Pre-stage perfusion and ultra-high seeding cell density in CHO fed-batch culture: a case study for process intensification guided by systems biotechnology.

Process intensification strategies are needed in the field of therapeutic protein production for higher productivities, lower cost of goods and improved facility utilization. This work describes an intensification approach, which connects a tangential-flow-filtration (TFF) based pre-stage perfusion process with a concentrated fed-batch production culture inoculated with an ultra-high seeding density (uHSD). This strategy shifted biomass production towards the pre-stage, reaching up to 45 × 106 cells/mL in perfusion mode. Subsequently, production in the intensified fed-batch started immediately and the product titer was almost doubled (1.9-fold) in an equivalent runtime and with comparable product quality compared to low-seeded cultures. Driven by mechanistic modelling and next-generation sequencing (NGS) the process had been optimized by selecting the media composition in a way that minimized cellular adaptation between perfusion and production culture. As a main feature, lactate feeding was applied in the intensified approach to promote cell culture performance and process scalability was proven via transfer to pilot-scale i.e., 20 L pre-stage perfusion and 80 L production reactor. Moreover, an earlier shift from a growth associated to a production stage associated gene expression pattern was identified for uHSD cultures compared to the reference. Overall, we showed that the described intensification strategy yielded in a higher volumetric productivity and is applicable for existing or already approved molecules in common, commercial fed-batch facilities. This work provides an in-depth molecular understanding of cellular processes that are detrimental during process intensification.

Integrity and Collaboration in Dynamic Sensor Networks.

Global Navigation Satellite Systems (GNSS) deliver absolute position and velocity, as well as time information (P, V, T). However, in urban areas, the GNSS navigation performance is restricted due to signal obstructions and multipath. This is especially true for applications dealing with highly automatic or even autonomous driving. Subsequently, multi-sensor platforms including laser scanners and cameras, as well as map data are used to enhance the navigation performance, namely in accuracy, integrity, continuity and availability. Although well-established procedures for integrity monitoring exist for aircraft navigation, for sensors and fusion algorithms used in automotive navigation, these concepts are still lacking. The research training group i.c.sens, integrity and collaboration in dynamic sensor networks, aims to fill this gap and to contribute to relevant topics. This includes the definition of alternative integrity concepts for space and time based on set theory and interval mathematics, establishing new types of maps that report on the trustworthiness of the represented information, as well as taking advantage of collaboration by improved filters incorporating person and object tracking. In this paper, we describe our approach and summarize the preliminary results.

Workflow for Target-Oriented Parametrization of an Enhanced Mechanistic Cell Culture Model.

The goal of this study is to develop a macroscopic mechanistic model describing growth and production within fed-batch cultivations of CHO cells. The model should be used for process characterization as well as for process monitoring including real-time parameter adaptations. The model proved to be able to describe a data-set of 40 processes differing in clones, scales, and process conditions with a normalized root mean square error of approximately 10%. However, due to limited parameter identifiability and limited knowledge about physiologically meaningful parameter values, a broad range of parameters could describe the data with similar quality. This hampered comparison of the model parameters as well as their real-time estimation. Therefore an iterative workflow combining techniques like sensitivity and identifiability analysis, analysis of the specific rates as well as structural adaptations of the parameter space is developed. By applying it the parameter variability could be reduced by 80% with similar predictive power as the original parameters. Summing up, based on a mechanistic CHO model, a generic and transferrable workflow is created for target-oriented parameter estimation in case of limited parameter identifiability. Finally, we suggest a methodology, which fits ideally into the frame of Process Analytical Technology aiming to increase process understanding.

The linkage between nutrient supply, intracellular enzyme abundances and bacterial growth: New evidences from the central carbon metabolism of Corynebacterium glutamicum.

Corynebacterium glutamicum serves as important production host for small molecular compounds that are derived from precursor molecules of the central carbon metabolism. It is therefore a well-studied model organism of industrial biotechnology. However, a deeper understanding of the regulatory principles underlying the synthesis of central metabolic enzymes under different environmental conditions as well as its impact on cell growth is still missing. We studied enzyme abundances in C. glutamicum in response to growth on: (i) one limiting carbon source by sampling chemostat and fed-batch cultivations and (ii) changing carbon sources provided in excess by sampling batch cultivations. The targeted quantification of 20 central metabolic enzymes by isotope dilution mass spectrometry revealed that cells maintain stable enzyme concentrations when grown on d-glucose as single carbon and energy source and, most importantly, independent of its availability. By contrast, switching from d-glucose to d-fructose, d-mannose, d-arabitol, acetate, l-lactate or l-glutamate results in highly specific enzyme regulation patterns that can partly be explained by the activity of known transcriptional regulators. Based on these experimental results we propose a simple framework for modeling cell population growth as a nested function of nutrient supply and intracellular enzyme abundances. In summary, our study extends the basis for the formulation of predictive mechanistic models of bacterial growth, applicable in industrial bioprocess development.

Combinatorial optimization of synthetic operons for the microbial production of p-coumaryl alcohol with Escherichia coli.

BACKGROUND: Microbes are extensively engineered to produce compounds of biotechnological or pharmaceutical interest. However, functional integration of synthetic pathways into the respective host cell metabolism and optimization of heterologous gene expression for achieving high product titers is still a challenging task. In this manuscript, we describe the optimization of a tetracistronic operon for the microbial production of the plant-derived phenylpropanoid p-coumaryl alcohol in Escherichia coli. RESULTS: Basis for the construction of a p-coumaryl alcohol producing strain was the development of Operon-PLICing as method for the rapid combinatorial assembly of synthetic operons. This method is based on the chemical cleavage reaction of phosphorothioate bonds in an iodine/ethanol solution to generate complementary, single-stranded overhangs and subsequent hybridization of multiple DNA-fragments. Furthermore, during the assembly of these DNA-fragments, Operon-PLICing offers the opportunity for balancing gene expression of all pathway genes on the level of translation for maximizing product titers by varying the spacing between the Shine-Dalgarno sequence and START codon. With Operon-PLICing, 81 different clones, each one carrying a different p-coumaryl alcohol operon, were individually constructed and screened for p-coumaryl alcohol formation within a few days. The absolute product titer of the best five variants ranged from 48 to 52 mg/L p-coumaryl alcohol without any further optimization of growth and production conditions. CONCLUSIONS: Operon-PLICing is sequence-independent and thus does not require any specific recognition or target sequences for enzymatic activities since all hybridization sites can be arbitrarily selected. In fact, after PCR-amplification, no endonucleases or ligases, frequently used in other methods, are needed. The modularity, simplicity and robustness of Operon-PLICing would be perfectly suited for an automation of cloning in the microtiter plate format.

Autoselection of cytoplasmic yeast virus like elements encoding toxin/antitoxin systems involves a nuclear barrier for immunity gene expression.

Cytoplasmic virus like elements (VLEs) from Kluyveromyces lactis (Kl), Pichia acaciae (Pa) and Debaryomyces robertsiae (Dr) are extremely A/T-rich (>75%) and encode toxic anticodon nucleases (ACNases) along with specific immunity proteins. Here we show that nuclear, not cytoplasmic expression of either immunity gene (PaORF4, KlORF3 or DrORF5) results in transcript fragmentation and is insufficient to establish immunity to the cognate ACNase. Since rapid amplification of 3' ends (RACE) as well as linker ligation of immunity transcripts expressed in the nucleus revealed polyadenylation to occur along with fragmentation, ORF-internal poly(A) site cleavage due to the high A/T content is likely to prevent functional expression of the immunity genes. Consistently, lowering the A/T content of PaORF4 to 55% and KlORF3 to 46% by gene synthesis entirely prevented transcript cleavage and permitted functional nuclear expression leading to full immunity against the respective ACNase toxin. Consistent with a specific adaptation of the immunity proteins to the cognate ACNases, cross-immunity to non-cognate ACNases is neither conferred by PaOrf4 nor KlOrf3. Thus, the high A/T content of cytoplasmic VLEs minimizes the potential of functional nuclear recruitment of VLE encoded genes, in particular those involved in autoselection of the VLEs via a toxin/antitoxin principle.

Absolute quantification of Corynebacterium glutamicum glycolytic and anaplerotic enzymes by QconCAT.

The soil bacterium Corynebacterium glutamicum is one of the best-studied production hosts for industrial biotechnology, and it is primarily used for the large-scale production of essential amino acids, such as l-lysine. For rational strain development, detailed knowledge of intracellular protein concentration is crucial to determine metabolic capacities and limitations. We developed a QconCAT approach for the accurate absolute quantification of key enzymes of C. glutamicum glycolysis and anaplerosis. Following well-defined batch cultivations, 10 metabolic enzymes were quantified, accounting for approximately 6% of the total cell dry weight. Copy numbers per cell ranged from 36,700±3500 for phosphofructokinase (PFK) to 507,700±40,300 for enolase (ENO), which is considerably lower than the corresponding data obtained from Saccharomyces cerevisiae. Moreover, accurate measurement of the biovolume permitted an estimation of molar concentrations of intracellular enzyme catalysts ranging from 7.6±1.9µM (PFK) to 105.2±28.6µM (ENO). Finally, model-assisted data evaluation demonstrates that our method provides an important cornerstone toward a more detailed mechanistic understanding of C. glutamicum metabolism. BIOLOGICAL SIGNIFICANCE: Determination of absolute protein amounts using quantitative concatemers (QconCAT's) has already been successfully demonstrated for various species including human, animal and yeast. Interestingly, application of the QconCAT methodology for the determination of cytoplasmic enzyme concentrations in a prokaryote has not been described so far. This study is concerned with a novel targeted approach for the absolute quantification of 10 key enzymes from the central carbon metabolism of the industrially important organism Corynebacterium glutamicum. We demonstrate a method that enables complete cell lysis of this robust soil bacterium, thus allowing for accurate quantification of cytoplasmic enzymes. By linking measured enzyme amounts with respective biovolume data, intracellular enzyme concentrations were estimated, which are of special importance for any systems biology approach studying C. glutamicum's metabolism at the mechanistic level. To our knowledge this is the first report of applying the QconCAT methodology for determining intracellular enzyme concentrations in a prokaryote.

Quantification of proteome dynamics in Corynebacterium glutamicum by (15)N-labeling and selected reaction monitoring.

Selected reaction monitoring allows quantitative measurements of proteins over several orders of magnitude in complex biological samples. Here we present a targeted approach for quantification of 19 enzymes from Corynebacterium glutamicum applying isotope dilution mass spectrometry coupled to high performance liquid chromatography (IDMS-LC-MS/MS). Investigations of protein dynamics upon growth on acetate and glucose as sole carbon source shows highly stable peptide amounts for enzymes of the central carbon metabolism during the transition phase and after substrate depletion. However significant adaptations of protein amounts are observed between both growth conditions well agreeing with known changes in metabolic fluxes. Time-resolved measurements of protein expression after metabolic switch from glycolytic to gluconeogenetic conditions reveal fast responses in protein synthesis rates for glyoxylate shunt enzymes.