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Biomedical subjects

H A Chase

Publications and source records attributed to H A Chase.

At least 37 records · Page 2Linked to original sources
PubMed1997

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.

Animals↗
PubMed1997

Characteristics of the adsorption of immunoglobulin M onto Q Sepharose Fast Flow ion-exchangers.

Measurement of adsorption breakthrough curves in packed beds has shown that the amounts and rates of uptake of immunoglobulin M (IgM) onto the commonly used anionic ion-exchanger Q Sepharose Fast Flow (based on 6% agarose) are severely limited as a result of the large molecular size of this adsorbate (RMM 950,000). A similar ion-exchanger based on a more porous 4% agarose, Q Sepharose 4 Fast Flow was evaluated as an alternative adsorbent for the purification of IgM. Equilibrium adsorption isotherms and the effective diffusivities of IgM within these two adsorbents were measured. Q-Sepharose 4 Fast Flow was found to have a maximum capacity for IgM 2.5 times greater than that of Q Sepharose 6 Fast Flow and the effective diffusivity of IgM was found to be between 6 and 7 times greater than with the latter material. Comparison of the breakthrough curves obtained for these adsorbents at a variety of flow velocities confirm that Q Sepharose 4 Fast Flow is a superior adsorbent for the capture and purification of large proteins.

Adsorption↗
PubMed1994

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.

Adsorption↗
PubMed1993

Novel affinity separations based on perfluorocarbon emulsions. Development of a perfluorocarbon emulsion reactor for continuous affinity separations and its application in the purification of human serum albumin from blood plasma.

Perfluorocarbon affinity emulsions are generated by the homogenisation of a perfluorocarbon oil with a polymeric fluorosurfactant previously derivatised with an affinity ligand and subsequently cross-linked in situ. This procedure gives rise to a novel liquid affinity adsorbent that can be used for continuous protein purification. Discrete emulsion droplets were found to be unstable when pumped for prolonged periods; however, when flocculated, the emulsion floccules with diameters of around 125 microns, were very stable and sedimented faster. A four-stage reactor unit (perfluorocarbon emulsion reactor for continuous affinity separations, PERCAS) was designed and constructed to carry out continuous separations, and exploited the unusual properties of the absorbent, i.e. liquid nature and high density. Each of the four stages of PERCAS consisted of a mixing tank, for contacting between emulsion phase and aqueous phase, adjacent to a settling tank for the subsequent separation of emulsion from the aqueous phase. Using PERCAS adsorption, washing, elution and re-equilibration of the emulsion could be carried out continuously with emulsion recycle. Using single-component adsorption of human serum albumin to a perfluorocarbon affinity emulsion derivatised with the triazine dye C.I. Reactive Blue 2, PERCAS was optimised with respect to flow-rates and input concentrations. The work was then extended to the continuous purification of essentially homogeneous human serum albumin from blood plasma.

Adsorption↗
PubMed1992

Affinity purification of proteins using expanded beds.

The use of expanded beds of affinity adsorbents for the purification of proteins from feedstocks containing whole or broken cells is described. It is demonstrated that such feedstocks can be applied to the bed without prior removal of particulate material by centrifugation or filtration thus showing considerable potential for this approach in simplifying downstream processing flow-sheets. A stable, expanded bed can be obtained using simple equipment adapted from that used for conventional packed bed adsorption and chromatography processes. Circulation and mixing of the adsorbent particles is minimal and liquid flow through the expanded bed shows characteristics similar to those of plug flow. Frontal analysis performed with the highly selective affinity system involving the adsorption of human polyclonal immunoglobulin G onto Protein A Sepharose Fast Flow indicate that the adsorption performance of the expanded bed is similar to that achieved when the same amount of adsorbent is used in a packed configuration at the same volumetric flow-rate. The adsorption performance of the expanded bed was not diminished when adsorption was carried out in the presence of intact yeast cells. Batch adsorption experiments also indicated that the adsorption characteristics of the affinity system were not greatly altered in the presence of cells in contrast to results from a less selective ion-exchange system. An expanded bed of Cibacron Blue Sepharose Fast Flow was used to purify phosphofructokinase from feedstock of disrupted yeast prepared by high pressure homogenisation without the need for prior removal of particulate material. The potential for the use of expanded beds in large scale purification systems is discussed.

Adsorption↗
PubMed1992

Novel affinity separations based on perfluorocarbon emulsions. Use of a perfluorocarbon affinity emulsion for the purification of human serum albumin from blood plasma in a fluidised bed.

A perfluorocarbon affinity emulsion has been generated by homogenisation of a saturated perfluorocarbon oil with a polymeric fluorosurfactant based on poly(vinyl alcohol) (relative molecular mass 9000-10,000) previously derivatised with the triazine dye CI Reactive Blue 4. This affinity emulsion has subsequently been cross-linked in situ and used in a fluidised bed for the purification of human serum albumin (HSA) from blood plasma. HSA was quantitatively recovered in a semi-continuous fashion from plasma at an average purity of 90 +/- 3.3%. The albumin binding capacity of the emulsion has been shown to be 0.59 mg/ml by frontal analysis corresponding to a mol/mol ligand usage of 13.5%. In all regards, when used in a fluidised bed, the emulsions have been shown to behave as a normal chromatographic material. They are stable under operational conditions with no coalescence being observed for periods greater than 1 year. These novel liquid affinity supports present an exciting opportunity to develop a range of unit operations for the continuous purification of proteins.

Chromatography, Affinity↗
PubMed1992

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.

Alcaligenes↗
PubMed1991

Liquid fluidized bed adsorption of protein in the presence of cells.

The adsorption, in a liquid fluidized bed, of Bovine Serum Albumin (BSA), onto an ion-exchange absorbent, Q-Sepharose Fast Flow, in the presence of Alcaligenes eutrophus cells, has been studied. The expansion of the fluidized bed is greater in the presence than in the absence of cells and obeys the laws of Richardson and Zaki. The effect of cell concentration on the equilibrium adsorption characteristics of the adsorbent has been assessed. The rate of adsorption of BSA onto the adsorbent has been studied in a batch stirred tank, and a fluidized bed system both in the presence and absence of cells. Comparisons have been made with the adsorption of human immunoglobulin G (human IgG), onto an affinity adsorbent, Protein A Sepharose CL-4B. The data from the fluidized bed breakthrough tests have been used to assess the validity of a theoretical model adapted from one that predicts the performance of the adsorption phase in the absence of cells in fixed bed systems. Tests have been done on the washing phase in the fluidized bed adsorption system to establish the most efficient method of washing cells and unadsorbed protein out of the bed.

Adsorption↗
PubMed1991

Combined chemical and mechanical processes for the disruption of bacteria.

Mechanical cell disruption by high pressure homogenisation or high speed bead mills is currently the general method of choice for the large scale disruption of micro-organisms; however, deleterious effects include the high energy requirement, the need for efficient cooling to prevent the excessive heating of the product and the micronisation of cell debris. Certain chemical treatments for microbial cell disruption alter the permeability of bacteria and yeasts, allowing partial release of soluble products. Such treatments are insufficient for the recovery of granular intracellular products. As cell wall strength has been cited as a major factor influencing the requirements for efficient mechanical disruption, the use of chemical pretreatment to decrease cell wall strength prior to mechanical breakage by homogenisation has been considered. The following treatments were shown to increase the sensitivity of the Gram-negative bacterium, Alcaligenes eutrophus, to disruption: alkaline pH shock, the addition of an anionic detergent, increase of the monovalent cation concentration, the addition of EDTA and enzymic lysis by lysozyme. These pretreatments allow equivalent disruption to be achieved at lower operating pressures or fewer passes through the homogeniser. Alkaline pH pretreatment at pH 10.5 allowed a 37.5% increase in soluble protein release on subsequent homogenisation. An increase of some 30% in soluble protein release was found following prior addition of 0.137 M monovalent cations (Na+ or K+) at 60 degrees C. Treatment with an anionic detergent showed a considerable decrease in the number of passes required through the homogeniser. Maximum cell rupture can thus be accomplished at reduced energy inputs.

Bacteria↗
PubMed1991

The disruption of Alcaligenes eutrophus by high pressure homogenisation: key factors involved in the process.

The disruption of the Gram-negative bacterium Alcaligenes eutrophus by high pressure homogenisation, using the APV Gaulin 15M 8BA and 30CD homogenisers is reported. The operating parameters such as operating pressure, number of passes, temperature and biomass concentration, mimicked trends previously reported for yeasts. Extension of the study to consider the effect of cell characteristics, including the growth rate, size and shape, illustrated the dominant effect of the growth phase. The improved disruption of bacterial cultures in the logarithmic phase with respect to stationary phase cultures was confirmed by an increased dependence of actively growing cultures on the operating pressure. An increase in size in excess of 30% on the accumulation of the storage product, PHB in the stationary phase caused little change in the ease of disruption. The use of transmission electron microscopy to directly monitor the disruption on multiple passes shed light on the two-stage nature of this disruption process.

Alcaligenes↗
PubMed1990

Two-component protein adsorption to the cation exchanger S Sepharose FF.

A model system, consisting of bovine serum albumin, lysozyme and the cation exchanger S Sepharose FF, was used to investigate multicomponent protein adsorption to ion exchangers. Two models, one based on a complete absence of competition between adsorbing molecules and the other a competitive model, based on the assumption that all adsorption sites are available to both proteins, have been compared to experimental results. Evidence for competitive adsorption was seen in experiments in which breakthrough curves and the profiles of adsorbed proteins in packed beds were determined. However, although the results for packed-bed experiments were more closely predicted by the fully competitive model, some discrepancies were found suggesting that when considering multicomponent protein adsorption to ion exchangers it may also be necessary to take account of factors such as the molecular size of the adsorbing proteins and any potential inter-protein interactions, factors which may hinder the development of a general model of multicomponent protein adsorption to ion exchangers.

Adsorption↗
PubMed1986

Adsorption of proteins on Sepharose affinity adsorbents of varying particle size.

The binding of bovine serum albumin (BSA) and lysozyme to a series of samples of Cibacron Blue F3G-A Sepharose CL-6B with different narrow-range mean particle diameters has been investigated. Significant variation with particle size has been found for the parameters measured. For both proteins the maximum capacities obtained increased with decreasing particle size. For lysozyme, this percentage increase in capacity is less than the percentage increase in external surface area as the particle size decreases. For BSA, however, the percentage increase in capacity is more than four times the increase in surface area. The dissociation constants and forward rate constants describing the affinity interaction also differ with particle size. Again the effects are more pronounced for the larger protein, BSA, than for lysozyme. The effect of these differing parameters on the performance of fixed beds has been assessed using a computer simulation of the adsorption process.

Adsorption↗
PubMed1986

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.

Adsorption↗
PubMed1984

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.

Adsorption↗
PubMed1980

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.

Bacillus megaterium↗