Applying intensified design of experiments to mammalian cell culture processes.

The analysis of data collected using design of experiments (DoE) is the current gold standard to determine the influence of input parameters and their interactions on process performance and product quality. In early development, knowledge on the bioprocess of a new product is limited. Many input parameters need to be investigated for a thorough investigation. For eukaryotic cell cultures, intensified DoE (iDoE) has been proposed as efficient tool, requiring fewer bioreactor runs by introducing setpoint changes during the bioprocess. We report the first successful application of iDoE to mammalian cell culture, performing sequential setpoint changes in the growth phase for the selected input parameters temperature and dissolved oxygen. The process performance data were analyzed using ordinary least squares regression. Our results indicate iDoE to be applicable to mammalian bioprocesses and to be a cost-efficient option to inform modeling early on during process development. Even though only half the number of bioreactor runs were used in comparison to a classical DoE approach, the resulting models revealed comparable input-output relations. Being able to examine several setpoint levels within one bioreactor run, we confirm iDoE to be a promising tool to speed up biopharmaceutical process development.

Hopanoids Confer Robustness to Physicochemical Variability in the Niche of the Plant Symbiont Bradyrhizobium diazoefficiens.

Rhizobia are a group of bacteria that increase soil nitrogen content through symbiosis with legume plants. The soil and symbiotic host are potentially stressful environments, and the soil will likely become even more stressful as the climate changes. Many rhizobia within the Bradyrhizobium clade, like Bradyrhizobium diazoefficiens, possess the genetic capacity to synthesize hopanoids, steroid-like lipids similar in structure and function to cholesterol. Hopanoids are known to protect against stresses relevant to the niche of B. diazoefficiens. Paradoxically, mutants unable to synthesize the extended class of hopanoids participate in symbioses with success similar to that of the wild type, despite being delayed in root nodule initiation. Here, we show that in B. diazoefficiens, the growth defects of extended-hopanoid-deficient mutants can be at least partially compensated for by the physicochemical environment, specifically, by optimal osmotic and divalent cation concentrations. Through biophysical measurements of lipid packing and membrane permeability, we show that extended hopanoids confer robustness to environmental variability. These results help explain the discrepancy between previous in-culture and in planta results and indicate that hopanoids may provide a greater fitness advantage to rhizobia in the variable soil environment than the more controlled environments within root nodules. To improve the legume-rhizobium symbiosis through either bioengineering or strain selection, it will be important to consider the full life cycle of rhizobia, from soil to symbiosis. IMPORTANCE Rhizobia, such as B. diazoefficiens, play an important role in the nitrogen cycle by making nitrogen gas bioavailable through symbiosis with legume plants. As climate change threatens soil health, this symbiosis has received increased attention as a more sustainable source of soil nitrogen than the energy-intensive Haber-Bosch process. Efforts to use rhizobia as biofertilizers have been effective; however, long-term integration of rhizobia into the soil community has been less successful. This work represents a small step toward improving the legume-rhizobium symbiosis by identifying a cellular component-hopanoid lipids-that confers robustness to environmental stresses rhizobia are likely to encounter in soil microenvironments as sporadic desiccation and flooding events become more common.

From nutritional wealth to autophagy: In vivo metabolic dynamics in the cytosol, mitochondrion and shuttles of IgG producing CHO cells.

To fulfil the optimization needs of current biopharmaceutical processes the knowledge how to improve cell specific productivities is of outmost importance. This requires a detailed understanding of cellular metabolism on a subcellular level inside compartments such as cytosol and mitochondrion. Using IgG1 producing Chinese hamster ovary (CHO) cells, a pioneering protocol for compartment-specific metabolome analysis was applied. Various production-like growth conditions ranging from ample glucose and amino acid supply via moderate to severe nitrogen limitation were investigated in batch cultures. The combined application of quantitative metabolite pool analysis, 13C tracer studies and non-stationary flux calculations revealed that Pyr/H+ symport (MPC1/2) bore the bulk of the mitochondrial transport under ample nutrient supply. Glutamine limitation induced the concerted adaptation of the bidirectional Mal/aKG (OGC) and the Mal/HPO42- antiporter (DIC), even installing completely reversed shuttle fluxes. As a result, NADPH and ATP formation were adjusted to cellular needs unraveling the key role of cytosolic malic enzyme for NADPH production. Highest cell specific IgG1 productivities were closely correlated to a strong mitochondrial malate export according to the anabolic demands. The requirement to install proper NADPH supply for optimizing the production of monoclonal antibodies is clearly outlined. Interestingly, it was observed that mitochondrial citric acid cycle activity was always maintained enabling constant cytosolic adenylate energy charges at physiological levels, even under autophagy conditions.

The Less the Better: How Suppressed Base Addition Boosts Production of Monoclonal Antibodies With Chinese Hamster Ovary Cells.

Biopharmaceutical production processes strive for the optimization of economic efficiency. Among others, the maximization of volumetric productivity is a key criterion. Typical parameters such as partial pressure of CO2 (pCO2) and pH are known to influence the performance although reasons are not yet fully elucidated. In this study the effects of pCO2 and pH shifts on the phenotypic performance were linked to metabolic and energetic changes. Short peak performance of qmAb (23 pg/cell/day) was achieved by early pCO2 shifts up to 200 mbar but followed by declining intracellular ATP levels to 2.5 fmol/cell and 80% increase of qLac. On the contrary, steadily rising qmAb could be installed by slight pH down-shifts ensuring constant cell specific ATP production (qATP) of 27 pmol/cell/day and high intracellular ATP levels of about 4 fmol/cell. As a result, maximum productivity was achieved combining highest qmAb (20 pg/cell/day) with maximum cell density and no lactate formation. Our results indicate that the energy availability in form of intracellular ATP is crucial for maintaining antibody synthesis and reacts sensitive to pCO2 and pH-process parameters typically responsible for inhomogeneities after scaling up.

Perfusion cultures require optimum respiratory ATP supply to maximize cell-specific and volumetric productivities.

Perfusion processes are an emerging alternative to common fed-batch processes in the growing biopharmaceutical industry. However, the challenge of maintaining high cell-specific productivities remains. In this study, glucose limitation was applied to two perfusion steady states and compared with a third steady state without any detectable limitation. The metabolic phenotype was enhanced under glucose limitation with a decrease of 30% in glucose uptake and 75% in lactate formation. Cell-specific productivities were substantially improved by 50%. Remarkably, the productivities showed a strong correlation to respiratory adenosine triphosphate (ATP) supply. As less reduced nicotinamide adenine dinucleotide (NADH) remained in the cytosol, the ATP generation from oxidative phosphorylation was increased by almost 30%. Consequently, the efficiency of carbon metabolism and the resulting respiratory ATP supply was crucial for maintaining the highly productive cellular state. This study highlights that glucose limitation can be used for process intensification in perfusion cultures as ATP generation via respiration is significantly increased, leading to elevated productivities.

Hyperosmotic stimulus study discloses benefits in ATP supply and reveals miRNA/mRNA targets to improve recombinant protein production of CHO cells.

Biopharmaceuticals are predominantly produced by Chinese hamster ovary (CHO) cells cultivated in fed-batch mode. Hyperosmotic culture conditions (≥ 350 mOsmol kg(∑1) ) resulting from feeding of nutrients may enhance specific product formation rates (qp ). As an improved ATP supply was anticipated to enhance qp this study focused on the identification of suitable miRNA/mRNA targets to increase ATP levels. Therefor next generation sequencing and a compartment specific metabolomics approach were applied to analyze the response of an antibody (mAB) producing CHO cell line upon osmotic shift (280 → 430 mOsmol kg(-1) ). Hyperosmotic culture conditions caused a ∼2.6-fold increase of specific ATP formation rates together with a ∼1.7-fold rise in cytosolic and mitochondrial ATP-pools, thus showing increased ATP supply. mRNA expression analysis identified several genes encoding glycosylated proteins with strictly tissue related function. In addition, hyperosmotic culture conditions induced an upregulation of miR-132-3p, miR-132-5p, miR-182, miR-183, miR-194, miR-215-3p, miR-215-5p which have all been related to cell cycle arrest/proliferation in cancer studies. In relation to a previous independent CHO study miR-183 may be the most promising target to enhance qp by stable overexpression. Furthermore, deletion of genes with presumably dispensable function in suspension growing CHO cells may enhance mAB formation by increased ATP levels.