Effects of phosphatidic acid on recombinant protein production by Chinese hamster ovary cells in serum-free culture

When recombinant Chinese hamster ovary (CHO) cells (ATCC CRL-8200) producing human interferon-γ (hIFN-γ) were incubated with exogenously supplied phosphatidic acid (PA) from egg yolk lecithin in a serum-free medium, PA induced increases in both the density of viable cells and concentration of hIFN-γ secreted into the medium. Dispersion of PA with a non-ionic surfactant, Tween 80, further enhanced both cell growth and hIFN-γ production. Replacement of the culture medium containing PA by fresh medium without PA in the course of a static culture did not influence cell growth indicating that PA is required to be continuously present in a serum-free medium to stimulate cell growth. Using a fresh medium containing PA for replacement resulted in significant enhancement of both cell density and hIFN-γ yield. These results suggest that PA is a promising constituent of low-protein serum-free media for the effective production of recombinant proteins.     

Two-stage depth filter perfusion culture for recombinant antibody production by recombinant Chinese hamster ovary cell

A rCHO cell line of DUKX origin 26*-320, producing recombinant antibody against the human platelet, was cultivated in a two-stage depth filter perfusion system (DFPS) for 20 days in order to attain high recombinant antibody concentration. The productivity of the first stage DFPS bioreactor reached 53 times that of the batch culture in a controlled stirred tank reactor and was showed 12.1 mg/L antibody concentration at a perfusion rate of 6.0 d⁻¹. Glucose concentration in the first DFPS was maintained at 1.5 g/L to avoid cell damage in the perfusion culture. A second stage DFPS system was attached to the first DFPS, which resulted in a low glucose concentration of 0.02 g/L and a high antibody concentration of 23.9 mg/L. The two-stage depth filter perfusion culture yielded 60% higher product concentration than the batch and 49-fold higher productivity of 69.3 mg/L/d in comparison with that (1.4 mg/L/d) in a batch system. Furthermore, antibody concentration of the second stage was 97% higher than that of the first stage, and the antibody productivities were comparable to that of the first stage. This two-stage DFPS system also showed potential for higher titer production of recombinant antibody and high volumetric productivity for long-term culture of bio-pharmaceutical substances.     

Adaptation of Chinese hamster ovary cells to low culture temperature: cell growth and recombinant protein production

Recombinant Chinese hamster ovary (rCHO) cells producing erythropoietin (EPO) and rCHO cells producing follicle-stimulating hormone (FSH) showed a significant increase in specific productivity (q) when grown at 32 degrees C compared to 37 degrees C. However, low culture temperature suppressed cell growth, and therefore, did not increase volumetric productivity as much as q. In an attempt to increase the volumetric productivity through improvement of hypothermic growth, EPO producing rCHO (CHO-EPO) cells and FSH producing rCHO (CHO-FSH) cells were adapted at 32 degrees C in a repeated batch mode using spinner flasks. Cell growth of both CHO-EPO and CHO-FSH gradually improved during adaptation at 32 degrees C. Specific growth rates of CHO-EPO and CHO-FSH cells at 32 degrees C, through adaptation, were increased by 73% and 20%, respectively. During adaptation at 32 degrees C, mRNA levels of cold-inducible RNA-binding protein (CIRP) of both rCHO cell lines did not change significantly, suggesting that CIRP expression may not be the only cause for growth suppression at low culture temperature. Unlike cell growth, the recombinant protein production of both rCHO cell lines was not increased during adaptation due to decreased specific productivities. The specific EPO productivity and specific FSH productivity were decreased by 49% and 22%, respectively. Southern blot analyses showed that the decreased specific productivities were not due to the loss of foreign gene copies. Taken together, improvement of hypothermic cell growth by adaptation does not appear to be applicable for enhanced recombinant protein production, since specific productivity decreases during adaptation to the low culture temperature.     

RNA interference technology to improve recombinant protein production in Chinese hamster ovary cells

RNA interference (RNAi) technology has become a novel tool for silencing gene expression in cells or organisms, and has also been used to develop new therapeutics for certain diseases. This review describes its other application of using RNAi technology to increase cellular productivity and the quality of recombinant proteins that are produced in Chinese hamster ovary (CHO) cells, the most important mammalian cell line used in producing licensed biopharmaceuticals in these days. The approaches reported include the silencing of apoptosis-associated gene expression, protein glycosylation-associated gene expression, lactate dehydrogenase involved in cellular metabolism, and dihydrofolate reductase used for gene amplification. All of these works belong to the single component approach therefore depends strongly on the identification of the down-regulation of the critical target gene which can markedly influence the cellular functions associated with recombinant protein expression in CHO cells. Future RNAi approaches can be extended to silence multiple targets involved in different cellular pathways for changing the global gene regulation in cells, as well as the targets related to microRNA molecules for cellular self regulation.     

Enhancement of recombinant protein production in Chinese hamster ovary cells through anti-apoptosis engineering using 30Kc6 gene

It was previously reported that silkworm hemolymph (SH) inhibits apoptosis and increases the production of recombinant human erythropoietin (EPO) in Chinese hamster ovary (CHO) cells. The apoptosis-inhibiting component in SH is a member of 30K protein family. In this study, the CHO cell line producing EPO was manipulated genetically to express the 30Kc6 gene encoding a 30K protein in the hemolymph of the silkworm, Bombyx mori. The transient expression of 30Kc6 significantly suppressed the cell death induced by serum deprivation. A stable cell line expressing 30Kc6 with an anti-apoptotic property was established. The stable expression of 30Kc6 inhibited serum-deprivation-induced apoptosis and increased the cell density and EPO titer by 5- and 10-fold, respectively. The positive effects of the 30Kc6 expression on cell viability and productivity were due to the stable maintenance of the mitochondrial activity. The 30Kc6 expression efficiently suppressed the depolarization of the mitochondrial membrane and subsequently balanced the generation/consumption of ATP. The use of the 30Kc6 gene is expected to provide a new method of host cell engineering for improving the productivity of the recombinant protein.     

Characterization of Chinese hamster ovary cell culture feed media precipitate

Process intensification of monoclonal antibody production is leading to more concentrated feed media causing issues with precipitation of solids from the media solution. This results in processing problems since components in the precipitate are no longer in solution, changing the media composition and leading to variability in cell culture performance. The goal of this work is to characterize the feed media precipitate, and in particular to identify the precipitated components so that mitigation strategies can be developed. From the conducted analysis, the precipitate was predominately found to be organic and was analyzed with liquid chromatography-mass spectrometry and inductively coupled plasma-optical emission spectroscopy (ICP-OES) to identify the constituent components. Up to ten amino acids were identified with tyrosine (approximately 77 wt.%) and phenylalanine (approximately 4 wt.%) being the most prevalent amino acids. Elemental analysis with ICP-OES revealed that inorganic components were accounted for less than one weight percentage of the solid precipitate with metal sulfates being the predominant inorganic components.     

Development of a serum-free culture medium for the large scale production of recombinant protein from a Chinese hamster ovary cell line

A serum-free medium, WCM5, has been developed for the large scale propagation of CHO (Chinese hamster ovary) cells which express recombinant protein using dihydrofolate reductase as a selectable marker. WCM5 was prepared by supplementing Iscoves medium without lecithin, albumin or transferrin with a number of components which were shown to benefit growth. WCM5 medium contained 5 mg l^–1 human recombinant insulin (Nucellin) but was otherwise protein-free. CHO 3D11^* cells which had been engineered to express a humanised antibody, CAMPATH^*-1H, were routinely grown using serum-containing medium. From a seeding density of 10⁵ cells ml^–1, cells grown in static culture with serum reached a maximal cell density of 6.5×10⁵ cells ml^–1 after 6 days in culture and produced a maximal antibody concentration of 69 mg l^–1 after 11 days in culture. CHO 3D11^* cells grown with serum were washed in serum-free medium then cultured in WCM5 medium. Following a period of adaptation the cell growth and product yield was superior to that achieved with serum-containing medium. CHO cells producing CAMPATH-1H grown in an 8000 l stirred bioreactor seeded with 2×10⁵ cells ml^–1 reached a maximal viable cell density of 2.16×10⁶ cells ml^–1 after 108 h in culture and a maximal antibody concentration of 131.1 mg l^–1 after 122 h in culture.Abbreviations CHO Chinese hamster ovary - dhfr dihydrofolate reductase - dhfr dihydrofolate reductase deficient - MTX methotrexate - H hypoxanthine - T thymidine - T/V trypsin versene - F12 Hams F12 medium - NEAA non essential amino acids     

Hyperosmolarity enhances transient recombinant protein yield in Chinese hamster ovary cells

The effect of hyperosmolarity on transient recombinant protein production in Chinese hamster ovary (CHO) cells was investigated. Addition of 90 mM NaCl to the production medium ProCHO5 increased the volumetric yield of recombinant antibody up to 4-fold relative to transfection in ProCHO5 alone. Volumetric yields up to 50 mg l⁻¹ were achieved in a 6 day batch culture of 3 l. In addition, hyperosmolarity reduced cell growth and increased cell size. The addition of salt to cultures of transiently transfected CHO cells is a simple and cost-effective method to increase TGE yields in this host.     

Glucose-limited chemostat culture of Chinese hamster ovary cells producing recombinant human interferon-gamma

A Chinese hamster ovary (CHO) cell line expressing recombinant human interferon-gamma (IFN-gamma) was grown under glucose limitation in a chemostate at a constant dilution rate of 0.015 h(-1) with glucose feed concentrations of 2.75 mM and 4.25 mM. The changes in cell concentration that accompanied changes in the glucose feed concentration indicated that the cells were glucose-limited. The cell yield on glucose remained constant, but there was a decline in residual glucose concentration and a reduced lactate yield from glucose in the latter stages of the culture. The consumption rates for many of the essential amino acids were increased later in the culture. The volumetric rate of interferon-gamma production was maintained throughout the course of this culture, indicating that IFN-gamma expression was stable under these conditions. However, the specific rate of IFN-gamma production was significantly lower at the higher glucose feed concentration. Under glucose limitation, the proportion of fully glycosylated IFN-gamma produced by these cells was less than that produced in the early stages of batch cultures. The proportion of fully glycosylated IFN-gamma increased during transient periods of glucose excess, suggesting that the culture environment influences the glycosylation of IFN-gamma.     

High-density culture of recombinant Chinese hamster ovary cells producing prothrombin in protein-free medium

A recombinant CHO cell line, CHO2DS, was immobilized on porous microcarrier Cytopore 1 and cultivated in 1 l modified Super-spinner and 2 l stirred tank bioreactor with the perfusion of a low-cost chemically defined protein-free medium DF6S. CHO2DS cells could enter into the inner space and grew both in the inner space and on the surface of Cytopore 1 in DF6S and produced prothrombin at 22 mg l^–1 after 10 days. From a seeding density of 5.7 × 10⁵ cells ml^–1, the highest viable cell density of CHO2DS was 1.12 × 10⁷ cells ml^–1.     

Microcarrier culture of recombinant Chinese hamster ovary cells for production of human immune interferon and human tissue-type plasminogen activator

Summary Recombinant Chinese hamster ovary cells were successfully cultured semi-continuously on microcarriers of gelatin or modified dextran under non-selective conditions for up to three weeks. High and constant production rates for human immune interferon and tissue-type plasminogen activator were obtained. For cells that produced interferon, the highest cell concentration and interferon production was obtained with gelatin microcarriers though the specific production when grown in the presence of 0.2% fetal calf serum was slightly higher for cells cultured on dextran microcarriers (0.12 U/cell day versus 0.11 U/cell day). For cells that produced plasminogen activator, a slightly higher cell concentration was obtained for cells grown on dextran microcarriers (9x10⁵ cells/ml versus 7x10⁵ cells/ml). However, the specific and total production rates were significantly higher for cells cultured on gelatin microcarriers (6.7 pg/cell day versus 2.1 pg/cell day). The maximum cell concentration and specific production rate could be increased to 2.3x10⁶ cells/ml and 3.4 pg/cell day for dextran microcarriers by adding 6-aminohexanoic acid to the medium. For gelatin microcarriers, the addition of 6-aminohexanoic acid increased the specific production rate to 14.4 pg/cell day. Cell growth, however, was inhibited.     

Hyperosmotic stress induces autophagy and apoptosis in recombinant Chinese hamster ovary cell culture

During recombinant Chinese hamster ovary (rCHO) cell culture, various events, such as feeding with concentrated nutrient solutions or the addition of base to maintain an optimal pH, increase the osmolality of the medium. To determine the effect of hyperosmotic stress on two types of programmed cell death (PCD), apoptosis and autophagy, of rCHO cells, two rCHO cell lines, producing antibody and erythropoietin, were subjected to hyperosmotic stress resulting from NaCl addition (310–610 mOsm/kg). For both rCHO cell lines, hyperosmolality up to 610 mOsm/kg increased cleaved forms of PARP, caspase‐3, caspase‐7, and fragmentation of chromosomal DNA, confirming the previous observation that apoptosis was induced by hyperosmotic stress. Concurrently, hyperosmolality increased the level of accumulation of LC3‐II, a widely used autophagic marker, which was determined by Western blot analysis and confocal microscopy. When glucose and glutamine concentrations were measured during the cultures, glucose and glutamine concentrations in the culture medium at various osmolalities (310–610 mOsm/kg) showed no significant differences. This result suggests that induction of PCD by hyperosmotic stress occurred independently of nutrient depletion. Taken together, autophagy as well as apoptosis was observed in rCHO cells subjected to hyperosmolality. Biotechnol. Bioeng. 2010;105: 1187–1192. © 2009 Wiley Periodicals, Inc.     

Promoter methylation and transgene copy numbers predict unstable protein production in recombinant Chinese hamster ovary cell lines

Mammalian cell lines for recombinant protein production need to maintain productivity over extended cultivation times. Long-term stability studies are time and resource intensive, but are widely performed to identify and eliminate unstable candidates during cell line development. Production instability of manufacturing cell lines can be associated with methylation and silencing of the heterologous promoter. We have identified CpG dinucleotides within the human cytomegalovirus major immediate early promoter/enhancer (hCMV-MIE) that are frequently methylated in unstable antibody-producing Chinese hamster ovary (CHO) cell lines. We have established methylation-specific real-time qPCR for the rapid and sensitive measurement of hCMV-MIE methylation in multiple cell lines and provide evidence that hCMV-MIE methylation and transgene copy numbers can be used as early markers to predict production instability of recombinant CHO cell lines. These markers should provide the opportunity to enrich stable producers early in cell line development and allow developers to put more emphasis on other criteria, such as product quality and bioprocess robustness.     

Evaluation of a novel pneumatic bioreactor system for culture of recombinant Chinese hamster ovary cells

Single use culture systems are a tool in research and biotechnology manufacturing processes and are employed in mammalian cell-based manufacturing processes. Recently, we characterized a novel bioreactor system developed by PBS Biotech. The Pneumatic Bioreactor System? (PBS) employs the Air-wheel?, which is a mixing device similar in structure to a water wheel but is driven by the buoyant force of gas bubbles. In this study, we investigated the physical properties of the PBS system, with which we performed biological tests. In 2 L PBS, the mixing times ranged from 6 (30 rpm, 0.175 vvm) to 15 sec (10 rpm, 0.025 vvm). The k_La value reached upto 7.66/h at 0.5 vvm, even without a microsparger, though this condition is not applicable for cell cultures. Also, when a 10 L PBS equipped with a microsparger was evaluated, a k_La value of upto approximately 20/h was obtained particularly in mild cell culture conditions. We performed cultivation of Chinese hamster ovary (CHO) cells in 2 and 10 L PBS prototypes. Results from the PBS were compared with those from an Erlenmeyer flask and conventional stirred tank type bioreactor (STR). The maximum cell density of 10.6 × 106 cells/mL obtained fromthe 2 L PBSwas about 2 times higher than that from the Erlenmeyer flask (5.6 × 10⁶ cells/mL) andwas similar to the STR (9.7 × 10⁶ cells/mL) when the CHO-S cells were cultured. These results support the general suitability of the PBS system using pneumatic mixing for suspension cell cultivation as a novel single-use bioreactor system.     

Application of a statistical design to the optimization of culture medium for recombinant interferon-gamma production by Chinese hamster ovary cells

Summary The importance of serum-free medium components on the growth of Chinese hamster ovary (CHO) cells and production of recombinant human interferon(IFN)-gamma was investigated. The complexity of the medium led to the adoption of a statistical optimization approach based on a Plackett-Burman design. From this analysis a set of nutritional components was identified as important for cell growth and recombinant protein production. Glycine was identified as an important determinant of specific growth rate, whereas for cell production bovine serum albumin (BSA), phenylalanine and tyrosine were also identified as important. BSA, sodium pyruvate, glutamate, methionine, proline, histidine, hydroxyproline, tyrosine and phenylalanine were shown to be important for IFN-gamma production. Other medium components, such as insulin, arginine, aspartate and serine produced an inhibitory effect on both cell growth and IFN-gamma production. The effect of the stimulatory nutrients as a whole group was tested by increasing their concentration in the medium. A significant improvement in specific cell growth rate, cell production and IFN-gamma production (up to 45%) was achieved on both shake-flask and fermentor cultures. An increase in the medium concentration of the negative variables had only a small inhibitory effect (approximately 10%) on the same parameters. Analysis of the effects of the group of stimulatory amino acids and BSA on CHO cell growth showed that the effect of the former was independent of BSA.Correspondence to: A. T. Bull     

Site- and branch-specific sialylation of recombinant human interferon-gamma in Chinese hamster ovary cell culture

Since sialic acid content is known to be a critical determinant of the biological properties of glycoproteins, it is essential to characterize and monitor sialylation patterns of recombinant glycoproteins intended for therapeutic use. This study reports site- and branch-specific differences in sialylation of human interferon-gamma (IFN-gamma) derived from Chinese hamster ovary (CHO) cell culture. Sialylation profiles were quantitated by reversed-phase HPLC separations of the site-specific pools of tryptic glycopeptides representing IFN-gamma's two potential N-linked glycosylation sites (i.e., Asn(25) and Asn(97)). Although sialylation at each glycosylation site was found to be incomplete, glycans of Asn(25) were more heavily sialylated than those of Asn(97). Furthermore, Man(alpha1-3) arms of the predominant complex biantennary structures were more favorably sialylated than Man(alpha1-6) branches at each glycosylation site. When the sialylation profile was analyzed throughout a suspension batch culture, sialic acid content at each site and branch was found to be relatively constant until a steady decrease in sialylation was observed coincident with loss of cell viability. The introduction of a competitive inhibitor of sialidase into the culture supernatant prevented the loss of sialic acid after the onset of cell death but did not affect sialylation prior to cell death. This finding indicated that incomplete sialylation prior to loss of cell viability could be attributed to incomplete intracellular sialylation while the reduction in sialylation following loss of cell viability was due to extracellular sialidase activity resulting from cell lysis. Thus, both intracellular and extracellular processes defined the sialic acid content of the final product. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 390-398, 1977.     

Metabolic effects on recombinant interferon-gamma glycosylation in continuous culture of Chinese hamster ovary cells

Asparagine linked (N-linked) glycosylation is an important modification of recombinant proteins, because the attached oligosaccharide chains can significantly alter protein properties. Potential glycosylation sites are not always occupied with oligosaccharide, and site occupancy can change with the culture environment. To investigate the relationship between metabolism and glycosylation site occupancy, we studied the glycosylation of recombinant human interferon-gamma (IFN-gamma) produced in continuous culture of Chinese hamster ovary cells. Intracellular nucleotide sugar levels and IFN-gamma glycosylation were measured at different steady states which were characterized by central carbon metabolic fluxes estimated by material balances and extracellular metabolite rate measurements. Although site occupancy varied over a rather narrow range, we found that differences correlated with the intracellular pool of UDP-N-acetylglucosamine + UDP-N-acetylgalactosamine (UDP-GNAc). Measured nucleotide levels and estimates of central carbon metabolic fluxes point to UTP depletion as the cause of decreased UDP-GNAc during glucose limitation. Glucose limited cells preferentially utilized available carbon for energy production, causing reduced nucleotide biosynthesis. Lower nucleoside triphosphate pools in turn led to lower nucleotide sugar pools and reduced glycosylation site occupancy. Subsequent experiments in batch and fed-batch culture have confirmed that UDP-sugar concentrations are correlated with UTP levels in the absence of glutamine limitation. Glutamine limitation appears to influence glycosylation by reducing amino sugar formation and hence UDP-GNAc concentration. The influence of nucleotide sugars on site occupancy may only be important during periods of extreme starvation, since relatively large changes in nucleotide sugar pools led to only minor changes in glycosylation.     

CHO工程细胞 (11G-S) 悬浮培养的无血清培养基的设计

以悬浮适应的表达重组尿激酶原 (Pro-urokinase,pro-UK) CHO工程细胞系11G-S为对象,采用Plackett-Burman实验设计及响应面分析法,设计支持CHO工程细胞 (11G-S) 悬浮生长的无血清培养基。以细胞密度为评价指标,在单因素实验的基础上采用Plackett-Burman实验设计对影响细胞生长的培养基添加成分进行考察,确定了3种对细胞生长明显促进作用的培养基添加成分：胰岛素、转铁蛋白及腐胺。继而利用响应面法分析了这3种添加成分的最佳水平范围,设计了一种适用于CHO工程细胞 (11G-S) 悬浮培养的无血清培养基SFM-CHO-S。11G-S细胞在SFM-CHO-S批次悬浮培养的细胞最大生长密度达到4.12×106 cells/mL,pro-UK的最大累积活性达到5 614 IU/mL,培养效果优于商品化的同类无血清培养基。