Metabolic control of recombinant monoclonal antibody N-glycosylation in GS-NS0 cells

Variable N-glycosylation at Asn(297) in the Fc region of recombinant therapeutic immunoglobulin G (IgG) molecules, specifically terminal galactosylation and sialylation, may affect both pharmacokinetic behavior and effector functions of recombinant therapeutic antibodies. We investigated the hypothesis that IgG Fc glycosylation can be controlled by manipulation of cellular nucleotide-sugar metabolism. In control cultures, N-glycans associated with the Fc domain of a recombinant humanized IgG1 produced by GS-NS0 cells in culture were predominantly biantennary, variably beta-galactosylated (average 0.3 mol galactose complex N-glycan(-1)) structures with no bisecting N-acetylglucosamine residues, sialylation, or alpha1,3-linked galactosylation evident. However, a variable proportion (5% to 15%) of high-mannose (Man5 to Man9) oligosaccharides were present. To manipulate the cellular content of the nucleotide sugar precursor required for galactosylation, UDP-Gal, we included either 10 mM glucosamine or 10 mM galactose in the culture medium. In the case of the former, a 17-fold increase in cellular UDP-N-acetylhexosamine content was observed, with a concomitant reduction (33%) in total UDP-hexose, although the ratio of UDP-Glc:UDP-Gal (4:1) was unchanged. Associated with these alterations in cellular UDP-sugar content was a significant reduction (57%) in the galactosylation of Fc-derived oligosaccharides. The proportion of high-mannose-type N-glycans (specifically Man5, the substrate for N-acetylglucosaminyltransferase I) at Asn(297) was unaffected. In contrast, inclusion of 10 mM galactose in culture specifically stimulated UDP-Gal content almost five-fold. However, this resulted in only a minimal, insignificant increase (6%) in beta1,4-galactosylation of Fc N-glycans. Sialylation was not improved upon the addition of the CMP-sialic acid (CMP-SA) precursor N-acetylmannosamine (20 mM), even with an associated 44-fold increase in cellular CMP-SA content. Analysis of recombinant IgG1 Fc glycosylation during batch culture showed that beta1,4-linked galactosylation declined slightly during culture, although, in the latter stages of culture, the release of proteases and glycosidases by lysed cells were likely to have contributed to the more dramatic drop in galactosylation. These data demonstrate: (i) the effect of steric hindrance on Fc N-glycan processing; (ii) the extent to which alterations in cellular nucleotide-sugar content may affect Fc N-glycan processing; and (iii) the potential for direct metabolic control of Fc N-glycosylation.     

Calnexin overexpression sensitizes recombinant CHO cells to apoptosis induced by sodium butyrate treatment

Sodium butyrate (NaBu) can enhance the expression of foreign genes in recombinant Chinese hamster ovary (rCHO) cells, but it can also inhibit cell growth and induce cellular apoptosis. In this study, the potential role of calnexin (Cnx) expression in rCHO cells treated with 5 mM NaBu was investigated for rCHO cells producing tumor necrosis factor receptor FC. To regulate the Cnx expression level, a tetracycline-inducible system was used. Clones with different Cnx expression levels were selected and investigated. With regard to productivity per cell (q_p), NaBu enhanced the q_p by over twofold. Under NaBu treatment, Cnx overexpression further enhanced the q_p by about 1.7-fold. However, under NaBu stress, the cells overexpressing Cnx showed a poorer viability profile with a consistent difference of over 25% in the viability when compared to the Cnx-repressed condition. This drop in the viability was attributed to increased apoptosis seen in these cells as evidenced by enhanced poly (ADP-ribose) polymerase cleavage and cytochrome C release. Ca²⁺ localization staining and subsequent confocal imaging revealed elevated cytosolic Ca²⁺ ([Ca²⁺]_c) in the Cnx-overexpressing cells when compared to the Cnx-repressed condition, thus endorsing the increased apoptosis observed in these cells. Taken together, Cnx overexpression not only improved the q_p of cells treated with NaBu, but it also sensitized cells to apoptosis.     

Influence of intracellular nucleotide and nucleotide sugar contents on recombinant interferon-gamma glycosylation during batch and fed-batch cultures of CHO cells

Both the macroheterogeneity of recombinant human IFN-gamma produced by CHO cells and intracellular levels of nucleotides and sugar nucleotides, have been characterized during batch and fed-batch cultures carried out in different media. Whereas PF-BDM medium was capable to maintain a high percentage of the doubly- glycosylated glycoforms all over the process, mono-glycosylated and non-glycosylated forms increased during the batch culture using SF-RPMI medium. Intracellular level of UTP was higher in PF-BDM all over the batch culture compared to the SF-RPMI process. UDP-Gal accumulated only during the culture performed in PF-BDM medium, probably as a consequence of the reduced UDP-Glc synthesis flux in SF-RPMI medium. When the recombinant CHO cells were cultivated in fed-batch mode, the UTP level remained at a relatively high value in serum-containing RPMI and its titer increased during the fed-phase indicating an excess of biosynthesis. Besides, an accumulation of UDP-Gal occurred as well. Those results all together indicate that UTP and UDP-Glc syntheses in CHO cells cultivated in SF-RPMI medium in batch process, could be limiting during the glycosylation processes of the recombinant IFN-gamma. At last, the determination of the energetic status of the cells over the three studied processes suggested that a relationship between the adenylate energy charge and the glycosylation macroheterogeneity of the recombinant IFN-gamma may exist.     

Oxidative stress-alleviating strategies to improve recombinant protein production in CHO cells

Large scale biopharmaceutical production of biologics relies on the overexpression of foreign proteins by cells cultivated in stirred tank bioreactors. It is well recognized and documented fact that protein overexpression may impact host cell metabolism and that factors associated with large scale culture, such as the hydrodynamic forces and inhomogeneities within the bioreactors, may promote cellular stress. The metabolic adaptations required to support the high-level expression of recombinant proteins include increased energy production and improved secretory capacity, which, in turn, can lead to a rise of reactive oxygen species (ROS) generated through the respiration metabolism and the interaction with media components. Oxidative stress is defined as the imbalance between the production of free radicals and the antioxidant response within the cells. Accumulation of intracellular ROS can interfere with the cellular activities and exert cytotoxic effects via the alternation of cellular components. In this context, strategies aiming to alleviate oxidative stress generated during the culture have been developed to improve cell growth, productivity, and reduce product microheterogeneity. In this review, we present a summary of the different approaches used to decrease the oxidative stress in Chinese hamster ovary cells and highlight media development and cell engineering as the main pathways through which ROS levels may be kept under control.     

Identification of cell culture conditions to control N‐glycosylation site‐occupancy of recombinant glycoproteins expressed in CHO cells

The effect of different cell culture conditions on N‐glycosylation site‐occupancy has been elucidated for two different recombinant glycoproteins expressed in Chinese hamster ovary (CHO) cells, recombinant human tissue plasminogen activator (t‐PA) and a recombinant enzyme (glycoprotein 2—GP2). Both molecules contain a N‐glycosylation site that is variably occupied. Different environmental factors that affect the site‐occupancy (the degree of occupied sites) of these molecules were identified. Supplementing the culture medium with additional manganese or iron increased the fraction of fully occupied t‐PA (type I t‐PA) by approximately 2.5–4%. Decreasing the cultivation temperature from 37 to 33°C or 31°C gradually increased site‐occupancy of t‐PA up to 4%. The addition of a specific productivity enhancer, butyrate, further increased site‐occupancy by an additional 1% under each cultivation temperature tested. In addition, the thyroid hormones triiodothyronine and thyroxine increased site‐occupancy of t‐PA compared to control conditions by about 2%. In contrast, the addition of relevant nucleoside precursor molecules involved in N‐glycan biosynthesis (e.g., uridine, guanosine, mannose) either had no effect or slightly reduced site‐occupancy. For the recombinant enzyme (GP2), it was discovered that culture pH and the timing of butyrate addition can be used to control N‐glycan site‐occupancy within a specific range. An increase in culture pH correlated with a decrease in site‐occupancy. Similarly, delaying the timing for butyrate addition also decreased site‐occupancy of this molecule. These results highlight the importance of understanding how cell culture conditions and media components can affect the product quality of recombinant glycoproteins expressed in mammalian cell cultures. Furthermore, the identification of relevant factors will enable one to control product quality attributes, specifically N‐glycan site‐occupancy, within a specific range when applied appropriately. Biotechnol. Bioeng. 2009;103: 1164–1175. © 2009 Wiley Periodicals, Inc.     

Model-based optimization of antibody galactosylation in CHO cell culture

Exerting control over the glycan moieties of antibody therapeutics is highly desirable from a product safety and batch-to-batch consistency perspective. Strategies to improve antibody productivity may compromise quality, while interventions for improving glycoform distribution can adversely affect cell growth and productivity. Process design therefore needs to consider the trade-off between preserving cellular health and productivity while enhancing antibody quality. In this work, we present a modeling platform that quantifies the impact of glycosylation precursor feeding – specifically that of galactose and uridine – on cellular growth, metabolism as well as antibody productivity and glycoform distribution. The platform has been parameterized using an initial training data set yielding an accuracy of ±5% with respect to glycoform distribution. It was then used to design an optimized feeding strategy that enhances the final concentration of galactosylated antibody in the supernatant by over 90% compared with the control without compromising the integral of viable cell density or final antibody titer. This work supports the implementation of Quality by Design towards higher-performing bioprocesses.     

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 (q_p). As an improved ATP supply was anticipated to enhance q_p 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 q_p by stable overexpression. Furthermore, deletion of genes with presumably dispensable function in suspension growing CHO cells may enhance mAB formation by increased ATP levels.     

Chemical inhibitors of hexokinase-2 enzyme reduce lactate accumulation,alter glycosylation processing,and produce altered glycoforms in CHO cell cultures

Chinese hamster ovary (CHO) cells, predominant hosts for recombinant biotherapeutics production, generate lactate as a major glycolysis by-product. High lactate levels adversely impact cell growth and productivity. The goal of this study was to reduce lactate in CHO cell cultures by adding chemical inhibitors to hexokinase-2 (HK2), the enzyme catalyzing the conversion of glucose to glucose 6-phosphate, and examine their impact on lactate accumulation, cell growth, protein titers, and N-glycosylation. Five inhibitors of HK2 enzyme at different concentrations were evaluated, of which 2-deoxy- d -glucose (2DG) and 5-thio- d -glucose (5TG) successfully reduced lactate accumulation with only limited impacts on CHO cell growth. Individual 2DG and 5TG supplementation led to a 35%–45% decrease in peak lactate, while their combined supplementation resulted in a 60% decrease in peak lactate. Inhibitor supplementation led to at least 50% decrease in moles of lactate produced per mol of glucose consumed. Recombinant EPO-Fc titers peaked earlier relative to the end of culture duration in supplemented cultures leading to at least 11% and as high as 32% increase in final EPO-Fc titers. Asparagine, pyruvate, and serine consumption rates also increased in the exponential growth phase in 2DG and 5TG treated cultures, thus, rewiring central carbon metabolism due to low glycolytic fluxes. N-glycan analysis of EPO-Fc revealed an increase in high mannose glycans from 5% in control cultures to 25% and 37% in 2DG and 5TG-supplemented cultures, respectively. Inhibitor supplementation also led to a decrease in bi-, tri-, and tetra-antennary structures and up to 50% lower EPO-Fc sialylation. Interestingly, addition of 2DG led to the incorporation of 2-deoxy-hexose (2DH) on EPO-Fc N-glycans and addition of 5TG resulted in the first-ever observed N-glycan incorporation of 5-thio-hexose (5TH). Six percent to 23% of N-glycans included 5TH moieties, most likely 5-thio-mannose and/or 5-thio-galactose and/or possibly 5-thio-N-acetylglucosamine, and 14%–33% of N-glycans included 2DH moieties, most likely 2-deoxy-mannose and/or 2-deoxy-galactose, for cultures treated with different concentrations of 5TG and 2DG, respectively. Our study is the first to evaluate the impact of these glucose analogs on CHO cell growth, protein production, cell metabolism, N-glycosylation processing, and formation of alternative glycoforms.     

Effect of doxycycline-regulated protein disulfide isomerase expression on the specific productivity of recombinant CHO cells: thrombopoietin and antibody

Protein disulfide isomerase (PDI), one of the ER-resident molecular chaperones, forms and isomerizes disulfide bonds. This study attempts to investigate the effect of PDI expression level on specific productivity (q) of recombinant Chinese hamster ovary (rCHO) cells producing thrombopoietin (TPO) and antibody (Ab). To regulate the PDI expression level, the Tet-Off system was introduced in TPO and Ab producing CHO cells, and stable Tet-Off cells (TPO-Tet-Off and Ab-Tet-Off) were screened using the luciferase assay. The doxycycline-regulated PDI expression system in Tet-Off rCHO cells (Tet-TPO-PDI and Tet-Ab-PDI) was established by the cotransfection of pTRE-PDI and pTK-Hyg expression vector into TPO-Tet-Off and Ab-Tet-Off cells, respectively. Subsequent screening was done by Western blot analysis of PDI and an enzyme-linked immunosorbent assay of the secreted TPO and antibody. We cultured two Tet-TPO-PDI and two Tet-Ab-PDI clones, and all these clones showed an average of 2.5-fold increase in PDI expression when compared to the basal level. In both these cell lines the PDI expression was tightly controlled by various concentrations of doxycycline. The q of TPO (q(TPO)) was unaffected but that of antibody producing cells was increased by 15-27% due to the PDI expression level.     

Methyl mercury uptake and associations with the induction of chromosomal aberrations in Chinese hamster ovary (CHO) cells

In order to evaluate possible health effects of environmental exposure of humans towards methyl mercury species, relevant exposure experiments using methyl mercury chloride in aqueous solution and Chinese hamster ovary (CHO) cells were performed. The solution was monitored for the presence of monomethyl, dimethyl and elemental mercury by several analytical techniques including chromatographic as well as atomic absorption and mass spectrometric methods. Methyl mercury induces structural chromosomal aberrations (CA) and sister chromatid exchanges (SCE) in CHO cells. At a concentration of methyl mercury in the culture medium of 1.0 x 10(-6) M where the frequencies of CA and SCE are significantly elevated, the intracellular concentration was 1.99 x 10(-16) mol/cell. Possible biochemical processes leading to the cytogenetic effects are discussed together with toxicological consequences, when humans (e.g. workers at waste deposits) are exposed to environmental concentrations of methyl mercury.     

Expression of GnTIII in a recombinant anti-CD20 CHO production cell line: Expression of antibodies with altered glycoforms leads to an increase in ADCC through higher affinity for FC gamma RIII.

The gene encoding the rat glycosylation enzyme beta1-4-N-acetylglucosaminyltransferase III (GnTIII) was cloned and coexpressed in a recombinant production Chinese hamster ovary (CHO) cell line expressing a chimeric mouse/human anti-CD20 IgG1 antibody. The new cell lines expressed high levels of antibody and have growth kinetics similar to that of the parent. Relative QPCR showed the cell lines to express varying levels of mRNA. High-performance liquid chromatography (HPLC) analysis showed the enzyme to have added bisecting N-acetylglucosamine (GlcNAc) residues in most (48% to 71%) of the N-linked oligosaccharides isolated from antibody preparations purified from the cell lines. In an ADCC assay the new antibody preparations promoted killing of CD20-positive target cells at approximately 10- to 20-fold lower concentrations than the parent. This activity was blocked using an anti-Fc gamma RIII antibody, supporting the role of Fc gamma RIII binding in this increase. In addition, cell binding assays showed the modified antibody bound better to Fc gamma RIII-expressing cells. The increase in ADCC activity is therefore likely due to an increased affinity of the modified antibody for the Fc gamma RIII receptor.     

Cellular and chemical reduction products of misonidazole

Misonidazole is readily reduced by zinc dust in aqueous solution in the presence of ammonium chloride. High pressure liquid chromatographic (HPLC) separation of the reduction mixture revealed the presence of three products. These were identified as the hydroxylamine, amine and the hydrazo derivative of misonidazole. There is evidence that the azoxy derivative was an intermediate in the reduction process. When the reduction was carried out in dilute solution (0.1 mg/ml), the hydroxylamine was the only product. In concentrated solution (20 mg/ml), the hydrazo derivative was the major product. When misonidazole was reduced with hydrogen using palladium as catalyst, the amine was the only detectable product. Of the three products, only the hydroxylamine was found to bind covalently to bovine albumin. In Chinese hamster ovary (CHO) cells under hypoxic conditions the amine was confirmed as one of the metabolites. There was no evidence for the presence of detectable amounts of the hydroxylamine in the cell extracts. These studies suggest that the hydroxylamine is probably the reactive reduction metabolite responsible for the in vivo and in vitro binding of misonidazole to cellular macromolecules.     

Design of serum-free medium for suspension culture of CHO cells on the basis of general commercial media

The design of serum-free media for suspension culture of genetically engineered Chinese hamster ovary (CHO) cells using general commercial media as a basis was investigated. Subcultivation using a commercial serum-free medium containing insulin-like growth factor (IGF)-1 with or without FCS necessitated additives other than IGF-1 to compensate for the lack of FCS and improve cell growth. Suspension culture with media containing several combinations of growth factors suggested the effectiveness of addition of both IGF-1 and the lipid signaling molecule lysophosphatidic acid (LPA) for promoting cell growth. Subcultivation of CHO cells in suspension culture using the commercial serum-free medium EX-CELL™302, which contained an IGF-1 analog, supplemented with LPA resulted in gradually increasing specific growth rate comparable to the serum-containing medium and in almost the same high antibody production regardless of the number of generations. The culture with EX-CELL™302 supplemented with LPA in a jar fermentor with pH control at 6.9 showed an apparently higher cell growth rate than the cultures without pH control and with pH control at 6.8. The cell growth in the medium supplemented with aurintricarboxylic acid (ATA), which was much cheaper than IGF-1, in combination with LPA was synergistically promoted similarly to that in the medium supplemented with IGF-1 and LPA. In conclusion, the serum-free medium designed on the basis of general commercial media could support the growth of CHO cells and antibody production comparable to serum-containing medium in suspension culture. Moreover, the possibility of cost reduction by the substitution of IGF-1 with ATA was also shown.     

A functional high‐content miRNA screen identifies miR‐30 family to boost recombinant protein production in CHO cells

The steady improvement of mammalian cell factories for the production of biopharmaceuticals is a key challenge for the biotechnology community. Recently, small regulatory microRNAs (miRNAs) were identified as novel targets for optimizing Chinese hamster ovary (CHO) production cells as they do not add any translational burden to the cell while being capable of regulating entire physiological pathways. The aim of the present study was to elucidate miRNA function in a recombinant CHO‐SEAP cell line by means of a genome‐wide high‐content miRNA screen. This screen revealed that out of the 1, 139 miRNAs examined, 21% of the miRNAs enhanced cell‐specific SEAP productivity mainly resulting in elevated volumetric yields, while cell proliferation was accelerated by 5% of the miRNAs. Conversely, cell death was diminished by 13% (apoptosis) or 4% (necrosis) of all transfected miRNAs. Besides these large number of identified target miRNAs, the outcome of our studies suggest that the entire miR‐30 family substantially improves bioprocess performance of CHO cells. Stable miR‐30 over expressing cells outperformed parental cells by increasing SEAP productivity or maximum cell density of approximately twofold. Our results highlight the application of miRNAs as powerful tools for CHO cell engineering, identified the miR‐30 family as a critical component of cell proliferation, and support the notion that miRNAs are powerful determinants of cell viability.     

Utilization and stability of vitamins in serum-containing and serum-free media in CHO cell culture

The concentrations of four vitamins, ascorbic acid, nicotinamide, choline and thiamine were evaluated in the culture supernatant of Chinese hamster ovary (CHO) cells. The media used were -modified Eagle's minimum essential medium (MEM-) supplemented with 10% fetal calf serum, and a 1:1 mixture of Ham's F12 and Dulbecco's modified Eagle's medium (DME/F12), containing neither serum nor protein. The reference experiment without cells revealed instability of ascorbic acid and thiamine. Moreover, a significant amount of each vitamin decreased in the culture supernatant. The possibility of growth limitation by vitamin depletion is strongly suggested.