Morphology engineering - Osmolality and its effect on Aspergillus niger morphology and productivity

<p>Abstract</p> <p>Background</p> <p>The filamentous fungus <it>Aspergillus niger </it>is a widely used strain in a broad range of industrial processes from food to pharmaceutical industry. One of the most intriguing and often uncontrollable characteristics of this filamentous organism is its complex morphology, ranging from dense spherical pellets to viscous mycelia depending on culture conditions. Optimal productivity correlates strongly with a specific morphological form, thus making high demands on process control.</p> <p>Results</p> <p>In about 50 2L stirred tank cultivations the influence of osmolality on <it>A</it>. <it>niger </it>morphology and productivity was investigated. The specific productivity of fructofuranosidase producing strain <it>A. niger </it>SKAn 1015 could be increased notably from 0.5 to 9 U mg^-1h^-1around eighteen fold, by increasing the culture broth osmolality by addition of sodium chloride. The specific productivity of glucoamylase producing strain <it>A. niger </it>AB1.13, could be elevated using the same procedure. An optimal producing osmolality was shown to exist well over the standard osmolality at about 3.2 osmol kg^-1depending on the strain. Fungal morphology of all cultivations was examined by microscope and characterized by digital image analysis. Particle shape parameters were combined to a dimensionless Morphology number, which enabled a comprehensive characterization of fungal morphology correlating closely with productivity. A novel method for determination of germination time in submerged cultivations by laser diffraction, introduced in this study, revealed a decelerated germination process with increasing osmolality.</p> <p>Conclusions</p> <p>Through the introduction of the versatile Morphology number, this study provides the means for a desirable characterization of fungal morphology and demonstrates its relation to productivity. Furthermore, osmolality as a fairly new parameter in process engineering is introduced and found to affect fungal morphology and productivity. Osmolality might provide an auspicious and reliable approach to increase the productivity in industrial processes. Because of the predictable behavior fungal morphology showed in dependence of osmolality, a customization of morphology for process needs seems feasible.</p

Robust cell culture performance facilitated by comprehensive equipment characterization – the importance of hydrodynamic stress

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Making large scale processes transparent – The application of CFD and classical engineering approaches to mitigate risk during cell culture process transfer

The demand for complex therapeutic proteins and especially monoclonal antibodies for treatment of various diseases has increased continuously in the last decades, prompting the continuous development of new processes. For the development of these cell culture processes, equipment knowledge is essential, especially since a process typically is scaled up and transferred numerous times in large pharmaceutical companies. The need for a thorough equipment understanding has been also recognized by the FDA in their PAT-publication. To gain such an understanding at full production scale, Boehringer Ingelheim has put tremendous effort in fully characterizing our multiple bioreactors at different scale across multiple sites. Computational fluid dynamics (CFD) modeling is utilized which allows the evaluation of integral parameters including energy input, mixing time, shear forces and mass transfer coefficient (kLa). The knowledge gained through this tool has been instrumental in understanding the bioreactor characteristics and establish appropriate process scale up and transfer strategies within and across sites. In addition, we have constructed a 15000L acrylic bioreactor model which provides opportunities to validate simulation results with experimental data. For example, local behavior of the reactor regarding the bubble size distributions and dead zones for mixing and gassing can be visualized in the at-scale acrylic bioreactor. One study that was conducted is to understand mixing and mass transfer behavior with respect to agitation rate and superficial gas flow rate. The interactions of these parameters in large scale, however, were in some cases found to be counterintuitive where higher gassing and agitation did not consistently result in higher kLa and better mixing. Although simple modifications and standardization of systems can lead to more similar hydrodynamic conditions, it is difficult to make modification in existing GMP facility due to rigid regulations in the biopharmaceutical sector. Therefore, it is crucial to rely on engineering principles and CFD simulations for transfer between different sites with different bioreactor systems to give additional confidence to ensure successful transfer. Please click Additional Files below to see the full abstract

Advanced cell culture performance by rational media design based on in depth process understanding

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Towards advanced understanding of scale-up: From computational fluid dynamics to systems biotechnology approaches

Scale-up of mammalian cell culture processes from development scale to commercial manufacturing scale is routinely performed in biopharmaceutical process development. For this purpose, well established biochemical engineering principles, empirical formula and scale-up criteria were developed. Considering well characterized equipment as well as company specific process and platform knowledge, scale-up typically is successfully achieved. Yet, improved understanding of scale-up phenomena is desirable for various reasons. Since miniaturized systems are increasingly used in biopharmaceutical process development and, at the same time, efforts with respect to resources and timelines to achieve final manufacturing scale are to be minimized, scale-up steps need to cope with larger bioreactor volume changes in the future. From a process science perspective, an integrated analysis of scale-up phenomena considering both the biochemical engineering aspects (e.g. power input, kLa) as well as cell-level data is needed. In order to gain more profound understanding of scale-up, comprehensive characterization of our cultivation systems using computational fluid dynamics (CFD) was achieved (Figure 1). To further improve and integrate the understanding of an antibody producing CHO cell in a bioreactor environment across scales, we performed thorough analysis of metabolic rates and fluxes in different cultivation scales. In addition, gene expression data using NGS were obtained (Figure 2). We designed a new method for preparation of liquid marbles by using hydrophilic particles [1] (Fig.1). Salt-hydrogel marbles were prepared by atomising droplets of hydrogel solution in a cold air column followed by rolling of the collected hydrogel microbeads in a bed of micrometre size salt particles. Evaporation of the water from the resulting salt marbles with a hydrogel core yielded hollow-shell salt microcapsules. The method is not limited to hydrophilic particles and could potentially be also applied to other materials, such as graphite, carbon, silica and others. The structure and morphology of the salt-hydrogel marbles were analysed with SEM and their particle size distributions were measured. We also tested the dissolution times of the dried salt marbles compared them to these for table salt samples at the same conditions. The high accessible surface area of the shell of salt microcrystals allows a faster initial release of salt from the hollow-shell salt capsules upon their dissolution in water than from the same amount of table salt. The results suggest that such hollow-shell particles could find applications as a table salt substitute in dry food products and salt seasoning formulations with reduced salt content without the loss of saltiness. Please click Additional Files below to see the full abstract

CFD supported scale up of perfusion bioreactors in biopharma

The robust scale up of perfusion systems requires comparable conditions over all scales to ensure equivalent cell culture performance. As cells in continuous processes circulate outside the bioreactor, performance losses may arise if jet flow and stirring cause a direct connection between perfusion feed and return. Computational fluid dynamics can be used to identify such short circuit flows, assess mixing efficiencies, and eventually adapt the perfusion setup. This study investigates the scale up from a 2 L glass bioreactor to 100 L and 500 L disposable pilot scale systems. Highly resolved Lattice Boltzmann Large Eddy simulations were performed in single phase and mixing efficiencies (Emix) furthermore experimentally validated in the 2 L system. This evaluation gives insight into the flow pattern, the mixing behavior and information on cell residence time inside the bioreactors. No geometric adaptations in the pilot scale systems were necessary as Emix was greater than 90% for all conditions tested. Two different setups were evaluated in 2 L scale where the direction of flow was changed, yielding a difference in mixing efficiency of 10%. Nevertheless, since Emix was confirmed to be &gt;90% also for both 2 L setups and the determined mixing times were in a similar range for all scales, the 2 L system was deemed to be a suitable scale down model. The results demonstrate how computational fluid dynamic models can be used for rational process design of intensified production processes in the biopharmaceutical industry

A highly tilted binding mode by a self-reactive T cell receptor results in altered engagement of peptide and MHC

A TCR derived from a patient with relapsing-remitting multiple sclerosis engages the self-peptide myelin basic protein in the context of HLA-DQ1 in a very unusual way

Implementation of N-1 process intensification in bioprocess development and production

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DNA fragility in the parallel evolution of pelvic reduction in stickleback fish

Evolution generates a remarkable breadth of living forms, but many traits evolve repeatedly, by mechanisms that are still poorly understood. A classic example of repeated evolution is the loss of pelvic hindfins in stickleback fish (Gasterosteus aculeatus). Repeated pelvic loss maps to recurrent deletions of a pelvic enhancer of the Pitx1 gene. Here, we identify molecular features contributing to these recurrent deletions. Pitx1 enhancer sequences form alternative DNA structures in vitro and increase double-strand breaks and deletions in vivo. Enhancer mutability depends on DNA replication direction and is caused by TG-dinucleotide repeats. Modeling shows that elevated mutation rates can influence evolution under demographic conditions relevant for sticklebacks and humans. DNA fragility may thus help explain why the same loci are often used repeatedly during parallel adaptive evolution

Transforming Ovarian Cancer Care by Targeting Minimal Residual Disease

Frontline treatment and resultant cure rates in patients with advanced ovarian cancer have changed little over the past several decades. Here, we outline a multidisciplinary approach aimed at gaining novel therapeutic insights by focusing on the poorly understood minimal residual disease phase of ovarian cancer that leads to eventual incurable recurrences