Manuel J.T. Carrondo

Virus-like particles in vaccine development

Virus-like particles (VLPs) are multiprotein structures that mimic the organization and conformation of authentic native viruses but lack the viral genome, potentially yielding safer and cheaper vaccine candidates. A handful of prophylactic VLP-based vaccines is currently commercialized worldwide: GlaxoSmithKline’s Engerix® (hepatitis B virus) and Cervarix® (human papillomavirus), and Merck and Co., Inc.’s Recombivax HB® (hepatitis B virus) and Gardasil® (human papillomavirus) are some examples. Other VLP-based vaccine candidates are in clinical trials or undergoing preclinical evaluation, such as, influenza virus, parvovirus, Norwalk and various chimeric VLPs. Many others are still restricted to small-scale fundamental research, despite their success in preclinical tests. This article focuses on the essential role of VLP technology in new-generation vaccines against prevalent and emergent diseases. The implications of large-scale VLP production are discussed in the context of process control, monitorization and optimization. The main up- and down-stream technical challenges are identified and discussed accordingly. Successful VLP-based vaccine blockbusters are briefly presented concomitantly with the latest results from clinical trials and the recent developments in chimeric VLP-based technology for either therapeutic or prophylactic vaccination.

Model for carbon metabolism in biological phosphorus removal processes based on in vivo13C-NMR labelling experiments

In vivo13C-NMR, 31P-NMR techniques were applied to study phosphorus and carbon metabolism in activated sludge during both the anaerobic and the aerobic stages. By supplying a 13C label on the methyl group of acetate at the beginning of the anaerobic stage, the fate of the label through the subsequent aerobic/anaerobic stages was traced in vivo. It was possible to follow the flux of label from acetate to hydroxybutyrate/hydroxyvalerate co-polymer in the first anaerobic stage, then to monitor the conversion of these units into glycogen in a subsequent aerobic stage, and afterwards, by submitting the same sludge to a second anaerobic stage, to observe the flux of labelled carbon from glycogen to the hydroxyvalerate and hydroxybutyrate units. The uptake/release of inorganic phosphate and the extracellular pH were monitored by 31P-NMR in the same experiments. The data provide an unequivocal demonstration of the involvement of glycogen in the biological phosphorus removal process. On the basis of these 13C labelling data, a biochemical model for the synthesis of polyhydroxyalkanoates from acetate and glycogen was elaborated in which the tricarboxylic acid cycle is proposed as an additional source of reduction equivalents. According to this study, from 1 C-mol acetate, 1.48 C-mol P(HBHV) are synthesized and 0.70 C-mol glycogen are degraded anaerobically, while 0.16 P-mol phosphate is released. In the aerobic stage, 1 C-mol of P(HBHV) is converted to 0.44 C-mol glycogen.

Advances in on-line monitoring and control of mammalian cell cultures: Supporting the PAT initiative

In recent years, much attention has been directed towards the development of global methods for on-line process monitoring, especially since the Food and Drug Administration (FDA) launched the Process Analytical Technology (PAT) guidance, stimulating biopharmaceutical companies to update their monitoring tools to ensure a pre-defined final product quality. The ideal technologies for biopharmaceutical processes should operate in situ, be non-invasive and generate on-line information about multiple key bioprocess and/or metabolic variables. A wide range of spectroscopic techniques based on in situ probes have already been tested in mammalian cell cultures, such as near infrared (NIR), mid infrared (MIR), 2D fluorescence and dielectric capacitance spectroscopy; similarly, the electronic nose technique based on chemical array sensors has been tested for in situ off-gas analysis of mammalian cell cultures. All these methods provide series of spectra, from which meaningful information must be extracted. In this sense, data mining techniques such as principal components regression (PCR), partial least squares (PLS) or artificial neural networks (ANN) have been applied to handle the dense flow of data generated from the real-time process analyzers. Furthermore, the implementation of feedback control methods would help to improve process performance and ultimately ensure reproducibility. This review discusses the suitability of several spectroscopic techniques coupled with chemometric methods for improved monitoring and control of mammalian cell processes.

Microencapsulation technology: a powerful tool for integrating expansion and cryopreservation of human embryonic stem cells.

The successful implementation of human embryonic stem cells (hESCs)-based technologies requires the production of relevant numbers of well-characterized cells and their efficient long-term storage. In this study, cells were microencapsulated in alginate to develop an integrated bioprocess for expansion and cryopreservation of pluripotent hESCs. Different three-dimensional (3D) culture strategies were evaluated and compared, specifically, microencapsulation of hESCs as: i) single cells, ii) aggregates and iii) immobilized on microcarriers. In order to establish a scalable bioprocess, hESC-microcapsules were cultured in stirred tank bioreactors. The combination of microencapsulation and microcarrier technology resulted in a highly efficient protocol for the production and storage of pluripotent hESCs. This strategy ensured high expansion ratios (an approximately twenty-fold increase in cell concentration) and high cell recovery yields (>70%) after cryopreservation. When compared with non-encapsulated cells, cell survival post-thawing demonstrated a three-fold improvement without compromising hESC characteristics. Microencapsulation also improved the culture of hESC aggregates by protecting cells from hydrodynamic shear stress, controlling aggregate size and maintaining cell pluripotency for two weeks. This work establishes that microencapsulation technology may prove a powerful tool for integrating the expansion and cryopreservation of pluripotent hESCs. The 3D culture strategy developed herein represents a significant breakthrough towards the implementation of hESCs in clinical and industrial applications.

Large-scale production and purification of VLP-based vaccines.

Abstract Virus-like particles (VLPs) hold tremendous potential as vaccine candidates. These innovative biopharmaceuticals present the remarkable advantages of closely mimicking the three-dimensional nature of an actual virus while lacking the virus genome packaged inside its capsid. As a result, an equally efficient but safer prophylaxis is anticipated as compared to inactivated or live attenuated viral vaccines. With the advent of successful cases of approved VLP-based vaccines, pharmaceutical companies are indeed redirecting their resources to the development of such products. This paper reviews the current choices and trends of large-scale production and purification of VLP-based vaccines generated through the baculovirus expression vector system using insect cells.

Improving expansion of pluripotent human embryonic stem cells in perfused bioreactors through oxygen control

The successful transfer of human embryonic stem cell (hESC) technology and cellular products into clinical and industrial applications needs to address issues of automation, standardization and the generation of relevant cell numbers of high quality. In this study, we combined microcarrier technology and controlled stirred tank bioreactors, to develop an efficient and scalable system for expansion of pluripotent hESCs. We demonstrate the importance of controlling pO(2) at 30% air saturation to improve hESCs growth. This concentration allowed for a higher energetic cell metabolism, increased growth rate and maximum cell concentration in contrast to 5% pO(2) where a shift to anaerobic metabolism was observed, decreasing cell expansion 3-fold. Importantly, the incorporation of an automated perfusion system in the bioreactor enhanced culture performance and allowed the continuous addition of small molecules assuring higher cell concentrations for a longer time period. The expanded hESCs retained their undifferentiated phenotype and pluripotency. Our results show, for the first time, that the use of controlled bioreactors is critical to ensure the production of high quality hESCs. When compared to the standard colony culture, our strategy improves the final yield of hESCs by 12-fold, providing a potential bioprocess to be transferred to clinical and industrial applications.

Effects of ammonia and lactate on growth, metabolism, and productivity of BHK cells☆

The aim of the present work was to study the effect of ammonia and lactate on growth, metabolism, and productivity of BHK cells producing a recombinant fusion protein. Results show that cell growth was reduced with the increase in ammonia or lactate: k(1/2) of 1.1 mM and 3.5 mM for stirred and stationary cultures, respectively, for ammonia and of 28 mM for both stationary and stirred cultures for lactate, were obtained. The cell-specific consumption rates of both glucose (q(Glc)) and glutamine (q(Gln)) increased, whereas that of oxygen (q(O2)) decreased, with the increase in ammonia or lactate concentrations. The cell-specific production rates of lactate (q(Lac)) increased with an increase in ammonia concentration; similarly for the cell-specific production rates of ammonia (q(Amm)), which also increased with an increase in lactate concentration; on the other hand, both q(Lac) and q(Amm) markedly decreased when lactate or ammonia concentrations were increased, respectively; lactate was consumed at lactate concentrations above 30 mM and ammonia was consumed at ammonia concentrations above 5 mM. In vivo (31)P NMR experiments showed that ammonia and lactate affect the intracellular pH, leading to intracellular acidification, and decrease the content in phosphomonoesters, whereas the cell energy state was maintained. The effect of lactate on cell growth and q(Gln) is partially due to osmolarity, on q(Glc) and q(Amm) is entirely due to osmolarity, but on q(Lac) is mainly due to lactate effect per se. An increase in ammonia from 0 to 20 mM induced a 50% reduction in specific productivity, whereas an increase in lactate from 0 to 60 mM induced a 40% decrease.

Human liver cell spheroids in extended perfusion bioreactor culture for repeated‐dose drug testing

Primary cultures of human hepatocyte spheroids are a promising in vitro model for long‐term studies of hepatic metabolism and cytotoxicity. The lack of robust methodologies to culture cell spheroids, as well as a poor characterization of human hepatocyte spheroid architecture and liver‐specific functionality, have hampered a widespread adoption of this three‐dimensional culture format. In this work, an automated perfusion bioreactor was used to obtain and maintain human hepatocyte spheroids. These spheroids were cultured for 3‐4 weeks in serum‐free conditions, sustaining their phase I enzyme expression and permitting repeated induction during long culture times; rate of albumin and urea synthesis, as well as phase I and II drug‐metabolizing enzyme gene expression and activity of spheroid hepatocyte cultures, presented reproducible profiles, despite basal interdonor variability (n = 3 donors). Immunofluorescence microscopy of human hepatocyte spheroids after 3‐4 weeks of long‐term culture confirmed the presence of the liver‐specific markers, hepatocyte nuclear factor 4α, albumin, cytokeratin 18, and cytochrome P450 3A. Moreover, immunostaining of the atypical protein kinase C apical marker, as well as the excretion of a fluorescent dye, evidenced that these spheroids spontaneously assemble a functional bile canaliculi network, extending from the surface to the interior of the spheroids, after 3‐4 weeks of culture. Conclusion: Perfusion bioreactor cultures of primary human hepatocyte spheroids maintain a liver‐specific activity and architecture and are thus suitable for drug testing in a long‐term, repeated‐dose format. (HEPATOLOGY 2012)

Animal Cell Technology

Classical rabies vaccine strains (genotype 1) protect inefficiently against rabies-related lyssaviruses (other genotypes) and in particular against the Mokola virus (genotype 3). DNA vaccine modem approach was used to produce Mokola valency. Purified Plasmid vector was injected into each anterior tibialis muscle of BALB/c mice. It appeared that the Mokola DNA vaccine has induced a sustained level of VNAb, a T helper cell response and confered protection against Mokola virus challenge. Therefore, it seems that the DNA vaccine approach is a very promising technology, which needs further investigations for a possible application.

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