Improving baculovirus production at high cell density through manipulation of energy metabolism

Carinhas, Nuno; Bernal, Vicente; Monteiro, Francisca; Carrondo, Manuel J.T.; Oliveira, Rui; Alves, Paula M.

doi:10.1016/j.ymben.2009.08.008

Cited by 82 publications

(93 citation statements)

References 48 publications

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“…Amino acid concentrations were analyzed by HPLC using the Waters AccQ.Tag Amino Acid Analysis Method (Waters, Milford, MA) as described elsewhere [9]. The extracellular concentrations of citrate and pyruvate were determined by 1 H-NMR spectroscopy using a 500 MHz Avance spectrometer (Bruker, Billerica, MA) with a 5 mm QXI inversed probe.…”

Section: Analysis Of Extracellular Metabolitesmentioning

confidence: 99%

Quantification of Metabolic Rearrangements During Neural Stem Cells Differentiation into Astrocytes by Metabolic Flux Analysis

et al. 2016

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Proliferation and differentiation of neural stem cells (NSCs) have a crucial role to ensure neurogenesis and gliogenesis in the mammalian brain throughout life. As there is growing evidence for the significance of metabolism in regulating cell fate, knowledge on the metabolic programs in NSCs and how they evolve during differentiation into somatic cells may provide novel therapeutic approaches to address brain diseases. In this work, we applied a quantitative analysis to assess how the central carbon metabolism evolves upon differentiation of NSCs into astrocytes. Murine embryonic stem cell (mESC)-derived NSCs and astrocytes were incubated with labelled [1-13 C]glucose and the label incorporation into intracellular metabolites was followed by GC-MS. The obtained 13 C labelling patterns, together with uptake/secretion rates determined from supernatant analysis, were integrated into an isotopic non-stationary metabolic flux analysis ( 13 C-MFA) model to estimate intracellular flux maps. Significant metabolic differences between NSCs and astrocytes were identified, with a general downregulation of central carbon metabolism during astrocytic differentiation. While glucose uptake was 1.7-fold higher in NSCs (on a per cell basis), a high lactate-secreting phenotype was common to both cell types. Furthermore, NSCs consumed glutamine from the medium; the highly active reductive carboxylation of alpha-ketoglutarate indicates that this was converted to citrate and used for biosynthetic purposes. In astrocytes, pyruvate entered the TCA cycle mostly through pyruvate carboxylase (81%). This pathway supported glutamine and citrate secretion, recapitulating well described metabolic features of these cells in vivo. Overall, this fluxomics study allowed us to quantify the metabolic rewiring accompanying astrocytic lineage specification from NSCs.

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Section: Analysis Of Extracellular Metabolitesmentioning

confidence: 99%

Quantification of Metabolic Rearrangements During Neural Stem Cells Differentiation into Astrocytes by Metabolic Flux Analysis

et al. 2016

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“…Böylece besinlerin hücre tarafından hızlı tüketimi sonucu oluşabilecek olumsuzluk ortadan kaldırılabilmektedir. Ulaşılabilen maksimum hücre yoğunluğu literatürde 10x10 6 hücre/ml olarak belirtilmiştir [55].…”

Section: Sf9 Hücresi Ile Protein üRetimi İçin Kullanılan Operasyon Mounclassified

Transfection of Spodoptera frugiperda Insect Cell and Production of Recombinant Protein

Aytekin¹

2015

Pamukkale J Eng Sci

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“…4.4). KGA and PYR were found to be limiting at high cell density infections and the supplement of either PYR or KGA at the time of infection has been previously shown to result in an improvement of budded virus yields by 6-7 fold (Carinhas et al, 2010). It is possible that the lower level of KGA in the high ICD case may contribute to the lower yield at the high ICD as it is a key metabolite that can be supplied into the TCA cycle through glutamine and glutamate.…”

Section: B 42 B) Were Typical For Racmnpvmentioning

confidence: 99%

“…These include the measurement of extracellular metabolites to identify the limiting nutrients in the medium (Caron et al, 1990;Drews et al, 1995;Reuveny et al, 1993); the measurement of consumption rates and fluxomics analysis ; and supplementing culture media with glucose, glutamine, complex nutrient mixtures, and key intermediate metabolites (Carinhas et al, 2010;Reuveny et al, 1993). However, a possible limitation of these approaches is that the metabolite levels in the media do not necessarily reflect intracellular metabolite levels.…”

Section: Introductionmentioning

confidence: 99%

“…The challenge for improving the yield of baculovirus insect cell systems is that protein yield (per cell basis) drops when cells are infected at a high cell density, which is known as "the cell density effect". Many research groups have investigated the extracellular environment through the measurement of extracellular metabolites to identify the limiting nutrients in the medium (Caron et al, 1990;Drews et al, 1995;Reuveny et al, 1993), the measurement of consumption rates and fluxomics analysis , the supplement of glucose, glutamine, complex nutrient mixtures, and key intermediate metabolites to culture media (Carinhas et al, 2010;Reuveny et al, 1993), in order to overcome the cell density effect.However, the limitation of these approaches is that the metabolite levels in the media might not necessarily reflect intracellular metabolite levels. For many reasons, some metabolites may not get into the cells at high cell densities post-infection, although they are available in the culture medium at reasonable levels.…”

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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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Improving baculovirus production at high cell density through manipulation of energy metabolism

Cited by 82 publications

References 48 publications

Quantification of Metabolic Rearrangements During Neural Stem Cells Differentiation into Astrocytes by Metabolic Flux Analysis

Quantification of Metabolic Rearrangements During Neural Stem Cells Differentiation into Astrocytes by Metabolic Flux Analysis

Transfection of Spodoptera frugiperda Insect Cell and Production of Recombinant Protein

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

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