the selectivity of this type of operation; a successful pro s one in which the proteins have markedly different mobilities( e.g. electrophoresis) In a number of protein n processes the residence time in the reactor is insufficient for equilibrium to be achieved and the kinetics of adsorption play an important role for example in affinity chromatography and in the CArE (continuous dsorption recycle extraction) process. New developments in materials have recently shown dramatic advances in overcoming mass transfer limitations in processes such as perfusion and membrane chromatography and adsorption resulting in extremely fast separations. Some recent examples of novel techniques, which exploit the principles discussed above and provide useful analyses for optimal design of operations, include expanded bed(fluidised bed)adsorption of proteins, which allows direct broth extraction; cross-flow electrofiltration of disrupted microbial cells and for improved ultrafiltration proteins; mathematical modelling of partitioning and phase behaviour in liquid-liquid extraction; mathematical modelling of chromatographic columns; perfusion and membrane chromatography; and advanced reversed phase chromatography using HPLC The potential for scale-up of many of these systems is analysed and discussed 7.3 LIQUID-LIQUID EXTRACTION: INTRODUCTION Liquid-liquid extraction as a technology has been used in the antibiotics industry for several decades and it is now beginning to be recognised as a potentially useful separation step in protein recovery and separation, particularly because it can readily be scaled-up and can, if necessary, be operated on a continuous basis. The physicochemica factors of the protein that determine partitioning are also starting to be understood. It is a easonably high-capacity process ar offer good selectivity for the desired protein product. However, poor solubility of the large protein molecules in typical organic solvents restricts the range of solvents available for use in such a separation process Two classes of solvents that appear to offer advantages for protein recovery for protein separations are aqueous polymer /salt(in some cases also polymer/polymer) systems and reverse micellar solutions. In both cases two phases are formed and the separation exploits the difference in partitioning of the proteins in the feed and extraction phases. In the aqueous polymer/salt separation systems the partitioning of the protein occurs between two immiscible aqueous phases; one rich in a polymer(usually polyethylene glycol, PEG)and the other in a salt(e. g. phosphate or sulphate). These systems show lon-denaturing solvent environment, small interfacial resistance to mass transfer, relatively high protein capacity and high selectivity. On the other hand reverse micelles exploit the solubilising properties of surfactants that can aggregate in organic solvents to form so-called inverted or reverse micelles. These aggregates consist of a polar core of

Innovative separation methods in bioprocessing 18 1 7.2.2 Kinetics and mass transfer The physical behaviour of the system has an effect on the development of novel separation processes. Processes can be divided into equilibrium and rate processes. In equilibrium processes selective separation depends on the attainment of a favourable equilibrium state. This, for example, includes liquid-liquid extraction and ion exchange chromatography, Rate processes, on the other hand, separate different proteins on the basis of their response to an imposed field (such as an electric field). Mobility and other similar properties determine the selectivity of this type of operation; a successful process is one in which the proteins have markedly different mobilities (e.g. electrophoresis). In a number of protein separation processes the residence time in the reactor is insufficient for equilibrium to be achieved and the kinetics of adsorption play an important role for example in affinity chromatography ana in the CARE (continuous adsorption recycle extraction) process. New developments in materials have recently shown dramatic advances in overcoming mass transfer limitations in processes such as perfusion and membrane chromatography and adsorption resulting in extremely fast separations, Some recent examples of novel techniques, which exploit the principles discussed above and provide useful analyses for optimal design of operations, include expanded bed (fluidised bed) adsorption of proteins, which allows direct broth extraction; cross-flow electrofiltration of disrupted microbial cells and for improved ultrafiltration of proteins; mathematical modelling of partitioning and phase behaviour in liquid-liquid extraction; mathematical modelling of chromatographic columns; perfusion and membrane chromatography; and advanced reversed phase chromatography using HPLC. The potential for scale-up of many of these systems is analysed and discussed. 7.3 LIQUID-LIQUID EXTRACTION: INTRODUCTION Liquid-liquid extraction as a technology has been used in the antibiotics industry for several decades and it is now beginning to be recognised as a potentially useful separation step in protein recovery and separation, particularly because it can readily be scaled-up and can, if necessary, be operated on a continuous basis. The physicochemical factors of the protein that determine partitioning are also starting to be understood. It is a reasonably high-capacity process and can offer good selectivity for the desired protein product. However, poor solubility of the large protein molecules in typical organic solvents restricts the range of solvents available for use in such a separation process. Two classes of solvents that appear to offer advantages for protein recovery for protein separations are aqueous polymer/salt (in some cases also polymer/polymer) systems and reverse micellar solutions. In both cases two phases are formed and the separation exploits the difference in partitioning of the proteins in the feed and extraction phases. In the aqueous polymer/salt separation systems the partitioning of the protein occurs between two immiscible aqueous phases; one rich in a polymer (usually polyethylene glycol, PEG) and the other in a salt (e.g. phosphate or sulphate). These systems show a non-denaturing solvent environment, small interfacial resistance to mass transfer, relatively high protein capacity and high selectivity. On the other hand reverse micelles exploit the solubilising properties of surfactants that can aggregate in organic solvents to form so-called inverted or reverse micelles. These aggregates consist of a polar core of

Innovative separation methods in bioprocessing 183 Most soluble and particulate material partitions to the lower, more polar (e.g. salt, phase and the protein of interest partitions to the top less polar phase, usually PEG Separation of actual proteins in such systems is based on manipulating the partition coefficient(k) by altering parameters such as average molecular weight of the polymer type of phase forming salt used for the heavy phase, the types of ions included in the system and ionic strength of added salts(e.g. NaCl)(Schmidt et al., 1994). Figure 7.2 shows that the partition coefficient of a-amylase is a strong function of the presence of NaCl in a PEG/ sulphate system. For extraction of the a-amylase from its contaminants a high concentration of NaCl is used in the first extraction stage, whereas a low concentra- tion of NaCl in the back-extraction stage will allow the recovery of a-amylase into the bottom sulphate phase as shown in Fig. 7.1 Concentration of NaCl ( w/w) Fig. 7. 2. Partition behaviour of a-amylase (log Ka and contaminant protein (log Ke)from industrial supernatant from B subtilis fermentation in PEG 4000/Sulphate systems as a function of added NaCi concentration at pH 7 and a phase volume ratio of The partition coefficient(k) is defined as the concentration of a particular protein in the lighter phase divided by the concentration in the heavier phase. The main factors that determine partition depend on the type of system used (1) Hydrophobicity. Differences in the surface hydrophobicity between proteins are exploited when partitioning them in PEG/salt two-phase systems. Typical systems that exploit a protein's hydrophobicity are PEG/ phosphate and PEG /sulphate with addition of a high concentration of NaCl(e.g. 10%) (2) Size-dependent partition. Molecular size of the proteins or surface area of the particles to be partitioned is the dominating factor. It has been shown that for PEG/ Dextran systems a protein's molecular weight is inversely proportional to its partition coefficient (3) Electrochemical. Electrical potential between the phases is used to separate molecules or particles according to their charge. This is demonstrated

Innovative separation methods in bioprocessing 183 Most soluble and particulate material partitions to the lower, more polar (e.g. salt) phase and the protein of interest partitions to the top less polar phase, usually PEG. Separation of actual proteins in such systems is based on manipulating the partition coefficient (K) by altering parameters such as average molecular weight of the polymer, type of phase forming salt used for the heavy phase, the types of ions included in the system and ionic strength of added salts (e.g. NaCI) (Schmidt et al., 1994). Figure 7.2 shows that the partition coefficient of a-amylase is a strong function of the presence of NaCl in a PEG/sulphate system. For extraction of the a-amylase from its contaminants a high concentration of NaCl is used in the first extraction stage, whereas a low concentration of NaCl in the back-extraction stage will allow the recovery of a-amylase into the bottom sulphate phase as shown in Fig. 7.1. 4- 2- -4 I I I I I 0 2 4 6 a 10 Concentration of NaCl (Yo w/W) Fig. 7.2. Partition behaviour of a-amylase (log KJ and contaminant protein (log K,) from industrial supernatant from E. subtilis fermentation in PEG 4000/Sulphate systems as a function of added NaCl concentration at pH 7 and a phase volume ratio of 1. The partition coefficient (K) is defined as the concentration of a particular protein in the lighter phase divided by the concentration in the heavier phase. The main factors that determine partition depend on the type of system used: (1) Hydrophobicity. Differences in the surface hydrophobicity between proteins are exploited when partitioning them in PEG/salt two-phase systems. Typical systems that exploit a protein’s hydrophobicity are PEG/phosphate and PEG/sulphate with addition of a high concentration of NaCl (e.g. 10%). Size-dependent partition. Molecular size of the proteins or surface area of the particles to be partitioned is the dominating factor. It has been shown that for PEG/Dextran systems a protein’s molecular weight is inversely proportional to its partition coefficient. Electrochemical. Electrical potential between the phases is used to separate molecules or particles according to their charge. This is demonstrated in (2) (3)

184 J, A. Asenjo and J B Chaudhuri PEG/Dextran systems with addition of small concentration of salts whose charged ions will partition between the phases (e. g. 0. 1 M NaCl or 0.05 M Na2SO4) and also by manipulating the pH of the system. In PEG/salt systems an increase in the pH usually increases the value of K. Figure 7.3 shows the increase in K with pH for a-amylase in a PEG/phosphate system. As the bottom phase has a high concentra tion of salt, it is the charges in the top phase that affect partitioning; thus the top (4) Biospecific affinity. The affinity between sites on the proteins and ligands attached to one of the phase polymers is used for separation. Dyes, inhibitors, fatty acids glutathione, Protein A and several other ligands have been used. Particularly impressive results for selective partitioning have been obtained with metal ions (e.g. Cu**)both in PEG/Dextran but also in PEG/salt systems(Wuenschell et al (5) Solubility dependent. In addition it is important to know the actual concentration of the protein in the extractant phase. In typical PEG/salt systems, protein solubility tends to be higher in the PEG and lower in the salt phase. It is clear that, in the region near saturation of the protein in one of the phases, a constant partition Fig. 7.3. Partition of pure a-amylase()in PEG 4000(10% w/w)/phosphate(11.5% w/w) According to this, it is possible to split the partition coefficient into different terms K=KhfobKe kmw ksol where hfob, el, mw, aff and sol stand for hydrophobicity, electrostatic, size(molecular weight), affinity and solubility contributions to the partition coefficient In practical terms partitioning in aqueous two-phase systems is influenced by many ystem variables. Generally, the higher the molecular weight of the polymers the lower the concentration needed for the formation of two phases. Also, the larger the molecular weight of the PEG, the lower the value of K. Work is presently being carried out on elucidating how the different physicochemical properties of individual proteins determine

Innovative separation methods in bioprocessing 185 their partition behaviour in two phase systems(Hachem, 1992, Asenjo et al., 1994 Schmidt, 1994). It has been possible to correlate the partition coefficient of a representative number of proteins to their hydrophobicity measured by precipitation. This correlation was not as ood if the hydrophobicity was evaluated by hydrophobic interaction chromatography HIC)or also by reverse phase -HPLC (RP-HPLC)(Hachem, 1992; Asenjo et al., 1994 Hachem et al., 1994). In a typical protein precipitation graph as that shown in Fig. 7.4 (S=protein in solution), the proteins solubility (and thus 'hydrophilicity) can be expressed by point m, which is oint at which the protein starts precipitating, Thus the hydrophobicity, P, was evaluated as 1/m. The correlation found between hydrophobicity and partition in PEG /salt systems with a high concentration of NaCl is shown in Fig. 7.5. This can be represented by the equati gK=D△ w log P-D△ w log Po co AW corresponds to the tie- line length of a system and is evaluated by the difference in oncentration of one component(e. g. PEG or salt) between the phases which is constan for one particular system. D is the'discrimination factor'and thus D Aw is the slope in Fig.7.5 which corresponds to the resolution of a particular system to exploit differences in hydrophobicity between proteins. Table 7. 2 gives values of resolution (D Aw) and trinsic hydrophobicity'(Po) found for PEG/phosphate systems with different concen trations of NaCl. Clearly the systems with higher concentrations of Nacl give a higher resolution to exploit the protein s hydrophobicity in partitioning 7.3.2 Reverse micelle extraction Water-in-oil microemulsions, or reverse micelles, are stable, monodisperse aggregates of surface-active molecules (1-10 nm diameter) in an organic solvent. Typically, the =-012330+0.8431x-8.83470e-2x22=0.972 y=87919-24846XP=0.985 08L Concentration of ammonium sulphate(M) Fig. 7. 4. The fitted curves o oulin a at 25c in a solution containing added ammonium the first represents the represe salting-in region and the equati

Innovative separation methods in bioprocessing 187 Reverse ·““ Protein Fig. 7.6. Protein partition into reverse There are two techniques for transferring proteins into the micellar phase. The m widely used method involves extraction of the protein with a biphasic liquid system,i.e iquid-liquid extraction. One phase is the aqueous solution of the protein, and the other the organic micellar solution, usually in equal volume. By gently shaking the two phases, he protein partitions from the aqueous into the micellar phase. In the second method solid state extraction of the protein, the protein powder is suspended in the micellar phase and gently stirred The protein solubilised in the reverse micellar solution can be transferred back into an aqueous solution, by contacting the micellar solution with an aqueous solution containing a high concentration of a particular salt(KCl, CaCl2), which has the capability to ex change with the protein in the micelles The basic idea is that the process of protein extraction by reverse micelles can be made pecific (i.e. tailored to a specific protein) and efficient (i.e. high extraction yield) by ontrolling the micellar parameters such as the water content, the type and concentration of surfactant, the type and concentration of salt, and the pH Leser et al.(1986)examined the transfer of ribonuclease-A, lysozyme, trypsin an pepsin, monitoring the protein concentration and the concentration of water found in the organic phase. It was observed that the transfer of water is generally moderate(beloy 4%), whereas, under certain conditions, the protein is quantitatively transferred. This fact demonstrated that the transfer of the protein into the micellar phase is not a passive process, i.e. is not simply due to the fact that water is transferred and with it the protein The conclusion was that there is a thermodynamic driving force for the hydrophilic protein to leave the aqueous environment and to transfer into the reverse micelles. In other words, it seems that under certain conditions the protein-reverse micelle complex is energetically favoured above the free protein and empty reverse micelles. Interactions can be electrostatic, when surfactants with charged head groups are used, or hydrophobic with the surfactant interface or the apolar solvent

Innovative separation methods in bioprocessing 187 Organic phase 4V& - .t nv Reverse 4 ' L micelle ?fiC%4k/ h 3 nc L.UVVaUL. V UdU UU!iiCUY u Protein 0 a a Aqueous phase Fig. 7.6. Protein partition into reverse micelles. There are two techniques for transferring proteins into the micellar phase. The most widely used method involves extraction of the protein with a biphasic liquid system, i.e. liquid-liquid extraction. One phase is the aqueous solution of the protein, and the other the organic micellar solution, usually in equal volume. By gently shaking the two phases, the protein partitions from the aqueous into the micellar phase. In the second method, solid state extraction of the protein, the protein powder is suspended in the micellar phase and gently stirred. The protein solubilised in the reverse micellar solution can be transferred back into an aqueous solution, by contacting the micellar solution with an aqueous solution containing a high concentration of a particular salt (KC1, CaC12), which has the capability to exchange with the protein in the micelles. The basic idea is that the process of protein extraction by reverse micelles can be made specific (Le. tailored to a specific protein) and efficient (Le. high extraction yield) by controlling the micellar parameters such as the water content, the type and concentration of surfactant, the type and concentration of salt, and the pH. Leser et al. (1986) examined the transfer of ribonuclease-A, lysozyme, trypsin and pepsin, monitoring the protein concentration and the concentration of water found in the organic phase. It was observed that the transfer of water is generally moderate (below 4%), whereas, under certain conditions, the protein is quantitatively transferred. This fact demonstrated that the transfer of the protein into the micellar phase is not a passive process, i.e. is not simply due to the fact that water is transferred and with it the protein. The conclusion was that there is a thermodynamic driving force for the hydrophilic protein to leave the aqueous environment and to transfer into the reverse micelles. In other words, it seems that under certain conditions the protein-reverse micelle complex is energetically favoured above the free protein and empty reverse micelles. Interactions can be electrostatic, when surfactants with charged head groups are used, or hydrophobic with the surfactant interface or the apolar solvent

188 J. A. Asenjo and J B Chaudhuri The fact that electrostatic interactions play an important role in the distribution of oteins over reverse micellar and aqueous phase is shown by the dependence of the aqueous phase pH and ionic strength The ph of the solution will affect the solubilisation characteristics of a protein prima rily in the way in which it modifies the charge distribution over the protein surface. With creasing pH the protein becomes less positively charged until it reaches its isoelectric point (pl). At pHs above the pI the protein will take on a net negative charge. If electro- static interactions play a significant role in the solubilisation process, partition with anionic surfactants should be possible only at pHs below the pl of the protein, where the protein is positively charged and electrostatic attractions between the protein and the surfactant head groups are favourable. At pHs above the pl, electrostatic repulsions ould inhibit protein solubilisation Goklen and Hatton(1987) have presented results on the effect of ph on solubilisation tochrome-c, lysozyme, and ribonuclease-A, in AOT/isooctane reverse micelle solu tions. The results were presented as the percentage of the protein transferred from a I mg/ml aqueous protein solution to an equal volume of isooctane containing 50 mM of the anionic surfactant AOT. A summary of their results is presented in Table 7.3 Table 73. Effect of pH on solubilisation Protel pH range of maximum solubilisation 10 5-10 l1,.1 6-11 As anticipated, only at pHs lower than the pI was there any appreciable solubilisation of a given protein, while above the pl the solubilisation appears to be totally suppressed However, at extremes of pH there is a drop in the degree of solubilisation of the proteins due to protein denaturation, observed as precipitate formation at the interface( Chaudhuri eral.,1993) Luisi et al.( 1979)used the quaternary ammonium salt methyl-trioctylammonium chloride(TOMAC) for the transfer of a-chymotrypsin from water to cyclohexane. It was found that the pH had to be reduced to values significantly below the pI (pI =8.)for there to be any appreciable solubilisation. The solubilisation occurred only over a very narrow ph range before decreasing rapidly again with further decreases in the ph of the aqueous feed phase, accompanied by precipitation at the interface Similar results have been obtained by Dekker et al. (1986) for the enzyme a-amylase Significant solubilisation of the enzyme was observed over a narrow pH range in the vicinity of 10-10.5(pI=5.1). In this pH range, all basic residues will be deprotonated d the only charged residues being the carboxyl groups bearing a negative charge

188 J. A. Asenjo and J. B. Chaudhuri The fact that electrostatic interactions play an important role in the distribution of proteins over reverse micellar and aqueous phase is shown by the dependence of the aqueous phase pH and ionic strength. The pH of the solution will affect the solubilisation characteristics of a protein primarily in the way in which it modifies the charge distribution over the protein surface. With increasing pH the protein becomes less positively charged until it reaches its isoelectric point (PI). At pHs above the PI the protein will take on a net negative charge. If electrostatic interactions play a significant role in the solubilisation process, partition with anionic surfactants should be possible only at pHs below the PI of the protein, where the protein is positively charged and electrostatic attractions between the protein and the surfactant head groups are favourable. At pHs above the PI, electrostatic repulsions would inhibit protein solubilisation. Goklen and Hatton (1987) have presented results on the effect of pH on solubilisation of cytochrome-c, lysozyme, and ribonuclease-A, in AOT/isooctane reverse micelle solutions. The results were presented as the percentage of the protein transferred from a 1 mg/ml aqueous protein solution to an equal volume of isooctane containing 50 mM of the anionic surfactant AOT. A summary of their results is presented in Table 7.3. Table 7.3. Effect of pH on solubilisation Protein PI pH range of maximum solubilisation cy tochrome-c 10.6 5-10 ribonuclease-A 7.8 1-7 lysozyme 11.1 6-1 1 As anticipated, only at pHs lower than the pl was there any appreciable solubilisation of a given protein, while above the PI the solubilisation appears to be totally suppressed. However, at extremes of pH there is a drop in the degree of solubilisation of the proteins due to protein denaturation, observed as precipitate formation at the interface (Chaudhuri et al., 1993). Luisi et al. (1979) used the quaternary ammonium salt methyl-trioctylammonium chloride (TOMAC) for the transfer of a-chymotrypsin from water to cyclohexane. It was found that the pH had to be reduced to values significantly below the PI (PI = 8.3) for there to be any appreciable solubilisation. The solubilisation occurred only over a very narrow pH range before decreasing rapidly again with further decreases in the pH of the aqueous feed phase, accompanied by precipitation at the interface. Similar results have been obtained by Dekker et al. (1986) for the enzyme a-amylase. Significant solubilisation of the enzyme was observed over a narrow pH range in the vicinity of 10-10.5 (PI = 5.1). In this pH range, all basic residues will be deprotonated and the only charged residues being the carboxyl groups bearing a negative charge