Investigation of relationship between EBNA-1 expression level and specific foreign protein productivity in transient gene expression of HEK293 cells

In an attempt to determine the relationship between the Epstein–Barr virus nuclear antigen-1 (EBNA-1) expression level and specific foreign protein productivity (q_p), EBNA-1-amplifed HEK293 cells, which achieved a higher EBNA-1 expression level than that achieved by HEK293E cells, were established using dihydrofolate reductase (dhfr)-mediated gene amplification. Compared with a control culture in a null pool, Fc-fusion protein production by transient transfection in the EBNA-1-amplified pool showed a significant improvement. q_p was linearly correlated with the EBNA-1 expression level in the transient transfection of EBNA-1-amplified clones, as indicated by the correlation coefficient (R² = 0.7407). The Fc-fusion protein production and q_p in a transient gene expression-based culture with EBNA-1-amplified HEK293 cells, E-amp-68, were approximately 2.0 and 3.2 times, respectively, higher than those in a culture with HEK293E cells. The increase in q_p by EBNA-1 amplification mainly resulted from an enhancement in the amount of replicated DNA and level of mRNA expression but not an improved transfection efficiency. Taken together, it was found that EBNA-1 amplification could improve the therapeutic protein production in an HEK293 cell-based transient gene expression system.     

Generating stable chinese hamster ovary cell clones to produce a truncated SARS‐CoV spike protein for vaccine development

The spike (S) protein of the severe acute respiratory syndrome coronavirus (SARS‐CoV) is important for vaccine development. S_TR2 (an 88 kDa truncated SARS‐CoV TW1 S protein carrying the S fragments S‐74‐253, S‐294‐739, and S‐1129‐1255) is capable of expressing a major form of glycoprotein as endo H‐sensitive (～115 kDa) in CHO cells. To establish stable expressing cell clones, we transfected CHO/dhFr‐cells with the amplifiable vectors ISID (IRES‐driven dhfr) and ISIZ (SV40‐driven dhfr) to select stepwise MTX, and observed enhanced ～115 kDa glycoform generation through gene amplification. Following stepwise MTX selection, we compared gene amplification levels between two vectors in engineered CHO cell chromosomes. These results confirm that the IRES‐driven dhfr promoter generates greater gene amplification, which in turn enhances S_TR2 expression. Our results indicate that the ～115 kDa glycoform of S_TR2 protein was capable of increasing after gene amplification. The S_TR2 glycoform did not change between suspension and serum‐free cultures, suggesting that the stable and amplified cell clones analyzed in this study have potential for producing homologous S_TR2 on a large scale. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

LC‐MS‐based metabolic characterization of high monoclonal antibody‐producing Chinese hamster ovary cells

The selection of suitable mammalian cell lines with high specific productivities is a crucial aspect of large‐scale recombinant protein production. This study utilizes a metabolomics approach to elucidate the key characteristics of Chinese hamster ovary (CHO) cells with high monoclonal antibody productivities (q_mAb). Liquid chromatography‐mass spectrometry (LC‐MS)‐based intracellular metabolite profiles of eight single cell clones with high and low q_mAb were obtained at the mid‐exponential phase during shake flask batch cultures. Orthogonal projection to latent structures discriminant analysis (OPLS‐DA) subsequently revealed key differences between the high and low q_mAb clones, as indicated by the variable importance for projection (VIP) scores. The mass peaks were further examined for their potential association with q_mAb across all clones using Pearson's correlation analysis. Lastly, the identities of metabolites with high VIP and correlation scores were confirmed by comparison with standards through LC‐MS‐MS. A total of seven metabolites were identified—NADH, FAD, reduced and oxidized glutathione, and three activated sugar precursors. These metabolites are involved in key cellular pathways of citric acid cycle, oxidative phosphorylation, glutathione metabolism, and protein glycosylation. To our knowledge, this is the first study to identify metabolites that are associated closely with q_mAb. The results suggest that the high producers had elevated levels of specific metabolites to better regulate their redox status. This is likely to facilitate the generation of energy and activated sugar precursors to meet the demands of producing more glycosylated recombinant monoclonal antibodies. Biotechnol. Bioeng. 2012; 109: 3103–3111. © 2012 Wiley Periodicals, Inc.     

Plasmid amplification-promoting sequences from the origin region of Chinese hamster dihydrofolate reductase gene do not promote position-independent chromosomal gene amplification

Initiation of DNA synthesis occurs with high frequency at oriß, a region of DNA from the amplified dihydrofolate reductase (DHFR) domain of Chinese hamster CHOC 400 cells that contains an origin of bidirectional DNA replication (OBR). Recently, sequences from DHFR oriß/OBR were shown to stimulate amplification of cis-linked plasmid DNA when transfected into murine cells. To test the role of oriß/OBR in chromosomal gene amplification, linearized plasmids containing these sequences linked to a DHFR expression cassette were introduced into DHFR- CHO DUKX cells. After selection for expression of DHFR, cell lines that contain a single integrated, unrearranged copy of the linearized expression plasmid were identified and exposed to low levels of the folate analog, methotrexate (MTX). Of seven clonal cell lines containing the vector control, three gained resistance to MTX by 5 to 15-fold amplification of the integrated marker gene. Of 16 clonal cell lines that contained oriß/OBR linked to a DHFR mini-gene, only 6 gained resistance to MTX by gene amplification. Hence, sequences from the DHFR origin region that stimulate plasmid DNA amplification do not promote amplification of an integrated marker gene in all chromosomal contexts. In addition to showing that chromosomal position has a strong influence on the frequency of gene amplification, these studies suggest that the mechanism that mediates the experiment of episomal plasmid DNA does not contribute to the early steps of chromosomal gene amplification.     

BiP Inducer X: An ER Stress Inhibitor for Enhancing Recombinant Antibody Production in CHO Cell Culture

Prolonged endoplasmic reticulum (ER) stress reduces protein synthesis and induces apoptosis in mammalian cells. When dimethyl sulfoxide (DMSO), a specific monoclonal antibody productivity (q_mAb)‐enhancing reagent, is added to recombinant Chinese hamster ovary (rCHO) cell cultures (GSR cell line), it induces ER stress and apoptosis in a dose‐dependent manner. To determine an effective ER stress inhibitor, three ER stress inhibitors (BiP inducer X [BIX], tauroursodeoxycholic acid, and carbazole) are examined and BIX shows the best production performance. Coaddition of BIX (50 μm ) with DMSO extends the culture longevity and enhances q_mAb. As a result, the maximum mAb concentration is significantly increased with improved galactosylation. Coaddition of BIX significantly increases the expression level of binding immunoglobulin protein (BiP) followed by increased expression of chaperones (calnexin and GRP94) and galactosyltransferase. Furthermore, the expression levels of CHOP, a well‐known ER stress marker, and cleaved caspase‐3 are significantly reduced, suggesting that BIX addition reduces ER stress‐induced cell death by relieving ER stress. The beneficial effect of BIX on mAb production is also demonstrated with another q_mAb‐enhancing reagent (sodium butyrate) and a different rCHO cell line (CS13‐1.00). Taken together, BIX is an effective ER stress inhibitor that can be used to increase mAb production in rCHO cells.     

Combinatorial treatment with lithium chloride enhances recombinant antibody production in transiently transfected CHO and HEK293E cells

Lithium chloride (LiCl), which induces cell cycle arrest at G2/M phase, is known as a specific production rate (q _p)-enhancing additive in recombinant Chinese hamster ovary (CHO) cell culture. To determine the potential of LiCl as a chemical additive that enhances transient gene expression (TGE), LiCl was added to the CHO-NK and human embryonic kidney 293E (HEK293E) cell cultures before and/or after transfection with polyethylenimine as a transfection reagent. The effect of this addition on transfection efficiency (pre-treatment) and q _p enhancement during TGE (post-treatment) was examined. For the TGE of monoclonal antibody (mAb) in CHO-NK cells, pretreatment alone with 10 mM LiCl and post-treatment alone with 5 mM LiCl resulted in 1.2- and 3.4-fold increase of maximum mAb concentration (MMC), respectively, compared with the TGE without LiCl treatment. Furthermore, combinatorial treatment with LiCl (10 mM for pre-treatment and 5 mM for post-treatment) synergistically increased the TGE of mAb (5.3-fold increase in MMC). Likewise, combinatorial treatment with LiCl (10 mM for pre-treatment and 15 mM for post-treatment) in HEK293E cells synergistically increased the TGE of mAb (4.9-fold increase in MMC). Taken together, the data obtained here demonstrate that combinatorial treatment with LiCl is a useful means to improve TGE in CHO as well as HEK293 cells.     

Effect of amplification ofdhfr andlac Z genes on growth and β-galactosidase expression in suspension cultures of recombinant CHO cells

Studies were conducted to characterize the effect of gene amplification and foreign gene expression on recombinant CHO cell growth. Chinese hamster ovary (CHO) cells were transfected with an expression vector containing the gene for dihydrofolate reductase (dhfr) and the gene for human β-interferon (β-IFN) or thelac Z gene which codes for β-galactosidase (β-gal). The recombinant genes in these CHO cells were amplified stepwise by growth in 0, 10⁻⁷, and 10⁻⁶ M methotrexate (MTX), and the β-gal expressing cells were adapted to suspension culture. Flow cytometric methods (FCM) were used to measure the distribution of amplifieddhfr gene content and foreign β-gal gene expression in the cell populations. A biochemical assay for β-gal was also used. Beta-gal expression was found to increase with increasing gene amplification. The growth rate of recombinant CHO cells at 10⁻⁷ M MTX was found to be 20% lower than that of recombinant CHO cells in MTX-free medium, and the cell growth rate at 10⁻⁶ M MTX was 20% lower than that of recombinant CHO cells at 10⁻⁷ M MTX. There was no effect of 10⁻⁵ M MTX on the growth of CHO-DG44 (dhfr-) cells. The reduction of growth rate in recombinant CHO cells is therefore thought to be mainly due to the effect ofdhfr and foreign gene amplification and increased β-galactosidase expression.     

Valeric acid induces cell cycle arrest at G1 phase in CHO cell cultures and improves recombinant antibody productivity

To find a more effective chemical reagent for improved monoclonal antibody (mAb) production, eight chemical reagents (curcumin, quercein, DL‐sulforaphane, thymidine, valeric acid, phenyl butyrate, valproic acid, and lithium chloride) known to induce cell cycle arrest were examined individually as chemical additives to recombinant CHO (rCHO) cell cultures producing mAb. Among these chemical additives, valeric acid showed the best production performance. Valeric acid decreased specific growth rate (μ), but increased culture longevity and specific mAb productivity (q_mAb) in a dose‐dependent manner. The beneficial effect of valeric acid on culture longevity and q_mAb outweighed its detrimental effect on μ, resulting in 2.9‐fold increase in the maximum mAb concentration when 1.5 mM valeric acid was added to the cultures. Furthermore, valeric acid did not negatively affect the mAb quality attributes with regard to aggregation, charge variation, and galactosylation. Unexpectedly, galactosylation of the mAb increased by the 1.5 mM valeric acid addition. Taken together, the results obtained here demonstrate that valeric acid is an effective chemical reagent to increase mAb production in rCHO cells.     

Relationship between cell size and specific thrombopoietin productivity in chinese hamster ovary cells during dihydrofolate reductase-mediated gene amplification

When parental Chinese hamster ovary (CHO) cell clones that are capable of producing thrombopoietin (TPO) were subjected to high methotrexate (MTX) concentrations, clonal variations in cell growth were apparent. In the clones that had no significant enhancement in specific TPO productivity (q _Tpo) when a higher level of MTX was administered, their growth was not depressed significantly nor their cell size changed significantly. On the other hand, those clones that showed a significant enhancement inq _Tpo at higher a MTX dosage, cell growth was depressed initially but recovered during successive sub-cultures. Furthermore, their cell size increased, which suggested that changes in cell size may be indicative of an enhancedq _Tpo. When the enhancement of theq _Tpo of 9 clones after a high MTX dosage was plotted against the extent of the increase of their size, there was a linear correlation (r ²=0.80,P<0.001, ANOVA), which suggested that an enhancement ofq _Tpo after high MTX administration can be measured by the increase in their cell size. Taken together, our data demonstrate that the selection of amplified CHO cell clones with enhancedq _Tpo can be done based upon their increased size and growth pattern. This facilitates the development of highly productive recombinant CHO cell lines.     

Correlation of antibody production rate with glucose and lactate metabolism in Chinese hamster ovary cells

A linear relationship was found between the antibody production rate (q _mAb) and the glucose and lactate consumption rate (q _GL) in Chinese hamster ovary cells. Under a series of q _mAb-perturbing conditions, q _GL was determined and a linear relationship between q _mAb and q _GL was further established (R ²  = 0.914). Mitochondrial dehydrogenase activity was monitored in all the q _mAb-perturbing conditions and showed a linear correlation with q _GL (R ² = 0.874) as well as with q _mAb. Taken collectively, our results establish that the metabolic parameter, q _GL, is linearly correlated with q _mAb; this finding strengthens our current understanding of process optimization for antibody production.     

Selection for methotrexate resistance in mammalian cells bearing a <Emphasis Type="Italic">Drosophila</Emphasis> dihydrofolate reductase transgene

Antifolates, such as methotrexate (MTX), are the treatment of choice for numerous cancers. MTX inhibits dihydrofolate reductase (DHFR), which is essential for cell growth and proliferation. Mammalian cells can acquire resistance to antifolate treatment through a variety of mechanisms but decreased antifolate titers due to changes in drug efflux or influx, or alternatively, the amplification of the DHFR gene are the most commonly acquired resistance mechanisms. In Drosophila, however, a resistant phenotype has only been observed to occur by mutation resulting in a MTX-resistant DHFR. It is unclear if differences in gene structure and/or genome organization between Drosophila and mammals contribute to the observed differences in acquired drug resistance. To investigate if gene structure is involved, Drosophila Dhfr cDNA was transfected into a line of CHO cells that do not express endogenous DHFR. These transgenic cells, together with wild-type CHO cells, were selected for 19 months for resistance to increasing concentrations of MTX, from 50- to 200-fold over the initial concentration. Since Drosophila Dhfr appears to have been amplified several fold in the selected transgenic mammalian cells, a difference in genome organization may contribute to the mechanism of MTX resistance.     

Similarities between human ataxia fibroblasts and murine SCID cells: High sensitivity to γ rays and high frequency of methotrexate-induced DHFR gene amplification,but normal radiosensitivity to densely ionizing α particles

Two-ray hypersensitive cell lines, human ataxia telangiectasia (AT) and murine severe combined immune deficiency (SCID) cells, proved to be very competent in amplifying their dihydrofolate reductase (DHFR) gene under methotrexate selection stress. Over a period of months, methotrexate-resistant clones were obtained which were able to grow in progressively increasing methotrexate concentrations up to 1 mM. By then methotrexate-resistant AT and SCID cells had amplified their DHFR gene 6- and 30-fold, respectively, and showed very high DHFR mRNA expression. In contrast, related cells with normal radiosensitivity (human GM637 and mouse BALB/c fibroblasts) did not show DHFR gene amplification under comparable conditions. This correlation of the capacity of DHFR gene amplification and-ray hypersensitivity in AT and SCID cells suggests that gene amplification may have a mechanism(s) in common with those involved in repair of-radiation-induced damage. No difference in cell killing could be observed following exposure to densely ionizing particles: AT and SCID cells exhibited comparable survival rates to GM637 and BALB/c cells, respectively.     

Isolation of methotrexate-resistant cell lines in Petunia hybrida upon stepwise selection procedure

Summary Cell suspensions of Petunia hybrida were subjected to a selection procedure in which the concentration of the selective agent, methotrexate (MTX), was gradually elevated. In mammalian cells, this procedure frequently results in MTX-resistant mutants due to amplification of the gene coding for dihydrofolate reductase (DHFR), the target protein of MTX.Five suspension lines were isolated, with degrees of resistance ranging from 10 to 500 M MTX (in wild type the LD_99.9 is 0.2 M). MTX^R phenotypes were unstable, as manifested by the loss of resistance upon prolonged growth in the absence of drug. All of the mutants also exhibited high values of MTX-binding protein (60- to 400-fold higher than that of the wild type), which declined to intermediate values upon MTX withdrawal. Finally, cellular extracts from all of the mutants also showed high specific staining of DHFR-activity in gels.The results suggest that the resistance of MTX in these plant cell-lines is mediated by the elevation of the amounts of DHFR, probably as a consequence of gene amplification.     

A mouse hybrid cell line that supports gene expression from a variety of promoters in amplifiable vectors

Summary A hybrid cell line was constructed by fusion of mouse L-cells with an NIH3T3 cell line derivative containing a hybrid gene consisting of the mouse immunoglobulin kappa (IgK) variable gene promoter linked to theEscherichia coli gpt gene. Such hybrids grew to a much higher density compared to either of the parental cell lines. The utility of this cell line as a host to express foreign genes was tested by the expression of TGF-β cDNA using the cytomegalovirus promoter. The vector also contained the human dihydrofolate reductase (DHFR) gene driven by SV40 early promoter, to allow for the amplification of the transfected gene. Initial transformants, selected at 100 nM methotrexate (MTX), were subsequently selected for resistance to a higher concentration of MTX (2 μM). Such clones expressed an increased level of TGF-β when compared to the initial transformants. Both the initial transformants and the clones with the amplified DHFR gene produced TGF-β in an acid-activatable precursor form. This mouse hybrid host cell line also allowed the expression of foreign genes cloned in an eukaryotic expression vector with the mouse IgK variable region promoter and human growth hormone as the reporter gene, whereas such vectors did not function in CHO cells. The mouse hybrid cell line was also found to be capable of being used with a broad range of promoters.     

Stable, recombinant expression of human insulin-like growth factor binding protein-1 (hIGFBP-1) in Chinese hamster ovary (CHO) cells

Stable expression of human insulin-like growth factor of binding protein-1 (hIGFBP-1)at high levels has been achieved in Chinese hamster ovary (CHO) cells by co-transfection and subsequent co-amplification of expression vectors containing the hIGFBP-1 cDNA and a dihydrofolate reductase (DHFR) cDNA gene into DHFR-deficient cells. Stepwise selection of the DHFR⁺ transformants in increasing concentrations of methotrexate (MTX) generated cells which had high copy numbers of the hIGFBP-1 gene (around 100 copies in cells amplified in medium containing 100 nM MTX). Expression of hIGFBP-1 in mixed clones was found to increase with increasing copy number and an apparent correlation between intra- and extracellular levels of hIGFBP-1 produced by these cells was observed. It was further observed that continuous cultivation over eight months in medium supplemented with 100 nM MTX increased the production of hIGFBP-1 25 times. The productivity did not increase further after five more months cultivation in MTX containing medium. A subcloning of this cell line gave clones with an even higher productivity. Further amplification in 500 nM or 1 uM MTX did not increase the hIGFBP-1 production. This revised version was published online in July 2006 with corrections to the Cover Date.     

Expression of heterologous oxalate decarboxylase in HEK293 cells confers protection against oxalate induced oxidative stress as a therapeutic approach for calcium oxalate stone disease

Oxalates stimulate alterations in renal epithelial cells and thereby induce calcium oxalate (CaOx) stone formation. Bacillus subtilis YvrK gene encodes for oxalate decarboxylase (OxdC) which degrades oxalate to formate and CO₂. The present work is aimed to clone the oxdC gene in a mammalian expression vector pcDNA and transfect into Human Embryonic Kidney 293 (HEK293) cells and evaluate the oxdC expression, cell survival rate and oxalate degrading efficiency. The results indicate cell survival rate of HEK293/pcDNAOXDC cells pre-incubated with oxalate was enhanced by 28%. HEK293/pcDNAOXDC cells expressing OxdC treated with oxalate, significantly restored antioxidant activity, mitochondrial membrane potential and intracellular reactive oxygen species (ROS) generation compared with HEK293/pcDNA. Apoptotic marker caspase 3 downregulation illustrates HEK293/pcDNAOXDC cells were able to survive under oxalate-mediated oxidative stress. The findings suggest HEK293 cells expressing oxdC capable of degrading oxalate protect cells from oxidative damage and thus serve as a therapeutic option for prevention of CaOx stone disease.