Nuno Carinhas

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

The cell density effect (i.e., the drop in the specific productivity in the baculovirus‐insect cells expression system when cells are infected at high cell densities) has been extensively described in the literature. In this article, a model for the central metabolism of serum‐free suspension cultures of Spodoptera frugiperda Sf9 cells is proposed and used to investigate the metabolic basis for this phenomenon. The main metabolic pathways (glycolysis, pentose phosphate pathway, tricarboxylic acids cycle, glutaminolysis, and amino acids metabolism), cellular growth and energetics were considered. The analysis of the stoichiometric model allowed further understanding of the interplay of the consumption of carbon and nitrogen sources in insect cells. Moreover, metabolic flux analysis revealed that Sf9 cells undergo a progressive inhibition of central metabolism when grown to high cell densities, for which the incorporation of amino acids carbon backbones into the TCA cycle (mainly glutamine) and the down‐regulation of glycolysis are partially responsible. Following infection by baculovirus and cellular division arrest, central energy metabolism depended on the infection strategy chosen (cell concentration at the moment of infection and multiplicity of infection), inhibition being observed at high cell densities. Interestingly, the energetic status of the culture correlated with the decrease in cellular production of baculovirus, meaning that there is room for process optimization through the application of metabolic engineering techniques. Biotechnol. Bioeng. 2009; 104: 162–180

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

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 alpha-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 alpha-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.

In situ 2D fluorometry and chemometric monitoring of mammalian cell cultures

The main objective of the present study was to investigate the use of in situ 2D fluorometry for monitoring key bioprocess variables in mammalian cell cultures, namely the concentration of viable cells and the concentration of recombinant proteins. All studies were conducted using a recombinant Baby Hamster Kidney (BHK) cell line expressing a fusion glycoprotein IgG1‐IL2 cultured in batch and fed‐batch modes. It was observed that the intensity of fluorescence signals in the excitation/emission wavelength range of amino acids, vitamins and NAD(P)H changed along culture time, although the dynamics of single fluorophors could not be correlated with the dynamics of the target state variables. Therefore, multivariate chemometric modeling was adopted as a calibration methodology. 2D fluorometry produced large volumes of redundant spectral data, which were first filtered by principal components analysis (PCA). Then, a partial least squares (PLS) regression was applied to correlate the reduced fluorescence maps with the target state variables. Two validation strategies were used to evaluate the predictive capacity of the developed PLS models. Accurate estimations of viable cells density (r2 = 0.95; 99.2% of variance captured in the training set; r2 = 0.91; 97.7% of variance captured in the validation set) and of glycoprotein concentration (r2 = 0.99 and 99.7% of variance captured in the training set; r2 = 0.99 and 99.3% of variance captured in the validation set) were obtained over a wide range of reactor operation conditions. The results presented herein confirm that 2D fluorometry constitutes a reliable methodology for on‐line monitoring of viable cells and recombinant protein concentrations in mammalian cell cultures. Biotechnol. Bioeng. 2009;102: 1098–1106.

Metabolic signatures of GS‐CHO cell clones associated with butyrate treatment and culture phase transition

Chinese hamster ovary (CHO) cells are preferred hosts for the production of recombinant biopharmaceuticals. Efforts to optimize these bioprocesses have largely relied on empirical experience and our knowledge of cellular behavior in culture is incomplete. More recently, comprehensive investigations of metabolic network operation have started to be used to uncover traits associated with optimal growth and recombinant protein production. In this work, we used 1H‐nuclear magnetic resonance (1H‐NMR) to analyze the supernatants of glutamine‐synthetase (GS)‐CHO cell clones expressing variable amounts of an IgG4 under control and butyrate‐treated conditions. Exometabolomic data revealed accumulation of several metabolic by‐products, indicating inefficiencies at different metabolic nodes. These data were contextualized in a detailed network and the cellular fluxomes estimated through metabolic flux analysis. This approach allowed comparing metabolic activity across different clones, growth phases and culture conditions, in particular the efficiency pertaining to carbon lost to glycerol and lactate accumulation and the characteristic nitrogen metabolism involving high asparagine and serine uptake rates. Importantly, this study shows that early butyrate treatment has a marked effect on sustaining high nutrient consumption along culture time, being more pronounced during the stationary phase when extra energy generation and biosynthetic activity is fueled to increase IgG formation. Collectively, the information generated contributes to deepening our understanding of CHO cells metabolism in culture, facilitating future design of improved bioprocesses. Biotechnol. Bioeng. 2013;110: 3244–3257.

Insect cells as a production platform of complex virus-like particles.

Virus-like particles (VLPs) are multiprotein structures that resemble the conformation of native viruses but lack a viral genome, potentiating their application as safer and cheaper vaccines. The production of VLPs has been strongly linked with the use of insect cells and the baculovirus expression vector system, especially those particles composed of two or more structural viral proteins. In fact, this expression platform has been extensively improved over the years to address the challenges of coexpression of multiple proteins and their proper assembly into complexes in the same cell. In this article, the role of insect cell technology in the development and production of complex VLPs is overviewed; recent achievements, current bottlenecks and future trends are also highlighted.

Hybrid metabolic flux analysis: combining stoichiometric and statistical constraints to model the formation of complex recombinant products

BackgroundStoichiometric models constitute the basic framework for fluxome quantification in the realm of metabolic engineering. A recurrent bottleneck, however, is the establishment of consistent stoichiometric models for the synthesis of recombinant proteins or viruses. Although optimization algorithms for in silico metabolic redesign have been developed in the context of genome-scale stoichiometric models for small molecule production, still rudimentary knowledge of how different cellular levels are regulated and phenotypically expressed prevents their full applicability for complex product optimization.ResultsA hybrid framework is presented combining classical metabolic flux analysis with projection to latent structures to further link estimated metabolic fluxes with measured productivities. We first explore the functional metabolic decomposition of a baculovirus-producing insect cell line from experimental data, highlighting the TCA cycle and mitochondrial respiration as pathways strongly associated with viral replication. To reduce uncertainty in metabolic target identification, a Monte Carlo sampling method was used to select meaningful associations with the target, from which 66% of the estimated fluxome had to be screened out due to weak correlations and/or high estimation errors. The proposed hybrid model was then validated using a subset of preliminary experiments to pinpoint the same determinant pathways, while predicting the productivity of independent cultures.ConclusionsOverall, the results indicate our hybrid metabolic flux analysis framework is an advantageous tool for metabolic identification and quantification in incomplete or ill-defined metabolic networks. As experimental and computational solutions for constructing comprehensive global cellular models are in development, the contribution of hybrid metabolic flux analysis should constitute a valuable complement to current computational platforms in bridging the metabolic state with improved cell culture performance.

Systems biotechnology of animal cells: the road to prediction.

A central concern of biopharmaceutical R&D is the production of sufficient quantities of recombinant products from manufacturing processes based on animal cell culture. The way in which bioprocess researchers have addressed this question experienced a tremendous shift over the years, progressing from almost empirical to more rational approaches. A step further is the application of systems biotechnology: recent technological advances for large-scale cell state characterization and creative methods for host cell modeling are becoming crucial for next-generation bioprocess optimization. Here we provide an overview of the main trends towards this goal, with a focus on metabolic models as central scaffolds for data integration and prediction of bioprocess outcomes.

How can measurement, monitoring, modeling and control advance cell culture in industrial biotechnology?

This report highlights the potential of measurement, monitoring, modeling and control (M(3) C) methodologies in animal and human cell culture technology. In particular, state-of-the-art of M(3) C technologies and their industrial relevance of existing technology are addressed. It is a summary of an expert panel discussion between biotechnologists and biochemical engineers with both academic and industrial backgrounds. The latest ascents in M(3) C are discussed from a cell culture perspective for industrial process development and production needs. The report concludes with a set of recommendations for targeting M(3) C research toward the industrial interests. These include issues of importance for biotherapeutics production, miniaturization of measurement techniques and modeling methods.

Combining metabolic flux analysis tools and 13C NMR to estimate intracellular fluxes of cultured astrocytes.

In this work, brain cell metabolism was investigated by (13)C NMR spectroscopy and metabolic flux analysis (MFA). Monotypic cultures of astrocytes were incubated with labeled glucose for 38 h, and the distribution of the label was analyzed by (13)C NMR spectroscopy. The analysis of the spectra reveals two distinct physiological states characterized by different ratios of pyruvate carboxylase to pyruvate dehydrogenase activities (PC/PDH). Intracellular flux distributions for both metabolic states were estimated by MFA using the isotopic information and extracellular rate measurements as constraints. The model was subsequently checked with the consistency index method. From a biological point of view, the occurrence of the two physiological states appears to be correlated with the presence or absence of extracellular glutamate. Concerning the model, it can be stated that the metabolic network and the set of constraints adopted provide a consistent and robust characterization of the astrocytic metabolism, allowing for the calculation of central intracellular fluxes such as pyruvate recycling, the anaplerotic flux mediated by pyruvate carboxylase, and the glutamine formation through glutamine synthetase.

Toward system-level understanding of baculovirus-host cell interactions: from molecular fundamental studies to large-scale proteomics approaches.

Baculoviruses are insect viruses extensively exploited as eukaryotic protein expression vectors. Molecular biology studies have provided exciting discoveries on virus–host interactions, but the application of omic high-throughput techniques on the baculovirus–insect cell system has been hampered by the lack of host genome sequencing. While a broader, systems-level analysis of biological responses to infection is urgently needed, recent advances on proteomic studies have yielded new insights on the impact of infection on the host cell. These works are reviewed and critically assessed in the light of current biological knowledge of the molecular biology of baculoviruses and insect cells.