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.     

Metabolic oligosaccharide engineering with N-Acyl functionalized ManNAc analogs: cytotoxicity, metabolic flux, and glycan-display considerations

Metabolic oligosaccharide engineering (MOE) is a maturing technology capable of modifying cell surface sugars in living cells and animals through the biosynthetic installation of non‐natural monosaccharides into the glycocalyx. A particularly robust area of investigation involves the incorporation of azide functional groups onto the cell surface, which can then be further derivatized using “click chemistry.” While considerable effort has gone into optimizing the reagents used for the azide ligation reactions, less optimization of the monosaccharide analogs used in the preceding metabolic incorporation steps has been done. This study fills this void by reporting novel butanoylated ManNAc analogs that are used by cells with greater efficiency and less cytotoxicity than the current “gold standard,” which are peracetylated compounds such as Ac₄ManNAz. In particular, tributanoylated, N‐acetyl, N‐azido, and N‐levulinoyl ManNAc analogs with the high flux 1,3,4‐O‐hydroxyl pattern of butanoylation were compared with their counterparts having the pro‐apoptotic 3,4,6‐O‐butanoylation pattern. The results reveal that the ketone‐bearing N‐levulinoyl analog 3,4,6‐O‐Bu₃ManNLev is highly apoptotic, and thus is a promising anti‐cancer drug candidate. By contrast, the azide‐bearing analog 1,3,4‐O‐Bu₃ManNAz effectively labeled cellular sialoglycans at concentrations ～3‐ to 5‐fold lower (e.g., at 12.5–25 µM) than Ac₄ManNAz (50–150 µM) and exhibited no indications of apoptosis even at concentrations up to 400 µM. In summary, this work extends emerging structure activity relationships that predict the effects of short chain fatty acid modified monosaccharides on mammalian cells and also provides a tangible advance in efforts to make MOE a practical technology for the medical and biotechnology communities. Biotechnol. Bioeng. 2012; 109:992–1006. © 2011 Wiley Periodicals, Inc.     

An investigation of intracellular glycosylation activities in CHO cells: Effects of nucleotide sugar precursor feeding

Controlling glycosylation of recombinant proteins produced by CHO cells is highly desired as it can be directed towards maintaining or increasing product quality. To further our understanding of the different factors influencing glycosylation, a glycosylation sub‐array of 79 genes and a capillary electrophoresis method which simultaneously analyzes 12 nucleotides and 7 nucleotide sugars; were used to generate intracellular N‐glycosylation profiles. Specifically, the effects of nucleotide sugar precursor feeding on intracellular glycosylation activities were analyzed in CHO cells producing recombinant human interferon‐γ (IFN‐γ). Galactose (±uridine), glucosamine (±uridine), and N‐acetylmannosamine (ManNAc) (±cytidine) feeding resulted in 12%, 28%, and 32% increase in IFN‐γ sialylation as compared to the untreated control cultures. This could be directly attributed to increases in nucleotide sugar substrates, UDP‐Hex (～20‐fold), UDP‐HexNAc (6‐ to 15‐fold) and CMP‐sialic acid (30‐ to 120‐fold), respectively. Up‐regulation of B4gal and St3gal could also have enhanced glycan addition onto the proteins, leading to more complete glycosylation (sialylation). Combined feeding of glucosamine + uridine and ManNAc + cytidine increased UDP‐HexNAc and CMP‐sialic acid by another two‐ to fourfold as compared to feeding sugar precursors alone. However, it did not lead to a synergistic increase in IFN‐γ sialylation. Other factors such as glycosyltransferase or glycan substrate levels could have become limiting. In addition, uridine feeding increased the levels of uridine‐ and cytidine‐activated nucleotide sugars simultaneously, which could imply that uridine is one of the limiting substrates for nucleotide sugar synthesis in the study. Hence, the characterization of intracellular glycosylation activities has increased our understanding of how nucleotide sugar precursor feeding influence glycosylation of recombinant proteins produced in CHO cells. It has also led to the optimization of more effective strategies for manipulating glycan quality. Biotechnol. Bioeng. 2010;107: 321–336. © 2010 Wiley Periodicals, Inc.     

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.     

Evaluation of the antioxidative and genotoxic effects of sodium butyrate on breast cancer cells

Oncogenic stimulation shows a rise in reactive oxygen species (ROS), and ROS can eventually induce carcinogenesis by causing DNA damage. In this context, this study aims to evaluate some biochemical and genotoxic changes in the control of cell death caused by NaBu (Sodium butyrate). treatment in breast cancer cells. NaBu’s impact on cell proliferation was determined via WST-1 assay. The lipid peroxidation (MDA), reduced glutathione (GSH), Nitric Oxide (NO), hydrogen peroxide (H₂O₂), and superoxide dismutase (SOD) enzyme levels were determined biochemically. NaBu-induced genotoxic damage was estimated via single-cell gel electrophoresis (SCGE). NaBu reduced cell viability and potentially induced GSH, but decreased SOD enzyme activity and the level of MDA and NO decreased also H₂O₂ decreased at different times and NaBu concentrations. Higher NaBu concentrations amplified DNA damage in MCF-7 cells compared to the control group. NaBu shows anticancer and genotoxic effects, especially through antioxidant enzymes, one of the oxidative stress parameters in breast cancer. However, the anticancer and genotoxic effects of NaBu is changed in the oxidative stress parameters with time and treatment concentration of NaBu in MCF-7 cells. Furthermore, his oxidative stress-dependent effect changes need to be clarified by further evaluation with molecular and more biochemical parameters.     

Effect of N-Acetylcystein on butyrate-treated Chinese hamster ovary cells to improve the production of recombinant human interferon-beta-1a

Sodium butyrate (NaBu) is used as a productivity enhancer for the production of therapeutic recombinant proteins in Chinese hamster ovary (CHO) cells. However, NaBu is well-known for having a cytotoxic effect, thereby inducing apoptosis. As an endeavor to reduce this defect, we studied 11 antioxidants known for inhibiting apoptosis, according to a Plackett-Burman statistical design on CHO cells producing recombinant interferon-beta-1a (IFN-beta). None of the antioxidants that we tested were as effective as N-acetylcystein (NAC) from the point of view of maintaining long-term survival of CHO cells and increasing the production of IFN-beta. In 7.5-L perfusion bioreactor cultures, the addition of NaBu and NAC elongated the culture period to almost 200 h throughout production phase and increased the production yield by 2-fold compared to control cultures containing only NaBu. Glycosylation patterns of produced IFN-beta at each run were also compared in IEF analysis. IEF profiles of where NaBu and NAC were added showed to be more isoforms with a lower pI than those of the control run. The sialic acid content was also increased by 17.7% according to HPLC analysis. Taken together, the data obtained demonstrate that the addition of NAC has positive effects on the elongation of the culture period, improving the production and increasing the sialylation of IFN-beta in NaBu-treated CHO cells.     

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.     

A functional analysis of N-glycosylation-related genes on sialylation of recombinant erythropoietin in six commonly used mammalian cell lines

Significant efforts have been made to improve the sialylation of recombinant glycoproteins with the aim of extending their in vivo circulation time. Here, we report a systematic functional analysis of 31 N-glycosylation-related genes on sialylation of recombinant EPO in six cell lines. BHK and CHO cells were found to sialylate recombinant EPO most effectively. None of the 31 genes, individually or in combination, was able to improve EPO sialylation in these cells. HEK293, Cos-7 and 3T3 cells showed intermediate sialylation capabilities, whereas NS0 cells sialylated recombinant EPO poorly. Overexpression of ST6GalI, ST3GalIII or ST3GalIV, but not ST3GalVI, was able to improve EPO sialylation in these four cell lines. qRT-PCR experiments revealed that ST3GalIII and ST3GalIV are indeed under expressed in HEK293, 3T3 and NS0 cells. Co-expression of upstream glycogenes failed to synergize with these sialyltransferases to further enhance sialylation, suggesting that the upstream glycogenes are all expressed at sufficient levels.     

Sodium butyrate protects against oxidative stress in HepG2 cells through modulating Nrf2 pathway and mitochondrial function

Sodium butyrate (NaBu) is a by-product of microbial fermentation of dietary fiber in the gastrointestinal tract and has been shown to increase the activity of antioxidant enzymes, such as catalase or heme oxidase-1, in vivo. However, the mechanism of this effect is still unclear. This study investigated the antioxidant effect of NaBu on HepG2 cells under H₂O₂-induced oxidative stress. NaBu (0.3 mM) attenuated cell death and accumulation of reactive oxygen species and improved multiple antioxidant parameters in H₂O₂-injured HepG2 cells. NaBu inhibited glycogen synthase kinase-3 beta (GSK-3β) by increasing the p-GSK-3β (Ser9) level and promoted nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2), which increased the expression of downstream antioxidant enzymes. Together with promotion of peroxisome proliferator-activated receptor gamma coactivator 1-alpha and mitochondrial DNA copy number, NaBu modulated energy metabolism and mitochondrial function, decreasing glycolysis, increasing β-oxidation, and enhancing the tricarboxylic acid cycle and oxidative phosphorylation. NaBu increased mitochondrial manganese-superoxide dismutase and glutathione peroxidase activity. In conclusion, NaBu protected HepG2 cells against oxidative stress by modulating Nrf2 pathway activity and mitochondrial function.     

Growth factor withdrawal in combination with sodium butyrate addition extends culture longevity and enhances antibody production in CHO cells

The effect of growth factor (GF) and sodium butyrate (NaBu) on Chinese hamster ovary (CHO) cell growth, cell viability and antibody production was investigated using shaking flasks in GF-containing and GF-deficient medium containing 0, 1 and 3 mM NaBu. The withdrawal of GF and the addition of NaBu suppressed cell growth, but they significantly increased specific antibody productivity, q_Ab. Interestingly, the withdrawal of GF in combination with the addition of NaBu markedly retarded cell death, leading to extended culture longevity. For instance, at 3 mM NaBu, cell viability fell below 80% after day 4 in GF-containing medium, but it remained over 80% until day 18 in GF-deficient medium. Due to the enhanced q_Ab and the extended culture longevity, approximately 2-fold increase in total antibody production was achieved in pseudo-perfusion culture with 1 mM NaBu in GF-deficient medium, compared to the culture in GF-containing medium. The effect of GF and NaBu on the change in the expression and activity of cellular proteins, c-Myc, Bcl-2 and pyruvate dehydrogenase (PDH), was also investigated. Both the withdrawal of GF and the addition of NaBu decreased the expression of c-Myc. The expression of Bcl-2 was enhanced by the addition of NaBu in a dose-dependent manner while it was not affected by the withdrawal of GF. In addition, both the withdrawal of GF and the addition of NaBu reduced metabolic rates, q_Glc, q_Lac and Y_Lac/Glc, and increased PDH activity while not affecting PDH expression, suggesting that they may reduce the glycolytic rates, but enhance the conversion rates of pyruvate to TCA intermediates. Taken together, the withdrawal of GF in combination with the addition of NaBu can be considered as a relevant strategy for alleviating NaBu-induced cell apoptosis and enhancing antibody production since it can be easily implemented as well as enhance q_Ab and extend culture longevity.     

Effect of sodium butyrate on autophagy and apoptosis in Chinese hamster ovary cells

Sodium butyrate (NaBu), which is widely used in recombinant Chinese hamster ovary cell (rCHO) cultures for high-level expression of therapeutic proteins, is known to induce apoptosis in a dose-dependent manner. Lately, the significance of autophagy has increased in the field of CHO cell culture due to the fact that autophagy is related to the programmed cell death mechanism. To determine the effect of NaBu on autophagy as well as apoptosis of rCHO cells, rCHO cells producing erythropoietin were subjected to NaBu treatment. NaBu treatment up to 5 mM increased cleaved forms of PARP, caspase-3, and Annexin V positive population, confirming the previous results that NaBu induces apoptosis. Concurrently, NaBu treatment increased the level of accumulation of the autophagic marker, LC3-II, independently of nutrient depletion, suggesting that NaBu induces autophagy. To elucidate the potential role of autophagy induced by NaBu, a representative autophagy inducer (rapamycin) or an inhibitor (bafilomycin A1) was added to cultures together with NaBu. It was found that autophagy had the potential role of a positive cell survival mechanism under NaBu treatment. Furthermore, gradual reduction in mitochondrial membrane potential/mass and recruitment of a mitophagy protein, Parkin, to the mitochondria were observed under NaBu treatment, suggesting that this positive function of autophagy might be mediated by the autophagic removal of damaged mitochondria. Taken together, autophagy was observed in rCHO cell culture under NaBu treatments and the results obtained here support the positive effects of autophagy induced by NaBu treatments.     

A Novel Approach to Decrease Sialic Acid Expression in Cells by a C-3-modified N-Acetylmannosamine

Due to its position at the outermost of glycans, sialic acid is involved in a myriad of physiological and pathophysiological cell functions such as host-pathogen interactions, immune regulation, and tumor evasion. Inhibitors of cell surface sialylation could be a useful tool in cancer, immune, antibiotic, or antiviral therapy. In this work, four different C-3 modified N-acetylmannosamine analogs were tested as potential inhibitors of cell surface sialylation. Peracetylated 2-acetylamino-2-deoxy-3-O-methyl-d-mannose decreases cell surface sialylation in Jurkat cells in a dose-dependent manner up to 80%, quantified by flow cytometry and enzyme-linked lectin assays. High-performance liquid chromatography experiments revealed that not only the concentration of membrane bound but also of cytosolic sialic acid is reduced in treated cells. We have strong evidence that the observed reduction of sialic acid expression in cells is caused by the inhibition of the bifunctional enzyme UDP-GlcNAc-2-epimerase/ManNAc kinase. 2-Acetylamino-2-deoxy-3-O-methyl-d-mannose inhibits the human ManNAc kinase domain of the UDP-GlcNAc-2-epimerase/ManNAc kinase. Binding kinetics of the inhibitor and human N-acetylmannosamine kinase were evaluated using surface plasmon resonance. Specificity studies with human N-acetylglucosamine kinase and hexokinase IV indicated a high specificity of 2-acetylamino-2-deoxy-3-O-methyl-d-mannose for MNK. This substance represents a novel class of inhibitors of sialic acid expression in cells, targeting the key enzyme of sialic acid de novo biosynthesis.     

Effects of culture conditions on N‐glycolylneuraminic acid (Neu5Gc) content of a recombinant fusion protein produced in CHO cells

CHO cells express glycoproteins containing both the N‐acetylneuraminic acid (Neu5Ac) and minor amounts of the N‐glycolylneuraminic acid (Neu5Gc) forms of sialic acid. As Neu5Gc is not expressed in humans and can be recognized as a foreign epitope, there is the potential for immunogenicity issues for glycoprotein therapeutics. During process development of a glycosylated fusion protein expressed by CHO cells, a number of culture conditions were identified that affected the Neu5Gc content of the recombinant glycoprotein. Sodium butyrate (SB), a well‐known additive reported to enhance recombinant protein productivity in specific cases, minimally affected product titers here, but did decrease Neu5Gc levels by 50–62%. A shift in culture temperature to a lower value after the exponential growth phase was used to extend the culture period. It was found that the Neu5Gc levels were 59% lower when the temperature shift occurred later near the stationary phase of the culture compared to an early‐temperature shift, near the end of the exponential growth phase. Studies on the effects of pCO₂ with this product showed that the Neu5Gc levels were 46% lower at high pCO₂ conditions (140 mmHg) compared to moderate pCO₂ levels (20–80 mmHg). Finally, a comparison of sodium carbonate versus sodium hydroxide as the base used for pH control resulted in a reproducible 33% decrease in Neu5Gc in bioreactors using sodium hydroxide. These results are of practical importance as SB is a commonly tested additive, and the other factors affecting Neu5Gc can conveniently be used to reduce or control Neu5Gc in processes for the manufacture of glycoprotein therapeutics. Biotechnol. Bioeng. 2010;105: 1048–1057. © 2009 Wiley Periodicals, Inc.     

N-acetylcysteine increases the biosynthesis of recombinant EPO in apoptotic Chinese hamster ovary cells

Sodium butyrate (NaBu) is known to enhance the rate of biosynthesis of recombinant proteins in Chinese hamster ovary cells (CHO). Here we demonstrate that supplementation with NaBu during rapid growth brings about abrupt death of the cells. The death of the cells is due to apoptosis, as assessed by intranucleosomal DNA fragmentation. The promotion of apoptotic death of the cells could be partially blocked by treatment with the well-known antioxidant, N-acetylcysteine (NAC). Strikingly, the NAC treatment enhanced the production of recombinant EPO two-fold compared with that of the culture without NAC supplementation. These results showed that NaBu treatment supplemented with NAC not only inhibits apoptosis, but also exerts a synergistic effect on the biosynthesis of recombinant EPO.     

Chinese hamster ovary (CHO) host cell engineering to increase sialylation of recombinant therapeutic proteins by modulating sialyltransferase expression

N‐Glycans of human proteins possess both α2,6‐ and α2,3‐linked terminal sialic acid (SA). Recombinant glycoproteins produced in Chinese hamster overy (CHO) only have α2,3‐linkage due to the absence of α2,6‐sialyltransferase (St6gal1) expression. The Chinese hamster ST6GAL1 was successfully overexpressed using a plasmid expression vector in three recombinant immunoglobulin G (IgG)‐producing CHO cell lines. The stably transfected cell lines were enriched for ST6GAL1 overexpression using FITC‐Sambucus nigra (SNA) lectin that preferentially binds α2,6‐linked SA. The presence of α2,6‐linked SA was confirmed using a novel LTQ Linear Ion Trap Mass Spectrometry (LTQ MS) method including MSn fragmentation in the enriched ST6GAL1 Clone 27. Furthermore, the total SA (mol/mol) in IgG produced by the enriched ST6GAL1 Clone 27 increased by 2‐fold compared to the control. For host cell engineering, the CHOZN^® GS host cell line was transfected and enriched for ST6GAL1 overexpression. Single‐cell clones were derived from the enriched population and selected based on FITC‐SNA staining and St6gal1 expression. Two clones (“ST6GAL1 OE Clone 31 and 32”) were confirmed for the presence of α2,6‐linked SA in total host cell protein extracts. ST6GAL1 OE Clone 32 was subsequently used to express SAFC human IgG1. The recombinant IgG expressed in this host cell line was confirmed to have α2,6‐linked SA and increased total SA content. In conclusion, overexpression of St6gal1 is sufficient to produce recombinant proteins with increased sialylation and more human‐like glycoprofiles without combinatorial engineering of other sialylation pathway genes. This work represents our ongoing effort of glycoengineering in CHO host cell lines for the development of “bio‐better” protein therapeutics and cell culture vaccine production. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:334–346, 2015     

N-acetylcysteine increases the biosynthesis of recombinant EPO in apoptotic Chinese hamster ovary cells

Sodium butyrate (NaBu) is known to enhance the rate of biosynthesis of recombinant proteins in Chinese hamster ovary cells (CHO). Here we demonstrate that supplementation with NaBu during rapid growth brings about abrupt death of the cells. The death of the cells is due to apoptosis, as assessed by intranucleosomal DNA fragmentation. The promotion of apoptotic death of the cells could be partially blocked by treatment with the well-known antioxidant, N-acetylcysteine (NAC). Strikingly, the NAC treatment enhanced the production of recombinant EPO two-fold compared with that of the culture without NAC supplementation. These results showed that NaBu treatment supplemented with NAC not only inhibits apoptosis, but also exerts a synergistic effect on the biosynthesis of recombinant EPO.     

Effect of Bcl‐xL overexpression on sialylation of Fc‐fusion protein in recombinant Chinese hamster ovary cell cultures

The sialic acid of glycoproteins secreted by recombinant Chinese hamster ovary (rCHO) cells can be impaired by sialidase under culture conditions which promote the extracellular accumulation of this enzyme. To investigate the effect of Bcl‐x_L overexpression on the sialylation of glycoproteins produced in rCHO cell culture, two rCHO cell lines producing the same Fc‐fusion protein, which were derived from DUKX‐B11 and DG44, respectively, were engineered to have regulated Bcl‐x_L overexpression using the Tet‐off system. For both cell lines, Bcl‐x_L overexpression improved cell viability and extended culture longevity in batch cultures. As a result, a maximum Fc‐fusion protein titer increased by Bcl‐x_L overexpression though the extent of titer enhancement differed between the two cell lines. With Bcl‐x_L overexpression, the sialylation of Fc‐fusion protein, which was assessed by isoelectric focusing gel and sialic acid content analyses, decreased more slowly toward the end of batch cultures. This was because Bcl‐x_L overexpression delayed the extracellular accumulation of sialidase activity by reducing cell lysis during batch cultures. Taken together, Bcl‐x_L overexpression in rCHO cell culture increased Fc‐fusion protein production and also reduced the impairment of sialylation of Fc‐fusion protein by maintaining high viability during batch cultures. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1133–1136, 2015     

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.     

Short‐ and long‐term effects on mAb‐producing CHO cell lines after cryopreservation

Cryopreservation provides the foundation for research, development, and manufacturing operations in the CHO‐based biopharmaceutical industry. Despite its criticality, studies are lacking that explicitly demonstrate that the routine cell banking process and the potential stress and damage during cryopreservation and recovery from thaw have no lasting detrimental effects on CHO cells. Statistics are also scarce on the decline of cell‐specific productivity (Q_p) over time for recombinant CHO cells developed using the glutamine synthetase (GS)‐based methionine sulfoximine (MSX) selection system. To address these gaps, we evaluated the impact of freeze‐thaw on 24 recombinant CHO cell lines (generated by the GS/MSX selection system) using a series of production culture assays. Across the panel of cell lines expressing one of three monoclonal antibodies (mAbs), freeze‐thaw did not result in any significant impact beyond the initial post‐thaw passages. Production cultures sourced from cryopreserved cells and their non‐cryopreserved counterparts yielded similar performance (growth, viability, and productivity), product quality (size, charge, and glycosylation distributions), and flow cytometric profiles (intracellular mAb expression). However, many production cultures yielded lower Q_p at increased cell age: 17 of the 24 cell lines displayed ≥20% Q_p decline after ～2–3 months of passaging, irrespective of whether the cells were previously cryopreserved. The frequency of Q_p decline underscores the continued need for understanding the underlying mechanisms and for careful clone selection. Because our experiments were designed to decouple the effects of cryopreservation from those of cell age, we could conclusively rule out freeze‐thaw as a cause for Q_p decline. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:463–477, 2018