Automated cell counting for Trypan blue-stained cell cultures using machine learning.

Cell counting is a vital practice in the maintenance and manipulation of cell cultures. It is a crucial aspect of assessing cell viability and determining proliferation rates, which are integral to maintaining the health and functionality of a culture. Additionally, it is critical for establishing the time of infection in bioreactors and monitoring cell culture response to targeted infection over time.

Development of an animal component free production process for Sabin inactivated polio vaccine.

Inactivated polio vaccine production using attenuated Sabin strains (sIPV) instead of wild type polio viruses (cIPV) is an initiative encouraged by the World Health Organization. This use of attenuated viruses is preferred as it reduces risks related to potential outbreaks during IPV production. Previously, an sIPV production process was set up based on the cIPV production process.

Proteome Analysis Is a Valuable Tool to Monitor Antigen Expression during Upstream Processing of Whole-Cell Pertussis Vaccines.

Physicochemical and immunochemical assays were applied to substantiate the relation between upstream processing and the quality of whole-cell pertussis vaccines. Bordetella pertussis bacteria were cultured on a chemically defined medium using a continuous cultivation process in stirred tank reactors to obtain uniform protein expression. Continuous culture favors the consistent production of proteins known as virulence factors.

Hematopoietic cancer cell lines can support replication of Sabin poliovirus type 1.

Viral vaccines can be produced in adherent or in suspension cells. The objective of this work was to screen human suspension cell lines for the capacity to support viral replication. As the first step, it was investigated whether poliovirus can replicate in such cell lines.

Assessment of mass transfer and mixing in rigid lab-scale disposable bioreactors at low power input levels.

Mass transfer, mixing times and power consumption were measured in rigid disposable stirred tank bioreactors and compared to those of a traditional glass bioreactor. The volumetric mass transfer coefficient and mixing times are usually determined at high agitation speeds in combination with sparged aeration as used for single cell suspension and most bacterial cultures. In contrast, here low agitation speeds combined with headspace aeration were applied.

Improved poliovirus D-antigen yields by application of different Vero cell cultivation methods.

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 1×10(6) cells mL(-1) during batch cultivation to 1.

Next generation inactivated polio vaccine manufacturing to support post polio-eradication biosafety goals.

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.

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.

Comparison of initial feasibility of host cell lines for viral vaccine production.

In order to reduce the time required for the development and production of viral vaccines, host cell lines should be available as expression systems for production of viral vaccines against groups of viral pathogens. A selection of cell lines was compared for their initial feasibility as expression system for the replication of polioviruses, influenza A viruses and respiratory syncytial virus (wild type strain A2). Six adherent cell lines (Vero, HEK-293, MRC-5, CHO-K1, BHK-21 c13, MDCK) and six single cell suspension cell lines (CAP, AGE1.

Isoelectric point determination of live polioviruses by capillary isoelectric focusing with whole column imaging detection.

Using a capillary isoelectric focusing-whole column imaging detection (CIEF-WCID) method, the isoelectric points (pI) of complete intact polioviruses were determined. The polioviruses that were analyzed are the commonly used viruses for the production of inactivated polio vaccines (IPV)-Mahoney (type 1), MEF (type 2), and Saukett (type 3)-as well as for attenuated oral polio vaccines (OPV) and Sabin types 1, 2, and 3. A method for analyzing biological hazardous components (biological safety level 2) was set up for the CIEF-WCID analyzer used.

Quantitative proteomics reveals distinct differences in the protein content of outer membrane vesicle vaccines.

At present, only vaccines containing outer membrane vesicles (OMV) have successfully stopped Neisseria meningitidis serogroup B epidemics. These vaccines however require detergent-extraction to remove endotoxin, which changes immunogenicity and causes production difficulties. To investigate this in more detail, the protein content of detergent-extracted OMV is compared with two detergent-free alternatives.

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.

Scale-down of the inactivated polio vaccine production process.

The anticipated increase in the demand for inactivated polio vaccines resulting from the success in the polio eradication program requires an increase in production capacity and cost price reduction of the current inactivated polio vaccine production processes. Improvement of existing production processes is necessary as the initial process development has been done decades ago. An up-to-date lab-scale version encompassing the legacy inactivated polio vaccine production process was set-up.

Identification and optimization of critical process parameters for the production of NOMV vaccine against Neisseria meningitidis.

Outer membrane vesicles (OMV) are used as a vaccine against Neisseria meningitidis serogroup B and are traditionally produced with detergent-extraction to remove toxic lipopolysaccharide. Engineered strains with attenuated lipopolysaccharide allowed the use of native vesicles (NOMV) with improved stability and immunogenicity. In the NOMV production process detergents are omitted and vesicle release is stimulated with EDTA extraction (a chelating agent) to enable a higher yield.

Inactivated polio vaccine development for technology transfer using attenuated Sabin poliovirus strains to shift from Salk-IPV to Sabin-IPV.

Industrial-scale inactivated polio vaccine (IPV) production dates back to the 1960s when at the Rijks Instituut voor de Volksgezondheid (RIV) in Bilthoven a process was developed based on micro-carrier technology and primary monkey kidney cells. This technology was freely shared with several pharmaceutical companies and institutes worldwide. In this contribution, the history of one of the first cell-culture based large-scale biological production processes is summarized.

Multivariate data analysis on historical IPV production data for better process understanding and future improvements.

Historical manufacturing data can potentially harbor a wealth of information for process optimization and enhancement of efficiency and robustness. To extract useful data multivariate data analysis (MVDA) using projection methods is often applied. In this contribution, the results obtained from applying MVDA on data from inactivated polio vaccine (IPV) production runs are described.

Expression of phosphofructokinase in Neisseria meningitidis.

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.

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.

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

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.

Online automatic tuning and control for fed-batch cultivation.

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.

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.

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.

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.