Metabolic responses of CHO cells to limitation of key amino acids.

Chinese hamster ovary (CHO) cells are the predominant host for production of therapeutic glycoproteins. In particular, the glutamine-synthetase (GS) expression system has been widely used in the biopharmaceutical industry for efficient selection of high-yielding clones. However, much remains unclear on how metabolic wiring affects culture performance. For instance, asparagine and serine have been observed to be the largest nitrogen sources taken up by GS-CHO cells, but their roles in biosynthesis and energy generation are poorly understood. In this work, a comprehensive profiling of extracellular metabolites coupled with an analysis of intracellular label distributions after 1-(13) C-pyruvate supplementation were used to trace metabolic rearrangements in different scenarios of asparagine and serine availability. The absence of asparagine in the medium caused growth arrest, and was associated with a dramatic increase in pyruvate uptake, a higher ratio of pyruvate carboxylation to dehydrogenation and an inability for de novo asparagine synthesis. The release of ammonia and amino acids such as aspartate, glutamate, and alanine were deeply impacted. This confirms asparagine to be essential for these GS-CHO cells as the main source of intracellular nitrogen as well as having an important anaplerotic role in TCA cycle activity. In turn, serine unavailability also negatively affected culture growth while triggering its de novo synthesis, confirmed by label incorporation coming from pyruvate, and reduced glycine and formate secretion congruent with its role as a precursor in the metabolism of one-carbon units. Overall, these results unfold important insights into GS-CHO cells metabolism that lay a clearer basis for fine-tuning bioprocess optimization.

Liquid films on shake flask walls explain increasing maximum oxygen transfer capacities with elevating viscosity.

In biotechnological screening and production, oxygen supply is a crucial parameter. Even though oxygen transfer is well documented for viscous cultivations in stirred tanks, little is known about the gas/liquid oxygen transfer in shake flask cultures that become increasingly viscous during cultivation. Especially the oxygen transfer into the liquid film, adhering on the shake flask wall, has not yet been described for such cultivations. In this study, the oxygen transfer of chemical and microbial model experiments was measured and the suitability of the widely applied film theory of Higbie was studied. With numerical simulations of Fick's law of diffusion, it was demonstrated that Higbie's film theory does not apply for cultivations which occur at viscosities up to 10 mPa s. For the first time, it was experimentally shown that the maximum oxygen transfer capacity OTRmax increases in shake flasks when viscosity is increased from 1 to 10 mPa s, leading to an improved oxygen supply for microorganisms. Additionally, the OTRmax does not significantly undermatch the OTRmax at waterlike viscosities, even at elevated viscosities of up to 80 mPa s. In this range, a shake flask is a somehow self-regulating system with respect to oxygen supply. This is in contrary to stirred tanks, where the oxygen supply is steadily reduced to only 5% at 80 mPa s. Since, the liquid film formation at shake flask walls inherently promotes the oxygen supply at moderate and at elevated viscosities, these results have significant implications for scale-up.

Metabolic signatures of GS-CHO cell clones associated with butyrate treatment and culture phase transition.

Chinese hamster ovary (CHO) cells are preferred hosts for the production of recombinant biopharmaceuticals. Efforts to optimize these bioprocesses have largely relied on empirical experience and our knowledge of cellular behavior in culture is incomplete. More recently, comprehensive investigations of metabolic network operation have started to be used to uncover traits associated with optimal growth and recombinant protein production. In this work, we used (1) H-nuclear magnetic resonance ((1) H-NMR) to analyze the supernatants of glutamine-synthetase (GS)-CHO cell clones expressing variable amounts of an IgG4 under control and butyrate-treated conditions. Exometabolomic data revealed accumulation of several metabolic by-products, indicating inefficiencies at different metabolic nodes. These data were contextualized in a detailed network and the cellular fluxomes estimated through metabolic flux analysis. This approach allowed comparing metabolic activity across different clones, growth phases and culture conditions, in particular the efficiency pertaining to carbon lost to glycerol and lactate accumulation and the characteristic nitrogen metabolism involving high asparagine and serine uptake rates. Importantly, this study shows that early butyrate treatment has a marked effect on sustaining high nutrient consumption along culture time, being more pronounced during the stationary phase when extra energy generation and biosynthetic activity is fueled to increase IgG formation. Collectively, the information generated contributes to deepening our understanding of CHO cells metabolism in culture, facilitating future design of improved bioprocesses.

Synchronous fluorescence spectroscopy as a novel tool to enable PAT applications in bioprocesses.

In this work, synchronous fluorescence spectroscopy (SFS) is evaluated as a new tool for real-time bioprocess monitoring of animal cell cultures. This technique presents several advantages over the traditional two-dimensional (2D) fluorometry since it provides data on various fluorescent compounds in a single spectrum, showing improved peak resolution and recording speed. Bioreactor cultures of three monoclonal antibody-producing CHO cell lines were followed in situ by both 2D and synchronous fluorometry techniques. The time profiles of the main spectral features in each data type present some differences, but principal component analysis indicated both as containing enough information to distinguish the cultures. Partial least squares regression models were then independently developed for viable cell density and antibody levels on the basis of the different fluorescence signals recorded, hiding half of the dataset for subsequent validation purposes. Regardless of the signal used, model predictions fit very well the off-line measurements; still, the synchronous spectra collected at a wavelength difference of 20 nm allowed comparable and superior performances for cell density and antibody titer, respectively, with validation accuracies higher than 91%. Therefore, SFS compares favorably with the traditional 2D approach, becoming an improved, faster option for real-time monitoring of cells and product titer over culture time. The readiness in data acquisition facilitates the design of process control strategies meeting the requirements of a PAT application.

¹H-NMR protocol for exometabolome analysis of cultured mammalian cells.

(1)H-Nuclear magnetic resonance ((1)H-NMR) spectroscopy is a powerful technique to analyze the composition of complex mixtures based on the particular proton fingerprint of each molecule. Here we describe a protocol for exometabolome analysis of mammalian cells using this technique, including sample preparation, spectra acquisition, and integration. The potential of this technique is exemplified by application to cultures of a Chinese hamster ovary (CHO) cell line. The average error associated to this method is under 3% and the limit of quantification for all metabolites analyzed is below 180 µM.

High-throughput analysis of animal cell cultures using two-dimensional fluorometry.

We report a new method which combines fluorescence spectroscopy at microtiter plate scale with multivariate statistical analysis for rapid and high-throughput analysis of secreted recombinant protein and viable cell growth in animal cell cultures. The potential of the method is demonstrated by application to cultures of three Chinese Hamster Ovary (CHO) cell clones with distinct IgG(4) antibody yields. Supernatant samples collected throughout culture time were analysed by two-dimensional fluorometry; significant changes were observed in the regions of tryptophan, metabolic cofactors and vitamins. Partial least squares regression was then used to correlate the entire fluorescence map with measured concentrations of antibody and viable cells. For both target variables, a model was calibrated with representative data from the two less productive clones and validated with data from the best producer clone; this allowed viable cell density to be predicted for the validation clone with an average error of 10%; even better, the secreted antibody could be predicted with an average error of 7%, proving the predictive capacity of the model beyond the calibration region. All the main spectral regions were required to establish the best correlations for both targeted variables. In conclusion, this method effectively analyzes cellular productivity in 96-well plate format, shortening the time spent in early phases of bioprocess development.