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.

Purification of proteins by adsorption chromatography in expanded beds.

Adsorption in stable expanded beds enables proteins to be recovered directly from particulate-containing feedstocks, such as fermentation broths and preparations of disrupted cells, without the need for prior removal of the suspended solids, which would normally result in the blockage of packed beds. The adoption of this technique will greatly reduce the complexity of downstream processing by eliminating certain filtration, centrifugation and concentration steps. Factors that are critical to the success of the procedure include the correct choice of adsorbent, together with careful design of the apparatus in which the separation is performed. The design, optimization and scale-up of appropriate operating protocols for expanded-bed procedures are very similar to those used for the operation of packed beds.

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.

Direct purification of lysozyme using continuous counter-current expanded bed adsorption.

We describe the application of a novel technique for the continuous counter-current chromatography of proteins. The unit operation has shown potential in extracting targeted species from unclarified feedstocks, delivering clarified streams of purified product. The adsorbent used in this equipment consists of a perfluorocarbon matrix, coated with poly(vinyl alcohol), and derivatized with the triazine dye Procion Red HE-7B. Purification of lysozyme from egg-whites and enriched bovine milk could be carried out continuously. The former was extracted in 90.5% yield at a rate of 7400 U/min, achieving a purification factor of 19.4. Lysozyme from the enriched milk sample was extracted continuously at a rate of 41000 U/min, in 66.0% yield. The continuous products streams in both cases were fully clarified, thus enabling their direct application to a final polishing step, if desired.

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.

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.

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.

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.

Applications of perfluorocarbon affinity emulsions for the rapid isolation of Staphylococcus aureus.

Human immunoglobulin G (IgG) has been immobilized to poly(vinyl alcohol)-stabilized liquid perfluorocarbon droplets. This affinity emulsion has been shown to adsorb Staphylococcus aureus cells from solutions with adsorption capacities of 32 x 10(9) and 48 x 10(9) cells/mL for affinity emulsions with immobilized IgG densities of 1.15 and 2.45 mg/mL, respectively. The kinetics of cell binding from solution were found to be rapid with clearance of S. aureus cells from a suspension (8 x 10(8) cell/mL) achievable in under 5 min. S. aureus cells (1.3 x 10(9) cells) could also be rapidly depleted from a suspension of Saccharomyces cerevisiae (3.4 x 10(10) cell/mL) in under 18 min with no cross-reactivity being observed. The affinity emulsion was stable for at least five cycles of operation with little loss in adsorption capacity when removal of adsorbed cells was carried out at pH 2.5.

Plasticization of poly(hydroxybutyrate) in vivo.

The influence of a variety of treatments on the mobility and crystallinity of poly(hydroxybutyrate) (PHB) in whole cells and native granules has been proved using 13C-n.m.r. spectroscopy and X-ray powder diffraction, and correlated with the known biological effects of these treatments. It was concluded that at least water is responsible for PHB plasticization in vivo, and that only native mobile PHB is susceptible to depolymerases. Another, probably hydrophobic, component appears to be involved either as plasticizer or nucleation inhibitor. Three states of the granule are identified in addition to the native, biologically-competent state: freeze-drying of whole cells leads to a partially-immobilized amorphous state which can be restored virtually to native mobility by rehydration; extended centrifugation of native granules in aqueous suspension, or treatment with hydrophobic detergents under certain conditions, leads to a crystalline state that is less susceptible to exogenous depolymerase; and heating to 95 degrees C or refrigeration has no detectable effect on mobility but leads to inactivation of the granule, presumably via damage to superficial membrane or protein.

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.

Prediction of the performance of preparative affinity chromatography.

This paper describes a theoretical approach to the prediction of the performance of preparative affinity separations for biological macromolecules in packed columns. The approach, which is applicable to conventional low-pressure packed column methods as well as high-performance liquid chromatography techniques, requires knowledge of certain parameters that describe the interactions between adsorbent and adsorbate during the affinity separation procedure. We have measured the parameters appropriate to the adsorption stages of affinity systems involving immobilised Cibacron Blue and immobilised monoclonal antibodies against beta-galactosidase. The theoretical predictions appear to agree well with the experimental performance of batch and packed column affinity systems. The influence of the factors that govern the performance of the adsorption stage of the separation procedure is explained in detail, and the possible advantages of using HPLC techniques in macropreparative affinity chromatography are discussed.

Rapid chromatographic monitoring of bioprocesses.

This paper describes the use of rapid chromatographic separation systems to monitor the level of specific proteins in various bioprocesses such as downstream processing and fermentation. In these monitoring systems, samples of the liquid are continuously extracted from the process and the proteins resolved from one another by a rapid chromatographic separation. The peak on the chromatogram corresponding to the protein of interest is identified and quantified to obtain on-line information on the level of that protein in the bioprocess. There are a number of advantages in using affinity separations as the rapid chromatographic principle. In particular, the use of immobilised monoclonal antibodies potentially allows a chromatographic sensor to be constructed for any protein against which a suitable antibody can be raised. The potential of this technique is illustrated with various examples, including measurement of the levels of monoclonal antibody in tissue culture supernatant using immobilised Protein A as the affinity adsorbent. A discussion of the inherent limitations of this type of protein biosensor is also included.

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.

Purification of four penicillin-binding proteins from Bacillus megaterium.

Four of the five penicillin-binding proteins in the cytoplasmic membranes of Bacillus megaterium have been purified to protein homogeneity. The method used involved the solubilization of the penicillin-binding proteins from the membranes by treatment with non-ionic detergent, followed by partial separation of the proteins by ion-exchange chromatography on DEAE-Sepharose CL-6B. Each protein was then purified to protein homogeneity by covalent affinity chromatography on ampicillin-affinose. The protein with the lowest molecular weight is a DD-carboxypeptidase. The other three proteins have previously been postulated to be peptidoglycan transpeptidases, endopeptidases or DD-carboxypeptidases in vivo, but it was not possible to demonstrate any of these activities with the purified proteins in various in vitro systems. Possible reasons for the observed lack of enzymic activity in vitro are discussed.

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

Affinity adsorption of Saccharomyces cerevisiae on concanavalin A perflurocarbon emulsions.

Perfluorocarbon affinity emulsions have been generated by immobilizing concanavalin A onto the surface of triazine-activated perfluorocarbon droplets. Immobilized concanavalin A densities of 0.1, 0.7 and 2.1 mg/ml were obtained by varying the concentration of cyanuric chloride used for activation. The affinity emulsions were found to adsorb Saccharomyces cerevisiae cells from solution with adsorption capacities of 1 x 10(9) cells, 4.6 x 10(9) and 6 x 10(9) cell/ml, respectively. Optimal conditions for the elution of bound cells were obtained by studying inhibition curves of concanavalin A-mannan precipitation using simple sugars. Methyl alpha-D-mannopyranoside showed the greatest inhibitory power with 50 per cent inhibition displayed at a concentration of 0.05 mM. Experiments carried out examining the concentrations of methyl alpha-D-mannopyranoside necessary to dissociate a concanavalin A-mannan precipitate demonstrated that at least a seven-fold higher concentration of methyl alpha-D-mannopyranoside was required for dissociation than that required for inhibition of its formation. The efficiency of elution of bound yeast cells was found to be dependent on the concentration of methyl alpha-D-mannopyranoside used, the time of elution and the immobilized ligand density. Thus, 100 per cent elution was obtained with a concanavalin A affinity emulsion (0.1 mg/ml) by incubation with 500 mM methyl alpha-D-mannopyranoside for 1 h.

Ion exchange purification of G6PDH from unclarified yeast cell homogenates using expanded bed adsorption.

The use of expanded beds of STREAMLINE ion exchange adsorbents for the direct extraction of an intracellular enzyme glucose-6-phosphate dehydrogenase (G6PDH) from unclarified yeast cell homogenates has been investigated. It has been demonstrated that such crude feedstocks can be applied to the bed without prior clarification steps. The purification of G6PDH from an unclarified yeast homogenate was chosen as a model system containing the typical features of a direct extraction technique. Optimal conditions for the purification were determined in small scale, packed bed experiments conducted with clarified homogenates. Results from these experiments were used to develop a preparative scale separation of G6PDH in a STREAMLINE 50 EBA apparatus. The use of an on-line rotameter for measuring and controlling the height of the expanded bed when operated in highly turbid feedstocks was demonstrated. STREAMLINE DEAE has been shown to be successful in achieving isolation of G6PDH from an unclarified homogenate with a purification factor of 12 and yield of 98% in a single step process. This ion exchange adsorbent is readily cleaned using simple cleaning-in-place procedures without affecting either adsorption or the bed expansion properties of the adsorbent after many cycles of operation. The ability of combining clarification, capture, and purification in a single step will greatly simplify downstream processing flowsheets and reduce the costs of protein purification. (c) 1996 John Wiley & Sons, Inc.