High cell density fed batch and perfusion processes for stable non-viral expression of secreted alkaline phosphatase (SEAP) using insect cells: comparison to a batch Sf-9-BEV system

The development of insect cells expressing recombinant proteins in a stable continuous manner is an attractive alternative to the BEV system for recombinant protein production. High cell density fed batch and continuous perfusion processes can be designed to maximize the productivity of stably transformed cells. A cell line (Sf-9SEAP) expressing high levels of the reporter protein SEAP stably was obtained by lipid-mediated transfection of Sf-9 insect cells and further selection and screening. The expression of the Sf-9SEAP cells was compared with the BEVS system. It was observed that, the yield obtained in BEVS was similar to the batch Sf-9SEAP at 8 and 7 IU/mL, respectively. The productivity of this foreign gene product with the stable cells was enhanced by bioprocess intensification employing the fed-batch and perfusion modes of culture to increase the cell density in culture. The fed batch process yielded a maximum cell density of 28 x 10(6) cells/mL and 12 IU/mL of SEAP. Further improvements in the productivity could be made using the perfusion process, which demonstrated a stable production rate for extended periods of time. The process was maintained for 43 days, with a steady-state cell density of 17-20 x 10(6) cells/mL and 7 IU/mL SEAP. The total yield obtained in the perfusion process (394 IU) was approximately 22 and 8 times higher than that obtained in a batch (17.6 IU) and fed batch (46.1 IU) process, respectively.     

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.     

Microcarrier culture of lepidopteran cell lines: implications for growth and recombinant protein production

Several microcarrier systems were screened with Sf-9 and High-Five cell lines as to their ability to support cell growth and recombinant (beta-galactosidase) protein production. Growth of both cell lines on compact microcarriers, such as Cytodex-1 and glass beads, was minimal, as cells detached easily from the microcarrier surface and grew as single cells in the medium. Cell growth was also problematic on Cytopore-1 and -2 porous microcarriers. Cells remained attached for several days inside the microcarrier pores, but no cell division and proliferation were observed. On the contrary, insect cells grew well in the interior of Fibra-Cel disks mainly as aggregates at points of fiber intersection, reaching final (plateau) densities of about 4 x 10(6) (Sf-9) and 2.7 x 10(6) (High-Five) cells mL(-1) (8 x 10(6) and 5.5 x 10(6) cells per cm(2) of projected disk area, respectively). Their growth was described well by the logistic equation, which takes into account possible inhibition effects. Beta-Galactosidase (beta-gal) production of Sf-9 cells on Fibra-Cel disks (infected at 3.3 x 10(6) cells mL(-1)) was prolonged (192 h), and specific protein production was similar to that of high-density free cell infection. Cultispher-S microcarriers were found to be a very efficient system for the growth of High-Five cells, whereas no growth of Sf-9 cells took place for the same system. Concentrations of about 9 x 10(6) cells mL(-1) were reached within 120 h, with cell growth in both microcarriers and aggregates, appearance of cellular bridges between microcarriers and aggregates, and eventual formation of macroaggregates incorporating several microcarriers. Specific protein productions after beta-gal baculovirus infection at increasing cell concentrations were almost constant, thus leading to elevated volumetric protein production: final beta-gal titers of 946, 1728, and 1484 U mL(-1) were obtained for infection densities of 3.4, 7.2, and 8.9 x 10(6) cells mL(-1), respectively.     

High cell density and high monoclonal antibody production through medium design and rational control in a bioreactor

A simple feeding strategy was developed and successfully employed for nutritional control in a 2-L fed-batch culture of hybridoma cells. A previously developed stoichiometric model for animal cell growth was used to design a supplemental medium for feeding. Undialyzed fetal bovine serum and trace metals (Fe(2+), SeO(3) (2-), Li(+), Zn(2+), and Cu(2+)) were fed to the cells periodically in addition to the automatic feeding of other nutrients in the supplemental medium. In this study, the maximum viable cell density was increased from 6.3 x 10(6) to 1.7 x 10(7) cells/mL, and the culture span was extended from 340 to 550 hours. The final monoclonal antibody titer achieved was 2400 mg/L. The specific production rates for ammonia and lactate were further reduced from 0.0045 and 0.0048 in our previous fed-batch experiments to 0.0028 and 0.0036 mmol/10(9) cell h, respectively. Only 3.4% of the total glucose consumption was converted into lactate, compared to 67% in a conventional batch culture.     

昆虫细胞（Sf21）悬浮培养过程中生长限制性基质间歇补加技术的 …

基于ｒ２１昆虫细胞在浮过程中所表现出的生长代谢特征,提出以培养液中残糖浓度作为控制参数,并利用限制性基质（葡萄糖和蛋白水解物）的间歇补加技术调控细胞生长的方案。实际控制表明：与批培养相比,Ｓ１ｆ２１细胞在两种具代表性的昆虫水解物）的间歇补加调控细胞生长的方案。实际控制表明：与批培养相比,Ｓｆ２１细胞在两种具代表性的昆虫细胞培养基（ＩＰＬ－４１和ＴＣ－１００）中的生长期和稳定期部都到有效的延长。ＴＣ     

An actively mixed mini-bioreactor for protein production from suspended animal cells

Biopharmaceutical production would benefit from rapid methods to optimize production of therapeutic proteins by screening host cell line/vector combination, culture media, and operational parameters such as timing of induction. Miniaturized bioreactors are an emerging research area aiming at improving the development speed. In this work, a 3 mm thick mini-bioreactor including two 12 mm wide culture chambers connected by a 5 mm wide channel is described. Active mixing is achieved by pressure shuttling between the two chambers. Gas-liquid phase exchange for oxygen and carbon dioxide is realized by molecular diffusion through 50 microm thick polymethylpentene membranes. With this unique design, a velocity difference between the middle area and the side areas at the interfaces of the culture chambers and the connecting channel is created, which enhances the mixing efficiency. The observed mixing time is on the order of 100 s. The combination of high permeability toward oxygen of polymethylpentene membranes and fluid movement during active pressure shuttling enables higher volumetric oxygen transfer coefficients, 5.7 +/- 0.4-14.8 +/- 0.6 h(-1), to be obtained in the mini-bioreactors than the values found in traditional 50 mL spinner flasks, 2.0-2.5 h(-1). Meanwhile, the calculated volume averaged shear stress, in the range of 10(-2)-10(-1) N/m(2), is within the typical tolerable range of animal cells. To demonstrate the applicability of this mini-bioreactor to culture suspended animal cells, the insect cell, Spodoptera frugiperda, is cultured in mini-bioreactors operated under a K(L)a value of 14.8 +/- 0.6 h(-1) and compared to the same cells cultured in 50 mL spinner flasks operated under a K(L)a value of 2.2 h(-1). Sf-21 cells cultured in the mini-bioreactors present comparable length of lag phases and growth rates to their counterparts cultured in 50 mL spinner flasks, but achieve a higher maximum cell density of 5.3 +/- 0.9 x 10(6) cell/mL than the value of 3.4 +/- 0.4 x 10(6) cell/mL obtained by cells cultured in 50 mL spinner flasks. Sf-21 cells infected with SEAP-baculovirus produce a maximum SEAP concentration of 11.3 +/- 0.7 U/mL when cultured in the mini-bioreactor. In contrast, infected Sf-21 cells cultured in 50 mL spinner flasks produce a maximum SEAP concentration of 7.4 +/- 0.9 U/mL and onset of production is delayed from 18 h in minibioreactor to 40 h in spinner flasks.     

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.     

Screening of transformed insect cell lines for recombinant protein production

Nine insect cell lines were evaluated for their potential as host systems for recombinant protein production using a new expression vector permitting the continuous high-level expression of secreted glycoproteins by transformed insect cells (Farrell et al., 1998). As a means of preliminary screening, all nine insect cell lines were transfected with the green fluorescence protein. Growth in static and suspension culture was then examined as a further method of screening. On the basis of their transfection efficiencies and cell growth characteristics, five insect cell lines, Bm5, High Five, IPLB-LdFB, IZD-MB-0503, and Sf-21, were selected for stable transformation to produce granulocyte-macrophage colony-stimulating factor (GM-CSF). These five cell lines were stably transformed using an antibiotic resistance scheme and evaluated as a polyclonal population. Increasing the antibiotic concentration was found to cause not only a decrease in the specific growth rate but also an increase in the specific protein production rate and final GM-CSF concentration. The transformed High Five cells exhibited by far the greatest specific protein production rate of 5.1 x 10(-)(6) microgram/(cell.h), resulting in the highest final GM-CSF concentration of 22.8 mg/L when grown in static culture. One cloned High Five cell line produced a GM-CSF concentration of 46 mg/L in static culture and 27 mg/L in suspension culture.     

High density culture of HeLa cells in a CelliGen perfusion system

A hollow fiber cartridge may be used in an extraneous recycle loop to facilitate perfusion operation of a stirred tank bioreactor. Retention of cells while removing waste products and replenishment with fresh nutrients allows higher than normal cell densities obtained in batch or continuous culture systems. This system successfully propagated HeLa cells to over 11 million viable cells per milliliter. Much higher perfusion rates (up to 4 vessel volumes per day) were necessary for high density culture of HeLa cells compared to BHK or a hybridoma cell line because of a much higher specific cellular metabolic rate. Cell specific glucose consumption rate, lactate production and ammonia production rates are several times higher for HeLa cells. Reproducible high cell densities and viabilities can be repeatedly obtained after harvest and dilution of a HeLa cell culture by partial drainage and reconstitution in the bioreactor.     

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.     

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.     

Evaluation and applications of optical cell density probes in mammalian cell bioreactors

On-line optical cell density probes were implemented to continuously monitor the cell densities in mammalian cell bioreactor and to achieve advanced bioreactor controls. We tested cell density probes from six manufacturers in high cell density bioreactors. When externally calibrated, Aquasant and Ingold backscattering probes produced the most linear probe responses (PR) versus cell density (CD), followed by the ASR and Cerex laser probes. Monitek and Wedgewood transmission probes had lower resolutions. All probes were tested in two murine hybridoma fermentations. Cell densities varied between 1 x 10(6) cells/mL to 20 x 10(6) cells/mL and the bioreactors were operated for 5 to 7 weeks. For our bioreactors, Aquasant, Ingold, ASR, Wedgewood, and Monitek probes gave satisfactory responses. Little fouling was observed with any probe at the end of 2 weeks. Fouling was a possibility after 3 weeks in one bioreactor but its effect can be easily corrected. Cell density control and specific perfusion control of bioreactors based on the Aquasant probe were achieved. Implementation of cell density probe based perfusion control, instead of "step perfusion adjustments" based on manual hemacytometer control, will result in smoother operation, healthier cultures, increased medium delivery efficiency, and reduced operational excursions. (c) 1995 John Wiley & Sons, Inc.     

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.     

Assessment of virus production and chloramphenicol acetyl transferase expression by insect cells in serum-free and serum-supplemented media using a temperature-sensitive baculovirus

Spodoptera frugiperda insect cells were grown in Sf-900 serum-free medium and two kinds of serum-supplemented media (IPL -41 and Grace's). The specific growth rates of uninfected cells were found to be 0.024, 0.35, and 0.034 h(-1) respectively, at 33 degrees C. The IPL -41 medium supported to highest maximum cell density (10.6 x 10(6) cells/mL) compared to 3.5 x 10(6) and 8.7 x 10(6) cells/mL with the Grace's and serum-free media, respectively. In temperature shifdown experiments with a temperature-sensitive baculo-virus (acts10YM1CAT), virus titer and chloramphenicol acetyl transferase (CAT) expression were highest in the IPL -41 (5.1 x 10(7) PFU/mL and 20000 U/mL). Use of Grace's medium gave higher virus titers than the serum-free medium (4.4 x 10(6) vs 4.1 x 10(5) PFU/mL) as well as higher CAT titers (7050 vs 1980 U/mL). Interestingly, in the three media used, the highest virus and CAT titers were obtained at MOI (multiplicity of infection) of 0.02 At MOI of 2.0 virtually no increase in virus of CAT titer was observed. This result is contrary to those obtained at constant-temperature (27 degrees C) infection and cell culture, in which higher virus titers and recombinant protein expression and obtained at higher MOI.     

Perfusion culture of baculovirus-infected BTI-Tn-5B1-4 insect cells: a method to restore cell-specific beta-trace glycoprotein productivity at high cell density

The impact of different cultivation-infection strategies on the productivity of baculovirus-infected BTI-Tn-5B1-4 (High Five) cells was investigated. Using beta-trace protein as the recombinant glycoprotein, the effects of multiplicity of infection (MOI) and time of infection (TOI) were studied on growth after infection as well as the degree of infection and recombinant protein productivity in batch culture. The highest productivities were found when infecting Tn5 cells at early exponential growth phase (EGP) (low cell density) using a high MOI. To increase the productive cell density of Tn5 cells after beta-trace-baculovirus infection, we performed studies infecting cells in the range of 1 to 5 x 10(6) cells/mL in fresh medium. Although the protein production was increased twofold, a strong negative cell density effect was still observed when maximal productive cell density exceeded 1 x 10(6) cells/mL. To verify whether the changing cell environment of the batch experiments was responsible for the decrease in protein productivity at increasing cell density at infection, several perfusion experiments were designed by infecting Tn5 cells at cell densities over 2 x 10(6) cells/mL under more steady-state conditions. The use of this experimental setup enabled successful infections at high cell densities with volumetric productivities of up to 1.2 g L(-1) day(-1) of beta-trace protein, which is very high for a glycoprotein expressed with the baculovirus expression vector system (BEVS). The cell specific protein productivity observed after infections at higher cell densities in perfusion mode was the same as in batch experiments at low cell concentrations, which clearly demonstrates that the cell density effect could be completely overcome with perfusion cultivation.     

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.     

Very high density of CHO cells in perfusion by ATF or TFF in WAVE bioreactor™. Part I. Effect of the cell density on the process

High cell density perfusion process of antibody producing CHO cells was developed in disposable WAVE Bioreactor? using external hollow fiber filter as cell separation device. Both “classical” tangential flow filtration (TFF) and alternating tangential flow system (ATF) equipment were used and compared. Consistency of both TFF‐ and ATF‐based cultures was shown at 20–35 × 10⁶ cells/mL density stabilized by cell bleeds. To minimize the nutrients deprivation and by‐product accumulation, a perfusion rate correlated to the cell density was applied. The cells were maintained by cell bleeds at density 0.9–1.3 × 10⁸ cells/mL in growing state and at high viability for more than 2 weeks. Finally, with the present settings, maximal cell densities of 2.14 × 10⁸ cells/mL, achieved for the first time in a wave‐induced bioreactor, and 1.32 × 10⁸ cells/mL were reached using TFF and ATF systems, respectively. Using TFF, the cell density was limited by the membrane capacity for the encountered high viscosity and by the pCO₂ level. Using ATF, the cell density was limited by the vacuum capacity failing to pull the highly viscous fluid. Thus, the TFF system allowed reaching higher cell densities. The TFF inlet pressure was highly correlated to the viscosity leading to the development of a model of this pressure, which is a useful tool for hollow fiber design of TFF and ATF. At very high cell density, the viscosity introduced physical limitations. This led us to recommend cell densities under 1.46 × 10⁸ cell/mL based on the analysis of the theoretical distance between the cells for the present cell line. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:754–767, 2013     

球形棕囊藻游离单细胞的密度与囊体形成的关系研究

球形棕囊藻(Phaeocystis globosa Scherffel)主要以囊体形态形成赤潮,由单细胞向囊体形态的转变是赤潮爆发的关键。本研究推测囊体形成的前提是游离单细胞达到一定密度阈值,当密度低于该阈值时,囊体无法形成。基于此,本文探究了不同条件(温度、营养充气搅动、摄食压力、初始密度)下囊体形成时游离单细胞的密度。结果显示:不同培养条件下,囊体形成所需的游离单细胞密度不一致,但都达到了104cells/mL的数量级;稀释试验表明,利用f/2培养基稀释使游离单细胞的密度小于104cells/mL时,囊体不能形成,而密度大于104cells/mL的游离单细胞对照组,在24 h内便有囊体形成。总的来说,游离单细胞在高密度情况下更容易形成囊体。