Evaluation of a critical process parameter: oxygen limitation during cultivation has a fully reversible effect on gene expression of Bordetella pertussis.

Modern (bio)pharmaceutical process development requires thorough investigation of all process parameters that are critical to product quality. The impact of a disturbance of such a parameter during processing needs to be known so that a rational decision can be made about the release of the product. In cultivation processes the dissolved oxygen (DO) concentration is generally accepted as being a critical parameter. In this article the impact of a 90 min period of oxygen limitation during the cultivation of the strictly aerobic Bordetella pertussis bacterium is investigated. The cultivation is the most important process step for the manufacturing of a vaccine against whooping cough disease. Samples were taken immediately before and after oxygen limitation and at the end of cultivation of four oxygen limited and three control cultivations. DNA microarray analysis of the full transcriptome of the B. pertussis bacterium revealed that a 90 min period of oxygen limitation has a substantial effect on overall gene expression patterns. In total 104 genes were identified as a significant hit at any of the sample points, of which 58 were directly related to oxygen limitation. The other genes were mainly affected towards the end of cultivation. Of all genes involved in oxygen limitation none were identified to show a significant difference between the oxygen limited and control cultivations at the end of the batch. This indicates a fully reversible effect of oxygen limitation on gene expression. This finding has implications for the risk assessment of dissolved oxygen concentration as a critical process parameter.

Modeling Neisseria meningitidis B metabolism at different specific growth rates.

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. At the Netherlands Vaccine Institute (NVI) a vaccine against serogroup B organisms is currently being developed. This study describes the influence of the growth rate of N. meningitidis on its macro-molecular composition and its metabolic activity and was determined in chemostat cultures. In the applied range of growth rates, no significant changes in RNA content and protein content with growth rate were observed in N. meningitidis. The DNA content in N. meningitidis was somewhat higher at the highest applied growth rate. The phospholipid and lipopolysaccharide content in N. meningitidis changed with growth rate but no specific trends were observed. The cellular fatty acid composition and the amino acid composition did not change significantly with growth rate. Additionally, it was found that the PorA content in outer membrane vesicles was significantly lower at the highest growth rate. The metabolic fluxes at various growth rates were calculated using flux balance analysis. Errors in fluxes were calculated using Monte Carlo Simulation and the reliability of the calculated flux distribution could be indicated, which has not been reported for this type of analysis. The yield of biomass on substrate (Y(x/s)) and the maintenance coefficient (m(s)) were determined as 0.44 (+/-0.04) g g(-1) and 0.04 (+/-0.02) g g(-1) h(-1), respectively. The growth associated energy requirement (Y(x/ATP)) and the non-growth associated ATP requirement for maintenance (m(ATP)) were estimated as 0.13 (+/-0.04) mol mol(-1) and 0.43 (+/-0.14) mol mol(-1) h(-1), respectively. It was found that the split ratio between the Entner-Doudoroff and the pentose phosphate pathway, the sole glucose utilizing pathways in N. meningitidis, had a minor effect on ATP formation rate but a major effect on the fluxes going through for instance the citric-acid cycle. For this reason, we presented flux ranges for underdetermined parts of metabolic network rather than presenting single flux values, which is more commonly done in literature.

Modeling Neisseria meningitidis metabolism: from genome to metabolic fluxes.

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 successfully 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.

Scale-up for bulk production of vaccine against meningococcal disease.

At the Netherlands Vaccine Institute (NVI) a vaccine against Neisseria meningitidis serogroup B organisms based on different porA subtypes contained in outer membrane vesicles (OMVs) is in advanced stage of development and will be evaluated in clinical trial studies in the near future. In order to meet the expected demand for product, the current biopharmaceutical production process is being scaled-up. This study describes the scale-up approach for the upstream process and the resulting bioreactor design and operation strategy leading towards a feasible solution for bulk production of a vaccine against meningococcal disease. The technically realized 1.2 m(3) bioreactor, equipped with a turbine impeller for gas dispersion, was complemented with an upward pumping impeller and a rotary plate foam breaker to contain foam inside the bioreactor. Aeration and ventilation in the culture broth were controlled by increasing the stirrer speed and gas flow rate simultaneously at increasing oxygen demand. The scale-up was successful and comparable growth curves and nutrient consumption profiles were reached on 0.06 and 1.2 m(3).

PAT for vaccines: the first stage of PAT implementation for development of a well-defined whole-cell vaccine against whooping cough disease.

Since variation in process time and process output is commonly accepted to be inevitable for biological processes, application of Process Analytical Technologies (PAT) on these processes is challenging. In this paper the applicability of PAT on the cultivation of Bordetella pertussis bacteria as part of the manufacture of a vaccine against whooping cough disease is investigated. Scrutinizing and eliminating the most prominent sources of variance make the cultivation process step highly reproducible. Furthermore, the use of DNA microarrays allows investigation of how disturbances influence cellular physiology and product quality. Marker genes for product quality were identified, providing the means to quantitatively assess product quality, which is hardly possible using the mandatory animal tests for product quality. The tools and results described in this paper, combined with suitable on line measurements, can make full PAT application for this process step possible. Ultimately, the process can be designed and controlled towards consistent end product quality.