Multidisciplinary Analysis of the Metabolic Shift to Lactate Consumption in CHO Cell Culture

Mammalian cells represent the most widely used host system for the industrial production of recombinant therapeutic proteins. In order to increase productivity, chemically defined media are often used and optimized to provide the cells with the necessary nutrients. However, a deregulated cell metabolism is often observed in these culture conditions. In particular, carbon sources are fast and inefficiently consumed with a consequent accumulation of byproducts, mainly lactate and ammonia. Lactate, in particular, causes a decrease of the medium pH which can be detrimental for cells growth and productivity. Its metabolic profile is normally characterized by a first production phase, which is associated to the cells exponential growth. Afterwards, when the cells enter into the stationary phase, a shift to net lactate consumption can occur under optimal culture conditions. However, such a metabolic shift is not easily controlled, since the mechanisms modulating lactate production in cell culture are still under investigation. This work aims to understand which factors could influence the lactate metabolic shift in CHO cell culture, focusing, in particular, on the mitochondrial role. To this purpose, cell lines with opposite lactate profiles were compared. The initial lactate production phase was common to all the fast growing cultures and was concomitant to a rapid glutamine consumption. After glutamine depletion, two different scenario occurred. In one case, the cells started to consume lactate until complete depletion. Alternatively, lactate continued to be accumulated, causing a decrease of media pH. The mitochondrial oxidative capacity was hypothesized as a possible cause of the different lactate profile. Indeed, mitochondrial membrane potential and oxygen consumption measurements highlighted a correlation between a reduced oxidative metabolism and a state of high lactate production. This correlation was confirmed by evaluating other cell lines or media compositions which resulted in the same divergence of lactate metabolism. Afterwards, the expression of selected genes was analysed in correlation with the observed lactate profile. Among 22 genes, two were identified as significantly downregulated (absolute fold change ≥2) in conditions of high lactate accumulation; namely, the mitochondrial aspartate-glutamate carrier (aralar1) and the translocase of the inner mitochondrial membrane 8 (timm8a). Aralar1, in particular, is an important component of the malate-aspartate shuttle (MAS). This system promotes the recycling of the cytosolic NADH pool, which is necessary for maintaining the glycolytic flux. Alternatively, NADH can be oxidised through pyruvate conversion into lactate, catalysed by the lactate dehydrogenase enzyme. Therefore, an inefficient MAS activity can engender an increased lactate accumulation. Finally, stable cell lines, which overexpressed aralar1 or timm8a, were generated. Clones derived from a lactate-producing cell line showed an improvement of lactate metabolism. In particular, they switched more easily to lactate consumption compared to the untransfected control, while maintaining a similar glucose consumption rate. On the other hand, no impact of the transgene expression was observed in the clones derived from the lactate-consuming cell line, since the switch to lactate consumption was maintained and no other effect on cell growth or glucose metabolism was detected. The data obtained indicate that lactate consumption was most likely promoted by a better NADH oxidation through the malate-aspartate shuttle, which resulted in a more efficient link between glycolysis and the mitochondrial oxidative metabolism. In conclusion, the results presented in this thesis indicate that the mitochondrial oxidative capacity plays a central role in the control of lactate production. Media composition and intrinsic cell characteristics have both an impact on mitochondrial activity. Moreover, the expression of specific genes can also be influenced by the culture media. In particular, aralar1 and timm8a have been identified as promising targets for the generation of a host cell line with an improved lactate metabolism.

Lactate metabolism shift in CHO cell culture: the role of mitochondrial oxidative activity

Martin Jordan, Mathieu Benjamin Stettler, Florian Maria Wurm, Francesca Zagari

Lactate production is monitored in industrial processes as a crucial metabolite for cultured mammalian cells. Typically lactate is strongly produced during the exponential growth phase, while its net consumption is frequently observed when cells enter into the stationary phase. Such a metabolic shift is desirable because it seems to favor optimal process performance. However, this shift is neither generic nor can it be easily controlled, as the mechanisms modulating lactate production/consumption in cell culture are still under investigation. In this study different lactate profiles were observed in a chemically defined medium for the parental CHO-S cells and a non-recombinant subclone. The initial lactate production phase, which is typical for fast growing cells, was similar for both cell lines. After glutamine depletion the situation changed: the parental cell line promptly switched to net lactate consumption, whereas the subclone continued to produce lactate until glucose was depleted as well. We speculated that the extra lactate production would be ascribed to a different mitochondrial oxidative capacity in the subclone. Therefore, the mitochondrial membrane potential and oxygen consumption were measured for both cell lines. Indeed, a correlation between high lactate production and a reduced oxidative metabolism was found. Interestingly, this particular metabolic phenotype was also strongly influenced by the medium composition: both cell lines underwent a switch to lactate consumption when cultivated in a second medium, while a third one promoted continuous lactate production even for the parental CHO cells. Again, the correlation between lactate profile and oxidative metabolism was confirmed, pointing to a central role of mitochondria on lactate metabolism.

Elsevier2013

Accelerating the development time of recombinant mammalian cell lines producing high titers of protein therapeutics

Sébastien Chenuet

The forthcoming arrival on the market of numerous protein therapeutics that require high clinical doses will foster the need for high-producing mammalian cell lines. Furthermore, pressures to reduce the development time for new therapeutics has meant more emphasis on efforts to accelerate the process that yields such cell lines. The objective of this thesis work was to develop methods to decrease the time needed to generate stable and high producing mammalian cell lines. This could be achieved by overcoming some of the major limitations that hamper the process of cell lines development. The host systems chosen for this study were Chinese hamster ovary (CHO DG44) and baby hamster kidney (BHK-21). The recombinant proteins used as reporters were the green fluorescent protein (GFP) and a human anti-Rhesus D IgG. One limitation hampering the development of cell lines producing high recombinant protein titers is the extensive variability of transgene expression within a transfected population as a result of the gene delivery process. This decreases the reproducibility of the generation of high producing cell lines. A direct comparison of two gene chemical delivery methods, polyethylenimine (PEI)- and calcium phosphate (CaPO4)-mediated transfection, with a mechanical method (microinjection) provided some insights into ways to overcome this limitation. Indeed, a correlation was observed between the amount of plasmid DNA delivered into cells and the specific productivity of the recovered recombinant cell lines. Hence, by increasing the amount of DNA delivered per cell, IgG-producing clones with specific productivities up to 22 pg/cell/day were recovered with a high efficiency. A second limitation is the time needed to assure the stability and clonality of cell lines by current analytical methods. This was overcome by using GFP as a reporter for the level of production of a co-expressed therapeutic protein. The co-transfection of plasmids individually carrying the GFP and IgG heavy (Hc)- and light chain (Lc) genes, resulted in integration of all three plasmids at the same site in the cell's genome. As a consequence, the stability of GFP expression correlated with that of IgG expression. This proved to be advantageous finding and eliminating unstable cell lines early in the process of selection. As a consequence, it was possible to derive in serum-free medium clones with a specific productivity almost 6 times higher than that of clones derived from transfection performed with the IgG Hc and Lc vectors only. A third limitation to the rapid recovery of stable cell lines is the time devoted to the cultivation of transfected cells in selective medium, normally one month. Here, the time of selection was shortened to one week by increasing the stringency of the selective pressure. The results from studies of plasmid integration kinetics by fluorescence in situ hybridization (FISH) showed that the increased stringency of selection decreased the time needed to enrich a transfected population with recombinant cells. Furthermore, stable clones were shown to be present in this population after one week of selection and could be uncovered by identifying patterns of GFP expression predictive of production stability. By limiting the screening to clones exhibiting these stable patterns of GFP expression, the number of clones needed in order to isolate stable cell lines producing high IgG titers could be dramatically reduced. In conclusion, these results combined together provided a new procedure to isolate within a week of the start of selection, stable and high-producing clones. 30-40% of the clones isolated using this procedure were stable for at least three months and their average specific productivity was 29 pg/cell/day.

EPFL2008

Development of high-titer transient gene expression processes for manufacturing recombinant proteins

Gaurav Backliwal

The market for recombinant therapeutic proteins (including antibodies) is estimated to be greater than $50-billion and has a compounded annual growth rate of around 20%. More than 50% of all approved processes for manufacturing recombinant proteins use mammalian cells as expression hosts due to their ability to carry out complex assembly and processing, human-like glycosylation and secretion of proteins. This percentage is not expected to change and should even increase with time. Classically, these manufacturing processes use stable cell lines for protein expression, wherein the plasmid coding for the protein of interest is stably integrated into the host cell chromosomes (Stable Gene Expression, SGE). An alternative method for expressing recombinant proteins is Transient Gene Expression (TGE). Herein, the expression of the protein of interest takes place from plasmids which are transfected into the cells and are maintained extra-chromosomally. TGE has become a rapid and easy method for obtaining proteins for structural, biochemical or pre-clinical studies, where only small amounts of one or more proteins might be required. As yet, TGE has not been used for manufacturing recombinant proteins for clinical testing or approved clinical use due to the requirement of large amounts of protein of consistent quality. Even though TGE has been scaled-up to the 100-liter scale and is being attempted at the 1000-liter scale, due to low specific productivity, low volumetric yields and the high cost of DNA, TGE processes are not able to economically produce sufficient amounts of proteins for such purposes. Therefore, the goal of this thesis was to improve TGE in order to create high-titer processes, which would be economically more feasible for manufacturing large-amounts of recombinant proteins. This was achieved by: Improving and simplifying gene delivery – by the combination of high cell density at time of transfection and in-situ complex formation (as apposed to a-priori complex formation), we could enhance protein expression levels and gene delivery. This made implementation more robust and easy, with greater choice of media which could be used and cell density which could be maintained. Improving gene expression – by using a combination of inhibitors of histone deacetylases like valproic acid, and growth factors like acidic fibroblast growth factor, we could enhance specific productivity by ∼20-fold. Enhancing the cell densities at which cells could be maintained after transfection – by the combination of cell cycle regulators like human p18 and p21 and treatment with inhibitors of histone deacetylases, we blocked cell growth which allowed cells to be maintained at significantly higher cell densities, allowing us to enhance volumetric productivity by ∼100-fold. Overall these improvements allowed antibody titers in excess of 1 g/l. The gram per liter range of volumetric productivity has previously only been reported for optimized SGE based manufacturing processes. We therefore improved the economic feasibility of TGE for manufacturing recombinant proteins and reduced the difference in manufacturing costs between TGE and SGE, for a given amount of protein, by an estimated 50-fold. With some further development work and study of related issues like protein quality, TGE based processes can be expected to become part of mainstream recombinant protein manufacturing in the future.

EPFL2008

Development of high-titer transient gene expression processes for manufacturing recombinant proteins

Gaurav Backliwal

EPFL2008

Accelerating the development time of recombinant mammalian cell lines producing high titers of protein therapeutics

Sébastien Chenuet

EPFL2008