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Biomedical subjects

N J Titchener-Hooker

Publications and source records attributed to N J Titchener-Hooker.

14 recordsLinked to original sources
PubMed2002

Hydrophobic interaction ligand selection and scale-up of an expanded bed separation of an intracellular enzyme from Saccharomyces cerevisiae.

A prototype Streamline-Phenyl matrix was evaluated in a hydrophobic interaction mode for the direct recovery of alcohol dehydrogenase (ADH) from yeast cell homogenate. At 5% breakthrough of ADH, a yield of 100% was obtained for a dynamic expanded bed capacity of 240 U(ADH)/ml matrix with a purification factor of 9.2. This compared with a dynamic capacity of 3013 U(ADH)/ml matrix for the packed bed equivalent and a purification factor of 18. In both systems the purification factor was found to increase simultaneously with a decrease in yield as the load of homogenate or breakthrough of ADH was increased. The expanded bed mode of operation conferred considerable robustness with respect to process fouling. No loss in yield was seen over five cycles of repeat loading with an unclarified homogenate. By contrast the packed bed media showed a decrease in yield from 86 to 56% over the same period. Successful scale up of the expanded bed protocol for a 20% breakthrough was demonstrated over a fourfold increase in column diameter. The application of hydrophobic interaction chromatography mediated expanded bed adsorption and its scale-up is discussed in the context of large-scale operations.

Adsorption↗
PubMed2001

Graphical method for the calculation of chromatographic performance in representing the trade-off between purity and recovery.

A simple engineering framework that enables the rapid representation of the performance of liquid chromatographic separations is provided in this paper. The fractionation diagram and its associated maximum purification factor versus product yield, and contamination index versus product yield diagrams, may be derived directly from chromatographic data. The fractionation diagram plots the relative change in the cumulative fractional mass of product eluted with the corresponding fractional total mass eluted, while the maximum purification factor versus yield diagram shows the degree of trade-off between the levels of purity and recovery achieved in the chromatographic step. The minimum contamination index versus yield plot is especially suitable for cases where the product and impurity are expressed in different units and shows how the extent of contaminant removal changes relative to product yield. These diagrams are more straightforward and easily interpretable compared to the basic conventional chromatograms and enable investigation of the degree of trade-off between purity and recovery for any set of operating conditions to be made. The approach is demonstrated for two different chromatographic systems. In the first, a set of simulation results from a verified size exclusion model is used to demonstrate the approach for product recovery. In the second, a set of experimental results for the removal of endotoxin from DNA is used. This demonstrates a problem where the product and impurity content are measured by different assay techniques and are expressed in different units, and also where the quality of process information is limited by the small number of fractions collected. The studies show how such an approach can help to identify the optimal operating conditions, in terms of acceptable yield and desired level of contaminant removal, and to redefine the location of product fractions needed to achieve these specifications.

Algorithms↗
PubMed2001

Economic comparison between conventional and disposables-based technology for the production of biopharmaceuticals.

Time to market, cost effectiveness, and flexibility are key issues in today's biopharmaceutical market. Bioprocessing plants based on fully disposable, presterilized, and prevalidated components appear as an attractive alternative to conventional stainless steel plants, potentially allowing for shorter implementation times, smaller initial investments, and increased flexibility. To evaluate the economic case of such an alternative it was necessary to develop an appropriate costing model which allows an economic comparison between conventional and disposables-based engineering to be made. The production of an antibody fragment from an E. coli fermentation was used to provide a case study for both routes. The conventional bioprocessing option was costed through available models, which were then modified to account for the intrinsic differences observed in a disposables-based option. The outcome of the analysis indicates that the capital investment required for a disposables-based option is substantially reduced at less than 60% of that for a conventional option. The disposables-based running costs were evaluated as being 70% higher than those of the conventional equivalent. Despite this higher value, the net present value (NPV) of the disposables-based plant is positive and within 25% of that for the conventional plant. Sensitivity analysis performed on key variables indicated the robustness of the economic analysis presented. In particular a 9-month reduction in time to market arising from the adoption of a disposables-based approach, results in a NPV which is identical to that of the conventional option. Finally, the effect of any possible loss in yield resulting from the use of disposables was also examined. This had only a limited impact on the NPV: for example, a 50% lower yield in the disposable chromatography step results in a 10% reduction of the disposable NPV. The results provide the necessary framework for the economic comparison of disposables and conventional bioprocessing technologies.

Antibodies, Bacterial↗
PubMed2001

Prediction of the pilot-scale recovery of a recombinant yeast enzyme using integrated models.

This article describes the rapid prediction of recovery process performance for a new recombinant enzyme product on the basis of a broad portfolio of computer models and highly targeted experimentation. A process model for the recombinant system was generated by linking unit operation models in an integrated fashion, with required parameter estimation and physical property determination accomplished using data from scale-down studies. This enabled the generic modeling framework established for processing of a natural enzyme from bakers' yeast to be applied. An experimental study of the same operations at the pilot scale showed that the process model gave a conservative prediction of recombinant enzyme recovery. The model successfully captured interactions leading to a low overall product yield and indicated the need for further study of precipitate breakage in the feed zone of a disc stack centrifuge in order to improve performance. The utility of scale-down units as an aid to fast model generation and the advantage of integrating computer modeling and scale-down studies to accelerate bioprocess development are highlighted.

Alcohol Dehydrogenase↗
PubMed2000

The use of rapid on-line monitoring of products and contaminants from within an expanded bed to control separations exhibiting fast breakthrough characteristics and to maximize productivity.

Conventional control of expanded-bed adsorption (EBA), like that of packed-bed chromatography, is based upon off-line measurements of the column eluant. The relatively high-void volumes in EBA systems means that this approach can lead to significant performance losses caused by the inability to achieve tight control of breakthrough. This problem is made worse if the product has a fast breakthrough characteristic or if it is necessary to operate to low levels of product loss. In this article we examine the utility of constant on-line monitoring from within the expanded bed using stopped-flow analysis (SFA) to provide data for the control of the expanded-bed operation. A modified Streamline 50 column with side ports that enable sampling along the expanded axis of the bed was used. Comparisons between off-line and on-line measurements are presented, showing how the advanced monitoring method can lead to better control and to an analysis of breakthrough development within the bed. The expanded bed was used to purify alcohol dehydrogenase from homogenized suspensions of bakers' yeast. Accurate control of breakthrough to 10% of the target enzyme was achieved using a SFA control system with a response time of 40 seconds. On-line data compared well to assays carried out off-line on the outlet stream for both the product enzyme (ADH), total protein, RNA, and cell debris levels (via UV 650 nm). This information was used to generate a series of graphs with which to track the EBA process in real-time. Results showed that bed utilization was not linear along the bed axis so that, for example, 60% of ADH is bound in the bottom 33% of the column during loading.

Alcohol Dehydrogenase↗
PubMed2000

Experimental measurement of particle size distribution and voidage in an expanded bed adsorption system.

This paper presents an experimental analysis of matrix bead size distribution and voidage variations with axial height in an expanded bed adsorption system. Use of a specially constructed expanded bed with side ports has enabled sampling from within the expanded bed along the vertical axis. Particles removed from within the bed were measured for their size distributions. Residence time distribution studies were used to estimate bed voidage. Measurements of axial and radial particle size distributions and axial voidage distribution have been made at different flow rates. Particle size was found to be radially constant, indicating constant stratification in the column. The particle size was found to decrease with increasing axial height. Voidage increased with axial height from a settled bed value of 0.39 to approaching unity for high liquid velocities and increased at a constant axial position with increased flowrate. This information provides key insight into bed stability and data for the improved modeling of this important unit operation.

Adsorption↗
PubMed1999

Visualizing integrated bioprocess designs through "windows of operation".

This paper demonstrates a simple graphical approach for the design and analysis of a bioprocess flowsheet in which process interactions are significant. Results are presented showing how the feasible space for operation can be simulated and used both to address key design and operating decisions and to identify suitable trade-offs between operating variables, such as fermentation growth rate and disruption conditions, in order to achieve prespecified levels of process performance. Using verified models to describe the production and isolation of an intracellular protein alcohol dehydrogenase (ADH) in yeast as a test bed, a series of so-called "windows of operation" are developed at growth rates in the range of 0.06-0.28 h(-1) and for a range of overall process specifications. The effects of altering the process design performance specification as defined by the level of cell debris removal and the overall process productivity on the size and position of the feasible space were investigated to demonstrate the sensitivity of the flowsheet to changes in process objectives. Using the approach it has been possible to visualise the processing trade-offs required to increase performance in terms of the level of cell debris removal by 50% and the overall process productivity by 400% from a defined base level. The approach provides a convenient tool when designing integrated bioprocesses by enabling process options to be compared visually and can help in achieving better process designs and accelerating process development for the biological process industry.

Alcohol Dehydrogenase↗
PubMed1999

Immobilised metal ion affinity chromatography purification of alcohol dehydrogenase from baker's yeast using an expanded bed adsorption system.

Alcohol dehydrogenase (ADH) from solutions of homogenised packed bakers' yeast has been successfully purified using immobilised metal-ion affinity chromatography in an expanded bed. Method scouting carried out using pure ADH solutions loaded onto 5-ml HiTrap columns charged with Zn2+, Ni2+ and Cu2+ and eluted using 0-50 mM EDTA gradient found that charging with Zn2+ gave the highest recovery and the lowest EDTA concentration required for elution. These results were used to develop a protocol for the expanded bed system and further tested using clarified yeast homogenate loaded onto XK16/20 packed beds (approximately 30 ml) packed with Chelating Sepharose FastFlow matrix in order to determine the optimum elution conditions using EDTA. The ADH was found to elute at 5 mM EDTA and the dynamic and total binding capacities of Streamline chelating for ADH were found to be 235 U/ml and 1075 U/ml matrix, respectively. Expanded bed work based on a step EDTA elution protocol demonstrated that ADH could be successfully eluted from unclarified homogenised bakers' yeast diluted to 10 mg/ml total protein content with a recovery of 80-100% that was maintained over five consecutive runs with a vigorous clean-in-place procedure between each run.

Adsorption↗
PubMed1998

Time course of SDS-alkaline lysis of recombinant bacterial cells for plasmid release.

SDS-alkaline lysis of recombinant Escherichia coli cell suspensions was carried out in a coaxial cylinder rheometer, and the data were used to establish the time course of lysis reaction. The results of the experiments showed that cell lysis reaction time depended on cell strain but was unaffected by plasmid size and plasmid copy number. The high molecular weight globular proteins and chromosomal DNA were denatured, and the resulting changes in rheometric measurements characterised the denaturation time.

Escherichia coli↗
PubMedDate not supplied

Selective flocculation and precipitation for the improvement of virus-like particle recovery from yeast homogenate.

The purification of an intracellular product from a complex mixture of contaminants after cell disruption is a common problem in processes downstream of fermentation systems. This is particularly challenging for the recovery of particulate (80 nm in diameter) multimeric protein products, named virus-like particles (VLPs), from cell debris and other intracellular components. Selective flocculation for debris removal followed by selective precipitation of the target protein can be used as a preclarification step to aid purification. In this paper, selective borax flocculation of cell debris in yeast homogenate, followed by selective poly(ethylene glycol) precipitation of VLPs are defined with a view to demonstrating their potential in aiding the initial clarification stages of the purification sequence. The translation from laboratory scale to pilot scale operation is addressed, demonstrating the challenge of scale-up of solid-liquid separation stages for biological particle processing.

Borates↗
PubMedDate not supplied

A tool for modeling strategic decisions in cell culture manufacturing.

The development of a prototype tool for modeling manufacturing in a biopharmaceutical plant is discussed. A hierarchical approach to modeling a manufacturing process has been adopted to confer maximum user flexibility. The use of this framework for assessing the impact of manufacturing decisions on strategic technical and business indicators is demonstrated via a case study. In the case study, which takes the example of a mammalian cell culture process delivering a therapeutic for clinical trials, the dynamic modeling tool indicates how manufacturing options affect the demands on resources and the associated manufacturing costs. The example illustrates how the decision-support software can be used by biopharmaceutical companies to investigate the effects of working toward different strategic goals on the cost-effectiveness of the process, prior to committing to a particular option.

Biotechnology↗
PubMedDate not supplied

A study of process interactions between cell disruption and debris clarification stages in the recovery of yeast intracellular products.

The effects of varying the levels of cell disruption achieved by high-pressure homogenization upon subsequent centrifugal separation of cell debris have been defined by measuring sedimentation velocity distributions of the cell debris. Using this data together with particle size distributions, a computer-based model of a disk stack centrifuge has been used to predict the efficiency of centrifugal separation. These predictions have been compared with both laboratory scale and pilot plant scale experimental results. A similar sedimentation velocity technique has been used to characterize the properties of the flocs formed by addition of poly(ethylene imine) to a borax-clarified homogenate supernatant. The variations of floc settling properties with respect to polymer concentration are defined.

Biotechnology↗
PubMedDate not supplied

Use of at-line spectrophotometry for the rapid definition of pilot-scale flocculation processes.

Traditionally most downstream bioprocesses have been operated without real-time knowledge of product and key contaminants, yielding little confidence in their operation and the impact on subsequent operations. A rapid UV-vis spectral prediction technique has been successfully demonstrated for the at-line characterization of a large scale continuous flocculation process in terms of RNA, key protein contaminants, and cell debris. A comparison was made between the spectral predictions and retrospective wet chemical assays, and a highly linear correlation was obtained. The spectral analysis technique allowed for real-time system information, which was applied to control the flocculation process to maintain satisfactory process performance, even when subjected to given possible process disturbances.

Alcohol Dehydrogenase↗