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.     

A two-stage bioreactor system for the production of recombinant proteins using a genetically engineered baculovirus/insect cell system

A two-stage bioreactor scheme was developed for the large-scale production of recombinant proteins using a genetically engineered baculovirus/insect cell system. The first bioreactor was employed for cell growth and the second for cell infection. Silkworm Bm5 cells were infected with a recombinant baculovirus, BmNPV/P5.cat, containing a bacterial chloramphenicol acetyltransferase (CAT) gene under the control of the polyhedrin gene promoter of Bombyx mori nuclear polyhedrosis virus (BmNPV). This recombinant baculovirus has been used as an expression vector for the production of recombinant CAT enzyme. A specific productivity of 82 to 90 mug CAT/(10(6) cells) was obtained using the BmNPV/Bm5 expression system, a yield similar to that achieved using the AcNPV/Sf expression system. Repeated infection of high-density cell cultures did not reduce the specific productivity of the CAT enzyme. Most importantly, the problems associated with the infection of high-density cell cultures were resolved by means of controlled infection conditions and appropriate replenishment of spent culture medium following infection. The glucose uptake rate by the cells following infection was 50% higher than that by the cells before infection. Not only did the infection of high-density cell cultures result in consistent yields of 250 mg/L of CAT enzyme, but also the two-stage bioreactor system was proven to be reliable for a long-term operation beyond 600 h. (c) 1993 John Wiley & Sons, Inc.     

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.     

Modeling and optimization of the baculovirus expression vector system in batch suspension culture

A mathematical model has been developed that predicts the cell population dynamics and production of recombinant protein and infective extracellular virus progeny by insect cells after infection with baculovirus in batch suspension culture. Infection in the model is based on the rate of virus attachment to suspended insect cells under culture conditions. The model links the events following infection with the sequence of gene expression in the baculovirus replicative cycle. Substrate depletion is used to account for the decrease in product yield observed when infecting at high cell densities. Model parameters were determined in shaker flasks for two media: serum-supplemented IPL-41 medium and serum free Sf900II medium. There was good agreement between model predictions and the results from an independent series of experiments performed to validate the mode. The model predicted: (1) the optimal time of infection at high multiplicity of infection: (2) the timing and magnitude of recombinant protein production in a 2-L bioreactor; and (3) the timing and magnitude of recombinant protein production at multiplicities of infection from 0.01 to 100 plaque-forming units per cell. Through its ability to predict optimal infection strategies in batch suspension culture, the model has use in the design and optimization of large-scale systems for the production of recombinant products using the baculovirus expression vector system. (c) 1994 John Wiley & Sons, Inc.     

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.     

Production of Choristoneura fumiferana Nucleopolyhedrovirus in C. fumiferana (CF-2C1 Cells) in a 3 Litre Bioreactor Using Serum-free Medium

The purpose of this study was to develop a cell culture process in a bioreactor for the production of a viral insecticide for the spruce budworm, Choristoneura fumiferana . Several cell lines were tested for their growth in serum-free medium suspension cultures. One cell line, CF-124T-2C1 (CF-2C1), was successfully adapted to grow in suspension cultures in SFM. Serum-free Ex-Cell 405 medium produced a much higher cell density (6.3 x 10 6 cells ml -1 ) than the Grace's medium supplemented with 10% fetal bovine serum (2.5 x 10 6 cells ml -1 ). Also, a higher yield of virus was obtained in the former medium. Ex-Cell 405, was used to study the growth of CF-2C1 cells and the production of C. fumiferana nucleopolyhedrovirus (CfMNPV) in a 3 l bioreactor. Under these conditions, a specific growth rate ( μ) of 0.027 h -1 was obtained during the exponential growth phase, and the specific carbon dioxide evolution rate, as determined by on-line measurement, was 0.9 x 10 -16 mol cell -1 s -1 and 1.78 x 10 -16 mol cell -1 s -1 during growth and infection phases, respectively. Virus production in bioreactor cultures infected at 1.3 x 10 6 cells ml -1 was consistently lower than that obtained in Erlenmeyer shake flasks. Only 26% of the cells were infected in the bioreactor compared to 44% in the shake flasks. However, a higher yield of occluded virus was obtained in the bioreactor cultures than in shake flasks. The production of occlusion bodies (OB) achieved in bioreactor cultures was 2 x 10 6 OB ml -1 .     

Production of recombinant protein using the HeLa S3-vaccinia virus expression system: bioreactor perfusion and effects of post-infection temperature

Adaptation of the vaccinia virus expression system to HeLa S3 suspension bioreactor culture for the production of recombinant protein was conducted. Evaluation of hollow fiber perfusion of suspension culture demonstrated its potential for increased cell density prior to infection. The hollow fiber was also used for medium manipulations prior to infection. Two process parameters, multiplicity of infection (MOI) and temperature during the protein production phase, were evaluated to determine their effect on expression of the reporter protein, enhanced green fluorescent protein (EGFP). An MOI of 1.0 was sufficient for infection and led to the highest level of intracellular EGFP expression. Reducing the temperature to 34 degrees C during the protein production phase increased production of the protein two-fold compared to 37 degrees C in spinner flask culture. Scaling up the process to a 1.5-liter bioreactor with hollow fiber perfusion led to an overall production level of 9.9 microg EGFP/10(6) infected cells, or 27 mg EGFP per liter.     

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.     

Inactivation of Bacteriophages by Thiol Reducing Agents

Adaptation of the vaccinia virus expression system to HeLa S3 suspension bioreactor culture for the production of recombinant protein was conducted. Evaluation of hollow fiber perfusion of suspension culture demonstrated its potential for increased cell density prior to infection. The hollow fiber was also used for medium manipulations prior to infection. Two process parameters, multiplicity of infection (MOI) and temperature during the protein production phase, were evaluated to determine their effect on expression of the reporter protein, enhanced green fluorescent protein (EGFP). An MOI of 1.0 was sufficient for infection and led to the highest level of intracellular EGFP expression. Reducing the temperature to 34 °C during the protein production phase increased production of the protein two-fold compared to 37 °C in spinner flask culture. Scaling up the process to a 1.5-liter bioreactor with hollow fiber perfusion led to an overall production level of 9.9 μg EGFP/10⁶ infected cells, or 27 mg EGFP per liter.     

Bioprocess development for the production of mouse-human chimeric anti-epidermal growth factor receptor vIII antibody C12 by suspension culture of recombinant Chinese hamster ovary cells

The mouse-human chimeric anti-epidermal growth factor receptor vIII (EGFRvIII) antibody C12 is a promising candidate for the diagnosis of hepatocellular carcinoma (HCC). In this study, 3 processes were successfully developed to produce C12 by cultivation of recombinant Chinese hamster ovary (CHO-DG44) cells in serum-free medium. The effect of inoculum density was evaluated in batch cultures of shaker flasks to obtain the optimal inoculum density of 5 × 10⁵ cells/mL. Then, the basic metabolic characteristics of CHO-C12 cells were studied in stirred bioreactor batch cultures. The results showed that the limiting concentrations of glucose and glutamine were 6 and 1 mM, respectively. The culture process consumed significant amounts of aspartate, glutamate, asparagine, serine, isoleucine, leucine, and lysine. Aspartate, glutamate, asparagine, and serine were particularly exhausted in the early growth stage, thus limiting cell growth and antibody synthesis. Based on these findings, fed-batch and perfusion processes in the bioreactor were successfully developed with a balanced amino acid feed strategy. Fed-batch and especially perfusion culture effectively maintained high cell viability to prolong the culture process. Furthermore, perfusion cultures maximized the efficiency of nutrient utilization; the mean yield coefficient of antibody to consumed glucose was 44.72 mg/g and the mean yield coefficient of glutamine to antibody was 721.40 mg/g. Finally, in small-scale bioreactor culture, the highest total amount of C12 antibody (1,854 mg) was realized in perfusion cultures. Therefore, perfusion culture appears to be the optimal process for small-scale production of C12 antibody by rCHO-C12 cells.     

Rapid development of clone-specific,high-performing perfusion media from established feed supplements

Perfusion cultivation of recombinant CHO cells is of substantial interest to the biopharmaceutical industry. This is due to increased space–time-yields (STYs) and a short residence time of the recombinant protein in the bioreactor. Economic processes rely on cultivation media supporting rapid growth in the exponential phase and high protein production in the stationary phase at minimal media consumption rates. To develop clone-specific, high-performing perfusion media we present a straightforward and rapid two-step approach combining commercially available basal media and feed supplements using design-of-experiment. First, the best performing feed supplements are selected in batch cultures. Then, the mixing ratio of selected feed supplements is optimized in small-scale semicontinuous perfusion cultures. The final media formulation is supported by statistical response surface modeling of a set of cultivation experiments with blended media formulations. Two best performing novel media blends were finally applied to perfusion bioreactor verification runs to reach 200 × 10⁶ c/ml within 2 weeks at minimum cell-specific perfusion rates as low as 10–30 pL/c/d. Obtained STYs of 0.4–1.2 g/L/d represent a 10-fold increase compared to batch cultures. This general workflow is universally applicable to any perfusion platform combining a specific cell line, basal medium, and established feed solutions.     

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.     

Scale-up of virus-like particles production: effects of sparging, agitation and bioreactor scale on cell growth, infection kinetics and productivity

The baculovirus-insect cells expression system was used for the production of self-forming Porcine parvovirus (PPV) like particles (virus-like particles, VLPs) in serum-free medium. At 2l bioreactor scale an efficient production was achieved by infecting the culture at a concentration of 1.5 x 10(6)cells/ml using a low multiplicity of infection of 0.05 pfu per cell. In a continuous bioreactor, it was shown that the uninfected insect cells were not sensitive to local shear stress values up to 2.25 N/m2 at high Reynolds numbers (1.5 x 10(4)) in sparging conditions. Uninfected insect cells can be grown at scaled-up bioreactor at high agitation and sparging rates as long as vortex formation is avoided and bubble entrapment is minimized. An efficient process scale-up to 25 l bioreactor was made using constant shear stress criteria for scale-up. The kinetics of baculovirus infection at low multiplicity of infection, either at different cell concentration or at different scales, are very reproducible, despite the different turbulence conditions present in the bioreactor milieu. The results suggest that the infection kinetics is controlled by the rate of baculovirus-cell receptor attachment and is independent of the bioreactor hydrodynamic conditions. Furthermore, the achieved specific and volumetric productivities were higher at the 25 l scale when compared to the smaller scale bioreactor. Different rates of cell lysis after infection were observed and seem to fully explain both the shift in optimal harvest time and the increase in cell specific productivity. The results emphasize the importance of integrated strategies and engineering concepts in process development at bioreactor stage with the baculovirus insect cell system.     

Use of a stationary bed reactor and serum-free medium for the production of recombinant proteins in insect cells.

Insect cells (Spodoptera frugiperda) have been cultured in a stationary bed reactor, packed with a fibrous polyester carrier. When the bioreactor was perfused with serum-supplemented medium, a cell density of 6 x 10(6) cells ml-1 packed carrier was reached. Scanning electron microscopy investigations have shown that the insect cells grew along the three-dimensionally oriented fibers of the Fibra-cel carrier. After infection of the logarithmically growing cells with a recombinant baculovirus (Autographa californica) containing the gene coding for beta-galactosidase, the medium in the bioreactor was changed to serum-free medium. At day 13 postinfection (p.i.), a beta-galactosidase level of 320 microgram ml-1 and, at day 17 p.i., a virus titer of 2.1 x 10(8) TCID50 units ml-1 (day 17 p.i.) were reached. In another bioreactor, operated in a similar way but with serum-containing medium, a beta-galactosidase concentration of 360 microgram ml-1 and a virus titer of 2.3 x 10(8) TCID50 units ml-1 were obtained. These results indicate the potential use of this production system for the production of recombinant protein and baculovirus in insect cells.     

Recombinant beta-galactosidase production in serum-free medium by insect cells in a 14-L airlift bioreactor.

Spodoptera frugiperda (Sf9) insect cells were successfully cultured in serum-free medium in a 14-L airlift bioreactor. Cell densities as high as 1 x 10(7) cells/mL were achieved with specific growth rates of approximately 0.0286 h-1 (doubling time of 24 h). This system was also used to demonstrate the expression of a reported gene, beta-galactosidase (beta-gal), when cells were infected with a recombinant baculovirus. Approximately 0.33 mg of beta-gal/mL (i.e., 104,000 units/mL) of medium were obtained at the 14-L scale, while about 0.95 mg of beta-gal/mL (i.e., 285,000 units/mL) of medium were obtained in small-scale shaker flasks. The difference was attributed to a suboptimal infection in the large scale. Specific oxygen consumption rates decreased from 5.58 x 10(-17) mol O2/cell.s in early exponential growth to 3.13 x 10(-17) mol O2/cell.s at 3 days post-infection.     

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.     

高渗胁迫和灌流培养策略提高HEK 293细胞生产重组腺病毒载体产量

由于各种疾病在全球范围内的肆虐,国际市场对重组腺病毒载体(adenoviral vector,Adv)疫苗的需求量急剧增加,而工艺研究是解决这一问题的有效手段之一。在细胞接毒前施加高渗胁迫可以提高分批培养模式下的Adv产量,新兴的灌流培养也可以显著提高Adv的产量。将高渗胁迫工艺与灌流培养相结合,有望进一步提升高细胞密度生产过程中的Adv产量。本研究利用摇瓶结合拟灌流培养作为生物反应器灌流培养的缩小模型,使用渗透压为300–405 mOsm的培养基研究了高渗胁迫对细胞生长和Adv生产的影响。结果显示,在细胞生长阶段使用370 mOsm的高渗透压培养基,在病毒生产阶段使用300 mOsm的等渗透压培养基的灌流培养工艺有效地提高了Adv的产量。进一步研究发现这可能归因于病毒复制后期HSP70蛋白的表达量增加。将这种工艺放大至生物反应器中,Adv的产量达到3.2×10¹⁰ IFU/mL,是传统灌流培养工艺的3倍。本研究首次将高渗胁迫工艺与灌流培养相结合的策略应用于HEK 293细胞生产Adv,同时揭示了高渗胁迫工艺增产Adv的可能原因,为HEK 293细胞生产其他类型Adv的工艺优化提供了借鉴。     

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.     

Insights into adenoviral vector production kinetics in acoustic filter-based perfusion cultures

One of the major limitations in the production of adenoviral vectors is the reduction in cell-specific productivity observed for increasing cell density at infection in batch cultures. This observation strongly suggests some nutrient depletion and/or metabolite inhibition in the media. These limitations have been partially overcome through other feeding strategies, such as fed-batch and sequential batch operations. To improve these results, we evaluated perfusion as a strategy to increase the volumetric productivity of HEK-293 cell cultures, by allowing productive infection at higher cell densities. An acoustic cell separator was employed in consideration of the increased shear sensitivity of the cells during the infection phase. The effects of perfusion rate and cell density at infection on the production of a recombinant adenovirus expressing the GFP were investigated. The perfusion mode allowed successful infection at cell densities in the range of 2.4-3 x 10(6) cell/mL, while maintaining a similar cell specific productivity (17,900 +/- 2400 VP/cell) to that of a batch infected at a low cell density (5 x 10(5) cell/mL). The highest virus concentrations (4.1 +/- 0.6 x 10(10) VP/mL) were attained for a feed rate of 2 vol/d and constituted a fivefold increase compared to a batch with medium replacement. Rapid assessment of the infection status was achieved through the use of on-line monitoring of respiration, fluorescence, and biovolume. Analysis of the kinetics of nutrient consumption and metabolite production revealed that a reduction in specific productivity is correlated with reduced metabolic activity.     

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)