Howard A. Chase

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.

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.

Reducing production of excess biomass during wastewater treatment

Excess biomass produced during the biological treatment of wastewaters requires costly disposal. As environmental and legislative constraints increase, thus limiting disposal options, there is considerable impetus for reducing the amount of biomass produced. This paper reviews biomass production during wastewater treatment and identifies methods for reducing the quantity of biomass produced. Efforts to reduce biomass production during aerobic metabolism must promote the conversion of organic pollutants to respiration products with a concomitant increase in the aeration requirements. Promoting further metabolism of assimilated organic carbon will release additional respiration products and reduce the overall biomass production e.g. by inducing cell lysis to form autochtonous substrate on which cryptic growth occurs or by encouraging microbial predation by bacteriovores. Uncoupling metabolism such that catabolism of substrate can continue unhindered while anabolism of biomass is restricted would achieve a reduction in the biomass yield. Metabolite overproduction in substrate excess conditions has been demonstrated in several bacterial species and can result in an increase in the substrate uptake while resulting in a decreased yield and increased carbon dioxide evolution rate. Addition of protonphores to uncouple the energy generating mechanisms of oxidative phosphorylation will stimulate the specific substrate uptake rate while reducing the rate of biomass production. Increasing the biomass concentration such that the overall maintenance energy requirements of the biomass within the process are increased can significantly reduce the production of biomass. Suitable engineering of the physical conditions and strategic process operation may result in environments in which biomass production may be reduced. It is noted that as biomass settling characteristics are a composite product of the microbial population, any changes which result in a shift in the microbial population may affect the settling properties. Reduced biomass production may result in an increased nitrogen concentration in the effluent. Anaerobic operation alleviates the need for costly aeration and, in addition, the low efficiency of anaerobic metabolism results in a low yield of biomass, its suitability for wastewater treatment is discussed. A quantitative comparison of these strategies is presented.

Modelling single-component protein adsorption to the cation exchanger S sepharose FF

Abstract The equilibrium and kinetic characteristics of the adsorption of bovine serum albumin (BSA) and lysozyme to the strong cation exchanger S Sepharose FF have been determined. The rates of protein adsorption have been compared to two different models, the first being based on a single lumped kinetic parameter, whilst the second model considers the individual transport processes occurring prior to the adsorption reaction, that is taking into account diffusion across the liquid film surrounding individual particles and also the diffusion within the ion-exchanger particle itself. It was found that the adsorption of lysozyme to S Sepharose FF, in both batch, agitated tanks and in packed beds was consistent with both models. In the case of BSA however, the agitated tank adsorption profile was consistent only with the pore diffusion model and neither model correctly predicted the latter part of the breakthrough profile observed in packed-bed experiments.

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.

Affinity separations utilising immobilised monoclonal antibodies—a new tool for the biochemical engineer

Abstract This review paper describes a new technique in biochemical engineering that is now being used for the purification of proteins for use as pharmaceuticals or in other applications where the purity of the product is an important consideration. The technique involves the immobilisation of antibodies to inert supports in order to form biospecific adsorbents that can be used in purification processes. The biospecific adsorbents that are formed have a high affinity for a single compound and are ideally suitable for the purification of proteins from a variety of biological sources. The main aim of the review is to introduce the biochemical principles on which the technique is based in order to lay the foundations for the adoption of the process in the near future as a new industrial unit operation. The review covers briefly the problems associated with the purification of proteins on a large scale and highlights the potential of affinity separations as industrial unit operations for this purpose. The reasons for using immobilised monoclonal antibodies are explained and advantages and disadvantages are discussed. The design of all stages of an affinity separation process is reviewed and a theoretical analysis of mass transfer in biospecific adsorption processes is presented. The performance of simple batch and fixed bed adsorption systems is compared and the suitability of their use in large scale separations is discussed together with techniques and procedures for the elution of the product and the regeneration of the adsorbent. Other aspects of the process design that are covered include the control of the repetitive operation of an affiinity separation process and the choice of support matrix and method of antibody coupling. Finally there is a brief consideration of other downstream processing stages likely to be necessary on both sides of the affinity separation process.

Development of operating conditions for protein purification using expanded bed techniques: The effect of the degree of bed expansion on adsorption performance

A strategy for the optimization of an expanded bed adsorption process has been developed by studying a model system involving the adsorption of lysozyme onto the adsorbent STREAMLINE SP. The hydrodynamic and adsorption properties of this ion exchange adsorbent in a variety of viscosities of feedstocks have been compared by analyzing bed expansion characteristics, liquid phase dispersion characteristics, equilibrium adsorption isotherms, and mass transfer characteristics. Additionally, the influences of the degree of bed expansion on adsorption performance have been investigated by frontal analysis. In these experiments, viscous feedstocks were simulated by the inclusion of glycerol in the adsorption buffers. Breakthrough curves for lysozyme were characterized and compared in terms of overall purification processing time and productivity. On the basis of these results, the relative productivities of different operating modes with the same process liquid were found to be almost the same. However, the processing time for each purification cycle decreased with increasing velocity of process liquid. It is demonstrated that an adsorption process carried out at a constant degree of bed expansion (twice its settled bed height, corresponding to bed voidage of 0.7) is more efficient, when characterized by the apparent dynamic binding capacity, than operation at a constant liquid velocity of 300 cm/h. These results have significant implications on the design and operation of the expanded bed adsorption procedures. The advantages and problems encountered in the use of expanded bed techniques for the direct extraction of proteins from unclarified feedstocks are also discussed.

Uncoupling of metabolism to reduce biomass production in the activated sludge process

Production of excess biomass during biological treatment of wastewaters requires costly disposal. Also with environmental and legislative constraints limiting disposal options, considerable impetus exists for reducing the amount of biomass produced. Uncoupling metabolism in activated sludge may reduce biomass production and this approach is investigated in conjunction with consequences upon substrate removal and population dynamics. To induce uncoupled metabolism, para-nitrophenol (pNP) a known protonphoric uncoupler of oxidative phosphorylation, was introduced to a bench-scale activated sludge process. Microbial populations were monitored by both microscopic and by three methods of molecular analyses. Presence of the protonphore caused a shift in the microbial population with protozoa being washed out of the system and filamentous bacteria proliferating. The molecular composition of the microbial community was determined by PCR amplification of 16SrRNA genes and subsequent denaturing gradient gel electrophoresis (DGGE). Band Patterns obtained by both a direct and nested approach were similar. However, profiles derived from nested PCR contained more bands, indicative of the increased sensitivity of this approach. Analysis of the active biomass by Polyacrylamide Gel Electrophoresis (PAGE) of small molecular weight RNA (5mwRNA) showed that a sustained shift in the diversity of the predominant, metabolically active species present occurred within two days of the introduction of the protonphore. Biomass production was reduced by 49%, but the total substrate removal rate was also reduced by 25%. The combined effect was a 30% decrease in the biomass yield. Introduction of the protonphore caused substrate removal efficiency to decrease from a consistent value of 96% to 68.5% with considerable variance. This decline in overall process performance was attributed to a surmountable effect arising from the design of apparatus that resulted in a decrease in the reactor biomass concentration. Although the specific biomass volume was consistent throughout, decreased sedimentation resulted in solids being removed in the final effluent which decreased the amount of biomass which could be recycled. The catalytic efficiency of the biomass increased as reflected by a 3.3 fold increase in the specific substrate uptake rate.

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.

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.