The combined effect of sodium butyrate and low culture temperature on the production,sialylation, and biological activity of an antibody produced in CHO cells

Cell cultures containing 0 ∼ 5 mM sodium butyrate (NaBu) and grown at 30 and 37°C were conducted to investigate the combined effect of NaBu and low temperature on the quantity and quality of an antibody production in CHO cells. Although NaBu addition decreased cell viability by apoptosis in a dose-dependent manner at both 30 and 37°C, the onset of significant apoptosis induced by NaBu was delayed by lowering culture temperature. The highest specific antibody productivity (q _p) of 23.26 pg/cell/day was obtained in the culture containing 2 mM NaBu at 30°C; however, the highest antibody concentration of 167.84 mg/L was achieved in the culture containing 1 mM NaBu at 30°C, as the detrimental effect of further NaBu addition on cell growth compromised its beneficial effect on q _p. Moreover, protein quality with respect to the total sialic acid content and Nglycolylneuraminic acid (Neu5Gc) level was evaluated. There were no apparent changes regarding the total sialic acid content of the antibody, but manipulation of cultures with NaBu treatment or (and) low culture temperature did decrease Neu5Gc levels by 5 ∼ 10%. Biological activity of the antibody was also assessed, and no obvious changes were observed. Collectively, the simultaneous application of NaBu and low culture temperature was an effective way to extend culture period and enhance final antibody concentration, without compromising the sialic acid content or biological activity.     

New cell line development for antibody-producing Chinese hamster ovary cells using split green fluorescent protein

Background

The establishment of high producer is an important issue in Chinese hamster ovary (CHO) cell culture considering increased heterogeneity by the random integration of a transfected foreign gene and the altered position of the integrated gene. Fluorescence-activated cell sorting (FACS)-based cell line development is an efficient strategy for the selection of CHO cells in high therapeutic protein production.

Results

An internal ribosome entry site (IRES) was introduced for using two green fluorescence protein (GFP) fragments as a reporter to both antibody chains, the heavy chain and the light chain. The cells co-transfected with two GFP fragments showed the emission of green fluorescence by the reconstitution of split GFP. The FACS-sorted pool with GFP expression had a higher specific antibody productivity (q _Ab) than that of the unsorted pool. The q _Ab was highly correlated with the fluorescence intensity with a high correlation coefficient, evidenced from the analysis of median GFP and q _Ab in individual selected clones.

Conclusions

This study proved that the fragment complementation for split GFP could be an efficient indication for antibody production on the basis of high correlation of q _Ab with reconstitution of GFP. Taken together, we developed an efficient FACS-based screening method for high antibody-producing CHO cells with the benefits of the split GFP system.     

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 co-down-regulation of caspase-3 and caspase-7 by siRNAs on sodium butyrate-induced apoptotic cell death of Chinese hamster ovary cells producing thrombopoietin

Previously, the expression of caspase-3 siRNA could not effectively inhibit sodium butyrate (NaBu)-induced apoptotic cell death of recombinant Chinese hamster ovary (rCHO) cells producing human thrombopoietin (hTPO). Caspase-3 siRNA expressing cells appeared to compensate for the lack of caspase-3 by increasing active caspase-7 levels. For the successful inhibition of NaBu-induced apoptosis of rCHO cells, both caspase-3 and caspase-7 were down-regulated using the siRNA expression vector system. Co-down-regulation of caspase-3 and caspase-7 increased cell viability and extended culture longevity in serum-free culture in the presence or absence of 1mM NaBu addition. In the cultures with 1mM NaBu addition, the maximum hTPO concentration in rCHO cells with down-regulation of both caspases was approximately 55% higher than that in rCHO cells without down-regulation of caspases and approximately 16% higher than rCHO cells with down-regulation of only caspase-3. However, in the culture with 3mM NaBu, this strategy could not dramatically enhance the culture longevity and hTPO production, compared to Bcl-2 overexpression. The different result in hTPO production between down-regulation of caspases and Bcl-2 overexpression may be because the down-regulation of caspase-3 and caspase-7, unlike Bcl-2 overexpression, could not maintain mitochondrial membrane potential in the presence of 3mM NaBu. Taken together, co-down-regulation of caspase-3 and caspase-7 is effective in regard to extension of culture longevity and enhancement of hTPO production in a serum-free culture in the presence or absence of 1mM NaBu addition.     

A DIGE approach for the assessment of differential expression of the CHO proteome under sodium butyrate addition: Effect of Bcl‐xL overexpression

Bcl‐x_L, a member of the Bcl‐2 family, is known to inhibit apoptosis of recombinant Chinese hamster ovary (rCHO) cells induced by the addition of sodium butyrate (NaBu), which is used for the elevated expression of recombinant protein. In order to understand the intracellular effects of Bcl‐x_L overexpression on CHO cells treated with NaBu, changes to the proteome caused by controlled Bcl‐x_L expression in rCHO cells producing erythropoietin (EPO) in the presence of 3 mM NaBu were evaluated using two‐dimensional differential in‐gel electrophoresis (2D‐DIGE) and MS analysis. The consequences of Bcl‐x_L overexpression were not limited to the apoptotic signaling pathway. Out of eight proteins regulated significantly by Bcl‐x_L overexpression in 3 mM NaBu addition culture, four proteins were related to cell survival (Iq motif‐containing GTPase‐activating protein 1), cell proliferation (dihydrolipoamide‐S‐acetyltransferase, guanine nucleotide binding protein alpha interacting 2), and repair of DNA damage (BRCA and CDKN1A interacting protein). Taken together, a DIGE approach reveals that overexpression of Bcl‐x_L not only inhibits apoptosis in the presence of NaBu but also affects cell proliferation and survival in various aspects. Biotechnol. Bioeng. 2010; 105: 358–367. © 2009 Wiley Periodicals, Inc.     

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.     

Enhanced human thrombopoietin production by sodium butyrate addition to serum-free suspension culture of bcl-2-overexpressing CHO cells

When sodium butyrate (NaBu) was added to serum-free suspension culture of recombinant CHO (rCHO) cells for enhanced expression of human thrombopoietin (hTPO), apoptotic cell death of rCHO cells was induced in a dose-dependent manner and hTPO quality was deteriorated in regard to sialic acid and acidic isoform contents. To overcome these problems, we overexpressed Bcl-2 protein, an antiapoptotic protein, in rCHO cells producing hTPO. Compared to serum-free suspension culture of control cells without Bcl-2 overexpression (R-neo cells) and NaBu addition, a more than 10-fold increase in the maximum hTPO concentration was obtained in serum-free suspension culture of cells with Bcl-2 overexpression (R-bc12-14 cells) and 3 mM NaBu addition. Both the enhanced specific productivity endowed by NaBu and the extended culture longevity provided by the antiapoptotic effect of Bcl-2 overexpression contributed to the enhancement of maximum hTPO concentration. The problem of quality reduction of hTPO induced by NaBu was not solved by Bcl-2 overexpression, but it was not that significant. Compared to the culture in the absence of NaBu, the percentage of hTPO isoforms in pI 3-5 with high in vivo biological activity produced by R-bc12-14 cells was decreased by approximately 18% in the presence of 3 mM. As a result, a more than 6-fold increase in the production of hTPO isoforms in pI 3-5 was achieved in R-bcl2-14 cell culture with 3 mM NaBu addition. Taken together, the data obtained suggest that Bcl-2 overexpression in rCHO cells and NaBu addition in serum-free suspension culture can be an effective means to enhance the production of highly glycosylated protein such as hTPO.     

H18杂交瘤抗凋亡能力的改造

利用PCR从pGEMTbcl-X_L质粒中获得bcl-X_L基因,构建真核表达载体pEF-bcl-X^{L,脂质体法转染杂交瘤细胞,G418筛选稳定表达株,Western blotting检测目的蛋白表达,流式细胞仪检测Bcl-X_L提高杂交瘤抗正丁酸钠诱导凋亡的功能。 将构建的编码鼠bcl-X_L基因的真核表达载体pEF-bcl-X_L,转染H18细胞后,获得稳定的表达株细胞;稳定表达Bcl-X_L的细胞具有抗正丁酸钠诱导凋亡的功能。鼠bcl-X_L基因在杂交瘤细胞中稳定表达,提高了杂交瘤抗凋亡的能力,对高密度大规模培养杂交瘤细胞具有重要意义。}     

Inhibition of sodium butyrate-induced apoptosis in recombinant Chinese hamster ovary cells by constitutively expressing antisense RNA of caspase-3

Sodium butyrate (NaBu) can enhance the expression of genes controlled by some of the mammalian promoters, but it can also inhibit cell growth and induce cellular apoptosis. Thus, the beneficial effect of using a higher concentration of NaBu on a foreign protein expression is compromised by its cytotoxic effect on cell growth. To overcome this cytotoxic effect of NaBu, the expression vector of antisense RNA of caspase-3 was constructed and transfected to recombinant Chinese hamster ovary (rCHO) cells producing a humanized antibody. Using this antisense RNA strategy, rCHO cells (B3) producing a low level of caspase-3 proenzyme were established. When batch cultures of both B3 cells and control cells transfected with antisense RNA-deficient plasmid were performed in the absence of NaBu, both cells showed similar profiles of cell growth and antibody production. Compared with control cell culture, under the condition of 5 mM NaBu addition at the exponential growth phase, expression of antisense RNA of caspase-3 significantly suppressed the NaBu-induced apoptosis of B3 cells and extended culture longevity by >2 days if the culture was terminated at cell viability of 50%. However, compared with control cell culture, the final antibody concentration of B3 cell culture was not increased in the presence of NaBu, which may be due to the loss of cellular metabolic capability resulted from the depolarization of mitochondrial membrane. Taken together, this study suggests that, although expression of antisense RNA of caspase-3 does not improve antibody productivity of rCHO cells, it can suppress NaBu-induced apoptotic cell death of rCHO cells and thereby may reduce problems associated with cellular disintegration.     

Inducible expression of Bcl-XL inhibits sodium butyrate-induced apoptosis in hybridoma, resulting in enhanced antibody production

Sodium butyrate (NaBu) can increase the specific Mab production rate of hybridomas by enhancing histone hyperacetylation and influencing the cell cycle, but it can also inhibit cell growth and induce apoptosis. Thus, the beneficial effect of NaBu on Mab secretion is compromised by its cytotoxic effect. In the present study, expression of the anti-apoptotic protein human Bcl-XL was made inducible in hybridoma H18 to overcome the cytotoxic effect of NaBu, circumventing the detrimental effects of constitutive high-level expression. We constructed an expression vector in which the promoter of a mammalian metallothionein (MT) gene drove the expression of bcl-XL in response to metal exposure. The vector was then used to exogenously control the expression of bcl-XL in H18 hybridoma cells. Our data showed that stably transfected H18.D4 cells expressed high levels of Bcl-X(L), which was induced within 24 h of addition of ZnSO4. NaBu (0.4 mM) increased antibody production by more than 3-fold in H18.D4. This effect resulted from the suppression of NaBu-induced apoptosis, allowing the H18.D4 cells to grow at higher viability and extending culture longevity by >3 days.     

Overexpression of bcl-2 inhibits sodium butyrate-induced apoptosis in Chinese hamster ovary cells resulting in enhanced humanized antibody production

Sodium butyrate (NaBu) can enhance the expression of genes from some of the mammalian promoters including cytomegalovirus (CMV) and simian virus 40 (SV40), but it can also inhibit cell growth and induce cellular apoptosis. Thus, the beneficial effect of using a higher concentration of NaBu on a foreign protein expression is compromised by its cytotoxic effect on cell growth. To overcome this cytotoxic effect of NaBu, a survival protein, human Bcl-2, was overexpressed in recombinant Chinese hamster ovary (CHO) cells (SH2-0.32), producing a humanized antibody directed against the S surface antigen of hepatitis B virus. When batch cultures of both control cells transfected with bcl-2-deficient plasmid (SH2-0.32-Deltabcl-2) and cells transfected with bcl-2 expression plasmid (14C6-bcl-2) were performed in the absence of NaBu, both cells showed similar profiles of cell viability and antibody production. Compared with the SH2-0.32-Deltabcl-2 culture, under the condition of NaBu addition at the exponential growth phase, overexpression of the bcl-2 gene considerably suppressed the NaBu-induced apoptosis of 14C6-bcl-2 by inhibiting caspase 3 activity and extending culture longevity by >2 days. As a result, the final antibody concentration of 14C6-bcl-2 culture was twofold higher than that of SH2-0.32-Deltabcl-2 culture in the presence of NaBu and threefold higher than that of SH2-0.32-Deltabcl-2 and 14C6-bcl-2 cultures in the absence of NaBu.     

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 sodium butyrate on the production, heterogeneity and biological activity of human thrombopoietin by recombinant Chinese hamster ovary cells

Human thrombopoietin (hTPO) is a heavily glycosylated protein with 6 and 24 potential N- and O-glycosylation sites, respectively. To determine the effect of sodium butyrate (NaBu) on the production and quality of hTPO in recombinant Chinese hamster ovary (rCHO) cells, NaBu (0-10 mM) was added to the cultures of exponentially growing cells. NaBu addition significantly increased both the specific and volumetric hTPO production, although it decreased the cell viability by apoptosis in a dose-dependent manner. The highest hTPO concentration of 82.2 +/- 5.6 microgml-1 was obtained in the culture with 3 mM NaBu addition. Compared with the culture without NaBu addition, the culture with 3 mM NaBu resulted in a 6.4-fold increase in qTPO and a 3.3-fold increase in the final hTPO concentration on day 7. However, NaBu deteriorated the quality of hTPO, resulting from increased heterogeneity, reduced acidic hTPO isoforms, reduced alpha(2 --> 3) sialylation, and decreased in vivo biological activity. We also found that the biological activity of hTPO in the culture with 3 mM NaBu addition collected on day 7 was 72% of that in the culture without NaBu addition. Taken together, the use of NaBu or its optimal concentration for high-level expression of a heavily glycosylated protein like hTPO should be determined by considering its detrimental effect on the quality of glycoprotein.     

Bcl‐xL overexpression does not enhance specific erythropoietin productivity of recombinant CHO cells grown at 33°C and 37°C

Overexpression of bcl‐xL in recombinant Chinese hamster ovary (rCHO) cells has been known to suppress apoptotic cell death and thereby extend culture longevity during batch culture. However, its effect on specific productivity (q) of rCHO cells is controversial. This study attempts to investigate the effect of bcl‐xL overexpression on q of rCHO cells producing erythropoietin (EPO). To regulate the bcl‐xL expression level, the Tet‐off system was introduced in rCHO cells producing EPO (EPO‐off‐bcl‐xL). The bcl‐xL expression level was tightly controlled by doxycycline concentration. To evaluate the effect of bcl‐xL overexpression on specific EPO productivity (q_EPO) at different levels, EPO‐off‐bcl‐xL cells were cultivated at the two different culture temperatures, 33°C and 37°C. The q_EPO at 33°C and 37°C in the presence of 100 ng/mL doxycycline (without bcl‐xL overexpression) were 4.89 ± 0.21 and 3.18 ± 0.06 μg/10⁶cells/day, respectively. In the absence of doxycycline, bcl‐xL overexpression did not affect q_EPO significantly, regardless of the culture temperature, though it extended the culture longevity. Taken together, bcl‐xL overexpression showed no significant effect on the q_EPO of rCHO cells grown at 33°C and 37°C. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

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.

     

Improvement of the culture stability of non-anchorage-dependent animal cells grown in serum-free media through immobilization

A murine hybridoma cell line producing a monoclonal antibody against penicillin-G-amidase and a murine transfectoma cell line secreting a monovalent chimeric human/mouse Fab-antibody fragment were cultivated in three different media (serum-containing, low protein serum-free, and iron-rich protein-free) in flask cultures, stirred reactors and a fixed bed reactor. In static batch cultures in flasks both cell lines showed similar good growth in all three media.In suspension in a stirred reactor, the hybridoma cell line could be cultivated satisfactory only in serum-containing medium. In low protein serum-free medium, Pluronic F68 had to be added to protect the hybridoma cells against shear stress. But even with this supplement only batch, not chemostat mode was possible. In iron-rich protein-free medium the hybridoma cells grew also in continuous chemostat mode, but the stability of the culture was low. The transfectoma cell line did not grow in stirred reactors in any of the three media.Good results with both cell lines were obtained in fixed bed experiments, where the cells were immobilized in macroporous Siran^®-carriers. The media, which were optimized in flask cultures, could be used without any further adaptation in the fixed bed reactor. Immobilization improved the stability and reliability of cultures of non-adherent animal cells in serum-free media tremendously compared to suspension cultures in stirred reactors. The volume-specific glucose uptake rate, an, indicator of the activity of the immobilized cells, was similar in all three media. Deviations in the metabolism of immobilized and suspended cells seem to be mainly due to low oxygen concentrations within the macroporous carriers, where the cells are supplied with oxygen only by diffusion.List of symbols c substrate or product concentration mmol l^–1 - c₀ substrate or product concentration in the feed mmol l^–1 - c_Glc glucose concentration mmol l^–1 - c_Gln glutamine concentration mmol l^–1 - c_Amm ammonia concentration mmol l^–1 - c_Lac lactate concentration mmol l^–1 - c_FAB concentration of Fab# 10 antibody fragment g l^–1 - c_MAb monoclonal antibody concentration mg l^–1 - D dilution rate d^–1 - q cell-specific substrate uptake or metabolite production rate mmol cell^–1 h^–1 - q_Glc cell-specific glucose uptake rate mmol cell^–1 h^–1 - q_Gln cell-specific glutamine uptake rate mmol cell^–1 h^–1 - q_MAb cell-specific MAb production rate mg cell^–1 h^–1 - q^* volume-specific substrate uptake or metabolite production rate mmol l^–1 h^–1 - q^*FB volume-specific substrate uptake or metabolite production rate related to the fixed bed volume mmol l_FB ^–1 h^–1 - q^*FB,Glc volume-specific glucose uptake rate related to the fixed bed volume mmol l_FB ^–1 h^–1 - q^*FB,Gln volume-specific glutamine uptake rate related to the fixed volume mmol l_FB ^–1 h^–1 - q^*FB,MAb volume-specific MAb production rate related to the fixed volume mg l_FB ^–1 h^–1 - q^*FB,02 volume-specific oxygen uptake rate related to the fixed bed volume mmol l_FB ^–1 h^–1 - t time h - U superficial flow velocity mm s^–1 - V medium volume in the conditioning vessel of the fixed bed reactor l - V_FB volume of the fixed bed l - x_v viable cell concentration cells ml^–1 - y_Amm,Gln yield of Ammonia from glutamine - y_Lac,Glc yield of lactate from glucose - specific growth rate h^–1 - _d specific death rate h^–1     

Stimulation of monoclonal antibody production by human-human hybridoma cells with an elevated concentration of potassium or sodium phosphate in serum-free medium

Potassium or sodium phosphate was found to stimulate the production of human monoclonal antibody by human-human hybridoma HB4C5. The addition of 15 mM Na-phosphate (pH 7.4) into serum-free culture medium increased the antibody production up to 4-fold, when seeded at cell density of 1×10⁵ cells/ml in dishes. At the higher cell density of 5×10⁵ cells/ml, K-phosphate was more effective than Na-phosphate, at the same concentration. In large-scale continuous culture, the addition of 10 mM Na-phosphate into serum-free culture medium stimulated antibody production by HB4C5 cells 6-fold.     

HDL3 binds to glycosylphosphatidylinositol-anchored proteins to activate signalling pathways

Previous studies have indicated that in HepG₂ cells HDL₃-signalling involves glycosylphosphatidylinositol (GPI) anchored proteins. HDL₃-binding to HepG₂ cells was found to be enhanced by cellular preincubation with PI-PLC inhibitors and sensitive to a cellular preincubation with exogenous PI-PLC, suggesting that HDL₃ binds directly on GPI-anchored proteins to initiate signaling. Moreover HDL₃-binding was found to be partly inhibited by antibodies against the HDL-binding protein (Ab_HBP).HDL₃, when binding to HepG₂ cells, promoted the release in the culture medium of a 110 kDa protein that binds Ab_HBP, while a cellular preincubation with antibodies against the inositol-phosphoglycan (IPG) moiety of GPI-anchor (Ab_IPG), used to block lipolytic cleavage of the GPI-anchor, inhibits HDL₃-induced release of the 110 kDa protein in the culture medium.In [³H]-PC prelabeled HepG₂ cells, Ab_HBP were found to stimulate PC-hydrolysis and DAG generation within 5 min as did HDL₃ stimulation. Cellular preincubation with Ab_IPG was found to inhibit only the HDL₃-signal and not the Ab_HBP-signal, while a prior cellular pretreatment with PI-PLC from Bacillus cereus was found to inhibit the HDL₃-and Ab_HBP-signal. Moreover cellular preincubation with Ab_HBP for 1 h at 37°C was found to inhibit HDL₃-signalling pathways.Our results suggest that in HepG2 cells a 110 kDa protein, which could be HBP, can be anchored to the membrane via GPI, and can function in HDL₃-signalling pathways as binding sites.