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A genetic approach based on the activation of interferon-regulated-factor-1 (IRF-1) has been applied to arrest BHK cell growth in spinner flasks through the presence of estradiol in the culture medium, leading to the activation of the constitutively expressed IRF-1/estrogen receptor fusion protein (IRF-1-hER). Two days after estradiol addition cell proliferation was inhibited; cell concentration being kept for the next 6 days, although a decrease in cell viability was observed. IRF-1 activation alters the cell energetic metabolism, as there is extra metabolic activity with higher glucose, glutamine and oxygen consumption rates. Although the proteolytic activity is higher than in the control cells, the cell protein content increases significantly after IRF-1 activation, suggesting an overall increase in protein synthesis. A significant increase in lactate dehydrogenase activity and a higher reduction of MTT (3-(4,5-dimethylthiazol-2-yl)-2–5-diphenyl tetrazolium bromide) were also observed, showing an increase in cell activity during cell growth inhibition. Changes in ATP and in cellular phospholipid content as well in glucose metabolism were detected, in real time by 31P and 13C nuclear magnetic resonance spectroscopy (NMR). The levels of ATP were unaffected but changes in cellular phospholipid content were observed. Although an increase on ADP and AMP content was observed 48 to 72 h after IRF-1 activation, both the ATP content and the energy charge (EC) remained constant. It can be concluded that the IRF-1 activation leads to a significant increase of the cell activity until the cell is no longer able to maintain the same energetic metabolism and starts to lose viability.

Activation of the constitutively expressed interferon-regulatory-factor-1/estrogen receptor fusion protein (IRF-1-hER) in BHK cells was accomplished through the addition of estradiol to the culture medium, which enabled IRF-1 to gain its transcriptional activator function and inhibit cell growth. With the addition of 100 nM estradiol at the beginning of the exponential phase of a cell suspension culture, IRF-1 activation led to a rapid cell growth inhibition but also to a significant decrease in cell viability. To apply this concept in industry, a reduction of the time span of estradiol exposure is required. Cycles of estradiol addition and removal were performed in 2-l stirred tank bioreactors operated under perfusion, where an initial step addition of 100 nM estradiol was performed, followed, after 48-72 h, by a slow dilution with estradiol-free fresh medium (perfusion rate varying between 0.7 and 1.4 per day). Cell growth inhibition was successfully achieved for three consecutive cycles. Diluting the estradiol by perfusing medium without estradiol to concentrations lower than 10 nM led to cell growth and viability recovery independently of the perfusion rate used. These observations permitted the definition of operational strategies for regulated IRF-1 BHK cell growth by pulse estradiol addition, followed by a period of 48 h in the presence of estradiol and by fast perfusion to estradiol concentrations lower than 10 nM. Cell growth response to IRF-1 activation and following estradiol removal by perfusion was also evaluated with an IRF-1-hER regulated clone expressing constitutively Factor VII, where the time of estradiol exposure and perfusion rate were varied. This clone presented a stronger response to IRF-1 activation without an increase in Factor VII specific productivity after cell growth inhibition; this clearly indicates that the stationary phase obtained is clone dependent. This work proves that it is possible to modulate the IRF-1 effect for cell growth control by the manipulation of cycles of addition and removal of estradiol, potentially representing a new generation of culture procedures for controlled growth production purposes.

The aim of this work was the optimisation of a fed-batch culture by metabolic confinement of BHK21 cells producing an antibody/cytokine fusion protein with potential application in tumour-targeted therapy. Previous results showed that at very low nutrient concentrations, a metabolic shift towards more efficient metabolic pathways occurs. The application of those results in the optimisation of a fed-batch culture resulted in higher cell growth (0.020 vs. 0.016 h(-1)) and cell viability, higher maximum cell concentration (2.5 vs. 1.1x10(6) cell ml(-1)), longer culture span (17 versus nine days) and higher product titre (60% increase), in relation to batch culture. This was achieved by maintaining glucose at 0.3 mM and glutamine at 0.2 mM through the addition of a concentrated solution based on the estimations of future nutrient consumption and growth rates through off line measurements. The production of toxic metabolites such as lactate and ammonia was reduced, especially the lactate production, which was markedly decreased due to the metabolic confinement of the cells. In conclusion, it was possible to increase the final titre of the recombinant antibody/cytokine fusion protein by confining the metabolism of the cells to an energetically more efficient state.

The influence of surface growth (two-dimensional microcarriers) and three-dimensional growth (aggregates and macroporous supports) in agitated, suspended batch culture systems upon growth and productivity of BHK was compared. Cultures using three porous microcarriers (CultiSpher G, Cellsnow EX, and Cytocell), one nonporous microcarrier (Cytodex 3) and natural aggregates were performed in stirred tanks using two different agitation rates (60 and 100 RPM). With the exception of Cytocell, cell growth, viability, and productivity were similar when three-dimensional structures (porous microcarriers and aggregates) were used. Nonporous microcarriers only compared well at 60 RPM as growth ceased under overagitation. These results suggest that cultures less susceptible to fluid shear are advantageous for scale-up. (c) 1996 John Wiley & Sons, Inc.

BHK-21 cells expressing a human IgG-IL2 fusion protein, with potential application in tumor-targeted therapy, were grown under different nutrient conditions in a continuous system for a time period of 80 days. At very low-glucose (< 0.5 mM) or glutamine (< 0.2 mM) concentrations, a shift toward an energetically more efficient metabolism was observed. Cell-specific productivity was maintained under metabolically shifted growth conditions and at the same time an almost identical intracellular ATP content, obtained by in vivo 31P NMR experiments, was observed. No significant differences in the oligosaccharide structures were detected from the IgG-IL2 fusion protein preparations obtained by growing cells under the different metabolic states. By using oligosaccharide mapping and MALDI/TOF-MS, only neutral diantennary oligosaccharides with or without core α1-6-linked fucose were detected that carried no, one or two β1-4-linked galactose. Although the O-linked oligosaccharide structures that are present in the IL2 moiety of the protein were studied with less detail, the data obtained from the hydrazinolysis procedure point to the presence of the classical NeuAcα2-3Galβ1-3GalNAc structure. Here, it is shown that under different defined cellular metabolic states, the quality of a recombinant product in terms of O- and N-linked oligosaccharides is stable, even after a prolonged cultivation period. Moreover, unaffected intracellular ATP levels under the different metabolic states were observed. © 2000 John Wiley & Sons, Inc. Biotechnol Bioeng 69: 129–139, 2000.

The activation of interferon-regulatory-factor-1 (IRF-1) hasbeen applied to regulate the cell growth of BHK cells. Theconstitutively expressed IRF-1-estrogen receptor fusion protein(IRF-1-hER) activated by the addition to the culture medium ofan estrogen analogue (estradiol), enabled IRF-1 to gain itstranscriptional activator function. By using a dicistronicstabilised self-selecting construct it was possible to controlcell proliferation. With the addition of 100 nM of estradiol at the beginning of the exponential phase, the IRF-1 activationled to a rapid cell growth inhibition. Two days after estradioladdition cell concentration was still maintained but a decreasein cell viability was observed. This cell response isindependent on clone (producer and non-producer) and culturesystem (static and stirred cultures). Specificrecombinant-protein productivity of the producer clone was notsignificantly altered. Control experiments confirmed that IRF-1activation effect was not due to the addition of estradiol per se, estradiol solvent or serum concentration. The extent ofcell growth inhibition is dependent on estradiol concentrationand estradiol addition time, although a decrease in cellviability was always observed. Reducing the time span ofestradiol exposure allowed the decrease in the cell viability tobe controlled and the stationary inhibited phase to be extended:when the time of contact between the cells and estradiol isreduced cell viability increases, archieving values similar tothose obtained if no estradiol is added. During this recoveryphase the cells passed two different phases: first a stationaryphase extension where cell growth was still inhibited, followedby an increase of cell concentration. The IRF-1 system isreversible. This pattern can be repeated for an extended period when estradiol addition and removal are repeated, showing acyclic response. Thus, it is possible to modulate the IRF-1effect by manipulating cycles of addition/removal of estradioland in this way the stationary phase can be maintained.

This work aimed at the characterisation and downstream processing of HIV-1 core and virus-like particles (CLPs and VLPs) produced in serum free medium. The produced core and virus-like particles have similar characteristics to those of the native immature HIV-1 virus in terms of protein composition, diameter, density and shape. The molecular mass (determined by gel filtration chromatography) and the diameter (determined by electron microscopy) were similar for CLPs and VLPs, this being correlated with the difference between the particle densities (1.14 g/cm 3 and 1.19 g/cm 3 , respectively, for CLPs and VLPs). After this characterization, several concentration and purification methods were tested to obtain enough material for further tests. For this purpose, a downstream processing strategy was developed, involving clarification by centrifugation and microfiltration, concentration and partial purification by ultrafiltration, and final purification by gel filtration chromatography. Quality analysis using Western immunoblot and gel filtration chromatography was performed to define the best operational conditions for each purification step. The particle recovery obtained was 87% with the main losses occurring in the ultrafiltration step. In addition, by evaluating the effect of operational conditions on product quality, it was possible to obtain a final product with high content of intact high molecular weight particles.

In this work the proteolytic activity in the supernatant and inside insect cells in culture was evaluated for different multiplicities of infection (MOI) and times of infection (TOI). Several methods to detect proteolytic activity in insect cells were tested and that using fluorescein thiocyanite-casein as a substrate was chosen. It was observed that infection caused not only a reduction in the concentration of proteases by decreasing their synthesis but also an inhibition of the intracellular proteolytic activity by increasing the intracellular ATP level (measured by in vivo nuclear magnetic resonance, NMR). The maximum proteolytic activity in the supernatant was observed at 72 hpi except when the cells were infected in the late exponential growth phase or with very low MOI, yielding a nonsynchronous infection. The proteolytic degradation of Pr55gag particles was studied during culture and after harvest. In this particular case it was concluded that the supernatant should be stored at low temperature or quickly purified, since the degradation after 24 h is only 3% at 4°C while at 27°C this value rises to 23%. There is a complex relationship between MOI, TOI, proteolytic activity, and product titer and quality. Thus, the optimal conditions for each case will be a compromise between the final product titer, the desired product quality, and operational issues like process time and capacity, requiring proper integration between bioreaction and downstream processing. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 65: 133–143, 1999.

Recombinant fusion proteins offer important new therapeutic approaches for the future. This report describes the use of three different genetic strategies (i.e. “mono-”, “bi-” and “tri-cistronic” vectors) to achieve stable secretion from BHK cells of a glycosylated antibody-cytokine fusion protein designed for use in anti-tumour therapy. It describes selection of a robust and effective production cell line based on stability of secretion of the product, quality of mRNA and protein products and performance in in vitro bioassays for potency. The data obtained at this stage were utilised in the selection of a suitable candidate production cell line. The relative productivity and general performance of the cells in stirred tank and fixed bed culture systems indicated that a variety of cell culture technologies provided robust tools for production of a highly selected cell clone. Consistency of the product glycosylation was determined by analysis of released oligosaccharides using matrix-assisted laser desorption ionisation – time of flight mass spectrometry and high-performance anion exchange chromatography. These investigations showed consistent expression of three glycoforms of the fusion protein which varied in their relative proportions in different culture systems and at different time points in a fixed bed reactor with continuous perfusion. In conclusion, this study dealt with a range of important scientific and technical issues which are essential for regulatory approval and commercial success of a recombinant protein and elucidates some useful markers for process development for similar recombinant biologicals.

In this work, a BHK21 clone producing a recombinant antibody/cytokine fusion protein was used to study the dependence of cell metabolism on the glucose and glutamine levels in the culture medium. Results obtained indicate that both glucose and glutamine consumptions show a Michaelis-Menten dependence on glucose and glutamine concentrations respectively. A similar dependence is also observed for lactate and ammonia productions. The estimated value of the Michaelis constant for the dependence of lactate production on glucose (KLacGlc) was 1.4 +/- 0.1 mM and for the dependence of ammonia production on glutamine (KAmmGln) was 0.25 +/- 0.11 mM and 0.10 +/- 0.03 mM, at glucose concentrations of 0.28 mM and 5.6 mM respectively. At very low glucose concentrations, the glucose to lactate yield decreased markedly, showing a metabolic shift towards lower lactate production. This metabolic shift was also confirmed by the significant increase in the specific oxygen consumption rate also observed at low glucose concentrations. Although it was highly dependent on glucose concentration, the oxygen consumption also increased with the increase in glutamine concentration. At very low glutamine concentrations, the glutamine to ammonia yield increased, showing a more efficient glutamine metabolism.

The present work aims at characterizing the regulatory mechanisms of metabolism and product formation of BHK cells producing a recombinant antibody/cytokine fusion protein. This work was carried out through the achievement of several steady-states in chemostat cultures, corresponding to different glucose and glutamine levels in the feed culture medium. Results obtained indicate that both glucose and glutamine consumptions show a Michaelis–Menten dependence on residual glucose and glutamine concentrations, respectively. Similar dependence was also observed for lactate and ammonia productions. K and K were estimated to be 0.4 and 0.15 mM, respectively, while q and q were estimated to be 1.8 and 0.55 nmol 10−6cells min−1, respectively. At very low glucose concentrations, the glucose-to-lactate yield decreased markedly showing a metabolic shift towards lower lactate production; also, the glucose-to-cells yield was increased. At very low-glutamine concentrations, the glutamine-to-ammonia and glutamine-to-cells yields increased, showing a more efficient glutamine metabolism. Overall, amino acid consumption was increased under low glucose or glutamine concentrations. Metabolic-flux analysis confirmed the metabolic shifts by showing increases in the fluxes of the more energetically efficient pathways, at low-nutrient concentrations. No effect of glucose or glutamine concentrations on the cell-specific productivity was observed, even under metabolically shifted metabolism; therefore, it is possible to confine the cells to a more efficient metabolic state maintaining the productivity of the recombinant product of interest, and consequently, increasing final product titers by increasing cell concentration and culture length. This work is intended to be a model approach to characterize cell metabolism in an integrated way; it is highly valuable for the establishment of operating strategies in mammalian cell fermentations in which cell metabolism is to be confined to a desired state. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 66: 104–113, 1999.

In this work the maximal operational hydrodynamic conditions (agitation and aeration rate) that cause no adverse effect in Sf-9 cells growth in SF900II serum-free medium were determined. Shear stresses higher than 1 N m-2 and aeration rates higher than 0.04 vvm affect cell growth and when these conditions increase to 1.5 N m-2 and 0.11 vvm, cell growth is completely inhibited with significant cell morphology changes and a strong decrease in viability. Although the pO2 did not show a significant effect upon cell growth in the range from 10 to 50%, cell infection and specific productivity were dramatically affected. The production was optimal at a pO2 of 25% with decreases higher than 50% being observed when the pO2 decreased to 10 or increased to 50%. The maximum product quality, i.e., the percentage of product in the form of high molecular weight particles, is not coincident with maximum product titer. Although the highest Pr55gag particle titer was obtained at 96 hours post infection (hpi) and at pO2 of 25%, the best product quality (defined by gel filtration chromatography and Western immunoblot) was obtained at 48 hpi, independently of the pO2 used. The effect of overcritical conditions upon productivity was also studied. As obtained for cell growth, cell infection is affected by shear stresses above 1 N m-2 and by aeration rates higher than 0.04 vvm, with decreases in Pr55gag particle titer higher than 70%, even when the overcritical values are still far from the limit at which cell death occurs. The results obtained and the optimization strategy used allowed the maximization of the oxygen supply without damaging the cells, with important consequences on the scale-up of a production process involving this insect cell/baculovirus expression system.

In this work Sf-9 cells previously adapted to SF900II and EXCELL 401 serum-free media (SFM) were grown and infected at different agitation rates in order to study the effects of power input upon cell growth, infection and production of Pr55gag particles in both SFM. Maximum cell concentration increased from 3 x 10 6 to 6 x 10 6 cells cm -3 and 6.5 x 10 6 cells cm -3 when the agitation increased from 80 to 250 rpm or sparged aeration (0.01 vvm at 170 rpm) is used, clearly indicating that cell growth is limited by gas transfer. The specific productivity increased 3.5-fold when the agitation rate was increased from 80 to 170 rpm, indicating that in SFM cell infection is also limited by gas transfer. The highest product concentration was obtained in SF900II at 120 h post-infection (hpi). The product quality analysis showed that SF900II is the best medium for production of Pr55gag particles and that a careful optimisation of the harvest time is required. The maximum product titre was obtained at 120 hpi, 48 h after the achievement of the highest quality.

In this work a recombinant BHK21 clone producing a fusion protein with potential application in tumour target therapy was adapted to five different serum-free media (SFM) and to a protein-free medium (PFM). Only the PFM did not require a gradual adaptation to cell growth in the absence of serum. All tested SFM required a gradual adaptation (up to 35 days). For the majority of the SFM tested, cell specific productivity was not affected by the decrease in serum concentration during adaptation; however, cell growth was significantly affected by the serum decrease. Both cell growth and productivity were increased when PFM SMIF6 was used instead of the control medium. Long term measurements (approximately 100 days) of cell specific productivity for PFM and the two best SFM showed that productivity was maintained. This indicates the media capability to be used in long term production processes.

Sf-9 insect cells were adapted to three different serum-free media (SF900II, EXCELL 401 and IPL/41 supplemented) in 125 ml stirred vessels by gradually reducing serum concentration from 10 to 0% (v/v). TC100 medium sup-plemented with 10% fetal bovine serum was used as control. With this procedure it was possible to obtain cells fully adapted to SF900II and EXCELL 401 in 5 weeks. The adapted cells could be frozen in serum-free medium and thawed without any decrease in specific growth rate or maximum cell concentration. Even after 4 months of culture in stirred vessels at 170 rpm the specific growth rate and maximum cell concentration (0.031 h and 4.8 × 10 cells/ml, respectively) remained constant.

Baby hamster kidney (BHK) cell aggregates grown in stirred vessels with different working volumes and impeller sizes were characterized. Using batch cultures, the range of agitation rates studied (25-100 rpm) led to aggregates with maximum sizes of 150 mum. Necrotic centers were not observed and cell specific productivity was independent of aggregate size. High cell viability was found for both single and adherent cells without an increase in cell death when agitation rate was increased. The increase in agitation rate affected aggregates by reducing their size and increasing their concentration and cell concentration in aggregates, while increasing the fraction of free cells in suspension. The experimental relationship between aggregate size and power dissipation rate per unit of mass was close to -1/4, suggesting a correlation with a critical turbulence microscale; this was independent of vessel scale and impeller geometry over the range investigated. Viscous stresses in the viscous dissipation subrange (below Kolmogoroff eddies) appear to be responsible for aggregate breakage. Under intense agitation BHK cells grown in the absence of microcarriers existed as aggregates without cell damage, whereas cells grown on the surface of microcarriers were largely reduced. This is a clear advantage for scaleup purposes if aggregates are used as a natural immobilization system in stirred vessels. (c) 1995 John WileySons, Inc.

Baby hamster kidney (BHK) cells were grown in stirred spinner flasks at 45 rpm, using both serum-containing and serum-free media. In both studies natural aggregates were formed. Aggregate formation is independent of cell growth or medium composition, but aggregate final properties are dependent on cell growth. Serum concentrations were varied between 0 and 20% without the addition of any serum substitutes. Aggregate characteristics (diameter, fraction of cells in aggregates, and compactness) remained constant above 5% serum, clearly indicating that there is no direct dependence of aggregate properties on serum above that concentration. Cell growth decreased with serum concentration below 5%. A serum-free medium with similar basal medium and supplemented with several additives was tested. Cell growth, cell viability, and aggregate characteristics remained unchanged when serum-free medium was compared with serum-containing medium with more than 5% serum at the same agitation rate. Using the serum-free medium, agitation rate was varied between 25 and 60 rpm. No differences in cell growth or viability were observed, but aggregate diameter was largely reduced with the increase in agitation rate (from 200 to 90 μm); this clearly indicates that the control of the size of aggregates by vessel hydrodynamics previously reported for serum-containing medium is independent of the medium composition including absence or presence of serum, for the two tested media.

Lactic acid production with cell recycling on an ultrafiltration tubular membrane reactor was studied; higher lactic acid concentrations as well as productivities were obtained under long-term fermentations compared with other high cell density systems. Different operational conditions, namely dilution rates and start-up modes, were assessed. Performances were very different at the three different dilution rates tested (D = 0.20 h(-1), D = 0.40 h(-1), or D = 0.58 h(-1)). The different behaviours are discussed and factors responsible for them are presented. The best way to operate for lactic acid production is chosen, the dilution rate of D = 0.40 h(-1) being the one providing the best overall performance. On the other hand, results show that of the two start-up modes tested, continuous start (membrane open) permits higher permeabilities throughout the operational runs than batch start (membrane closed). Operational stability was found to be directly associated with membranes that work at "steady state," the membrane permeability being kept around 15 L/m(2) h. Optimized cell bleed can improve time of operation if such membrane permeability can be maintained for a longer time. A comparison of results with those obtained in other lactic acid production systems is presented; such comparison shows that this tubular ultrafiltration membrane cell recycle reactor presents three important advantages: (1) concomitant lactic acid concentrations and productivities; (2) long periods of operation at reasonable permeabilities; and (3) good mechanical stability permitting the use of steam sterilization. (c) 1995 John Wiley & Sons, Inc.

Large-scale animal cell operation is costly both in terms of facilities and consumables. Hence developmental studies with animal cells normally start at laboratory scale, often using small stirred tanks. In order to better optimise cell performance, it is necessary to know the physical conditions under which the cells are grown. In this study a laboratory-scale vessel (2 dm 3 working volume) with two large-bladed paddle impellers was characterised hydrodynamically. Three different aeration methods (surface, sparging and membrane aeration) were investigated and compared. Power input and oxygen transfer rates to culture medium were determined as a function of agitation and gas flow rates. Nondimensional correlations were established for each case, which can be useful for scale-up purposes. The results obtained indicate that power input is quite dependent on the vessel accessories: for the same agitation rate, the maximum power is required for the membrane structure and the minimum for surface aeration, with the addition of the sparger leading to an intermediate situation. Predictions found in the literature can be used for simple vessels, but may not be applicable when accessories are added to the vessel structure; in such cases, the use of experimental relationships are required. Oxygen transfer rate was dependent on the aeration method and working conditions (agitation and gas flow rates), particularly for sparger aeration. Membrane aeration gave larger oxygen transfer but higher gas pressure and flow rates were required. Surface aeration was the least effective method, nevertheless requiring gas flow rates similar to those used for membrane aeration. The aeration method of choice depends upon the culture and work specificities: surface aeration is limited to small cell concentrations and low oxygen consumption rates. For higher cell concentrations and oxygen consumption rates, both membrane and sparger aeration methods can be applied: the use of the sparger is limited to cells that are not affected by the presence of bubbles or the addition of surfactants, whereas the membrane aeration basket should not be used when a hydrodynamically controlled stirred tank is required

Some anchorage-dependent animal cells can form natural aggregates in stirred tanks. Baby hamster kidney (BHK) natural aggregates are described and characterized. Total cell concentration and viability could be obtained after aggregate mechanical dissociation, with negligible cell lysis and no change in cell membrane permeability. During a normal batch run, aggregates were formed immediately after inoculation, a few spherical aggregates increasing in size during the initial growth phase. At the end of the growth phase, an increase in aggregate concentration was observed, without a considerable increase in aggregate diameter. At the end of the batch run, 160 h after inoculation, aggregates disintegrated into smaller, non-spherical units, following a sharp viability decrease. Cell concentrations of 1.2 x 10(6) cells/ml were obtained, with 60% of the total cells being in aggregates; the cell concentration in aggregates achieved 5 x 10(8) cells/ml, with a porosity of 55%. Viability was consistently in the range 85-90%, both for aggregate and suspended cells.

The growth and productivity of an Sp2/0 cell line, F3b10, expressing a recombinant antibody (rAb) and BHK21 cells expressing either the same rAb from the same plasmids (BHK.IgG) or secreted alkaline phosphatase (SEAP) (BHK.SEAP) were investigated. The F3b10 line was grown as a single cell suspension. The BHK lines were grown either as suspended natural aggregates or on Cytodex 3 microcarriers. The data for F3b10 showed that the cell-specific rAb production rate (QsrAb) increased in parallel with increases in the specific growth rate (mu). A similar result was obtained for suspended aggregate cultures of both recombinant BHK cell lines. In contrast, for microcarrier cultures of both BHK cell lines, Qsproduct increased as mu decreased. This report shows that the relationship between cell growth and Qsproduct for the cell lines and products studied is dependent upon the culture process. In systems where recombinant cells are growing as a single cell suspension or within a natural suspension aggregrate, Qsproduct increased with increases in mu. In such systems, the cells have a rounded morphology. When cells were grown on microcarriers. Qsproduct decreased as mu increased. Cells growing attached to a surface are flat and elongated. The observed differences in the relationship of Qsproduct to mu are correlated with changes in cell morphology. The relationship between Qsproduct and mu is also affected by the choice of cell line.

The production of a flocculent strain of Lactobacillus plantarum was performed in a high cell density reactor: a fluidized bed reactor (FBR) with a settler and an external cell recirculation. Two variables were assessed, the recirculation rate (R) and the dilution rate (D). The effect of the latter is much more important than the effect of the former in ensuring a quick start up in the flocculation process. The cell volumetric productivities obtained with this system increase directly with dilution rate and recirculation rate. The values of cell volumetric productivities obtained are considerably higher than those obtained in continuous stirred tank reactors (CSTR) and much higher than in batch reactors.

Mammalian cell cultures are the preferred production system for secreted pharmaceutical proteins (Hauser, 1997). Life of a mammal begins with fertilization of the oocyte that divides and proliferates until the animal reaches its mature size. Further cell growth is tightly controlled. Proliferation and cell death are balanced to keep the total cell mass essentially constant, while the synthesis and secretion of cellular products continues. Despite rapid progress in the understanding of growth regulatory mechanisms, tumorigenic growth due the loss of proliferation control of a single body cell initially is still a leading cause of disease and death.

A genetic approach, based on the growth regulatory protein interferon-regulated-factor- 1 (IRF-1), activated by the addition of estradiol, has been applied to regulate BHK cell growth. With the addition of 100 nM of estradiol 48 hours after inoculation, growth inhibition occurs within 24 hours but is followed by a significant decrease in cell viability, whereas rec-protein specific productivity is not significantly altered. Viability decrease is not due to estradiol effect and is independent upon the culture system. In order to define strategies to extend the stationary growth phase, several parameters have been studied: estradiol concentration, time post inoculation for estradiol addition and time span of estradiol exposure. When the time of contact between the cells and estradiol is reduced the cell viability increases, achieving similar values of the control, without the estradiol, and leading to a stationary growth phase extension.

Goat jugular vein endothelial cells (cje), involved on the in vivo regulation of a large number of important physiological processes, are essential for the in vitro production of candidate heartwater vaccines based on its infection with Rickettsia Cowdria ruminantium.

The production, purification and stability of quality (in terms of integrity and glycosylation) of an antibody/interleukin-2 fusion protein with potential application in tumour-targeted therapy expressed in BHK21 cells are described. Consistency of the product throughout time was determined by analysis of glycosylation of the fusion protein using MALDI-TOF mass spectroscopy and HPAEC-PAD combined with product integrity studies by SDS-PAGE and Western blotting. These investigations showed consistent expression in terms of integrity and of three major oligosaccharide structures of the fusion protein after 62 generations. The data obtained at this stage indicated the suitability of the cell line for production purposes. Different approaches for the production of this protein were subsequently carried out. The relative productivity of the recombinant fusion protein and general performance of the cells in two different protein-free medium (PFM) culture systems, continuous chemostat and continuous perfusion using a Centritech centrifuge as a cell retention device, were studied. The results indicate that the chemostat culture resulted in more stable and controllable nutrient environment, which could indicate better product consistency, in accordance with what has been observed under serum-containing conditions, in relation to the perfusion culture. Finally, product obtained from the chemostat culture was analysed and purified. The purification process was optimised with an increase in the overall yield from 38 to 70% being obtained, a significant improvement with important consequences for the implementation of an industrial-scale culture system. In conclusion, it was possible to produce and purify the recombinant antibody/interleukin-2 fusion protein assuring the quality and stability of the product in terms of integrity and glycosylation. Therefore, a candidate production process was established.

The aim of the present work was to study the effect of ammonia and lactate on growth, metabolism, and productivity of BHK cells producing a recombinant fusion protein. Results show that cell growth was reduced with the increase in ammonia or lactate: k(1/2) of 1.1 mM and 3.5 mM for stirred and stationary cultures, respectively, for ammonia and of 28 mM for both stationary and stirred cultures for lactate, were obtained. The cell-specific consumption rates of both glucose (q(Glc)) and glutamine (q(Gln)) increased, whereas that of oxygen (q(O2)) decreased, with the increase in ammonia or lactate concentrations. The cell-specific production rates of lactate (q(Lac)) increased with an increase in ammonia concentration; similarly for the cell-specific production rates of ammonia (q(Amm)), which also increased with an increase in lactate concentration; on the other hand, both q(Lac) and q(Amm) markedly decreased when lactate or ammonia concentrations were increased, respectively; lactate was consumed at lactate concentrations above 30 mM and ammonia was consumed at ammonia concentrations above 5 mM. In vivo (31)P NMR experiments showed that ammonia and lactate affect the intracellular pH, leading to intracellular acidification, and decrease the content in phosphomonoesters, whereas the cell energy state was maintained. The effect of lactate on cell growth and q(Gln) is partially due to osmolarity, on q(Glc) and q(Amm) is entirely due to osmolarity, but on q(Lac) is mainly due to lactate effect per se. An increase in ammonia from 0 to 20 mM induced a 50% reduction in specific productivity, whereas an increase in lactate from 0 to 60 mM induced a 40% decrease.

In this work a model envisaging the integrated optimization of bioreaction and downstream processing is presented. This model extends the work presented in part 1 of this pair of papers by adding ultrafiltration to process optimization. The new operational parameters include ultrafiltration time, pressure, and stirring rate. For global optimization, the model uses as constraints the final product titer and quality to be achieved after downstream processing. This extended model was validated with the same system used in part 1, i.e., PA317 cells producing a recombinant retrovirus containing the LacZ gene as a marker in stirred tanks using porous supports. Optimization of the extended model led to the conclusion that bioreaction should have two steps, batch and perfusion, similar to what was found in part 1. Ultrafiltration in a stirred cell should be performed at low pressures and stirring rates to reduce the losses of infective retroviruses. Sensitivity analysis performed on the results of the integrated optimization showed that under optimal conditions the productivity is less sensitive to the parameters related to ultrafiltration than to those associated with bioreaction. These results were interpreted as reflecting the high yield of ultrafiltration (90%). The relevance of the model extension to perform integrated optimization was also demonstrated since a restriction in the specific ultrafiltration area in downstream processing conditioned perfusion duration and perfusion rate in bioreaction. This clearly indicates that overall process optimization cannot be achieved without integrated optimization.

Although retroviruses are a promising tool for gene therapy, there are two major problems limiting the establishment of viable industrial processes: retrovirus stability and low final yield in the supernatant. This fact emphasizes the need for an effective process optimization, not only at a genetic level but also at a bioprocess engineering level. In part 1 of this paper a mathematical model was developed to optimize the bioreaction yield by determining the best retrovirus harvest strategy in perfusion cultures. PA317 cells producing recombinant retroviruses were used to develop and test this model. Cell culture was performed in stirred tanks using porous supports. The parameters of the proposed model were experimentally determined for batch and perfusion cultures at 32 and 37 degrees C both with and without additives to enhance production; the model was then validated. This model allowed the determination of the optimal values of all operational variables included: batch and perfusion duration and perfusion rate. The highest productivity (2682 virus cm(-)(3) h(-)(1)) was obtained under the following conditions: batch at 37 degrees C for 53 h followed by perfusion at 32 degrees C for 23 h with a perfusion rate of 0.107 h(-)(1). This value was 3.5-fold higher than the best result obtained in batch cultures for the same conditions of titer and quality. A sensitivity analysis of the parameters showed that the parameters that affect most the final productivity depend on the bioreaction phase: cell growth in batch culture and production and product degradation in perfusion culture. In part 2 of this paper, this model is extended to the first step of downstream processing, and the addition of further steps to the process is discussed in order to achieve global process optimization.

Aggregation can only be used as a natural immobilisation system in stirred tanks if aggregate size can be controlled, in order to avoid nutrient and product transport limitations. In this work several cell lines have been studied and their aggregate characteristics compared: BHK, Vero, CHO and 293. 250 ml spinner flasks (Corning) were used varying the agitation rate from 25 to 120 rpm and maintaining the media composition. The aggregates of each cell line showed a different behaviour, particularly in aggregate size, morphology and fraction of single cells in suspension. However, in all cases it was possible to observe a decrease in aggregate size when the agitation rate was increased. A model including the entire range of isotropic eddies was applied to the experimental data, confirming the aggregate size control through the hydrodynamics imposed to the vessel.

In the present work, BHK cells producing a recombinant protein with potential application in tumor target therapy were used in order to study the effect of operating conditions upon growth, production and product quality. Cells were grown in a protein-free medium, SMIF6, as spinner batch cultures with cells immobilized in porous and nonporous supports. Two different support concentrations and two distinct agitation rates were used. Results obtained indicate that increasing support concentration led to slightly higher titers, particularly for non porous supports; increasing agitation rates always led to lower final titers. Product quality decreased with increasing support concentration and agitation rates, this being more evident for non porous support cultures. No significant difference in specific growth rates for the different supports was observed, although higher cell concentrations were obtained with porous supports. Specific productivities were higher for non porous supports, but final titers as well as product quality were superior with porous supports.

In this work Sf-9 cells previously adapted to SF900II serum free medium were grown in 125 cm3 stirred tanks at different agitation rates in order to study the effects of both medium and power input in cell growth, infection and production. The maximum cell concentration in serum free medium increased from 3 x 106 to 5 x 106 cells/cm3 when the agitation rate increased from 80 to 170 rpm, clearly indicating that cell growth is limited by oxygen. The product concentration at 120 hours post infection (hpi) was higher in SF900II medium at 170 rpm than in serum containing medium. It is expected that the specific productivity in SF900II can still be increased by increasing oxygen transfer; this hypothesis results from the observation that the productivity increased 3.5 fold when agitation rate was increased from 80 to 170 rpm. Thus, in serum free medium both cell growth and cell infection are oxygen limited and can be further optimized.

In this work, a BHK21 clone producing a fusion protein with potential application in tumor target therapy was adapted to several serum-free media. Three different serum-free media (SFM) were tested (QBSF 56, CHO-S-SFMH, DMEM/Ham’s F12 derived medium) as well as a protein-free medium (PFM) (SMIF6). Only SMIF6 medium did not require a gradual adaptation to cell growth in the absence of serum. For all SFM tested, cell productivity was not affected by the decrease in serum concentration during adaptation; however, cell growth was largely affected. Regarding the PFM SMIF6 both cell growth and productivity increased compared to DMEM with 10% FCS; thus, it was chosen for the development of future work.

The effect of medium viscosity upon cell growth and aggregate characteristics of baby hamster kidney (BHK) cells cultivated in stirred tanks was evaluated. Two thickening agents were tested, 9300 MW dextran and a low-viscosity sodium (carboxymethyl)-cellulose; both were used in two different sets of experiments: (i) 250 cm3 Wheaton spinner flasks with a ball impeller operated at 45 rpm; (ii) 500 cm3 Corning spinner flasks with a paddle impeller, operated at constant power dissipation (88 cm2 s-3). Aggregate diameter and the fraction of cells in aggregates increased with the increase in viscosity. Power laws were applied to the experimental results. A dependence of aggregate size upon power dissipation of the order of -0.19 and kinematic viscosity of 0.34 and 0.49 for the constant agitation and constant power dissipation tests were obtained, respectively. A model based upon the entire universal-equilibrium range (i.e., the entire spectrum of isotropic eddies) was used to predict theoretical relationships between the variables studied. The model leads to a power dependence of -0.25 for the energy dissipated in the entire universal-equilibrium range and between 0.25 and 0.5 for the kinematic viscosity in the viscous dissipation subrange, depending on the energy correlation used; it also gives a good explanation for the dependence of aggregate size on the hydrodynamics of the vessel.

Natural aggregates of Baby Hamster Kidney cells were grown in stirred vessels operated as repeated-batch cultures during more than 600 hours. Different protocols were applied to passaging different fractions of the initial culture: single cells, large size distributed aggregates and large aggregates. When single cells or aggregates with the same size distribution found in culture are used as inoculum, it is possible to maintain semi-continuous cultures during more than 600 hours while keeping cell growth and viability. These results suggest that aggregate culture in large scale might be feasible, since a small scale culture can easily be used as inoculum for larger vessels without noticeable modification of the aggregate characteristics. However, when only the large aggregates are used as inoculum, it was shown that much lower cell concentrations are obtained, cell viability in aggregates dropping to less than 60%. Under this 'selection' procedure, aggregates maintain a constant size, larger than under batch experiments, up to approximately 400 hours; after this time, aggregate size increases to almost twice the size expected from batch cultures.