Scalable transient gene expression in Chinese hamster ovary cells in instrumented and non-instrumented cultivation systems

Cell expansion, gene transfer and protein production were all executed with a single serum-free, animal protein-free commercial medium designed for suspension-adapted Chinese hamster ovary cells (CHO DG44). This is a most important process to consider for clinical production of recombinant proteins. The transfection with polyethylenimine (PEI) was shown here to be scalable using both stirred-tank bioreactors of 3- and 150-l and novel agitated cultivation vessels (50 ml ventilated centrifuge tubes and 1-l square-shaped glass bottles) that lack any instrumentation. The transient transfections spanned a range of working volumes from 2 ml to 80 l. The maximum transient recombinant antibody yield was 22 mg/l, the highest ever reported for a multiliter transfection in CHO. The transiently expressed protein had the same extent of glycosylation as the same antibody produced from a stably transfected recombinant CHO cell line.     

Butyrated ManNAc analog improves protein expression in Chinese hamster ovary cells

The chemical additive sodium butyrate (NaBu) has been applied in cell culture media as a direct and convenient method to increase the protein expression in Chinese hamster ovary (CHO) and other mammalian cells. In this study, we examined an alternative chemical additive, 1,3,4‐O‐Bu₃ManNAc, for its effect on recombinant protein production in CHO. Supplementation with 1,3,4‐O‐Bu₃ManNAc for two stable CHO cell lines, expressing human erythropoietin or IgG, enhanced protein expression for both products with negligible impact on cell growth, viability, glucose utilization, and lactate accumulation. In contrast, sodium butyrate treatment resulted in a ～20% decrease in maximal viable cell density and ～30% decrease in cell viability at the end of cell cultures compared to untreated or 1,3,4‐O‐Bu₃ManNAc treated CHO cell lines for both products. While NaBu treatment enhanced product yields more than the 1,3,4‐O‐Bu₃ManNAc treatment, the NaBu treated cells also exhibited higher levels of caspase 3 positive cells using microscopy analysis. Furthermore, the mRNA levels of four cell apoptosis genes (Cul2, BAK, BAX, and BCL2L11) were up‐regulated more in sodium butyrate treated wild‐type, erythropoietin, or IgG expressing CHO‐K1 cell lines while most of the mRNA levels of apoptosis genes in 1,3,4‐O‐Bu₃ManNAc treated cell lines remained equal or increased only slightly compared to the levels in untreated CHO cell lines. Finally, lectin blot analysis revealed that the 1,3,4‐O‐Bu₃ManNAc‐treated cells displayed higher relative sialylation levels on recombinant EPO, consistent with the effect of the ManNAc component of this additive, compared to control while NaBu treatment led to lower sialylation levels than control, or 1,3,4‐O‐Bu₃ManNAc‐treatment. These findings demonstrate that 1,3,4‐O‐Bu₃ManNAc has fewer negative effects on cell cytotoxicity and apoptosis, perhaps as a result of a more deliberate uptake and release of the butyrate compounds, while simultaneously increasing the expression of multiple recombinant proteins, and improving the glycosylation characteristics when applied at comparable molarity levels to NaBu. Thus, 1,3,4‐O‐Bu₃ManNAc represents a highly promising media additive alternative in cell culture for improving protein yields without sacrificing cell mass and product quality in future bioproduction processes.

     

Methylation of human ornithine decarboxylase gene before transfection abolishes its transient expression in Chinese hamster ovary cells

Different methylations of cloned human ornithine decarboxylase gene with restriction methylases in vitro before transfection greatly reduced the transient expression of ODC in Chinese hamster ovary cells. Single methylation of the gene with Hpa II (CCGG) methylase decreased the transiently expressed peak activity by about 50%, single methylation with Hha I (CCGG) methylase by about 80% whilst a double methylation at both Hpa II and Hha I restriction sites virtually abolished any transiently expressed ornithine decarboxylase activity. These results together with our earlier circumventing evidence indicate that the expression of mammalian ornithine decarboxylase is critically influenced by the methylation state of the gene.     

Engineering mRNA translation initiation to enhance transient gene expression in chinese hamster ovary cells

To increase transient expression of recombinant proteins in Chinese hamster ovary cells, we have engineered their protein synthetic capacity by directed manipulation of mRNA translation initiation. To control this process we constructed a nonphosphorylatable Ser(51)Ala site-directed mutant of eIF2alpha, a subunit of the trimeric eIF2 complex that is implicated in regulation of the global rate of mRNA translation initiation in eukaryotic cells. Phosphorylation of eIF2alpha by protein kinases inhibits eIF2 activity and is known to increase as cells perceive a range of stress conditions. Using single- and dual-gene plasmids introduced into CHO cells by electroporation, we found that transient expression of the eIF2alpha Ser(51)Ala mutant with firefly luciferase resulted in a 3-fold increase in reporter activity, relative to cells transfected with reporter only. This effect was maintained in transfected cells for at least 48 h after transfection. Expression of the wild-type eIF2alpha protein had no such effect. Elevated luciferase activity was associated with a reduction in the level of eIF2alpha phosphorylation in cells transfected with the mutant eIF2alpha construct. Transfection of CHO cells with the luciferase-only construct resulted in a marked decrease in the global rate of protein synthesis in the whole cell population 6 h post-transfection. However, expression of the mutant Ser(51)Ala or wild-type eIF2alpha proteins restored the rate of protein synthesis in transfected cells to a level equivalent to or exceeding that of control cells. Associated with this, entry of plasmid DNA into cells during electroporation was visualized by confocal microscopy using a rhodamine-labeled plasmid construct expressing green fluorescent protein. Six hours after transfection, plasmid DNA was present in all cells, albeit to a variable extent. These data suggest that entry of naked DNA into the cell itself functions to inhibit protein synthesis by signaling mechanisms affecting control of mRNA translation by eIF2. This work therefore forms the basis of a rational strategy to generically up-regulate transient expression of recombinant proteins by simultaneous host cell engineering.     

Monitoring gene expression in Chinese hamster ovary cells using secreted apoaequorin.

A luminescence method for monitoring gene expression in Chinese hamster ovary cells using apoaequorin as a secreted reporter enzyme is described. In this method, the cell is not disrupted prior to assay as in the earlier aequorin procedure and in the firefly method. The apoaequorin secretion vector is constructed by fusing the DNA fragment of the signal peptide sequence of human follistatin to the apoaequorin gene. Transfection of Chinese hamster ovary cells with the vector causes the apoaequorin to be secreted directly into the culture medium. Assay is carried out by removing a small aliquot of the culture medium, incubating it with coelenterazine, and adding Ca2+ to trigger light emission from the regenerated aequorin. The light intensity is measured with a photomultiplier photometer and is proportional to the amount of apoaequorin present. The method is highly specific and sensitive and can be carried out in a relatively short period of time.     

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.     

Gadd45-induced cell cycle G2/M arrest for improved transient gene expression in Chinese hamster ovary cells

Gadd45 is a p53-regulated protein and is involved in cell cycle arrest in the G2/M phase. In an effort to improve transient gene expression (TGE) in Chinese hamster ovary (CHO) cells, the effect of Gadd45-induced cell cycle arrest on TGE in CHO cells was investigated using the two different expression vectors encoding Fcfusion protein and recombinant antibody. To regulate the expression of Gadd45 in CHO cells, the CHO-TREx-gadd45 cell line was established using the T-REx system controlled by doxycycline. During the cultures for TGE, Gadd45 overexpression severely inhibited cell growth, but significantly enhanced TGE. Compared with the culture without Gadd45 overexpression, the TGE of Fc fusion protein and humanized antibody were increased by 111 and 93%, respectively. The enhanced TGE, despite the cell growth arrest induced by Gadd45 overexpression, was due to the significantly increased specific productivity, resulting from enhanced transfection efficiency, increased cell size, and active DNA demethylation. Taken together, the data obtained here demonstrate that Gadd45-induced cell cycle arrest in G2/M phase can significantly enhance TGE in CHO cells.     

Insulin-induced gene 33 mRNA expression in Chinese hamster ovary cells is insulin receptor dependent

Gene 33 (g33) is a non-tissue-specific gene regulated in rat liver and hepatoma cells by insulin and other agents. It is thought to participate in the transition from quiescence to proliferation in mitogen-treated cells. The mechanism(s) by which insulin exerts its action on g33 are not totally understood; it is unclear whether a functional insulin receptor is required for this action. In this study, we evaluate the mechanism for insulin induction of g33 mRNA in Chinese hamster ovary (CHO) cells transfected with the neomycin-resistant plasmid (CHONeoB), human insulin receptor (CHONewIRa), and a kinase-defective insulin receptor mutated at the ATP-binding site (CHOK1018A). Transfected cells had higher levels of insulin binding than that of CHONeoB cells; insulin-induced phosphorylation of the insulin receptor and its intracellular substrates were impaired in CHOK1018A cells. Maximal insulin induction of mRNA(g33) occurred 3 h after hormonal exposure in all cell lines. The degree of insulin stimulation of g33 mRNA levels was four- to sixfold higher in CHONewIRa than in CHONeoB or CHOK1018A cells, which had minimal levels of insulin-stimulated g33 mRNA levels. Half-maximal stimulation of g33 mRNA levels was observed at 0.06 +/- 0.01 nM in CHONewIRa cells, consistent with insulin interaction with its own receptor. Wortmannin, an inhibitor of phosphatidyl inositol 3-kinase (PI3K), had some effects on insulin stimulation of g33 mRNA in CHO NewIRa cells. PD98059, an inhibitor of mitogen-activated kinase kinase (MAPKK), and rapamycin, a p70 S6 kinase inhibitor, had minimal effect on insulin stimulation of g33 mRNA in all cells tested. By contrast, hydroxy-2-naphthalenylmethyl)phosphonic acid triacetoxymethyl ester (HNMPA(AM)(3), a selective inhibitor of the insulin receptor tyrosine kinase, caused complete inhibition of insulin stimulation of g33 mRNA levels. These data indicate that the insulin receptor with intact kinase activity is required for insulin stimulation of g33 mRNA levels. They also suggest that AKT, a PI 3-kinase downstream effector molecule, could mediate insulin stimulation of g33 mRNA. The mechanism(s) of insulin regulation of g33 expression downstream of receptor do not seem to rely entirely on the classic insulin receptor transduction pathway, as a minor effect was observed upon inhibition of MAPKK, suggesting that multiple pathways may be involved.     

Evidence of gene amplification in tunicamycin-resistant Chinese hamster ovary cells

Restriction digests of genomic DNA from tunicamycin-resistant Chinese hamster ovary cells, 3E11, were probed with the yeast transferase gene, ALG7. The data presented suggest moderate amplification of the N-acetylglucosaminyl-1-phosphate transferase gene occurred in these cells, consistent with the previously observed chromosomal translocations and increased enzymatic activity. This is the first example of gene amplification as a mechanism for aberrations in N-linked glycosylation.     

rhVEGF165在中国仓鼠卵巢细胞中的稳定表达、纯化及其生物学活性

利用哺乳动物细胞表达系统,稳定表达和纯化高生物学活性的人重组血管内皮生长因子 (VEGF165) 蛋白。将VEGF165克隆于表达载体pCDNA4.0,与T-GS载体共同转染CHO-S (中国仓鼠卵巢细胞) 细胞,MSX (Methionine sulphoximine) 加压筛选高表达细胞株,5 L发酵罐培养,细胞培养上清液通过三步纯化得到rhVEGF165蛋白,通过Western blotting、Biacore和人脐静脉内皮细胞增殖实验等对表达蛋白的特异性、亲和力及生物学活性等进行检测。所建立的细胞     

RNA interference of cofilin in Chinese hamster ovary cells improves recombinant protein productivity

RNA interference (RNAi) has been recently applied to improve the yield and quality of recombinant proteins produced in Chinese hamster ovary (CHO) cells, the most commonly used mammalian cell line for production of complex biopharmaceuticals. Proteomic profiling of CHO cells undergoing gene amplification identified cofilin, a key regulatory protein of actin cytoskeletal dynamics, as a cellular target for genetic engineering studies. Transient reduction of cofilin by small interfering RNA (siRNA) enhanced specific productivity in recombinant CHO cells by up to 80%. CHO cell lines expressing cofilin-specific short hairpin RNA (shRNA) vectors showed up to a 65% increase in specific productivity. These results suggest that modulation of cofilin, and its regulatory pathways, may be a new approach to enhance recombinant protein productivity in CHO cells.     

Change of insulin-like growth factor gene expression in Chinese hamster ovary cells cultured in serum-free media

Although the sera used in animal cell culture media provide the macromolecules, nutrients, hormones, and growth factors necessary to support cell growth, it could also be an obstacle to the production of recombinant proteins in animal cell culture systems used in many sectors of the biotechnology industry. For this reason, many research groups, including our laboratory, have been trying to develop serum-free media (SFM) or serum-supplemented media (SSM) for special or multi-purpose cell lines. The Chinese hamster ovary (CHO) cell, for example, is frequently used to produce proteins and is especially valuable in the large-scale production of pharmaceutically important proteins, yet information about its genome is lacking. Also, SFMs have only been evaluated by comparing growth patterns for cells grown in SFMs with those grown in SSM or by measuring the titer of the target protein obtained from cells grown in each type of medium. These are not reliable methods of obtaining the type of information needed to determine whether an SFM should be replaced with an SSM. We carried out a cDNA microarray analysis to evaluate MED-3, an SFM developed in our laboratory, as a CHO culture medium. When CHO cells were cultured in MED-3 instead of an SSM, several genes associated with cell growth were down-regulated, although this change diminished over time. We found that the insulin-like growth factor (IGF) gene was representative of the proteins that were down-regulated in cells cultured in MED-3. When several key supplements-including insulin, transferrin, ethanolamine, and selenium-were removed from MED-3, theIGF expression was consistently down-regulated and cell growth decreased proportionately. Based on these results, we concluded that when an SFM is used as a culture medium, it is important to supplement it with substances that can help the cells maintain a high level ofIGF expression. The data presented in this study, therefore, might provide useful information for the design and development of SFM or SSM, as well as for the design of genome-based studies of CHO cells to determine how they can be used optimally for protein production in pharmaceutical and biomedical research.     

Kinetics of micronucleus expression in synchronized irradiated Chinese hamster ovary cells

The kinetics of expression of radiation-induced micronuclei (MN) in synchronized Chinese hamster cells (CHO) was examined. The purpose of the study was to determine if the cell cycle distribution of a population significantly influences the levels of radiation induced MN, thereby obscuring the exact quantification of the radiation effect. Cells were synchronized by centrifugal elutriation, irradiated, and then different phases of the cell cycle were examined for: cell cycle progression, division probability, and temporal expression of MN. The results demonstrate that the time interval for maximal MN expression is long enough that the position of cells in the cell cycle and radiation induced division delays do not prevent the majority of cells from completing their first post-irradiation mitosis, therefore, expressing MN. By following the progression of synchronized cell populations by flow cytometry and also examining the time of division of individual cells for 24 hr after irradiation, we observed that the maximum number of cells from all phases of the cell cycle are in their first post-irradiation interphase at that time, thus explaining the MN results.     

Pesticide clastogenicity in Chinese hamster ovary cells

Paraquat, alachlor, butachlor, phorate and monocrotophos, several of the most extensively used pesticides in Taiwan, were investigated for their clastogenicity using chromosome aberration (CAb) induction in Chinese hamster ovary (CHO) cells. Significance levels of the binomial trend analysis and binomial mutagenicity data test were two criteria for the summary judgement of the pesticide clastogenicity. Except for phorate, all pesticides tested were clastogenic to CHO cells in the absence of in vitro metabolic activation by S9. 5 microliters/ml rat-liver extract, S9, were used as the source of in vitro metabolic activation. 3 different outcomes were found after the addition of S9. Paraquat: significant decrease in induced CAbs. Monocrotophos: concomitant occurrence of decreased cytotoxicity and increased clastogenicity. Alachlor, butachlor and phorate: increased cytotoxicities with no sign of enhancement in clastogenicity.