High density culture of insect cells using rational medium design and feeding strategy

The objective of this study is to achieve high density cell culture by a rational medium design and feeding strategy. Insect cell/baculovirus expression system is one of the widely used methods for the production of heterologous proteins in the cell culture domain. Insect cell Spodoptera frugiperda Sf-21 and a recombinant baculovirus with encoded gene for human interleukin-5 were chosen as the model system in this study. A stoichiometric model was established to study the demand of nutrients, including glucose, 20 amino acids, and yeastolate, for the synthesis of cell mass. The coefficients for individual nutrients in the stoichiometric equation governing insect cell growth were determined from the information of cell mass and compositions. Based on the stoichiometric coefficients, the initial and supplemental media for fed-batch cell cultures were designed. The experiments began with the inoculation of Sf-21 cells into a spinner flask with the initial medium, which provided a starting environment for achieving optimum cell growth. This was followed by the periodic feeding of supplemental medium designed by utilizing the stoichiometric equation that governs insect cell growth. With this strategy, it was demonstrated that the Sf-21 cell culture reached a cell density in excess of 1.9᎒⁷ cells/ml. During the cultivation process, the utilization of various nutrients and the production of metabolites were also monitored. Further experiments proved that high concentration of recombinant product (such as human interleukin-5) could be achieved by infecting the high density cells (resulting from the designed medium) with recombinant baculoviruses.     

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.     

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.     

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.     

Glutamine is not an essential amino acid for Sf-9 insect cells

Summary Spodoptera frugiperda (Sf-9) insect cells are fully capable of growth and proliferation in a glutamine, glutamate and aspartate-free medium, provided that ammonium ions are supplied. S. frugiperda (Sf-21) and Mamestra brassicae cells (IZD-MB-0503) also grow in glutamine-free media but not Trichoplusia ni cells (BTI-TN 5B1-4). The yield of -galactosidase in Sf-9 cells infected with a recombinant baculovirus under glutamine-free conditions was even higher than the yield obtained in glutamine containing cultures.     

High-level recombinant protein production in bioreactors using the baculovirus-insect cell expression system

In order to develop an efficient process for large-scale production of recombinant protein, various factors were studied which affect the productivity of Sf-9 (Spodoptera frugiperda) insect cells when using the baculovirus expression system. It was shown that upon infection with the Bac-BRV6L recombinant baculovirus, the level per cell of VP6 (a bovine rotavirus nucleocapsid protein) would drop 10-fold when host cell density at the time of infection increased from 2 x 10(6) to 3 x 10(6) cells/mL. The decrease was found to be totally reversible by culture medium renewal after infection, even when cells were infected at the stationary phase. Recombinant protein production was 4-6 times higher using TNMFH medium supplemented with 10% fetal bovine serum (FBS) than in IPL/41 serum-free medium. Fine-tuning of infection parameters in a 4-L surface-aerated bioreactor resulted in the production of typically 350 mg/L of VP6 protein, representing more than 25% of total cell proteins.     

Optimization of protein-production by the baculovirus expression vector system in shake flasks

Summary Shake flasks were successfully employed for the cultivation of Spodoptera frugiperda (Sf-9) insect cells and for the production of \-galactosidase, a recombinant model protein, utilizing the baculovirus expression vector system. The culture doubling time and maximal cell density were 20 h and 5 × 10⁶ cells/ml respectively. The optimal liquid volumes for flasks rotating at 100 rpm were 25–40% of the flask total volume. Enzyme production (about 600 mg/l) was best at a multiplicity of infection of between 1 and 20 and at a cell density at time of infection of 0.7 × 10⁶ cells/ml. At a rotation speed of 100 rpm, Pluronic F-68 had no effect on growth and enzyme production. Offprint requests to: Y. Shoham     

Quantification of cell culture factors affecting recombinant protein yields in baculovirus-infected insect cells

An experimental study was undertaken to quantify the effects of infection cell density, medium condition, and surface aeration on recombinant protein yields in insect cells. In the absence of surface aeration and fresh medium, insect cells generated higher product yields (on a per cell basis) when infected with recombinant baculovirus at low cell densities, LCD (3 x 10(5)-4 x 10(5) cells/mL), than at high cell densities, HCD (>0.9 x 10(6) cells/mL), for two distinct baculovirus types. Surface aeration of a HCD culture infected in spent medium improved beta-glactosidase yields 5-fold over the nonaerated case. Surface aeration and medium replenishment improved beta-galactosidase yields of a HCD culture by 20-fold (compared to a 1.6-fold improvement for a LCD culture), resulting in cultures with productivties that were independent of the cell density at infection.     

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.     

Investigation of sequential behavior of carboxyl protease and cysteine protease activities in virus-infected Sf-9 insect cell culture by inhibition assay

Proteases produced during the culture of Spodoptera frugiperda Sf-9 cells infected with Autographa californica nuclear polyhedrosis virus (AcNPV) were assayed with various protease inhibitors. This inhibitory analysis revealed that: (1) carboxyl and cysteine proteases were predominantly produced by the insect cells infected with recombinant AcNPV, the gene of which encoded a variant of green fluorescent protein in a portion of the polyhedrin gene of the baculovirus, and (2) the protease activity was almost completely blocked by pepstatin A (carboxyl protease inhibitor) and E64 (cysteine protease inhibitor) in an additive manner in the presence of EDTA. Utilizing the additive property of the inhibitors, the inhibition-based protease assay discriminated between the two protease activities and elucidated the sequential behavior of the carboxyl and cysteine proteases produced in the virus-infected Sf-9 cell culture. The carboxyl protease(s) existed in the virus-infected cells all the time and their level in the medium continuously increased. Uninfected cells also contained a carboxyl protease activity, the level of which was similar to that of the virus-infected cells. At a certain time after virus infection, the cysteine protease activity was largely increased in the virus-infected cells and a significant amount of the protease(s) was released into the medium, due to the cell membranes losing their integrity. The behavior of intracellular and extracellular cysteine protease activities coincided with that of a recombinant protein whose expression was under the control of the viral polyhedrin promoter. Similar examinations with wt-AcNPV-infected and uninfected insect cells showed that the inhibition-based protease assay was useful for analyzing the carboxyl protease and cysteine protease activities emerging in the insect cell (Sf-9)/baculovirus expression system.     

Recombinant protein synthesis in Trichoplusia ni BTI-Tn-5B1-4 insect cell aggregates.

The Trichoplusia ni BTI-Tn-5B1-4 (Tn-5B1-4) insect cell line has received considerable attention as a host for the baculovirus expression vector system. In the present study, suspension cultures were used to compare Tn-5B1-4 cell aggregates and cells selected to grow predominantly as individual cells. No significant difference was found between cell aggregates and cells growing predominantly individually in regard to cell growth rate, glucose consumption and lactate accumulation, and specific recombinant protein synthesis levels. In addition, the levels of recombinant protein synthesis were considerably higher than those produced by the commonly used Spodoptera frugiperda Sf-9 insect cell line.     

Secretion of functional papain precursor from insect cells. Requirement for N-glycosylation of the pro-region

The synthetic gene coding for the precursor of the cysteine protease papain (EC 3.4.22.2) has been expressed using the baculovirus/insect cell system. The prepropapain gene was cloned into the transfer vector IpDC125 behind the polyhedrin promoter. The recombinant construct was then incorporated by homologous recombination into the Autographa californiaca nuclear polyhedrosis virus genome. The host Spodoptera frugiperda Sf9 cells infected with the recombinant baculovirus secrete an enzymatically inactive N-glycosylated papain precursor. This zymogen could be activated in vitro to yield about 400 nmol of active papain per liter of culture. The recombinant active mature papain was enzymatically indistinguishable from natural papain but the precursor was not processed to the same amino acid residue. The insect cells also accumulated prepropapain and glycosylated propapain intracellularly. This accumulation was an indication that there are rate-limiting steps in the secretion of proteins from insect cells in this expression system. Characterization of mutants of the precursor has shown that entry into the secretory pathway and addition of carbohydrate are prerequisite conditions for the production and secretion of functional propapain.     

Recombinant protein production in insect cell cultures infected with a temperature-sensitive baculovirus

Spodoptera frugiperda (IPLB-SF-21) insect cells were grown in shake-flasks and infected with a temperature-sensitive baculovirus to express the gene of chloramphenicol acetyl transferase (CAT) in serum-free medium (SF-900) and two serum-supplemented media (IPL-41 and Grace's). In temperature-shift experiments (cell growth at 33°C followed by virus replication at 27°C 3–4 days later), virus and CAT production were much poorer in the serum-free medium than in serum-supplemented media, though cell growth was virtually the same in the different media tested. In all the three media, highest virus and CAT titers were obtained at the lowest MOI (multiplicity of infection 0.02). This result is contrary to that obtained in constant-temperature culture (27°C for both cell growth and virus replication). Virus and CAT production was greatly improved when the entire culture was run at constant temperature. It appeared that infected cells were severely damaged at 33°C (6°C above the optimal 27°C), resulting in little or no virus and protein production. As a result of these temperature-shift experiments, a larger-scale (141 air-lift bioreactor) serum-free culture of Sf-9 insect cells was conducted at constant temperature (27°C) to produce recombinant protein (β-galactosidase). A cell density as high as 1×10⁷ cells.ml⁻¹, and a β-gal concentration of up to 104,000 unit.ml⁻¹ were achieved.     

Culture in the rotating-wall vessel affects recombinant protein production capability of two insect cell lines in different manners

Summary The production of recombinant secreted alkaline phosphatase protein in virally infected insect cells was studied in shaker flask and high aspect rotating-wall vessel (HARV) culture. Two commonly used cell lines, Spodoptera frugiperda Sf-9 (Sf-9) and a nonaggregating isolate of the Trichoplusia ni BTI-Tn-5B1-4 (Tn-5B1-4) cell line, Trichoplusia ni Tn-5B1-4-NA (TN-5B1-4-NA), were used and monitored for 120-h postinfection. Different responses to culture in the HARV were seen in the two cell lines. While the Sf-9 cell line was able to produce slightly greater amounts of recombinant protein in the HARV than in shaker flask controls, the Tn-5B1-4-NA cell line produced significantly lesser amounts in the HARV than in the shaker flasks. Both cell lines exhibited longer life spans and longer periods of protein production in HARV culture than in shaker flask culture, presumably due to lower levels of shear encountered in the HARV. The important difference was in the protein production rate responses of the two cell lines. While the protein production rates of Sf-9 cells were comparable in both HARV and shaker flask cultures, the protein production rates of Tn-5B1-4-NA cells were much lower in HARV culture than in shaker flask cultures. The conclusion is drawn that cell line-specific adaptation to the HARV strongly influences recombinant protein production.     

Propagation ofSpodoptera frugiperda cells (Sf9) and production of recombinant proteins with the baculovirus expression system using improved spinner flasks with membrane aeration

Summary It has been shown that the growth of Spodoptera frugiperda cells is significantly reduced or ceased under oxygen limiting culture conditions. This paper describes the use of a new membrane-aerated spinner flask which was compared to conventional surface-aerated spinner flasks with regard to growth of the insect cell line Sf9 and recombinant protein production after infection with baculovirus. Using a commercially available serum-free culture medium Sf9 cells reached highest cell densities (3×10⁶ ml^–1) in the membrane-aerated spinner flask. Production of recombinant protein was also influenced by the oxygen supply. In the membrane-aerated spinner flask and in a surface-aerated spinner flask with reduced filling volume more than 20000 U ml^–1 of a recombinant interleukin-2 variant were accumulated whereas only 100 U ml^–1 were produced in a surface-aerated spinner flask with insufficient oxygen supply. Sufficient oxygenation appears to be essential for proliferation of Sf9 cells as well as recombinant protein production after infection with baculovirus. Membrane oxygenation allows sufficient oxygen supply at high cell density and an at least 2.5 fold higher filling volume per spinner unit.     

Evidence of Pluronic F-68 direct interaction with insect cells: impact on shear protection, recombinant protein, and baculovirus production*

Pluronic F-68 has been widely used to protect animal cells from hydrodynamic stress, but its mechanism of action is still debatable. Published evidence indicates that Pluronic F-68 interacts with cells, yet scarce information exists of its effect on recombinant protein and virus production by insect cells. In this work, the effect of Pluronic F-68 on production of recombinant baculovirus and rotavirus protein VP7 was determined. Evidence of Pluronic F-68 direct interaction with Sf-9 insect cells also was obtained. Maximum recombinant VP7 concentration and yield increased 10x, whereas virus production decreased by 20x, in spinner flask cultures with 0.05% (w/v) Pluronic F-68 compared to controls lacking the additive. No differences were observed in media rheology, nor kinetics of growth and infection (as inferred from cell size) between both cultures. Hence, Pluronic F-68 influenced cell physiology independently of its shear protective effect. Cells subjected to a laminar shear rate of 3000 s(-1) for 15 min, without gas/liquid interfaces, were protected by Pluronic F-68 even after its removal from culture medium. Furthermore, the protective action was immediate in vortexed cells. The results shown here indicate that Pluronic F-68 physically interacts with cells in a direct, strong, and stable mode, not only protecting them from hydrodynamic damage, but also modifying their capacity for recombinant protein and virus production.     

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.     

Maximization of recombinant protein yield in the insect cell/baculovirus system by one-time addition of nutrients to high-density batch cultures

Suspension cultures of Sf-9 cells at different stages of growth were infected with a recombinant baculovirus expressing -galactosidase, using a range of multiplicities of infection (MOI) of 0.05 to 50. Following infection, the cells were resuspended either in the medium in which they had been grown or in fresh medium. Specific -galactosidase yields were not markedly affected by either MOI or medium change in cultures infected in early exponential phase (3×10⁶ cells mL^–1). In cultures infected at later growth stages, -galactosidase yields could only be maintained by medium replacement. The possibility that this requirement for medium replacement is due either to the accumulation of an inhibitory byproduct or nutrient limitation was examined. Alanine, a major byproduct of cultured insect cell metabolism, did not significantly reduce recombinant protein yield when added to infected cultures in concentrations of up to 40 mM. Following a factorial design, various nutrient concentrates were added alone or in combination to cultures infected in late exponential phase. Additions that included both yeastolate ultrafiltrate and an amino acid mixture restored specific -galactosidase yields to levels observed at earlier growth stages or in late stages with medium replacement; the addition of these concentrates, by permitting production at higher cell density, led to increases in the volumetric yield of recombinant protein. Together or separately, the concentrates when added to uninfected late exponential phase cultures, lead to a doubling of the maximum total cell protein level normally supported by unamended medium.     

Characterisation of Tetraploid and Diploid Clones of Spodoptera frugiperda Cell Line

We have isolated and characterised diploid and tetraploid clones from the normally heterologous Spodoptera frugiperda (Sf-9)cell line by dilution cloning technique. Tetraploid clones were found to have cell sizes in excess of 35% larger than that of the diploid clones. In contrast, the maximum cell numbers achieved in batch cultures of diploid clones were on average 185% higher than the tetraploid cell numbers. Growth rates and metabolic quotients during the exponential phase were similar for both clones. Tetraploid cells infected with wild-type and recombinant green fluorescent protein (GFP) baculovirus, resulted in more polyhedra or GFP product per cell. Importantly, the difference between the clones either completely diminished or reduced to 50% when the yield was assessed in terms of the amount of polyhedra or GFP per mL of medium, respectively. These results indicate that the existing heterogeneity in insect cell populations with respect to ploidy level, are correlated to cell growth and product yield.