Enhanced growth of Sf-9 cells to a maximum density of 5.2 x 10(7) cells per mL and production of beta-galactosidase at high cell density by fed batch culture

Significant improvement in cell growth and protein production has been achieved in Sf-9 insect cell cultures using pulse additions of multicomponent nutrient feed concentrates (Bédard et al., 1994; Chan et al., 1998). The present work focuses on investigating an alternative feeding strategy wherein the nutrients are fed in a semi continuous manner. Fed batch culture experiments were carried out to compare the two different feeding strategies, pulse and semi continuous and a process developed to achieve a cell density of 5.2 x 10(7) cells/mL of Sf-9 cells in a 3.5 L bioreactor. Production of recombinant protein beta-galactosidase was carried out by infecting the cells with baculovirus at a MOI of 10 at cell densities of 17 x 10(6)cells/mL. Specific productivity could be maintained at cell densities as high as 14 x 10(6) cells/mL. The results presented indicate that the feeding method can provide significant improvements in the performance with a reduction in the amount of total nutrients added. On-line monitoring of the culture using the capacitance probe showed that the capacitance probe can be used successfully to monitor the biomass and infection process even at higher cell densities.     

On-line monitoring of physiological parameters of insect cell cultures during the growth and infection process

On-line monitoring of insect cell cultures used for the production of recombinant proteins with the baculovirus expression vector system (BEVS) provides valuable tools for the optimization, operation, and control of the production process. The relative permittivity (epsilon') and CO(2) evolution rates (CER) were measured on-line using the biomass monitor and the infrared CO(2) analyzer, respectively. The growth and infection phases of two different cell lines, Spodoptera frugiperda (Sf-9) and Trichoplusia ni(High-5), were monitored using the above measurements. These in turn were correlated to the progress of the culture by using the off-line measurements of protein produced, virus titer, and biovolume, which is the product of viable cell density and mean cell volume. The epsilon', CER, and the biovolume profiles were closely matched during the growth phase of cells when grown in a batch or fed batch culture. The relationship became more complex when the cultures were either in stationary phase or in the postinfection phase. The epsilon' profile was found to be a good indicator of the process of synchronous baculoviral infection, showing a plateau between 18 and 24 h postinfection (hpi), the period during which budded virus is produced, and a peak at approximately 48 hpi correlated to the onset of accelerated cell lysis. The CER profile continues to increase after the growth period with a peak around the 24 hpi period, after which there is a decline in the profile corresponding to release of virus as seen from virus titer determinations. This was examined for Sf-9 cultures under conditions of cell densities from 3 to 50 x 10(6) cells/mL and MOI values ranging from 0.001 to 1000. The profiles were found to be similar also in the case of the High-5 cells. Thus both measurements give reliable information regarding the physiological status of the cells as seen from their correlation to virus and protein production. A further combination of these with the off-line measured parameters such as the biovolume and metabolite concentrations can give a more detailed understanding of the process and help in the better design and automation of these processes.     

The use of culture redox potential and oxygen uptake rate for assessing glucose and glutamine depletion in hybridoma cultures

Culture redox potential (CRP) and oxygen uptake rate (OUR) were monitored on-line during glucose- and glutamine-limited batch cultures of a murine hybridoma cell line that secretes a neutralizing monoclonal antibody specific to toxin 2 of the scorpion Centruroides noxius Hoffmann. It was found that OUR and CRP can be used for assessing the viable cell concentration and growth phases of the culture. Before nutrient depletion, OUR increased exponentially with viable cell concentration, whereas CRP decreased monotonically until cell viability started to decrease. During the death phase, CRP gradually increased. A sudden decrease in OUR occurred upon glucose or glutamine depletion. CRP traced the dissolved oxygen profile during a control action or an operational eventuality, however, during nutrient depletion it did not follow the expected behavior of a system composed mainly by the O(2)/H(2)O redox couple. Such a behavior was not due to the accumulated lactate or ammonia, nor to possible intracellular redox potential changes caused by nutrient depletion, as inferred from respiration inhibition by rotenone or uncoupled respiration by 2,4-dinitrophenol. As shown in this study, operational eventualities can be erroneously interpreted as changes in OUR when using algorithms based solely on oxygen balances. However, simultaneous measurements of CRP and OUR may be used to discriminate real metabolic events from operational failures. The results presented here can be used in advanced real-time algorithms for controling glucose and glutamine at low concentrations, avoiding under- or over-feeding them in hybridoma cultures, and consequently reducing the accumulation of metabolic wastes and improving monoclonal antibody production. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 555-563, 1997.     

Design of an efficient medium for insect cell growth and recombinant protein production

We report the development of a new serum-free medium based on the use of factorial experiments. At first, a variety of hydrolysates were screened using a fractional factorial approach with High-Five cells. From this experiment yeastolate ultrafiltrate was found to have, by far, the most important effect on cell growth. Furthermore, Primatone RL was found to remarkably prolong the stationary phase of Sf-9 and High-Five cell cultures. The optimal concentrations for yeastolate and Primatone were determined to be 0.6 and 0.5%, respectively, on the basis of a complete factorial experiment. This new medium, called YPR, supported good growth of both Sf-9 and High-Five cells in batch cultures, with maximal densities of 5.4 and 6.1 x 10(6) cells/ml, respectively. In addition, both cell lines achieved good growth in bioreactor batch culture and had a prolonged stationary phase of 3-4 d in YPR medium compared to Insect-XPRESS medium. The ability of the new medium to support recombinant protein expression was also tested by infecting Sf-9 or High-Five cells at high density (2 x 10(6) cells/ml) with a baculovirus expressing secreted placental alkaline phosphatase (SEAP). The maximum total SEAP concentration after 7 d was about 43 lU/ml (58 mg/L) and 28 lU/ml (39 mg/L) for High-Five and Sf-9 cells, respectively.     

On-line monitoring of the progress of infection in Sf-9 insect cell cultures using relative permittivity measurements

The use of on-line relative permittivity (epsilon') measurements for monitoring cultures of Sf-9 cells was evaluated in a batch culture and a batch infected with a baculovirus expressing beta-galactosidase. It was found that viable cell density and volume essentially accounted for all the variation in epsilon' in both non-infected and synchronously infected cultures, indicating that the epsilon' of a cell suspension was sensitive only to changes in the viable cell population. Additionally the parameter provided clearly defined signposts of the progress of the infection.     

Comparison of growth and recombinant protein expression in two different insect cell lines in attached and suspension culture

Culture conditions required for obtaining maximum recombinant protein concentrations from two cell lines, Spodoptera frugiperda (IPLBeta-Sf21-AE) and Trichoplusia ni (Tn 5Beta-1-4), were determined in this work. Conditions studied include mode of culture (suspended vs attached), agitation rates, inoculum sizes, cell concentration at the time of infection, and various serum-free media (SFM). Results were compared with the performance of attached cultures in TnM-FH with 10% fetal bovine serum. Growth rates in the different culture media tested were similar, but the cell numbers achieved (i.e., yield) improved 2 to 2.7-fold in SFM over cultures in TnM-FH. Agitation rates of 150-160 rpm were necessary for maximum growth of suspended Tn 5Beta-1-4 cells compared to 125-150 rpm for Sf-21 cells. An inoculum size of 5 x 10(5) cells/mL gave good growth rates and optimum biomass yields for both cell lines. Cultures of both cell lines were infected with viruses encoding for beta-galactosidase or human secreted alkaline phosphatase (seAP). Protein expression in TnM-FH in attached culture showed that Tn 5Beta-1-4 cells are 2-4.5 times more productive on a per cell basis than Sf-21 cells grown under similar conditions. Production of beta-galactosidase in Sf-21 cells increased 50% in suspension cultures with SFM compared to attached cultures in TnM-FH, but seAP expression was essentially unchanged by culture techniques. The Tn 5Beta-1-4 cells produced 2.6-4.4 and 2.7-3 times more beta-galactosidase and seAP, respectively, in SFM in suspension compared to Sf-21 cells. EX-CELL 401 and Sf900-II were formulated as optimized SFM for Sf cell lines. However, in Sf-21 cultures EX-CELL 400 performed better than the other two media, as it increased the beta-galactosidase yield up to 25%. Surprisingly, EX-CELL 401 was the best medium for the production of beta-galactosidase by Tn 5Beta-1-4 cells, resulting in 25% and 69% higher volumetric and specific yields, respectively, compared to EX-CELL 405 which was formulated for this specific cell line. These results show that even when culture media are designed for maximal growth of a specific cell line, other media may provide the best conditions for protein production.     

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.     

Baculovirus production for gene therapy: the role of cell density,multiplicity of infection and medium exchange

One of the major concerns regarding the use of insect cells and baculovirus expression vectors for the production of recombinant proteins is the drop in production observed when infecting cultures at high cell densities; this work attempts to understand this so-called cell density effect in the scope of baculovirus production for gene therapy purposes. A Spodoptera frugiperda insect cell line (Sf-9) was cultured and infected in serum-free medium, and the patterns of production of a recombinant baculovirus expressing the green fluorescent protein (GFP) were analyzed at different cell concentrations at infection (CCIs) and multiplicities of infection (MOIs). The results confirm that a cell density effect on productivity occurs which is dependent on the MOI used, with a high MOI “delaying” the drop in production to higher cell densities. Medium replacement at the time of infection using a high MOI considerably improved baculovirus production, with the different production indicators, namely the titer, specific yield, amplification factor, and time of harvesting, increasing with cell concentration for the CCI range tested. Virus titers as high as 2.6 × 10¹⁰ IP.mL⁻¹ were obtained in cultures infected at 3.5 × 10⁶ cells.mL⁻¹, while the amplification factor was roughly 19 times higher than the highest value obtained without medium exchange.     

TubeSpin bioreactor 50 for the high-density cultivation of Sf-9 insect cells in suspension

Here we present the TubeSpin bioreactor 50 (TubeSpins) as a simple and disposable culture system for Sf-9 insect cells in suspension. Sf-9 cells had substantially better growth in TubeSpins than in spinner flasks. After inoculation with 10⁶ cells/ml, maximal cell densities of 16 × 10⁶ and 6 × 10⁶ cells/ml were reached in TubeSpins and spinner flasks, respectively. In addition the cell viability in these batch cultures remained above 90% for 10 days in TubeSpins but only for 4 days in spinner flasks. Inoculation at even higher cell densities reduced the duration of the lag phase. After inoculation at 2.5 × 10⁶ cells/ml, the culture reached the maximum cell density within 3 days instead of 7 days as observed for inoculation with 10⁶ cells/ml. Infection of Sf-9 cells in TubeSpins or spinner flasks with a recombinant baculovirus coding for green fluorescent protein (GFP) resulted in similar GFP-specific fluorescence levels. TubeSpins are thus an attractive option for the small-scale cultivation of Sf-9 cells in suspension and for baculovirus-mediated recombinant protein production.     

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.     

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.     

Culture of insect cells in helical ribbon impeller bioreactor

An 11-L helical ribbon impeller (HRI) bioreactor was tested for the culture of Spodoptera frugiperda (Sf-9) cells. This impeller and surface baffling ensured homogeneous mixing and high oxygen transfer through surface aeration and surface-induced babble generation. Serum-supplemented and serum-free cultures, using TNMFH and IPL/41 media, respectively, grew a similar specific growth rates(0.031 and 0.028 h(-1)) to maximum cell densities of 5.5 x 10(6)-6.0 x 10(6) cells. mL(-1) with viability exceeding 98% during exponential growth phase. Growth limitation coincided with glucose and glutamine depletion and production of significant amounts of alanine. The bioreactor was further tested under more stringent conditions by infecting a serum-free medium culture with a recombinant baculovirus. Heterologous protein production of approximately 35 mug per 10(6) cells was comparable to yields obtained in serum-free cultures grown in spinner flasks and petri dishes. Average specific oxygen up-take and carbon dioxide production rates of the serum-free culture prior to infection as measured by on-line mass spectroscopy were 0.20 mumol O(2)mu.(10(6) cells)(-1) h(-1) and 0.22 mumol CO(2) . (10(6) cells)(-1)h(-1) and increased by 30-40% during infection. Therefore, the mixing and oxygenation conditions of this bioreactor were suitable for insect cell culture and recombinant protein production, with limitation being mainly attributed to nutrient depletion and toxic by-product generation.     

Computer-aided on-line monitoring of physiological variables in suspended cell cultures of Perilla frutescens in a bioreactor

A computer-aided on-line real-time monitoring system for plant cell bioprocesses was established and applied to the cultivation of Perilla frutescens plant cells in a bioreactor. This system calculated several informative process variables which were useful for the identification of the physiological states of the plant cells during cultivation. Some variables, such as the respiratory quotient (RQ), pH, and specific carbon dioxide evolution rate (SCER), could be used for the identification of the growing phase of cell cultures. The results also suggest that the oxygen uptake rate (OUR) and the specific OUR (SOUR) may depend on the accumulation of anthocyanin (a secondary metabolite) in P. frutescens cell cultures.     

Factors influencing recombinant protein yields in an insect cell-bacuiovirus expression system: multiplicity of infection and intracellular protein degradation

The insect cell (Sf9)-baculovirus (AcNPV) expression system was employed for the synthesis of beta-galactosidase, a model heterologous protein. In the recombinant virus studied, the lacZ gene is fused to a portion of the polyhedrin structural gene and is under the control of the polyhedrin promoter. The effect of the multiplicity of infection (MOI) on product titer was determined by infecting cells with MOI values ranging from 0 to 100 and monitoring the production of beta-galactosidase with time. The relationship between final product titer and MOI was dependent on the growth phase of the cells prior to infection. The final product titer from cells infected in the early exponential phase was relatively independent of MOI. For cells infected in late-exponential phase there was a logarithmic relationship between the final beta-galactosidase titer and the MOI used, with the highest MOI studied resulting in greatest protein synthesis. The synthesis and degradation rates of beta-galactosidase were investigated by a pulse-chase technique using L-[(35)S]-methionine. At 24 h postinfection, the degradation rate is of the same order of magnitude as the synthesis rate. However, the synthesis rate of beta-galactosidase increases dramatically at 96 h postinfection. During this later period, the degradation rate is negligible. Although degradation of recombinant protein occurs in this system, degradation activity declines as infection proceeds and is insignificant late in intention when recombinant protein expression is intense.     

Improving performance of mammalian cells in fed-batch processes through "bioreactor evolution"

The amount of recombinant product obtained from mammalian cells grown in a bioreactor is in part limited by achievable cell densities and the ability of cells to remain viable over extended periods of time. In an attempt to generate cell lines capable of better bioreactor performance, we subjected the DG44 Chinese Hamster Ovary (CHO) host cell line and a recombinant production cell line to an iterative process whereby cells capable of surviving the harsh conditions in the bioreactor were selected. This selective process was termed "bioreactor evolution". Following the selective process, the "evolved" host cells attained a 2-fold increase in peak cell density and a 72% increase in integral cell area. Transient transfection experiments demonstrate that the evolved cells have the same transfection efficiency and the same secretory potential as the initial cells. The "evolved" host was also found to contain a large subpopulation of cells that did not require insulin for growth. From this, a new population of growth-factor-independent cells was obtained. These improvements in host properties should prove beneficial in the expression of recombinant proteins in fed-batch processes. The selective process was also applied to a recombinant production cell line. The evolved cells from this selection exhibited a 38% increase in peak cell density, a 30% increase in integral cell area, and a 36% increase in product titer. These increases were obtained without any appreciable impact on product quality, demonstrating the usefulness of this simple approach to improve the performance of recombinant cell lines.     

A simple and modified manometric method for measuring oxygen uptake rate of plant cells in flask cultures

Summary A simple and convenient technique was developed based on the principle of Warburg manometric method to measure O₂ uptake rate (OUR) and CO₂ evolution rate (CER) of suspended cells in a shake flask culture. It was successfully applied to suspension cultures of rice (Oryza sativa) and Panax notoginseng cells, and some important bioprocess parameters, such as OUR, CER, respiratory quotient (RQ), specific OUR (SOUR) and specific CER (SCER), were quantitatively obtained. The measuring system is easy to operate, able to treat many samples simultaneously and is economical.     

Production of recombinant adeno-associated viral vectors using a baculovirus/insect cell suspension culture system: from shake flasks to a 20-L bioreactor

Production of recombinant adeno-associated viral vectors using a baculovirus/insect cell system at various scales is presented. Shake flask studies were conducted to assess conditions to be used in bioreactors. Two insect cell lines, Trichoplusia ni (H5) and Spodoptera frugiperda (Sf9), were compared for their ability to produce rAAV-2 after infection with recombinant baculoviruses coding for the essential components of the vector. The effect of varying the ratio between individual baculoviruses and the effect of the overall multiplicity of infection (MOI), as well as the cell density at infection, were also examined. Infectious rAAV-2 particles were proportionally produced when increasing the individual MOI of BacRep virus up to 1.6. When equal amounts of each virus were used, a leveling effect occurred beyond an overall MOI of 5 and a maximum titer was obtained. Increasing the cell density at infection resulted in higher yields when infecting the cells in fresh medium; however, for the production of bioactive particles, an optimal peak cell density of approximately 1 x 10(6) cells/mL was observed without medium exchange. Infection in 3- and 20-L bioreactors was done at an overall MOI of 5 with a ratio of the three baculoviruses equal to 1:1:1. Under these conditions and infecting the cells in fresh medium, a total of approximately 2.2 x 10(12) infectious viral particles (bioactive particles) or 2.6 x 10(15) viral particles were produced in a 3-L bioreactor. Without replacing the medium at infection, similar titers were produced in 20 L. Our data demonstrates the feasibility of rAAV-2 production by BEVS at various scales in bioreactors and indicates that further optimization is required for production at high cell densities.     

Physiological aspects of surface-immobilized Catharanthus roseus cells

Summary Growth and alkaloid production of surface-immobilized C. roseus cells were studied in a 2-1 bioreactor. Media designed to maximize cell growth or alkaloid production were employed. Nitrate and carbohydrate consumption rates as well as growth rates and biomass yields of immobilized cultures were equal or somewhat lower than for cell suspension cultures. Respiration rate (O₂ consumption and CO₂ production rates) of immobilized C. roseus cell cultures was obtained by on-line analysis of inlet and outlet gas composition using a mass spectrometer. Respiration rate increased during the growth phase and decreased once the nitrogen or the carbon source was depleted from the medium. The respiration rate of immobilized C. roseus cells resembled rates reported in the literature for suspension cultures. Offprint requests to: Denis Rho     

Rational scale-up of a baculovirus-insect cell batch process based on medium nutritional depth

We have developed a serum-free cell culture process utilizing a recombinant baculovirus (AcNPV) expression vector to infect Trichoplusia ni insect cells for the production of the human lysosomal enzyme, glucocerebrosidase. The enzyme, which is harvested as a secreted protein in this process, can serve as a replacement therapy for the genetic deficiency Gaucher disease. In the course of pilot scale-up of a batch glucocerebrosidase process from 25-mL working volume shaker flask units to 25-L working volume stirred bioreactor units, a semi-empirical model was developed for the rational determination of scaleable process parameters, including host cell density at infection, multiplicity of infection (MOI), and harvest time. A key assumption of the model is that maximum protein production is limited by the serum-free medium's nutritional capacity, which can, in turn, be determined from the growth of uninfected cells. For the host cell/medium combination used in this study, the nutritional limit was determined to be 1.3 x 10(7) to 1.7 x 10(7) viable-cell-days/mL. Based on this, the model predicts that optimal protein expression is consistent with a 4-day batch process where the host cell density at the time of infection is 1.5 x 10(6) to 2.0 x 10(6) cells/mL and the MOI is 0.09-0.3. These parameters were empirically confirmed to give the highest achievable batch product yield, first in shaker flasks and then at larger scales. The low MOI allows at least one population doubling to take place post viral addition, so that the effective infected cell density producing product generally exceeds 4 x 10(6) cells/mL. It was also interesting to note that this process consistently achieved the same level of maximum protein production at the 25-L bioreactor scale in 4 days compared to 5 days at the shaker flask scale. This may be attributable to better control of the culture environment in the bioreactor. Unlike some other lepidopteran insect cells, such as Sf-9, T. ni cells were found to produce significant levels of the inhibitory metabolites ammonia and lactate. Our results suggest that reduction and/or removal of inhibitory metabolites might be beneficial for infection of high-density cultures of these cells and might also facilitate application of more sophisticated culture strategies, including fed-batch. (c) 1996 John Wiley & Sons, Inc.