Handbook of Methods and Instrumentation in Separation Science: Volume 1

By Academic Press

Handbook of Methods and Instrumentation in Separation Science, Volume 1 provides concise overviews and summaries of the main methods used for separation. It is based on the Encyclopedia of Separation Science. The handbook focuses on the principles of methods and instrumentation. It provides general concepts concerning the subject matter; it does not present specific procedures.

This volume discusses the separation processes including affinity methods, analytical ultracentrifugation, centrifugation, chromatography, and use of decanter centrifuge and dye. Each methodology is defined and compared with other separation processes. It also provides specific techniques, principles, and theories concerning each process. Furthermore, the handbook presents the applications, benefits, and validation of the processes described in this book.

This handbook is an excellent reference for biomedical researchers, environmental and production chemists, flavor and fragrance technologists, food and beverage technologists, academic and industrial librarians, and nuclear researchers. Students and novices will also find this handbook useful for practice and learning.

• One-stop source for information on separation methods
• General overviews for quick orientation
• Ease of use for finding results fast
• Expert coverage of major separation methods
• Coverage of techniques for all sizes of samples, pico-level to kilo-level

• Language: English
• Publisher: Academic Press
• Release date: Nov 11, 2009
• ISBN: 9780123757272

Book preview

instructions.

Affinity Membranes

K. Haupt    Lund University, Lund, Sweden

S.M.A. Bueno    Universidade Estadual de Campinas, Brazil

The rapid development in biotechnology and the large potential of biomolecules for applications in medicine, food industry and other areas, result in an increasing demand for efficient and reliable tools for the purification of proteins, peptides, nucleic acids and other biological substances. This situation is being additionally enforced by the increasing number of recombinant gene products that have arrived on the market or that are currently being investigated, such as insulin, erythropoietin and interferons. The recovery of fragile biomolecules from their host environments requires their particular characteristics to be taken into account for the development of any extraction or separation process. On the other hand, there is a demand for techniques that can easily be scaled up from laboratory to industrial production level.

In this context, the use of affinity methods has the advantage that coarse and fine purification steps are united through the introduction of a specific recognition phenomenon into the separation process. The most widely used method for preparative affinity separation of biomolecules is liquid chromatography on beaded resins (soft gels). Despite the commercial availability of many affinity ligands immobilized on to gel beads for use in column chromatography, there are some drawbacks in a large scale application of these supports. Flow rates and thus performance are limited by the compressibility of the resins and pore diffusion. Because of these intrinsic limitations, other chromatographic techniques, such as perfusion chromatography, or different separation techniques, such as affinity precipitation and affinity phase partitioning, have been suggested as possible alternatives. Another technique that is gaining increasing importance is membrane-based separation. Adsorptive membrane chromatography was introduced as a purification method in the mid 1980s. Microporous membranes have been successfully coupled with biological or biomimetic ligands, yielding affinity membrane chromatography supports. Several of them, with for example protein A and G, dye or metal chelate ligands, are commercially available. Affinity membrane chromatography is in fact a hybrid technique combining affinity gel chromatography and membrane filtration, with the advantages of the two technologies.

The purpose of the present review is to discuss relevant aspects and developments that are important for the design of an affinity membrane chromatography process, including the choice of the membrane material, coupling chemistry, affinity ligands, membrane configurations, operation modes and scale-up. In a wider sense, membrane-based affinity fractionation also comprises affinity filtration methods where the target molecule binds to an affinity ligand coupled to nanoparticles, which can then be separated by filtration through a membrane. However, this application will not be discussed here in detail.

General Characteristics of Membrane Chromatography

In contrast to chromatographic supports based on beaded resins with dead end pores, membrane chromatographic supports have through-pores and lack interstitial space. Mass transfer is mainly governed by forced convection and pore diffusion is negligible. The observed back-pressures are normally quite low, and high flow rates and thus high throughputs and fast separations become possible without the need for high pressure pumps or equipment. As the association time for an antibody–antigen complex is typically about 1 s or less, but the diffusion of a protein molecule to the centre of a 50 μm porous bead takes tens of seconds, in a membrane support, the low diffusional limitation leads to faster adsorption kinetics and higher throughput efficiency. Little deterioration of the separation efficiency occurs even at elevated flow rates. On the other hand, with affinity membranes the formation of the affinity complex can become the rate-limiting process at high flow rates.

A problem often encountered in membrane chromatography is extra-cartridge back-mixing, which can severely degrade membrane performance. This phenomenon is due to dead volumes outside the membrane, in tubing, fittings and valves, and leads to peak broadening and dilution. It is more pronounced in membrane chromatography systems compared to conventional columns packed with beaded supports, owing to the larger throughput/bed volume ratio.

Although the specific surface area of membranes is typically only 1% of that of conventional chromatographic resins, microporous membrane systems have high internal surface areas and reasonably high capacities. The open-pore structure of membranes increases the accessibility of affinity ligands and reduces steric hindrance compared to small-pore adsorbents.

Membrane Geometry

Just like filtration membranes in general, affinity membranes can be produced in different configurations, and membrane modules of various geometries are commercially available or have been manufactured in research laboratories (Figure 1).

Figure 1 Different geometries of affinity membranes. (A) Flat sheet; (B) stack of flat discs; (C) hollow fibre; (D) spiral-wound flat sheet; (E) continuous rod. The arrows indicate flow directions. (Adapted from Journal of Chromatography 702, Roper DK and Lightfoot EN, Separation of biomolecules using adsorptive membranes, pp. 3–26, Copyright 1995, with permission from Elsevier Science.)

Flat sheet or disc membranes can be mounted as individual membranes in specially designed cartridges or in commercial ultrafiltration units for use in dead-end filtration mode. This allows for the production of inexpensive single- or multiple-use devices for the rapid adsorption of a target molecule from dilute samples in batch or continuous recycling mode. Cartridges are also available that allow for operation in cross-flow filtration mode.

Stacks of flat membrane discs have been employed for affinity membrane chromatography in column-like devices, the main purpose being to increase the adsorption capacity. Another configuration is continuous rod-type membranes which can be directly cast in a chromatographic column. Both types of membrane columns are compatible with conventional high performance liquid chromatography or fast protein liquid chromatography systems and have advantages over columns packed with beaded resins, as described above. Being highly porous with a mean pore diameter of 0.1–10 μm, they allow for efficient separations even at high flow rates.

If the target molecule is to be recovered from complex feed solutions such as cell homogenates or blood plasma, or from solutions containing high molecular mass additives such as antifoam agents or even particulate material, the use of membranes in dead-end filtration mode is often impossible due to membrane fouling. A remedy to this problem is the operation in cross-flow filtration mode where the build-up of a polarization layer at the membrane surface is avoided or diminished. Hollow-fibre membranes are well adapted for such applications. They are usually mounted as bundles in tubular cartridges. Another configuration are flat-sheet membranes that are spiral-wound around a cylindrical core. Both systems have the advantage of high surface area/cartridge volume ratios and high operational capacities.

Membrane Material, Activation and Ligand Coupling

Membrane Material

Due to the specific properties of biomolecules, the membrane materials to be used for their separation should ideally possess the following characteristics:

• Macroporosity: This will allow biomolecules to cross the membrane and to access the affinity sites.

• Hydrophilicity: Using hydrophilic supports, nonspecific adsorption by hydrophobic interactions and denaturation of biomolecules can be avoided.

• Presence of functional groups: These are required for the coupling of an affinity ligand.

• Chemical and physical stability: The material has to withstand the sometimes harsh conditions during derivatization, operation and regeneration.

• Biocompatibility: This is particularly important if the membranes are used in extracorporeal devices, for example for blood treatment.

• Large surface area relative to membrane volume: This will allow for the construction of small, integrated devices with high operational capacities.

Cellulose and cellulose acetate were among the first materials that have been used for affinity membrane preparation. They are hydrophilic and biocompatible, and due to the presence of hydroxyl groups, ligand coupling can be easily achieved using for example CNBr or carbonyldiimidazole activation. In order to improve the mechanical and chemical stability of cellulose membranes, chemical cross-linking with epichlorohydrin is sometimes carried out. Cellulose membranes normally have a rather small pore size, resulting in a high pressure drop. Attempts to produce membranes with larger pores using coarse cellulose fibres have resulted in a less uniform membrane structure.

Polysulfone is another suitable membrane material which has good film-forming properties. It is of sufficient physical, chemical and biological stability, and ligands can be coupled after chloromethylation-amination or acrylation-amination.

Microporous polyamide (nylon) membranes have also been used for the preparation of affinity membranes. This material is mechanically stable and has a rather narrow pore size distribution. It contains only a small number of terminal amino groups for ligand coupling, which can, however, be increased by partial hydrolysis of the amide functions.

A suitable membrane material is polyvinyl alcohol, in particular because of its hydrophilicity and biocompatibility. Poly(ethylene-co-vinyl alcohol), which has a somewhat higher chemical stability, has also been used. Both materials contain hydroxyl groups and can be activated by the CNBr method, allowing immobilization of affinity ligands having an amino function. Ligands can also be coupled using epichlorohydrine or butanediol diglycidyl ether-activation.

Other materials that have been used for affinity membranes are poly(methyl methacrylate), poly (hydroxyethyl dimethacrylate), polycaprolactam, poly (vinylidene difluoride), poly(ether-urethane-urea) and silica glass. Table 1 shows a list of membrane materials and the appropriate ligand-coupling chemistries.

Table 1

Membrane materials and possible chemistries for ligand coupling

Composite Membranes

The main difficulty when choosing a membrane for affinity separation of biomolecules is sometimes to find a material that fulfils several or all of the abovementioned requirements. For example, a chemically stable material might be too hydrophobic and lead to nonspecific and irreversible adsorption of the protein to be separated, whereas a hydrophilic material that is compatible with the fragile protein molecules might not withstand the conditions required for ligand coupling and for regeneration and sterilization of the membrane. Therefore, the choice of a membrane material will sometimes be a compromise. The use of a composite membrane consisting of two or more different materials may often be the only solution to a particular separation problem. This approach consists of the grafting of hydrophilic polymers on to a chemically and mechanically stable microporous membrane. The result is an increased biocompatibility as well as the introduction of suitable functional groups for ligand coupling. One example is the radiation-induced graft polymerization of 2-hydroxyethyl methacrylate or glycidyl methacrylate on to a polyethylene hollow fibre membrane. This increases the hydrophilicity of the material and introduces active hydroxyl groups or reactive epoxy groups.

Activation and Ligand Coupling

From a practical point of view, apart from the chemical compatibility of the membrane material with the activation and coupling solutions, an important aspect is that these solutions need to access the pores of the membrane. In many cases it will therefore be necessary to do the activation in dynamic mode, that is, by forced convection. This is especially important if the membrane material is hydrophilic and the activation and coupling solutions are based on nonpolar solvents, since in that case the wettability of the membrane by the solutions will be low.

Spacer Arms

Occasionally, affinity membranes may show poor performance if the ligand, and in particular a small ligand, is coupled directly to the membrane. This is often due to a low steric availability of the ligand, a problem that can be overcome by the use of a suitable spacer arm. In that way, the ligand accessibility for the molecule to be separated is improved, resulting in an increase in membrane-binding capacity. For example, 1,6-diminohexane or 6-aminohexanoic acid are often used as spacers. In other cases, the coupling method itself provides a spacer, as is the case with butanediol diglycidyl ether. If composite membranes with crafted flexible copolymer chains are used, spacer arms are not normally required.

Affinity Ligands

Biologicial Ligands

Just like other affinity separation techniques, affinity membrane technology uses biomolecules as the affinity ligands, thus taking advantage of the specificity of biological recognition. One of the most common applications is the use of immobilized monoclonal antibodies against natural or recombinant proteins as the ligand for immunoaffinity separation. Another important example are membranes with covalently coupled protein A or protein G for immunoglobulin purification from plasma, serum or cell culture supernatants. Immobilized lectines have been used for the purification of glycoproteins. The use of inhibitors or coenzymes for the purification of enzymes is also possible. Although biomolecules are widely used as ligands for their selectivity, they do have drawbacks. Their poor stability and sometimes high price can make them problematic for use in large scale affinity separation. Drastic conditions are often necessary for elution of the ligate, for example with high affinity antibody–antigen interactions. This can lead to partial inactivation of the molecule to be purified. Ligand denaturation and inactivation, in particular with protein ligands, can occur during regeneration and sterilization of the membrane. Another important issue is the possible leaching of the affinity ligand, leading to a contamination of the final product, which is particularly problematic if the product is to be used in medical applications.

Pseudobiospecific Ligands

An alternative approach involves the use of biomimetic or pseudobiospecific affinity ligands. These are usually smaller and simpler molecules with higher chemical and physical stability than biomolecules. The working principle of pseudobiospecific ligands relies on the complementarity of structural features of ligand and ligate rather than on a biological function, whereas biomimetic ligands have a certain structural resemblance with a biological ligand. For example, textile dyes can be used for the separation of proteins, and in particular Cibacron Blue F3GA has been employed as ligand in affinity membranes for the purification of dehydrogenases, since it often binds specifically to the nucleotide-binding site. Other dyes may adsorb proteins less specifically, but by selection of the right dye (a large number of different dyes is currently available) and the appropriate adsorption and elution conditions, highly efficient separations can be obtained.

Proteins carrying accessible histidine residues on their surface have been shown to have affinity for transition metal–chelate ligands. Typical examples are the iminodiacetate–copper(II) complex (IDA-Cu(II)) and the nitrilotriacetate–nickel (NTA-Ni(II)) ligand widely used for purification of recombinant proteins with genetically attached poly-His tails.

A third group are amino acids such as phenylalanine, tryptophane and histidine. Being the least selective, they have nevertheless been successfully employed for protein purification. However, fine-tuned adsorption and elution conditions are necessary to achieve efficient separation. Mention should also be made of the thiophilic affinity system that has been used with affinity membranes. It is based on the salt-promoted adsorption of proteins via thiophilic regions (containing aromatic amino acids) on to sulfone or thioether-containing heteroaliphatic or aromatic ligands.

Molecularly Imprinted Membranes

A completely different approach for the preparation of affinity membranes is the use of molecularly imprinted polymeric materials. These are produced by polymerization of functional and cross-linking monomers in the presence of the target molecule (the molecule to be separated later), which acts as a molecular template. In this way, binding sites are introduced in the polymer that are complementary in shape and functionality to the target molecule, and that often have specificities comparable to those of antibodies. At the same time, the cross-linked polymeric material provides a porous, chemically and physically very stable support. Even though the technology is in principle applicable to larger biomolecules such as proteins, it has mainly been used for the separation of small molecules like amino acids and peptides. The molecular imprinting technique is reviewed in more detail elsewhere.

Scale-up

Process scale-up tends to be rather easy in adsorptive membrane chromatography, at least compared to the use of conventional beaded resins as the chromatographic support. It has been demonstrated that the diameter of a stack of disc membranes can be increased by up to one order of magnitude and more, with the dynamic capacity remaining constant. This allows for the processing of considerably larger sample volumes at higher flow rates. With radial flow membranes, when both the height and diameter of the cartridge were increased and the flow rate adjusted proportionally to the increased cartridige volume, the apparent specific capacity decreased only slightly.

Applications

Several different applications of affinity membranes have been described. Typical examples of their use for the separation and purification of biomolecules are shown in Table 2.

Table 2

Examples for the use of affinity membranes for isolation and purification of biomolecules

The most common application is the separation and purification of biomolecules and especially proteins for large scale production. A common example is the separation of immunoglobulins from blood-serum or plasma or from cell culture supernatants. Hollow-fibre cartridges with immobilized protein A or pseudobiospecific ligands have been used for this purpose. Figure 2 shows a chromatogram from a case study of immunoglobulin G separation from human plasma using a small, developmental-scale (28 cm² surface area) poly(ethylene-co-vinyl alcohol) hollow-fibre membrane cartridge. The pseudobiospecific affinity ligand histidine was immobilized on to the membrane after activation with butanediol diglycidyl ether, thus introducing a spacer arm. Serum was injected 10-fold diluted in cross-flow filtration mode. Weakly retained and entrapped proteins were then removed by washing the lumen and the outer shell of the fibres, as well as the pores in back-flushing mode. Adsorbed immunoglobulins were subsequently eluted with a buffered solution of 0.4 mol L−1 NaCl in back-flushing mode. The eluted fraction contained 93% immunoglobulins (82% IgG, 10.8% IgM). The dynamic binding capacity of the membrane for immunoglobulin G was determined to be 1.9 gm−2. The process could then be scaled up by using a cartridge with 1 m² membrane surface area.

Figure 2 Separation of immunoglobins from human serum using a poly(ethylene-co-vinyl alcohol) hollow-fibre cartridge with immobilized L-histidine. (a) Immunoglobulin adsorption in cross-flow filtration mode; (b) lumen wash; (c) shell wash; (d) back-flush wash; (e) back-flush elution. (Adapted from Journal of Membrane Science 117, Bueno SMA, Legallais C, Haupt K and Vijayalakshmi MA, Experimental kinetic aspects of hollow-fiber membrane-based pseudobioaffinity filtration: Process for IgG separation from human plasma, pp. 45–56, Copyright 1996, with permission from Elsevier Science.)

A related application is the final polishing of an already pure product. For example, the removal of bacterial endotoxins from contaminated solutions of monoclonal antibodies has been demonstrated using membrane-bound pseudobiospecific ligands.

Affinity membranes have also been suggested for use in extracorporeal circuits, for the removal of toxic substances such as certain metabolites or antibodies from blood. For example, exogenous human serum amyloid P component, a substance associated with Alzheimer’s disease, has been removed from whole rat blood in an extracorporeal circulation system. This model system used a polyclonal antibody coupled to cellulose flat-sheet membranes. The biocompatibility of the membrane was also demonstrated. A similar application is the removal of autoantibodies from human plasma, using membrane-bound affinity ligands in extracorporeal circuits.

Apart from preparative applications, small cartridges with membrane discs or continuous membrane rods should be useful for analytical-scale separations and affinity solid-phase extraction, for example for immunoextraction.

Conclusions

Affinity membrane separation techniques combine the specificity of affinity adsorption with the unique hydrodynamic characteristics of porous membranes. They provide low pressure separation systems which are easy to scale up and ideal for the processing of large volumes of potentially viscous feed solutions (e.g. microbial broth, bacterial cell extract, conditioned media) often involved in the production of recombinant proteins. The additional microfiltration effect of membranes allows for the processing even of unclarified, particle-containing feed solutions. The high performance of this separation technique is due to the presence of through-pores and the absence of diffusional limitations; mass transfer is mainly governed by forced convection. Affinity membranes are used in applications such as purification of biomolecules, final product polishing, removal of unwanted substances from patients’ blood in extracorporeal circuits, but also for smaller scale analytical separations. Biological affinity ligands and biomimetic or pseudobiospecific ligands are currently employed, as well as different membrane configurations such as flat sheets, hollow fibres or continuous rods. The technology is now in the process of being adapted more and more for large scale industrial separation and purification.

See also: Centrifugation; Countercurrent Chromatography: Large-Scale; Countercurrent Chromatography: Overview; Liquid Chromatography: Mechanisms: Gradient Polymer Chromatography.

Further Reading

Brandt S, Goffe RA, Kessler SB, O'Connor JL, Zale SE. Membrane-based affinity technology for commericial scale purifications. Bio/Technology. 1988;6:779.

Charcosset C. Purification of proteins by membrane chromatography. Journal of Chemical Technology and Biotechnology. 1998;71:95.

Klein E. Affinity Membranes: Their Chemistry and Performance in Adsorptive Separation Processes. New York: John Wiley; 1991.

Roper DK, Lightfoot EN. Separation of biomolecules using adsorptive membranes. Journal of Chromatography. 1995;702:3.

Suen S-J., Etzel MR. A mathematical model of affinity membrane bioseparations. Chemical Engineering Science. 1992;47:1355.

Thömmes J, Kula MR. Membrane chromatography – an integrative concept in the downstream processing of proteins. Biotechnology Progress. 1995;11:357.

Affinity Partitioning in Aqueous Two-Phase Systems

G. Johansson    Center for Chemistry and Chemical Engineering, Lund University, Lund, Sweden

Aqueous Two-phase Systems in General

The division of water into non-miscible liquid layers (phases) by addition of two polymers has led to the remarkable possibility of being able to partition proteins and other cell components between phases of nearly the same hydrophilicity. Proteins can be separated by partitioning if they have unequal distribution between the phases, i.e. when their partition coefficients, K (the concentration in top phase divided by the concentration in bottom phase), differ. Usually the difference in the K value of many proteins is not very large and then repeated extractions have to be carried out to get a reasonable purification. If, however, the protein of interest (the target protein) has a very high K value and is mainly in the upper phase and all the contaminating proteins have very low K values so that they are in the bottom phase, an effective and selective extraction can be obtained in a single or a few partitioning steps. This type of partitioning has been made possible by using affinity ligands restricted to the upper phase.

The composition of the phases when two polymers like dextran and polyethylene glycol (PEG) are dissolved together in water depends on the amount of the polymers and their molecular weights. The concentration of the polymers in two phases of a given system can be found in the phase diagram for the temperature being used. A typical phase diagram is shown in Figure 1.

Figure 1 ) give systems with more top phase (three to five times) than bottom phase.

The line that connects the points in the diagram representing the compositions of the top and bottom phases of a system is called the tie-line. Each system with a total composition (percentage of each polymer) belonging to the same tie-line will have the same phase compositions. The smaller the tie-line, the more similar are the two phases in their composition. The greatest difference in composition of the top and bottom phases is therefore obtained by using high polymer concentrations.

The partitioning of proteins and also of membranes and particles depends on the polymer concentration of the system. The K value of a protein will be the same for all systems belonging to the same tie-line. The partition coefficient will, in most cases, decrease with the length of the tie-line, i.e. by using higher concentrations of the two polymers the material will accumulate more in the lower phase. Another way to affect the partitioning of proteins is by addition of salts to the system. Their effect depends on the type of cation and anion introduced with the salt. Negatively charged proteins show increasing K values when the cation is changed in the series:

For the anion the partition coefficient increases in the following order:

The highest K value of negatively charged proteins will then be obtained with the salt tetrabutylammonium hydrogenphosphate and the lowest K value with potassium perchlorate. Proteins with zero net charge (at their isoelectric points) are not affected by salts while positively charged proteins behave in an opposite manner to the negatively charged ones. For a number of proteins the log K values are nearly a linear function of their net charge (Figure 2).

Figure 2 Log K of the protein ribonuclease-A as function of its net charge, Z , 50mM). System compositions: (A) 6.2% w/w dextran 500 and 4.4% w/w PEG 8000; (B) 9.8% w/w dextran 500 and 7.0% w/w PEG 8000. Protein concentration, 2gL −1 . Temperature, 20°C. (Reprinted from Johansson G (1984) Molecular Cell Biochemistry 4: 169–180, with permission from Elsevier Science.)

Affinity Partitioning

The principle of affinity partitioning is to localize an affinity ligand in one phase to make it attract ligand-binding proteins. Since the phase-forming polymers are in each phase, either one can be used as ligand carrier. The standard system for affinity partitioning has been the one composed of dextran, PEG and water. Dextran is then used for localizing the ligand in the bottom phase while PEG can be used to concentrate the ligand to the top phase. PEG has often been chosen as ligand carrier because bulk proteins can be effectively partitioned into the dextran-rich lower phase by using high concentrations of polymers and a suitable salt. Thus, the target protein is extracted towards the upper phase leaving contaminating proteins in the bottom phase. PEG has two reactive groups (the terminal hydroxyl groups) which can be used as points of ligand attachment. In many cases only one ligand molecule is attached per PEG molecule. If the ligand is a large molecule (e.g. an antibody protein) several PEG chains may be attached to the one ligand molecule. Normally, only a fraction (1–10%) of the PEG in the two-phase system has to carry the ligand to reach maximal extraction efficiency. The more extreme the partitioning of a ligand–polymer is toward a phase the more effective it will be in extracting a ligand-binding protein into this phase. The partitioning of the ligand–polymer should be in the same range as the non-derivatized polymer but it may, in some cases, be more extreme. The higher the polymer concentrations are in the system, i.e. the longer the tie-line of the system, the more extreme is the partitioning of PEG to the top phase and dextran to the bottom phase. This can be expressed by the partition coefficients of the two polymers:

where c is the respective polymer concentration in top or bottom phase. Table 1 shows the KPEG and Kdextran values for systems containing PEG 8000 and dextran 500. Dextran has a more extreme value of K than PEG, i.e. KPEG < 1/Kdextran. Dextran should therefore, in principle, be a better ligand carrier than PEG. The concentration ratio for dextran is roughly the square of the ratio for PEG in the same system.

Table 1

Partition coefficients of PEG (KPEG) and dextran (Kdextran) and their logarithmic values (log) at various tie-line lengths of the system in Figure 1

A Simple Theory for Affinity Partitioning

A basic theory for affinity partitioning was elaborated by Flanagan and Barondes in 1975. They analysed the combined binding and partition equilibria taking place in and between the two phases, respectively (Figure 3).

Figure 3 Scheme for affinity partitioning of a protein (P) with two binding sites for a ligand attached to PEG (L). The complexes between protein and ligand–PEG are PL and PL 2 , respectively.

In this scheme the ligand–PEG(L), the free protein (P) and the two complexes (PL and PL2) have each their own partition coefficient (KL, KP, KPL and KPL2). Furthermore, in both phases association between protein and ligand–PEG takes place which can be described by the association constants:

one set for each phase.

A total association constant for the equilibrium:

can also be used: Ktot = K1K2.

The association constants, Ktot, K1 and K2 may differ between the two phases. According to Flanagan and Barondes, the measured log K value of a protein, log Kprotein, will, theoretically, give rise to a saturation curve when plotted versus the concentration of polymer-bound ligand in the system (compare Figure 4).

Figure 4 Increase in the logarithmic partition coefficient of phosphofructokinase (PFK) from bakers’ yeast as function of the concentration of Cibacron blue F3G-A PEG (Cb-PEG). System composition: 7%w/w dextran 500, 5%w/w PEG 8000 including Cb–PEG, 50mM sodium phosphate buffer pH 7.0, 0.5 mM EDTA, 5mM 2-mercaptoethanol and 4 nkat g −1 enzyme. Temperature, 0°C. The inverse plot inserted is used to determine the Δ log K max .

The log Kprotein value reaches a plateau when the concentration of L–PEG is so high that practically all the protein is present as the fully saturated complex PL2. The protein molecule is then surrounded by two PEG chains and outwardly shows a PEG atmosphere.

, is related to KP, KL and the K values via the following equations:

or:

The maximum increase in the logarithmic partition coefficient, Δ log Kmax, is consequently given by:

If Ktot,T = Ktot,B then Δ log Kmax = 2 log KL.

From the values in Table 1 it may therefore be assumed that for proteins with two binding sites Δ log Kmax can be as high as 3.57 (an increase of 3700 times in K) when PEG is used as ligand carrier with KL = 61. If dextran is used as carrier, in the same system, the Δ log Kmax should theoretically be around – 8 corresponding to a one hundred million times increase in the affinity of the protein for the lower phase if KL is 0.0001. A higher number of binding sites (n) should then give strongly increasing Δ log Kmax values with Δ log Kmax = n log KL. However, the affinity extraction effect may be reduced by a reduction of individual binding strengths.

Experimental Results

The extraction curves of a protein, here exemplified with phosphofructokinase (PFK) from baker’s yeast, using Cibacron Blue F3G-A PEG, closely follows the predicted behaviour (Figure 4). The inverse plot makes it possible to estimate the value of Δ log Kmax.

The dependence of Δ log Kmax of PFK on the polymer concentration is shown in Figure 5. Increasing concentration of polymers corresponds to longer tie-line length (and greater KL value) and this makes the affinity partitioning, measured as Δ log Kmax, more efficient.

Figure 5 (A) Log K ), 3% of total PEG; or without Cb–PEG (Δ). (B) Δ log K ) and log K ) as function of the tie-line length. System composition: dextran 500 and PEG 8000 (including Cb–PEG) in weight ratio 1.5 : 1, 50mM sodium phosphate buffer pH 7.0, 0.5 mM EDTA, 5mM 2-mercaptoethanol, and 4 nkat g −1 enzyme. Temperature, 0°C.

In addition to the concentration of polymers and ligand–PEG the actual Kprotein obtained also depends on pH value, the salt added to the system and the temperature. Two salts which have little or no effect on the affinity partitioning are phosphates and acetates in concentrations up to 50mM. In the case of PEG the Δ log Kmax is reduced with increasing temperature.

The detachment of ligand from the enzyme can be achieved either by using a high concentration of salt or by the addition of an excess of free ligand. For PFK the addition of adenosine triphosphase (ATP) to the system containing ligand–PEG strongly reduces the partition coefficient of the enzyme (Figure 6).

Figure 6 The effect of adenosine triphosphase (ATP) and of ATP + Mg ²+ on the partitioning of phosphofructokinase from bakers’ yeast in a system containing Cibacron blue F3G-A PEG (Cb–PEG). Δ log K ). System composition: 7% w/w dextran 500 and 5% w/w PEG 8000 including 0.5% Cb–PEG (of total PEG). 50mM sodium phosphate buffer pH 7.0, 0.5mM EDTA, 5mM 2-mercaptoethanol, and 4 nkat g −1 enzyme. Temperature, 0°C.

Types of Affinity Ligands Used

A number of affinity ligands have been used and some are presented in Table 2. The attachment of ligand to polymers and the purification of the ligand–polymer differs from case to case. Some ligands such as reactive texile dyes can be bound directly to PEG and to dextran in water solution of high pH. Other ligands are introduced by reactions in organic solvent, such as the attachment of acyl groups to PEG by reaction with acyl chloride in toluene. PEG may also be transformed into a more reactive form such as bromo-PEG, tosyl-PEG or tresyl-PEG. Some reaction pathways are shown in Figure 7. A number of methods to synthesize polymer derivatives have been published by Harris.

Table 2

Examples of affinity partitioning

Figure 7 Some reactions used for the covalent linkage of ligands to polymers, preferentially to PEG. The encircled ‘L’ represents the ligand and the open circles the polymer chain.

Preparative Extractions

The following steps may be useful for a high degree of purification by affinity partitioning.

1. Pre-extraction in a system without ligand–PEG to remove proteins with relatively high partition coefficients. The target protein stays in the bottom phase by adjusting the choice of polymer concentration, salt and pH.

2. Affinity partitioning is carried out by changing the top phase for one containing ligand–PEG. The target protein will now be in the top phase.

3. Washing the top phase with bottom phase to remove co-extracted proteins.

4. ‘Stripping’ of protein from the affinity ligand by addition of highly concentrated phosphate solution (50% w/w) to the separated upper phase. This generates a PEG-salt two-phase system with PEG and ligand–PEG in the top phase and target protein in the salt-rich bottom phase. An alternative stripping procedure can be carried out by adding a new pure dextran phase to the recovered top phase and supplying the system with free ligand. In this case the target protein will be collected in the lower phase.

For each step the number of extractions and the most suitable volume ratios for yield and purity can be optimized. The procedure is summarized in Figure 8.

Figure 8 ) by using four partitioning steps and PEG–dextran two-phase systems with PEG-bound ligand. This approach has been used for the purification of lactate dehydrogenase (LDH) from meat juice by affinity partitioning with Procion yellow HE-3G PEG. The inserted SDS-PAGE patterns of the original meat extract and the final product (obtained in the phosphate-rich phase) show the removal of contaminating proteins. Recovery of enzyme = 79%. System composition: 10%w/w dextran 500 and 7.1% w/w PEG 8000 including 1% Procion yellow HE-3G PEG (of total PEG), 50mM sodium phosphate buffer pH 7.9, and 25% w/w muscle extract. Temperature, 0°C. (Reprinted from Johansson G and Joelsson M (1986) Applied Biochemistry Biotechnology 13: 15–27, with permission from Elsevier Science.)

The yield in the top phase, YT, can be calculated from the K value of target protein and the volumes of top and bottom phase, VT and VR, respectively, using the following equation:

and the yield in the bottom phase, YB

A considerable concentration of the target protein, in addition to purification, can be achieved by choosing an extreme volume ratio with a small collecting phase.

An example of preparative extraction of an enzyme by applying the method given in Figure 8 is the purification of lactate dehydrogenase (LDH) using a PEG-bound textile dye. Crude extract of pig muscle, cleared by centrifugation, is mixed with PEG, dextran and Procion yellow HE-3G PEG. After the first partitioning the top phase is washed twice with pure lower phases and then it is mixed with a 50% w/w salt solution (25% NaH2PO4 + 25% Na2HPO4⋅H2O). The protein content of the final product in the salt-rich phase compared with that of the initial extract is demonstrated by the polypeptide pattern in sodium dodecyl sulfate-polyacryl amide gel electrophoresis (SDS-PAGE) shown in Figure 8. The L–PEG (and PEG) recovered in the final top phase is ≥ 95% of the initially introduced amount.

Purification of PFK in combination with a precipitation step with PEG before the affinity partitioning step greatly reduces the original volume of enzyme solution. The extraction included both preextraction and washing steps. The final polishing of the enzyme was made by ion exchanger and desalting with gel chromatography. The results can be seen in Table 3.

Table 3

Purification of phosphofructokinase from 1 kg (wet weight) bakers′ yeast

a In the presence of the protease inhibitor phenylmethylsulfonyl fluoride.

The effectiveness of affinity partitioning depends on the binding strength between ligand and protein. Good extraction is obtained with association constants of 10⁴ M−1 or more (Figure 9). The capacity, based on the amount of ligand in the system, is in the range of several hundred grams of protein per kilogram of system. Affinity extractions with 150 g of protein per kilogram of system have been carried out, and in these cases the two-phase systems strongly change the phase volume ratio while the bulk protein acts as a phase-forming component. In systems with high protein concentration the amount of dextran can be reduced or even excluded.

Figure 9 Affinity extraction into the top phase, by using increasing amount of PEG-bound ligand, calculated for an enzyme with the mole mass 100 000 g mol −1 , containing two binding sites for the ligand, and with K P  = 0.01. The value for the partition coefficient, K L , of the ligand is 100. The association constant, K , +) and 500gL −1 (Δ).

Countercurrent Distribution

A convenient way of multiextraction is countercurrent distribution (CCD). Here a number of top phases are sequentially moved over a set of bottom phases and equilibration takes place after each transfer. The process can be seen as a step-wise chromatography. The original two-phase system, number 0, contains the sample and after that a number (n) of transfers have been carried out n + 1 systems are obtained and the various proteins in the sample are distributed along the CCD train. The CCD process is visualized in Figure 10(A).

Figure 10 ). System composition: 7%w/w dextran 500 and 5%w/w PEG 8000 including ligand–PEG, 50mM sodium phosphate buffer pH 7.0, 0.2mM EDTA, and 5mM 2-mercaptoethanol. Temperature, 3°C. Systems in chamber 0–2 were initially loaded with yeast extract. (Reprinted from Johansson G, Andersson M and Akevland HE (1984) Journal of Chromatography 298: 485–495. With permission from Elsevier Science.)

The distribution of a pure substance can be calculated from the K value of the substance and the volumes of the phases, VT and VB. Assuming that all of the top phase volume is mobile and all bottom phase stationary, the fractional amount, Tn,i, in tube number i (i goes from 0 to n) after n transfers will be given by:

This makes it possible to calculate the theoretical curve for a substance and to make comparisons with the experimental distribution curve. Such an analysis may reveal the presence of several components even if they are not separated into discrete peaks. Figure 10(B) shows an example of a CCD of a yeast extract using PEG-bound affinity ligands. The distribution of a number of enzyme activities has been traced.

Use of Dextran as a Ligand Carrier

Dextrans of the molecular weights normally used (40 000 and 500 000 Da) contain many thousands of reactive hydroxyl groups per molecule. The affinity partitioning effect achieved by introducing one or just a few dye ligands is shown in Figure 11. Since the dye ligands used here carries seven to ten charged groups per molecule they also add a considerable (negative) net charge to the ligand dextran. Its partitioning will then be sensitive to the presence of salt and the choice of salt. The ligand–dextran can be directed either to the bottom phase or the top phase. This steering is more effective the greater the number of ligands per dextran molecule.

Figure 11 (A) Partitioning of Procion yellow HE-3G dextran 70 (PrY–Dx) depending on the degree of substitution, n ), at pH 7.9. Arrow indicates K of unsubstituted dextran. System composition: 8% w/w dextran 70 and 4.5% w/w PEG 8000 including PrY–Dx (50 μM bound dye), and indicated salt. Temperature, 22°C and pH of system adjusted to 7.9. (Reprinted from Johansson G and Joelsson M (1987)Journal of Chromatography 411: 161–166. With permission from Elsevier Science.) (B) Effect of the concentration of PrY–Dx on the partitioning of the enzyme glucose-6-phosphate dehydrogenase (G6PDH) using PrY–Dx with n . System as in (A) with 50mM sodium phosphate buffer.

The effect of ligand–dextran on the partitioning of an enzyme, glucose-6-phosphate dehydrogenase, is shown in Figure 11. There is also a tendency towards affinity precipitation when the concentration of ligand molecules is equal to the concentration of enzyme binding sites in the system. This is seen as a shallow dip in the extraction curve.

Use of a Third Polymer as Ligand Carrier

The ligand can be bound to a third polymer chosen in such a way that it will be mainly concentrated in one phase. Alternatively, if it is carrying enough charged groups, it may be steered to one phase by using salts. The efficiency, measured as Δ log K/log KL, equal to the apparent number of binding sites of the protein, has in several cases showed that the most effective polymer for carrying the ligand is neither of the two phase-forming polymers. The effect of a dye ligand, bound to various polymers, on the partitioning of lactate dehydrogenase in a dextran–PEG system is presented in Table 4.

Table 4

The effect of ligand carrier on its efficiency in producing affinity partitioning

(Reprinted from Johansson and Joelsson M (1984) Journal of Chromatography 411: 161–166. With permission from Elsevier Science.)

Lactate dehydrogenase (LDH) was partitioned in systems containing 7% (w/w) dextran 500, 5% w/w PEG 8000, 25mM sodium phosphate buffer, pH 7.5, and Procion yellow HE-3G polymer of dye concentration of 42μM. Temperature, 22°C.

Chiral Affinity Partitioning

For separation of low molecular weight substances into their enantiomeric forms a system may be used where one of the phases contains a high molecular weight substance which binds one of the enantiomers. Bovine serum albumin as well as cyclodextrin have been used for this purpose.

Analytical Uses

Besides the preparative use of aqueous two-phase systems, they have been applied to a number of analytical studies of the properties of biological macromolecules and particles. Some of these uses are binding studies, conformational changes, studies of antibodies, and homogeneity studies of protein, nucleic acids, membranes, organelles and cells.

Multiphase Systems

By using more than two polymers, multiphase systems can be obtained. In principle, the number of phases can be as many as the kinds of polymers used. A three-phase system of PEG, Ficoll, and dextran has been used with two ligands (in different phases) for directing the partitioning of blood serum proteins.

Semi-organic Systems

Part of the water in a two-phase system may be replaced by certain solvents. Often dextran cannot be used because of low solubility but it may be replaced by Ficoll. The log K of a protein may change drastically by introducing the organic solvent. Also the Δ log K may in some cases be reduced while in other cases has been found to remain relatively uneffected.

Affinity Partitioning of Nucleic Acids and Bioparticles

Affinity partitioning in aqueous two-phase systems is not restricted to proteins, but has been also used for purification of DNA, using base-pair specific ligands, membrane fragments, and cells, such as erythrocytes. Some examples of such affinity extractions are found in Table 2.

Future Prospects

More specific ligands will certainly come into use for affinity partitioning and systems with much larger partition coefficients will be developed. This will allow not only specific extraction of biomaterials but also their many-fold concentration. Effective recycling processes of ligand–polymers will make it economically feasible to use affinity partitioning for extraction of enzymes on a technical scale. Successive extraction of several components from one and the same source by using a number of ligands in series extraction can be foreseen.

Conclusions

Affinity partitioning is a method of selective liquid–liquid extraction for purification and studies of proteins and other ‘water stable’ cell constituents. The scaling up of this process is uncomplicated and the recovery of ligand polymer reduces the cost.

See also: Affinity Membranes; Centrifugation.

Further Reading

Albertsson PA. In: Partition of Cell Particles and Macromolecules. 3rd edn. New York: John Wiley; 1986:334–340.

Albertsson PA, Birkenmeier G. Affinity separation of proteins in aqueous three-phase systems. Analytical Biochemistry. 1988;175:154–161.

Flanagan SD, Barondes SH. Affinity partitioning – a method for purification of proteins using specific polymer-ligands in aqueous polymer two-phase systems. Journal of Biological Chemistry. 1975;250:1484–1489.

Harris JM. Laboratory synthesis of polyethylene glycol derivatives. Journal of Macromolecular Science. Reviews of Polymer Chemistry and Physics. 1985;C25:325–373.

Johansson G. Multistage countercurrent distribution. In: Townshend A, ed. The Encyclopedia of Analytical Science. London: Academic Press; 1995:4709–4716.

Johansson G, Joelsson M. Affinity partitioning of enzymes using dextran-bound Procion yellow HE-3G. Influence of dye-ligand density. Journal of Chromatography. 1987;393:195–208.

Johansson G, Kopperschläger G, Albertsson PA. Affinity partitioning of phosphofructokinase from baker's yeast using polymer-bound Cibacron blue F3G-A. European Journal of Biochemistry. 1983;131:589–594.

Kopperschläger G, Birkenmeier G. Affinity partitioning and extraction of proteins. Bioseparation. 1990;1:235–254.

Tjerneld F, Johansson G, Joelsson M. Affinity liquid-liquid extraction of lactate dehydrogenase on a large scale. Biotechnology and Bioengineering. 1987;30:809–816.

Walter H, Johansson G, eds. San Diego, CA: Academic Press; . Methods in Enzymology, Aqueous Two-phase Systems. 1974;228.

Affinity Separations

K. Jones    Affinity Chromatography Ltd, Freeport, Ballsalla, Isle of Man, UK

Introduction

Of the collection of separation technologies known as ‘affinity’, affinity chromatography is by far the most widely used variant. Affinity chromatography is becoming increasingly important as the speed of the revolution taking place in biotechnology processing increases. The concept of an ‘affinity’ separation results from a naturally occurring phenomenon existing within all biological macromolecules. Each biological macromolecule contains a unique set of intermolecular binding forces, existing throughout its internal and external structure. When alignment occurs between a specific site of these forces in one molecule with the site of a set of forces existing in another (different) molecule, an interaction can take place between them. This recognition is highly specific to the pair of molecules involved. The interactive mechanism can be converted into a universal mutual binding system, where one of the binding pair is attached to an inert matrix, packed into a column and used exclusively to capture the other matching molecule. When used in this (affinity) mode, the technique is probably the simplest of all chromatographic methods. It is, however, restricted almost exclusively to the separation and purification of biological macromolecules, and is unsuitable for small molecules.

Affinity chromatography or bioselective adsorption chromatography was first used in 1910, but it was only in the 1960s that affinity chromatography as practised today was developed as a purification technique. By the late 1970s the emergence of recombinant DNA technology for the manufacture of protein pharmaceuticals provided a new impetus for this highly specific chromatographic method, implemented by the demand for ever-increasing product purity implicit in regulatory frameworks devised by (amongst others) the USA’s Food and Drug Administration (FDA). Finally, the need to reduce the cost of drugs is under constant scrutiny by many Governments, particularly those with controlled health schemes funded by revenue raised by taxation. These mutually incompatible pressures indicate the need for more efficient separation systems; the affinity technique provides the promise of meeting all necessary requirements.

Separation and purification methods for biological macromolecules vary from the very simple to the esoteric. The type of technique adopted is basically a function of source, the fragility of the molecule and the purity required. Traditionally, high purity protein pharmaceuticals have used multistage processing, but this is very inefficient as measured by the well-documented fact that 50–80% of total production costs are incurred at the separation/purification stage. In contrast, the highly selective indigenous properties of the affinity method offer the alternative of very elegant single-step purification strategies. The inherent simplicity and universality of the method has already generated a wide range of separation technologies, mostly based upon immobilized naturally occurring proteinaceous ligands. By comparing the ‘old’ technologies of ‘natural’ ligands or multistage processing with the ‘new’, exemplified by synthetic designed ligands, the most recent advances in affinity processing can be described.

Biological Recognition

As nature evolved, life forms had to develop a protective mechanism against invading microorganisms if they were to survive. Thus there is a constant battle between the cell’s defence mechanism and the attacking microorganisms, a battle resolved by the cells generating antibodies (the immunoglobulins) able to recognize the protein coat of attacking microorganisms and signal killer cells to destroy the invaders before they cause harm to the host. Equally, if microorganisms were to survive, they had continually to mutate and change their protein coats to avoid detection by existing antibodies. The ‘attack and destroy’ process is a function of changes in the molecular structure in a specific part of the protein, with only the most minute of changes occurring at the surface of the protein. Evolution has thus designed a system where every protein has a very precise structure, but one which will always be recognized by another. One element of the interacting pair can be covalently bonded onto an inert matrix. The resulting chromatographic medium can then be packed into a column, and used to separate exclusively its matching partner from an impure mixture when added as a solution to the top of the column. This fact can be stated as follows – for every protein separation problem there is always an affinity solution. The process of producing a satisfactory medium is quite difficult. The matching pair must be identified, and one of them isolated in a pure form. Covalent bonding onto an inert matrix in a stable manner must always allow the ‘docking’ surface of the protein to be positioned to make it available to the target protein. The whole also has to be achieved at an acceptable cost.

This technique has resulted in many successful applications, often using antibodies as the affinity medium (immunoaffinity chromatography), but large scale separations using these ‘natural’ ligands are largely restricted by cost and regulatory reasons. Although immunoaffinity chromatography is still widely practised, in recent years the evolution of design technologies has provided powerful new approaches to mimic protein structures, resulting in the development of synthetic ligands able to work in harsh operational environments and at low cost.

The Affinity Process

The affinity method is critically dependent upon the ‘biological recognition’ existing between species. By permanently bonding onto an inert matrix a molecule (the ligand) that specifically recognizes the molecule of interest, the target molecule (the ligate) can be separated. The technique can be applied to any biological entity capable of forming a dissociable complex with another species. The dissociation constant (Kd) for the interaction reflects the complementarity between ligand and ligate. The optimal range of Kd for affinity chromatography lies between 10–4 and 10–8molL–1. Most biological ligands can be used for affinity purposes providing they can be immobilized, and once immobilized continue to interact successfully with their respective ligates. The ligand can be naturally occurring, an engineered macromolecule or a synthetic molecule. Table 1 provides some examples of immobilized ligands used to purify classified proteins. The affinity method is not restricted to protein separations; nucleic acids and whole cells can also be separated.

Table 1

Affinity ligands and purified proteins

The simplicity of the chromatographic process is shown in Figure 1. The ligand of interest, covalently bonded onto the inert matrix, is contained in the column, and a solution containing the target (the ligate) is passed through the bed. The ligand recognizes the ligate to the exclusion of all other molecules, with the unwanted materials passing through the column packing while the ligate is retained. Once the bed is saturated with the target molecule (as measured by the breakthrough point), contaminating species are washed through, followed by collection of the target molecule as a very pure fraction using an eluting buffer solution. Finally, the column is cleansed from any strongly adsorbed trace materials, usually by regeneration with a strong alkali or acid, making it available for many more repeat runs. An outstanding advantage of the affinity process is an ability to concentrate very dilute solutions while stabilizing the captured protein once adsorbed onto the column. Many of the in-demand proteins manufactured by genetically engineered microorganisms are labile, allowing only minute quantities to be present in the fermentation mix before they begin to deteriorate. An ability to capture these very small quantities while stabilizing them in the adsorbant phase results in maximization of yield, making massive savings in total production costs.

Figure 1 Schematic diagram of affinity chromatography.

Although the technical processing advantages are clear there is a major difficulty in the application of affinity chromatography as understood by most practitioners today. Most ligands described in Table 1 suffer from two primary disadvantages: a lack of stability during use; and high cost. Fortunately these problems have now been overcome, and affinity chromatography is now accepted as the major separations technology for proteins.

Matrices

By definition matrices must be inert and play no part in the separation. In practice most play a (usually) negative role in the separation process. To minimize these disadvantages matrices have to be selected with great care. There is a theoretically perfect matrix, defined as consisting of monodispersed perfectly shaped spheres ranging from 5 to 500 μm in diameter, of high mechanical strength, zero nonspecific adsorption and with a range of selectable pore sizes from 10–500 nm, a very narrow pore size distribution and low cost. This idealized matrix would then provide the most efficient separation under all experimental conditions. As always, a compromise has to be reached, the usual approach being to accentuate the most attractive characteristics while minimizing the limitations, usually by manipulating the experimental conditions most likely to provide the optimum result.

The relative molecular masses of proteins vary from the low thousands to tens of millions, making pore size the most important single characteristic of the selected matrix. Very large molecules need very open and highly porous networks to allow rapid and easy penetration into the core of the particle. Structures of this type must therefore have very large pores, but this in turn indicates low surface areas per unit volume, suggesting relatively low numbers of surface groups to which ligands can be covalently attached. The matrix must also be biologically and chemically inert. A special characteristic demanded from biological macromolecular separations media is an ability to be sanitized on a routine basis without damage. This requires resistance to attack by cleansing reagents such as molar concentrations of strong alkali, acids and chaotropes. In contrast to analytical separations, where silica-based supports are inevitably used, silica cannot meet these requirements and is generally not favoured for protein separations. Table 2 contains examples of support matrices used in affinity separations.

Table 2

Support matrices

PTFE, polytetrafluoroethylene.

The beaded agaroses have captured over 85% of the total market for biological macromolecule separations, and are regarded as the industry standard to