Effect of elevated oxygen and glutamine levels on foreign protein production at high cell densities using the insect cell-baculovirus expression system

Taticek, Ronald A.; Shuler, Michael L.

doi:10.1002/(sici)1097-0290(19970420)54:2<142::aid-bit6>3.0.co;2-l

Cited by 69 publications

(65 citation statements)

References 37 publications

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“…As shown in Figure 4, DO varied between 20 and 70% air saturation depending on the timing of measurement and medium exchange. These values remained within the optimal range for insect cell culture (Taticek and Shuler 1997), thus oxygen was not a limiting factor, particularly when the cells were exposed in the gas phase for 'breathing'. This hypothesis can be further realized by the profile of the molar ratio of lactate production to glucose utilization (L/G, solid squares in Figure 4).…”

Section: Oxygenationmentioning

confidence: 65%

“…The cell number during the reactor operation was estimated indirectly by measuring the glucose consumption rate as described previously . A specific glucose consumption rate of 0.2 mg per 10 6 cells per day, which was determined previously (Taticek and Shuler 1997) and confirmed in our laboratory in spinner flask studies (not shown), was used to calculate the cell number.…”

Section: Cell Countmentioning

confidence: 95%

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High-density cultivation of insect cells and production of recombinant baculovirus using a novel oscillating bioreactor

Chung

2003

Cytotechnology

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Section: Oxygenationmentioning

confidence: 65%

Section: Cell Countmentioning

confidence: 95%

High-density cultivation of insect cells and production of recombinant baculovirus using a novel oscillating bioreactor

Chung

2003

Cytotechnology

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“…The phenomenon has been observed also for recombinant proteins produced by genetically modified baculovirus. Several causes have been proposed to explain the "cell density effect", including nutrient limitation (Bédard et al, 1994), accumulation of toxic by-products (Taticek & Shuler, 1997), autocrine factors and cell cycle distribution (Braunagel et al, 1998;Calles et al, 2006), and inhibition of the central energy metabolism (Bernal et al, 2009), among others, but the causes remain to be identified. The cell density effect, that could be observed in several insect cell lines infected with different baculoviruses, both wild-type and recombinant, can be overcome, at least partially in Sf9 cell cultures by adopting alternative strategies for cell culture and viral infection, such as fedbatch or perfusion, as will be explained hereinafter.…”

Section: Parameters Of Infectionmentioning

confidence: 99%

Production of Insecticidal Baculoviruses in Insect Cell Cultures: Potential and Limitations

Claus¹,

Gioria²,

Micheloud³

et al. 2012

Insecticides - Basic and Other Applications

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Section: The Cell Density Effectmentioning

confidence: 99%

“…The cell density effect has been reported mainly for Sf9 cells Caron et al, 1990;Doverskog et al, 2000;Jesionowski and Ataai, 1997;Radford et al, 1997;Reuveny et al, 1993;Taticek and Shuler, 1997;Wong et al, 1996;Yamaji et al, 1999). There are a few investigations conducted in other cell lines such as Anticarsia gemmatalis (saUFL-AG-286) (Micheloud et al, 2009), Trichopulsia ni (Hi5) (Chico and Jager, 2000;Ikonomou et al, 2004;Taticek and Shuler, 1997;Yang et al, 1996), andHeliothis zea (HzAM1) (Chakraborty et al, 1996), which show that the cell density effect applies generally to baculovirus infected insect cell systems.There have been many speculations on the cause of the cell density effect including nutrient limitation, toxic compound accumulation, cell-to-cell contact inhibition, but the reasons for this drawback remain unidentified (Doverskog et al, 1997;Taticek and Shuler, 1997). It has been suggested that the cause of the drop in productivity at high cell densities for baculovirus infection processes is related to nutrient limitations rather than accumulation of toxins (Drews et al, 1995;Radford et al, 1997;Wong et al, 1996).…”

mentioning

confidence: 99%

Metabolomic study of baculovirus infected insect cells to identify metabolite based strategies for improving virus yields

Tran¹

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The baculovirus insect cell system has been used widely for biopesticide applications and for the production of recombinant proteins, vaccines and vectors for gene delivery. To be commercially viable, especially for the low margin applications of biopesticides and veterinary vaccines, the yield of this production system needs to be improved. One of the important approaches is the infection of insect cell cultures at high cell densities in order to maximize volumetric production. However, the cell specific yield gradually reduces with increasing infection cell density (ICD). As a result, the volumetric production can not improve further regardless of efforts to increase the uninfected cell density in batch or fedbatch cultures. The phenomenon known as "the cell density effect" appears not only during the late stage of protein production, but also during the early stage of virus replication and transcription. Discovering the changes in intra and extracellular metabolites between low and high ICDs may reveal evidence for improving yields. This PhD thesis focuses on the development of an intracellular metabolite extraction protocol for infected insect cell cultures and for measuring intra and extracellular metabolites of infected cells of different baculovirus infected insect cell systems.Prior to analyzing the intracellular metabolites of low and high ICDs, an extraction protocol was developed and validated for insect cell cultures, especially infected insect cells. The ice-cold saline quenching solution developed for mammalian cells did not work well for insect cells, especially for infected cultures. Therefore, a modification of the quenching solution was made by an addition of 0.2% Pluronic® F-68 that successfully protected the cell membrane during quenching and washing procedures. In addition, one wash was found to be enough for removal of medium originated metabolites adhering on the surface of cell membranes. The ATP recovery (over the direct extract) of the extraction protocol using ice-cold saline quenching solution plus 0.2% Pluronic® F-68 after one wash was almost 90% for Spodoptera frugiperda (Sf9) cells infected with a recombinant Autographa californica multiple nucleopolyhedrovirus (rAcMNPV) and Helicoverpa zea (HzAM1) cells infected with a wild-type Helicoverpa armigera single nucleopolyhedrovirus (HearNPV) at 24 hours post infection (hpi).The intracellular metabolite patterns and substrate consumption rates of low versus high ICDs was first investigated for the Sf9/rAcMNPV system in Sf900™III medium. A preliminary study was conducted to identify how late post-infection the cell membrane integrity was maintained during the quenching and washing processes. The result showed that the extraction protocol is efficient up to 48 hpi for infected Sf9 cells. Levels of intracellular nucleotides (tri-phosphates), amino acids (AA), and TCA cycle intermediates iii (α-ketoglutarate, pyruvate) during an infection at a low ICD (2×10 6 cells/mL) were shown to be higher than those during an infection at a high...

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Effect of elevated oxygen and glutamine levels on foreign protein production at high cell densities using the insect cell-baculovirus expression system

Cited by 69 publications

References 37 publications

High-density cultivation of insect cells and production of recombinant baculovirus using a novel oscillating bioreactor

High-density cultivation of insect cells and production of recombinant baculovirus using a novel oscillating bioreactor

Production of Insecticidal Baculoviruses in Insect Cell Cultures: Potential and Limitations

Metabolomic study of baculovirus infected insect cells to identify metabolite based strategies for improving virus yields

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