A biphasic cultivation strategy to optimize protein expression and minimize aggregation of the final product

The effects of specific culture parameters are cell line-dependent and need to be investigated to allow optimization of productivity and product quality. In this study, a biphasic cultivation strategy for a Chinese hamster ovary (CHO) cell line expressing an erythropoietin fusion protein (Epo-Fc) was developed. In biphasic cultivation, culture conditions are changed after accumulating biomass during an initial growth phase to allow maximum recombinant protein expression in the production phase. Cultures were run in batch mode, and after an initial growth phase, cultivation temperature and pH were shifted to allow optimized protein production and quality. Using a design of experiments (DoE) approach, the individual and synergistic effects of cultivation temperature and pH on the responses cell growth, recombinant protein production, and protein aggregation could be systematically evaluated. The results show that all three responses were influenced by the cultivation temperature. Additionally, an interaction effect between pH and temperature was found to be associated with protein aggregation. By lowering culture temperature and pH from initial 37°C and pH 7.05, a 2.5-fold increase in final product concentration and a reduction in aggregates from 75% to less than 1% could be achieved. Furthermore, lowering temperature and pH substantially reduced cell-specific glucose and glutamine consumption as well as lactate and ammonium production. Increasing cell-specific protein production by optimizing culture conditions significantly contributes to a more economic bioprocess

Genotype of CHO host cell line has higher impact on mAb production and quality than process strategy or cell culture medium

Chinese hamster ovary (CHO) cells comprise a variety of lineages, including CHO-DXB11, CHO-K1, CHO-DG44 and CHO-S. Despite the fact that CHO cell lines share a common ancestor, extensive mutagenesis and clonal selection have resulted in substantial genetic heterogeneity among them. Data from sequencing shows that different genes are lacking from individual CHO cell lines and that each cell line harbors a unique set of mutations that are relevant to the bioprocess. However, literature outlining how the observed genetic differences affect CHO cell performance during bioprocess operations remains scarce. In this study, we examined host cell-specific differences among three widely used CHO cell lines (CHO-K1, CHO-S and CHO-DG44) and recombinantly expressed the same monoclonal antibody (mAb) in an isogenic format in all cell lines by using bacterial artificial chromosomes (BACs) as transfer vector. Cell-specific growth, product formation and heavy and light chain mRNA levels were studied in batch, fed-batch and perfusion cultures. Furthermore, two different cell culture media were investigated. Product quality was studied through glycoprofiling, and the thermal denaturation was analyzed using differential scanning calorimetry (DSC). We found CHO cell line-specific preferences for mAb production or biomass synthesis that were determined by the host cell line rather than product-specific mRNA levels. Additionally, quality attributes of the expressed mAb were influenced by the host cell line and medium used

PI3K p110 gamma overexpression in idiopathic pulmonary fibrosis lung tissue and fibroblast cells: in vitro effects of its inhibition

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Neuromatch Academy: a 3-week, online summer school in computational neuroscience

Neuromatch Academy (https://academy.neuromatch.io; (van Viegen et al., 2021)) was designed as an online summer school to cover the basics of computational neuroscience in three weeks. The materials cover dominant and emerging computational neuroscience tools, how they complement one another, and specifically focus on how they can help us to better understand how the brain functions. An original component of the materials is its focus on modeling choices, i.e. how do we choose the right approach, how do we build models, and how can we evaluate models to determine if they provide real (meaningful) insight. This meta-modeling component of the instructional materials asks what questions can be answered by different techniques, and how to apply them meaningfully to get insight about brain function

Biphasic cultivation strategy to avoid Epo-Fc aggregation and optimize protein expression

AbstractIn biphasic cultivations, the culture conditions are initially kept at an optimum for rapid cell growth and biomass accumulation. In the second phase, the culture is shifted to conditions ensuring maximum specific protein production and the protein quality required. The influence of specific culture parameters is cell line dependent and their impact on product quality needs to be investigated. In this study, a biphasic cultivation strategy for a Chinese hamster ovary (CHO) cell line expressing an erythropoietin fusion protein (Epo-Fc) was developed. Cultures were run in batch mode and after an initial growth phase, cultivation temperature and pH were shifted. Applying a DoE (Design of Experiments) approach, a fractional factorial design was used to systematically evaluate the influence of cultivation temperature and pH as well as their synergistic effect on cell growth as well as on recombinant protein production and aggregation. All three responses were influenced by the cultivation temperature. Additionally, an interaction between pH and temperature was found to be related to protein aggregation. Compared with the initial standard conditions of 37°C and pH 7.05, a parameter shift to low temperature and acidic pH resulted in a decrease in the aggregate fraction from 75% to less than 1%. Furthermore, the synergistic effect of temperature and pH substantially lowered the cell-specific rates of glucose and glutamine consumption as well as lactate and ammonium production. The optimized culture conditions also led to an increase of the cell-specific rates of recombinant Epo-Fc production, thus resulting in a more economic bioprocess

Life hung by a thread: endurance of Antarctic fauna in glacial periods

Today, Antarctica exhibits some of the harshest environmental conditions for life on Earth. During the last glacial period, Antarctic terrestrial and marine life was challenged by even more extreme environmental conditions. During the present interglacial period, polar life in the Southern Ocean is sustained mainly by large-scale primary production. We argue that during the last glacial period, faunal populations in the Antarctic were limited to very few areas of local marine productivity (polynyas), because complete, multiannual sea-ice and ice shelf coverage shut down most of the Southern Ocean productivity within today’s seasonal sea-ice zone. Both marine sediments containing significant numbers of planktonic and benthic foraminifera and fossil bird stomach oil deposits in the adjacent Antarctic hinterland provide indirect evidence for the existence of polynyas during the last glacial period. We advocate that the existence of productive oases in the form of polynyas during glacial periods was essential for the survival of marine and most higher-trophic terrestrial fauna. Reduced to such refuges, much of today’s life in the high Antarctic realm might have hung by a thread during the last glacial period, because limited resources available to the food web restricted the abundance and productivity of both Antarctic terrestrial and marine life.<br/