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 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.

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.

Biotechnology and BioengineeringVolume 116, Issue 5 p. 945-949 ISSUE INFORMATIONFree Access Biotechnology and Bioengineering: Volume 116, Number 5, May 2019 First published: 01 April 2019 https://doi.org/10.1002/bit.26757AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Volume116, Issue5May 2019Pages 945-949 RelatedInformation

Market demands for monoclonal antibodies (mAbs) are steadily increasing worldwide. As a result, production processes using Chinese hamster ovary cells (CHO) are in the focus of ongoing intensification studies for maximizing cell-specific and volumetric productivities. This includes the optimization of animal-derived component free (ADCF) cultivation media as part of good cell culture practice. Dipeptides are known to improve CHO culture performance. However, little or even conflicting assumptions exist about their putative import and functionality inside the cells. A set of well-known performance boosters and new dipeptide prospects was evaluated. The present study revealed that dipeptides are indeed imported in the cells, where they are decomposed to the amino acids building blocks. Subsequently, they are metabolized or, unexpectedly, secreted to the medium. Monoclonal antibody production boosting additives like L-alanine-L-glutamine (AQ) or glycyl-L-glutamine (GQ) can be assigned to fast or slow dipeptide uptake, respectively, thus pinpointing to the need to study dipeptide kinetics and to adjust their feeding individually for optimizing mAb production.

Conventional analysis and optimization procedures of mammalian cell culture processes mostly treat the culture as a homogeneous population. Hence, the focus is on cell physiology and metabolism, cell line development, and process control strategy. Impact on cultivations caused by potential variations in cellular properties between different subpopulations, however, has not yet been evaluated systematically. One main cause for the formation of such subpopulations is the progress of all cells through the cell cycle. The interaction of potential cell cycle specific variations in the cell behavior with large‐scale process conditions can be optimally determined by means of (partially) synchronized cultivations, with subsequent population resolved model analysis. Therefore, it is desirable to synchronize a culture with minimal perturbation, which is possible with different yield and quality using physical selection methods, but not with frequently used chemical or whole‐culture methods. Conventional nonsynchronizing methods with subsequent cell‐specific, for example, flow cytometric analysis, can only resolve cell‐limited effects of the cell cycle. In this work, we demonstrate countercurrent‐flow centrifugal elutriation as a useful physical method to enrich mammalian cell populations within different phases of a cell cycle, which can be further cultivated for synchronized growth in bioreactors under physiological conditions. The presented combined approach contrasts with other physical selection methods especially with respect to the achievable yield, which makes it suitable for bioreactor scale cultivations. As shown with two industrial cell lines (CHO‐K1 and human AGE1.HN), synchronous inocula can be obtained with overall synchrony degrees of up to 82% in the G1 phase, 53% in the S phase and 60% in the phase, with enrichment factors ( ) of 1.71, 1.79, and 4.24 respectively. Cells are able to grow with synchrony in bioreactors over several cell cycles. This strategy, combined with population‐resolved model analysis and parameter extraction as described in the accompanying paper, offers new possibilities for studies of cell lines and processes at levels of cell cycle and population under physiological conditions. © 2014 American Institute of Chemical Engineers Biotechnol. Prog ., 31:165–174, 2015