New methods for separation and recovery of biomolecules Magnus Glad and Per-Olof Larsson Lund University, Lund, Sweden New methods and applications in the separation of biomolecules are reviewed, with an emphasis on the large-scale recovery of proteins. Highlights include the advent of flow-through particles in perfusion chromatography, which allows for very high flow rates, while retaining a high chromatographic efficiency. Current Opinion in Biotechnology 1991, 2:413-418

Introduction The products of biotechnological processes are often obtained in a very dilute state, contaminated with large amounts of substances with similar physical and chemical characteristics. Thus, the cost of purification may amount to 50-90 % of the total cost of the product. Clearly there is a considerable incentive to improve the methods of purification. A computer survey of the literature published within the period of review revealed prolific activity in this area of research. Out of the m a w hundreds of relevant papers, we have chosen to include only those that describe new separation methods, new applications of known methods, or particularly notable achievements using known technology. The field has been reviewed extensively and we have selected a good number of overviews to allow the reader easy access to various specialities. In line with the general theme of this issue, we have emphasized the selection of preparative and largescale applications. In most cases, biomolecutes are understood to mean macromolecules, particularly proteins.

Tailoring proteins for isolation The importance of an integrated approach in biotechnological processes is often emphasized. An obvious example of the benefits of upstream efforts to facilitate downstream processing is the genetic fusion of special sequences to one end of the target protein. These so-called 'tails', 'tags' or 'handles' are selected to be easily recognizable in the purification scheme. A short, authoritative overview of this area has been given by Sassenfeld [1.]. Double handles have been described by Jansson et aL [2,] for improving the expression and isolation of an easily degradable mutant protein A. The amino-terminus of the target protein was provided by an albumin-binding protein G domain and the carboxy-terminus by a polyhistidine peptide. This dual affinity system allowed the sequential use of two specific purifications (albuminand Zn 2 +-alFmity chromatography). The isolation of nondegraded, full length, target protein was vastly improved by the presence of the flanking sequences and the double purification scheme.

Recently, Lilius et aL [3"] have described the use of a pentahistidine tail for the rapid purification of galactokinase by affinity precipitation using Zn2+-EGTA as the precipitant. The galactokinase was precipitated at a much lower Zn2 + concentration than other proteins because of the high concentration of histidine residues located on its surface.

Precipitation techniques Fractional precipitation with salts is often used as a first step in purification schemes. The purification achieved may be less dramatic, but it yields a material suitable for subsequent, more efficient purification steps, such as column chromatography. Richardson et aL [4.] have reinvestigated this important unit operation from a bioengineering point of view. They have developed a systematic approach to optimize yield and purity with a special view to large-scale processing. Affinity precipitation has been given considerable attention during recent years as a means of specifically isolating or enriching proteins at a very early stage of a purification sequence. The subject is covered extensively by Chen [5"] in an overview of novel affinity techniques. Van Dam et al. [6.] have investigated thoroughly metal affinity precipitation of proteins. Important factors governing the efficiency and selectivity of the method were the availability of histidine residues on the protein surface, pH, total protein concentration, and the structure of the metal ligand (free ion, chelated ion or polymer-bound chelated ion). If the target protein lacks surface-bound histidine residues, this may be remedied by genetically introducing a polyhistidine tail, as discussed above [3"].

Aqueous polymeric two-phase systems Separations based on aqueous polymeric two-phase systems have a number of attractive features, such as mild separation conditions, rapid mass transfer, high capacity and easy, linear scale-up. In spite of this, the technique is not used widely in processes. The reasons may be a mixture of real limitations and of misconceptions as discussed by Huddleston and Lyddiatt [7"]. An outstanding

Abbreviations HPLC--high-performance liquid chromatography; HSA--human serum albumin.

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Biochemicalengineering coverage of the field appeared in the December 1990 issue of Bioseparation [8.o]. The whole issue is devoted to aqueous polymeric two-phase systems and the contributors are well-known experts. The original two-phase systems as described by Albertsson relied on dextran and polyethylene glycol. Later developments include cheap substitutes for the expensive, fractionated dextran, which are often based on starch materials. Sturesson et at [9] describe a number of applications using hydroxypropyl starch which behaves essentially as dextran. Another means of avoiding dextran involves the use of polyethylene glycol-salt systems. Disposal of concentrated salt solutions may, however, be a problem in large-scale operations. A remedy would be to recycle the salt solutions as demonstrated by Greve and Kula [10o]. The standard way of separating phases is by centrifugation techniques. Alternatives may be interesting, for example, in multistep separations and where the phases are very viscous or have similar densities. Rat et al. [11 °] showed that the time required for demixing was reduced by a factor of three on applying an electric field with the proper field strength (around 15 V c m - 1 ) and proper field direction. The basis of the improved demixing is the unequal distribution of ions between the phases. Dispersed phase droplets thus carry a net charge, making them responsive in an electric field. Flygare et al. [12 o] have used a magnetic technique with a number of phase systems. A small amount of micronsized iron oxide was added to the phase mixture and completely partitioned into one of the phases, thus rendering it magnetically susceptible. Improvement in separation time by a factor of 5-50 was often observed. In one case (near isopycnic phase system), a 250 000-fold improvement was observed.

Membranes Separations based on membranes (ultrafiltration or microfiltration) have Several advantages compared with column techniques, such as speed and easy scale-up. The inherent separation property of membranes, that is, separation according to size, is not usually sufficient for the separation of a mixture of proteins. Preferably, the molecular weight should differ by an order of magnitude or more. This limited selectivity may be improved by the introduction of functional groups such as ion-exchange ligands or affinityligands. Sometimes the separation process relies both on the size-discriminating ability and on the specificity of the ligands. In other cases the membrane structure serves only as an efficient carrier of functional groups. Unarska et al. [13] have studied the adsorption of immunoglobulin to Protein A bound to microfdtration nylon membranes and to standard agarose beads. The membranes proved to have superior adsorption kinetics, as expected from an adsorbent where mass transport is effectuated mainly by convective flow and not by diffusion. Interestingly, large-pore membranes (3 Ixm) gave the same kinetics as small.pore membranes (0.45 Ilm)

but at 10 times less pressure drop. Tennikova [14] prepared thick membranes (1 mm) and used them as short chromatography columns in 'high-performance membrane chromatography' (invoMng hydrophobic interactions). As expected, the thick membrane gave an advantageously high capacity; in this case, 40g ovalbumin m - 2 of membrane ( = 40 g litre-1). Ling and Mattiasson [15 o] combined membranes with a separate adsorbent. The filtered process stream was mixed with adsorbent particles before entering an arrangement of cross-flow ultraffltration membrane devices. Molecules that became affinity-bound to the adsorbent particles were retained by the membrane. The adsorbent consisted of non-porous silica nanoparticles (10--20 run in diameter) derivatized with cibacron blue. Such particles will very rapidly equilibrate with the surrounding medium, both in the adsorption and the desorption step, which is important for the efficient operation of a combined membrane-particle unit. When alcohol dehydrogenase was purified from a yeast homogenate using 1.5 m 2 membrane modules, the productivity was 2 g of enzyme per h. Membranes are often used in combination with crude bioextracts. The construction of membranes that minimize the tendency to fouling is therefore of prime importance. Capannelli et al. [16] have studied this problem and have related nltrafiltration characteristics with porosimetry and contact angle measurements.

Chromatography Chromatography is the dominant separation technique, at least when it comes to the publication of new resuits. Although the focus here will be on some new developments in the chromatography of biomolecules, the usefulness of standard techniques should not be overlooked. A convincing example is the work byJohansson et al. [17,] who have achieved a 25 000-fold purification of staphylococcal enterotoxin B on a large scale, with a total recovery of 74 %, using a sound combination of one size exclusion and two ion-exchange steps. New support materials

In attempts to reduce the impact of diffusional limitations in chromatographic supports, the use of small particles has gained recognition. An alternative approach, which might revolutionize separation science in the future, is the development of flow-through particles by A f e y a n e t al [18..,19oo]. These specially prepared polystyrene particles (10-20 lam in diameter) are suitable for use in socalled perfusion chromatography. Perfusion chromatography seems to be the ultimate way to solve the mass transfer problems associated with chromatography, in that the mobile phase actually flows through each individual particle via very large pores (6000-8000A diameter). The solutes enter the interior of the particles mainly through convective transport, and diffusion is responsible only for their transport through the interconnecting micropores (500-1500A diameter with a maximum depth of less than 1 lam). These pores are also responsi-

New methods for separation and recovery of biomolecules Glad and karsson 415 ble for the relatively large surface area which is needed to obtain the high binding capacity necessary for economical preparative applications. The resulting performance equals that achieved using particles one order of magnitude smaller, without having to cope with the normal increase in back pressure. This new type of chromatographic support is suitable for both analytical and preparative applications. Fast analytical applications, for instance a reversed-phase separation of five standard proteins using acetonitrile-gradient elution, could be accomplished within 40s. The column packing is also excellent for scaling-up. A 50ml anion-exchange column could process 120mg protein from a supernatant of a hybridoma cell culture in 2 rain. Czok and Guiochon [20] have described columns packed with bundles of aligned porous silica fibers (181am diameter, 270/~. average pore diameter) in size exclusion chromatography. Fibers can be packed more densely than conventional beads, resulting in smaller interstitional volume and larger pore volume, which should improve the performance. Traditional gel-filtration chromatography using polysaccharide packings is also under development. Kfigedal et al. [21] have described Superdex, a crosslinked agarose bead modified with dextran, which has been used to obtain both high flow rates and the desired selectivity. Another composite material, which also combines different advantageous properties, has been described by Glad and Schomack [22]. They used a polystyrene-cellulose composite exhibiting both high flow rates (up to 2500cmh -1) and high biocompatibility in a large-scale ion-exchange chromatography process in which immunoglobulins and albumin were purified from bovine plasma. IJ et al. [23] have used an interesting type of non-porous

agarose beads, prepared by shrinkage and cross-linking in organic solvents, to immobilize cibacron blue. These beads were further deformed during packing at high flow rates and then used for the purification of various dehydrogenases in affinity high-performance liquid chromatography (HPLC). The compressed bed of agarose beads could tolerate flow rates up to more than 1000 cmh-1. Affinity chromatography The term 'affinity chromatography' is used in a broad sense to include, not only biological interactions, but also various types of chemical and physical interactions. Scouten [24.] has covered recent progress in affinity chromatography techniques, including both analytical and preparative developments. In the work presented by Ohlson et al. [25], the emphasis was on high-performance methods. One spectacular achievement using affinity chromatography has been demonstrated by Takeya et a/.[26] who managed to obtain a 27 million-fold purification of a glycosyl transferase. The process included three afiqnity steps: binding to underivatized Sepharose 4B; one immunoafflnity step; and one cibacron blue-affinity step. Immunoaffinityadsorption seems to be the most popu-

lar form of allqnity chromatography today. One example of this is the use of immobilized bacterial Fc-receptors, such as protein A and Protein G, for immunoglobulin purification. In a recent review, Desai [27.] evaluates immunoaftinitypurification as a tool for process-scale isoiation of biochemical therapeutic agents. Various solid supports, coupling methods, desorption methods, scale-up considerations, as well as automation, are discussed extensively. One major problem when using immunoafiqnity for the isolation of biomolecules is that the conditions required for elution are normally drastic (e.g. buffers at pH < 3). This is destructive not only for the eluted biomolecule (low activityyields) but also for the chromatographic sotbent (support or ligand), the lifetime of which could decrease markedly. To facilitate elution from immunosorbents, Velander et al. [28o] have used specially selected monoclonal antibodies showing metal-dependent binding to human plasma protein C or Factor IX. Protein C bound in a metal-free environment to its antibody (elution with 25 mM calcium chloride) and Factor IX bound in the presence of magnesium chloride (elution with EDTA). Besides the high yields of active protein achieved with this method, it is likely that unwanted plasma proteins, which may interact non-specifically with the affinity sorbent, will not be eluted under these mild and 'selective' conditions. Another promising approach, for obtaining easy elution and high yields is so-called weak-affinity chromatography, as described byZopfand Ohlson [29"]. In this method, antibodies with a relatively low binding constant (10z---104M - t) are selected for immobilization. The chromatographic process is run isocratically, producing rapid and specific separations under near physiological conditions. Metal-affinitychromatography is another important technique which has been reviewed recently byAmold [30"]. Stable and inexpensive chelating sorbents are used which bind metal ions and can then interact with metal-binding residues on protein surfaces. The binding strength is usually high but e~cient elution can be achieved by relatively mild means, such as a slight decrease in pH. A thorough mechanistic investigation was performed by Belew and Porath [31], who also noticed that metal ion transfer can arise because the immobilized Cu2+ has a higher afllnity for certain histidine-fich peptides than for the chelator group in the matrix, thus inhibiting retardation of the desired protein on the column. Mimetic ligands are usually synthetic, non-biological, lowmolecular-weight molecules that in some way mimic biological interactions. The main benefits of these ligands are their low price and their stability in the presence of the strong chemical agents used for regeneration and sanitation. One well-known example is the reactive dye, cibacron blue, which binds to certain enzymes, such as dehydrogenases, in their dinucleotide fold, thus mimicking a coenzyme. Burton et al. [32"] have shown that a proper design of these dyes can improve their binding to biomolecules. In an application of immunoafiinity chromatography, Welling el a l [33"] have ingeniously constructed a syn-

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thetic antibody using known structural data. They immobilized a 13-residue synthetic peptide with an amino acid sequence similar to that of certain hypervariable segments In anti-lysozyme,which is responsible for the binding to lysozyme. Kauvar et al. [34 •] have described paralog chromatography, which is based on the use of synthetic paratope (antigen-recognition site) analogs for the construction of chromatographic sorbents. These peptide analogs can be selected to mimic the specificity of moderate atfinity antibodies. Kits comprising random peptides whose binding characteristics can be elucidated in micro-scale before scaling up are now available. The so-called thiophilic ligands can be used for groupspecific adsorption of immunoglobulins, C3, C4 and ct-2-macroglobulins. They possess an affinity resembling protein A and protein G, but have the advantage of being cheaper and more stable. They are based on divinytsulphone-activated supports to which thiols have been coupled. The sulphone group together with a thioether seem to be essential for the salt-promoted adsorption of proteins. In a recent investigation, Oscarsson and Porath [35] classified several pyridine- and alkylthioether adsorbents according to their selectivity and their hydrophobic-thiophilic behavior. Thiophilic adsorption has also been transformed to the more efficient HPLC mode by Nopper et al. [36] who made similar derivatives of epoxy-silanized, macroporous, microparticulate silica. The sorbent was useful for rapid, highcapacity, single-step purification of all subclasses of monoclonal and polyclonal antibodies. Ngo et al. [37"] have developed an alternative type of immunoglobulinselective ligand, based on pyridine derivatives. The binding mechanism is not thiophilic (no sulphur is present) but may be based on the formation of conjugated systems. The use of molecular imprinting to prepare specific sorbents is to some extent related to synthetic sorbents mimicking biological interactions. A recent review by Ekberg and Mosbach [38] covers various types of imprinting systems. Interacting monomers are allowed to prearrange around a print molecule before polymerization. The resulting polymer with specifically arranged, interacthag entities then shows selectivity for the print molecule, which can be separated from a mixture of closely resembling substances such as D/L amino acid derivatives. To date, small print molecules have been used most extensively in this methodology, but it is to be expected that macromolecules of biological interest will also be employed in the future.

Other techniques Electrophoretic separations are standard methods for analyzing protein mixtures. On the other hand, their preparative use is very limited in spite of the high resolution they afford. A prime reason for this is that the heat generated during the preparative separations is difficult to dissipate and therefore leads to deteriorated resolution. However, equipment that allows the separation to occur in thin flowing layers in combination with efficient cooling has become available recently. Using this method, Os-

trem et al. [39] have isolated S1 nuclease with a preparative, recycling isoelectrophoresis aparatus. Up to about 1 g of a crude protein mixture could be separated in 3 h. Nath et al. [40] have used continuous-flow zone electrophoresis for the direct isolation of the three enzymes, formate dehydrogenase, formaldehyde dehydrogenase and methanol oxidase. The sturdiness of the method and the equipment was illustrated by the fact that cell homogenates could be electrophoresed without prior clearing of the cell debris. Many affinity interactions, especially immunoaffinityinteractions, are very strong. Standard desorption procedures im'olve lowering the pH or adding agents that can change protein conformation. An interesting and radical method is described by Wallace et al. [41.] who have made use of conducting, electroactive affinity adsorbents. Human serum albumin (HSA) was bound to polypyrrol-coated carbon fibers and used for the adsorption of anti-HSA. Interestingly, the adsorbent could be manipulated electrochemically to release the antibodies. Another new method of releasing strongly adsorbed protein from an affinity matrix is described by Olson et aL [42]. By using high pressure (above 100MPa), they showed that bovine serum albumin, adsorbed to its immobilized antibody, could be released without any dam: age to either the albumin or the antibody. This was in contrast to the standard elution method (glycin buffer; pH 2.5) which destroyed the antibody. Even repeated pressurizations proved to be harmless. When very pure proteins are required, the ultimate step is usually crystallization. Protein crystallization is no trivial task and may require considerable time and effort, even when solutions of pure, or almost pure, proteins are available. Visuri et al. [43"] have found that this process may be highly facilitated ff high pressure is used during the crystallization. Thus, at 200 MPa, microcrystals of glucose isomerase were rapidly and consistently formed from saturated solutions of the enzyme.

References and recommended reading Papers of special interest, published within the annual period o f resiew, have been highlighted as: • o f interest •• of outstanding interest 1. ShSSEaXTEI.D HM: Engineering Proteins for Purification. Trends • Biotecbnol 1990, 8:88-93. A short, excellent overview o f the technique of genetically adding 'tags', 'tails' or 'handles' to proteins to fascilitate their purification. 2. •

JANSSONB, PAL~tCR&'rrzC, UIIL~N ISt, NIL.c6ONB: A Dual-AIFmity Gene Fusion System to Express Small Recombinant Proteins in a Soluble Form: Expression and Characterization o f Protein A Deletion Mutants. Protein Eng 1989, 2:555-561. An albumin-binding domain gas fused to the amino-terminus o f the target protein and a Zn2+-binding sequence (pob~aistidine) t o the carboxy.terminus. The target protein (a protein A mutant) yeas protected from protease action by the flanking sequences and x~as subsequently purified on two affinity columns (albumin and Zn 2+ ), ensuring the recovery of a full length target protein. 3. •

LIt/us G, PERSSONM, BfJLOWL, MOSBACHK: Metal Affinity Precipitation o f Proteins Carrying Genetically Attached Polyhistidine Affinity Tails. E u r J Bit~Jem 1991, in press.

N e w m e t h o d s for separation a n d r e c o v e r y of b i o m o l e c u l e s Glad a n d Larsson A pentahistidine tail v~ts genetically fused to the carboxy-terminus of galactose dehydrogenase. The modified enzD~e could be purified easily by a/Iinity precipitation with a bifuncdonal Zn2+ complex, EGTA(Zn2 +). The tail v-as removed by carboxTpeptidase A. Interestingly, F_..w./~ridg/acol/cells producing the recombinant protein showed increased zinc tolerance. 4. •

l~c~N P, tlOARE M, Dtm'lXqLLF: A New Biochemical Engineering Approach to the Fractional Precipitation of Proteins. Biotecbnol Bioeng 1990, 36:354-366. An optimized fractional precipitation of proteins is important for the subsequent steps in a purification scheme. A ~'stematic approach for finding the interval that gives optimum ~Seld and purity is described. The experimental data are obtained from precipitation of alcohol dehydrogenase from baker's yeast. 5. CHEN J-P: Novel Affinity-Based Processes for Protein Purifi"~'he cation. J Ferment Bioeng 1990, 70:199-209. review gives a thorough-description of aft'ratty precipitation, af~nity cross-flow filtration and affinity partitioning. 6. •

VAN D&~t ME, WtmNSCHEU. GE, ARNOLD FIt: Metal AtFmity Precipitation of Proteins. BiotedDnol Appl Biocbem 1989, 11:492-502. A careful investigation of metal (Cu z + ) affinity precipitation of proteins. Several factors influencing the yield are discussed. Examples are the number of histidine residues located on the surface, the protonation of histidine residues, the character of the complexing agent (free ion, chelated ion or chelated pobmer-bound ion) and the concentration of reactants. A simple mathematical model is proposed. The experimental data relate to human hemoglobin and sperm-whale myoglobin. 7. •

HUDDLESTONJG, LYDDIATI"A: Aqueous Two-Phase Systems in Biochemical Recovery. Appl Biocbem BiotedJnol 1990, 26:249--279. A general overview of aqueous t-~o-phase sTstems , including scale-up considerations. Comments are made on the real limitations of the technique and on the misconceptions associated with it. 8. ••

HUSTEDTH, JOHANSSON G, TJER,NELD F (EDS): Aqueous 'IM,'oPhase Separation Systems. In Bioseparation (special issue) 1990, 1:177-324. The December issue is an excellent and very up-to-date over,Sew of preparath-e aqueous tv'o-phase separation. Ten individual contributions from experts in the field cover important aspects such as theoretical background, choice of pobTners, economy and enviromental aspects, as well as many applications. 9.

STURESSONS, TJEIZ\'ELDF, JOH.ANSSONG: Partition of Macromolecules and Cell Particles in Aqueous Two-Phase Systems Based on ttydroxypropyl Starch and Poly(Ethylene Glycol). Appl Biodoem Biotecbnol 1990, 26:281L295.

10. GREVEA, KULAM-R: RecTcling of Salts in Partition Protein • Extraction Processes. J Cbem Tech Biotedmo11991, 50:27-42. Several ~ of recD~clinga pob,-(ethylene gb'col)-potassium phosphate system were im'estigated. The sD~tems were used for the fractionation of Saccbarom)w.es ceret,isiae and Bacillus ceretm homogenates. Under certain conditions, 95% of the salt could be recycled in an economic way by counter-current extraction v-ith aliphatic alcohols. 11. •

RAt KS~LSP,,STEWARTP,M)TODD P: Electrokinetic Demixing of Two-Phase Aqueous Pol~,aner Systems. I. Separation Rates of Polyethylene-Glycol-Dextran Mixtures. Sep Sci Tedmol 1989, 25:985-995. By appb-ing an electric field of the appropriate direction and strength (around 15 Vcm - 1 ) over a ~'o-phase mixture, the demixing time could be decreased by a factor of about three. 12. •

FLYGARES, \VIKSTROMP, JOHANSSONG, I.AP,SSON P-O: Magnetic Aqueous Two-Phase Separation in Preparative Applications. EnzD~ne Microb Tecbnol 1990, 12:95-103. Several tv'o-phase sD~tems were made magnetically susceptible by the addition of micron-sized iron oxide particles. The iron oxide did not adversely affect the extracted en~anes. In the presence of a magnetic field, the phases separated 5-240 000 times faster than reference sD"sterns. The magnetic technique should be particularly useful in high viscosity sD~tems and in sD"stemswhere the phases have similar densities.

13.

UNARSKAM, DAVIESPA, ESXOUFMP, BELLHOUSEBJ: Comparative Study of Reaction Kinetics in Membrane and Agarose Bead Affinity Systems. J GDrornatogr 1990, 519:53-67.

14.

TEh~'IKOVATB, BELENmIBG, SVECF: High-Performance Membrane Chromatography. A Novel Method of Protein Separation. J Liquid C13romatogr 1990, 13:63--70.

15. •

I.t'qG TGI, M_~Trl.J,SSON B: Membrane Filtration Affinity Purification (MFAP) of Dehydrogenases using Cibacron Blue. Biotecbnol Bioeng 1989, 34:1321-1325. Cross-flow ultrafiltration units were combined with silica nanoparticles (10-20 run) coated v-ida immobilized cibacron blue. Enz)rnes adsorbed by the particles were retained by the membrane. The rapid equilibration possible v'ida the small afiqnity particles gives the technique high operational capacity (e.g. 2 g h - 1 alcohol dehydrogenase from a yeast homogenate using 1.5 m 2 membrane units). 16.

CAPA.\~'ELUG, BoTrLNo A, GEKASV, TRAGARDItG: Protein Fouling Behaviour of UItrafiltration Membranes Prepared with Varying Degrees of Hydrophilicity. Process Biocl:em Int 1990, 25:221-224.

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JOH.~.NSSONHJ, P E T I E ~ N TN, BERGLOFJH: Development of a Chromatographic Process for Large-Scale Purification of Staphylococcal Enterotoxin B. J Cbem TedJ BiotedJnol 1990, 49:233-241. Staphylococcal enterotoxin B was purified in a large-scale chromatographic process in ~ ' o ion-exchange steps and a final gel filtration step. Starting with 2000 litres of crude extract, the protein toxin v'as subject to a 25 000-fold purification with an impressive total recovery of 74 %. 18. to

AFEY&'qNB, GORDON NF, MAZSAROFF I, VARADY lo FULTON SP, YANG YB, REGNIER FE: Flow-Through Particles for the High-Performance Liquid Chromatographic Separation of Biomolecules: Perfusinn Chromatography. J Cbromatogr 1990, 519:1-29. A new matrix with very interesting so-called 'darough-pores' is described. The mobile phase viii flow through these large pores to enter the interior of the particles. The surface area is enhanced by using a network of smaller, inter-connecting pores between the through-pores. Bandspreading, resolution and binding capacity are unaffected by mobile phase velocities up to several thousands of cm per hour. The theoretical background is given, and basic performance studies as well as several applications are described. 19.

ArE'CANNB, FULTON SP, GORDON NF, M.~tROFF I, VARADY in REGNIERFE: Perfusion Chromatography. An Approach to Purifying Biomolecules. BiotedJnology 1990, 8:203-206. The properties and the benefits of the flow-through particles in perfusion chromatography applications, such as high-speed anab~is, on-line monitoring, rapid method development and dilute feed (large volumes) adsorption are discussed (see also [18••]). ,,

20.

CZOKM, GUIOCHON G: Aligned Fiber Columns for Size-Exclusion Chromatography. J Ctjromatogr 1990, 506:303-317.

21.

KAGEDALL, ENGSTROMB, ELLEGRENH, LIEBERA-K, Lt~'DSTROM H, SKOLD A, SCPLEN~'LNG/~l: Chemical, Physical and Chromatographic Properties of Superdex 75 Prep Grade and Superdex 200 Prep Grade Gel Filtration Media. J Cbromatogr 1991, 537:17-32.

22.

GLADM, SCHORNACKS: Composite High Flow Rate Ion Exchangers for Economical Scale-up. In Production of Biolog~ icals from Animal Cells hz Culture edited by Spier RE, Griffiths JB, Meignier B [book]. London: Butterworths 1991 pp 561-565.

23.

la J, ERIKSSON K-O, HJERT~N S: High-Performance Liquid Chromatography of Proteins on Deformed Nonporous Agarose Beads. Affinity Chromatrography of Dehydrogenases on Cibaeron Blue-Derivatized Agarose. Prep Biocbem 1990, 20:107-121.

24. ScOtrlENWH: AtFmity Chromatography for Protein Isolation. . Cttrr Opin BiotedJnol 1991, 2:37-43. Recent developments in affinity chromatography for protein isolation are discussed.

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OHLSONS, HANSSON L, GLAD M, MOSBACH K, IAKS.5ON P-O: High Performance Liquid Affinity Chromatography: A New Tool in Biotechnology. Trends Biotecbnol 1989, 7:179-186.

26.

TAKEYAA, HOSOMI O, ISHIURA M: Complete Purification and Characterization of cx-3-N-Ace~lgalactosaminyltratmferase Encoded by the Human Blood Group A Gene. J Biochem 1990, 107:360-368.

27. •

DESAI MA: I m m u n o ~ t y Adsorption: Process Scale Isolation of Therapeutic-Grade Biochemicals. J Chem Tech Biotecbnol 1990, 48:105-126. A review covering imnunoaffmity purification and its application to process-scale purification of high ~'alue, therapeutic-grade biochemicals from complex mixtures. Solid-phase requirements, methods of activation, antigen coupling, human IgG-coupling, the application of bioselective methods on the early stages in a purification process, and the stringent regulatory requirements are discussed.

The binding of b~soz3rne to an immobilized fragment of an antibody w~as demonstrated. The fragment consisted of a 13-residue s3aathetic peptide analogous to the hyper~ariable segment of a monoclonal antibody directed toveards b~ozyme. 34. KAUVARLM, CHEUNG PYK, GOMER RH, FLEISCHERAA: Paralog • Chromatography. Biotecbniques 1990, 8:204-209. A small peptide can mimic a part of the hyper~riable region of an antibody and function as a mixed-mode ligand. This ~as exemplified by the separation of a complex mixture of proteins in a yeast extract. 35.

~ N S, PORAIH J: Protein Chromatography with P~Tidine- and Alkyl-Thioether-Bascd Agarose Adsorbents. J Cbromatogr 1990, 499:235-247.

36.

NOPPERB, KOHEN F, WItCHEK M: A ThiophUic Adsorbent for the One-Step High-Performance Liquid Chromatography Purification of Monoclonal Antibodies. Anal Biochem 1989, 180:66-71.

VELANDER"~-I, ORTtLN'ER CL~ TtLMRAKANJP, MADURAWE RD, RAISTONAH, STR1CKIAND DK, DROHAN WN: Process Impli. cations for Metal-Dependent ImmunoatTmity Interactions. Biotecbnol Prog 1989, 5:119-125. Immunosorbents with immobilized, specially selected, monoclonal antibodies sho~ing metal-dependent binding were used for the purification of human plasma protein C and Factor IX. Gentle elution could be achieved by changing the metal ion concentration in the eluent_

37. •

29. ZOPF D, OHLSON S: Weak-Affmity Chromatography'. Nature • 1990, 346:87-88. Describes a new approach in affinity chromatography using rexersible, weak biospecific interactions that allow separations under |socratic conditions. Its implications for anabxical and preparative separations are described.

39.

28. •

30. ARNOLDFH: Metal-Affinity Separations: A New Dimension in • Protein Processing. Biotecbnolog), 1991, 9:151-156. A re~iew on recent progress in metal-affinity chromatography primarily aimed at understanding the mechanism of metal-protein recognition. Integration of the expression and bioprocessing of recombinant proteins is also discussed. 31.

BEI.EWM, PORATtl J: ImmobiliTed Metal Ion A/Fruity Chromatography. Effect of Solute Structure, Ligand Density and Salt Concentration on the Retention of Peptides. J Cbromatogr 1990, 516:333-354.

32. •

BURTONSJ, STEADCV, LOWE CR: Design and Applications of Biota|tactic Anthraquinone Dyes. IlL Anthraquinone-Immobilised C.I Reactive Blue 2 Analogues and Their Interaction with Horse Liver Alcohol Dehydrogenase and Other Adenine Nucleotide-Binding Proteins. J Cbromatogr 1990, 508:109-125. It was shown by chromatography of adenine nucleotide-reqniring enzTmes that the new analogues of cibacron blue, which were immobilized via the anthraquinone ring, gave more efficient purification than earlier dem~a~'es, vdaich were immobilized via the triazine ring. 33. ••

WELL~G GW, GEURTS T, VAN GORKUM J, DAMHOF RA, DmJFHOtrr. ~ , BLOE~,~IOFFW, WELHNG-WESTERS: Synthetic Antibody Fragment as Ligand in ImmunoatFmity Chromatography. J Cbromatogr 1990, 512:337-343.

NGO "IF, Krt~TrrR N: Chemistry and Preparation of Afireity Ligands Useful in ImmunoglobuUn Isolation and Serum Protein Separation. J Chromatogr 1990, 510:281-290. Synthetic affinity ligands showing alTmity for immunoglobulins and albumin were prepared using ~'-ious p~aidine d e r i ~ ' e s .

38.

EKBERGB, MOSBACH K: Molecular Imprinting: A Technique for Producing Specific Separation Materials. Trends Biotecl:~ nol 1989, 7:92-96. OSTRE.~tJA, VANOOSBREETI~ MARQUEZR, BARSTOWL: Purification of Sl Nuclease from Aspergillus oryzae by RecTcling Isoelectric Focusing. Electropboresis 1990, 11:953-957.

40.

NATHS, SCHfYITEH, WEBER G, HUSTEDT 11, DECKWERW-D: Separation of Enz)anes from Candida botdinii Crude Extract by Continuous Flow Zone Electrophoresis. Electrophoresis 1990, 11:937-941. 41. WALtACEGG, MAXWELLKE, LEWISTW, HODGSON AJ, SPENCER • MJ: New Conducting Polymer AtTmity Chromatography Stationary Phases. J Liquid Cbromatogr 1990, 13:3091-3110. A conducting, electrochemically ac~'e support (carbon fibers coated with polypyrrol) was packed in a column fitted with electrodes. HSA was adsorbed on the column. It w~ts shov,,n that the subsequent adsorption and desorption of anti-HSA could be controlled to a cemain extent by • the applied potential. 42.

Ol.SOy C'W, LEUNG SK, YARMUSHMIA Recovery of Antigens from Immunoadsorbents Using High Pressure. Biotechnology 1989, 7:369-373. 43. VtsuPdK, KAIPALN'ENE, KrvL~t,~J, NIXr~uH, LEtSOLAM, PALOSAAm • S: A New Method for Protein Co'stallization Using High Pressure. Biotechnology 1990, 8:547-549. High pressure (200 MPa) dramatically accelerated crystallization of glucose isomerase. The onset of cr~ralliT~ation from a supersaturated solution began within 2minutes and the yield of crystals (microcrystals) ~as much higher than at 0.1 MPa. Negligable inactivation occurred.

M Glad and P-O Larsson, Pure and Applied Biochemistry, Chemical Center, Ltmd University, PO Box 124, S-221 IX),Lurid, Sweden.