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the unclarified feedstock, to investigate the effects of operating variables on mixing Bruce et al. 1999 Karau et al. 1997 Thommes et al. 1995b , or, more recently, to evaluate the effects of the feedstock itself on the bed Fernandez-Lahore et al. 1999 . Operational Procedure. In principle, expanded bed processes are operated in a similar fashion to packed bed processes, the main difference being the direction of liquid flow. The standard sequence of frontal chromatography is generally followed...
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Figure 21 Principle of the preparative continuous annular chromatography. Figure 21 Principle of the preparative continuous annular chromatography. If the substance zones are made visible by the use of colored substances, the impression of helical bands is given Fig. 21 . In spite of this appearance, however, the migration of the substances occurs exclusively in an axial direction and no flow in radial direction takes place. An investigation of radial mass transfer and rotation speed on the van...
Membrane Operation
As the cross-flow filtration technique plays a more important role in protein isolation and purification, this technique will be described in more detail. As was mentioned, the big advantage of cross-flow filtration is the formation of a nearly constant permeate flux over time Fig. 8 . During dead-end filtration a continuous formation of a filter cake occurs. With the thickness of this filter cake the pressure drop of the membrane increases whereas the permeate flux decreases. Better results...
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Vermeer AW, Norde W 2000 The thermal stability of immunoglobulin unfolding and aggregation of a multi-domain protein. Biophys. J. 78 1 , 394-404. Vielle C, Zeikus JG 1996 Thermoenzymes identifying molecular determinants of protein structural and functional stability. Trends Biotechnol. 14, 183-191. Vogt G, Argos P 1997 Protein thermal stability hydrogen bonds or internal packing Fold. Des. 2, S40-46. Volkin DB, Burke CJ, Sanyal G, Middaugh CR 1996 Analysis of vaccine stability. Dev. Biol....
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Figure 3 A typical grade efficiency curve for a a tubular centrifuge and b a scroll-type centrifuge, showing the cut size d50 . Figure 3 A typical grade efficiency curve for a a tubular centrifuge and b a scroll-type centrifuge, showing the cut size d50 . separated due to the flow ratio. A typical reduced grade efficiency curve is shown in Fig. 4. This curve was plotted based on the data from Fig. 3b. In analogy with the grade efficiency curve, the particle size that corresponds to G' 50 is...
Chromatofocusing
Chromatofocusing is a chromatographic technique in which proteins are eluted from an ion exchange column by a pH gradient. The proteins are separated according to their isoelectric points, pi. The protein with the highest pi elutes first. This technique was developed by Sluyterman and co-workers in late 1970s Sluyterman and Elgersma 1978a,b Sluyterman and Wijdenes 1981 . As mentioned in the above section, protein elution may be achieved by changing the pH. When ordinary buffers are used, sudden...
Chromatography With Nonporous Media
Nonporous supports increase the speed of separation by completely eliminating the problem of slow diffusion of proteins in the interior of the chromatography support. The main drawback of nonporous supports is their low capacity, attributable to the fact that only the outer surface is available for protein binding. Nonporous supports are available in many formats where particles and various forms of membranes are the most common. The term nonporous is somewhat misleading in case of membranes...
I Dav
Saitoh T., Hinze W.L. 1991 Concentration of hydrophobic organic compounds and extraction of protein using alkylammoniosulfate zwitterionic surfactant mediated phase separations cloud point extractions . Anal. Chem. 63, 2520-2525. Sanchez-Ferrer A., Gilabert-Perez M., N ez E., Bru R., Garc a-Carmona F. 1994 Triton X-114 phase partitioning in plant protein purification. J. Chromatogr. A 668, 75-83. Sch tte H., Kroner K.H., Hummel W., Kula M.R. 1983 Recent developments in separation and...
Streaming Potential
Streaming potential is a method based on the principle that if a solid surface comes in contact with a solution, there is an electrochemical potential built up between the bulk solution and the surface. This potential is dependent on the charge distribution on the surface, the flow rate, and the buffer used. The charge distribution over an affinity column can be monitored, and one can thereby register binding of target molecules to the affinity ligand. The system can therefore be used to study...
Displacement Chromatography
People tend to associate a separation as shown in Fig. 1 with chromatography, i.e., an approach called elution chromatography. The components of the sample mixture are separated into individual peaks separated by volumes of empty mobile phase. This facilitates detection because most biopolymers, such as proteins and DNA, can be followed by a UV detec- Figure 1 Schematic presentation of a separation by elution chromatography. Figure 1 Schematic presentation of a separation by elution...
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Figure 4 Purification of recombinant ketosteroid isomerase from crude E. coli lysate using centrifugal precipitation chromatography in the absence a and b and in the presence of affinity ligand, -estradiol-17-methyl-PEG-5000 c and d . Reproduced from Ito 1999 with permission. Figure 4 Purification of recombinant ketosteroid isomerase from crude E. coli lysate using centrifugal precipitation chromatography in the absence a and b and in the presence of affinity ligand,...
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Per-Olof Larsson Lund University, Lund, Sweden E. K. Lee Hanyang University, Ansan, Korea Bo Mattiasson Lund University, Lund, Sweden Ricardo A. Medronho Federal University of Rio de Janeiro, Rio de Dieter Melzner Sartorius AG, G ttingen, Germany Joakim Nilsson Royal Institute of Technology, Stockholm, Sweden Per-Ake Nygren Royal Institute of Technology, Stockholm, Sweden Kerstin Plate University of Hannover, Hannover, Germany Oskar-Werner Reif Sartorius AG, G ttingen, Germany Ipsita Roy Indian...
Continuous MSMPR Crystallizer
A continuous mixed suspension mixed product removal MSMPR crystal-lizer is also widely used in industrial crystallizations for large-scale separation and in laboratory experiments for crystallization kinetics analysis. Agitation of suspension with baffles is usually enough to attain the MSMPR condition inside a crystallizer. In an industrial crystallizer a draft-tube baffle is commonly used. Then we can suppose that the suspension is homogeneous in terms of the external space, and it allows us...
Ionic StrengthConductivity and pH
Elution from a chromatography adsorbent often involves a change in ionic strength or pH. By registering changes in conductivity and pH, one gets good information on whether chromatography is run under desired conditions and when to expect the eluted material. This allows one to modify the elution conditions, if necessary. Registration of these parameters is normally carried out using a flowthrough conductivity sensor and pH electrode, respectively, that are integrated components of the...
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containing - OH group, is essentially correct. Methods are also available for activating matrices with other groups like amide, -SH, or -SiOH. An interesting underexploited method is the use of Ugi's reaction Carlsson et al. 1989 . In this, the ligand to be attached contains one or more functional groups out of the amino, carboxyl, carbonyl, or isocyanide groups. The matrix should contain any of the others. The remaining functional groups from the list of four have to be added in the form of...
BiotinAvidin
An analogous approach, which does not utilize recombinant methods, is the exploitation of avidin-biotin interaction, known to be the affinity pair with the highest binding constant. It has given rise to a technology of its own Wilchek and Bayer 1990 . Biotinylated proteins enzymes binding to avidin streptavidin columns have been frequently used, with positive results in many cases Kim et al. 1999a Pouny et al. 1998 . It should be added that fusion proteins with biotin binding domains have also...
Disruption of Microbial Cells Using Lytic Agents
Disruption processes utilizing chemicals or enzymes as lytic agents have also been extensively investigated. In comparison with other disruption methods, these tend to be expensive and also require subsequent removal of the lytic agents from the process stream. On scale-up, disruption by lytic agents can be effected within a sealed vessel and contained to the same degree as a fermentor. Different classes of chemicals can permeabilize membrane layers of the microbial cells by different...
Affinity Extraction
Aqueous two-phase affinity partitioning has been mostly applied to PEG dextran systems containing PEG derivatives Andrews et al. 1990 . Only a few affinity two-phase studies have been performed in PEG salt systems due to the fact that biospecific interactions are usually obstructed by high salt concentrations. Several ligands have been developed and applied to the purification of proteins. The high purity required for molecular biology diagnostic and therapeutic proteins has spurred efforts to...
Nomenclature
b intensity of the field of forces m s 2 c concentration of solids as mass of solids by mass of liquid Cv feed volumetric concentration Cvu underflow volumetric concentration km filter medium permeability m2
Objectives Of Protein Crystallization
In general, protein crystallization is performed for one of two distinct reasons 1 X-ray crystallography for structural biology studies and 2 purification as a means of bioseparation. In the first application, the protein crystals are used to ascertain the three-dimensional structure of the molecule, which is then used to study the various properties of a protein such as optical, spectroscopic, electrical, thermal, and mechanical. The information on the biological functions and on the exact...
In vivo Biotinylated Affinity Tags
The strong binding Kd 10 15 M between biotin and avidin or streptavidin Green 1975 Wilchek and Bayer 1990 is frequently used in biochemistry and molecular biology for immobilization and detection purposes. For use in such applications biotinylation of proteins is usually achieved through cova-lent coupling of biotin to the protein by the use of biotin-ester reagents that preferentially modify lysine residues Savage et al. 1992 . However, such coupling is difficult to direct to a specific...
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After approximately 30 min the bed should have stabilized and only small circulatory movements of the adsorbent beads are observed. Prior to sample application, bed stability may be assessed in order of increasing accuracy by the degree of bed expansion, visual inspection, or an RTD test. After expansion, equilibration, and stabilization, the biomass-contain-ing broth is applied to the bed. The properties of the feedstock are usually very different to those of the equilibration buffer, often...
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Ohlson S, Bergstr m M, Leickt L, Zopf D 1998 . Weak affinity chromatography of small saccharides with immobilized wheat germ agglutin and its application to monitoring of carbohydrate transferase activity. Bioseparation 7 101-105. Ohlson S, Lundblad A, Zopf D 1988 . Novel approach to affinity chromatography using weak monoclonal antibodies. Anal. Biochem. 169 204-208. Olivera BM, Hillyard DR, Marsh M, Yoshikami D 1995 . Combinatorial peptide libraries in drug design lesons from venomous cone...
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Figure 9 Types of the polymer enriched phase formed after the phase separation of the polymer during affinity precipitation. a, loose gel with high water content b, compact hydrophobic phase with low water content and c, suspension of compact polymer. Figure 9 Types of the polymer enriched phase formed after the phase separation of the polymer during affinity precipitation. a, loose gel with high water content b, compact hydrophobic phase with low water content and c, suspension of compact...
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gant selection strategy based on fusion of the peptides to a plasmid binding lac repressor protein Schatz 1993 . From the series of sequences found to be in vivo biotinylated at a central invariant lysine residue, a consensus 13-amino-acid stretch was defined. Thus, this sequence could replace the larger in vivo biotinylated fusion partners used earlier by other groups. Recently, this tag was used in combination with a His6 peptide and ABP into a novel affinity fusion partner See below Nilsson...
Requirements In Dsp Monitoring
The high proportion of the process cost attributed to downstream processing stage for many proteins suggests the importance of performing the purification sequence within specified limits at high yields and knowing rapidly when such limits are crossed so as to make alterations without delay. For example, adsorption to a chromatography matrix is traditionally monitored by assaying the levels of key components in the effluent stream. However, in order to control the loading, it is important to...
Ionic Strength
increasing ionic strength of the aqueous phase reduces electrostatic interactions as a result of the Debye screening effect, which is stronger for larger ions. Thus, at a higher ionic strength, the interactions between hydrophilic biomolecules and the surfactant polar groups are reduced, and smaller micelles are formed. As a consequence of this effect, the solubilization capacity of the organic phase for water and biomolecules decreases. Complete solubilization of three proteins cytochrome c,...
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accessible for site-specific cleavage of SPA fusion proteins to release the target protein Forsberg et al. 1992 . 6 The SPA fusion partner is secretion competent, and SPA fusions can be efficiently secreted to the E. coli periplasm and in some cases also to the culture medium, using the SPA promoter and signal sequence Abrahmsen et al 1985, 1986 Moks et al. 1987a,b Stahl et al. 1990 Hansson et al. 1994 . To allow chemical cleavage of SPA fusion proteins with both CNBr and hydroxylamine, an...
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Katakam M, Robillard P, Brown J, Tolman G 1998 Effects of moisture and lyophilization cycle on aggregation of a monoclonal antibody product. Pharm. Sci. Suppl. 1, S-543. Keck RG 1996 The use of t-butyl hydroperoxide as a probe for methionine oxidation in proteins. Anal. Biochem. 236, 56-62. Kendrick BS, Chang BS, Arakawa T, Peterson B, Randolph TW, Manning MC, Carpenter JF 1997 Preferential exclusion of sucrose from recombinant interleukin-1 receptor antagonist role in restricted conformational...
Filtration Using a Centrifugal Pump
Most pressure filtration systems use a centrifugal pump for delivering the slurry to the filter. In this situation, the filtration follows neither a constant-pressure filtration nor a constant-rate filtration. This occurs because the flow rate delivered by the pump is a function of the resistance offered by the system, as shown schematically in Fig. 22. At the beginning of the filtration, Figure 21 Filtration of yeast using perlite as filter aid precoat-bodyfeed method in a plate-and-frame...
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Govrin E, Levine A 1999 . Purification of active cysteine proteases by affinity chromatography with attached E-64 inhibitor. Protein Expr. Purif. 15 247-250. Greenwood JM, Gilkes NR, Miller RC Jr., Kilburn DG, Warren RAJ 1994 . Purification and processing of cellulose-binding domain-alkaline phosphatase fusion proteins. Biotechnol. Bioeng. 44 1295-1305. Gupta MN, Kaul R, Guoqiang D, Dissing U, Mattiasson B 1996 . Affinity precipitation of proteins. J. Mol. Recognit. 9 356-359. Gupta MN,...
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cake. The membrane has to be changed when the membrane pores are clogged by solids. Dead-end mode is only applicable when liquids with very low particle content are processed and no concentration polarization takes place. Typically it is used in microfiltration or prefiltration steps. Dead-end systems are mainly used in laboratory scale separations. The membranes are available as ready-to-use devices with different membrane areas for one-time application see also Table 1 . For process...
Bb Tag Serum Albumin-binding Region
Figure 2 Schematic representation of streptococcal protein G SPG and the extensions of various serum albumin binding subfragments described in the text. S, signal peptide processed during secretion W, cell wall anchoring region. The regions responsible for serum albumin and immunoglobulin binding, respectively, are marked. BB 25 kDa was the first isolated serum albumin-binding affinity tag. More recently, the new tags ABP 15 kDa and ABD 5 kDa , containing two or one serum albumin-binding...
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Roser B 1991 Trehalose drying a novel replacement for freeze-drying. Biopharm. 4 9 , 47-53. Rossi S, Buera MP, Moreno S, Chirife J 1997 Stabilization of the restriction enzyme EcoRI dried with trehalose and other selected glass-forming solutes. Biotechnol. Prog. 13, 609-616. Roos Y 1993 Melting and glass transitions of low molecular weight carbohydrates. Carbohydr. Res. 238, 39-48. Roos Y, Karel M 1991 Phase transitions of mixtures of amorphous polysaccharides and sugars. Biotechnol. Prog. 7,...
Regeneration
Figure 6a Continuous countercurrent contactor operation a a schematic of the process and b a photograph of the four-stage unit just prior to homogenate application. Reproduced with permission from Owen 1998. adsorbent. The adsorbent used was a solid perfluoropolymer particle Table 3 , coated with poly vinyl alcohol PVA , cross-linked and deriva-tized with the dye-ligand Procion red HE-7B. The contactor was used successfully with a variety of unclarified feedstocks such as egg whites, yeast Owen...
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Guerra P, Acklin C, Kosky AA, Davis JM, Treuheit MJ, Brems DN 1998 PEGylation prevents the N-terminal degradation of megakaryocyte growth and development factor. Pharm. Res. 15, 1822-1827. Hancock GE, Smith JD, Heers KM 2000 The immunogenicity of subunit vaccines for respiratory syncytial virus after co-formulation with aluminum hydroxide adjuvant and recombinant interleukin-12. Viral Immunol. 13 1 , 57-72. Hatley RHM, Franks F 1991 Applications of DSC in the development of improved...
OnePolymer Systems
These systems can either be polymer water two-phase systems or polymer low molecular weight solute water two-phase systems. The polymer water ATPS is formed due to temperature changes and is described further in Sec. 2.3. Phase separation of nonionic polymer inorganic salt is known to occur at room temperatures, and most are composed with hydrophilic salts. The higher the valency of the anion, the lower is the salt concentration required for phase formation. The higher the polymer MW, the lower...
Fundamentals of Filtration Techniques
During a membrane separation process the low-viscosity feed suspension is usually applied on one side of a membrane. The stream that passes through the membrane under the influence of the pressure force is termed the permeate filtrate . After removal of the required amount of permeate, the remaining material is termed retentate concentrate . The extent of the concentration is characterized by the concentration factor f , which is the ratio of the feed volume to the final volume Eq. 1 .
CEBased Enzyme Microassays
Small amounts of enzyme can be detected by the performance of assays in capillary electrophoretic systems Bao and Regnier 1992 Wu and Regnier 1993 Wu et al. 1994 Harmon et al. 1996 . The electrophoretically mediated microassay EMMA is based on the fact that mobility of the enzyme, reagents, and product is different under applied potential and may be used to mix the reactants and separate the enzyme from the product after catalysis. At constant potential, the enzyme and the product are...
Textile Dyes
The principal application of immobilized dye column chromatography has been to purify proteins, a role that has enjoyed spectacular success largely due to the economy, stability, and capacity of immobilized reactive dye columns Lowe et al. 1992 Stellwagen 1993 Labrou and Clonis 1994 . Most applications employ positive immobilized dye chromatography. One case in which negative immobilized dye affinity chromatography has been used is in the removal of albumin up to 98 for the isolation of...
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nozzle centrifuge followed by a further concentration in a scroll-type centrifuge. In this case, dry solids of 25-35 can be obtained. Scroll-Type Centrifuges. Figure 12 shows a scroll-type machine, also known as a decanter centrifuge. It is equipped with a screw conveyor that continuously discharges the deposited solids. The bowl and the screw conveyor rotate in the same direction, but the latter rotates with a velocity slightly lower or higher than the former. This speed difference is usually...
Peg Precipitation Mode
to perturbations in heat and pH thus, some proteins, such as adenylate kinase and trypsin and certain proteins of thermophilic organisms are relatively heat stable in comparison with the majority of the other enzymes. Similarly some proteins are stable and even biologically active outside the pH range of pH 5-10, where many of the proteins undergo denaturative changes in confirmation. The precipitation strategy therefore involve the option to use heat and pH to denature and precipitate the...
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Heftmann E. 1973 History of chromatography and electrophoresis. In Chromatography. A Laboratory Handbook of Chromatographic and Electrophoretic Methods E. Heftmann, ed. , Van Nostrand-Reinhold, New York, pp. 19-26. Heftmann E. 1983 Chromatography Fundamentals and Applications of Chromatographic and Electrophoretic Methods Part A Fundamentals and Techniques, Elsevier, Amsterdam. Horvath C.G., Preiss B.A., Lipsky S.R. 1967 Fast liquid chromatography an investigation of operation parameters and...
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Buera MP, Levi G, Karel M 1992 Glass transition in polyvinylpyrrolidone effect of molecular weight and diluents. Biotechnol. Prog. 8, 144-148. Bush L, Webb C, Bartlett L, Burnett B 1998 The formulation of recombinant factor IX stability, robustness, and convenience. Semin. Hematol. 35, 18-21. Carbeck JD, Severs JC, Gao J, Wu Q, Smith RD, Whitesiobs GM 1998 Correlation between the charge of proteins in solution and in the gas phase investigated by protein charge ladders, capillary...
Protein Purification In Postgenomic Era
As researchers attempt to understand the vast amount of genetic information, proteomics has become a major focus in the biotechnology industry since gene function is derived from the protein product it encodes. The challenge of studying proteins holistically is driving the development of analytical and preparative tools that allow the resolution and characterization of complex sets of protein mixtures in a high-throughput mode and the subsequent purification of target therapeutic protein....
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De Bern rdez Clark E 2001 Protein refolding for industrial processes. Curr. Opin. Biotechnol. 12, 202-207. Eriksson H, Sandahl K, Forslund G and Osterlund B 1991a Knowledge-based planning for protein purification. Chemom. Intell. Lab Syst. 13, 173-184. Eriksson H, Sandahl K, Brewer J, Osterlund B 1991b Reactive planning for chromatography. Chemom. Intell. Lab Syst. 13, 185-194. Guerrier L, Girot P, Schwartz W, Boschetti E 2000 New method for the selective capture of antibodies under...
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Rongxiu L, Dowd V, Stewart DJ, Burton SJ, Lowe CR 1998 . Design, synthesis and application of a protein A mimetic. Nature Biotechnol. 16 190-195. Roy I, Pai A, Lali A, Gupta MN 1999 . Comparison of batch, packed bed and expanded bed purification of A. niger cellulase using cellulose beads. Bioseparation 8 317-326. Roy I, Sardar M, Gupta MN 2000 . Exploiting unusual affinity of usual polysaccharides for separation of enzymes on fluidized beds. Enzyme Microb. Technol. 27 53-65. Russell AJ,...
Axial Dispersion
Axial dispersion is longitudinal mixing or back mixing in a column. If axial dispersion is high, the efficiency of the column is low. Hence, in chromatographic separations, axial dispersion should be at a minimum. According to Fick, molecular diffusion is defined as where D is the molecular diffusion coefficient, x is the direction of diffusion. Similarly, in a column where molecular diffusion is outweighed by the turbulent flow, where Da is the axial dispersion coefficient, C is concentration,...
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discharge method, 179 filtration, 131, 161, 162 porosity, 163 Calmodulin, 60 -SPA fusion protein, 114 Canavalia ensiformis, 70 Candida utilis, 18 Canola protein extract, 235, 240 Capacitance, 534, 539 Capacity factor k , 34, 39, 44 Capacity signal, 539 Capillary electrophoresis CE , 531, 533, 541, 581, 582 based enzyme microassay, 533 immunoassay, 533 -MS, 581 Capillary zone electrophoresis CZE , 519, 531, 533 Carbodiimides, 62, 63 Carbonic anhydrase, 357 Carbonylimidazole, 62...
Mild Elution of Protein A and Protein G Fusions
Protein A Z and ABP fusion proteins are most conveniently eluted from affinity columns using low pH e.g., HAc of pH 3 . The eluant is usually removed either by vaporization or by the use of a desalting column. However, a low pH can, in some instances, be destructive for target protein function and must in such cases be avoided. In addition, the use of low-pH eluants following an initial capture of a fusion protein at physiological pH could result in passage of the isolectric point pI of the...