Characterization of apoptosis in PER.C6® batch and perfusion cultures.

Preventing or delaying cell death is a challenge in mammalian cell cultures for the development and optimization of production processes for biopharmaceuticals. Cell cultures need to be maintained highly viable for extended times in order to reach maximum production yields. Moreover, programmed cell death through apoptosis is often believed to occur without being detected by classical viability measurements. In this study, we characterized cell death in PER.C6® batch and perfusion cultures using three flow cytometry techniques measuring different steps of the apoptosis cascade: DNA fragmentation, caspases activation and phosphatidylserine externalization. We showed that apoptosis is the main pathway of PER.C6® cell death in batch cultures after depletion of main carbon sources. In high cell density perfusion cultures fed at a constant specific perfusion rate, both high viability and very limited apoptosis were observed. When extending this perfusion process far beyond standard operations, cultures were exposed to suboptimal process conditions, which resulted in an increase of apoptotic cell death. Moreover, we showed that the reference viability measurement using trypan blue exclusion properly assesses the level of cell death in PER.C6® cultures. This study is a first step in understanding the mechanisms of PER.C6® cell death, which will be helpful to support applications of the cell line.

Photosynthetic efficiency and oxygen evolution of Chlamydomonas reinhardtii under continuous and flashing light.

As a result of mixing and light attenuation in a photobioreactor (PBR), microalgae experience light/dark (L/D) cycles that can enhance PBR efficiency. One parameter which characterizes L/D cycles is the duty cycle; it determines the time fraction algae spend in the light. The objective of this study was to determine the influence of different duty cycles on oxygen yield on absorbed light energy and photosynthetic oxygen evolution. Net oxygen evolution of Chlamydomonas reinhardtii was measured for four duty cycles (0.05, 0.1, 0.2, and 0.5) in a biological oxygen monitor (BOM). Oversaturating light flashes were applied in a square-wave fashion with four flash frequencies (5, 10, 50, and 100 Hz). Algae were precultivated in a turbidostat and acclimated to a low photon flux density (PFD). A photosynthesis-irradiance (PI) curve was measured under continuous illumination and used to calculate the net oxygen yield, which was maximal between a PFD of 100 and 200 µmol m⁻² s⁻¹. Net oxygen yield under flashing light was duty cycle-dependent: the highest yield was observed at a duty cycle of 0.1 (i.e., time-averaged PFD of 115 µmol m⁻² s⁻¹). At lower duty cycles, maintenance respiration reduced net oxygen yield. At higher duty cycles, photon absorption rate exceeded the maximal photon utilization rate, and, as a result, surplus light energy was dissipated which led to a reduction in net oxygen yield. This behavior was identical with the observation under continuous light. Based on these data, the optimal balance between oxygen yield and production rate can be determined to maximize PBR productivity.

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.

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.

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.

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.

Selection of chemically defined media for CHO cell fed-batch culture processes.

Two CHO cell clones derived from the same parental CHOBC® cell line and producing the same monoclonal antibody (BC-G, a low producing clone; BC-P, a high producing clone) were tested in four basal media in all possible combinations with three feeds (=12 conditions) in fed-batch cultures. Higher amino acid feeding did not always lead to higher mAb production. The two clones showed differences in cell physiology, metabolism and optimal medium-feed combinations. During the phase transitions of all cultures, cell metabolism showed a shift represented by lower specific consumption and production rates, except for the specific glucose consumption rate in cultures fed by Actifeed A/B. The BC-P clone fed by Actifeed A/B showed a threefold cell volume increase and an increase of the specific consumption rate of glucose in the stationary phase. Since feeding was based on glucose this resulted in accumulation of amino acids for this feed, while this did not occur for the poorer feed (EFA/B). The same feed also led to an increase of cell size for the BC-G clone, but to a lesser extent.

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.

Configuration of bioreactors.

Lab-scale stirred-tank bioreactors (0.2-20 l) are used for fundamental research on animal cells and in process development and troubleshooting for large-scale production. In this chapter, different configurations of bioreactor systems are shortly discussed and setting up these different configurations is described. In addition, online measurement and control of bioreactor parameters is described, with special attention to controller settings (PID) and online measurement of oxygen consumption and carbon dioxide production. Finally, methods for determining the oxygen transfer coefficient are described.

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.

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.

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.

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.