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Biomedical subjects

M Hoare

Publications and source records attributed to M Hoare.

At least 19 recordsLinked to original sources
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

Metabolic control of recombinant protein N-glycan processing in NS0 and CHO cells.

Chinese hamster ovary and murine myeloma NS0 cells are currently favored host cell types for the production of therapeutic recombinant proteins. In this study, we compared N-glycan processing in GS-NS0 and GS-CHO cells producing the same model recombinant glycoprotein, tissue inhibitor of metalloproteinases 1. By manipulation of intracellular nucleotide-sugar content, we examined the feasibility of implementing metabolic control strategies aimed at reducing the occurrence of murine-specific glycan motifs on NS0-derived recombinant proteins, such as Galalpha1,3Galbeta1,4GlcNAc. Although both CHO and NS0-derived oligosaccharides were predominantly of the standard complex type with variable sialylation, 30% of N-glycan antennae associated with NS0-derived TIMP-1 terminated in alpha1,3-linked galactose residues. Furthermore, NS0 cells conferred a greater proportion of terminal N-glycolylneuraminic (sialic) acid residues as compared with the N-acetylneuraminic acid variant. Inclusion of the nucleotide-sugar precursors, glucosamine (10 mM, plus 2 mM uridine) and N-acetylmannosamine (20 mM), in culture media were shown to significantly increase the intracellular pools of UDP-N-acetylhexosamine and CMP-sialic acid, respectively, in both NS0 and CHO cells. The elevated UDP-N-acetylhexosamine content induced by the glucosamine/uridine treatment was associated with an increase in the antennarity of N-glycans associated with TIMP-1 produced in CHO cells but not N-glycans associated with TIMP-1 from NS0 cells. In addition, elevated UDP-N-acetylhexosamine content was associated with a slight decrease in sialylation in both cell lines. The elevated CMP-sialic acid content induced by N-acetylmannosamine had no effect on the overall level of sialylation of TIMP-1 produced by both CHO and NS0 cells, although the ratio of N-glycolylneuraminic acid:N-acetylneuraminic acid associated with NS0-derived TIMP-1 changed from 1:1 to 1:2. These data suggest that manipulation of nucleotide-sugar metabolism can promote changes in N-glycan processing that are either conserved between NS0 and CHO cells or specific to either NS0 cells or CHO cells.

Animals↗
PubMed2001

The formation of plasmid DNA loaded pharmaceutical powders using supercritical fluid technology.

The invention of novel drugs based on biological macromolecules requires the development of specialized formulation methods. Supercritical fluid technology offers the possibility to produce dry powder formulations suitable for inhalation or needle-free injection. In this article we describe the first application of a process involving supercritical carbon dioxide for the production of plasmid DNA-loaded particles. The technique of solution enhanced dispersion by supercritical fluids (SEDS) is used to coformulate the 6.9 kb plasmid pSV beta with mannitol as excipient. After initial experiments showed a high degradation of the plasmid during powder formation, a systematic investigation of the process revealed pH effects to be crucial for the recovery of intact DNA. The application of high-buffer concentration led to an increase of the recovered supercoiled proportion from 7% to 80%.

Biotechnology↗
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

Rapid monitoring for the enhanced definition and control of a selective cell homogenate purification by a batch-flocculation process.

Downstream-bioprocess operations, for example, selective flocculation, are inherently variable due to fluctuations in feed material, equipment performance, and quality of additives such as flocculating agents. Due to these fluctuations in operating conditions, some form of process control is essential for reproducible and satisfactory process performance and hence, product quality. Both product (alcohol dehydrogenase) and key contaminants (RNA, protein, cell debris) within a Saccharomyces cerevisiae system were monitored in real-time adopting an at-line enzymatic reaction and rapid UV-VIS spectral-analysis technique every 135 seconds. The real-time measurements were implemented within two control configurations to regulate the batch-flocculation process according to prespecified control objectives, using the flocculant dose as the sole manipulative variable. An adaptive, model-based control arrangement was studied, which combined the rapid measurements with a process model and two model parameter-identification techniques for real-time prediction of process behavior. Based on an up-to-date mathematical description of the flocculation system, process optimization was attained and subsequent feedback control to this optimum operating set point was reproducibly demonstrated with a 92% accuracy. A simpler control configuration was also investigated adopting the cell debris concentration as the control variable. Both control arrangements resulted in superior flocculation-process performances in terms of contaminant removal, product recovery, and excess flocculant usage compared to an uncontrolled system.

Alcohol Dehydrogenase↗
PubMed2000

Laboratory scaledown of protein purification processes involving fractional precipitation and centrifugal recovery.

The ability to predict the performance of large-scale processes is central to the rapid development of successful operations at the pilot and industrial scale. In this article, we examine the operation, at laboratory scale, of precipitation reactors and centrifuges for protein precipitate recovery and dewatering and how they might best mimic large-scale reactors and centrifuges, in this case, a pilot-scale batch stirred-tank reactor and a multichamber-bowl centrifuge. Novel approaches to bench-top centrifuge operation are provided, in particular with a view to delivery of material for subsequent high-resolution purification, which would be obtained at full pilot scale. Results are presented in terms of properties of the protein precipitates, the fraction of solids recovered, and the extent of dewatering achieved. Good agreement was obtained at bench scale (a 1000-fold scale down factor) for all of these parameters for pilot-scale, batch-feed operation. In addition, the methodology developed allows identification of the extent of break-up that occurs in continuous-feed centrifuges when processing shear-sensitive materials such as the protein precipitates studied here.

Alcohol Dehydrogenase↗
PubMed2000

An interlaboratory comparison of physiological and genetic properties of four Saccharomyces cerevisiae strains.

To select a Saccharomyces cerevisiae reference strain amenable to experimental techniques used in (molecular) genetic, physiological and biochemical engineering research, a variety of properties were studied in four diploid, prototrophic laboratory strains. The following parameters were investigated: 1) maximum specific growth rate in shake-flask cultures; 2) biomass yields on glucose during growth on defined media in batch cultures and steady-state chemostat cultures under controlled conditions with respect to pH and dissolved oxygen concentration; 3) the critical specific growth rate above which aerobic fermentation becomes apparent in glucose-limited accelerostat cultures; 4) sporulation and mating efficiency; and 5) transformation efficiency via the lithium-acetate, bicine, and electroporation methods. On the basis of physiological as well as genetic properties, strains from the CEN.PK family were selected as a platform for cell-factory research on the stoichiometry and kinetics of growth and product formation.

Journal Article↗
PubMed2000

Accelerated prediction of recombinant protein production in Saccharomyces cerevisiae by using rapid monitoring techniques.

The use of a stopped-flow analyser for the monitoring of the production of a secreted recombinant protein, a wild-type cutinase, from S. cerevisiae CEN.PK111-32D pUR7320 is described. Induction is through use of a galactose promoter, and the monitoring facility is used to record the formation of the cutinase and cell density with optical density measurements. A range of induction conditions was studied with a view to using the monitoring to predict the likely level of cutinase formation. Results achieved within 4 to 5 h of induction were of sufficient quality to allow the use of simple modelling relating cutinase formation and cell production to predict likely final specific activities of the product. The utility of such monitoring and prediction is discussed with regards to improved process confidence and definition during fermentation production.

Journal Article↗
PubMed2000

The use of laboratory centrifugation studies to predict performance of industrial machines: studies of shear-insensitive and shear-sensitive materials.

A method for using a bench-top centrifuge is described in order to mimic the recovery performance of an industrial-scale centrifuge, in this case a continuous-flow disc stack separator. Recovery performance was determined for polyvinyl acetate particles and for biological process streams of yeast cell debris and protein precipitates. Recovery of polyvinyl acetate particles was found to be well predicted for these robust particles. The laboratory centrifugation scale-down technique again predicted the performance of the disc stack centrifuge for the recovery of yeast cell debris particles although there was some suggestion of over-prediction at high levels of debris recovery due to the nature of any cell debris aggregates present. The laboratory centrifuge scale-down technique also proved to be an important investigative probe into the extent of shear-induced breakup of shear-sensitive protein precipitate aggregates during recovery in continuous high speed centrifuges. Such breakup can lead to over 10-fold reduction in separator capacity.

Acceleration↗
PubMed1999

Distribution of the receptor EphA7 and its ligands in development of the mouse nervous system.

EphA7 is a receptor tyrosine kinase of the Eph family. We have mapped EphA7 immunoreactivity and ligand binding in mouse embryo heads and developing brain. Immunoreactivity for the full-length receptor is found in all the cell populations that express EphA7 mRNA. In particular, it is located on growing axons from EphA7-expressing neurons, both in the trigeminal nerve and in developing brain. In many cases it persists in terminal fields in adult brain. Ligand is detected in a largely complementary distribution in embryos, but is surprisingly weak or undetectable in the target regions of many EphA7-positive axons postnatally.

Animals↗
PubMed1999

Near-infrared spectroscopy for bioprocess monitoring and control.

This article describes the calibration of a spectroscopic scanning instrument for the measurement of selected contaminants in a complex biological process stream. Its use is for the monitoring of a process in which contaminants are to be removed selectively by flocculation from yeast cell homogenate. The main contaminants are cell debris, protein, and RNA. A low-cost instrument has been developed for sensitivity in the region of the NIR spectrum (from 1900 to 2500 nm) where preliminary work found NIR signatures from cell debris, protein, and RNA. Calibration models have been derived using a multivariate method for concentrations of these contaminants, such as would be found after the flocculation process. Two strategies were compared for calibrating the NIR instrument. In one case, samples were prepared by adding materials representative of the contaminants to clarified yeast homogenate so the contaminant levels were well known but outside the range of interest. In the other case, where samples were like those from the process stream after flocculation and floc removal, there was uncertainty of analysis of contaminant level, but the calibration was in the range of interest. Calibration using process stream samples gave results close to those derived from traditional assays. When the calibration models were used to predict the contaminant concentrations in previously unseen samples, the correlation coefficients between measurements and predictions were above 90% in all cases but one. The prediction errors were similar to the errors in the traditional assays.

Alcohol Dehydrogenase↗
PubMed1999

Virus-like particle analysis in yeast homogenate using a laser light-scattering assay.

Virus-like particles (VLPs) expressed intracellularly by the yeast S. cerevisiae have helped set the framework of a wide range of biologicals, particularly as carriers for viral antigens. This article investigates the use of dynamic light scattering (DLS) for the rapid evaluation of the concentration and purity of VLPs to aid the complex purification strategy. Development of the assay was performed in a high background process stream (yeast homogenate) and involved a change in the signal proportional to the VLP concentration by addition of antibodies that bind on the VLP surface and detection of that size change by DLS. Overall, the assay was found to provide a significant improvement of rapid monitoring alternatives for VLPs, exhibiting good sensitivity and speed of measurement. Data are given for the use of the DLS-based assay for optimization of VLP release during a yeast cell disruption treatment.

Antibodies, Monoclonal↗
PubMed1998

Monitoring recombinant human interferon-gamma N-glycosylation during perfused fluidized-bed and stirred-tank batch culture of CHO cells.

Chinese hamster ovary cells producing recombinant human interferon-gamma were cultivated for 500 h attached to macroporous microcarriers in a perfused, fluidized-bed bioreactor, reaching a maximum cell density in excess of 3 x 10(7) cells (mL microcarrier)-1 at a specific growth rate (mu) of 0.010 h-1. During establishment of the culture, the N-glycosylation of secreted recombinant IFN-gamma was monitored by capillary electrophoresis of intact IFN-gamma proteins and by HPLC analysis of released N-glycans. Rapid analysis of IFN-gamma by micellar electrokinetic capillary chromatography resolved the three glycosylation site occupancy variants of recombinant IFN-gamma (two Asn sites occupied, one Asn site occupied and nonglycosylated) in under 10 min per sample; the relative proportions of these variants remained constant during culture. Analysis of IFN-gamma by capillary isoelectric focusing resolved at least 11 differently sialylated glycoforms over a pI range of 3.4 to 6.4, enabling rapid quantitation of this important source of microheterogeneity. During perfusion culture the relative proportion of acidic IFN-gamma proteins increased after 210 h of culture, indicative of an increase in N-glycan sialylation. This was confirmed by cation-exchange HPLC analysis of released, fluorophore-labeled N-glycans, which showed an increase in the proportion of tri- and tetrasialylated N-glycans associated with IFN-gamma during culture, with a concomitant decrease in the proportion of monosialylated and neutral N-glycans. Comparative analyses of IFN-gamma produced by CHO cells in stirred-tank culture showed that N-glycan sialylation was stable until late in culture, when a decline in sialylation coincided with the onset of cell death and lysis. This study demonstrates that different modes of capillary electrophoresis can be employed to rapidly and quantitatively monitor the main sources of glycoprotein variation, and that the culture system and operation may influence the glycosylation of a recombinant glycoprotein.

Animals↗
PubMed1998

Bioprocess monitoring: an optical biosensor for rapid bioproduct analysis.

The use of an optical biosensor for rapid bioproduct analysis is described. The biosensor, which is sensitive to changes in the concentration of bioproduct at its biologically active surface, has been shown to provide concentration data within 10 s of sample addition to the device. This has been achieved through the use of linear regression analysis to extract information from the early part of the biosensor interaction profiles. The system has been used to monitor both the production and purification of antibody fragments expressed during batch fermentation of recombinant Escherichia coli. Data obtained using the biosensor have been used to provide real-time profiles describing the location of antibody fragments during bioprocessing. Biosensor data have also been compared with those obtained from ELISA, the traditional method of retrospective analyses of samples collected during bioprocessing.

Animals↗
PubMed1998

An optical biosensor for real-time chromatography monitoring: breakthrough determination.

The use of an optical biosensor for immunorecognition of protein products during affinity chromatography is discussed to provide rapid data describing the loading and subsequent breakthrough, followed by elution and fraction collection. The optical biosensor works by following in real-time the interaction of soluble ligate with an appropriate ligand attached to the optically active surface. The initial rate of interaction between soluble ligate and immobilized ligand has been shown to correlate well with ligate concentration. This method of analysis has also been shown to agree well with ELISA, the traditionally employed technique for immunoassay of protein products lacking, for example, catalytic activity. Forward prediction, using models of the breakthrough fitted to the real-time data, has enabled the column saturation point to be determined before it has been reached, thus enabling appropriate action to ensure minimal loss of protein product while improving column utilization efficiency. The biosensor, operated within a flow injection analysis regime, has been demonstrated to provide concentration data within 10 s, with a total assay turnaround of 30 s.

Animals↗
PubMed1998

Stability of a single-chain Fv antibody fragment when exposed to a high shear environment combined with air-liquid interfaces.

The effect of shear on the antigen binding activity of a recombinant scFv antibody fragment was investigated in the presence of air-liquid interfaces using a stirred vessel that was incompletely filled. Changes in binding activity of the scFv to its antigen were monitored using an optical biosensor which had been sensitized with hen egg lysozyme (the antigen). The biosensor response was used as a measure of scFv binding activity. In buffer solution (mean velocity gradient approximately 20,000 s-1), loss of binding activity followed a first-order model with a mean rate constant of 0.83 h-1. In unstirred buffer solution, no such loss was observed. Similarly, in sheared fermentation broth there was no loss of binding activity and protective effects were attributed to the antifoam PPG.

Biosensing Techniques↗
PubMed1998

Rapid monitoring of virus-like particles using an optical biosensor: a feasibility study.

Virus-like particles (VLPs) are multimeric proteins expressed by Saccharomyces cerevisiae. The particles are approximately 80 nm in diameter and they are used as a framework for a range of biological products; for example as carriers of viral antigens. Rapid monitoring of purified VLPs was investigated using an optical biosensor. The aim was to develop an assay which may be employed for real-time bioprocess monitoring of VLPs. Problems of mass transfer of analyte were overcome through selection of a planar biosensor surface, in preference to the traditional polymer-coated surface. To prolong the surface activity for interaction analysis, a sandwich assay was developed which involved the use of a secondary capture species. It was shown that VLP concentration in pure solution could be determined within 10 min.

Biosensing Techniques↗