Journal of Chromatography B (v.790, #1-2)

Continuous annular chromatography by Frank Hilbrig; Ruth Freitag (1-15).
The principle of continuous annular chromatography (CAC) has been known for several decades. CAC is a continuous chromatographic mode, which lends itself to the separation of multi-component mixtures as well as of bi-component ones. In CAC, the mobile and stationary phases move in a crosscurrent fashion, which allows transformation of the typical one-dimensional batch column separation into a continuous two-dimensional one. With the exception of linear gradient elution, all chromatographic modes have at present been applied in CAC. This review focuses on the capacity of CAC for preparative bioseparation. The historical developments and the predecessors of modern CAC are briefly summarized. The state-of-the-art in the theoretical prediction and simulation of CAC separations is discussed, followed by an overview of current CAC instrumentation and example applications, especially for the isolation of proteins and other bio(macro)molecules. In this context, issues of scale up as well as method development and transfer from batch to continuous CAC columns are discussed using recent bioseparation efforts as pertinent examples.
Keywords: Proteins; DNA;

This review describes the performance of various column designs available to process-scale users of low-pressure chromatography for protein purification. By carrying out a range of ion-exchange separations using Whatman microgranular ion-exchange celluloses we are able to compare and contrast the practical performance issues associated with several designs of axial and radial flow columns.
Keywords: Proteins;

Comparison of protein A affinity sorbents by Rainer Hahn; Robert Schlegel; Alois Jungbauer (35-51).
Protein A is a popular generic ligand for purification of monoclonal and recombinant antibodies. The performance of 15 commercially available protein A media was studied. Equilibrium and dynamic binding capacity for human IgG was determined and the capture of IgG from a crude feed-stock was investigated. For initial screening the dynamic binding capacity was determined at small scale. Media with good performance were further tested with increased column height. Comparing the data from the two different column heights it could be shown that the dynamic capacity strongly depends on the residence time. Agarose based media exhibited higher binding capacity at residence times longer than 3 min whereas polymeric media or media based on porous glass showed a lesser dependence on the flow velocity and the residence time. A quantitative description of this behavior was derived by determination of the adsorption isotherms and fitting the breakthrough profiles with the Thomas solution. Agarose based media exhibited higher maximum equilibrium binding capacities and the dissociation constants derived from adsorption isotherms were smaller. The other media exhibited higher apparent rate constants, indicating a faster mass transfer. This can be explained by the smaller particle diameter of these media and it can be assumed that constant pattern conditions are thereby obtained more quickly. Selectivity was tested by performing antibody purification under standardized conditions. Polyclonal human IgG in cell culture supernatant containing 2.5% fetal calf serum was used as a representative feed-stock. Under the applied conditions several sorbents showed very tight binding of IgG and in some cases most of the sample remained on the sorbent. The study can be useful as a guide for optimization of large-scale purification processes.
Keywords: Protein A; Proteins;

Isolation of antigens and antibodies by affinity chromatography by Vladimir I. Muronetz; Timo Korpela (53-66).
Antibody–antigen binding constants are commonly strong enough for an effective affinity purification of antibodies (by immobilized antigens) or antigens (by immobilized antibodies) to work out a straightforward purification method. A drawback is that antibodies are large protein molecules and subject to denaturation under conditions required for the elution from the complex. Structures of antigens can vary but usually antigens are also equally subject to similar problems. The lability of the components can sometimes make the procedure sophisticated, but usually in all cases it is possible to find a satisfactory approach. In certain cases, specific interactions of the Fc part of antibodies are more facile to exploit for their purification.
Keywords: Antibodies; Antigens;

Affinity chromatography is potentially the most selective method for protein purification. The technique has the purification power to eliminate steps, increase yields and thereby improve process economics. However, it suffers from problems regarding ligand stability and cost. Some of the most recent advances in this area have explored the power of rational and combinatorial approaches for designing highly selective and stable synthetic affinity ligands. Rational molecular design techniques, which are based on the ability to combine knowledge of protein structures with defined chemical synthesis and advanced computational tools, have made rational ligand design feasible and faster. Combinatorial approaches based on peptide and nucleic acid libraries have permitted the rapid synthesis of new synthetic affinity ligands of potential use in affinity chromatography. The versatility of these approaches suggests that, in the near future, they will become the dominant methods for designing and selection of novel affinity ligands with scale-up potential.
Keywords: Proteins; Peptides;

Protein purification by affinity precipitation by Frank Hilbrig; Ruth Freitag (79-90).
Developing the most efficient strategy for the purification of a (recombinant) protein especially at large scale remains a challenge. A typical problem of the downstream process of mammalian cell products is, for instance, the early capture of the highly diluted product from the complex process stream. Affinity precipitation has been suggested in this context. The technique is known for over 20 years, but has recently received more attention due to the development of new materials for its implementation, but also because it seems ideally suited to specific product capture at large scale. The present review gives a comprehensive overview over this technique. Besides an introduction to the basic principle and a brief summary of the historical development, the main focus is on the current state-of-art of the technique, the available materials, important recent applications, as well as process design strategies and operating procedures. Special consideration is given to affinity precipitation for product recovery at large scale.
Keywords: Proteins;

Role of mass spectrometry in the purification of peptides and proteins by Cecilia B. Mazza; Jie Y. Cavanaugh; Uwe D. Neue; Dorothy J. Phillips (91-97).
Experiments were carried out to evaluate the fractionation of proteins and peptides according to mass. Model mixtures were separated by either reversed-phase or ion-exchange chromatography with mass spectrometry-compatible mobile phase additives. Fraction collection was triggered by the mass/charge ratio of each one of the components of the mixture. Chromatography was additionally monitored with a UV–Vis detector in order to compare the new technique with generally accepted in separations. The results indicated that adequate purification is achieved by this new technique. Fraction collection triggered by changes in the mass/charge ratio reduces sample handling and analysis time. This study demonstrates the utility of mass-directed fractionation of peptides and proteins when mass spectrometry-compatible mobile phase additives are used.
Keywords: Peptides; Proteins;

Hydrophobic interaction chromatography of proteins by Rainer Hahn; Karin Deinhofer; Christine Machold; Alois Jungbauer (99-114).
Hydrophobic interaction chromatography media suited for large scale separations were compared regarding dynamic binding capacity, recovery and mass transfer properties. In all cases, pore diffusion was the rate limiting step. Reduced heights equivalent to a theoretical plate for bovine serum albumin derived from breakthrough curves at reduced velocities between 60 and 1500 ranged from 10 to 700. Pore diffusion coefficients were derived from pulse response experiments for the model proteins α-lactalbumin, lysozyme, β-lactoglobulin, bovine serum albumin and immunoglobulin G. Diffusivity of lysozyme did not follow the trend of decreasing diffusivity with increasing molecular mass, as observed for the rest of the proteins. In general, mass transfer coefficients were smaller compared to ion-exchange chromatography. Dynamic binding capacities for the model protein bovine serum albumin varied within a broad range. However, sorbents based on polymethacrylate showed a lower dynamic capacity than media based on Sepharose. Some sorbents could be clustered regarding binding capacity affected by salt. These sorbents exhibited a disproportional increase of binding capacity with increasing ammonium sulfate concentration. Recovery of proteins above 75% could be observed for all sorbents. Several sorbents showed a recovery close to 100%.
Keywords: Proteins; Lactalbumins; Albumin; Lysozyme; Immunoglobulins;

The adsorption of different proteins in a single biospecific and hydrophobic adsorbent particle for preparative protein chromatography has been observed directly by confocal laser scanning microscopy as a function of time at a constant bulk concentration c b. The bulk concentration was in the non-linear part of the adsorption isotherm. At all times the concentration of free protein at the particle surface was almost equal to the bulk content indicating that external mass transfer resistance is not rate limiting for the adsorption under these conditions. Inside the particles a distinct maximum in adsorbed and free protein concentration that moved inside to a distance of ≈0.2 R (R particle radius) from the particle surface, was observed. This is due to a decreasing solid-phase density and adsorptive capacity in the particle between 0.8 R and R indicating that the fraction of macropores (or void space) is larger in the outer than in the inner part of the adsorbent particles. By increasing the bulk concentration by a factor of 10 the equilibration time was reduced by about the same magnitude. This is in agreement with the concentration dependence of the effective pore diffusion coefficient D p,eff=D p/{ϵ p[1+nK/(K +c)2]} derived from the mass conservation relations describing the adsorption process. The time dependence protein adsorption up to ≈90% of the equilibration value q* could be described by a bilinear free driving force model. The rapid equilibration in the outer part of the particle with a half-life time of ≈100 s in the studied systems accounted for 0.3–0.4 q*. The slower equilibration with a up to ten times longer half-life time, was the adsorption in the inner part of the particle that outside 0.5 R accounts for 0.5–0.6 q*. These data were compared with literature data for batch adsorption of proteins in biospecific, hydrophobic and ion-exchange adsorbents. They could also be described by a bilinear free driving force model, with about the same quantitative results as obtained for similar conditions in the single particle experiments. The static adsorption parameters, maximum binding site concentration n, and dissociation constant for the protein binding to a binding site K, were determined from Scatchard plots. For the same protein–adsorbent system the plots changed from linear to non-linear with increasing n. This change occurred when the average distance between adjacent binding sites become of the same order of magnitude as the size of the binding site or adsorbed protein. This causes a shielding of free binding sites increasing with n and the concentration of adsorbed protein, yielding a concentration dependence in K. These results show that for a high throughput and rapid adsorption in preparative chromatography, the adsorption step should be carried out in the non-linear part of the adsorption isotherm with concentrations up to c b where q*/c b≥10 to obtain high protein recoveries. To avoid tailing due to the flow of adsorbed proteins in the inner part of the particles further into the particles at the start of the desorption, and to speed up desorption rates, protein adsorption in the particle within 0.5 R from the particle center should be avoided. This requires the further development of suitable pellicular particles for preparative protein chromatography that meet this requirement.
Keywords: Proteins;

The major limitations associated with conventional packed bed chromatography for protein separation and purification can be overcome by using adsorptive microporous membranes as chromatographic media. Microporous membranes have advantages as support matrices in comparison to conventional bead supports because they are not compressible and they eliminate diffusion limitations. As a result, higher throughput and shorter processing times are possible using these membrane systems. In this paper, we review the current state of development in the area of attaching functionalized polymer brushes onto a microporous membrane to form a novel chromatographic medium for protein separation and purification. The functionalized polymer brushes were appended onto the pore surface of a microporous hollow-fiber membrane uniformly across the membrane thickness by radiation-induced graft polymerization and subsequent chemical modifications. We review various applications of this adsorptive membrane chromatography by focusing on polymer brushes bearing ion-exchange, hydrophobic and affinity groups. Proteins were captured in multilayers by the ion-exchange group-containing polymer brushes due to the formation of a three-dimensional space for protein binding via the electrostatic repulsion of the polymer brushes. In contrast, proteins were captured in a monolayer at most by the polymer brushes containing hydrophobic or affinity ligands. By permeating a protein solution through the pores rimmed by the polymer brushes, an ideal capturing rate of the proteins with a negligible diffusional mass-transfer resistance was achieved by the functionalized polymer brushes, based on ion-exchange, hydrophobic, and affinity interactions.
Keywords: Proteins;

The primary objective of work was to characterize, optimize and model a chromatographic process based on ethylenediamine-N,N,N′,N′-tetra(methylphosphonic) acid (EDTPA)-modified zirconia particles. Zirconia particles were produced by spray-drying colloidal zirconia. Zirconia spheres produced were further classified, calcined and modified with EDTPA to yield a solid-phase support for use in bio-chromatography (r_PEZ). Specifically, the ability of r_PEZ to selectively bind and enrich IgG, IgA, and IgM from biological fluids was evaluated and demonstrated. To better understand the force of interaction between the IgG and the r_PEZ, the equilibrium disassociation constant (K d) was determined by static binding isotherms, as a function of temperature and by frontal analysis at different linear velocities. The maximum static binding capacity (Q max) was found to be in the range 55–65 mg IgG per ml of beads, and unaffected by temperature. The maximum dynamic binding capacity (Q x ) was found to be in the range 20–12 mg IgG per ml of beads. The adsorption rate constant (k a) was determined by a split-peak approach to be between 982 and 3242 l mol−1 s−1 depending on the linear velocity. The standard enthalpy and entropy values were estimated for this interaction of IgG with this novel support.
Keywords: Antibodies;

The aim of this work was to test a recycling method for imidazole used in immobilized metal affinity chromatography (IMAC) as eluent for recombinant histidine-tag (His-tag) protein. After evaluating two supports, the method was optimized with a mixture of bovine serum albumin, sodium chloride and imidazole. Recycling was performed with an eluate fraction from IMAC of His-tag enhanced green fluorescent protein produced in our laboratory and pure imidazole was recovered in water and was analyzed after being freeze-dried. The imidazole was then reused as eluent in IMAC without any modification in its structure or behavior. This procedure can be used for large-scale chromatography.
Keywords: Imidazole; Proteins; Enhanced green fluorescent protein;

Whey proteins as a model system for chromatographic separation of proteins by Linda Pedersen; Jørgen Mollerup; Ernst Hansen; Alois Jungbauer (161-173).
Although chromatographic separation of whey proteins has been considered too expensive, whey may serve as an excellent model mixture to investigate and validate the use of simulation tools in the development and optimization of chromatographic separations and the outcome could easily be utilized since the model system has an intrinsic value. Besides, milk from transgenic animals could be an attractive source of pharmaceuticals which must be separated from the other proteins in the milk. Several whey proteins are of interest especially, α-lactalbumin, β-lactoglobulins, immunoglobulins, lactoperoxidase, and lactoferrin. The scope of the project is to develop a consistent set of chromatographic data for whey proteins including isotherms, transport properties and scale-up studies and to develop the appropriate models for the anion exchangers Q-Sepharose XL, Source 30Q, Ceramic Q-HyperD F, and Merck Fractogel EMD TMAE 650 (S). In this work we have determined and correlated gradient and isocratic retention volumes in the linear range of the isotherm for α-lactalbumin, β-lactoglobulin A and B, and bovine serum albumin at a pH from 6 to 9 at various NaCl concentrations.
Keywords: Proteins; Whey proteins; Lactalbumin; Lactoglobulin; Albumin;

Application of semi-industrial monolithic columns for downstream processing of clotting factor IX by K. Branović; A. Buchacher; M. Barut; A. Strancar; Djuro Josic (175-182).
It has been shown in a previous study that monolithic columns can be used for downstream processing of different concentrates of clotting factor IX [K. Branović et al., J. Chromatogr. A 903 (2000) 21]. This paper demonstrates that such supports are useful tools also at an early stage of the purification process of factor IX from human plasma. Starting with the eluate after solid-phase extraction with DEAE-Sephadex, the use of monolithic columns has allowed much better purification than that achieved with conventional anion-exchange supports. The period of time required for separation is also much reduced. In up-scaling experiments, separations are carried out with 8, 80 and 500 ml columns. A volume of 1830 ml of DEAE-Sephadex eluate, containing a total of 27.6 g of protein and 48 500 IU of factor IX is applied to the 500 ml monolithic column. This corresponds to a separation on a pilot scale. The results of this separation after up-scaling are comparable to those obtained with the 8 ml column on a laboratory scale.
Keywords: Factor IX; Vitronectin;

A review is given of preparative methods for the isolation of the vitamin K-dependent clotting factors II, VII, IX, X and clotting inhibitor protein C, all derived from human plasma. Factor II, activated factor VII and activated protein C are also obtained from recombinant animal cells. The methods for their purification are described. The problem of difference in posttranslational modifications between plasma derived and recombinant protein is discussed with regard to therapeutic proteins.
Keywords: Proteins; Protein C; Thrombin; Protein S; Factor II; Factor VII; Factor IX; Factor X;

Chromatographic purification and properties of a therapeutic human protein C concentrate by M. Radosevich; F.-L. Zhou; J.-J. Huart; T. Burnouf (199-207).
Protein C deficiency (inherited and acquired) has a relatively high incidence rate in the general population worldwide. For many years, protein C deficient patients have been treated with fresh frozen plasma, prothrombin complex concentrates, heparin or oral anticoagulants, which all have clinical drawbacks. We report the production process of a highly purified human protein C concentrate from 1500 l of cryo-poor plasma by a four-step chromatographic procedure. After DEAE-Sephadex adsorption, protein C was separated from clotting factors II, VII and IX by DEAE-Sepharose FF and further purified, using a new strategy, by an on-line chromatographic system combining DMAE-Fractogel and heparin-Sepharose CL-6B. In addition, the product was treated against viral risks by solvent-detergent and nanofiltration on 15-nm membranes. The protein C concentrate was essentially free of other vitamin K-dependent proteins. Proteolytic activity was undetectable. Neither activated protein C, prekallikrein activator, nor activated vitamin K-dependent clotting factors were found resulting in good stability of the protein C activity. In vitro and in vivo animal tests did not reveal any sign of potential thrombogenicity. The final freeze-dried product had a mean protein C concentration of 58 IU/ml and a mean specific activity of 215 IU/mg protein, corresponding to over 12 000-fold purification from plasma. Therefore, this concentrate appears to be of potential benefit for the treatment of protein C deficiency.
Keywords: Protein C; Proteins; Factor II; Factor VII; Factor IX;

Purification of human plasma haptoglobin by hemoglobin-affinity column chromatography by Chun Yi Liau; Tsai Mu Chang; Ju Pin Pan; Wen Liang Chen; Simon J.T. Mao (209-216).
Haptoglobin (Hp) is an acute-phase protein; its plasma levels increase consistently in response to infection and inflammation. The concentration of human plasma Hp is ranged between 1 and 1.5 mg/ml. Similar to blood type, individual human Hp is classified as Hp 1-1, 2-1, or 2-2. The structural and functional analysis of the Hp, however, has not been studied in detail due to its difficult isolation procedure. Previously, we reported a single step for the purification of porcine Hp. In this study, we established a purification method using a high capacity hemoglobin-affinity column. Briefly, DEAE-purified human hemoglobin was first coupled to Sepharose 4B to prepare an affinity column in a 15-ml bed volume. Following a flow through of human plasma and an extensive wash, the bound material was eluted with a solution of 0.15 M NaCl, pH 11 (adjusted by ammonium), to remove low-affinity bound proteins. The high-affinity bound Hp was then eluted with 0.15 M NaCl containing 5 M urea, pH 11, and collected in tubes containing 100 μl of 1 M Tris buffer, pH 7.0. The biological activity of dialyzed Hp was retained as it formed a complex with hemoglobin on a sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). Using this procedure, approximately 10 mg of Hp 1-1, with homogeneity greater than 96%, was obtained from 15 ml of human plasma. Affinity purified Hp 2-1 or 2-2, however, contained trace amounts of apoA-I with the similar approach. The Hp could be further purified by HPLC using a Superose 12 gel-permeation chromatography, if desired, to achieve 100% purity. All the phenotypes of purified Hp consisted of α and β chains on SDS–PAGE in the presence of a reducing reagent, further confirmed by a Western blot analysis. We conclude that human hemoglobin-affinity column was most suitable for the isolation of Hp 1-1 in large quantities. Whereas, one additional step using a gel-permeation was necessary for that of Hp 2-1 and 2-2.
Keywords: Haptoglobin; Proteins; Glycoproteins;

Purification of an oxidation-sensitive enzyme, pI258 arsenate reductase from Staphylococcus aureus by Joris Messens; José C. Martins; Ingrid Zegers; Karolien Van Belle; Elke Brosens; Lode Wyns (217-227).
Arsenate reductase (ArsC) from Staphylococcus aureus pI258 is extremely sensitive to oxidative inactivation. The presence of oxidized ArsC forms was not that critical for NMR, but kinetics and crystallization required an extra reversed-phase purification to increase sample homogeneity. The salt ions observed in the X-ray electron density of ArsC were investigated. Carbonate was found to have the lowest dissociation constant for activation (K a=1.1 mM) and potassium was stabilizing ArsC (ΔT m=+6.2 °C). Also due to the use of these salt ions, the final yield of the purification had improved with a factor of four, i.e. 73 mg/l culture.
Keywords: Arsenate reductase; Enzymes;

Fractionation and purification of the enzymes stored in the latex of Carica papaya by Mohamed Azarkan; Anouar El Moussaoui; Delphine van Wuytswinkel; Géraldine Dehon; Yvan Looze (229-238).
The latex of the tropical species Carica papaya is well known for being a rich source of the four cysteine endopeptidases papain, chymopapain, glycyl endopeptidase and caricain. Altogether, these enzymes are present in the laticifers at a concentration higher than 1 mM. The proteinases are synthesized as inactive precursors that convert into mature enzymes within 2 min after wounding the plant when the latex is abruptly expelled. Papaya latex also contains other enzymes as minor constituents. Several of these enzymes namely a class-II and a class-III chitinase, an inhibitor of serine proteinases and a glutaminyl cyclotransferase have already been purified up to apparent homogeneity and characterized. The presence of a β-1,3-glucanase and of a cystatin is also suspected but they have not yet been isolated. Purification of these papaya enzymes calls on the use of ion-exchange supports (such as SP-Sepharose Fast Flow) and hydrophobic supports [such as Fractogel TSK Butyl 650(M), Fractogel EMD Propyl 650(S) or Thiophilic gels]. The use of covalent or affinity gels is recommended to provide preparations of cysteine endopeptidases with a high free thiol content (ideally 1 mol of essential free thiol function per mol of enzyme). The selective grafting of activated methoxypoly(ethylene glycol) chains (with M r of 5000) on the free thiol functions of the proteinases provides an interesting alternative to the use of covalent and affinity chromatographies especially in the case of enzymes such as chymopapain that contains, in its native state, two thiol functions.
Keywords: Enzymes; Papain; Lysozyme; Enzyme inhibitors;

An overview of the purification of an oligomeric enzyme, an extramitochondrial acetyl-coenzyme A hydrolase from rat liver, is presented. The enzyme has been purified to homogeneity using two successive size-exclusion chromatography runs, first for the monomeric and second for the oligomeric form of the enzyme. The sequential gel-filtration steps efficiently removed the contaminants of any molecular size, first of different size from that of the monomeric form of the enzyme (K av=0.47 on Superdex 200) and second of different size from that of the oligomeric form (K av=0.33), allowing us to purify the enzyme in high purity. This strategy provides an excellent model for purifying many other oligomeric proteins including key enzymes or allosteric enzymes regulating metabolism.
Keywords: Oligomeric enzymes; Cystolic acetyl-coenzyme A hydrolase;

Preparative procedures and purity assessment of collagen proteins by Z. Deyl; I. Mikšı́k; A. Eckhardt (245-275).
Collagens represent a large family (25 members identified so far) of closely related proteins. While the preparative procedures for the members that are ubiquitous and present in tissues in large quantities (typically fibre and network forming collagens types I, II, III, IV and V) are well established, the procedures for more recently discovered minor collagen types, namely those possessing large non-collagenous domain(s) in their molecule, are mostly micropreparative and for some collagenous proteins even do not exist. The reason is that the proof of their existence is based on immunochemical staining of tissue slices and nucleic database searching. Methods of preparation and identification of constituting α-polypeptide chains as well as collagenous and non-collagenous domains are also reviewed. Methods for revealing non-enzymatic posttranslational modifications (particularly of the fibre forming collagen types) are briefly described as well.
Keywords: Collagens; Proteins;

Simple and rapid chromatographic purification of type V collagen from a pepsin digest of porcine intestinal connective tissue, an unmanageable starting material for conventional column chromatography by Kenji Sato; Tamae Tanahashi-Shiina; Feng Jun; Atsuko Watanabe-Kawamura; Masami Ichinomiya; Yutaka Minegishi; Yasuyuki Tsukamasa; Yasushi Nakamura; Makoto Kawabata; Kozo Ohtsuki (277-283).
A chromatographic method is described for purification of type V collagen, a minor constituent in extracellular matrix, from a pepsin digest of porcine intestinal connective tissue. The starting material was a viscous and turbid solution even after centrifugation. Direct application of the sample to a commercially available DEAE-cellulose column resulted in clogging. On the other hand, type V collagen, [α1(V)]2α2(V) form, was successfully captured by a filter paper-based DEAE-cellulose column chromatography and purified by a subsequent commercially available cation-exchange medium without clogging. This is a vast improvement over previously described salt fractionation methods.
Keywords: Collagens; Proteins; Cellulose;

Synthesis and chromatographic purification of recombinant human pituitary hormones by Maria Teresa C.P. Ribela; Peter W. Gout; Paolo Bartolini (285-316).
Recombinant DNA-derived proteins and, in particular, human pituitary hormones, are increasingly used for research, diagnostic and therapeutic purposes. This trend has demanded new synthetic approaches and improved purification techniques. The type and sequence of the purification steps have to be selected in accordance with the cloning and protein expression strategy, the host organism and cellular localization of the protein of interest, with a view to producing the desired product at a required purity, biological activity and acceptable cost. This review article describes and analyzes the main synthetic and purification strategies that have been used for the production of recombinant human growth hormone, prolactin, thyrotropin, luteinizing hormone and follicle-stimulating hormone, giving special consideration to the few published downstream processes utilized by the biotechnology industry. Practically all types of prokaryotic and eukaryotic organisms utilized for this purpose are also reviewed.
Keywords: Hormones; Polypeptides; Peptides; Growth hormone; Prolactin; Thyrotropin; Luteinizing hormone; Follicle stimulating hormone;

Preparative purification of B-phycoerythrin from the microalga Porphyridium cruentum by expanded-bed adsorption chromatography by Ruperto Bermejo; F. Gabriel Acién; Mª.José Ibáñez; José M. Fernández; Emilio Molina; José M. Alvarez-Pez (317-325).
B-Phycoerythrin (B-PE) is a major light-harvesting pigment of microalgae. Due to its high fluorescence efficiency and its intense and unique pink color, it is widely used as a fluorescent probe and analytical reagent as well as being employed as a natural dye in foods and cosmetics. Tedious methodologies for B-PE purification have been published. In this work we present a new, fast, preparative and scaleable two-step chromatographic method for B-PE purification from the red microalga Porphyridium cruentum. Initially, phycobiliproteins were released from the microalga cells by osmotic shock and captured by applying the centrifuged cell suspension to a column containing 74 ml Streamline-DEAE equilibrated with 50 mM acetic acid–sodium acetate buffer, pH 5.5, using expanded-bed adsorption chromatography at an upward flow of 200 cm h−1. After adsorption, washing was carried out in the expanded-bed mode. Having removed unbound proteins and cellular debris, the bed was allowed to sediment and a B-PE-rich solution was eluted with a downward flow of the same 250 mM buffer. In order to obtain pure B-PE, we utilized conventional ion-exchange chromatography with a column of DEAE-cellulose loaded directly with the eluate from Streamline-DEAE and developed using a discontinuous gradient of acetic acid–sodium acetate buffer, pH 5.5. With this new methodology, 66% of B-PE contained in the biomass of the microalgae was recovered, a value significantly higher than those obtained following other methodologies. The B-PE purity was tested using sodium dodecyl sulfate–polyacrylamide gel electrophoresis and spectroscopic characterization.
Keywords: Phycoerythrins; Pigments; Phycobiliproteins; Proteins;

Endostatin capture from Pichia pastoris culture in a fluidized bed by Joseph Shiloach; Patrick Santambien; Loc Trinh; Anthony Schapman; Egisto Boschetti (327-336).
One of the characteristics of the methylothrophic yeast Pichia pastoris is its ability to grow to a very high cell density. Biomass concentrations of 300–400 g wet mass/l are common. It is therefore obvious that the recovery processes of extracellular proteins from this microorganism should take into account the effect of high biomass content. Separation by filtration and/or centrifugation is possible but these steps are cumbersome and can affect the protein recovery. The use of fluidized beds is attractive proteins capture option since it eliminates the biomass while capturing the desired protein. Zirconia-based resins possess unique properties which make them appropriate for processing high biomass concentrations in an expanded bed mode. The beads are particularly heavy (density is 3.2 g/ml) and small (75 μm) and therefore can accommodate high fluidization velocity and high mass transport. Specific operating conditions for effective capture of expressed protein have to be determined. This determination is generally time consuming and requires relatively large amount of feedstock for the lab trials. To avoid multiple chromatographic trials in columns, optimal conditions of adsorption and elution were determined by ProteinChip technology coupled with mass spectrometry. This technology involves flat chip surfaces functionalized as chromatographic beads where it is possible to adsorb and desorb proteins. Four different functional groups (strong anion-exchange, weak cation-exchange, hydrophobic and metal chelate) were tested and the retained proteins were analyzed directly by mass spectrometry. The weak cation-exchange group was chosen for further work. The Zirconia-based weak cation-exchange sorbent (CM HyperZ) was evaluated for binding capacity in a packed column and then for capturing endostatin from crude feed stock. Based on the previously determined conditions; 45 l of culture containing approximately 15 kg of biomass (wet mass) and 3 g endostatin were applied on an expanded bed at a flow-rate of 535 cm/h, yielding 80% of the endostatin and removing approximately 80% of foreign proteins.
Keywords: Endostatin; Proteins;

MspA is the prototype of a new family of tetrameric porins and provides the main general diffusion pathway for hydrophilic compounds through the outer membrane of Mycobacterium smegmatis. Structural analysis was hampered by the scarce amount of pure protein. After replacement of the GC-rich codons of the mspA gene by codons optimal for high-level expression in Escherichia coli, the mature MspA protein was overproduced in E. coli. The recombinant MspA (rMspA) monomer (M r 20 000) was purified by anion exchange and hydrophobic interaction chromatography yielding 2.6 mg pure protein per liter of culture. This exceeded the yield of the native protein 10-fold. Circular dichroism revealed that rMspA is folded in a native-like structure. rMspA assembled partially to the channel-forming tetramer both during expression in E. coli and after purification in vitro. Thus, overexpression in E. coli and chromatographic purification are key steps towards a high resolution structure of MspA.
Keywords: Proteins;

Purification of a full-length recombinant glucocorticoid receptor by Kazuki Okamoto; Naoya Suematsu; Fumihide Isohashi (349-353).
We described a novel purification method for a recombinant glucocorticoid receptor (GR) in detail. The purification procedure consists of sequential chromatographies using common ion-exchange columns (Mono Q and Mono S). This procedure is based upon a new finding that the activated GR binds both to a Mono Q column and to a Mono S column at the same pH. The entire chromatographies took about 3 h and GR represented 97% of the purified protein sample. This purification protocol will be applicable to the purification of native GR, point-mutated recombinant GR and other nuclear receptors.
Keywords: Proteins;

Serum amyloid A protein was subjected to one-step octyl-Sepharose extraction in three different dimensions. Elution was performed partly without UV recording, and with urea or guanidine-based buffers. The eluent was applied directly to denaturing two-dimensional electrophoresis with immobilised pH gradient, or octyl-Sepharose extracted fractions were pooled and lyophilised before application. Proteins were characterised by N-terminal analysis or mass spectrometry. In most of the species that were studied, previously undescribed serum amyloid proteins were detected. Compared to conventional strategies, the presented techniques are more rational and yield more comprehensive information. The presented data also provide a basis for novel perspectives regarding certain inflammatory conditions.
Keywords: Proteins;

Preparative purification of soybean agglutinin by affinity chromatography and its immobilization for polysaccharide isolation by Laura Franco-Fraguas; Alicia Plá; Fernando Ferreira; Hugo Massaldi; Norma Suárez; Francisco Batista-Viera (365-372).
Optimized procedures for the affinity purification of soybean agglutinin (SBA) from soybean flour, and its further immobilization, were developed. Lectin purification on galactosyl-Sepharose yielded 44.5±3.5 mg of pure SBA/50 g of flour. To prepare SBA adsorbents, the lectin was immobilized onto 1-cyano-4-(dimethylamino)pyridinium tetrafluoroborate (CDAP) activated Sepharose with high yields (77%). Feasibility of the use of this improved SBA adsorbent for affinity purification of Streptococcus pneumoniae capsular polysaccharides from strain 14 (CPS-14) at laboratory scale was demonstrated. Using SBA-Sepharose adsorbent (7.0 mg lectin per ml), amounts of 6.3 mg of pure CPS-14 per cycle were produced, the adsorbent being reused up to four times without loss of capacity.
Keywords: Polysaccharides; Bicinchoninic acid; Cyan(dimethylamino)pyridinium tetrafluoroborate; Lectins; Proteins; Agglutinin;