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Schistosomiasis affects 200 to 250 million people worldwide. Hepatic schistosomiasis is a well-recognized cause of chronic liver disease and portal hypertension. There are no previous reports of schistosomiasis post liver transplantation. We report on 2 cases of schistosomiasis in liver transplant recipients--a case of gastric schistosomiasis and a case of hepatic schistosomiasis. A discussion of the pathology of schistosomal infection and a rationale for screening potential liver transplant recipients from endemic areas follows.

The risk of a pandemic with a virulent form of influenza is acknowledged by the World Health Organization (WHO) and other agencies. Current vaccine production facilities would be unable to meet the global requirement for vaccine. As a possible supplement a DNA vaccine may be appropriate, and bioprocess engineering factors bearing on the use of existing biopharmaceutical and antibiotics plants to produce it are described. This approach addresses the uncertainty of timing of a pandemic that precludes purpose-built facilities. The strengths and weaknesses of alternative downstream processing routes are analyzed, and several gaps in public domain information are addressed. The conclusion is that such processing would be challenging but feasible.

First-line therapy for hepatitis C virus (HCV) infection comprises interferon-alpha (IFN-alpha) and ribavirin for 6 or 12 months. Mild complications of therapy are common, but more serious complications are rare. Three patients with chronic HCV infection, acquired through injecting drug use, developed idiopathic facial paralysis (Bell's palsy) during therapy, with spontaneous resolution after withdrawal of treatment. Large-scale cohort studies reveal that IFNs are associated rarely with neurologic complications, and only one previous report has linked IFN-alpha therapy and Bell's palsy. We postulate that IFN-alpha therapy led to a breakdown of peripheral tolerance to myelin sheath antigens, leading to neuropathy, just as IFN-alpha therapy can cause autoimmune thyroiditis through breakdown of tolerance to native thyroid antigens.

We have developed a simple and robust transient expression system utilizing the 25 kDa branched cationic polymer polyethylenimine (PEI) as a vehicle to deliver plasmid DNA into suspension-adapted Chinese hamster ovary cells synchronized in G2/M phase of the cell cycle by anti-mitotic microtubule disrupting agents. The PEI-mediated transfection process was optimized with respect to PEI nitrogen to DNA phosphate molar ratio and the plasmid DNA mass to cell ratio using a reporter construct encoding firefly luciferase. Optimal production of luciferase was observed at a PEI N to DNA P ratio of 10:1 and 5 mug DNA 10(6) cells(-1). To manipulate transgene expression at mitosis, we arrested cells in G2/M phase of the cell cycle using the microtubule depolymerizing agent nocodazole. Using secreted human alkaline phosphatase (SEAP) and enhanced green fluorescent protein (eGFP) as reporters we showed that continued inclusion of nocodazole in cell culture medium significantly increased both transfection efficiency and reporter protein production. In the presence of nocodazole, greater than 90% of cells were eGFP positive 24 h post-transfection and qSEAP was increased almost fivefold, doubling total SEAP production. Under optimal conditions for PEI-mediated transfection, transient production of a recombinant chimeric IgG4 encoded on a single vector was enhanced twofold by nocodazole, a final yield of approximately 5 microg mL(-1) achieved at an initial viable cell density of 1 x 10(6) cells mL(-1). The glycosylation of the recombinant antibody at Asn297 was not significantly affected by nocodazole during transient production by this method.

Computational fluid dynamics was used to model the high flow forces found in the feed zone of a multichamber-bowl centrifuge and reproduce these in a small, high-speed rotating disc device. Linking the device to scale-down centrifugation, permitted good estimation of the performance of various continuous-flow centrifuges (disc stack, multichamber bowl, CARR Powerfuge) for shear-sensitive protein precipitates. Critically, the ultra scale-down centrifugation process proved to be a much more accurate predictor of production multichamber-bowl performance than was the pilot centrifuge.

The use of biosensor technology is described to address in real-time the production and subsequent purification of a bioactive recombinant protein product. The product, D1.3 Fv antibody fragment, was expressed in Escherichia coli and purified via two process routes, one for extracellular and one for intracellular product material. The cells were harvested by centrifugation in a solid bowl CARR Powerfuge and stored at -70 degrees C. Clarification of the supernatant was performed by depth filtration, followed by affinity chromatography for final purification of the extracellular product. To purify the intracellular product the harvested cells were resuspended and homogenised. Removal of debris in the CARR Powerfuge was followed by depth filtration and affinity chromatography. In this work we have shown the rapid determination of bioactive product levels, and the impact this has on improved accountability and confidence is demonstrated in process mass balances on the product using the data acquired during process operation.

The early specification of bioprocesses often has to be achieved with small (tens of millilitres) quantities of process material. If extensive process discovery is to be avoided at pilot or industrial scale, it is necessary that scale-down methods be created that not only examine the conditions of process stages but also allows production of realistic output streams (i.e., streams truly representative of the large scale). These output streams can then be used in the development of subsequent purification operations. The traditional approach to predicting filtration operations is via a bench-scale pressure filter using constant pressure tests to examine the effect of pressure on the filtrate flux rate and filter cake dewatering. Interpretation of the results into cake resistance at unit applied pressure (alpha) and compressibility (n) is used to predict the pressure profile required to maintain the filtrate flux rate at a constant predetermined value. This article reports on the operation of a continuous mode laboratory filter in such a way as to prepare filter cakes and filtrate similar to what may be achieved at the industrial scale. Analysis of the filtration rate profile indicated the filter cake to have changing properties (compressibility) with time. Using the insight gained from the new scale-down methodology gave predictions of the flux profile in a pilot-scale candle filter superior to those obtained from the traditional batch filter used for laboratory development., (Copyright 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 83: 454-464, 2003.)

The use of an optical biosensor for monitoring antibody fragment accumulation following induction in a batch fermentation of recombinant E. coli is compared to the more traditional method of ELISA quantification. Using the biosensor, concentration data can be obtained within minutes of sample addition to the device, compared to an average assay time of 3-4 h for the ELISA. We describe two biosensor assays developed as an alternative to ELISA and compare them with ELISA in the ability to provide quantitative product accumulation profiles during fermentation. Discrepancies in titers recorded by the assays are explained by a combination of differences in product variants detected by each assay and interference from sample contaminants. Method of sample preparation is also shown to be important if accurate concentration data is required. Both biosensor assays are shown to be capable of providing product accumulation profiles comparable to those obtained by ELISA. The use of a rapid extraction technique would allow such data to be obtained during process operation, enabling improved fermentation control and more rapid process development.

Specific measurement of recombinant protein titer in a complex environment during industrial bioprocessing has traditionally relied on labor-intensive and time-consuming immunoassays. In recent years, however, developments in analytical technology have resulted in improved methods for protein product monitoring during bioprocessing. The choice of product-monitoring technology for a particular bioprocess will depend on a variety of assay factors and instrument-specific factors. In this article, we have compiled an overview of the advantages and disadvantages of the most commonly used technologies used: electrochemiluminescence, optical biosensors, rapid chromatography and nephelometry. The advantages of each technology for measuring both small and large recombinant therapeutic proteins are compared with a conventional enzyme-linked immunosorbent assay (ELISA) technique.

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.

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., (Copyright 2001 John Wiley & Sons, Inc.)

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%., (Copyright 2001 John Wiley & Sons, Inc.)

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., (Copyright 2000 John Wiley & Sons, Inc.)

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., (Copyright 2000 John Wiley & Sons, Inc.)

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., (Copyright 2000 John Wiley & Sons, Inc.)

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.

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.

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.

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., (Copyright 2000 John Wiley & Sons, Inc.)

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.

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., (Copyright 1999 John Wiley & Sons, Inc.)

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., (Copyright 1999 John Wiley & Sons, Inc.)

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., (Copyright 1998 John Wiley & Sons, Inc.)

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.

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.

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., (Copyright 1998 John Wiley & Sons, Inc.)

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.

Bulk crystallization is emerging as a new industrial operation for protein recovery. Characterization of bulk protein crystallization is more complex than protein crystallization for structural study where single crystals are grown in flow cells. This is because both nucleation and crystal growth processes are taking place while the supersaturation falls. An algorithm is presented to characterize crystallization using the rates of the two kinetic processes, nucleation and growth. The values of these rates allow ready comparison of the crystallization process under different operating conditions. The crystallization, via adjustment to the isoelectric pH of a fungal lipase from clarified fermentation broth, is described for a batch stirred reactor. A maximum nucleation rate of five to six crystals formed per microliter of suspension per second and a high power dependency ( approximately 11) on the degree of supersaturation were found. The suspended protein crystals were found to grow at a rate of up to 15-20 nm/s and also to exhibit a high power dependency ( approximately 6) of growth rate on the degree of supersaturation., (Copyright 1998 John Wiley & Sons, Inc.)

Downstream processing operations are often carried out blind in the process timescale since product monitoring on-line is not common. Knowledge of the location and concentration of the product and key contaminants is complementary to other process information for process development and, if available on-line in conjunction with a suitable model, control. This article sets out to demonstrate a model describing a two-cut fractional protein precipitation process and how this may be used for control of the process to maximize yield in the face of variable process stream conditions. Estimation of the model parameters is achieved by means of data-fitting by least squares and in comparison prediction by a Kalman filter algorithm. A description and error analysis of equipment for at-line monitoring of the soluble product in a pilot plant environment is presented which includes a micro-centrifuge necessary to clarify small volumes of sample prior to analysis. Finally, an account of the successful implementation of this equipment and the Kalman filter algorithm for control at bench scale is given where conditions in the process stream are deliberately disturbed to test the control operation. (c) 1997 John Wiley & Sons, Inc.

Sample clarification is a common operation in biochemical analytical methods for removing interfering or unwanted particulates from an analyte sample. Filtration provides one option for removal of particulates. However, in many cases the loss of soluble protein due to filter adsorption is unacceptable and an alternative must be sought. In this paper a microcentrifuge designed to automatically sample, spin, deliver supernatant to an analyser and wash out solids from the bowl is described. The performance of the system is assessed in terms of its clarification efficiency and the time required to achieve satisfactory clarification. Additionally, the effects of different protein precipitating agents on yeast homogenate samples separated using the microcentrifuge are studied where the system is used to deliver supernatant to a flow injection analyser. The paper demonstrates that the microcentrifuge may be used to separate rapidly such samples on a time scale between 10-60 s depending upon the type and size of sample and be successfully used as a component of an at-line monitoring system.

An optical biosensor system employing a resonant mirror (RM), with a stirred cuvette has been used to follow the interaction of a recombinant antibody fragment with its antigen, hen egg lysozyme. The data generated by the biosensor were analysed in order to determine the kinetic constants for the interaction using a linear transform (derivative analysis). For comparison the data were also analysed using an exponential curve fitting routine. It was demonstrated that the exponential curve fitting method produced results which were in agreement with the existing linear transform method. It was also shown that early fitting of the association phase response, using the exponential curve fitting routine between 0 and 70 s after sample addition, yielded sufficient information to provide a prediction of Kon. The potential use of the optical biosensor for the rapid monitoring of protein production and purification is discussed.

We have characterized the heterogeneity of recombinant human interferon-gamma (IFN-gamma) produced by three expression systems: Chinese hamster ovary cells, the mammary gland of transgenic mice, and baculovirus-infected Spodopera frugiperda (Sf9) insect cells. Analyses of whole IFN-gamma proteins by electrospray ionization-mass spectrometry (ESI-MS) from each recombinant source revealed heterogeneous populations of IFN-gamma molecules resulting from variations in N-glycosylation and C-terminal polypeptide cleavages. A series of more specific analyses assisted interpretation of maximum entropy deconvoluted ESI-mass spectra of whole IFN-gamma proteins; MALDI-MS analyses of released, desialylated N-glycans and of deglycosylated IFN-gamma polypeptides were combined with analyses of 2-aminobenzamide labeled sialylated N-glycans by cation-exchange high-performance liquid chromatography. These analyses enabled identification of specific polypeptide cleavage sites and characterization of associated N-glycans. Production of recombinant IFN-gamma in the mammalian expression systems yielded polypeptides C-terminally truncated at dibasic amino acid sites. Mammalian cell derived IFN-gamma molecules displayed oligosaccharides with monosaccharide compositions equivalent to complex, sialylated, or high-mannose type N-glycans. In contrast, IFN-gamma derived from baculovirus-infected Sf9 insect cells was truncated further toward the C-terminus and was associated with neutral (nonsialylated) N-glycans. These data demonstrate the profound influence of host cell type on posttranslational processing of recombinant proteins produced in eukaryotic systems.

Methods based on the use of optical biosensors have recently become available to provide a convenient means of determining the rate and equilibrium constants for bimolecular interactions between immobilized ligands and soluble ligate molecules. However, the association data that these methods provide are not always accurately described by the expected pseudo-first-order reaction mechanism, particularly when the ligand is immobilized on a dextran matrix. We show that a better description of the association data, especially at higher ligate concentrations, is achieved with a double exponential function, indicating that at least two rate-limiting processes are involved. Various models are considered in order to explain these observations: the presence of two (or more) distinct populations of immobilized ligand; a change, possibly conformational, in the immobilized ligand before or after ligate binding; or the hindrance of ligate binding to immobilized ligand. We suggest that steric hindrance caused by ligate binding to the dextran-coated sensor surface seems the most likely explanation for the observed biphasic association kinetics and that the faster initial phase should be used in oder to determine association constants that can be compared to those in solution.

Recombinant human interferon-gamma (IFN-gamma) was expressed in Chinese hamster ovary cells, baculovirus-infected Sf9 insect cells and the mammary gland of transgenic mice. The N-linked carbohydrate populations associated with both Asn25 and Asn97 glycosylation sites were characterized by matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) in combination with exoglycosidase array sequencing. A site-specific analysis of dual (2N) and single (1N) site-occupancy variants of IFN-gamma derived from Chinese hamster ovary cells showed that N-glycans were predominantly of the complex bi- and triantennary type. Although Asn25-linked glycans were substituted with a core fucose residue, Asn97 N-glycans were predominantly non-fucosylated, and truncated complex and high-mannose oligosaccharide chains were also evident. Transgenic mouse derived IFN-gamma exhibited considerable site-specific variation in N-glycan structures. Asn25-linked carbohydrates were of the complex, core fucosylated type, Asn97-linked carbohydrates were mainly of the oligomannose type, with smaller proportions of hybrid and complex N-glycans. Carbohydrates associated with both glycosylation sites of IFN-gamma from Sf9 insect cells were mainly tri-mannosyl core structures, with fucosylation confined to the Asn25 site. These data demonstrate the profound influence of host cell type and protein structure on the N-glycosylation of recombinant proteins.

Recombinant human interferon-gamma (IFN-gamma) glycoform populations produced by Chinese hamster ovary cells have been resolved by micellar electrokinetic capillary chromatography (MECC). Separations were performed in uncoated fused silica capillaries at alkaline pH in the presence of micellar concentrations of the anionic detergent sodium dodecyl sulfate (SDS). Maximum resolution was obtained reproducibly with high-ionic-strength borate/SDS electrophoresis buffer. Under the conditions described, glycoform migration time was inversely related to the amount of carbohydrate associated with the protein. Digestion of IFN-gamma with peptide-N-glycosidase F allowed virtual real-time monitoring of glycosidase digests by capillary electrophoresis. Analysis of other digestions with either neuraminidase or endoglycosidase H (endo H) showed most IFN-gamma glycoforms to be sialylated and a minor proportion of glycoforms to be associated with oligomannose structures. While both bovine pancreas ribonuclease B and horse-radish peroxidase glycoforms were separated by this technique, proteins glycosylated at multiple sites such as bovine serum fetuin and human alpha 1-acid glycoprotein were not well resolved by MECC.

The novel benzoxazolophenanthridine antibiotic, jadomycin B, is produced by Streptomyces venezuelae ISP5230 following a 42 degrees C heat shock or exposure to ethanol. To further characterize these unusual culture conditions, studies were carried out using different media, varying nutrient content and concentrations, initial pH, and time of application of heat or ethanol stress. Highest titers of jadomycin B accumulated 48 h after S. venezuelae ISP5230 was inoculated into a D-galactose-L-isoleucine production medium (pH 7.5) which was supplemented with ethanol (6%, v/v) between 6 and 13 h. Cultures supplemented with ethanol later than 17 h post inoculation into the production medium produced little or no jadomycin B. Among other heat-shock inducing treatments examined, infection with phage SV1 was associated with increased jadomycin B production. Although jadomycin B titers showed little change with variations in the concentration of phosphate in the production medium, the nature of the nitrogen source was found to be important. Different colored pigments, presumed to be jadomycin B analogs, were formed when other amino acids replaced L-isoleucine in the medium as the sole nitrogen source. Increased jadomycin B titers accompanied increased L-isoleucine and D-galactose concentrations in the production medium.

We describe a new personal computer based image transfer and storage system. We prospectively assessed the uses and benefits of ImLink (Electronic Imaging Ltd.) in a regional neuroradiology department linked by the system to six referring district general hospitals. One hundred and fifty consecutive CT scans transferred by the system were analysed (142 head, four spine and four body scans). Sixty-eight CT scans were of acute neurosurgical cases. A significant change in management occurred in 55 of these cases (81%), principally as a result of prevention of transfer of head-injured patients when no neurosurgically correctable lesion was evident. The high quality of transferred images achieved by the system allows the provision of specialist radiological advice to colleagues in linked district hospitals. A further 50 non-acute head CT scans were transferred by district hospital radiologists seeking the second opinion of a neuroradiologist. Twenty-three out-of-hours acute head CT scans were transferred from clinicians in our own hospital to the home of the on-call neuroradiologist. Overall 63% of the 150 scans were transferred during normal working hours. We discuss the multiple uses and future potential for such an image transfer and storage system.

The use of a scroll decanter centrifuge for the removal and dewatering of affinity-flocculated yeast cell debris from a crude homogenate is described. Laboratory shear modulus measurements were used to compare the structure of flocculated and nonflocculated sediments and to indicate the dewatering conditions under which the sediment could be discharged from the centrifuge. The structure of the flocculated sediment was such that a dry beach could be used within the centrifuge while still being able to discharge the solids. The scroll decanter performance for recovery and dewatering of the flocculated homogenate was found to be independent of feed flow rate and differential scroll rate. Eighty-five percent of the solid material was recovered from the flocculated homogenate while the extent of sediment dewatering resulted in the loss of only 7% of the soluble protein in the sediment. The supernatant clarity matched that achieved by low-gravity laboratory centrifugation studies.

A biochemical engineering framework for optimizing the design and operation of fractional protein precipitation has been developed. The method utilizes a fractionation diagram to represent the purification of a product protein relative to total contaminating protein. The purification factor for a single or double-cut fractional precipitation is obtained as the gradient of an appropriate operating tie-line. A computer algorithm has been devised to maximize the tie-line gradient for a given yield enabling a plot of optimum purification factor versus yield to be constructed. The recovery of the enzyme alcohol dehydrogenase from clarified bakers homogenate using saturated ammonium sulphate has been examined. Fractionation and purification versus yield diagrams were used to investigate the effects of such process parameters as pH, temperature, and initial total protein concentration on fractionation efficiency. The results are discussed in terms of the underlying solubility and mixing phenomena and the industrial application of fractional precipitation.

Nucleic acids, lipid, and colloidal particulate material can be selectively flocculated from a yeast cell homogenate by the cationic polymer polyethyleneimine (PEI). Flocculation can occur from a crude homogenate, a homogenate clarified centrifugally, or by the prior use of sodium tetraborate (borax). Flocculation from a homogenate previously clarified by the use of borax is best suited for large-scale operation. The supernatant obtained following centrifugation is effectively free of nucleic acid, lipid, and particulate material with essentially 100% soluble enzyme recovery. Enzyme specific activity increases by approximately 45% compared to a zero PEI control.

The acid precipitation of soya protein was studied in a continuous-flow tubular reactor under conditions of turbulent flow. Preliminary batchwise experiments of a semiquantitative nature were also carried out on a bench-scale reactor to better define the parameters affecting precipitate growth. The experiments indicated the dominant growth mechanism to be the aggregation of primary precipitate particles produced by the contacting of the protein and acid streams. The rate of particle growth was observed to rise with an increase in the protein concentration as well as with greater intensity of turbulence. The final mean particle size decreased with increased intensity of turbulence. A theoretical model was set up to simulate the growth of the precipitate particles.

The kinetics of precipitation of soya protein has been examined in a tubular flow reactor with the precipitants, ammonium sulfate, ethanol, divalent calcium, and sulfuric acid. The precipitate growth profiles obtained in all cases showed the rapid formation of a precipitate phase and the attainment of a final size within 16 s. The final mean particle diameter d(m) varied with precipitating agent used in the order: sulfuric acid (12.5 mum) > ethanol (7.5 mum) approximately calcium ion (7.2 mum) > ammonium sulfate (3.1 mum). In the case of ethanol precipitation, changes in the design of the contacting section of the reactor led to differences in the precipitate growth curve. Protein solubility curves are also presented, and with the reactor data, they provide a convenient method for assessing the effect of precipitating agent on the design of protein precipitation reactors.

One hundred fifty patients over the age of 30 who had undergone major abdominal operations were stratified according to the risk of deep venous thrombosis and randomized into three groups to receive different prophylactic regimens: group A, electrical calf stimulation; group B, low-dose subcutaneous heparin; group C, intermittent sequential compression and thromboembolism-deterrent (TED) stockings. All the patients were scanned with the 125I-fibrinogen test for the whole stay in hospital. The incidence of 125I-fibrinogen detected deep venous thrombi was 18% in group A, 9% in group B, and 4% in group C. The results indicate that the regimen of intermittent sequential compression and TED stockings is as effective as low-dose subcutaneous heparin. Electrical calf stimulation is less effective.

The effect of acoustic conditioning on the particle size distribution of isoelectric and calcium-ion-precipitated soya protein has been examined in low-residence-time chambers. In a previous study a beat frequency of 5 Hz obtained using a dual-source system of opposing vibrators was determined as giving optimal improvement in particle-settling characteristics for isoelectric soya protein precipitate. In this study the effect of amplitude of vibration, a measure of acoustic power input, and residence time of acoustic conditioning has been examined. Acoustic power input changed the flow pattern in the conditioning chamber from laminar streamline flow to a well-mixed, turbulent pattern. Such a mixing effect promoted the rapid aggregation of fine particles, a process that was modeled on the basis of orthokinetically controlled collisions. The rate of removal of fine particles due to acoustic conditioning was shown to be proportional to a mixing effect that was related to the acoustic power dissipated per unit volume. The consequences of fine-particle aggregation on the centrifugal recovery of the precipitate are discussed.

The use of microporous membranes has been examined for the recovery of precipitated protein suspensions and related soluble protein. Membrane flux rates and soluble protein transmissions are reported for unstirred batch-cell studies and cross-flow experiments. The unstirred batch-cell gave soluble protein transmissions in the range 80-100% for feeds containing either soluble protein or a mix of soluble and isoelectrically precipitated protein. In all cases a sharp decline in flux was observed which was, for example, considerably greater for soluble protein at its isoelectric point, pH 4.6, than at pH 8.8. The presence of precipitated protein led to a further decrease in flux rate. In cross-flow studies, flux decline was eventually accompanied by a significant decline in soluble protein transmission. The flux protein-transmission characteristics of microporous membranes are discussed in terms of the interaction of the soluble and precipitated protein with the membrane.

The study examines the use of ultrafiltration and microfiltration membranes for concentrating isoelectric soya protein. Experiments with an unstirred batch cell indicate that the flux is limited by the protein which remains in solution after precipitation of the major proportion. The porosity of the precipitate cake formed is shown to be a second important factor. A significant improvement in flux can be obtained by using membranes which permit passage of the soluble protein and by increasing the precipitate particle size. The results are shown to be within the range predicted theoretically by the two limiting cases of a particulate model and a soluble protein model.

Isoelectric soya-protein precipitate densities were measured for mean particle sizes ranging from 3.4-65 microm by gradient centrifugation, centrifugation in water-immiscible solvents, tracerdilution, gravity sedimentation of isolated particles. Coulter counter volume determination, and a comparison of Coulter counter and centrifugal sedimentation size distributions. The immiscible system and tracer dilution methods were both found to be unreliable due to experimental uncertainties. The Coulter counter volume measurement indicated the existence of a density-size relationship with the aggregate density decreasing as the size increased. Comparison with sedimentation measurements showed that the Coulter counter measures 80% of the total aggregate volume for 6-microm particles. The relation between aggregate density (rho(a), kg m (-3)) and size (d, microm) was measured for isoelectric soya protein and casein precipitated by ammonium sulfate, using a comparison of the Coulter counter size distribution and centrifugal sedimentation. The functions were described for soya by [rho(alpha) - 1001 = 246d-0.408 and for casein by rho(alpha) - 1136 = 31d-0.441]. The gradient centrifugation method measured the buoyant density of hydrated protein precipitate which was independent of size, and is consistent with an aggregate structure consisting of primary particles. However, the aggregate structure was not described for all sizes by the theoretical cubic packing of hard-sphere primary particles, nor by the successive random addition of primary particles. The density-size functions indicated up to a fivefold difference in Stokes settling velocities compared to those calculated assuming a constant density difference.

Hollow fiber ultrafiltration and microfiltration membranes are examined for the processing of isoelectric soya protein precipitate suspensions. A model based on the various resistances to permeate flux is used to describe membrane performance. The main resistance to permeate flux is due to the interaction between the active membrane and the soluble and precipitated protein; that is, as compared with resistances due to the active membrane itself or the membrane support structure, or arising from concentrated soluble or precipitated protein layers over the membrane surface. Soluble protein rejection and precipitate mean particle diameter are correlated with observed values of this main resistance.In contract to the ultrafiltration of soluble proteins, the flux rates observed when processing protein precipitate suspensions under a similar range of operating conditions do not approach a limiting value with increased transmembrane pressure. At high protein concentrations, greater flux rates may be achieved for precipitated as compared with soluble proteins. The use of a microfiltration membrane does not give further improvement in flux rate; this may be attributed to problems of pore fouling with precipitate particles.