Mathieu Streefland

Improved OMV vaccine against Neisseria meningitidis using genetically engineered strains and a detergent-free purification process

The use of detergent-extracted outer membrane vesicles (OMVs) is an established approach for development of a multivalent PorA vaccine against N. meningitidis serogroup B. Selective removal of lipopolysaccharide (LPS) decreases toxicity, but promotes aggregation and narrows the immune response. Detergent-free OMV vaccines retain all LPS, which preserves the native vesicle structure, but result in high toxicity and lower yield. The present study assessed the effects of gene mutations that attenuated LPS toxicity (lpxL1) or improved OMV yield (rmpM) in combination with the available OMV purification processes. The results substantiate that OMVs from a strain with both mutations, produced with a detergent-free process provide better vaccine characteristics than the traditional detergent-based approach. With comparable toxicity and yield, no aggregation and cross-protection against other PorA subtypes, these OMV vaccines are potentially safe and effective for parenteral use in humans.

Photosynthetic efficiency of Chlamydomonas reinhardtii in flashing light.

Efficient light to biomass conversion in photobioreactors is crucial for economically feasible microalgae production processes. It has been suggested that photosynthesis is enhanced in short light path photobioreactors by mixing‐induced flashing light regimes. In this study, photosynthetic efficiency and growth of the green microalga Chlamydomonas reinhardtii were measured using LED light to simulate light/dark cycles ranging from 5 to 100 Hz at a light‐dark ratio of 0.1 and a flash intensity of 1000 µmol m−2 s−1. Light flashing at 100 Hz yielded the same photosynthetic efficiency and specific growth rate as cultivation under continuous illumination with the same time‐averaged light intensity (i.e., 100 µmol m−2 s−1). The efficiency and growth rate decreased with decreasing flash frequency. Even at 5 Hz flashing, the rate of linear electron transport during the flash was still 2.5 times higher than during maximal growth under continuous light, suggesting storage of reducing equivalents during the flash which are available during the dark period. In this way the dark reaction of photosynthesis can continue during the dark time of a light/dark cycle. Understanding photosynthetic growth in dynamic light regimes is crucial for model development to predict microalgal photobioreactor productivities. Biotechnol. Bioeng. 2011;108: 2905–2913.

Extraction and stability of selected proteins in ionic liquid based aqueous two phase systems

Ionic liquid-based aqueous two-phase extraction of a plant protein, Rubisco (Ribulose-1,5-biphosphate carboxylase oxygenase), using Iolilyte 221 PG and sodium potassium phosphate buffer, was investigated as a new alternative extraction method and compared with a conventional PEG-based two-phase system. The influence of various factors, such as the concentration of phase components, pH and temperature, on partitioning of Rubisco was evaluated by Design of Experiments. Rubisco partitions to the ionic liquid (IL) phase and the partition coefficients for the IL based two-phase system were 3–4 times higher than in a PEG-based system. Additionally, studies were done in aqueous solution of IL with varying concentrations to establish a relationship between IL concentration and protein stability. In addition to Rubisco, the stability of BSA and IgG1 was investigated in aqueous solution of two ionic liquids: Iolilyte 221PG and Cyphos 108. No fragmentation or aggregation was observed at 10% w/w concentration of the ionic liquid. However, all three proteins studied formed aggregates at 50% w/w concentration of ionic liquid. This indicates a narrow range of IL concentration for their application in protein extraction.

Photosynthetic efficiency of Chlamydomonas reinhardtii in attenuated, flashing light†

As a result of mixing and light attenuation, algae in a photobioreactor (PBR) alternate between light and dark zones and, therefore, experience variations in photon flux density (PFD). These variations in PFD are called light/dark (L/D) cycles. The objective of this study was to determine how these L/D cycles affect biomass yield on light energy in microalgae cultivation. For our work, we used controlled, short light path, laboratory, turbidostat‐operated PBRs equipped with a LED light source for square‐wave L/D cycles with frequencies from 1 to 100 Hz. Biomass density was adjusted that the PFD leaving the PBR was equal to the compensation point of photosynthesis. Algae were acclimated to a sub‐saturating incident PFD of 220 µmol m−2 s−1 for continuous light. Using a duty cycle of 0.5, we observed that L/D cycles of 1 and 10 Hz resulted on average in a 10% lower biomass yield, but L/D cycles of 100 Hz resulted on average in a 35% higher biomass yield than the yield obtained in continuous light. Our results show that interaction of L/D cycle frequency, culture density and incident PFD play a role in overall PBR productivity. Hence, appropriate L/D cycle setting by mixing strategy appears as a possible way to reduce the effect that dark zone exposure impinges on biomass yield in microalgae cultivation. The results may find application in optimization of outdoor PBR design to maximize biomass yields. Biotechnol. Bioeng. 2012; 109: 2567–2574.

Multivariate PAT solutions for biopharmaceutical cultivation: current progress and limitations

Increasingly elaborate and voluminous datasets are generated by the (bio)pharmaceutical industry and are a major challenge for application of PAT and QbD principles. Multivariate data analysis (MVDA) is required to delineate relevant process information from large multi-factorial and multi-collinear datasets. Here the key role of MVDA for industrial (bio)process data is discussed, with a focus on progress and limitations of MVDA as a PAT solution for biopharmaceutical cultivation processes. MVDA based models were proven useful and should be routinely implemented for bioprocesses. It is concluded that although the highest level of PAT with process control within its design space in real-time during manufacturing is not reached yet, MVDA will be central to reach this ultimate objective for cell cultivations.

Process analytical technology (PAT) tools for the cultivation step in biopharmaceutical production

The process analytical technology (PAT) initiative is now 10 years old. This has resulted in the development of many tools and software packages dedicated to PAT application on pharmaceutical processes. However, most applications are restricted to small molecule drugs, mainly for the relatively simple process steps like drying or tableting where only a limited number of parameters need to be controlled. A big challenge for PAT still lies in applications for biopharmaceuticals and then especially in the cultivation process step, where the quality of a biopharmaceutical product is largely determined. This review gives an overview of the currently available tools for monitoring and controlling the biopharmaceutical cultivation step and of the main challenges for the most common cell platforms (i.e. Escherichia coli, yeast, and mammalian cells) used in biopharmaceutical manufacturing. The real challenge is to understand how intracellular mechanisms (from synthesis to excretion) influence the quality of biopharmaceuticals and how these mechanisms can be monitored and controlled to yield the desired end product quality. Modern “omics” tools and advanced process analyzers have opened up the way for PAT applications for the biopharmaceutical cultivation process step.

Multivariate data analysis as a PAT tool for early bioprocess development data

Early development datasets are typically unstructured, incomplete and truncated, yet they are readily available and contain relevant process information which is not extracted using classical data analysis techniques. In this paper, we illustrate the power of multivariate data analysis (MVDA) as a Process Analytical Technology tool to analyze early development data of a PER.C6® cell cultivation process. MVDA increased our understanding of the process studied. Principal component analysis enabled a thorough exploration of the dataset, identifying causes for batch deviations and revealing sensitivity of the process to scale. These findings were previously undetected using traditional univariate analysis. The lack of structure and gaps in the early development datasets made it impossible to fit them to more advanced partial least square regression models. This paper clearly shows that MVDA should be routinely used to analyze early development data to reveal relevant information for later development and scale-up. The value of these early development runs can be greatly enhanced if the experiments are well-structured and accompanied with full process analytics. This up-front investment will result in shorter and more efficient process development paths, resulting in lower overall development costs for new biopharmaceutical products.

A practical approach for exploration and modeling of the design space of a bacterial vaccine cultivation process

A licensed pharmaceutical process is required to be executed within the validated ranges throughout the lifetime of product manufacturing. Changes to the process, especially for processes involving biological products, usually require the manufacturer to demonstrate that the safety and efficacy of the product remains unchanged by new or additional clinical testing. Recent changes in the regulations for pharmaceutical processing allow broader ranges of process settings to be submitted for regulatory approval, the so‐called process design space, which means that a manufacturer can optimize his process within the submitted ranges after the product has entered the market, which allows flexible processes. In this article, the applicability of this concept of the process design space is investigated for the cultivation process step for a vaccine against whooping cough disease. An experimental design (DoE) is applied to investigate the ranges of critical process parameters that still result in a product that meets specifications. The on‐line process data, including near infrared spectroscopy, are used to build a descriptive model of the processes used in the experimental design. Finally, the data of all processes are integrated in a multivariate batch monitoring model that represents the investigated process design space. This article demonstrates how the general principles of PAT and process design space can be applied for an undefined biological product such as a whole cell vaccine. The approach chosen for model development described here, allows on line monitoring and control of cultivation batches in order to assure in real time that a process is running within the process design space. Biotechnol. Bioeng. 2009; 104: 492–504

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. Many process parameters may change the EDTA extraction efficiency, but it is unknown what the optimal ranges for these parameters are in terms of quality. The present study systematically optimized EDTA extraction and was representative for production at large-scale. Two critical process parameters were identified, harvest point of the cultivation (harvest) and pH of the extraction buffer (pH), which significantly affected yield (7-fold) and bacterial lysis (35-fold). The other quality attributes remained unchanged. Optimization of harvest and pH revealed that the desired low bacterial lysis coincided with intermediate but sufficient yield. High functional immunogenicity and low toxicity of the optimized vaccine were also confirmed. The EDTA extraction is therefore a robust process step which produces high quality OMV if harvest and pH are controlled accurately.

Gene-expression-based quality scores indicate optimal harvest point in Bordetella pertussis cultivation for vaccine production†

The evolution of vaccine product quality during batch cultivation of Bordetella pertussis, the causative agent of whooping cough, was investigated with the goal to determine the optimal harvest point. The process was explored by measuring mRNA expression at frequent intervals during cultivation. The genes that are involved in virulence are already known for this product and changes in their expression levels are proposed to be indicative for product quality. A quantitative product quality score is calculated based on the expression levels of these virulence genes, which allows comparison of expected product quality between culture samples. Product quality scores were maximal throughout the logarithmic growth phase, but dropped significantly at the start of the stationary phase. This showed that the decreasing lactate and glutamate concentrations towards the end of the batch are critical for product quality. On‐line measurement of these nutrients allows the cultivation process to be harvested at the optimal harvest point, increasing process robustness and consistency. Biotechnol. Bioeng. 2009;103: 900–908.