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
180 J. A Asenjo and J B Chaudhuri relation to efficiency in a bioseparation. Some properties(e.g. charge and affinity)can show extremely high resolution in purification operations, whereas others(e. g. molecular weight) show much lower resolution 7.2 SYSTEM CHARACTERISTICS 7.2.1 Physicochemical basis for separation operations Development of new and efficient separation processes will be based on more effectively exploiting differences in the actual physicochemical properties of the product such as surface charge/ titration curve, surface hydrophobicity, molecular weight, biospecificity towards certain ligands(e.g. metal ions, dyes), pI and stability, compared to those of th contaminant components in the crude broth. The main physicochemical factors involved in the development of separation processes are shown in Table 7. 1(Asenjo, 1993) Table 7.1. Physicochemical basis for the development of separation processes ochemical basis Separation process Ion-exchange chromatography Electrodialysis Aqueous two-phase partitioning Reverse micelle extraction Hydrophobicity Hydrophobic interaction chromatography Reversed phase chromatography Precipitation Aqueous two-phase partitioning Specific binding Affinity chromatography Gel filtr Ultrafiltration lysis Electrophoresis Isoelectric point Chromatofocusing Isoelectric focusing Sedimentation rate Surface activity Adsorption Foam fractionation Solid-liquid extraction Supercritical fluid extraction From Asenjo, 1993)
180 relation to efficiency in a bioseparation. Some properties (e.g. charge and affinity) can show extremely high resolution in purification operations, whereas others (e.g. molecular weight) show much lower resolution. 7.2 SYSTEM CHARACTERISTICS 7.2.1 Physicochemical basis for separation operations Development of new and efficient separation processes will be based on more effectively exploiting differences in the actual physicochemical properties of the product such as surface charge/titration curve, surface hydrophobicity, molecular weight, biospecificity towards certain ligands (e.g. metal ions, dyes), PI and stability, compared to those of the contaminant components in the crude broth. The main physicochemical factors involved in the development of separation processes are shown in Table 7.1 (Asenjo, 1993). J. A. Asenjo and J. B. Chaudhuri Table 7.1. Physicochemical basis for the development of separation processes Physicochemical basis Separation process Charge Ion-exchange chromatography Electrodialysis Aqueous two-phase partitioning Reverse micelle extraction Hydrophobicity Hydrophobic interaction chromatography Reversed phase chromatography Precipitation Aqueous two-phase partitioning Specific binding Affinity chromatography Size Gel filtration Ultrafiltration Dialysis Electric mobility Electrophoresis Isoelectric point Chromatofocusing Isoelectric focusing Sedimentation rate Centrifugation Surface activity Adsorption Solubility Solid-liquid extraction Foam fractionation Supercritical fluid extraction (From Asenjo, 1993)
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
182 J. A Asenjo and J B Chaudhuri water and the solubilised protein stabilised by a surfactant shell layer. For protein extraction, one phase is the aqueous feed solution, the other the reversed micellar phase that acts as the extractant. They have several of the advantages quoted for aqueous two- phase systems The suitability of using foam separation as well as gas aphrons as novel separation techniques for proteins are presently under investigation 7.3.1 Aqueous two-phase separation Partitioning in two aqueous phases can be used for the separation of proteins from cell debris as well as for purification from other proteins. Partitioning can be done in a single step or as a multistage process. Differences in partition coefficients, however, between the different proteins can be high, hence one step tends to be sufficient (usually one for extraction and one for elution or back-extraction). The use of affinity partitioning can eatly enhance the specificity of the extraction. A typical process for extraction of a protein into the top PEG phase in a first stage and the back extraction into a bottom salt phase(e.g. phosphate or sulphate) in a second 'back extraction' step from a cell homogenate that includes recycle of the PEG phase is shown in Fig. 7. 1(Hustedt et al 1985) PEG +salt Sal ⊙ Glass-bead mixer eat Separator Tc hase phase Separator Cell debris Fig. 7. 1. Scheme of enzyme purification by liquid-liquid extraction. The cells are disrupted by et milling, and after passing through a heat exchanger, PEG and salts are added into the process stream of broken cells. After mixing and obtaining of equilibrium the phase system is separated the outflowing bottom phase is going aining PEG-rich top a second mixer after addition of more salt to ocess stream. The product is recovered in the resulting bottom phase while the concentrated PEG solution (upper phase) goes to waste or is led. From Hustedt et al, 1985)
182 water and the solubilised protein stabilised by a surfactant shell layer. For protein extraction, one phase is the aqueous feed solution, the other the reversed micellar phase that acts as the extractant. They have several of the advantages quoted for aqueous twophase systems. The suitability of using foam separation as well as gas aphrons as novel separation techniques for proteins are presently under investigation. 7.3.1 Aqueous two-phase separation Partitioning in two aqueous phases can be used for the separation of proteins from cell debris as well as for purification from other proteins. Partitioning can be done in a single step or as a multistage process. Differences in partition coefficients, however, between the different proteins can be high, hence one step tends to be sufficient (usually one for extraction and one for elution or back-extraction). The use of affinity partitioning can greatly enhance the specificity of the extraction. A typical process for extraction of a protein into the top PEG phase in a first stage and the back extraction into a bottom salt phase (e.g. phosphate or sulphate) in a second ‘back extraction’ step from a cell homogenate that includes recycle of the PEG phase is shown in Fig. 7.1 (Hustedt et al., 1985). J. A. Asenjo and J. B. Chaudhuri .................................................................... Fig. 7.1. Scheme of enzyme purification by liquid-liquid extraction. The cells are disrupted by wet milling, and after passing through a heat exchanger, PEG and salts are added into the process stream of broken cells. After mixing and obtaining of equilibrium the phase system is separated, the outflowing bottom phase is going to waste. The product-containing PEG-rich top phase goes to a second mixer after addition of more salt to the process stream. The product is recovered in the resulting bottom phase while the concentrated PEG solution (upper phase) goes to waste or is recycled. (From Hustedt et al., 1985)
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)