A physiological product‐release model for baculovirus infected insect cells

Haas, Richard; Nielsen, Lars K.

doi:10.1002/bit.20565

Cited by 9 publications

(3 citation statements)

References 22 publications

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“…The S. frugiperda clone 9 cell-line (Sf9; ATCC CRL 1711) and recombinant b-Galactosidase-expressing A. californica multi-capsid nucleopolyhedrovirus (rb-Gal AcMNPV) have been described previously [26]. Cell stocks were propagated in the commercial serumfree medium Sf-900 II (Invitrogen, Carlsbad, CA), and maintained by regular passaging every 3-4 days in 25 mL shaker flask cultures (125 mL single-use Erlenmeyer flasks; Corning, Lowell, MA) at seeding densities of 3-5 Â 10 5 cells/mL.…”

Section: Sf9-racmnpv Systemmentioning

confidence: 99%

“…The passage 1 (P1) virus stock was prepared by infecting Sf9 cells in Sf-900 II medium, at a Time of Infection (TOI) of 1.5 Â 10 6 cells/ mL and a Multiplicity of Infection (MOI) of 0.1 Plaque Forming Units (PFU)/cell, using frozen virus stocks [26]. At 4 days post-infection (d.p.i.…”

Section: Sf9-racmnpv Systemmentioning

confidence: 99%

“…The HaSNPV virus stocks were titered using an in-house suspension culture (SC) titration assay [32], which was informed by the PowerNielsen baculovirus infection model [33] and HaSNPV-HzAM1 infection kinetics [34]. The product of rAcMNPV infections (rbGal) was quantified using a well-established colorimetric assay based on the enzymatic hydrolysis of o-nitrophenyl-galactopyranoside (ONPG), as described previously [26]. The product of wtHaSNPV infections (OBs) was quantified via triplicated hemocytometer counts under a phase-contrast microscope, after liberation from infected cells using 0.5% w/v SDS, as described previously [35].…”

Section: Cell Virus and Product Quantificationmentioning

confidence: 99%

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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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Section: Sf9-racmnpv Systemmentioning

confidence: 99%

Section: Sf9-racmnpv Systemmentioning

confidence: 99%

Section: Cell Virus and Product Quantificationmentioning

confidence: 99%

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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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Baculovirus Kinetics, Insect Cell Culture

Chan

Reid

Nielsen

2010

Encyclopedia of Industrial Biotechnology

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Viral‐based processes continue to represent an important class of cell culture processes. Indeed, the number of viral‐based cell culture processes is increasing with (a) viral vaccine production moving from primary cells to continuous cell lines, (b) increased use of viral biopesticides, (c) increased use of viral expression systems, and (d) the use of viral gene therapy vectors. The inherent complexity combined with the problems of working with human or animal pathogens has meant that the academic treatment of viral production kinetics has lagged behind that of other cell culture systems. The emergence of the baculovirus (an innocuous insect virus) for protein expression changed this situation and the baculovirus is used throughout this article to illustrate how to handle complex viral kinetics. Emphasis is placed on the development of mathematical models, which are essential tools when studying viral production kinetics.

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Decline in baculovirus-expressed recombinant protein production with increasing cell density is strongly correlated to impairment of virus replication and mRNA expression

Huynh

Tran

Chan

et al. 2013

Appl Microbiol Biotechnol

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The cell density effect is a well-established constraint in the baculovirus-insect cell expression platform, in which cell-specific productivity declines with increasing cell density, hence limiting the maximum achievable volumetric yield of protein product. A deeper elucidation of this phenomenon is sought in this study, by tracking the peak production of viral DNA (vDNA), recombinant LacZ mRNA, and β-galactosidase (β-gal) protein, over a wide range of cell densities. Sf9 suspension cell cultures were propagated in Sf-900 III serum-free medium and synchronously infected with rAcMNPV at multiple infection cell densities (ICDs) of between 0.5 and 8 × 10(6) cells/mL. There was a strong negative linear correlation between the specific β-gal yield and the peak cell density (PCD) post-infection, but contrary to previous reports, the yield decline started at a lower PCD of around 1 × 10(6) cells/mL. Most interestingly, there also was a corresponding strong negative linear correlation between the specific vDNA or LacZ mRNA yield, and the PCD. Comparing the infections at the highest and lowest PCDs tested, the yield decline was most dramatic for β-gal protein (95 %) and LacZ mRNA (90 %), while it was more moderate for vDNA (50 %). These declines were significantly reduced but not completely arrested, when spent medium was replaced with fresh at the ICD. These findings suggest that protein yield deterioration with increasing cell density originated from limitations during upstream events such as virus gene replication or transcription, rather than during the translational phase. Such limitations may be largely nutritional, but a more complex mechanism may be implicated.

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A physiological product‐release model for baculovirus infected insect cells

Cited by 9 publications

References 22 publications

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

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

Baculovirus Kinetics, Insect Cell Culture

Decline in baculovirus-expressed recombinant protein production with increasing cell density is strongly correlated to impairment of virus replication and mRNA expression

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