Validation of a CFD model for cell culture bioreactors at large scale and its application in scale-up.

Among all the operating parameters that control the cell culture environment inside bioreactors, appropriate mixing and aeration are crucial to ensure sufficient oxygen supply, homogeneous mixing, and CO2 stripping. A model-based manufacturing facility fit approach was applied to define agitation and bottom air flow rates during the process scale-up from laboratory to manufacturing, of which computational fluid dynamics (CFD) was the core modeling tool. The realizable k-ε turbulent dispersed Eulerian gas-liquid flow model was established and validated using experimental values for the volumetric oxygen transfer coefficient (kLa). Model validation defined the process operating parameter ranges for application of the model, identified mixing issues (e.g., impeller flooding, dissolved oxygen gradients, etc.) and the impact of antifoam on kLa. Using the CFD simulation results as inputs to the models for oxygen demand, gas entrance velocity, and CO2 stripping aided in the design of the agitation and bottom air flow rates needed to meet cellular oxygen demand, control CO2 levels, mitigate risks for cell damage due to shear, foaming, as well as fire hazards due to high O2 levels in the bioreactor gas outlet. The recommended operating conditions led to the completion of five manufacturing runs with a 100% success rate. This model-based approach achieved a seamless scale-up and reduced the required number of at-scale development batches, resulting in cost and time savings of a cell culture commercialization process.

High throughput screening data for a case study of CHO cell culture process development.

In this article, we present four sets of data from high-throughput screening (HTS) studies of different chemically defined media using an industrially relevant Chinese hamster ovary (CHO) cell line. While complex hydrolysate media was used in the early phase process development and manufacturing of a monoclonal antibody (mAb), here we seek to determine an appropriate chemically defined media for late phase process development. Over 150 combinations of chemically defined basal media, feed media, and basal and feed media supplements, such as polyphenolic flavonoid antioxidants (including rosmarinic acid (RA)), were evaluated in four HTS studies to replace the complex hydrolysate media. Specifically, these four screening studies incorporated custom design of experiment (DOE), one-factor-at-a-time (OFAT), and definitive screening design methodologies for titer improvement. Titer was improved two fold compared to the early phase process using the addition of RA to chemically defined media. This dataset exemplifies how HTS can be used as an effective approach to systematically and statistically determine media composition and supplementation to increase mAb titer. These data were presented in connection with a published paper [1].

Micafungin-Induced Cell Wall Damage Stimulates Morphological Changes Consistent with Microcycle Conidiation in Aspergillus nidulans.

Fungal cell wall receptors relay messages about the state of the cell wall to the nucleus through the Cell Wall Integrity Signaling (CWIS) pathway. The ultimate role of the CWIS pathway is to coordinate repair of cell wall damage and to restore normal hyphal growth. Echinocandins such as micafungin represent a class of antifungals that trigger cell wall damage by affecting synthesis of ß-glucans. To obtain a better understanding of the dynamics of the CWIS response and its multiple effects, we have coupled dynamic transcriptome analysis with morphological studies of Aspergillus nidulans hyphae in responds to micafungin. Our results reveal that expression of the master regulator of asexual development, BrlA, is induced by micafungin exposure. Further study showed that micafungin elicits morphological changes consistent with microcycle conidiation and that this effect is abolished in the absence of MpkA. Our results suggest that microcycle conidiation may be a general response to cell wall perturbation which in some cases would enable fungi to tolerate or survive otherwise lethal damage.

Dynamic Transcriptomic and Phosphoproteomic Analysis During Cell Wall Stress in Aspergillus nidulans.

The fungal cell-wall integrity signaling (CWIS) pathway regulates cellular response to environmental stress to enable wall repair and resumption of normal growth. This complex, interconnected, pathway has been only partially characterized in filamentous fungi. To better understand the dynamic cellular response to wall perturbation, a ß-glucan synthase inhibitor (micafungin) was added to a growing A. nidulans shake-flask culture. From this flask, transcriptomic and phosphoproteomic data were acquired over 10 and 120 min, respectively. To differentiate statistically-significant dynamic behavior from noise, a multivariate adaptive regression splines (MARS) model was applied to both data sets. Over 1800 genes were dynamically expressed and over 700 phosphorylation sites had changing phosphorylation levels upon micafungin exposure. Twelve kinases had altered phosphorylation and phenotypic profiling of all non-essential kinase deletion mutants revealed putative connections between PrkA, Hk-8-4, and Stk19 and the CWIS pathway. Our collective data implicate actin regulation, endocytosis, and septum formation as critical cellular processes responding to activation of the CWIS pathway, and connections between CWIS and calcium, HOG, and SIN signaling pathways.

Comprehensive Analysis of Aspergillus nidulans PKA Phosphorylome Identifies a Novel Mode of CreA Regulation.

In filamentous fungi, an important kinase responsible for adaptation to changes in available nutrients is cyclic AMP (cAMP)-dependent protein kinase (protein kinase A [PKA]). This kinase has been well characterized at a molecular level, but its systemic action and direct/indirect targets are generally not well understood in filamentous fungi. In this work, we used a pkaA deletion strain (ΔpkaA) to identify Aspergillus nidulans proteins for which phosphorylation is dependent (either directly or indirectly) on PKA. A combination of phosphoproteomic and transcriptomic analyses revealed both direct and indirect targets of PKA and provided a global perspective on its function. One of these targets was the transcription factor CreA, the main repressor responsible for carbon catabolite repression (CCR). In the ΔpkaA strain, we identified a previously unreported phosphosite in CreA, S319, which (based on motif analysis) appears to be a direct target of Stk22 kinase (AN5728). Upon replacement of CreA S319 with an alanine (i.e., phosphonull mutant), the dynamics of CreA import to the nucleus are affected. Collectively, this work provides a global overview of PKA function while also providing novel insight regarding significance of a specific PKA-mediated phosphorylation event.IMPORTANCE The cyclic AMP (cAMP)-dependent protein kinase A (PKA) signaling pathway is well conserved across eukaryotes, and previous work has shown that it plays an important role in regulating development, growth, and virulence in a number of fungi. PKA is activated in response to extracellular nutrients and acts to regulate metabolism and growth. While a number of components in the PKA pathway have been defined in filamentous fungi, current understanding does not provide a global perspective on PKA function. Thus, this work is significant in that it comprehensively identifies proteins and functional pathways regulated by PKA in a model filamentous fungus. This information enhances our understanding of PKA action and may provide information on how to manipulate it for specific purposes.

Phosphoproteomic and transcriptomic analyses reveal multiple functions for Aspergillus nidulans MpkA independent of cell wall stress.

The protein kinase MpkA plays a prominent role in the cell wall integrity signaling (CWIS) pathway, acting as the terminal MAPK activating expression of genes which encode cell wall biosynthetic enzymes and other repair functions. Numerous studies focus on MpkA function during cell wall perturbation. Here, we focus on the role MpkA plays outside of cell wall stress, during steady state growth. In an effort to seek other, as yet unknown, connections to this pathway, an mpkA deletion mutant (ΔmpkA) was subjected to phosphoproteomic and transcriptomic analysis. When compared to the control (isogenic parent of ΔmpkA), there is strong evidence suggesting MpkA is involved with maintaining cell wall strength, branching regulation, and the iron starvation pathway, among others. Particle-size analysis during shake flask growth revealed ΔmpkA mycelia were about 4 times smaller than the control strain and more than 90 cell wall related genes show significantly altered expression levels. The deletion mutant had a significantly higher branching rate than the control and phosphoproteomic results show putative branching-regulation proteins, such as CotA, LagA, and Cdc24, have a significantly different level of phosphorylation. When grown in iron limited conditions, ΔmpkA had no difference in growth rate or production of siderophores, whereas the control strain showed decreased growth rate and increased siderophore production. Transcriptomic data revealed over 25 iron related genes with altered transcript levels. Results suggest MpkA is involved with regulation of broad cellular functions in the absence of stress.

A fast and simple method to estimate relative, hyphal tensile-strength of filamentous fungi used to assess the effect of autophagy.

Fungal hyphal strength is an important phenotype which can have a profound impact on bioprocess behavior. Until now, there is not an efficient method which allows its characterization. Currently available methods are very time consuming, thus, compromising their applicability in strain selection and process development. To overcome this issue, a method for fast and easy, statistically verified quantification of relative hyphal tensile strength was developed. It involves off-line fragmentation in a high shear mixer followed by quantification of fragment size using laser diffraction. Particle size distribution (PSD) is determined, with analysis time on the order of minutes. Plots of PSD 90th percentile versus time allow estimation of the specific fragmentation rate. This novel method is demonstrated by estimating relative hyphal strength during growth in control conditions and rapamycin-induced autophagy for Aspergillus nidulans (parental strain) and a mutant strain (ΔAnatg8) lacking an important autophagy gene. Both strains were grown in shake flasks and relative hyphal tensile strength was compared. The mutant strain grown in control conditions appears to be weaker than the parental strain, suggesting that Anatg8 may play a role in other processes involving cell wall biosynthesis. Furthermore, rapamycin-induced autophagy resulted in apparently weaker cells even for the mutant strain. These findings confirm the utility of the developed method in strain selection and process development.

Insights regarding fungal phosphoproteomic analysis.

Protein phosphorylation is a major means of regulation for cellular processes, and is important in cell signaling, growth, and cell proliferation. To study phosphorylated proteins, high throughput phosphoproteomic technologies, such as reverse phase protein array, phospho-specific flow cytometry, and mass spectrometry (MS) based technologies, have been developed. Among them, mass spectrometry has become the primary tool employed for the identification of phosphoproteins and phosphosites in fungi, leading to an improved understanding of a number of signaling pathways. Using mass spectrometry techniques, researchers have discovered new kinase substrates, established connections between kinases and fungal pathogenicity, and studied the evolutionary lineage of kinases between different fungal species. Further, many specific phosphorylation sites recognized by individual kinases have been described. In this review, we will focus on recent discoveries made in yeast and filamentous fungi using phosphoproteomic analysis.