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The first vaccine against poliovirus (PV), the causative agent of poliomyelitis, was developed in the 1950s by Jonas Salk. The vaccine (IPV) consists of an injected dose of purified and inactivated wild-type PVs (all three serotypes). Soon after this discovery, at the Rijks Instituut voor de Volksgezondheid (RIV) in Bilthoven, an industrial-scale production process for IPV was developed based on micro-carrier technology and primary monkey kidney cells. In the 1970s, the manufacturing of IPV was scaled up to 300-L by the development of well-controlled bioreactors for cell culture, the so-called “Bilthoven Units” (originally used for bacterial fermentations). In 2004, the Vero cell line was introduced to replace the then used tertiary monkey kidney cells followed by a scale-up from 700 to 1,500-L (from two 350-L to two 750-L bioreactors). IPV manufacturing has been part of the regular vaccine manufacturing activities in Bilthoven ever since the establishment of the IPV production process. With polio eradication on our doorstep, the World Health Organization (WHO) is pursuing a new IPV based on non-wild-type strains to increase the biosafety of vaccine manufacturing. In addition, and due to the pending cessation of oral polio vaccines (OPV), the global demand for affordable IPV is increasing. To accommodate these questions two research programs were started at the Netherlands Vaccine Institute (now Institute for Translational Vaccinology). One concerned optimization of the current conventional IPV production process, the other the manufacturing of an affordable sIPV, an IPV based on the attenuated Sabin PV strains normally used for OPV production. The technology of the sIPV production process, developed in the latter project, is also aimed to be transferred to developing countries manufacturers (Chapter 2). From the substantial history in polio vaccine production in Bilthoven, a valuable dataset has been generated. Data from over 50 batches at two different production

Vero cells were grown adherent to microcarriers (Cytodex 1; 3 g L-1) using animal component free media in stirred-tank type bioreactors. Different strategies for media refreshment, daily media replacement (semi-batch), continuous media replacement (perfusion) and recirculation of media, were compared with batch cultivation. Cell densities increased using a feed strategy from 1 × 106 cells mL-1 during batch cultivation to 1.8, 2.7 and 5.0 × 106 cells mL-1 during semi-batch, perfusion and recirculation, respectively. The effects of these different cell culture strategies on subsequent poliovirus production were investigated. Increased cell densities allowed up to 3 times higher d-antigen levels when compared with that obtained from batch-wise Vero cell culture. However, the cell specific d-antigen production was lower when cells were infected at higher cell densities. This cell density effect is in good agreement with observations for different cell lines and virus types. From the evaluated alternative culture methods, application of a semi-batch mode of operations allowed the highest cell specific d-antigen production. The increased product yields that can easily be reached using these higher cell density cultivation methods, showed the possibility for better use of bioreactor capacity for the manufacturing of polio vaccines to ultimately reduce vaccine cost per dose. Further, the use of animal-component-free cell- and virus culture media shows opportunities for modernization of human viral vaccine manufacturing.

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.

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.

Worldwide efforts to eradicate polio caused a tipping point in polio vaccination strategies. A switch from the oral polio vaccine, which can cause circulating and virulent vaccine derived polioviruses, to inactivated polio vaccines (IPV) is scheduled. Moreover, a manufacturing process, using attenuated virus strains instead of wild-type polioviruses, is demanded to enhance worldwide production of IPV, especially in low- and middle income countries. Therefore, development of an IPV from attenuated (Sabin) poliovirus strains (sIPV) was pursued. Starting from the current IPV production process based on wild type Salk strains, adaptations, such as lower virus cultivation temperature, were implemented. sIPV was produced at industrial scale followed by formulation of both plain and aluminium adjuvanted sIPV. The final products met the quality criteria, were immunogenic in rats, showed no toxicity in rabbits and could be released for testing in the clinic. Concluding, sIPV was developed to manufacturing scale. The technology can be transferred worldwide to support post polio-eradication biosafety goals.

Neisseria meningitidis serogroup B is a pathogen that can infect diverse sites within the human host. According to the N. meningitidis genomic information and experimental observations, glucose can be completely catabolized through the Entner–Doudoroff pathway and the pentose phosphate pathway. The Embden–Meyerhof–Parnas pathway is not functional, because the gene for phosphofructokinase (PFK) is not present. The phylogenetic distribution of PFK indicates that in most obligate aerobic organisms, PFK is lacking. We conclude that this is because of the limited contribution of PFK to the energy supply in aerobically grown organisms in comparison with the energy generated through oxidative phosphorylation. Under anaerobic or microaerobic conditions, the available energy is limiting, and PFK provides an advantage, which explains the presence of PFK in many (facultatively) anaerobic organisms. In accordance with this, in silico flux balance analysis predicted an increase of biomass yield as a result of PFK expression. However, analysis of a genetically engineered N. meningitidis strain that expressed a heterologous PFK showed that the yield of biomass on substrate decreased in comparison with a pfkA-deficient control strain, which was associated mainly with an increase in CO2 production, whereas production of by-products was similar in the two strains. This might explain why the pfkA gene has not been obtained by horizontal gene transfer, since it is initially unfavourable for biomass yield. No large effects related to heterologous expression of pfkA were observed in the transcriptome. Although our results suggest that introduction of PFK does not contribute to a more efficient strain in terms of biomass yield, achievement of a robust, optimal metabolic network that enables a higher growth rate or a higher biomass yield might be possible after adaptive evolution of the strain, which remains to be investigated

De kwaliteit van farmaceutische producten wordt geborgd door een op Good Manufacturing Practice gebaseerd kwaliteits systeem, waarbij kwaliteitscontrole van het eindproduct een belangrijke rol vervult. Recentelijk is hierin verandering gekomen en vragen regelgevers bij overheden steeds meer om een kwaliteitssysteem dat al tijdens het proces operationeel is. Voor chemische farmaca wordt dit al steeds meer door de industrie toegepast, maar voor biologische producten blijkt die nog steeds moeilijk. In dit proefschrift wordt een methode beschreven die on line monitoring en kwaliteitscontrole voor een complex biologisch product (een cellulair vaccin) mogelijk maakt. Door te identificeren welke procesparameters invloed hebben op de kritieke eigenschappen van het product, kan een controle systeem worden ingericht dat deze kritische parameters tijdens het proces monitort en beheerst. Hiermee verschuift de kwaliteitscontrole van achteraf naar tijdens het proces. Dit maakt de productie van vaccins flexibeler, goedkoper en bovenal veiliger voor de patient.

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 production

Performance of controllers applied in biotechnological production is often below expectation. Online automatic tuning has the capability to improve control performance by adjusting control parameters. This work presents automatic tuning approaches for model reference specific growth rate control during fed-batch cultivation. The approaches are direct methods that use the error between observed specific growth rate and its set point; systematic perturbations of the cultivation are not necessary. Two automatic tuning methods proved to be efficient, in which the adaptation rate is based on a combination of the error, squared error and integral error. These methods are relatively simple and robust against disturbances, parameter uncertainties, and initialization errors. Application of the specific growth rate controller yields a stable system. The controller and automatic tuning methods are qualified by simulations and laboratory experiments with Bordetella pertussis.

Background - Neisseria meningitidis is a human pathogen that can infect diverse sites within the human host. The major diseases caused by N. meningitidis are responsible for death and disability, especially in young infants. In general, most of the recent work on N. meningitidis focuses on potential antigens and their functions, immunogenicity, and pathogenicity mechanisms. Very little work has been carried out on Neisseria primary metabolism over the past 25 years. Results - Using the genomic database of N. meningitidis serogroup B together with biochemical and physiological information in the literature we constructed a genome-scale flux model for the primary metabolism of N. meningitidis. The validity of a simplified metabolic network derived from the genome-scale metabolic network was checked using flux-balance analysis in chemostat cultures. Several useful predictions were obtained from in silico experiments, including substrate preference. A minimal medium for growth of N. meningitidis was designed and tested succesfully in batch and chemostat cultures. Conclusion - The verified metabolic model describes the primary metabolism of N. meningitidis in a chemostat in steady state. The genome-scale model is valuable because it offers a framework to study N. meningitidis metabolism as a whole, or certain aspects of it, and it can also be used for the purpose of vaccine process development (for example, the design of growth media). The flux distribution of the main metabolic pathways (that is, the pentose phosphate pathway and the Entner-Douderoff pathway) indicates that the major part of pyruvate (69%) is synthesized through the ED-cleavage, a finding that is in good agreement with literature.

In current industrial biotechnology, animal-cell culture is an important source of therapeutic protein products. The conventional animal-cell production processes, however, include many unit operations as part of the fermentation and downstream processing strategy. The research described in this thesis focuses on exploring the opportunities for physical integration of fermentation and part of the downstream processing by means of extractive fermentations in Aqueous Two-Phase Systems (ATPSs). This in situ recovery concept, where the product is extracted from the bioreactor during fermentation, combines fermentation, primary recovery and part of the purification in a single step. The subsequent reduction of process steps and of processing time may provide an opportunity to improve the process economy or even the product quality. ATPSs are thought to be appropriate, because they form a relatively mild environment to cells and they are suitable for protein extractions. ATPSs can be formed by dissolving two polymers, e.g. PEG and dextran in an aqueous solution. Above certain concentrations two aqueous phases will form, each enriched in one of the polymers.In chapter 2 of this thesis, first the influence of the individual ATPS-forming polymers PEG and dextran on the relevant physical culture medium parameters and on the growth of a model (hybridoma) cell line were characterized. It was found that the polymers raised the osmotic pressure. This could be compensated for, however, by reducing the Na-chloride concentration of the culture medium. Subsequently, it was found that cell growth was possible in culture media containing up to 0.025 g/g of PEG or up to 0.15 g/g of dextran. Using these findings, ATPSs of PEG and dextran were selected. In ATPSs of PEG 35.000, dextran 40.000 and culture medium, the hybridoma cells partitioned to the dextran-rich phase and could be cultured over prolonged periods of time. The IgG product, however, partitioned along with the cells in the lo

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 s