On-line monitoring of respiration in recombinant-baculovirus infected and uninfected insect cell bioreactor cultures

Respiration rates in Spodoptera frugiperda (Sf-9) cell bioreactor cultures were successfully measured on-line using two methods: The O(2) uptake rate (OUR) was determined using gas phase pO(2) values imposed by a dissolved oxygen controller and the CO(2) evolution rate (CER) was measured using an infrared detector. The measurement methods were accurate, reliable, and relatively inexpensive. The CER was routinely determined in bioreactor cultures used for the production of several recombinant proteins. Simple linear relationships between viable cell densities and both OUR and CER in exponentially growing cultures were used to predict viable cell density. Respiration measurements were also used to follow the progress of baculoviral infections in Sf-9 cultures. Infection led to increases in volumetric and per-cell respiration rates. The relationships between respiration and several other culture parameters, including viable cell density, cell protein, cell volume, glucose consumption, lactate production, viral titer, and recombinant beta-galactosidase accumulation, were examined. The extent of the increase in CER following infection and the time postinfection at which maximum CER was attained were negatively correlated with the multiplicity of infection (MOI) at multiplicities below the level required to infect all the cells in a culture. Delays in the respiration peak related to the MOI employed were correlated with delays in the peak in recombinant protein accumulation. DO levels in the range 5-100% did not exert any major effects on viable cell densities, CER, or product titer in cultures infected with a baculovirus expressing recombinant beta-galactosidase. (c) 1996 John Wiley & Sons, Inc.     

Effects of oxygen on recombinant protein production by suspension cultures of Spodoptera frugiperda (Sf-9) insect cells.

Spodoptera frugiperda (Sf-9) insect cells have been grown in serum-free medium in 250-ml spinner flasks. The maximum cell density obtained in these cultures was dependent on the aeration rate of the culture. Similar yields of uninfected cells were obtained when cultures were stirred in spinner flasks at 80 rev min-1 and in a 4-1 stirred-tank bioreactor and the dissolved oxygen in the bioreactor was controlled at 20% of air saturation. Cells were infected with a recombinant baculovirus at different multiplicities of infection: the timing and maximum level of expression of the recombinant protein were dependent on the multiplicity of infection, the cell density at infection, and on the aeration rate of the culture. Oxygen-limited growth resulted in undetectable levels of recombinant protein (< 6 ng recombinant protein 10(-7) cells). Compared with the maximum yields observed in spinner flask cultures, higher levels of recombinant protein were produced when cells were grown and infected in the bioreactor. The level of dissolved oxygen in the bioreactor was controlled at 50% of air saturation.     

Using cell size kinetics to determine optimal harvest time for <Emphasis Type="Italic">Spodoptera frugiperda</Emphasis> and <Emphasis Type="Italic">Trichoplusia ni</Emphasis> BTI-TN-5B1-4 cells infected with a baculovirus expression vector system expressing enhanced green fluorescent protein

Infecting insect cells with a baculovirus expression vector system (BEVS) is an increasingly popular method for the production of recombinant proteins. Due to the lytic nature of the system, however, determining the optimal harvest time is critical for maximizing protein yield. We found that measuring the change in average diameter during the progress of infection with an automated cell analysis system (Cedex HiRes, Innovatis AG) could be used to determine the time of maximum protein production and, thus, optimal harvest time. As a model system, we use insect cells infected with a baculovirus expressing enhanced green fluorescent protein (EGFP). We infected two commonly used insect cell lines, Spodoptera frugiperda (Sf-9) and Trichoplusia ni BTI-TN-5B1-4 (Hi5) with an Autographa californica nuclear polyhedrosis virus (AcNPV) encoding EGFP at various multiplicities of infection (MOI). We monitored the progress of infection with regard to viability, viable cell density and change in average cell diameter with a Cedex HiRes analyzer and compared the results to the EGFP produced. Peak protein production was reached one to two days after the point of maximum average diameter in all conditions. Thus, optimal harvest time could be determined by monitoring the change in average cell diameter during the course of an infection of a cell culture.     

Low multiplicity infection of insect cells with a recombinant baculovirus: The cell yield concept

In vitro infection of insect cells with baculoviruses is increasingly considered a viable means for the production of biopesticides, recombinant veterinary vaccines, and other recombinant products. Batch fermentation processes traditionally employ intermediate to high multiplicities of infection necessitating two parallel scale-up processes-one for cells and one for virus. In this study, we consider the use of multiplicities of infection as low as 0.0001 plaque-forming units per cell, a virus level low enough to enable infection of even large reactors (e.g., 10 m(3)) directly from a frozen stock. Using low multiplicities in the Sf9/beta-gal-AcNPV system, recombinant protein titers comparable with the maximum titer observed in high multiplicity infections were achieved. Cultures yielding the maximum titer were characterized by reaching a maximum cell density between 3 and 4 x 10(9) cell L(-1). This optimal cell yield did not depend on the multiplicity of infection, supporting the existing view that batch cultures are limited by availability of substrate. Up to a certain cell density, product titer will increase almost linearly with availability of biocatalyst, that is, cells. Beyond this point any further cell formation comes at the expense of final product titer. Low multiplicity infections were found not to cause any significant dispersion of the protein production process. Hence, product stability is not a major issue of concern using low multiplicities of infection. The sensitivity to initial conditions and disturbances, however, remains an issue of concern for the commercial use of low multiplicity infections. (c) 1996 John Wiley & Sons, Inc.     

Metabolite profiling of CHO cells with different growth characteristics

Mammalian cell cultures are the predominant system for the production of recombinant proteins requiring post-translational modifications. As protein yields are a function of growth performance (among others), and performance varies greatly between culture medium (e.g., different growth rates and peak cell densities), an understanding of the biological mechanisms underpinning this variability would facilitate rational medium and process optimization, increasing product yields, and reducing costs. We employed a metabolomics approach to analyze differences in metabolite concentrations of CHO cells cultivated in three different media exhibiting different growth rates and maximum viable cell densities. Analysis of intra- and extracellular metabolite concentrations over the course of the cultures using a combination of HPLC and GC-MS, readily detected medium specific and time dependent changes. Using multivariate data analysis, we identified a range of metabolites correlating with growth rate, illustrating how metabolomics can be used to relate gross phenotypic changes to the fine details of cellular metabolism.     

Relationship between oxygen uptake rate and time of infection of Sf9 insect cells infected with a recombinant baculovirus

Oxygen uptake rates (OUR) of Sf9 insect cells propagated in a serum-free medium (SF900II, Gibco) and of cells infected with a recombinant AcNPV were investigated before and after infection in a laboratory-scale bioreactor. The volumetric OURs of uninfected and exponentially growing cells were found to be proportional to the cell density. For infected cultures, the specific OUR of cells increased immediately after addition of virus and a maximum of 1.3 times the value of uninfected cells was noted for all the cultures between 8 to 30 hours post infection, which coincides with the period at which most viral replication and the majority of DNA synthesis takes place. It was observed that the rate of rise in the specific OUR decreased as the cell density at the time of infection increased, which meant that the later the infection, the later the maximum sOUR was observed. We therefore suggest that OUR measurement can be used to reflect the efficiency of a batch infection. Carbohydrate and amino acid consumption rates from an infected run were analysed in an effort to identify substrate(s) that may be used at increased rates to fuel the rise in oxygen demand observed early in the infection cycle. No observable rise in the consumption rates of glucose or glutamine, which are the major energy sources for animal cells, were seen after infection but an increase in the consumption rates of some amino acids suggests that infected Sf9 cells may utilise amino acids at an enhanced rate for energy post infection.     

Metabolic flux analysis of HEK-293 cells in perfusion cultures for the production of adenoviral vectors

To meet increasing needs of adenovirus vectors for gene therapy programs, development of efficient and reproducible production processes is required. Perfusion cultures were employed to allow infection at greater cell concentrations. In an effort to define culture conditions resulting in enhanced productivities, experiments performed at different feed rates and infected at various cell densities were compared using metabolic flux analysis. The highest specific product yields were achieved in experiments performed at high perfusion rates and/or low cell concentrations. The intracellular flux analysis revealed that these experiments exhibited greater glycolytic fluxes, slightly higher TCA fluxes, and greater ATP production rates at the time of infection. In contrast, cultures infected at high cell density and/or low medium renewal rates were characterized by a more efficient utilization of glucose at the time of infection, but the specific product yields achieved were lower. The intracellular flux analysis provided a rational basis for the implementation of a feeding strategy that allowed successful infection at a density of 5x10(6)cells/ml.     

Analysis of mammalian viable cell biomass based on cellular ATP

Analysis of cellular ATP as a means of measuring viable biomass loading was investigated in hybridoma cell culture. ATP analysis by the luciferin-luciferase assay was compared with trypan blue-stained hemocytometer counts. The cell-specific ATP content varied between 2 and 6 fmol per viable cell over a batch culture. ATP levels were highest during exponential growth, and decreased during the stationary and decline phases. Electronic counting and volume measurements were performed to assay the viable cell biomass. Cell sorting, using fluorescein diacetate, was used to separate viable and nonviable cells in cultures with between 35% and 90% viable cells. Viable cells contained over 2 orders of magnitude greater cell-specific ATP than nonviable cells. Cell-specific ATP correlated directly with the viable cell volume rather than viable cell numbers. Over the range of batch culture conditions, ATP analysis should provide a more accurate measurement of hybridoma viable biomass than hemocytometer counts.     

Improved yields of the extracellular domain of the epidermal growth factor receptor produced using the baculovirus expression system by medium replacement following infection

The extracellular domain of the epidermal growth factor receptor (EGFR) was expressed using the baculovirus expression vector system. The maximum level of the EGFR extracellular domain secreted into the medium in Sf-9 (Spodoptera frugiperda or fall armyworm) cell batch culture was approximately 2.5 g ml^–1. In order to increase this yield, a process was developed that included the following sequence of steps: batch growth to maximum cell density, infection of the cells with recombinant virus, and replacement of spent medium. By using this process, the specific yield of recombinant protein, which in batch culture drops when infection is carried out at densities greater than 3 × 10⁶ cells ml^–1, can be maintained at a maximum in cultures infected at densities of 10⁷ cells ml^–1 or greater. The process, when applied to 3-1 and 11-1 bioreactor cultures, allowed a maximum volumetric yield of triple the maximum value attainable in batch culture. Spent-medium analysis indicates that medium replacement provides certain nutrients that could otherwise be limiting for recombinant protein production.     

The role of the cell cycle in determining gene expression and productivity in CHO cells

Understanding the relationships between cell cycle and protein expression is critical to the optimisation of media and environmental conditions for successful commercial operation of animal cell culture processes. Using flow cytometry for the analysis of the early phases of synchronised batch cultures, the dependency of product expression on cell cycle related events has been evaluated in a recombinant CHO cell line. Although the production of recombinant protein is initially found to be cell cycle related, the maximum specific protein productivity is only achieved at a later stage of the exponential phase which also sees a maximum in the intracellular protein concentration. Subsequent work suggests that it is the batch phase/medium composition of cultures which is the major determinant of maximum specific productivity in this cell line. Furthermore the effect of the positive association between S phase and specific productivity is subordinate to the effect of batch phase/medium composition on the specific productivity of batch cultures. This revised version was published online in July 2006 with corrections to the Cover Date.     

On-line estimation of viable cells in a hybridoma culture at various DO levels using ATP balancing and redox potential measurement

Two on-line methods for the estimation of viable cell number in hybridoma cultivation were investigated. One used an empirical correlation between redox potential and animal cell density. The other was based on an ATP balance with ATP steady-state assumption. Oxygen uptake rate measurement provided the amount of ATP which was produced by oxidation of NADH. Oxygen uptake rate was measured either by stationary liquid phase balance with surface aeration or by gas balance during bubble aeration with headspace flushing with an inert gas. The amount of ATP produced through the glycolysis was estimated based on the amount of lactate produced. In cultures, in which pH was controlled via manipulation of the gas phase composition, the flow of CO(2) was linearly correlated with the lactate concentration. At constant dissolved oxygen levels, the viable cell density was proportional to the estimated ATP production rate, during exponential growth and during later phases. The estimated specific ATP production rate, however, varied from 2.2 pmol cell(-1) h(-1) at 10% air saturation to 4.5 pmol cell(-1) h(-1) at 100% air saturation. Specific rates of glutamine, glucose, and lactate followed the shape of the specific ATP production rate, whereas the specific oxygen uptake rate was minimal at around 50% air saturation. (c) 1996 John Wiley & Sons, Inc.     

Baculovirus expression system scaleup by perfusion of high-density Sf-9 cell cultures

A perfusion system based on a 4-L stirred tank bioreactor and a custom-designed tangential (cross-flow) filter was assembled to realize a scaleup of the Baculovirus Expression Vector System (BEVS). When perfused with 1 to 1.5 vol/day, Spodoptera frugiperda (Sf-9) insect cell cultures grew from 4 x 10(6) to 15 x 10(6) cells/mL over 3 to 4 days. The possibility of maintaining high specific production of recombinant VP6 protein (from bovine rotavirus) after baculovirus infection of the high-density cultures was then assessed. The process consisted of a growth phase in TNMFH + 10% FBS, followed by infection with Bac-BRV6L recombinant baculovirus and a shift to a low-serum (0 to 1%) medium for perfusion during the production phase. Multiple runs were executed, each including a battery of shaker flask controls at various cell densities and serum concentrations. On average, specific rVP6 production in the bioreactor amounted to 76% of that found in 20-mL shaker cultures simulatingthe bioreactor's high cell density, low serum concentration, and medium renewal rate. Mechanical stress generated by cell/medium separation in theperfusion process reduced cell growth rate but had minimal effect on rVP6production. Our results also indicated that serum concentration during the infection phase affected the rVP6 specific production in a cell density-dependent fashion. Although the feasibility of the cell density scale up was demonstrated, optimization is still needed to achieve a truly cost-effective process.     

Suppression of apoptosis in perfusion culture of Myeloma NS0 cells enhances cell growth but reduces antibody productivity

A spin filter perfusion systems was used to achieve a high cell density culture for two NS0 cell lines in 2 litres bioreactors. One cell line is transfected with the bcl-2 gene (NS0 Bcl-2) encodes the 'anti-apoptotic' human Bcl-2 protein and the other cell line (NS0 Control) with a blank vector. The runs started as batch cultures for two days and were perfused with fresh medium at 0.5 volumes per day (day(-1)) for 4 days, increasing gradually to 2 day(-1) at day 7. The increase of the viable cell density of Bcl-2 cell line was far greater than the control cell line, although they were perfused with the same amount of medium. At the end of the period of each perfusion rate, the viable cell densities of Bcl-2 culture were 30%, 120%, 160% and 220% higher than its control cell line corresponding values. Overall, there was a roughly 9 fold increase in viable cell density from the inoculum for the control culture, but almost a 30 fold increase for the Bcl-2 culture. The mode of cell death in the control culture was initially predominantly by necrosis (viability higher than 80%), but apoptotic cell death became more significant after day 8 of the culture. Cell death in the Bcl-2 culture was almost entirely by necrosis, although it remained at a very low level (less than 5%) to the termination time. The cell cycle distributions for both cell lines were very much similar indicating they have a similar doubling time and G1 to S progression rate. Interestingly, the Bcl-2 cultures exhibited reduced antibody specific production rate with increasing viable cell number and time. The volumetric production rate was, however, similar in both cultures. Bcl-2 as an anti-death protein allowed cells to survive and thus divide to higher cell densities without the need for additional nutrients. Most of the cellular energy in a producer cell line is used for biomass production rather than for antibody production, as was the case with the control cell line.     

Analyzing the dynamics of cell growth and protein production in mammalian cell fed-batch systems using logistic equations

The logistic modeling approach was used to describe experimental viable cell density (X) and product concentration (P) data from two industrial fed-batch mammalian cell culture processes with maximum product concentrations in the 3.0-9.4 g/l range. In both cases, experimental data were well described by the logistic equations and the resulting specific growth rate and protein productivity profiles provided useful insights into the process kinetics. Subsequently, sensitivity equations for both the X and P models were analyzed which helped characterize the influence of model parameters on X and P time courses. This was augmented by conventional sensitivity analyses where five values of each model parameter, 25% apart, were used to generate X and P time courses. Finally, results from sensitivity analysis were used to simulate X and P time courses that were reflective of typical early- and late-stage fed-batch cell culture processes. Different combinations of the logistic model parameters were used to arrive at the same final product concentration demonstrating the ability of the logistic approach to describe the multitude of process paths that result in the same final product concentration. Overall, the capability of the logistic equations to well describe X and P data from fed-batch cultures, coupled with their ability to simulate the multitude of paths leading up to the desired cell density and product concentration profiles, make them a useful tool during mammalian cell fed-batch process development.     

Simple and efficient control of CHO cell cultures

Cell cultures must tightly be kept under control in order to guarantee a sufficiently small variability in the protein product quality. A simple and efficient technique for CHO-cell cultures is presented that allows keeping the viable cell count X(v) and the specific growth rate μ of the cells on predefined trajectories. As X(v) and μ cannot directly be measured online, they are controlled indirectly via the total mass of oxygen consumed. Online values of the latter can precisely be estimated from off gas analysis, i.e. from the O? volume ratio measured in the vent line and air flow rate measurements. In glutamine-limited fed-batch cultivations, the glutamine feed rate can be manipulated in such a way that the viable cell density and the specific growth rate are kept on predefined profiles for nearly the entire cultivation time. The viability of the cells is not affected by the closed loop control actions. The technique was validated with CHO-cells cultured in a 2.5-L fully instrumented stirred tank bioreactor. It is shown that the controller is able to run the process exactly on predefined tracks with a high batch-to-batch reproducibility. By means of six fed-batch cultivations of CHO cells it was shown that a remarkable reproducibility of viable cell concentration could be achieved throughout 140 h cultivation time.     

补料速度对工程菌生长和产物表达的影响研究

通过研究各培养阶段补料速度对温敏启动子控制的rIL-2工程菌E.coliK802(pLY-4)培养密度和rIL-2表达的影响,发现在各培养阶段控制不同的补料速度有利于提高菌密度和rIL-2表达,缩短培养周期。确定了rIL-2工程菌高密度培养方案,三批重复实验,平均菌密度为58OD_(600);菌干重18.0g/L,rIL-2表达水平为42.4％。     

The enumeration of Leptospira interrogans serovar pomona by a bioluminescence ATP assay

A rapid method for enumerating viable Leptospira interrogans serovar pomona cells was investigated using a bacterial adenosine triphosphate (ATP) assay. The ATP was assayed by the luciferin-luciferase bioluminescence reaction. Samples of serovar pomona grown in liquid polysorbate 80-bovine albumin (P80-BA) medium for 1-3 days were analysed for ATP content, culture density (nephelometry), direct cell count and most probable number of viable cells (MPNVC) as determined by the dilution tube technique. A linear relationship was found between ATP content and the number of viable cells over the range of 4 X 10(8) to 8 X 10(9) leptospires/ml. Over this range the correlation coefficient for ATP content versus viable cells (0.96) was similar to the coefficient for culture density versus the number of viable cells. The coefficient for direct counts versus the number of viable cells was smaller. The bioluminescence assay of bacterial ATP is a promising method for enumerating viable leptospires in pure culture.     

Scale-up of the adenovirus expression system for the production of recombinant protein in human 293S cells

Human 293S cells, a cell line adapted to suspension culture, were grown to 5×10⁶ cells/mL in batch with calcium-free DMEM. These cells, infected with new constructions of adenovirus vectors, yielded as much as 10 to 20% recombinant protein with respect to the total cellular protein content. Until recently, high specific productivity of recombinant protein was limited to low cell density infected cultures of no more than 5×10⁵ cells/mL. In this paper, we show with a model protein, Protein Tyrosine Phosphatase 1C how high product yield can be maintained at high cell densities of 2×10⁶ cells/mL by a medium replacement strategy. This allows the production of as much as 90 mg/L of active recombinant protein per culture volume. Analysis of key limiting/inhibiting medium components showed that glucose addition along with pH control can yield the same productivity as a medium replacement strategy at high cell density in calcium-free DMEM. Finally, the above results were reproduced in 3L bioreactor suspension culture thereby establishing the scalability of this expression system. The process we developed is used routinely with the same success for the production of various recombinant proteins and viruses.Abbreviations CFDMEM calcium-free DMEM - CS bovine calf serum - hpi hours post-infection - J+ enriched Joklik medium - MLP major late promoter - MOI multiplicity of infection (# of infectious viral particle/cell) - q specific consumption rate (mole/cell.h) - pfu plaque forming unit (# of infectious viral particle) - Y yield (g/E6 cells or mole/cell)     

Comparison of Trichoplusia ni BTI-Tn-5B1-4 (high five) and Spodoptera frugiperda Sf-9 insect cell line metabolism in suspension cultures

Nutrient utilization and byproduct accumulation were monitored in Spodoptera frugiperda Sf-9 and Trichoplusia ni BTI-Tn-5B1-4 (High Fivetrade mark) cell lines during growth and following viral infection in suspension cultures in order to develop a better understanding of cell metabolism and to acquire information relevant to large scale fed-batch bioreactors. The utilization of glucose, dissolved oxygen, and amino acids were monitored in Sf-9 cell cultures grown in Sf-900 II serum-free medium (SFM) and in High Fivetrade mark cell cultures grown in both Sf-900 II and Express Five SFM. Using the optimal medium for each cell line, i.e., Sf-900 II SFM for Sf-9 cells and Express Five SFM for High Fivetrade mark cells, the cell growth rate, maximum cell density, specific glucose and glutamine utilization rates, and specific alanine production rate were comparable during cell growth. In addition, the expression level of recombinant human tissue plasminogen activator was comparable in the two cell lines on a per cell basis. It was found, however, that lactate and ammonia accumulated in High Fivetrade mark cell cultures, but not in Sf-9 cell cultures. In addition, High Fivetrade mark cells utilized asparagine more rapidly than glutamine, whereas Sf-9 cells consumed only minimal asparagine, and the oxygen utilization rate was significantly higher in High Fivetrade mark cell cultures. It was also found that the medium had a significant effect on High Fivetrade mark cell metabolism, e.g., the specific glucose utilization rate and the specific lactate and alanine production rates were significantly higher in Sf-900 II SFM than in Express Five SFM. In addition, the maximum cell density and specific asparagine utilization rate were significantly higher in Express Five SFM. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55:909-920, 1997.     

Methyl Mercury Stimulates Protein 32P Phospholabeling in Cerebellar Granule Cell Culture

Cultures of cerebellar granule neurons have been utilized to examine morphological and biochemical consequences of methyl mercury (MeHg). Exposure to MeHg for 24 h was found to exert toxic effects at concentrations below 1 microM characterized by neuron degeneration and neuritic varicosities. Dose-response and time course profiles for cell death were established using the 51Cr release assay, which revealed that 1 microM MeHg produced 15% cell death at 24 h, progressing to 50% at 48 h. Labeling of cultures with [32P]orthophosphate following 24-h exposure to 1 microM MeHg disclosed abnormalities in both protein and lipid phosphorylation. After 24-h exposure to 5 microM MeHg, phospholabeling of protein and lipid increased 174 and 128%, respectively, compared with controls. This stimulation of phosphorylation appeared to be neuron specific since cultures enriched in cerebellar glial cells and devoid of granule neurons displayed dose-dependent inhibition of total phosphorylation. Measurement of 32P labeling of ATP using a cyclic AMP-dependent protein kinase assay in conjunction with the firefly luciferase assay for ATP indicated no significant change in either total ATP levels or [32P]ATP specific activity at 1 or 4 h as a function of [MeHg]. Studies measuring 32P-phosphoprotein turnover indicated that MeHg had no effect on intracellular protein phosphatase activity. We conclude that one of the manifestations associated with in vitro cerebellar granule cell neurotoxicity is an abnormality in protein phosphorylation that is independent of [32P]ATP specific activity and protein phosphatase activity.