Scaling-up vaccine production: implementation aspects of a biomass growth observer and controller

Abstract This study considers two aspects of the implementation of a biomass growth observer and specific growth rate controller in scale-up from small- to pilot-scale bioreactors towards a feasible bulk production process for whole-cell vaccine against whooping cough. The first is the calculation of the oxygen uptake rate, the starting point for online monitoring and control of biomass growth, taking into account the dynamics in the gas-phase. Mixing effects and delays are caused by amongst others the headspace and tubing to the analyzer. These gas phase dynamics are modelled using knowledge of the system in order to reconstruct oxygen consumption. The second aspect is to evaluate performance of the monitoring and control system with the required modifications of the oxygen consumption calculation on pilot-scale. In pilot-scale fed-batch cultivation good monitoring and control performance is obtained enabling a doubled concentration of bulk vaccine compared to standard batch productio

Sparging - shear sensitivity of animal cells

Biopharmaceuticals are increasingly produced by modern biotechnological techniques. The in-vitro culture of animal cells in stirred tanks is one of the feasible systems, especially for proteins that require specific post-tanslational modifications to evoke a desired respons in patients. Animal cell are usually capable to perform these modifications in contrast to bacteria and yeast. Another advantage of animal cells is that they secrete their product into the culture medium, which is greatly facilitating the purification of the product.As a disadvantage of animal cells is mentioned their sensitivity for shear stress. The high energy containing regions of liquid flow that can develop in a stirred tank can damage the cells because of their relative large size compared to yeast cells and bacteria and their lack of a protective cell wall. As a result of the scale-up of stirred tank cultures with animal cells the first detrimental shear phenomenon to become manifest is the shear stress associated with the sparging of the cell suspension. Sparging is the most simple and efficient method to provide cells in large-scale stirred tanks with sufficient oxygen. The cell killing event of sparging is the bursting of bubbles at the surface of the suspension.In chapter 1 an overview is given of the status of the research on the shear sensitivity of animal cells untill about 1990, the starting point of the work on this thesis. The major conclusion of this overview is that because of a lack of a generic approach many fragmented data were gathered that allowed few generalized conclusions. The culture media used for shear testing was highly variable and often considered as of moderate importance. Later the central item of this thesis, the composition and features of the culture medium, emerged as the key factor to control shear sensitivity of animal cells.Chapters 2 and 3 describe the effect of a reduced serum content of the culture medium on the shear sensitivity of animal cells for sparging. The use of culture media without serum has become a requirement for the production of biopharmaceuticals. The protective effect of serum was quantified in a bubble column under standardized conditions and the protection by serum was characterized as an immediate non-metabolic effect. In addition, in these chapters, the "killing volume" hypothesis as developed for insect cells, is validated for hybridoma cells.In chapter 4 the effects of silicone antifoam are described. Just like serum, antifoam is preferably omitted during animal cell fermentations. However, for some sparging experiments in the bubble column the addition of antifoam was necessary and therefore its effects needed to be quantified.The protective effect of the polymer polyethylene-glycol (PEG) against cell death caused by sparging is described in chapter 5. The protection by PEG is correlated with the surface tension of the culture medium. It seems that the presence of PEG in the boundary layer of bubbles is essential for its protective effect.The subject of chapter 6 is the protective effect of dextran. This polymer provides protection for animal cells against the bursting of bubbles by increasing the viscosity of the liquid. To establish the protective effect, high concentrations of high molecular weight dextran are required.In chapter 7 an overview is given of the status of the research on the shear sensitivity of animal cells. Just like in this thesis, the focus in this trend article is determined by the composition and features of the culture medium. Trends in the production of biopharmaceuticals with animal cells, like the use of culture media without components from human or animal source, are discussed in this last chapter.</p

Improved Production Process for Native Outer Membrane Vesicle Vaccine against Neisseria meningitidis

An improved detergent-free process has been developed to produce vaccine based on native outer membrane vesicles (NOMV) against Neisseria meningitidis serogroup B. Performance was evaluated with the NonaMen vaccine concept, which provides broad coverage based on nine distinct PorA antigens. Scalable aseptic equipment was implemented, replacing undesirable steps like ultracentrifugation, inactivation with phenol, and the use of preservatives. The resulting process is more consistent and gives a higher yield than published reference processes, enabling NOMV production at commercial scale. Product quality met preliminary specifications for 9 consecutive batches, and an ongoing study confirmed real-time stability up to 12 months after production. As the NOMV had low endotoxic activity and induced high bactericidal titres in mice, they are expected to be safe and effective in humans. The production process is not limited to NonaMen and may be applicable for other N. meningitidis serogroups and other gram-negative pathogens. The current results therefore facilitate the late-stage development and clinical evaluation of NOMV vaccines

Cysteine Depletion Causes Oxidative Stress and Triggers Outer Membrane Vesicle Release by Neisseria meningitidis Implications for Vaccine Development

Outer membrane vesicles (OMV) contain immunogenic proteins and contribute to in vivo survival and virulence of bacterial pathogens. The first OMV vaccines successfully stopped Neisseria meningitidis serogroup B outbreaks but required detergent-extraction for endotoxin removal. Current vaccines use attenuated endotoxin, to preserve immunological properties and allow a detergent-free process. The preferred process is based on spontaneously released OMV (sOMV), which are most similar to in vivo vesicles and easier to purify. The release mechanism however is poorly understood resulting in low yield. This study with N. meningitidis demonstrates that an external stimulus, cysteine depletion, can trigger growth arrest and sOMV release in sufficient quantities for vaccine production (61500 human doses per liter cultivation). Transcriptome analysis suggests that cysteine depletion impairs iron-sulfur protein assembly and causes oxidative stress. Involvement of oxidative stress is confirmed by showing that addition of reactive oxygen species during cysteine-rich growth also triggers vesiculation. The sOMV in this study are similar to vesicles from natural infection, therefore cysteinedependent vesiculation is likely to be relevant for the in vivo pathogenesis of N. meningitidis

ENIGMA and global neuroscience: A decade of large-scale studies of the brain in health and disease across more than 40 countries

This review summarizes the last decade of work by the ENIGMA (Enhancing NeuroImaging Genetics through Meta Analysis) Consortium, a global alliance of over 1400 scientists across 43 countries, studying the human brain in health and disease. Building on large-scale genetic studies that discovered the first robustly replicated genetic loci associated with brain metrics, ENIGMA has diversified into over 50 working groups (WGs), pooling worldwide data and expertise to answer fundamental questions in neuroscience, psychiatry, neurology, and genetics. Most ENIGMA WGs focus on specific psychiatric and neurological conditions, other WGs study normal variation due to sex and gender differences, or development and aging; still other WGs develop methodological pipelines and tools to facilitate harmonized analyses of "big data" (i.e., genetic and epigenetic data, multimodal MRI, and electroencephalography data). These international efforts have yielded the largest neuroimaging studies to date in schizophrenia, bipolar disorder, major depressive disorder, post-traumatic stress disorder, substance use disorders, obsessive-compulsive disorder, attention-deficit/hyperactivity disorder, autism spectrum disorders, epilepsy, and 22q11.2 deletion syndrome. More recent ENIGMA WGs have formed to study anxiety disorders, suicidal thoughts and behavior, sleep and insomnia, eating disorders, irritability, brain injury, antisocial personality and conduct disorder, and dissociative identity disorder. Here, we summarize the first decade of ENIGMA's activities and ongoing projects, and describe the successes and challenges encountered along the way. We highlight the advantages of collaborative large-scale coordinated data analyses for testing reproducibility and robustness of findings, offering the opportunity to identify brain systems involved in clinical syndromes across diverse samples and associated genetic, environmental, demographic, cognitive, and psychosocial factors

Factors Associated with Revision Surgery after Internal Fixation of Hip Fractures

Background: Femoral neck fractures are associated with high rates of revision surgery after management with internal fixation. Using data from the Fixation using Alternative Implants for the Treatment of Hip fractures (FAITH) trial evaluating methods of internal fixation in patients with femoral neck fractures, we investigated associations between baseline and surgical factors and the need for revision surgery to promote healing, relieve pain, treat infection or improve function over 24 months postsurgery. Additionally, we investigated factors associated with (1) hardware removal and (2) implant exchange from cancellous screws (CS) or sliding hip screw (SHS) to total hip arthroplasty, hemiarthroplasty, or another internal fixation device. Methods: We identified 15 potential factors a priori that may be associated with revision surgery, 7 with hardware removal, and 14 with implant exchange. We used multivariable Cox proportional hazards analyses in our investigation. Results: Factors associated with increased risk of revision surgery included: female sex, [hazard ratio (HR) 1.79, 95% confidence interval (CI) 1.25-2.50; P = 0.001], higher body mass index (fo