Elsevier

Metabolic Engineering

Volume 12, Issue 1, January 2010, Pages 39-52
Metabolic Engineering

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

https://doi.org/10.1016/j.ymben.2009.08.008Get rights and content

Abstract

The insect cells/baculovirus system is well recognized as a safe and suitable technology to produce heterologous proteins, vaccines and vectors for gene therapy. Efficient and robust production processes, able to deliver higher product concentrations, are however still needed to cope with increased requirements for large-scale manufacture. The work herein presented describes a combined experimental and modelling effort to quantify and environmentally manipulate the metabolism of Spodoptera frugiperda cells, targeting high cell density production of baculovirus vectors with potential application in human gene therapy. Culture medium supplementation with pyruvate or α-ketoglutarate at the time of infection resulted in 6–7-fold higher specific baculovirus yields at high cell density when compared to control cultures. This pushed volumetric titers to levels higher than classical low cell density infections. A quantitative description of intracellular pathways is provided using metabolic flux analysis; a direct stimulation of carbon flow through the tricarboxylic acids cycle was observed. Analysis of flux partitioning coefficients at the pyruvate and α-ketoglutarate branch-points further revealed a metabolic transition to a more energetically active state, which was confirmed by increased intracellular adenosine triphosphate generation rates. These results represent a cost-efficient and scalable strategy for high cell density production of recombinant baculovirus vectors.

Introduction

Baculovirus (BV) expression vectors are widely used for expressing a myriad of heterologous proteins with economic and biomedical interest (Summers, 2006). Recent achievements in engineering human-like glycosylation pathways in the host insect cells reinforced the advantages of this system (Jarvis, 2003). In addition to its value as a recombinant protein production platform, the development of virus-like particles (VLPs) for vaccination (Noad and Roy, 2003), BV surface-bound recombinant antigens (Oker-Blom et al., 2003) and modified BV vectors for transduction of diverse mammalian cells (Airenne et al., 2009), have brought extra attention and pressure to this technology. Efforts have been directed at optimizing upstream processing so as to meet product demands in industrially attractive processes. A bottleneck that has been recognized for several years is the loss in productivity after infection above 1×106 cells mL−1, resulting not only in poor specific product yields, but also considerably lower volumetric titers (Caron et al., 1990; Carinhas et al., 2009). This is a bottleneck common to different biological systems and especially relevant in animal cell bioprocesses (Ferreira et al., 2005; Henry et al., 2005). Most attempts to overcome this cell density effect in insect cells consisted in supplementing culture media with glucose, glutamine, a range of different complex nutrient mixtures, as well as total or partial medium exchange (for a review, see Ikonomou et al., 2003). However, only modest and/or cost-inefficient improvements were attained. A detailed understanding of how the infection process impacts the intracellular metabolic fluxes would give us important clues to increase productivity in high cell density cultures.

An efficient strategy to optimize the output in a relevant cellular process is to manipulate the carbon and nitrogen pathways of host cells (Stephanopoulos, 1999). Given the complexity of such biological systems, the rapid expansion of metabolic engineering approaches to attain these objectives has been associated with the development of modelling tools aiming to simulate and analyze the outcome of such manipulations. Stoichiometric analysis based on metabolite balancing is an established framework for the quantification of intracellular fluxes and their distribution within a network representative of the main cellular metabolic events. Despite being a steady-state approach (meaning that kinetic information on enzymatic activities is not considered), it has been of noteworthy importance to study the capabilities of industrially relevant microorganisms (Forster et al., 2003; Reed et al., 2003; Lee et al., 2005), as well as finding targets for metabolic engineering (Khannapho et al., 2008; Asadollahi et al., in press). In particular, Metabolic Flux Analysis (MFA) is an enabling tool to estimate a metabolic state constrained by exchange fluxes of nutrients between cells and the environment (Stephanopoulos et al., 1998). It has been also successfully exploited to identify principal nodes in the primary metabolism, which are characterized by significant changes in flux partitioning under different conditions, and thus can be regarded as potential control points for manipulation (van Gulik et al., 2000).

The application of MFA to BV-infected insect cells was previously carried out by our group to address the post-infection adaptations of cellular metabolism (Bernal et al., 2009). The identification of the energetic state as a reliable sensor of system productivity seems conclusive from these results, making possible the design of rational strategies for bioprocess optimization. In the present work, we hold to the same hypothesis and go a step further by manipulating the metabolic pathways of Spodoptera frugiperda Sf-9 cells through medium supplementation in order to boost recombinant BV production at high cell density. After testing key compounds as culture supplements, the stimulatory effects on the host energetic metabolism are quantified, confirming the potential of the strategy herein presented.

Section snippets

Cell culture and maintenance

Cultivation of the host insect cell line Sf-9 (ECACC 89070101) was performed in 500 mL Erlenmeyer flasks (Corning, USA) with a working volume of 50 mL. Sf900II serum- and protein-free medium (Gibco, Invitrogen, USA) was used throughout this work. For maintenance, cells were re-inoculated every 3 days at a cell density of 4–5×105 cells mL−1, and kept in a humidified incubator operated at 27 °C and 90 rpm. Cell density and viability were determined by cell counting using a Fuchs-Rosenthal chamber after

Uptake of pyruvate and α-ketoglutarate

The rational followed in our work arises from the possibility to enhance energy production through purposeful alteration of the cellular environment, attempting to sustain consistently high ATP generation rates even after infection at high cell densities. Pyruvate, a central metabolite connecting glycolysis with the TCA cycle, and α-ketoglutarate, a TCA cycle intermediate bridging this pathway with glutamine utilization and nitrogen metabolism, were selected to directly enhance TCA cycle fluxes

Discussion

The importance of the high-energy molecule ATP in industrial production processes has received much attention during the past decades, extending the border line of metabolic engineering (Zhou et al., 2009). Both mitochondrial respiration and ATP synthesis play a fundamental role in metabolism, largely determining the throughput and ability of cells to thrive in different environments. In fact, a central aspect determining cellular responsiveness to virus replication seems to be the capacity to

Conclusions

Optimization of virus-based bioprocesses is a daunting task due to the number of culture parameters affecting product delivery and quality. When to infect, how many infectious particles to be added, what medium composition to use and when to harvest are questions to which any answer represents a compromise between quantity and quality of the final product.

This work comprises a comprehensive selection of metabolic-directed strategies to improve recombinant baculovirus production in Sf-9 insect

Acknowledgments

This work was partially supported by the European Projects BACULOGENES (FP6 LHSB-CT-2006-037541) and CLINIGENE-NoE (FP6 LSHB-CT-2004-018933). Nuno Carinhas acknowledges Fundação para a Ciência e a Tecnologia (FCT) for his Ph.D. grant (SFRH/BD/36676/2007). Vicente Bernal holds a post-doctoral fellowship from Fundación Séneca (Murcia, Spain). The authors are thankful to Ana Luísa Simplício for her advice during the implementation of the HPLC protocol for amino acids analysis.

References (51)

  • D.-Y. Lee et al.

    Complementary identification of multiple flux distributions and multiple metabolic pathways

    Metab. Eng.

    (2005)
  • R. Noad et al.

    Virus-like particles as immunogens

    Trends Microbiol.

    (2003)
  • A. Roldão et al.

    Stochastic simulation of protein expression in the baculovirus/insect cells system

    Comput. Chem. Eng.

    (2008)
  • G. Stephanopoulos

    Metabolic fluxes and metabolic engineering

    Metab. Eng.

    (1999)
  • M.D. Summers

    Milestones leading to the genetic engineering of baculoviruses as expression vector systems and viral pesticides

    Adv. Virus Res.

    (2006)
  • J. Zhou et al.

    ATP in current biotechnology: regulation, applications and perspectives

    Biotechnol. Adv.

    (2009)
  • K.J. Airenne et al.

    Baculovirus-mediated gene transfer: an emerging universal concept

  • Asadollahi, M.A., Maury, J., Patil, K.R., Schalk, M., Clark, A., Nielsen, J., 2009. Enhancing sesquiterpene production...
  • C. Bédard et al.

    Growth, nutrient consumption, and end-product accumulation in Sf-9 and BTI-EAA insect cell cultures: insights into growth limitation and metabolism

    Biotechnol. Prog.

    (1993)
  • C. Bédard et al.

    Maximization of recombinant protein yield in the insect cell/baculovirus system by one-time addition of nutrients to high-density batch cultures

    Cytotechnology

    (1994)
  • V. Bernal et al.

    Cell density effect in the baculovirus-insect cells system: a quantitative analysis of energetic metabolism

    Biotechnol. Bioeng.

    (2009)
  • R. Bhatia et al.

    Insect cell physiology

    Cytotechnology

    (1997)
  • A. Burgener et al.

    Intracellular ATP and total adenylate concentrations are critical predictors of reovirus productivity from Vero cells

    Biotechnol. Bioeng.

    (2006)
  • N. Carinhas et al.

    Baculovirus production for gene therapy: the role of cell density, multiplicity of infection and medium exchange

    Appl. Microbiol. Biotechnol.

    (2009)
  • A.W. Caron et al.

    High-level recombinant protein production in bioreactors using the baculovirus-insect cell expression system

    Biotechnol. Bioeng.

    (1990)
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    Current address: Departamento de Bioquímica y Biología Molecular B e Inmunología, Facultad de Química, Universidad de Murcia, E-30100 Murcia, Spain.

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