The affinity technology in downstream processing

journal of biotechnology ELSEVIER

Journal of Biotechnology36 (1994)95-119

Minireview

The affinity technology in downstream processing N. Labrou, Y.D. Clonis * Enzyme Technology Laboratory, Department of Agricultural Biology and Biotechnology, Agricultural University of Athens, lera Odos 75, 118 55 - Athens, Greece

Received 21 October 1993; revision accepted 18 February 1994

Abstract

The quality criteria imposed on several biochemicals are stringent, thus, high-separation purification technology is important to downstream processing. Affinity-based purification technologies are regarded as the finest available, and each one differs in its purifying ability, economy, processing speed and capacity. The most widely used affinity technology is classical affinity chromatography, however, other chromatography-based approaches have also been developed, for example, perfusion affinity chromatography, hyperdiffusionT M affinity chromatography, high-performance affinity chromatography, centrifugal affinity chromatography, affinity repulsion chromatography, heterobifunctional ligand affinity chromatography and the various chromatographic applications of 'affinity tails'. On the other hand, non-chromatographic affinity technologies aim at high throughput and seek to circumvent problems associated with diffusion limitations experienced with most chromatographic packings. Continuous affinity recycle extraction, aqueous two-phase affinity partitioning, membrane affinity filtration, affinity cross-flow ultrafiltration, reversible soluble affinity polymer separation and affinity precipitation are all non-chromatographic technologies. Several types of affinity ligands are used to different extents; antibodies and their fragments, receptors and their binding substances, avidin/biotin systems, textile and biomimetic dyes, (oligo)peptides, antisense peptides, chelated metal cations, lectins and phenylboronates, protein A and G, calmodulin, DNA, sequence-specific DNA, (oligo)nucleotides and heparin. Likewise, there are several support types developed and used; natural, synthetic, inorganic and composite materials. Key words: Downstream processing; Bioseparation; Enzyme purification; Biocatalyst purification; Affinity technol-

ogy; Affinity chromatography; Affinity ligand; Affinity adsorbent; Dye ligand

I. Introduction

The key-factor for the commercial development of biotechnology is downstream processing, which purifies bioproducts and often accounts for

* Corresponding author.

at least 50% of total costs. Critical steps in downstream processing are the 'high-separation' technologies, of which chromatography is the most widely used. However, where substantial purity is necessary, for example, with molecular biology, diagnostic and therapeutic proteins, the finest of technologies is often employed: affinity chromatography (Cuatrecasas and Anfinsen, 1971; Lowe, 1979; Clonis, 1987a; Clonis and Lowe, 1988;

0168-1656/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0168-1656(94)00054-G

96

N. Labrou, Y.D. Clonis/Journal of Biotechnology 36 (1994) 95-119

Clonis, 1990). The affinity concept relies on the specific reversible complexes formed between the (macro)molecule to be purified and the 'ligand' bound on a suitable polymer support, e.g., porous beads. The affinity adsorbent is packed in a chromatographic column and the sample is applied. Ideally, only the substance that selectively recognises the ligand binds the adsorbent, whereas all other species pass unbound through the bed. After washing the column, adsorbed substances are eluted by employing either a non-specific technique (change of ionic strength, pH, temperature, or adding chaotropic agents, urea, ethylene glycol, detergents, ethanol, etc.) or a specific technique (adding a competing substance; substrate, inhibitor, cofactor, metal-chelator, imidazole, etc.). The development of affinity separation technology paralleled that of immobilisation chemistry, support materials and affinity-ligand design. Various types of beaded supports are used which are derived from specific manufacturing processes (Arshady, 1991a), for example, natural polymers (e.g., agarose, dextran and cellulose), synthetic polymers (e.g., perfluorocarbons, polyacrylamide, polyacryloyltrihydroxym ethylacrylamide, polyhydroxyethylmethacrylate, plastics, latex), inorganics (e.g., silica, controlled pore glass, titania), and composite materials (e.g., silica-polysaccharides, polyacrylamide-agarose, core-shell graft composites). Following the revolution of monosized/monodispersed beads, a breakthrough is now realised by the 'flow-through particles' exploited in perfusion chromatography (Afeyan et al., 1990). Perfusion takes place when the rate of convective mass transfer within chromatography particles exceeds the rate of diffusive mass transfer of a given solute. In perfusion technolo~y the pores are wide enough (6 x 103-8 )< 103 Angstr6m) to allow convective flow through the particles. Smaller diffusive pores along the throughpore channels of the particles provide high adsorption area with diffusion path lengths less than 1/zm. The binding capacity of perfusive media is therefore limited by the fact that binding sites (e.g., affinity ligands) must be confined to the pore wall surface or a surface coating, in other words, the affinity ligand is not present within the entire pore volume. This new particle

design, without significant losses in separation and capacity, reduces process time by improving mass transfer. Probably the most recent particle design is the one introduced by Sepracor Inc. called HyperD TM and it is exploited in hyperdiffusionT M chromatography (Sepracor Inc., 1993). The latter technique is differenciated from perfusion chromatography as it uses hyperdiffusive particles with their entire pore volume filled by a homogeneous flexible and soft hydrogel. Capacity is achieved by a space-filling rather than a surface area approach, therefore, it appears that in hyperdiffusion media the hydrogel prevents perfusion from taking place. Accordingly, protein molecules can diffuse freely into hyperdiffusive media (throughout the hydrogel), yet convective transport of those molecules through the beads cannot occur at a rate greater than that of diffusive transport. Other materials for affinity-ligand coupling are non-porous fibres, hollow fibres, flat membranes, phase-forming water soluble polymers, perfluorocarbon emulsions and solid particles. The coupling chemistry of ligands is diverse and established (Dean et al., 1985; Clonis, 1989; Arshady, 1991b; Clonis, 1992), however, a most unusual ligand-coupling approach is one where immobilisation is effected via adsorption. For example, considering the di-chlorotriazine dye ligand Procion ® Blue MX-R, immobilisation by adsorption can be effected as follows. First, a hydrophobic 1H,1H-pentadecafluorooctylamine tail is anchored to the reactive dye by a nucleophilic substitution reaction (two tails per dye molecule). Afterwards, the dye-tail conjugate is physically adsorbed on Teflon ® fluorocarbon particles by simple hydrophobic interactions (Stewart et al., 1989). Likewise, hydrophobic fluorocarbon supports can be turned into hydrophilic-coated materials. In this case, hydrophobic perfluorocarbon tails are chemically anchored to hydrophilic polyvinylalcohol (PVA). Afterwards, the PVA-tail congugate is physically adsorbed on the hydrophobic surface of Teflon ® particles. Therefore, the new Teflon ® surface exhibits hydrophilic character and is rich in hydroxyl groups via which several affinity ligands can be immobilised (Stewart et al., 1990). Perfluorooctanoyl PVA-bound ligands also can be immobilised on

N. Labrou, YD. Clonis/Journal of Biotechnology 36 (1994) 95-119 Table 1 Affinity pairs and their strength of interaction (dissociation constant) Affinity pair Target protein

Ligand

Avidin Streptavidin Receptors Antibodies Transport proteins Lectins Enzymes

biotin biotin hormones, toxins, etc. antigens vitamins, sugars, etc. carbohydrates substrates

Strength of interaction k D (M) approx. 10-15 approx. 10-15 10-9-10 -12 10- 7-10 - 11 10 -6 -10 -8 10- 3 - - 10- 6 10- 3_10-5

perfluorocarbon emulsions (McCreath et al., 1990), whereas, the principle of immobilisation by adsorption has been exploited in systems of surfactant-ligands and octadecyl-bonded silica (Torres et al., 1988). Turning now to the most important component of an affinity system, the ligand, one distinguishes two categories (Table 1). In the first category belong ligands of k D values ranging from 10 -7 up to 10 -15 M, therefore, these are ligands of high affinity. The second category collects ligands of broader spectrum of interaction and selectivity. These ligands are known as general-affinity ligands and inevitably exhibit higher k D values (typically, 10-4-10 -6 M). Nevertheless, a factor which is often overlooked in the development of an affinity process is the immobilised ligand concentration of the adsorbent. This factor may influence not only the binding capacity of the affinity adsorbent but also its selectivity and purifying ability (Lowe, 1979; Boyer and Hsu, 1992).

2. High-affinity ligands The highly specific and strong interactions observed between antigens and antibodies are exploited in immunoaffinity chromatography (IAC). The technique may be used either with polyclonals or monoclonals (MAb), although the latter are often prefered. This is due to various reasons, for example, MAbs offer the possibilities of employ-

97

ing impure antigen (sera) for their preparation, selecting a suitable MAb of desired binding strength (usually 5 × 10-7-5 × 10-8 M) and producing them at the scale required by growing as ascites or in tissue culture. Numerous examples of IAC applications may be found in the literature, whereas theoretical and practical aspects have been discussed (Ehle and Horn, 1990; Pepper, 1992). An alternative approach to whole-antibody ligand, is to employ antibody fragments. For example, Fv antibody fragments are cloned and expressed in Escherichia coli. and the resulted binding domains in their immobilised form, purified hen-egg lysozyme (Berry et al., 1991). The same enzyme is purified on immobilised synthetic amino acid sequences that mimic antigenbinding Ab sites (Welling et al., 1990). Antipeptide antibody ligands, which are prepared against synthetic amino acid sequences of antigens, present another approach to ligand design. Thus, the eight amino acid sequence of the Cterminal region of rice a-amylase is employed as antigen fragment to develop an antibody affinity ligand (Kato and Terashima, 1993). Cross-reactive antibodies offer an alternative solution in IAC technology. For example, anti-barley aamylase antibody ligand is used to purify rice a-amylase which is cloned, expressed and secreted by yeast cells. This is possible because of the homology between barley and rice a-amylases (Kato and Terashima, 1993). Receptors and receptor-binding substances are highly specific affinity systems. Thus, employment of receptor-ligand adsorbents is an effective high-affinity method for isolating receptor-binding molecules. Recombinant interleukin-2 (rlL-2) is purified to homogeneity from mammalian and microbial sources on its immobilised receptor, following elution with 0.2 N acetic acid containing 0.2 M NaC1 (Weber and Bailon, 1990). The isolated product is an active monomeric form of the lymphokine, in contrast to that obtained by immunoaffinity chromatography where several active molecular forms of rlL-2 were shown (Bailon et al., 1987). The same affinity principle is applied, in homogeneous-phase system, to purify estrogen receptors from cytosols of rat uteri and human fibroid uterine tissue. In this case, the

98

N. Labrou, Y.D. Clonis/Journal of Biotechnology 36 (1994)95-119

Table 2 Some general-affinity ligands used in affinity separation technology Triazine dyes Commercial textile dyes Purpose-designed biomimetic dyes Amino acid-based ligands Antisense peptides Paralog peptides Oligopeptides Lysine, histidine Nucleotide-based ligands Nucleotides Coenzymes PolyU and polyA DNA (ss and ds) (Multi)sequence-specific DNA Heparin Chelated metal cations Iminodiacetic acid-chelated metal cations Nitrilotriacetic acid-chelated metal cations Carbohydrate-binding ligands Lectins Phenylboronic acid Protein ligands Staphylococcal protein A Streptococcal protein G Calmodulin

target receptors interacted with a soluble estradiolpolylysine ligand. The highly charged soluble conjugate receptor-estradiolpolylysine is separated from other proteins on a DEAE-cellulose column. After desorption and dissociation of the conjugate, the receptors are separated from the affinity ligand on a CM-Sephadex ® column (Bhattachacharjee and Ali, 1992). Useful high-affinity systems are also those

based on the avidin/streptavidin-biotin complexes (Bayer and Wilchek, 1990; Desarnaud et al., 1992). Biotin is a vitamin and avidin is a glycoprotein of egg-white, which together form a strong complex of k o value of approx. 10 -15 M. Streptavidin is the bacterial analogue to avidin and is isolated from Streptomyces avidinii. The major differences between avidin and streptavidin lie in the fact that the former is alkaline and usually consists of a single oligosaccharide chain per subunit, whereas the latter is a neutral nonglycosylated protein. Therefore, in terms of suitability to affinity separations, the avidin system may be less suitable than the streptavidin, since the former is expected to show higher non-specific interactions due to association with negatively charged molecules. Among several applications of the biotin-avidin system, an interesting one is the purification of solid-phase synthesised peptides. During the chemical synthesis, biotinylated methionine is added as terminal amino acid. After removal of the synthetic peptides from the solid-phase resin, the product is first purified on an anti-hapten antibody column to remove undesired truncated peptides containing the hapten. Afterwards, the unbound product obtained from the first column is finally purified on an avidin affinity column (Bayer and Wilchek, 1990).

3. General-affinity ligands General or group-specific affinity ligands, as the name implies, exhibit wide spectrum of interaction with protein molecules, therefore, their selectivity is reduced and they can be used in different purification cases. Table 2 summarises the most important types of such affinity ligands.

Table 3 A comparison between immobilised dye-ligands and biological-ligands Criterion

Dye-ligand adsorbent

Biological-ligand adsorbent

Economy Synthesis Stability Capacity/ligand utilisation Specificity Scaling-up

inexpensive, commodity chemicals easy chemically and biologically stable high moderate (dyes) to high (biomimetic dyes) large-scale potential

c a n be expensive, especially coenzymes and antibodies often lengthy routes and use of dangerous chemicals biodegradable and chemically labile moderate to low high to moderate, depending on ligand selection limited application

N. Labrou, Y.D. Clonis /Journal of Biotechnology 36 (1994) 95-119

3.1. Textile and biomimetic triazine dyes

Perhaps these are the most promising ligands of large-scale potential, and their corresponding adsorbents find wide application to protein purification (Clonis, 1987a, 1991; Clonis et al., 1987a; Scawen and Atkinson, 1987; Clonis and Lowe, 1988). The originally employed dyes were commercial textile chlorotriazine polysulphonated molecules which are readily immobilised on various supports bearing hydroxyl groups. Unlike various biological affinity adsorbents, the stability of dye adsorbents is limited only by the support itself. As shown in Table 3, dyes offer clear advantages over biological ligands, in terms of economy, ease of immobilisation, safety, stability and adsorbent capacity. Apparently, the only drawback of commercial textile dyes seems to be their moderate selectivity. However, the overall size, shape and distribution of ionic and hydrophobic groups, enable these molecules to interact with the binding site(s) of numerous target proteins. Although the binding is often dominated by electrostatic forces, the dye-protein interaction should not be compared to simple ionexchange type, since binding is frequently possible at pHs greater than the p I of the protein. Furthermore, elution is often effected by soluble competing ligands (Bouriotis and Dean, 1981; Clonis and l_owe, 1981; Clonis et al., 1981; Makriyannis and Clonis, 1993), which suggests interaction with the protein at discrete sites. This view is supported by chromatographic, spectra difference, kinetic, affinity labelling and inactivation studies. All these approaches point to the fact that many triazine dyes interact, at least with several dehydrogenases and kinases, in a specific manner at the nucleotide binding site of the protein (Clonis and Lowe, 1980a, 1981; Clonis et al., 1981). Although the most widely used dye Cibacron ® blue 3GA (CI Reactive blue 2), as well as other dyes, generally bind proteins with moderate specificity, this has not prevented them from acting as effective ligands in numerous purification cases (Scawen and Atkinson, 1987), including several large-scale applications (Clonis, 1991). For example, Trisacryl Plus®-immobilised Cibacron blue 3GA is used in the large-scale

99

purification of human albumin (Allary et al., 1991). During this process, trace contaminants of leached dye may be quantified by an ELISA method (Santambien et al., 1992). The amount of albumin (98-100% pure) produced per cycle reached 250 g on a 50 lit column, corresponding to 82% yield (Allary et al., 1991). Several recent protocols are published where proteins are purified on immobilised dyes, for example, NAPD ÷linked alcohol dehydrogenase from anaerobic extreme thermophiles on Blue-Sepharose ® CL-6B adsorbent (Nagata et al., 1992), L-lactate dehydrogenase and pyruvate kinase from rabbit muscle, both suitable for analytical application, on Cibacron ® blue 3GA and Procion ® yellow MX4G (Tsamadis et al., 1992; Makriyannis and Clonis, 1993), fumarase from Saccharomyces cerevisiae suitable for X-ray studies on Sepharose ® CL-6B-immobilised triazine dyes (Keruchenko et al., 1992), and mammalian glycoprotein nerve growth factor from the venom of Vipera russelli russelli on amino derivatives of dyes immobilised on polyvinylpolypyrrolidone particles (Koyama et al., 1992). Nevertheless, to address the problem of moderate dye selectivity, two approaches are available. According to the first, it takes place screening of a large number of immobilised commercial dyes. Subsequently, the adsorbent exhibiting the highest purification factor for the target macromolecule is employed to purification protocols for the protein of interest (Bouriotis and Dean, 1981; Clonis and Lowe, 1981; Vlatakis et al., 1987; Kroviarski et al., 1988; Giuliano, 1992). The second approach reflects a new trend in this area of biotechnology and signalled the beginning of a new era in affinity separation. This approach is based on a new generation of dyes, the biomimetic dyes. Purpose-made structural changes of the parent dye lead to new redesigned dyes which mimic naturally occurring biological ligands. Therefore, the biomimetic dyes exhibit increased selectivity towards target enzymes. Fig. 1 shows the structures of the parent dye Cibacron ® Blue 3GA (a) and three biomimetic dyes (b, c and d). The first step towards designing entirely novel biomimetic dyes was to study the enzyme alcohol dehydrogenase (ADH) and the monochloro-triazine dye Cibacron ® blue 3GA

100

N. Labrou, Y.D. Clonis/Journal of Biotechnology 36 (1994) 95-119

(Lowe et al., 1986). By comparing the conformations that N A D ÷ and Cibacron Blue 3 G A adopt during binding to A D H (Fig. 2), one realises that the adenine, adenosine ribose and pyrophosphate groups of N A D ÷ adopt similar positions in binding to A D H as the anthraquinone, diaminobenzene sulphonate and the triazine groups of Cibacron ® Blue 3GA. However, the terminal animobenzene sulphonate ring of the dye ligand binds very differently to A D H than the ribose and nicotinamide groups of the biological ligand N A D +. Consequently, a novel biomimetic blue dye analogue is designed exhibiting increased length, flexibility and selectivity, thus, mimicking

(a)

~

SO3"

Phe

~

•

B

"O35

(b)

~

SO3"

O

NH

NHCH2CH2NH'~

"O3S

N NH--~ SO3

(c) Fig. 2. Schematic presentation depicting the mode of binding of coenzyme NAD ÷ (top) and dye Cibacron® Blue 3GA (bottom) to the nucleotide binding site of horse liver alcohol dehydrogenase.

P2 S03"

/,~cl NH"~ "O3S

#N N @ ' - - - C H 2 P O 3"

(d)

H2N\

./~---~ N=N ./~--~

H3c

N

/N==~NCI

cl

Fig. 1. Structures of parent dye Cibacron® Blue 3GA (a) followed in sequence by three biomimetic dyes: two blue-analogues (b and c) and a benzamidino-cationicyellow (d).

better the biological ligand N A D +. This is achieved by inserting a central ethyl-spacer to the parent dye and replacing the terminal o-isomer for a m-isomer ring (Fig. lb). These modifications are critical since horse liver alcohol dehydrogenase displayed a 7-fold decrease of its dissociation constant with the new biomimetic dye ligand (Lowe et al., 1986). A similar approach is adopted for designing a biomimetic ligand for alkaline phosphatase. In this case, the new dye

N. Labrou, Y.D. Clonis/Journal of Biotechnology 36 (1994) 95-119

ligand is designed by substituting the terminal 2-aminobenzene sulphonate for a 4-aminobenene phosphonate ring (Fig. lc). The corresponding dye adsorbent is able to purify calf intestinal alkaline phosphatase by 330-fold in a single step (Lindner et al., 1989). The third biomimetic dye (Fig. ld) is a benzamidino-cationic yellow and is designed for a different application. The two main proteolytic constituents of crude bovine pancreatic extract, typsin and chymotrypsin, are totally separated on agarose-immobilised benzamidino-cationic dye, where only trypsin bound (Clonis et al., 1987b). This is because the cationic dye bears a guanidino-group same as the potent trypsin inhibitor benzamidine. Other trypsin-like proteases can be purified on this novel affinity ligand (Clonis et al., 1987b). The field of biomimetic dye technology is expected to follow rapid development and solve problems associated with the selectivity of commercial textile dye ligands. 3.2. A m i n o acid-based ligands

Amino acid sequences encoded in the antisense strand of DNA are called 'antisense peptides' (Fig. 3) and are used as affinity ligands of appreciable selectivity against the corresponding 'sense peptides' (Chaiken, 1992) or 'sense re-

Sense peptide H2N. . . . . . . . . . . . . . . .

COOH

3'-0-0-0-0-0-0-0-0-0-0-0-5'

Sense DNA Antisense DNA

5'-0-0-0-0-0-0-0-0-0-0-0-3'

HOOC . . . . . . . . . . . . . . .

Antisense peptide Fig. 3. The conceptof antisense peptides.

NH 2

101

gions' of proteins (Scapol et al., 1992). For example, recombinant human interferon from mammalian cell culture is purified by antisense peptide affinity chromatography. Following computer-predicted exposure of protein fragments on the surface of interferon, the corresponding antisense peptides of the fragments are selected. These are encoded in the antisense strand of DNA corresponding to the 1-14, 42-54 and 103115 fragments of the target protein. The synthesised antisense peptide adsorbents afforded 10fold purification of recombinant interferon (Scapol et al., 1992). A somewhat different approach is offered by the so-called 'paralog' peptide ligands. This technique employs immobilised short peptides, and it is based on molecular scale multi-mode adsorbents of appropriately diversified characteristics in terms of charge and hydrophobicity (Benedek et al., 1992). Individual amino acids, synthetic (poly)amino acids and oligopeptides of appropriate recognition sequences for the target enzymes, are employed to protein separation. Trypsin is probably the most widely studied protein in this area, however, several other proteolytic activities are also purified on immobilised peptides. Among several di- and tri-peptides, the tri-peptide HGly-AIa-Arg-OH is proved to be the ligand of the highest affinity for trypsin (Kasai, 1992). Likewise, several solid-phase synthesised tri-peptides are tested as affinity ligands for urokinase and tissue plasminogen activator (tPA). Accordingly, hydrophobic residues, especially aromatics, flanking the N-side of argininal gave strong binding with tPA. The most effective oligopeptide affinity ligand is shown to be the transition-state analogue D-Phe-o-Phe-Argal, where arginine is replaced by its aldehyde derivative. This affinity ligand is able to purify tPA from culture media at multi-gram quantities (Patel et al., 1990). Peptide antibiotics can also be used as affinity ligands, for example, the cyclic oligopeptide bacitracin is useful for the purification of various proteinases (Van Noort et al., 1991; Fadeev et al., 1992). Poly(L-aspartic acid) is reported to be an effective ligand for selective removal of microtubular-associated proteins from rat brain (Fujii et al., 1990), whereas L-histidine immobilised to aminohexyl-

102

N. Labrou, KD. Clonis /Journal of Biotechnology 36 (1994) 95-119

agarose can purify subclasses 1 and 2 of IgG from human placenta (El-Kak and Vijayalakshmi, 1992). Finally, an adsorbent bearing amide bonds, dimethylaminopropyl-carbamylpentyl-agarose, is used successfully in the purification of human factor VIII (Te Booy et al., 1990). The effectiveness of specifically designed molecules as affinity ligands, prompted consideration of the possibility of de novo design and synthesis of biomimetic ligands for protease purification. The trypsin-like family of enzymes forms one of the largest groups of enzymes requiring cationic substrates and includes several proteolytic proteins. These proteins possess similar catalytic mechanisms and bind, strongly and with high affinity, the side chains of arginine and lysine in a primary pocket, at the bottom of which is lying an aspartic acid residue. Secondary interactions with the side chains of other nearby amino acids determine the overall affinity strength of the ligand. Kallikrein is a trypsin-like protease, but it differs from pancreatic trypsin in that it prefers phenylalanine in the secondary interaction site. This is because the side chain of phenylalanine slips into an adjacent hydrophobic pocket between tryptophane-215 and tyrosine-99 residues. Therefore, the prefered natural ligand for kallikrein should possess the dipeptide Arg-Phe (Fig. 4, bottom). Consequently, it is almost predictable what a synthetic biomimetic ligand for kallikrein should be like. Fig. 4 (top) provides the structure of this ligand; a benzamidine is linked to a reactive triazine ring and on this structure is then attached a phenethylamine. The former moiety of the synthetic ligand mimics the side chain of Arg, whereas the latter mimics that of Phe. In fact, this synthetic ligand, when immobilised via a 6-aminohexyl-spacer, purifies kallikrein by llO-fold from crude pancreatic extract (Lowe et al., 1992). 3.3. Nucleotide-based ligands and coentymes Immobilised nucleotides and nucleotide coenzymes (Lowe, 19811, in spite of their cost, stability and binding-capacity problems, are useful affinity adsorbents and still find application in enzyme purification (Mosbach et al., 1972; Lowe, 1977;

a’ Fig. 4. Structures of the purpose-designed synthetic biomimetic ligand for kallikrein binding, comprising p-aminobenzamidine and phenethylamine moieties substituted on a triazine ring (bottom), and the biological di-peptide ligand Arg-Phe (top).

Clonis and Lowe, 1980b). For example, the enzyme fructose-6-phosphate,2-kinase : fructose-2,6bis-phosphatase from rat skeletal muscle is purified on two affinity columns, Cibacron@ blueagarose and ATP-agarose (Kitamura et al., 19891, whereas on the same nucleotide adsorbent, the membrane-associated diacylglycerol kinase from Drosophila heads is also purified (Inoue et al., 1992). Immobilised thiamin monophosphate, a non-nucleotide coenzyme affinity adsorbent, is able to purify thiamin pyrophosphokinase and adenylate kinase from human blood (Egi et al., 1992). The dramatic development of molecular biology and recombinant DNA technology has led to increasing interest in purification methods for DNA-binding proteins. Several affinity chromatography methods are based on immobilisedDNA adsorbents. A typical example is the largescale purification of E. coli Rep protein, a helicase that is involved in the replication of the microbial chromosome. This protein is purified on two consecutive affinity columns of celluloseimmobilised ssDNA and dsDNA (Lohman et al., 1989). An alternative and original idea (Kadonaga and Tjian, 19861, that of applying sequencespecific DNA synthetic fragments as ligands for isolating DNA-binding proteins and enzymes,

N. Labrou, Y.D. Clonis /Journal of Biotechnology 36 (1994) 95-119

nowadays attracts increasing attention. A sequence-specific transcription factor, adenovirus-2 late promoter (MLP) upstream element factor, is purified from HeLa cells and yeast on Sepharose-immobilised 35-mer synthetic oligonucleotide strands. These strands were previously hybridised and ligated with T4 DNA ligase (Moncollin et al., 1990). The same principle is applied to the purification of two DNA-binding proteins from nuclear extracts of HeLa cells implicated in transcription control. The adsorbent employed is Sepharose CL-4B-immobilised 26mer duplex obtained form synthetic oligonucleotide sequence-specific ligand (Zhang et al., 1992). Likewise, DNA-binding multiple polypeptides are purified from nuclear extracts of HeLa cells on two affinity columns made of latex particles. The affinity ligands present on the two columns are hemically synthesised and immobilised duplexes obtained form complementary oligonucleotide oligomers ranging from 150 to 250 bp (Inomata et al., 1992). A group of enzymes where this technology holds great potential is that of restriction endonucleases. For example, HpaI, SphI, EcoRV and EcoRI, are purified to near homogeneity by DNA-cellulose and sequencespecific DNA affinity chromatography (Vlatakis and Bouriotis, 1991a). The sequence-specific affinity adsorbents are designed so as to possess two enzyme-recognition sequences and, therefore, be able to purify two enzymes. The sequence of events for synthesising a sequencespecific ligand is shown in Fig. 5. Three self-complementary oligonucleotides are synthesised, each one is annealed with itself, 5' phosphorylated and ligated. Each of the resulting sequence-specific dsDNA oligomers, after purification, is immobilised to CNBr-activated agarose (Vlatakis and Bouriotis, 1991a). Likewise, an agarose-immobilised sequence-specific dsDNA synthetic ligand, containing recognition sequences for 34 different restriction enzymes, is proved effective in purifying, so far, six restriction endonucleases (Pozidis et al., 1993). A totaly different affinity ligand, namely heparin, is also employed for interacting with DNA-binding proteins. Immobilised heparin is regarded as a group-specific affinity adsorbent with application to the purification of (poly)nu-

103

5'-GATCGCATGCCGCGGATCCCGGGCCCAGGTGGCCAGCTGTCGAC-Y + 3'-CGTACGGCGCCTAGGGCCCGGGTCCACCGGTCGACAGCTGCTAG-5' U (annealing) 5'-GATCGCATGCCGCGGATCCCGGGCCCAGGTGGCCAGCTGTCGAC-Y 3'-CGTACGGCGCCTAGGGCCCGGGTCCACCGGTCGACAGCTGCTAG-5'

phosphorylationat the 5'-protrudingends by T4 polynucleotidekinase and ATP

polymerizationby T4 DNA ligase and ATP

multisequenee-specificdsDNA affinityligand Fig. 5. Steps for synthesis of d s D N A l i g a n d b e a r i n g recognition s e q u e n c e s for 34 d i f f e r e n t restriction e n d o n u c l e a s e s .

cleotide/DNA-binding proteins, for example, restriction endonucleases (Vlatakis and Bouriotis, 1991a; Pozidis et al., 1993), factor TIF-IB which confers promoter specificity to mouse RNA polymerase I (Schnapp et al., 1990) and casein kinases from bovine spermatozoa (Ruzzene et al., 1992).

3.4. Chelated metal cations The technology based on such ligands is referred to as metal chelate affinity chromatography (MCAC) or immobilised metal affinity chromatography (IMAC) and finds application in protein and enzyme processing (Arnold, 1991). It may be classified as an intermediate between highly specific, high-affinity methods and wider spectrum, low-specificity adsorption methods, such as ion exchange. The IMAC adsorbent is designed so as to chelate transition metal cations that interact with specific groups, for example, the imidazole of histidine residues of peptides and proteins. Although a variety of immobilised metal-chelating structures are available, iminodiacetic acid (IDA) remains the most popular adsorbent. Some of the alternatives are: (a) tris (carboxymethyl)ethylenediamine (TED), which is used for proteins with too high affinity for IDA-

N. Labrou, Y.D. Clonis/Journal of Biotechnology 36 (1994) 95-119

104

metal conjugates (Yip and Hutchens, 1992); (b) nitrilotriacetic acid (Hochuli et al., 1987; Grice and Gruninger-Leitch, 1990); and (c) 8-hydroxyquinoline, which is used with less usual metals (e.g., Fe 3+, Al 3+, Yb 3+, Ca 2+) (Zachariou and Hearn, 1992). In IDA-supports, the metal-ion is 'immobilised' by chelation via the nitrogen and the two carboxylate oxygen atoms, whereas the remaining coordination sites are occupied by water or buffer molecules which can be displaced by appropriate protein functional groups (Fig. 6). One adjusts the strength of interaction between protein and IMAC adsorbent by varying the type of chelated metal-ion, where Cu 1+ generally provides the highest binding strength. Elution of bound proteins from IMAC adsorbents is effected by pH change, inclusion of EDTA or competing substances (e.g., imidazole). This affinity technology is employed in several occasions, for example, the purification of Fel d I cat's major allergen from house dust extract (Dandeu et al., 1990), the fractionation of microsomal cytochrome P-450 isoenzymes (Roos, 1991), the adsorption studies of serum albumins from several sources (Andersson et al., 1991), the interaction studies with peptides and proteins lacking histidyl groups (Andersson et al., 1992) or with phosphorylated analogues of target molecules (Muszynska et al., 1992), the separation of photosystems I and II from thermophilic cyanobacteria (Ritter et al., 1992), the single-step purification of solid-phase

SOLIDPHASE

O=C I

CH2~ ) I'I'z-CHOH N/" CH

c:o

x

oj X

×

Fig. 6. Structure of the chelation complex formed between immobilised iminodiacetic acid and metal cation Me. Remaining metal coordination sites are occupied by water or buffer molecules X which can be displaced by protein functional groups.

synthesised peptides via interaction of the unprotonated a-amino group (Hansen et al., 1992) and the oriented immobilisation of covalently modified monoclonal antibodies with a chelating peptide, Lys-Gly-(His)6, on Ni2+-nitrilotriacetic acid adsorbent (Loetscher et al., 1992). Recently, chelated-Cu 2÷ adsorbent is used in the purification of variants of leech hirudin cloned, expressed and secreted in yeast. Wild-type hirudin is a potent inhibitor to thrombin and consists of 65 amino acids, having just only one His-51 residue. Therefore, hirudin variants are engineered so as to contain additional surface histidines, facilitating the purification process by IMAC (Chung et al., 1993).

3.5. Carbohydrate-binding ligands Glycoconjugates are ubiquitous in nature and exist as glycoproteins, proteoglycans, enzymes, antibodies, receptors, hormones, glycolipids, sugars and polysaccharides, nucleosides and nucleotides. The common feature of all is the existence of carbohydrate moiety on their molecular structure, which differ from one another. Two types of ligands, lectins and boronates, exhibit affinity against the carbohydrate moiety of target molecules and, therefore, are used in affinity purifications (O'Shannessy and Wilchek, 1990). Lectins are glycoproteins whose affinity for the sugar moiety varies with their origin. Immobilised concanavalin A (Con A) for years finds application, for example, to the purification of galactosyltransferase isoforms from human malignant ascitic fluid (Boyle and Peters, 1988), study of the cell surface glycoproteins of goat epididymal maturing spermatozoa (Sarkar et al., 1991), extraction of two glycoproteins from bovine milk (Kim et al., 1992) and purification of cathepsin A, a lysosomal carboxypeptidase, from pig kidney (Miller et al., 1992). Other immobilised lectins have also been reported as useful purification tools (West and Golding, 1992). Immoblised boronic acid is used for the same purpose as lectins (Bouriotis et al., 1981; Hageman and Kuehn, 1992), however, its mechanism of binding is very different. Aminobenzeneboronic acid is immobilised via its arylamino

N. Labrou, Y.D. Clonis /Journal of Biotechnology 36 (1994) 95-119 Support--CH2CH2CO--NH

/~'"'OH HO OH

diol--ligand

1L Support--CH2CH2CO--NH

carbohydrate Fig. 7. Diester formation, in alkaline environment, between diol-bearing ligand and immobilised aminobenzeneboronic acid.

group to suitably activated supports, giving rise to affinity adsorbents which form stable complexes with sugar diols (Fig. 7). The pK a of the acid is about 9, therefore, the working pH cannot be much lower than 8.0-8.5 in order for the boronic acid to be in its functional boronate anion form. Aliphatic boronates also used in affinity technology, for example, to separate various catechols (Adamek et al., 1992), whereas immobilised maminophenyl boronate is useful for measuring glycated albumin in serum and urine (Silver et al., 1991). In certain purification cases of glycosylated macromolecules, individual sugars can be employed as affinity ligands. Lactose, for example, is immobilised to epoxy-activated agarose and the corresponding adsorbent used in the purification of three toxins and two agglutinins from seeds of Abrus precatorius (Hegde et al., 1991).

105

3.6. Protein ligands Protein A has affinity for IgG molecules, therefore the corresponding adsorbent is useful for the preparation of high-purity antibodies. Among numerous examples, a typical one is that of the purification of murine IgG 1 monoclonal antibodies from hybridoma supernatants (Schuler and Reinacher, 1991). A new cross-linked porous hydrophilic reactive support of improved flow properties and stable bed-volume is reported as being a suitable matrix for direct protein A immobilisation (Coleman et al., 1990). Calmodulin is a calcium-dependent protein and it is exploited in its capacity to act as a group-specific ligand. It has been used in several protein purifications as, for example, with recombinat cAMP-dependent protein kinase. This enzyme is fused, by genetic engineering techniques, to bear a calmodulinbinding peptide, which acts as recognition tail and binding site to calmodulin affinity adsorbent. The peptide-tail is derived from the C-terminus of skeletal muscle myosin light-chain kinase (Stofko-Hahn et al., 1992). The same affinity adsorbent is used to purify simultaneously synapsin I and synaptophysin from calf brain cortex homogenate (Liona et al., 1992).

4. Chromatography-based affinity technologies There is no doubt that affinity chromatography remains the most favoured and widely used of all

Table 4 Main chromatography-based affinity technologies used in downstream processing Affinity chromatography Perfusion affinity chromatography Hyperdiffusion affinity chromatography The genetic engineering approach: affinity tails High-performance affinity chromatography Centrifugal affinity chromatography Affinity-repulsion chromatography Heterobifunctional affinity ligand chromatography

106

N. Labrou, Y.D. Clonis/Journal of Biotechnology 36 (1994) 95-119

affinity technologies. Notably, all examples cited until now are affinity chromatography applications. Nevertheless, several other chromatography-based approaches have been developed and are currently in use (Table 4).

4.1. Affinity tails The 'affinity tail' concept is the genetic engineering approach to protein purification via affinity chromatpgraphy. The large-scale production and downstream processing of heterologous proteins can be facilitated by a genetic approach which is based on the attachment of an 'affinity tail' at the target protein by covalent bond. In principle, the target-protein gene is fused after a synthetic fragment encoding the affinity tail, for example, binding domains derived from staphylococcal protein A which are known to exhibit affinity for IgG. The fusion product is secreted to the growth medium, e.g., of E. coli, and purified by affinity chromatography on an IgG-affinity column which binds specifically the affinity tail of the fused product. Afterwards, the affinity tail is disconnected by chemical (e.g., hydroxylamine) or enzymatic (e.g., enterokinase) cleavage from the the fused protein. For example, this approach is used in the purification of human native insulinlike growth factor, IGF-I (Monks et al., 1987). Taking this concept further, a dual affinity fusion technique is designed for the purification of insulin-like growth factor II (Hammarberg et al., 1989). Accordingly, the gene encoding the target-peptide hormone (product) is fused between two flanking heterologous genes encoding IgG- and albumin-binding domains of protein A and protein G, respectively. The tripartite fusion product is purified by affinity chromatography on HSA-agarose and IgG-agarose columns using both the N- and the C-terminal domains (Monks et al., 1987). It is believed that the latter approach offers some advantages, for example, it ensures recovery of full-length products, the product is purified using either or both terminal regions and subsequently is characterised by protein sequencing and analysis, and one gains information about proteolysis sites in any inserted protein. Immunoaffinity chromatography is useful

for the separation of tail-fused proteins where a small peptide, e.g., of eight amino acids, is engineered onto the N-terminal of the target product. An antibody specific for the first four amino acids of the engineered sequence is used as immobilised affinity ligand to purify the target protein by binding at its tail (Hopp et al., 1988). Following the purifucation step, removal of the peptide tail is accomplished by proteolytic cleavage using enterokinase. Another affinity-tail group is the one collecting all ligands bearing histidine residues. In this case, the affinity adsorbent employed to purify the fused protein is an immobilised metal chelator. For example, mouse dihydrofolate reductase expressed as fused product in E. coli is purified on a Ni2+-nitrilotriacetic acid column (Hochuli et al., 1988). The fused enzyme comprised two to six histidine residues either at the amino- or the carboxy-terminus. Following the purification procedure, removal of the affinity tail from the product is effected by enzymatic digestion in the presence of carboxypeptidase C. Protein A and /3-galactosidase fused with the affinity peptide Ala-His-Gly-His-Arg-Pro at their C- and N-terminal, respectively, are purified on a Zn2+-IMAC column (Ljunquist et al., 1989). Table 5 lists several affinity tail types along with the corresponding ligands/adsorbents used in the separation of the fused product.

4.2. High-performance affinity chromatography Support materials suitable for affinity chromatography applications are expected to consist of macroporous hydrophilic beaded particles, usually bearing free hydroxyl groups for ligand attachment. When the support is made of noncompressible spherical particles of small diameter (e.g., 5-20 tzm) and narrow size distribution (e.g., 0.2-2.0 p~m) the technique is termed high-performance affinity chromatography (HPAC) (Ohlson et al., 1978; Clonis, 1989, 1992). Silica-based supports are used widely in HPAC, although they present serious drawbacks, such as poor chemical stability in alkaline conditions, need for derivatization with organofunctional silanes prior to ligand immobilisation, and relatively small pore size. It appears that such problems are partly circum-

N. Labrou, Y.D. Clonis/Journal of Biotechnology 36 (1994) 95-119 Table 5 Types of affinity tails and corresponding ligands/adsorbents used in the purification of heterologous proteins (fused products) Tail type Ligand/adsorbent Enzymes /3-Galactosidase

substrate analog, antibody Glutathione S-transferase glutathione Chloramphenicol acetyltransferase substrate analog

Polypeptide-binding proteins Staphylococcal protein A (domain) IgG antibodies (Fv) Streptococcal protein G (domain) albumin calmodulin Calmodulin-bindingpeptide Carbohydrate-binding domains x-linked amylose Maltose-binding protein starch granules Glucoamylase starch-binding domain Cellulase cellulose-binding domain cellulose Antigenic epitopes Hydrophilic (aa)8-peptide

anti-peptide MAb

Charged amino acids Poly(Arg + ) Poly(Asp - )

cation exchanger anion exchanger

Poly(His) (His)2_ 6

Poly(amino acids) Poly(Phe ) Poly(Cys)

immobilised transition metal cations; Zn +2, Ni +2 phenyl-agarose thiopropyl-agarose

vented with the introduction of suitably coated wide-pore silica and purpose-designed synthetic high-performance particles bearing hydroxyl groups (e.g., Separon ® H1000, TSK®-PW, Dynospheres ~ XR-3507, Asahipak ® GS-520) or activated groups (e.g., Eupergit ® C30N, Affi-Prep ® 10). The chemistry of support activation and ligand immobilisation, as well as adsorbent handling and packing, are well established and have been presented in detail (Clonis, 1989, 1992). A new activation method is useful for rapid coupling of unstable ligands (Hill and Arrio, 1992). This method employs 4,6-diphenylthieno(3,4-d)-l,3-dioxol-2-one-5,5'-dioxide (TDO) esters and supports bearing hydroxyl groups. The excellent book-chapter by Unger et al. (1991) on advanced

107

silica-based packing materials is recommended to the reader. In spite of its problems, silica is still used in HPAC mainly on an analytical scale. Notably, there is just only one large-scale example of HPAC on silica support, that of the purification of rabbit muscle lactate dehydrogenase on a 3.3-1 column of silica-immobilised triazine dye Procion ® blue MX-R (Clonis et al., 1986). Several other HPAC separations on silica-based adsorbents were reported recently. Glycolipids in crude extract from rat liver are separated quantitatively from neutral lipids and phospholipids on a silica-phenylboronic acid column (Tomono et al., 1990). The affinity ligand 8-((6-aminohexyl) amino)-2'-phosphoadenosine-5'-diphosphoriboseimmobilised on epoxy-activated silica is employed to the purification of two NADP+-dependent enzymes, glutathione reductase and glucose-6-phosphate dehydrogenase from various sources (A1hama et al., 1991). Resolution of adenylic acid o l i g o m e r s , (dA)19_24 , and poly(A) mRNA from yeast, is accomplished on a 50-mer thymidylic acid ligand, (dT)50, immobilised to silica (Gross et al., 1991). High-performance immunoaffinity chromatography (HPIAC) is applied to the purification of glutamine synthetase. The adsorbent comprises silica-immobilised polyclonal anti-enzyme antibodies (Alhama, 1992). HPIAC is also applied to the simultaneous extraction of albumin and transferrin from immunoglobulin G. In this case the adsorbent is mixed-function, combining immobilised anti-albumin and anti-transferrin antibodies (Wheatley, 1992). Human erythrocyte sialoglycoproteins A, B and C (glycophorins) are purified from glycophorin E and other proteins on silica-wheat germ agglutinin, whereas the three individual components are separated on silica-iminodiacetic acid-Cu ÷2 (Corradini et al., 1988). High-performance immobilised metal affinity chromatography (HPIMAC) is also effective for purifying phenobarbital-induced rat liver microsomal cytochrome P-450 on Superose®-iminodiacetic acid (Kastner and Neubert, 1991). Synthetic supports offer a potential alternative to silica. Evaluation of high-performance affinity supports for their ability to purify lactate dehydrogenase revealed that two synthetic materials exhibited superior performance to silica (Clonis,

108

N. Labrou, Y.D. Clonis/Journal of Biotechnology 36 (1994) 95-119

1987b). High-performance IMAC with synthetic TSK®-PW is proved to be useful technique for evaluating the adsorption properties of 67 synthetic biologically active peptides of different molecular size (Yip et al., 1989). A synthetic material also bearing free hydroxyl-groups for ligand coupling is Asahipak ® GS-520. This support is activated with epichlorohydrin and subsequently derivatised with D-glucose. The derived affinity adsorbent is then employed to separate tetravalent and divalent molecular species of concanavalin A (Abe and Ishii, 1990). Dynospheres ® XR3507 is a monosized (monodispersed) hydrophilic macroporous synthetic material bearing free hydroxyl-functions where several ligands can be immobilised. Concanavalin A is immobilised on this support by the 1,1'-carbonyldiimidazole activation method, and the corresponding HPAC adsorbent used in isolating allergen Ag7 from mugwort pollen (Nilsen et al., 1990). Two familiar biomimetic triazine dyes, the terminal ring phosphonate analogue of Cibacron blue and the benzamidino-cationic yellow (Fig. 1), are also exploited in HPAC enzyme purifications. After the dyes are immobilised on Dynospheres ®, the corresponding adsorbents are employed for the purification of alkaline phosphatase and urokinase, respectively (Clonis and Lowe, 1991). A polystyrene-based support appears to be an effective alternative for certain HPAC applications. After its chlorosulphonated form is derivatised with the ligand serine, the target biomolecule, basic fibroblast growth factor, bound strongly the affinity adsorbent and it is purified from bovine brain crude extract (Jacquot-Dourges et al., 1990). A different approach to column packings is introduced by the concept of deformed non-porous high-performance agarose support (Hjerten and Li, 1990). This support consists of 12-15 /xm diameter beads, which by being compressed and non-porous result in superior resolution at high flow rates. For example, when aminophenylboronic acid is attached to such an epoxyactivated support, the corresponding affinity adsorbent separates glycosylated from non-glycosylated haemoglobin. Turning now to non-beaded supports, a non-porous fibre-form material is proposed for HPAC applications (Wikstrom and

Larssen, 1987). Non-porous quartz fibres of mean diameter 0.5 /zm are first coated by silylation to introduce mercapto groups. Subsequently, the introduced nucleophile groups reacted with preactivated dextran on which the ligand NAD ÷ is then immobilised via the remaining reactive functions of the carbohydrate. This affinity adsorbent is able to purify ox heart lactate dehydrogenase, exhibiting good binding capacity (15 mg enzyme per g of support).

4.3. Other affinity chromatography-based technologies Centrifugal affinity chromatography (CAC) combines the high flow rate, created by centrifugation force, with the specificity of affinity chromatography. It is regarded as the less expensive alternative to HPAC. Its applicability is demonstrated by the purification of human IgG on immobilised protein A (Slinerland and Scouten, 1990), the removal of albumin from human serum using Blue-Trisacryl ® gel (Berg and Scouten 1990), and the rapid screening of a large number of immobilised dyes for binding goat IgG (Berg and Scouten, 1990). Affinity-repulsion chromatography is an unusual type of bioseparation technology. The support material should be charged and of the same sign as that of the target protein. The strength of such electrostatic interactions between target protein and adsorbent are minimized in the presence of salt. Therefore, the protein mixture is loaded on the column in a buffered salt solution and adsorbed species are eluted at low ionic strength or with deionized water (Teichberg, 1990). This affinity technique is used in binding peanut agglutinin on lactosylagarose modified with ethylenediamine (elution with water and lactose) and in adsorbing concanavalin A on maltosyl-agarose modified with ethylenediamine (elution with water and methyl a-glucoside) (Teichberg, 1990). In heterobifunctional ligand affinity chromatography it is necessary to be available a heterobifunctional affinity soluble ligand possessing two different affinity moieties. One moiety is interacting in-solution with the target protein to be purified, while the second one is left to bind to a suitable affinity

N. Labrou, Y.D. Clonis/Journal of Biotechnology 36 (1994) 95-119 adsorbent (Mattiasson and Olsson, 1986; Olsson and Mattiasson, 1986). A model system based on this principle is developed for purifying lactate dehydrogenase (LDH). Its soluble heterobifunctional ligand consisted of Cibacron blue-soy bean trypsin inhibitor, whereas immobilised trypsin is used as affinity column for binding the complex LDH-heterobifunctional ligand (Mattiasson and Olsson, 1986). Affinity chromatography is a useful tool in elucidating more basic research problems, for example, the quantification and explanation of molecular recognition phenomena occurring between immobilised ligands and macromolecules (Mayes et al., 1991; Chaiken et al., 1992).

109

interior vessel is made of a supported filter. The filter porosity is such that it does not permit passage of the adsorbent but allows the liquid stream and its soluble contents to flow through. The affinity adsorbent is placed in the space between the cocentric cylinders and it is continuously recirculated between the two contactors. In one contactor the continuous adsorption of protein to the affinity adsorbent takes place, whereas unbound materials pass through the filter to waste or recycle. In the second contactor the continuous elution of product from the adsorbent takes place, through the filter, to the collection apparatus. The effectiveness of this technology is demonstrated with the purification of /3-galactosidase from E. coli using as affinity adsorbent PABTG-agarose (Gordon et al., 1990).

5. Non-chromatographic affinity technologies 5.2. Aqueous two-phase affinity partitioning In spite of the wide application of chromatography-based affinity technologies, several nonchromatographic approaches attrack inreasing attention ('Fable 6). Therefore, various affinity purification processes are developed aiming to circumvent problems encountered with chromatographic technologies. These efforts are directed towards minimizing diffusion limitations presented by chromatographic packings, increasing throughput and affinity-ligand usage. 5.1. Continuous affinity recycle extraction In continuous affinity recycle extraction (CARE), instead of packing conventional adsorbent particles in a fixed bed (column), solid/liquid contact is achieved in well-mixed reactors. The adsorbent is continuously recirculated between two contactors (tanks). Each contactor consists of two cocentric cylinder vessels. The wall of the Table 6 Non-chromatographicaffinitytechnologies Continuousaffinityrecycleextraction Aqueous two-phaseaffinitypartitioning Membrane affinityfiltration Affinitycross-flowultrafiltration Reversible solubleaffinitypolymers Affinityprecipitation

Affinity partitioning is regarded as the non-expensive approach to affinity-based purification technologies. When aqueous solutions of two high-molecular weight polymers, e.g., dextran and polyethylene glycol (PEG), are mixed and then left to settle, depending on the molecular weight of the polymers, their concentration and temperature, two phases may be formed. The upper phase is PEG-rich and the bottom phase is dextran-rich. By appropriate adjustment of the ionic strength, pH a n d / o r hydrophobicity of the system, many proteins partition to the bottom dextran-rich phase. However, when some of the PEG is replaced by PEG-immobilised affinity ligand, the target protein binds the ligand-PEG conjugate and selectively partitions to the upper PEGrich phase, leading to some purification. The advantage of affinity partitioning, especially important to industrial scale, lies in the fact that it can be used at the first stage of a purification process, immediately after cell disintegration, without need of centrifugation or debris removal. Nevertheless, the PEG molecules carried along at subsequent chromatography separation stages may interfere with column performance. The area of affinity partitioning is reviewed and several application are reported (Johansson, 1987; Kopperschl~iger and Birkenmeier, 1990; Waiters et

110

N. Labrou, Y.D. CIonis/Journal of Biotechnology 36 (1994) 95-119

al., 1991). PEG-immobilised triazine dyes are widely exploited in two-phase affinity systems for partial purification of enzymes, especially those binding nucleotides (Johansson, 1987). For example, glucose-6-phosphate dehydrogenase (Delgado et al., 1990) and phosphofructokinase (Tejedor et al., 1992) are partially purified from rat erythrocyte haemolysates at the upper PEG-rich phase containing PEG-Cibacron ® blue 3GA. The specific binding of rabbit muscle lactate dehydrogenase is studied with several PEG-bound triazine dyes and the effect of cupper (II) complexes examined in order to establish optimum purification conditions (Zutautas et al., 1992). Affinity partitioning with PEG-immobilised triazine dyes and herring DNA is reported for restriction endonucleases (Vlatakis and Bouriotis, 199lb). Three such enzymes, EcoRI, EcoRV and BamHI, are partially purified by two-phase affinity partitioning. However, when the two-phase affinity step is coupled to a ion-exchange chromatography, high purification factors are obtained (37-52-fold), leading to enzymes free from contaminating nuclease activities (Vlatakis and Bouriotis, 1991b). Alkaline phosphatase is a commercially important enzyme and is purified at large-scale in three steps; non-affinity two-phase partitioning, DEAE-cellulose and agarose-immoblised Procion ® Navy HE-R chromatography. The purified enzyme is free from DNAses and DNA-nicking activities, with phosphodiesterase I activity present in less than 0.01% (Kirchberger and Kopperschl~iger, 1990). Recently, the receptor-ligand recognition principle is exploited in affinity partitioning. Thus, partitioning of rat erythrocytes, naturally bearing transferrin receptors, towards the top PEG-rich phase increased after the cells, prior to partitioning, are incubated with transferrin-modified monomethoxy-PEG (Delgado et al., 1992).

5.3. Membrane affinity filtration cross-flow ultrafiltration

and affinity

Covalent attachment of an affinity ligand to a filtration membrane provides the basis of membrane affinity filtration purifications. Central component of this technology is a hollow-fibre

microporous membrane to the body of which the ligand is immobilised. The feed stream moves along the interior free space of the fibre whose pores are large enough (0.5-1.0 /zm) to permit convective flow through its body. Therefore, while binding of the target protein to the immobilised ligand occurs without diffusion limitations, unbound substances pass through the fibre body and are collected from the outer surface. An additional advantage offered by this technology, particularly important to industrial-scale application, is that there is no need for solids removal from the feed stream. It is possible that two functions, liquid-solid separation and affinity purification of the product, can be performed simultaneously. It is reported that this affinity technology scales linearly from laboratory to production scale (Spalding, 1991). Affinity columns often are exploited at the last stages of a process, mainly for two reasons. One is that by loading the affinity adsorbent with less inpure, partially purified, material the adsorbent's life-span is extented. The second is that, generally, affinity columns handle relatively small volumes. In contrast, affinity membranes allow rapid processing of large volumes even, sometimes, right after the bioreactor harvesting, eliminating concentration and partial-purification steps. For example, protein A-membrane is used in the purification of mouse IgG monoclonal antibodies from serum. A module of 10-ml hollow fibre affinity membrane processed in 15 rain 1.2-1 crude monoclonal, where 95% of nucleic acids and 99.9% albumin are removed, leading to a 97% yield (Spalding, 1991). a-Globulin is purified using a hollow-fibre affinity membrane which is prepared by radiation-induced grafting of glycidyl methacrylate onto the porous polyethylene hollow fibre, followed by attachment of either amino acid ligands Phe or Trp (Kim et al., 1991). Membrane configurations other than hollow fibres are developed and used in biomolecule separation. Thus, membrane discs and modules of flat membrane sheets bearing immobilised ligands are effective tools in downstream processing (Briefs and Kula, 1992). The purification of formate dehydrogenase from Candida boidinii is realised in a three-step membrane filtration process; cation exchange, Procion ® red

N. Labrou, Y.D. Clonis/ Journal of Biotechnology 36 (1994) 95-119

HE-3B and anion exchange, showing a scalability factor of 40 from laboratory to large-scale (Champluvier and Kula, 1992). Cibacron ® blue 3GA immobilised on microporous flat membrane is used in the purification of glucose-6-phosphate dehydrogenase from yeast. Desorption of the enzyme is effected specifically by NAD ÷ and ethylene glycol, leading to an affinity filtration step performance of 27-fold purification and > 82% yield (Champluvier and Kula, 1992a). Immunoaffinity membrane adsorbents are proved to be useful purification tools for three recombinant proteins, interferon-a, 2a, interleukin, and interleukin-2 receptors (Nachman et al., 1992). There are several membrane materials suitable for affinity-based separations, for example, hydrophilic modified-polyvinylidene difluoride, activated nylon, cellulosic polymers, polysulphone and polyacrolein mixtures. To these is now added a new one, obtained from electrostatically-spun poly (ether-urethane-urea), permitting a variety of chemical activation and functionalisation methods to be applied. This membrane material, after derivatisation with protein A, it is used to purify IgG (Bamford et al., 1992). Another non-chromatographic technology is the affinity cross-flow ultrafiltration. Essential for applying this concept is the availability of an affinity macroligand capable of reversible selective binding of the target protein. Also required is a membrane having pores large enough to allow passage of all unwanted substances as well as the target protein, but not of the affinity macroligand. When the crude sample is filtered in the presence of the affinity macroligand, only the target protein will be retained, as a complex with the macroligand. Following the washing step, bound target protein is desorbed, passed through the membrane and collected. However, the macroligand remains retained by the membrane and is recycled and reconditioned if necessary (Luong et al., 1987). Heat-killed cells of Saccharomyces cerevisiae are used as 'macroligand' in the purification of concanavalin A from crude extract of Jack beans (Mattiasson and Ramstrop, 1984). Dextran-p-aminobenzamidine is used as macroligand to partially separate trypsin from chymotrypsin (the retanate contained 35% of the

111

chymotrypsin applied) (Adamiski-Medda et al., 1981), whereas for the same purpose, polymerized N-acryloyl-rn-aminobenzamidine is proved to be a much better macroligand (98% purity of trypsin) (Luong et al., 1987). As with membrane affinity filtration, the affinity cross-flow ultrafiltration technology offers high mass transfer speed and throughput, along with its capability of processing unclarified and viscous reaction mixtures. 5.4. Reversible soluble affinity polymers

This technology involves binding of the target protein to a suitable ligand precoupled to a polymer. Following a change in a property of the liquid medium (pH, temperature, etc.) precipitation/separation of the ligand-polymer conjugate occurs; hence coprecipitation of the ligand-bound target protein is effected. For example, trypsin is purified on poly(N-vinylcaprolactam)-trypsin inhibitor conjugate, which exhibits a lower critical solution temperature of 32°C. On heating to temperatures higher than this, two phased are separated; a polymer-rich phase and an aqueous phase containing practically no polymer. This phase separation is completely reversible on cooling (Galaev and Mattiasson, 1993). However, it appears that there are still problems to be solved with this technology. Coupling of small-size affinity ligands to the polymer resulted in a 1001000-fold decrease in the affinity of the target-enzyme, leading to ineffective purification system (Galaev and Mattiasson, 1993). 5.5. Affinity precipitation

Affinity precipitation is a technique whereby an insoluble protein network is formed by crosslinking a mutifunctional affinity ligand with the target protein, resulting in selective precipitation of the target protein from solution. Such a ligand, in principle, consists of two recognition moieties which are able to bind simultaneously, whereas the network-forming protein should exhibit three or more ligand-binding sites. It is believed that this technique offers certain advantages, e.g., no need for solid supports, high ligand utilisation, low capital cost, and the possibility for sequential

112

N. Labrou, YD. Clonis /Journal of Biotechnology 36 (1994) 95-119

precipitation. Although there are several reports claiming affinity precipitation, only a few should be regarded as true affinity precipitation cases (Pearson, 1987). Fig. 8 shows the structures of two affinity precipitation ligands, bis-NAD ÷ and a Reactive Blue 2-analogue. Bis-NAD + precipitates several NAD+-dependent dehydrogenases (Larsson and Mosbach, 1979), whereas the triazine ring-methoxylated analogue of p-sulphonated isomer of Reactive Blue 2 selectively precipitates and purifies to homogeneity lactate dehydrogenase from crude extracts of rabbit muscle (Pearson et al., 1989). Affinity precipitation is demonstrated with bis-Cibacron Blue 3GA and bis-boronic acid ligands, respectively, for the purification of oxidoreductases and erythrocyte agglutination via interactions with cell surface glycoproteins (Burnett et al., 1980). Furthermore, affinity precipitation is shown to work with highmolecular weight ligands. For example, potato starch, oyster glycogen, waxy maise starch and

high amylose maise starch, are affinity ligands capable of precipitating and purifing a-amylase from Bacillus amyloliquefaciens (Buur et al., 1993).

6. Affinity purification technologies: large-scale potential vs. purifying ability One would face a difficult, if not impossible, task by attempting to generalise and arrange the affinity-based purification technologies in order of their large-scale potential. The risk taken in doing so is mainly due to the fact that several factors are implicated and must be considered seriously before a decision is reached as to which affinity technology is the optimun choice for a particular large or industrial scale purification problem. Inevitably some affinity technologies exhibit superior purifying ability but are expensive to apply at large-scale (e.g., HPAC). On the other

Table 7 Evaluation map of main affinitypurifiactiontechnologiesaccordingto their large-scale potential vs. purifyingability LARGE-SCALEPOTENTIAL

high

APr AC, AT AU

AP

RSAP

CARE

medium

MAF

CAC

low

HPAC

HBLAC

low

medium

high

PURIFYING ABILITY

AC, affinitychromatography;AP, aqueous two-phaseaffinitypartitioning;APr, affinityprecipitation; AT, affinitytails; AU, affinity cross-flow ultrafiltration; CAC, centrifugal affinity chromatography; CARE, continuous affinity recycle extraction; HBLAC, heterobifunctional ligand affinity chromatography;HPAC, high-performanceaffinity chromatography;MAF, membrane affinity filtration; RSAP, reversible soluble affinitypolymers.

N. Labro~ Y.D. Clonis/ Journal of Biotechnology 36 (1994) 95-119

hand, some others are more economical and ideal for industrial application but their purifying ability is poor (e.g., aqueous two-phase affinity partitioning). Which affinity technology is the best choice for a particular purification case depends on several factors: e.g., required product purity, market price of product, process costs, instrumentation and engineering aspects, process capacity for the particular pair product-affinity adsorbent, life-time and cost of the affinity material employed, etc. An arrangement of main affinity purification technologies has been attempted in terms of their large-scale potential vs. purifying ability (Table 7). It appears that, inherently, aqueous two-phase affinity partitioning exhibits low to medium purifying ability, nevertheless it holds high large-scale potential (Johansson, 1987). Consequently, it may be a good choice for medium purity products but not a good one for therapeutic proteins or diagnostic enzymes. Reversible soluble affinity polymers are the basis for an emerging affinity technique which still faces problems of non-specific interactions raised by the support-polymer itself (Galaev and Mattiasson, 1993). Therefore, at this stage it has to be classified as an affinity technique of low purifying ability. The remaining affinity technologies are all

113

of potentially medium to high purifying ability, always depending on the specificity of the affinity ligand. Affinity chromatography, including its various new particle technologies (e.g., perfusive and hyperdiffusive particles), is the most widely used affinity technique at large-scale, and its purifying ability has been well established (Clonis, 1987a, 1990, 1991). Although one finds only a few references on affinity precipitation, it is probably the technique of the highest large-scale potential (Pearson, 1987; Pearson et al., 1989) and if it is operated on a true-affinity basis, which is difficult and rare to achieve, it can work wonders. Affinity cross-flow ultrafiltration and, to a lesser extend, membrane affinity filtration are mainly designed for medium to large-scale operation (Spalding, 1991), however, sometimes the latter is compromised by the cost factor. It seems that cross-flow ultrafiltration is at an advantageous position as the liquid phase requires no clarification prior to processing, and the technique by design is process-scale oriented. CARE, although it can hold good purifying ability, requires a special engineering set-up not available on the market (Gordon et al., 1990) which can turn out to be costly. Likewise, CAC may also be expensive at large-scale due to equipment costs, and until now has found

NHCH2CO(NH)2CO(CH2)4CO(NH)2COCH2NH adenine nicotinamide I I R--P--P--R

H2NOC I R--P--P--R

÷'?

~

sof

NH.._..~N~NOCH3

Fig. 8. Structures of two ligands for affinity precipitation: N2,N~-adipodihydrazido-bis-N6-carbonylmethyi-NAD + (b/s-NAD ÷) (top) and triazine ring-methoxylated p-sulphonated isomer of Reactive Blue 2 (bottom).

114

N. Labrou, Y.D. Clonis /Journal of Biotechnology 36 (1994) 95-119

no such application. HPAC, undoubtedly very effective in analytical and preparative operations (Clonis et al., 1986; Unger et al., 1991; Clonis, 1992), generally is of limited importance to large-scale application due to packing material and equipment costs. Heterobifunctional ligand affinity chromatography has so far attracted limited attention as it requires both an affinity column and a soluble bifunctional affinity ligand (Mattiasson and Olsson, 1986). This double-affinity approach, although in principle may lead to a better selectivity, very much limits its large-scale potential due to costs related to ligand synthesis and operational complexity. References Abe, Y. and Ishii, S.I, (1990) High performance affinity chromatography of concanavalin A. J. Chromatogr. 510, 95100. Adamek, V., Liu, X.-C., Zhang, Y., Adamkova, K. and Scouten, W. (1992) New aliphatic boronate ligands for affinity chromatography. J. Chromatogr. 625, 91-99. Adamiski-Medda, D., Nguyen, Q.T. and Dellacherie, E. (1981) Biospecific ultrafiltration: a promising purification technique for proteins. J. Membr. Sci. 9, 337-340. Afeyan, N., Gordonni, N., Mazsaroff, I., Varady, L., Fulton, S., Yang, Y.B. and Regnier, F. (1990) Flow-through particles for the high performance liquid chromatographic separation of biomolecules: perfusion chromatography. J. Chromatogr. 519, 1-29. Alhama, J., Lopez-Barea, J. and Toribio, F. (1991) High performance affinity chromatography of NADP dehydrogenases from cell free extracts using a nucleotide analogue as general ligand. J. Chromatogr. 586, 51-59. Alhama, J., Lopez-Barea, J., Toribio, F. and Roldan, J.M. (1992) Purification and determination of glutamine synthetase by hige performance immunoaffinity chromatography. J. Chromatogr. 589, 167-175. Allary, M., Saint-Blancard, S., Boschetti, E. and Girot, P. (1991) Large scale production of human albumin: Three years experience of an affinity chromatography process. Bioseparation 2, 167-175. Andersson, L., Sulkowski, E. and Porath, P. (1991) Immobilized metal ion affinity chromatography of serum albumins. Bioseparation 2, 15-22. Andersson, L. and Sulkowski, E. (1992) Evaluation of the interaction of protein a-amino groups with Me II by immobilized metal ion affinity chromatography. J. Chromatogr. 604, 13-17. Arnold, F. (1991) Metal-affinity separations: a new dimension in protein processing. Bio/Technology 9, 151-156.

Arshady, R. (1991) Beaded polymer supports and gels. I. Manufacturing techniques. J. Chromatogr. 586, 181-197. Arshady, R. (1991a) Beaded polymer supports and gels. II. Physicochemical criteria and functionalization. J. Chromatogr. 586, 199-219. Bailon, P., Weber, D., Keeney, R., Frederics, J., Smith, C., Familleti, C. and Smart, J. (1987) Receptor-affinity chromatography: a one step purification for recombinant interleukin-2. Bio/Technology 5, 1195-1198. Bamford, C., Lamee, K.A., Purbrik, M. and Wear, T.J. (1990) Studies of a novel membrane for affinity separations. I. Functionalization and protein coupling. J. Chromatogr. 606, 19-31. Bayer, E. and Wilchek, E. (1990) Applications of a avidin-biotin technology to affinity based separations. J. Chromatogr. 510, 3-11. Benedek, K., Varkonyi, A., Hughes, B., Zabel, K. and Kauvar, L. (1992) 'Paralogs', sorbent families for protein separations. J. Chromatogr. 627, 51-61. Berg, A. and Scouten. W. (1990) Dye-ligand centrifugal affinity chromatography. Bioseparation 1, 23-31. Berry, M., Davies, J., Smith, S. and Smith, I. (1991) Immobilized of Fv antibody fragments on porous silica and their utility in affinity chromatography. J. Chromatogr. 587, 161-169. Bhattachacharjee, M. and Ali, E. (1992) Protein purification using a soluble affinity matrix: Purification of estrogen receptor with estradiol-polylysine conjugate. Anal. Biochem. 201, 233-236. Briefs, K.G. and Kula, R.-M. (1992) Fast protein chromatography on analytical and preparative scale using modified microporous membranes. Chem. Engin. Sci. 47, 141-149. Bouriotis, V. and Dean, P.D.G. (1981) Use of triazyne dye in the affinity chromatographic purification of alkaline phosphatase from calf intestine. J. Chromatogr. 206, 521-530. Bouriotis, V., Galpin, I. and Dean, P.D.G. (1981) Application of immobilized phenylboronic acids as supports for groupspecific ligands in the affinity chromatography of enzymes. J. Chromatogr. 210, 267-278. Boyer, P.M. and Hsu, J.T. (1992) Effects of ligand concentration on protein adsorption in dye-ligand adsorbents. Chem. Engin. Sci. 47, 241-251. Boyle, F. and Peters, T. (1988) Characterisation of galactosyltransferase isoforms by ion-exchange and lectin affinity chromatography. Clin. Chim. Acta 178, 289-296. Burnett, T., Peebles, H. and Hageman, H. (1980) Synthesis of fluorescent boronic acid which reversible binds to cell walls and a diboronic acid which agglutinates erythrocytes. Biochem. Biophys. Res. Commun. 96, 157-165. Burton, S., Stead, C.V. and Lowe, C. (1988) Design and application of biomimetic anthraquinone dyes. II. The interaction of C.I. reactive blue 2 analogues bearing terminal ring modifications with horse liver alcohol dehydrogenase. J. Chromatogr. 455, 201-216. Buur, S., Biedermann, K. and Jorgensen, O.B. (1993) Affinity precipitation of a-amylase-starch complexes. 6th Euro-

N. Labrou, Y.D. Clonis/Journal of Biotechnology 36 (1994) 95-119 pean Congress in Biotechnology, Florence, Italy, Vol. 1, MO300. Chaiken, I. (1992) Analysis of macromolecular interaction using immobilized ligands. Anal. Biochem. 201, 197-210. Champluvier, B. and Kula, M.-R. (1992a) Sequential membrane-based purification of proteins, applying the concept of multidimensional liquid chromatography. Bioseparation 2, 343-351. Champluvier, B. and Kula, M.-R. (1992b) Dye-ligand membranes as selective adsorbents for rapid purification of enzymes: a case study. Biotech. Bioeng. 40, 33-40. Chung, B.H., Chu, C.W., Chang, Y.K., Sohn, J.H., Rhee, S.K. and Park, Y.H. (1993) Purification of recombinant hirudin using metal-chelate affinity chromatography. 6th European Congress in Biotechnology, Florence, Italy, 1993, Vol. 1, MO097. Clonis, Y.D. and Lowe, C.R. (1980a) Triazine dyes, a new class of affinity labels for nucleotide-dependent enzymes. Biochem. J. 191,247-251. Clonis, Y.D. and Lowe, C.R. (1980b) Affinity chromatography on immobilised nucleotides: the symthesis, specificity, and applications of immobilised IMP. Eur. J. Biochem. 110, 279-288. Clonis, Y.D. (1987a) Large-scale affinity chromatography. Bio/Technology 5, 1290-1293. Clonis, Y.D. (1987b) Matrix evaluation for preparative highperformance affinity chromatography. J. Chromatogr. 407, 179-187. Clonis, Y.D. and Lowe, C.R. (1981) Affinity chromatography on immobilized triazine dyes. Studies on the interaction with multinucleotide-dependent enzymes. Biochim. Biophys. Acta 659, 86-98. Clonis, Y.D., Goldfinch, M. and Lowe, C.R. (1981) The interaction of yeast hexokinase with Procion Green H-4G. Biochem. J. 197, 203-212. Clonis, Y.D., Jones, K. and Lowe, C.R. (1986) Process-scale high performance liquid affinity chromatography. J. Chromatogr. 363, 31-36. Clonis, Y.D., Atkinson, A., Bruton, C. and Lowe, C.R. (Eds.) (1987a) Reactive dyes in protein and enzyme technology, Macmillan Press, Basingstoke, UK. Clonis, YD,, Stead, C.V. and Lowe, C.R. (1987b) Novel cationic Triazine dyes for protein purification. Biotechn. Bioeng. 30, 621-627. Clonis, Y.D. and Lowe, C.R. (1988) Affinity chromatography, In: D. William and V. Marks (Eds.), Principles of Clinical Biochemistry, Heinemann, Oxford, UK, pp. 383-390. Clonis, Y.D. (1989) High performance affinity chromatography. Ill: R.W. Oliver (Ed.), HPLC of Macromolecules: a Practical Approach, IRL Press, Oxford, UK, pp. 157-182. Clonis, Y.D. (1990) Process-scale affinity chromatography. In: J. Asenjo (Ed.), Separation Processes in Biotechnology, Mercel-Dekker, New York, pp. 401-445. Clonis, Y.D. (1991) Preparative dye-ligand chromatography. In: M. Hearn (Ed.), HPLC of Proteins, Peptides and Polynucleotides, VCH, New York, pp. 453-468.

115

Clonis, Y.D. and Lowe, C.R. (1991) Monosized adsorbents for high-performance affinity chromatography: application to the purification of calf intestinal alkaline phosphatase and human urine urokinase. J. Chromatogr. 540, 103-111, Clonis, Y.D. (1992) High performance affinity chromatography for protein separation and purification. In: A. Kenney and S. Fowell (Eds.), Practical Protein Chromatography, Humana Press, Totowa, pp. 105-123. Coleman, P., Walker, M., Milbrath, D., Stauffer, D., Rasmusen, J., Krepski, L. and Heilmann, M. (1990) Immobilization of protein A at high density on azlactone-functional polymeric beads and their use in affinity chromatography. J. Chromatogr. 458, 1-11. Corradini, D., Rassi, Z., Horvath, C., Guerra, G. and Horne, W. (1988) Combined lectin-affinity and metal-interaction chromatography for the separation of glycophorins by high performance liquid chromatography. J. Chromatogr. 458, 1-11. Cuatrecasas, P. and Anfinsen, C.B. (1971) Affinity chromatography. In: W.B. Jakoby (Ed.) Methods in Enzymology, Academic Press, Inc., London, UK, Vol. XXII, pp. 345378. Dandeu, P.J., Rabillon, J., Beltrand, M., Lux, M., Duval, R. and David, B, (1990) Immobilized metal ion affinity chromatography for the purification of Fel dI, a cat major allergen from a house dust extract. J. Chromatogr. 512, 177-188. Dean, P.D.G., Johnson, W. and Midlle, F. (Eds.) (1985) Affinity Chromatography: a Practical Approach, IRL Press, Oxford, UK. Delgado, C., Tejetor, M. and Luque, J. (1990) Partial purification of glucose 6-phoshate dehydrogenase and phosphofructokinase from rat erythrocyte haemolysate by partitioning in aqueous two-phase systems. J, Chromatogr. 498, 159-168. Delgado, C., Sancho, P., Mendietta, J. and Luque, J. (1992) Ligand receptor interactions in affinity cell partitioning: studies with transferrin covalently linked to monomethoxypoly(ethylene glucol) and rat reticulocytes. J. Chromatogr. 594, 97-103. Desarnaud, F., Marie, J., Larguier, R., Lombard, C., Jard, S. and Bonnafous, J.-C. (1992) Protein purification using combined streptavidin (or avidin)-Sepharose and thiopropyl-Sepharose affinity chromatography. J. Chromatogr. 603, 95-104. Egi, Y., Koyama, S., Shioda, T., Yamada, K. and Kawasaki, T. (1992) Identification, purification and reconstitution of thiamin metabolizing enzymes in human red blood cells. Biochim. Biophys. Acta 1160, 171-178. Ehle, H. and Horn, A. (1990) Immunoaffinity chromatography of enzymes. Bioseparation 3, 47-53. E1-Kak, A. and Horn, A. (1990) Separation and purification of IgG1 and IgG2 from IgG-Cohn-II fraction of human plasma by pseudobioaffinity chromatography on histidylAH-Sepharose. Bioseparation 3, 47-53 Fadeev, A., Mingalyov, P., Staroverov, S., Lunina, E.,

116

N. Labrou, Y.D. Clonis /Journal of Biotechnology 36 (1994) 95-119

Lisichkin, G., Gaida, A. and Monastyrsky, V. (1992) Silica sorbents with one- and two-site attached bacitracin in affinity chromatography. J. Chromatogr. 596, 114-117. Fujii, T., Nakamura, A., Ogoma, Y., Kondo, Y. and Arai, T. (1990) Selective purification of microtubule: associated protein 1 and 2 from rat brain using poly(L-aspartic acid). Anal. Biochem. 184, 268-273. Galaev, I.Y. and Mattiasson, B. (1993) Affinity thermoprecipitation: the critical role of polymer in ligand-protein interactions. 6th European Congress on Biotechnology, Florence, 13-17 June 1993, Italy, Vol. I, MO099 Giuliano, K. (1992) Chromatography of protein on colums of polyvinylpolypyrrolidone using adsorbed textile dyes as affinity ligands. Anal Biochem. 200, 370-375. Gordon, N., Tsujimura, H. and Cooney, C. (1990) Optimization and simulation of continuous affinity-recycle extractions. Bioseparation 1, 9-21. Gross, T., Bard, M. and Jarret, H.W. (1991) High performance affinity chromatography of messenger RNA. J. Chromatogr. 588, 157-164. Hageman, J. and Kuehn, G. (1992) In: A. Kenney and S. Fowell (Eds.), Practical Protein Chromatography, Humana Press, Totowa, pp. 45-71. Hammarberg, B., Nygren, P.-A., Holmgren, E., Elmbland, A., Tally, M., Hellman, U., Momks, T. and Uhlen, M. (1989) Dual affinity fusion approach and its use to express recombinant human insulin-like growth factor II. Proc. Natl. Acad. Sci. USA 86, 4367-4371. Hansen, P., Lindeberg, G. and Andersson, L. (1992) Immobilized metal ion affinity chromatography of synthetic peptides. J. Chromatogr. 627, 125-135. Hegde, R., Maiti, T. and Podder, S. (1991) Purification and charactirization of three toxins and two agglutinins from Adrus precatorius seed by using lactamyl-Sepharose affinity chromatography. Anal. Biochem. 194, 101-109. Hill, M. and Arrio, B. (1992) Activation of matrices by 4,6-diphenylthieno(3,4-d)-l,3-dioxol-2-one-5,5-dioxide: high performance liquid affinity chromatographic separations. J. Chromatogr. 589, 101-108. Hjerten, S. and Li, J.-P. (1990) High performance liquid chromatography of proteins on deforment non porous agarose beads: fast boronate affinity chromatography of haemoglobin at neutral pH. J. Chromatogr. 500, 543-553. Hochuli, E., Dodeli, H. and Schacher, A. (1987) New metal chelate adsorbent selective for proteins and peptides containing neighbouring histidine residues. J. Chromatogr. 411, 177-184. Hochuli, E., Bannwarth, W., Dodeli, H., Gentz, R. and Stuber, D. (1988) Genetic approach to facilitate purifications of recombinant protein with a novel metal chelate adsorbent. Bio/Technology 6, 1321-1325. Hopp, T., Prickett, K., Price, V., Libby, R., March, C., Cerretti, D., Urdal, D. and Conlon, P. (1988) A short polypeptide marker sequence useful for recombinant protein identification and purification. Bio/Technology 6, 1204-1209. Inomata, Y., Kawaguchi, H., Hiramoto, M., Wada, T. and

Handa, H. (1992). Direct purification of multiple ATF/E4TF3 polypeptides from HeLa cell crude nuclear extracts using aDNA affinity latex particles. Anal. Biochem. 206, 109-114. Inoue, H., Yoshioka, T. and Hotta, Y. (1992) Partial purification and charactirization of membrane associated diacylglycerol kinase of Drosophila heads. Biochim. Biophys Acta 1122, 219-224. Jacquot-Dourges, M., Muller, D., Barritault, D. and Jozefonvicz, J. (1990) High performance affinity chromatography of basic fibroblast growth factor on polystyrene sulphonate resins modified with serine. J. Chromatogr. 510, 141-147. Johansson, G. (1987) Aqueous two-phase affinity partitioning. In: Y. Clonis, A. Atkinson, S. Bruton and C. Lowe (Eds.), Reactive Dyes in Protein and Enzyme Technology, Macmillan Press, Basingstoke, UK, pp. 101-124. Kadonaga, J. and Tjian, R. (1986) Affinity purification of sequence-specific DNA binding proteins. Proc. Natl. Acad. Sci. USA 83, 5889-5893. Kasai, K. (1992) Trypsin and affinity chromatography. J. Chromatogr. 597, 3-18. Kastner, M. and Neubert, D. (1991) High performance metal chelate affinity chromatography of cytochromes P-450 using chelating Superose. J. Chromatogr. 587, 43-54. Kato, S. and Terashima, M. (1993) Purification of secreted proteins by immunoaffinity chromatographywith cross-reactive and peptide-antibodies. 6th European Congress of Biotechnology, Florence, Italy, Vol. 1,539-542. Kim, M., Saito, K., Furusaki, S., Sato, T., Sugo, T. and Ishigaki, I. (1991) Adsorption and elution of bovine globulin using an affinity membrane containing hydrophobic amino acids as ligands. J. Chromatogr. 585, 45-51. Kim, D.H., Kanno, C. and Mizokani, Y. (1992) Purification and characterization of major glucoproteins, PAS-6 and PAS-7, from bovine milk fat globule membrane. Biochim. Biophys. Acta 1122, 203-211. Kirchberger, J. and Kopperschliiger, G. (1990) An improved purification procedure of alkaline phosphatase from calf intestine by applying partition in aqueous two-phase systems and dye ligand chromatography. Bioseparation 1, 33-41. Keruchenko, J., Keruchenko, I., Gladilin, K., Zaitsev, V. and Chirgadze, N. (1992) Purification, characterization and preliminary X-ray study of fumarase from Saccharomyces cerevisiae. Biochim. Biophys. Acta 1122, 85-92. Kitamura, K., Uyeda, K., Kangawa, K. and Matsuo, H. (1989) Purification, characterization of rat skeletal muscle fructose-6,2-kinase: fructose-2,6- bisphospatase. J. Biol. Chem. 246, 9799-9806. Kopperschl/iger, G. and G. Birkenmeier (1990) Affinity partition and extraction of proteins. Bioseparation 1,235-254. Koyama, J., Inoue, S., Ikeda, K. and Hayaski, K. (1992) Purification and amino acid sequence of a nerve growth factor from the venom of Vipera russeli russelli. Biochim. Biophys. Acta 1160, 287-292. Kroviarski, Y., Cochet, S., Vadon, C., Truskolaski, A., Boivin,

N. Labrou, Y.D. Clonis /Journal of Biotechnology 36 (1994) 95-119 P. and Bertrand, O. (1988) New strategies for the screening of a large number of immobilized dyes for purification of enzymes. J. Chromatogr. 449, 403-412. Kroviarski, Y., Cochet, S., Vadon, C., Truskolaski, A., Boivin, P. and Bertrand, O. (1988) Purification of human 6-phosphogluconate dehydrogenase from human haemolysatewith chromatography on an immobilized dye as the essential step and use of automotion. Simultaneus purification of lactate dehydrogenase. J. Chromatogr. 449, 413-422. Larsson, O.-P. and Mosbach, K. (1979) Affinity precipitation of enzymes. FEBS Lett. 98, 333-338. LeGrice, S.F. and F. Gruninger-Leitch, F. (1990) Rapid purification of homodimer and heterodimer HIV-1 reverse transcriptase by metal chelate affinity chromatography. Eur. J. Biochem. 187, 307-314. Lindner, N., Jeffcoat, R. and Lowe, C.R. (1989) Design and application of biomimetic anthraquinone dyes: purification of calf intestinal alkaline phosphatase with immobilized terminal ring analogue of C.I. Reactive Blue 2. J. Chromatogr. 473, 227-240. Ljungquist, C., Breitholtz, A., Brink-Nilsson, H., Monks, T., Uhlen, M. and Niisson, M. (1989) Immobilization and affinity purification of recombinant proteins using histidine peptide fusions. Eur. J. Biochem. 186, 563-569. Liona, I., Annaert W., and De Potter, W. (1992) Simultaneous purification of the neuroproteins synapsin I and synapsisin. J. Chromatogr. 596, 51-58. Loetscher, P., Mottlau, L. and Hochuli, E. (1992) Immobilization of monoclonai antibodies for affinity chromatography using a chelating peptide. J. Chromatogr. 595, 113-119. Lohman, T., Chao, K., Green, J., Sage, S. and Runyon, G. (1989) Large-scale purification and characterization of Escherichia coli rep gene product. J. Biol. Chem. 264, 1013910147. Lowe, C.R. (1977) The synthesis of adenosine 5'-monophosphate to study the effect of the nature of spacer arm in affinity chromatography. Eur. J. Biochem. 73, 267-275. Lowe, C.R., Bruton, S.J., Pearson, J., Clonis, Y.D. and Stead, C.V. (1986) Design and application of biomimetic dyes in biotechnology. J. Chromatogr. 376, 121-130. Lowe, C.R. (1979) Affinity chromatography. In: T.S. Work and E. Works (Eds.), Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 7 II, pp. 269-522, Elsevier, Amsterdam, The Netherlands. Lowe, C.R. (1981) The applications of immobilised nucleotides in biotechnology. In: A. Wiseman (Ed.), Topics in Enzyme and Fermeatation Biotechnology, Vol. 5, pp. 13-146, Ellis Horwood, Chichester, UK. Lowe, C.R., Burton, S.J., Burton, N.P., Alderton, K.W., Pitts, J.P. and Thomas, J.A. (1992) Designr dyes: biomimetic ligands for the purification of pharmaceutical proteins by affinity chromatography. Trends Biotechnol. 10, 442-448. Makriyannis, T. and Clonis, Y.D. (1993) Simultaneous separation and purification of pyruvate kinase and lactate dehydrogenase by dye-ligand chromatography. Process Biochem. 28, 179-185. Mattiasson, B. and Olsson, U. (1986) General chromato-

117

graphic purification procedure based on the use of heterobifunctional affinity ligands. J. Chromatogr. 370, 21-28. Mattiasson, B. and Ramstrop, M. (1984) Ultrafiltration affinity purification: Isolation of Concanavalin A from seeds of Canavalia ensiformis. J. Cbromatogr. 283, 322-330. Mayes, A., Hubble, J. and Eisenthal, R. (1991) Investigation of liquid phase cooperative binding interactions on the capacity of insoluble affinity adsorbents. J. Chromatogr. 539, 245-254. Miller, J., Changaris, D. and Levy, R. (1992) Purification, subunit structure and inhibitor profile of cathepsin A.J. Chromatogr. 627, 153-162. Moncollin, V., Gerard, M. and Egle, J.-M. (1990) Purification of the upstream element factor of the adenovirus-2 major late promoter from HeLa and yeast by sequence-specific DNA affinity chromatography. J. Chromatogr. 510, 243250. Moks, T., Abrahmsen, L., Osterlof, B., Josephson, S., Ostling, M., Enfors, S.-O., Persson, I., Nilsson, B. and Uhlen, M. (1987) Large-scale affinity purification of human insulinlike growth factor I from culture medium of Escherichia coli. Bio/Technology 5, 379-382. Mosbach, K., Guilford, H., Ohlsson, R. and Scott, M. (1972) General ligands in affinity chromatography: cofactor-substrate elution of enzymes bound to the immobilized nucleotides adenosine 5'-monophosphate amd nicotinamideadenine binucleotide. Biochem. J, 127, 625-631. Muszynska, G., Dobrowolska, G., Medin, A., Ekmam, P, and Porath, M. (1992) Model studies on iron (lid ion affinity chromatography. Interaction of immobilized iron (lid ions with phosporylated amino acids, peptides and proteins. J. Chromatogr. 604, 19-28. McCreath, G., Chase, H., Purvis, D. and Lowe, C.R. (1992). Novel affinity separation based on perfluorocarbon emulsions: use of perfluorocarbon affinity emulsion for the purification of human serum albumin from blood plasma in a fluidised bed. J. Chromatogr. 597, 189-196. Nachman, M., Azad, A.R.M. and Bailon, P. (1992) Efficient recovery of recombinant proteins using membrane-based immunoaffinity chromatography. Biotech. Bioeng. 40, 564-571. Nagata, Y., Maeda, K. and Scopes, R. (1992) NADP-linked alcohol dehydrogenases from extreme thermophiles: simple affinity purification schemes and comparative properties of the enzymes from different strains. Bioseparation 2, 353-362. Ngo, T. and Khatter, N. (1990) Chemistry and preparation of affinity ligands useful in immunogiobulin and serum protein separation. J. Chromatogr. 510, 281-291. Van Noort, J.M., Van der Berg, P. and Mattern, E.I. (1991) Visualization of proteases within a compex sample following their selective retention on immobilized bacitracin, a peptide antibiotic. Anal. Biochem. 198, 385-390. Nilsen, B., Paulsen, B., CIonis, Y.D. and Mellbye, K. (1990) Purification of the glycoprotein allergen Ag7 from mugwort pollen by Concanavalin A affinity chromatography. J. Biotechnol. 16, 305-316.

118

N. Labrou, Y.D. Clonis/Journal of Biotechnology 36 (1994) 95-119

Ohson, S., Hansson, L., Larsson, P-O., and Mosbach, K. (1978) High performance liquid affinity chromatography (HPLAC) and its application to the separation of enzymes and antigens. FEBS Lett. 93, 5-9. Olsson, U. and Mattiasson, B. (1986) Theoretical and experimental evaluation of the use of heterobifunctional affinity ligands in general chromatographic purification systems. J. Chromatogr. 370, 29-37. O'Shannessy, D. and Wilchek, M. (1990). Immobilization of glycoconjugates by their oligosaccharides: use of hydrazido-drivatized matrices. Anal. Biochem. 191, 1-8. Petel, A., O'Hara, M., Callaway, J., Greene, D., Martin, J. and Nishikawa, A. (1990) Affinity purification of tissue plasminogen activator using transition-state analogues. J. Chromatogr. 510, 83-93. Pearson, J. (1987) Dye-ligand affinity precipiration, In: Y. Clonis, A. Atkinson, S. Bruton and C. Lowe (Eds.), Reactive Dyes in Enzyme and Protein Technology, Macmillan Press, Basingstoke, UK, pp. 187-191. Pearson, J., Clonis, Y.D. and Lowe, C.R. (1989) Preparative affinity precipitation of L-lactate dehydrogenase. J. Biotechnol. 11, 267-274. Pepper, D.S. (1992) Selection of antibodies for immunoaffinity chromatography. In: A. Kenney and S. Fowell (Eds.), Practical Protein Chromatography, Humana Press, Totowa, pp. 135-196. Pozidis, C., Vlatakis, G. and Bouriotis, V. (1993) Sequencespecific DNA affinity chromatography: application of a group-specific adsorbent for the isolation of restriction endonucleases. J. Chromatogr. 630, 151-157. Ritter, S., Komenta, J., Setlikova, E., Setlik, I. and Wetle, W, (1992) Immobilized metal affinity chromatography for the separation of photosystems I and II from the thermophilic cyanoacterium Synechococcus elongatus. J. Chromatogr. 625, 21-31. Roos, P.H. (1991) Immobilized metal affinity chromatography as a means of fractionating microsomal cytochrome P450 isozymes. J. Chromatogr. 587, 33-42. Ruzzene, M., Vianello, F., Donella-Deana, A. and Deana, R. (1992) Purification and characterization of two casein kinases from ejaculated bovine spermatozoa. J. Biochem. 112, 768-774. Santambien, P., Hulak, I., Girot, P. and Boschetti, E. (1992) Elisa-based quantification of Cibacron Blue F3GA used as ligand in affinity chromatography. Bioseparation 2, 327334. Sarkat, M., Majumder, G. and Chatterjee, T. (1991) Goat sperm membrane: lectin-binding sites of sperm surfase and lectin affinity chromatography of mature sperm membrane antigens. Biochim. Biophys. Acta 1970, 198-204. Scawen, M.D. and Atkinson, A. (1987) Large-scale dye-ligand chromatography. In: Y. Clonis, A. Atkinson, C. Bruton and C. Lowe (Eds.), Reactive Dyes in Enzyme and Protein Technology, Macmillan Press, Basingstoke, UK, pp. 51-85. Schnapp, A., Clos, J., Hadelt, W., Schreck, R., Cvekl, A. and Grummt, I. (1990) Isolation and functional characterization of TIF-IB, a factor that confers promoter specificity

to mouse RNA polymerase I. Nucleic Acids Res. 18, 1385-1393. Schuler, G. and Reinacher, M. (1991) Development and optimization of a single step procedure using protein A affinity chromatography to isolate murine IgG1 monoclonal antibodies from hybridoma supernatants. J. Chromatogr. 587, 61-70. Sepracor, Inc. (1993) Convection vs. diffusion in HyperDiffusion chromatography. Literature code PB04, Marlborough, MA, USA. Silver, A., Lamb, E., Cattell, W. and Dawnay, A. (1991) Investigation and validation of the affinity chromatography method for measuring glycated albumin in serum and urine. Clin. Chim. Acta 202, 11-22. Slingerland, R, and Scouter, W. (1990) Centrifugal affinity chromatography. J. Chromatogr. 510, 205-211. Spalding, B.J. (1991) Downstream processing: key to slashing production cost 100-fold. Bio/Technology 9, 229-233. Stewart, D., Hughes, P. and Lowe, C.R. (1989) Affinity chromatography on triazine dyes immobilised on novel perfluorocarbon supports. J. Biotechnol. ll, 253-266. Stewart, D., Purvis, D. and Lowe, C.R. (1990) Affinity chromatography on novel perfluorocarbon supports: immobilization of C.I. reactive blue-2 on a polyvinyl alcohol-coated perfluoropolymer support and its application in affinity chromatography. J. Chromatogr. 510, 177-187. Stofko-Hahn, R., Carr, D. and Scott, J. (1992) A single-step purification for recombinant proteins: characterization of a microtubule associated protein (MAP 2) fragment which associates with the type II cAMP-dependent protein kinase. FEBS Lett. 302, 274-278. Te Booy, M., Faber, A., Jonge, E., Wolterink, E., Riethorst, W., Beugeling, T., Bantjes, A., Over, J. and Koning, B. (1990) Large-scale purifation of factor VIII by affinity chromatography: optimization of process parameters. J. Chromatogr. 503, 103-114. Teichberg, V.I. (1990) Affinity-repulsion chromatography. J. Chromatogr. 510, 49-57. Tejetor, M., Delgado, C., Grupeli, M. and Luque, J. (1992) Affinity partitioning of erythrocytic phosphofructokinase in aqueous two-phase systems containing poly(ethyleneglycol)-bound Cibacron Blue. J. Chromatogr. 589, 127134. Tomono, Y., Abe, K. and Watanabe, K. (1990) High performance liquid affinity chromatography and in situ fluorescen labeling on thin-layer chromatography of glycosphingolipids. Anal. Biochem. 184, 360-371. Torres, J., Guzman, R., Carbonel, R. and Kilpatrick, P. (1988) Affinity surfactants as reversible bound ligands for high performance affinity chromatography. Anal. Biochem. 171, 411-418. Tsamadis, G., Papageorgakopoulou, N. and Clonis, Y.D. (1992) Downstream processing of diagnostic enzymes: optimised protocols for the simultaneous separation and purification of lactate dehydrogenase and pyruvate kinase from rabbit muscle. Bioproc. Engin. 7, 213-218. Unger, K.K., Lork, K.D. and Wirth, H.-J. (1991) Development

N. Labrou, Y.D. Clonis/Journal of Biotechnology 36 (1994) 95-119 of advanced silica-based packing materials. In: Hearn, M.T.W. (ed.), HPLC of Proteins, Peptides and Polynucleotides, VCH Publishers, Inc., New York, Chapt. 3, pp. 59-117. Vilter, H. (1990) Aqueous two-phase extraction of plant enzymes from sources containing large amounts of tannins and anionic mucilages. Bioseparation 1, 283-292. Vlatakis, G. and Bouriotis, V. (1991a) Sequence-specific DNA affinity chromatography: application to the purification of EcoRI and SphI. Anal. Biochem. 195, 352-357. Vlatakis, G. and Bouriotis, V. (1991b) Affinity partitioning of restriction endonucleases: application to the purification of EcoRI and EcoRV.J. Chromatogr. 538, 311-321. Walters, H , Johansson, G. and Brooks, D. (1991) Partitioning in aqueous two-phase systems: recent results. Anal. Biochem. 197, 1-18. Weber, D. and Bailon, P. (1990) Application of receptor-affinity chromatography to bioaffinity purification. J. Chromatogr. 510, 59-69. Welling, G., Geurts, T., Gorkum, J.V., Damhof, R., WouterDrijfhout, J., Bloemhoff, W. and Welling-Wester, S. (1990) Synthetic antibody fragment as ligand in immunoaffinity chromatography. J. Chromatogr. 512, 337-343. West, I. and Goldring, O. (1992) Lectin affinity chromatography. In: A. Kenny and S. Fowell (Eds.), Practical Protein Chromatography, Humana Press, Totowa, pp. 81-89. Wheatley, J.W. (1992) Multiple ligand application in high

119

performance immunoaffinity chromatography. J. Chromatogr. 603, 273-278. Wikstrom, P. and Larsson, P.-O. (1987) Affinity fibre: a new support for rapid enzyme purification by high performance liquid affinity chromatography. J. Chromatogr. 388, 123134. Yip, T.T., Nakagawa, Y. and Porath, J. (1989) Evaluation of the interaction of peptides with Cu(lI), Ni (II) and Zn (II) by high performance immobilized metal ion affinity chromatography. Anal. Biochem. 183, 159-171. Yip, T.T. and Hutchens, T.W. (1992) lmmobilised metal ion affinity chromatography. In: A. Kenney and S. Fowell (Eds.), Practical Protein Chromatography, Humana press, Totowa, pp. 17-31. Zachariou, M. and Hearn, M.T.W. (1992) High performance liquid chromatography of amino acids, peptides and proteins: 8-hydroxyquinoline-metal chelate chromatographic support. J. Chromatogr. 599, 171-177. Zhang, X.Y., Asiedu, C.K., Supakar, P. and Ehrlich, M. (1992) Increasing the activity of affinity purified DNAbinding proteins by adding high concentrations of nonspecific proteins. Anal. Biochem. 201, 366-374. Zutautas, V., Baskeviciute, B. and Pesliakas, H. (1992) Affinity partitioning of enzymes in aqueous two-phase systems containing dyes and their copper (II) complexes bound to poly(ethylene glycol). J. Chromatogr. 606, 55-64.