Rapid intensification of an established CHO cell fed-batch process

Currently, the mammalian biomanufacturing industry explores process intensification (PI) to meet upcoming demands of biotherapeutics while keeping production flexible but, more importantly, as economic as possible. However, intensified processes often require more development time compared with conventional fed-batches (FBs) preventing their implementation. Hence, rapid and efficient, yet straightforward strategies for PI are needed. In this study we demonstrate such a strategy for the intensification of an N-stage FB by implementing N-1 perfusion cell culture and high inoculum cell densities resulting in a robust intensified FB (iFB). Furthermore, we show successful combination of such an iFB with the addition of productivity enhancers, which has not been reported so far. The conventional CHO cell FB process was step-wise improved and intensified rapidly in multi-parallel small-scale bioreactors using N-1 perfusion. The iFBs were performed in 15 and 250 ml bioreactors and allowed to evaluate the impact on key process indicators (KPI): the space–time yield (STY) was successfully doubled from 0.28 to 0.55 g/L d, while product quality was maintained. This gain was generated by initially increasing the inoculation density, thus shrinking process time, and second supplementation with butyric acid (BA), which reduced cell growth and enhanced cell-specific productivity from ~25 to 37 pg/(cell d). Potential impacts of PI on cell metabolism were evaluated using flux balance analysis. Initial metabolic differences between the standard and intensified process were observed but disappeared quickly. This shows that PI can be achieved rapidly for new as well as existing processes without introducing sustained changes in cellular and metabolic behavior.publishedVersio

Dynamics of triacylglycerol and EPA production in Phaeodactylum tricornutum under nitrogen starvation at different light intensities

publishedVersio

Automation of high CHO cell density seed intensification via online control of the cell specific perfusion rate and its impact on the N-stage inoculum quality

publishedVersio

Bioengineering bacterial outer membrane vesicles as vaccine platform

publishedVersionPaid Open Acces

Production of bacterial outer membrane vesicles as vaccine platform

Bacterial outer membrane vesicles (OMVs) are non-infectious but highly immunogenic particles. These vesicles are used as vaccines against the disease of the source bacteria. Fascinatingly, the addition of heterologous antigens to these vesicles creates a versatile vaccine platform. Such a platform can be used as an alternative to subunit vaccines, during infectious disease outbreaks or for the development of vaccines against pathogens that require high containment. A unique aspect of this platform is the reusability of the production process for many different vaccines. This in turn could reduce the time to market for new vaccines significantly. We designed a heterologous OMV vaccine concept for Lyme disease based on spontaneous released OMVs from Neisseria meningitidis that express the Outer surface protein A (OspA) of Borrelia burgdorferi on the surface. The productivity of spontaneously released OMVs was improved by the introduction of oxidative stress to the bacterial culture. Increased dissolved oxygen concentrations during cultivation showed to be an excellent process parameter for enhanced release of OMVs, while the bacterial culture remains viable. This presentation will cover the development of the OMV-based vaccine platform and the impact of changes in the upstream process on the downstream process of the investigational OMV-based Lyme disease vaccine. Please click Additional Files below to see the full abstract

Continuous production of Neisseria meningitidis outer membrane vesicles

Outer membrane vesicles (OMVs) are nanoparticles secreted by Gram-negative bacteria that can be used for diverse biotechnological applications. Interesting applications have been developed, where OMVs are the basis of drug delivery, enzyme carriers, adjuvants, and vaccines. Historically, OMV research has mainly focused on vaccines. Therefore, current OMV production processes have been based on batch processes. The production of OMVs in batch mode is characterized by relatively low yields and high costs. Transition of OMV production processes from batch to continuous processes could increase the volumetric productivity, reduce the production and capital costs, and result in a higher quality product. Here, we study the continuous production of Neisseria meningitidis OMVs to improve volumetric productivity. Continuous cultivation of N. meningitidis resulted in a steady state with similar high OMV concentrations as are reached in current batch processes. The steady state was reproducible and could be maintained for at least 600 h. The volumetric productivity of a continuous culture reached 4.0 × 1014 OMVs per liter culture per day, based on a dilution rate of 1/day. The tested characteristics of the OMVs did not change during the experiments showing feasibility of a continuous production process for the production of OMVs for any application.publishedVersionPaid Open Acces

Modeling Neisseria meningitidis metabolism: from genome to metabolic fluxes

A genome-scale flux model for primary metabolism of Neisseria meningitidis was constructed; a minimal medium for growth of N. meningitidis was designed using the model and tested successfully in batch and chemostat cultures

Metabolic modeling of Chlamydomonas reinhardtii: energy requirements for photoautotrophic growth and maintenance

In this study, a metabolic network describing the primary metabolism of Chlamydomonas reinhardtii was constructed. By performing chemostat experiments at different growth rates, energy parameters for maintenance and biomass formation were determined. The chemostats were run at low irradiances resulting in a high biomass yield on light of 1.25 g  mol−1. The ATP requirement for biomass formation from biopolymers (Kx) was determined to be 109 mmol g−1 (18.9 mol mol−1) and the maintenance requirement (mATP) was determined to be 2.85 mmol g−1 h−1. With these energy requirements included in the metabolic network, the network accurately describes the primary metabolism of C. reinhardtii and can be used for modeling of C. reinhardtii growth and metabolism. Simulations confirmed that cultivating microalgae at low growth rates is unfavorable because of the high maintenance requirements which result in low biomass yields. At high light supply rates, biomass yields will decrease due to light saturation effects. Thus, to optimize biomass yield on light energy in photobioreactors, an optimum between low and high light supply rates should be found. These simulations show that metabolic flux analysis can be used as a tool to gain insight into the metabolism of algae and ultimately can be used for the maximization of algal biomass and product yield

Breakthrough in marine invertebrate cell culture : Sponge cells divide rapidly in improved nutrient medium

Sponges (Phylum Porifera) are among the oldest Metazoa and considered critical to understanding animal evolution and development. They are also the most prolific source of marine-derived chemicals with pharmaceutical relevance. Cell lines are important tools for research in many disciplines, and have been established for many organisms, including freshwater and terrestrial invertebrates. Despite many efforts over multiple decades, there are still no cell lines for marine invertebrates. In this study, we report a breakthrough: we demonstrate that an amino acid-optimized nutrient medium stimulates rapid cell division in 9 sponge species. The fastest dividing cells doubled in less than 1 hour. Cultures of 3 species were subcultured from 3 to 5 times, with an average of 5.99 population doublings after subculturing, and a lifespan from 21 to 35 days. Our results form the basis for developing marine invertebrate cell models to better understand early animal evolution, determine the role of secondary metabolites, and predict the impact of climate change to coral reef community ecology. Furthermore, sponge cell lines can be used to scale-up production of sponge-derived chemicals for clinical trials and develop new drugs to combat cancer and other diseases.publishedVersio

The role of starch as transient energy buffer in synchronized microalgal growth in Acutodesmus obliquus

publishedVersionPaid Open Acces