A study of the influence of yeast cell debris on protein and alpha-glucosidase adsorption at various zones within the expanded bed using in-bed sampling.

Expanded bed adsorption chromatography is used to capture products directly from unclarified feedstocks, thus combining solid-liquid separation, product concentration and preliminary purification into a single step. However, when non-specific ion-exchangers are used as the adsorbent in the expanded bed, there is the possibility that electrostatic interactions of cells or cell debris with the adsorbent may interfere with the adsorption of soluble products. These interactions depend on the particle size of the cell debris and its surface charge, which in turn depend on the extent of disruption used to release the intracellular products. The interactions occurring during expanded bed adsorption between the anionic ion-exchanger STREAMLINE DEAE and particulate yeast homogenates obtained by high pressure homogenisation at different intensities of disruption achieved by operating at different pressures were studied, while maintaining all other parameters constant. In-bed sampling from the expanded bed using ports fitted up the height of expanded bed was used to study the retention of yeast cells and cell debris within the bed and its influence on the adsorption of total soluble protein and alpha-glucosidase within various zones of the expanded bed. The retention of the biomass present in the homogenate obtained at a lower intensity of disruption was found to be high at the lower end of the column (17% from 13.8 MPa sample compared to 1% from 41.4 MPa sample). This interaction of the particulate material with the adsorbent was found to reduce the dynamic binding capacity of the adsorbent for total soluble protein from 3.6 mg/mL adsorbent for 41.4 MPa sample to 3.0 mg/mL adsorbent for 13.8 MPa sample. The adsorption of alpha-glucosidase was found to increase with an increase in the concentration of the enzyme in the feed, which increased with the intensity of disruption. Selective adsorption of 6,732 U alpha-glucosidase per mg of total protein bound, was noticed for the feedstock prepared at a higher disruption intensity at 41.4 MPa compared to adsorption of 1,262 U/mg of total protein bound for that prepared at 13.8 MPa. The selective adsorption of alpha-glucosidase due to its high concentration together with simultaneous high specific activity of the enzyme in the feed indicated the significance of selective release of enzymes during microbial cell disruption for efficient expanded bed adsorption processes.

Removal of poly-histidine fusion tags from recombinant proteins purified by expanded bed adsorption.

Enzymatic methods have been used to cleave the C- or N-terminus polyhistidine tags from histidine tagged proteins following expanded bed purification using immobilized metal affinity chromatography (IMAC). This study assesses the use of Factor Xa and a genetically engineered exopeptidase dipeptidyl aminopeptidase-1 (DAPase-1) for the removal of C-terminus and N-terminus polyhistidine tags, respectively. Model proteins consisting of maltose binding protein (MBP) having a C- or N-terminal polyhistidine tag were used. Digestion of the hexahistidine tag of MBP-His(6) by Factor Xa and HT15-MBP by DAPase-1 was successful. The time taken to complete the conversion of MBP-His(6) to MBP was 16 h, as judged by SDS-PAGE and Western blots against anti-His antibody. When the detagged protein was purified using subtractive IMAC, the yield was moderate at 71% although the overall recovery was high at 95%. Likewise, a yield of 79% and a recovery of 97% was obtained when digestion was performed with using "on-column" tag digestion. On-column tag digestion involves cleavage of histidine tag from polyhistidine tagged proteins that are still bound to the IMAC column. Digestion of an N-terminal polyhistidine tag from HT15-MBP (1 mg/mL) by the DAPase-I system was superior to the results obtained with Factor Xa with a higher yield and recovery of 99% and 95%, respectively. The digestion by DAPase-I system was faster and was complete at 5 h as opposed to 16 h for Factor Xa. The detagged MBP proteins were isolated from the digestion mixtures using a simple subtractive IMAC column procedure with the detagged protein appearing in the flowthrough and washing fractions while residual dipeptides and DAPase-I (which was engineered to exhibit a poly-His tail) were adsorbed to the column. FPLC analysis using a MonoS cation exchanger was performed to understand and monitor the progress and time course of DAPase-I digestion of HT15-MBP to MBP. Optimization of process variables such as temperature, protein concentration, and enzyme activity was developed for the DAPase-I digesting system on HT15-MBP to MBP. In short, this study proved that the use of either Factor Xa or DAPase-I for the digestion of polyhistidine tags is simple and efficient and can be carried out under mild reaction conditions.

Isolation of monocytes from human peripheral blood using immuno-affinity expanded-bed adsorption.

A novel technique for the separation of monocytes from human peripheral blood preparations has been developed. The technique is based on the use of expanded-bed adsorption and a solid perfluorocarbon derivatized with avidin or streptavidin for the indirect positive or negative capture of cells labeled with biotinylated monoclonal antibodies. The perfluorocarbon support was prepared and characterized and the contactor design and operating conditions, that enable cells to be selectively isolated, were investigated. Experiments consisted of applying an immunolabeled pulse of 1 x 10(8) peripheral blood mononuclear cells (PBMCs), isolated by density gradient centrifugation, directly onto a refrigerated expanded bed. The major cell types remaining were T-lymphocytes, B-lymphocytes, and monocytes. Monocytes could be positively adsorbed, following labeling with anti-CD14 mAb, with a clearance of up to 89% and a depletion factor of 7.6. They could also be "eluted" using mechanical shear, with a 77% yield of the applied cells at a purity of 90% and >/= 65% viability. Negative isolation of monocytes, following labeling of the other cells present with anti-CD2, CD7, CD16, CD19, and CD56 mAbs, resulted in lymphocyte depletions of up to 81% with a monocyte enrichment factor of 3.8 and purity of 71%. The monocyte viability in the flowthrough was assessed to be > 95%. This combination of expanded-bed adsorption and fluidizable affinity supports shows significant potential for the intensification of cell separations.

Impact of operating variables on the expanded bed adsorption of Saccharomyces cerevisiae cells using a concanavalin A derivatized perfluorocarbon.

The use of fluidizable affinity adsorbents for the adsorption of cells in expanded mode is investigated. Affinity adsorbents have been synthesized by immobilizing the lectin Concanavalin A onto the surface of triazine-activated perfluorocarbon-solids. The adsorbents were found to adsorb Saccharomyces cerevisiae cells from solution with adsorption capacities of up to 6.8 x 10(9) cells mL(-1). Adsorption kinetics were rapid with a time constant of

Direct recovery of glutathione S-transferase by expanded bed adsorption: anion exchange as an alternative to metal affinity fusions.

The use of expanded beds of ion-exchange adsorbents for the direct recovery of a recombinant intracellular protein, glutathione S-transferase (GST), from unclarified Escherichia coli homogenates is described. The results form the basis for a comparison between this approach for purifying GST and a chelating fusion strategy and highlight the need to consider the additional costs entailed by these more-complicated approaches. The separation performance was investigated with respect to choice of anion or cation exchanger, adsorption pH, load volume, sample preparation, and stepwise elution protocol. Anion exchange was found to be more appropriate than cation exchange, as the low pHs involved in the latter caused a loss of activity. The optimal pH for adsorption was found to be 9 with a dynamic capacity from clarified homogenate in packed mode of 112 U mL(-1) (11.2 mg GST mL(-1)). As increasing volumes of unclarified homogenate were applied to the expanded bed, the yield of GST in the eluate decreased, and the purification factor was found to increase and then decrease. This was due to the displacement of weakly bound proteins by GST and then its displacement by even more strongly binding proteins. The dynamic capacity of the anion exchanger, STREAMLINE DEAE, from unclarified homogenate in expanded mode decreased slightly to 85 U mL(-1) (8.5 mg GST mL(-1)). The elution protocol for GST from the anion exchanger was then adjusted to try to maximize the degree of purification. Anion exchange expanded bed adsorption of GST from unclarified E. coli homogenate gave an eluted yield of 95.7% and 1.64-fold purification. Interestingly, a decrease in the expression level of GST in the feedstream from 23 down to 13% caused a decrease in the dynamic capacity from 85 to 14.5 U mL(-1) whereas the degree of purification remained similar.

Immobilised metal affinity chromatography of beta-galactosidase from unclarified Escherichia coli homogenates using expanded bed adsorption.

The development of an expanded bed process for the direct extraction and partial purification of beta-galactosidase from unclarified Escherichia coli homogenates using its natural affinity for metal loaded STREAMLINE Chelating is described. Small packed beds were used to determine the effect of chelated metal ion (Cu2+, Ni2+, Co2+ or Zn2+), loading pH and ionic strength on the selective binding capacity, and recovery of beta-galactosidase from clarified homogenates. An elution protocol was developed using the competitive displacer, imidazole, to recover beta-galactosidase in 87% yield and 3.4-fold purification. These results were then used to develop a separation for the recovery of beta-galactosidase from unclarified homogenates in a 2.5-cm diameter expanded bed. Although Ni2+ loaded STREAMLINE Chelating had a 5% dynamic capacity for beta-galactosidase of just 118 U ml(-1) (0.39 mg ml(-1)), the low capacity was thought to be due to the large size of the target (464,000) relative to the exclusion limit of the macroporous adsorbent. Despite this low capacity, Ni2 STREAMLINE Chelating was used successfully to recover beta-galactosidase from an unclarified homogenate in 86.4% yield and at 5.95-fold purification. The degree of purification relative to a commercial standard, as assessed using the purification factor and sodium dodecyl sulphate-polyacrylamide gel electrophoresis was high suggesting that this pseudo-affinity procedure compared favourably with alternative methods.

Facilitated downstream processing of a histidine-tagged protein from unclarified E. coli homogenates using immobilized metal affinity expanded-bed adsorption.

The facilitated downstream processing of an intracellular, polyhistidine-tagged protein, glutathione S-transferase [GST-(His)(6)], direct from unclarified E. coli homogenates using expanded beds of STREAMLINE chelating, has been investigated. A series of pilot experiments were used to develop preparative-scale separations of GST-(His)(6), initially in packed and then in expanded beds. Packed beds of Ni(2+)-loaded STREAMLINE chelating proved to have the highest 5% dynamic capacity for GST-(His)(6), of 357 U mL(-1) (36 mg mL(-1)). When using immobilized Cu(2+) or Zn(2+), metal ion transfer was observed from the iminodiacetate ligands to the high-affinity chelator, GST-(His)(6). The subsequent metal affinity precipitation of this homodimer resulted in operational problems. An equilibrium adsorption isotherm demonstrated the high affinity of GST-(His)(6) for immobilized Ni(2+), with a q(m) of 695 U mL(-1) (70 mg mL(-1)) and a K(d) of 0.089 U mL(-1) (0.0089 mg mL(-1)). Ni(2+)-loaded STREAMLINE chelating was therefore selected to purify GST-(His)(6) from unclarified E. coli homogenate, resulting in an eluted yield of 80% and a 3.34-fold purification. The high dynamic capacity in the expanded mode of 357 U mL(-1) (36 mg mL(-1)) demonstrates that this specific interaction was not affected by the presence of E. coli cell debris.

On-line monitoring of the purification of GST-(His)6 from an unclarified Escherichia coli homogenate within an immobilised metal affinity expanded bed.

The use of a rapid chromatographic assay to monitor the level of a specific protein during its downstream processing by expanded bed adsorption is described. An expanded bed column (5 cm diameter) has been modified to allow the abstraction of liquid samples at various heights along the bed, in an automated, semi-continuous manner throughout the separation. The withdrawn samples were filtered in-line and the level of the target protein assayed by a rapid on-line chromatographic method. Using this technique it was possible to monitor the development of adsorbate profiles during the loading, washing and elution phases of the application of an unclarified feedstock. The potential of the technique is demonstrated using the separation of histidine tagged glutathione s-transferase (GST-(His)6) from an unclarified Escherichia coli homogenate using an expanded bed of Ni2+ loaded STREAMLINE Chelating. The level of GST-(His)6 in the abstracted homogenate samples was measured using Zn2+ loaded NTA-silica as an affinity chromatographic sensor. The approach described demonstrates potential for the on-line monitoring and control of expanded bed separations and for providing a greater understanding of adsorption/desorption and hydrodynamic processes occurring within the bed.

The effect of column verticality on separation efficiency in expanded bed adsorption.

The effect of column verticality on liquid dispersion and separation efficiency in expanded bed adsorption columns was investigated using 1 and 5 cm diameter columns. Column misalignment of only 0.15 degree resulted in the reduction of the Bodenstein number from 140 to 50 for the 1 cm dia. column and from 75 to 45 for the 5 cm dia. column. This degree of misalignment was not detectable by visual assessment of adsorbent particle movement within the column. Depending on the relative importance of transport limitations, kinetic limitations and dispersion to any specific separation, this increase in dispersion with column alignment can significantly affect separation efficiency. Pure protein breakthrough profiles resulting from the application of bovine serum albumin onto STREAMLINE Q XL demonstrated that, at 10% breakthrough, 7.8% more protein could be applied to a vertical 1 cm dia. column compared to the same column misaligned by 0.15 degree. When an unclarified yeast homogenate was applied to a 1 cm dia. vertical column packed with STREAMLINE DEAE, 10% breakthrough of glucose-6-phosphate dehydrogenase (G6PDH) corresponded to a load 55% greater compared to the same column aligned 0.185 degree off-vertical. The G6PDH breakthrough curves for vertical and 0.15 degree off-vertical runs performed using a 5 cm column were essentially indistinguishable.

Evaluation of the effect of in-bed sampling on expanded bed adsorption.

An expanded bed adsorption (EBA) column (5 cm diameter) has been modified to allow the abstraction of liquid samples from various positions along the height of an expanded bed. As the adsorbent particles were fluidized, in-bed monitoring of key component concentrations during feedstock application, washing and elution was achieved by the withdrawal of liquid samples from the voids within the expanded bed through ports along the wall of the column. Component levels in the withdrawn streams can be assayed using on-line analytical chromatography or samples can be collected and assayed off-line. On-line monitoring can be used to control the duration of the loading stage and as a tool to provide information about the hydrodynamic and adsorption/desorption processes that occur during expanded bed adsorption. Studies of residence time distributions indicated that the modifications to the column do not significantly affect liquid dispersion. Using the adsorption of glucose-6-phosphate dehydrogenase from yeast homogenate on Streamline DEAE as a model system, comparison of breakthrough curves for runs when in-bed monitoring was and was not performed also suggested that separation efficiency is not appreciably affected by in-bed sampling.

Immobilization of alpha-amylase on poly(vinyl alcohol)-coated perfluoropolymer supports for use in enzyme reactors.

The suitability and potential for the use of poly(vinyl alcohol) (PVA)-coated solid perfluoropolymers in immobilized-enzyme engineering have been evaluated by using alpha-amylase from Bacillus licheniformis for the hydrolysis of starch. alpha-Amylase was covalently immobilized on PVA-coated poly(tetrafluoroethylene-hexafluoropropylene) (PVA-FEP) by covalent coupling with the use of p-beta-sulphate-(ethyl sulphonide)-aniline, 2,4,6-trichloro-1,3,5-triazine, 1.1'-carbonyldi-imidazole and 2,2, 2-trifluoroethanesulphonyl chloride activation procedures, and also for comparison with cyanogen bromide-activated Sepharose 4B. In all cases, immobilization greatly improved the thermostability of the alpha-amylase and its resistance to inactivation by 6 M urea. Also the enhancements of enzymic activities with increased temperature were higher for the immobilized enzymes than for the soluble enzyme, and the immobilized alpha-amylases were well suited to the continuous hydrolysis of starch conducted at elevated temperatures. Although the specific activities of the enzymes immobilized on PVA-FEP were lower than for that immobilized to Sepharose 4B, these novel supports showed far superior strength. The enzymes immobilized on PVA-FEP were able to be readily recovered from stirred batch bioreactors for repeated reuse, whereas the enzymes immobilized to Sepharose were fractured and fragmented under similar conditions of stirring. A conventional fixed-bed bioreactor was found to be unsuitable for continuous starch hydrolysis owing to an unacceptable build-up of pressure drop across the bed. However, an expanded bed reactor containing alpha-amylase immobilized on solid PVA-coated perfluorocarbon showed great potential for the continuous hydrolysis of starch. Only 20% of the enzyme activity was lost after use for 3 weeks at 72 degrees C. It is concluded that PVA-coated solid perfluorocarbon is a highly promising support for use in immobilized enzyme engineering.

Comparison of diffusion and diffusion-convection matrices for use in ion-exchange separations of proteins.

A comprehensive study has been undertaken to characterise a range of chromatographic properties for a series of modified polystyrene-divinylbenzene (PS-DVB) chromatography matrices. The matrices studied included diffusion matrices and matrices that allowed convective mass transfer of liquid into the particles at high flow-rates, so-called "perfusion" matrices. The matrices tested included the following: CG1000sd 20-50 microns (TosoHaas), PLRP4000s 15-25 microns, 50-70 microns (Polymer Labs.), Source 15RPC and 30RPC, 15S, 30S, (Pharmacia Biotech), POROS 20SP type 1 matrix and OH activated POROS 20 type 2 matrix (PerSeptive Biosystems) and SP Sepharose Fast Flow (Pharmacia Biotech). A Van Deemter equation was used to determine bead tortuosities and split ratios. Frontal analysis, resolution studies, ionic capacities and isotherms were measured. It was found that diffusion-convection chromatographic particles had smaller plate heights to comparable diffusion particles. The smallest diffusion bead, Source 15, had the lowest plate heights at low superficial velocities, but the small particle size resulted in a high back pressure at high flow-rates. The equilibrium binding capacities for lysozyme and IgG on the diffusion-convection matrices were substantially lower than the equilibrium binding capacities on the diffusion matrices. The dynamic capacities for these proteins were also lower on the diffusion-convection particles, compared to the diffusion particles, over the tested flow-rates. At high protein loading, resolution between proteins was higher on diffusion particles than on diffusion-convection particles. Diffusion-convection particles showed low or no resolution at high protein loading. At analytical level loadings, the diffusion-convection particles achieved a high resolution over the whole flow-rate range tested and were more suitable for this application than diffusion particles.

The use of affinity adsorbents in expanded bed adsorption.

The potential for the use of affinity ligands in expanded bed adsorption (EBA) procedures is reviewed. The use of affinity ligands in EBA may improve its use in direct recovery operations, as the enhanced selectivity of the adsorbent permits selective capture of the target from complex feedstocks and high degrees of purification. The properties of ligands suitable for use in EBA processes are identified and illustrated with examples. In addition to its use in the recovery of soluble products, such as proteins and nucleic acids, from particulate feedstocks, EBA can also be used to recover particulate entities, such as cells and packaged DNA (viruses and phages), from feedstocks. Affinity ligands coupled to appropriate chosen support materials will be required for such processes in order to achieve the necessary selectivity for the required particulate entity. The latter point is illustrated by the use of proteinaceous ligands immobilized to perfluorocarbon emulsions to achieve separations of microbial cells.

Methanotrophs, Methylosinus trichosporium OB3b, sMMO, and their application to bioremediation.

One of the most problematic groups of the USEPA and EU priority pollutants are the halogenated organic compounds. These substances have a wide range of industrial applications, such as solvents and cleaners. Inadequate disposal techniques and accidental spillages have led to their detection in soil, groundwater, and river sediments. Persistence of these compounds in the environment has resulted from low levels of biodegradation due to chemical structural features that preclude or retard biological attack. Research has indicated the idea that treatment systems based on methanotrophic co-metabolic transformation may be a cost-effective and efficient alternative to physical methods because of the potential for high transformation rates, the possibility of complete compound degradation without the formation of toxic metabolites, applicability to a broad spectrum of compounds, and the use of a widely available and inexpensive growth substrate. A substantial amount of work concerning methanotrophic cometabolic transformations has been carried out using the soluble form of methane monooxygenase (sMMO) from the obligate methanotroph Methylosinus trichosporium OB3b. This NADH-dependent monooxygenase is derepressed when cells are grown under copper stress. sMMO has a wider specificity than the particulate form. sMMO has been shown to degrade trichloroethylene (TCE) at a rate of at least one order of magnitude faster than obtained with other mixed and pure cultures, suggesting it has a wider application to bioremediation. Furthermore, sMMO catalyzes an unusually wide range of oxidation reactions, including the hydroxylation of alkanes, epoxidation of alkenes, ethers, halogenated methanes, cyclic and aromatic compounds including compounds, that are resistant to degradation in the environment. However, the practical application of methantrophs and Methylosinus trichosporium OB3b to the treatment of chlorinated organics has met with mixed success. Although oxidation rates are rapid, compound oxidation with M. trichosporium OB3b is difficult. This fastidious organism grows relatively slowly, which limits the speed with which sMMO expressing biomass can be generated. Furthermore, product toxicity toward the cell, affecting the stability of the enzyme when transforming certain compounds has been observed, for example, by the products of 1,2,3 trichlorobenzene hydroxylation (2,3,4- and 3,4,5-trichlorophenol) and of TCE degradation (chloral hydrate). Because of this toxicity and the inability of sMMO to further oxidize its own hydroxylation products, the ability of methane monoxygenase to carry out the monooxygenation of a wide variety of substituted aromatics and polyaromatics cannot be fully exploited in M. trichosporium OB3b. Many of these problems could be overcome by the use of either a mixed downstream heterotrophic population of organisms that could accommodate the products of hydroxylation or to express sMMO in an organism that could metabolize the products of hydroxylation. The latter of these two approaches would have several advantages. The main benefit would be the removal of the need for methane, which is required to induce sMMO in M. trichosporium OB3b, and supply carbon and energy to the cells that continuously oxidise the target compound, but also acts as a competitive inhibitor of sMMO. Instead, the recombinant could utilize the products of sMMO-mediated hydroxylation as a carbon source.

Rate-limiting mass transfer in immunosorbents: characterisation of the adsorption of paraquat-protein conjugates to anti-paraquat Sepharose 4B.

A series of paraquat-protein conjugates of different molecular size has been prepared by the coupling of paraquat hexanoate to the proteins lysozyme, ovalbumin, bovine serum albumin. The characteristics of the adsorption of these conjugates to an immunosorbent consisting of monoclonal anti-paraquat antibodies covalently immobilised to Sepharose 4B have been determined. Equilibrium adsorption isotherms were found to obey the Langmuir equation and indicated that 80% or more of the antibody binding sites were accessible to the conjugates. The rates of mass transfer of the conjugates to their adsorption sites on the immobilised antibodies was well described by a model in which mass transfer is controlled by transfer across the external film and diffusion within the porous adsorbent bead. The effective diffusivities of the conjugates within the immunosorbent were measured and has allowed the effect of the size of the adsorbing molecule on the rate of adsorption to be considered. The amount of paraquat that could be adsorbed and the rates of adsorption decreased as the size of the protein to which it is conjugated increased. The diffusivity of the conjugates within the pores of the adsorbent is reduced between two and five times compared to their diffusivities in free solution. The reduction is greater for the larger proteins and the variations of the effective diffusivities and the pore diffusivities with the molecular weight of the conjugate can be well described with simple correlations.

Modification of polystyrenic matrices for the purification of proteins. II. Effect of the degree of glutaraldehyde-poly(vinyl alcohol) crosslinking on various dye ligand chromatography systems.

A poly(styrene-divinylbenzene) chromatography matrix, CG1000sd (TosoHaas) has been modified by the adsorption and crosslinking of poly(vinyl alcohol) (PVA) to create a matrix suitable for the attachment of dye ligands for the adsorption of lysozyme. However, it is shown that there was limited recovery and repeated drops in capacity with adsorption of human serum albumin (HSA). The effect of changing the nature of the PVA crosslinking on the HSA binding characteristics was studied, as well as the effect of using differing dye ligands. The total amount of irreversible HSA binding decreased with greater crosslinking and there were large differences in HSA adsorption characteristics between differing dye types.

Modification of polystyrenic matrices for the purification of proteins. III. Effects of poly(vinyl alcohol) modification on the characteristics of protein adsorption on conventional and perfusion polystyrenic matrices.

Poly(styrene-divinylbenzene) (PS-DVB) chromatography matrices, CG1000sd 20-50 microns (TosoHaas), PLRP4000s 15-25 microns, PLRP4000s 50-70 microns (Polymer Laboratories) have been modified by the adsorption and crosslinking of poly(vinyl alcohol) (PVA) to create a matrix suitable for the attachment of dye ligands. The adsorption capacities of lysozyme and HSA on these Procion Yellow HE-3G dyed PVA modified PS-DVB matrices were measured at various flow-rates and the capacities were compared with a Procion Yellow HE-3G dyed OH-activated POROS 20, 20-micron matrix (PerSeptive Biosystems). The adsorption of small proteins was not hindered by the smaller pores of the CG1000sd beads, but as protein size increased, and at high flow-rates, a high mass transfer rate became more dependent on large pore size and small particle diameter.

Preparation and use of ion-exchange chromatographic supports based on perfluoropolymers.

A poly(vinyl alcohol) (PVA) coated particulate perfluoropolymer (FEP) support has been functionalised with ion-exchange groups for use in ion-exchange chromatography of proteins. Anion-exchange (DEAE and Q) and cation-exchange (SP) groups were introduced to PVA-FEP which had previously been activated using cyanuric chloride. The equilibrium adsorption capacities of SP-PVA-FEP were 31.8 and 25.2 mg ml-1 for lysozyme and IgG respectively while for DEAE-PVA-FEP, the equilibrium adsorption capacities were 14.9 and 9.7 mg ml-1 for beta-lactoglobulin and HSA respectively. The equilibrium adsorption capacities for Q-PVA-FEP were determined to be 17.2 and 13.5 mg ml-1 for beta-lactoglobulin and HSA respectively. Experiments carried out to investigate the resolving power of materials showed that both SP and Q-PVA-FEP were able to resolve proteins with only small differences in their isoelectric points and that this resolution could be maintained at a flow-rate of 1500 cm h-1. SP-PVA-FEP was used to purify lysozyme from egg whites where a 50-fold purification, to homogeneity, was achieved in 98% yield. The anion exchanger, Q-PVA-FEP could be used to purify G6PDH from a clarified homogenate of bakers' yeast 14.3-fold in 81% yield.

A new approach to continuous counter-current protein chromatography: Direct purification of malate dehydrogenase from a Saccharomyces cerevisiae homogenate as a model system.

A novel technique for protein chromatography has been developed, which can be used to extract proteins from particulate-containing solutions (such as fermentation broths or preparations of disrupted cells) on a continuous basis, and delivers clarified streams of purified product. Adsorbents deployed in this type of contactor are based on PVA-coated perfluorocarbons derivitized with affinity ligands such as triazine dyes. In this article, we describe the application of this equipment for the continuous purification of malate dehydrogenase from an unclarified homogenate of Saccharomyces cerevisiae, using a Procion Red HE-7B-derivitized adsorbent. Although operating conditions were not optimized to produce a product of maximized purification factor, concentration, and yield, we have shown that MDH can be purified continuously in 78% yield at a rate of 70 U/min, with a purification factor of approximately 10. This corresponds to specific productivity of approximately 0.35 U/min per milliliter of settled adsorbent, a higher specific productivity than was feasible with the same adsorbent using expanded bed adsorption (EBA). (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 427-441, 1997.

Modification of polystyrenic matrices for the purification of proteins. Effect of the adsorption of poly(vinyl alcohol) on the characteristics of poly(styrene-divinylbenzene) beads for use in affinity chromatography.

A poly(styrene-divinylbenzene) (PSDVB) chromatography matrix, CG1000-sd (TosoHaas), has been modified using poly(vinyl alcohol) (PVA) to create a matrix suitable for the attachment of functional groups for the selective purification of proteins. The characteristics of the modified matrix have been studied using a BET nitrogen adsorption/desorption technique and it has been found that the adsorption of PVA results in the bead micropores being filled whilst the bead macropores are left essentially unaltered. There was no protein adsorption onto the modified matrices. A dye ligand (Procion Blue MX-R) has been covalently attached to PVA-PSDVB matrix and the lysozyme capacities of the PVA-PSDVB matrix have been determined. The matrix compares well with commercial Blue Sepharose Fast Flow, an affinity matrix on cross-linked agarose. The dye-PVA-PSDVB matrix is stable when subjected to sanitisation with sodium hydroxide.