Increase In Specific Productivity Research Articles

Enhancing the productivity and proliferation of CHO-K1 cells by oncoprotein YAP (Yes-associated protein)

CHO cells are extensively employed in biological drug industry to manufacture therapeutic proteins. Nevertheless, production of biopharmaceuticals faces obstacles such as limited growth and inadequate productivity. Employing host cell engineering techniques for CHO cells serves as a valuable approach to address the constraints encountered in biologics manufacturing. Despite advancements, most techniques focus on specific genes to address individual cellular challenges. The significance of YAP, transcriptional co-activator, cannot be overstated due to its involvement in regulating organ size and tumor formation. YAP’s influence extends to various cellular processes and is regulated by kinase cascade in the Hippo pathway, which phosphorylates serine residues in specific LATS recognition motifs. Activation of YAP has been observed to impact both the size and quantity of cells. This research investigates the effects of YAP5SA on proliferation, apoptosis, and productivity in CHO-K1 cells. YAP5SA, with mutations in all five LATS-target sites, is selected for its heightened activity and resistance to repression through the Hippo-LATS1/2 kinase signaling pathway. Plasmid harboring YAP5SA was transfected into EPO-CHO and the influence of YAP5SA overexpression was investigated. According to our findings, transfection of EPO-CHO cells with YAP5SA exhibited a substantial enhancement in CHO cell productivity, resulting in a 3-fold increase in total protein and EPO, as well as a 1.5-fold increase in specific productivity. Additionally, it significantly contributes in augmenting viability, size, and proliferation. Overall, the findings of this study exemplify the potential of utilizing YAP5SA to impact particular cellular mechanisms, thereby presenting an avenue for customizing cells to fulfill production demands.Key points• YAP5SA in CHO cells boosts growth, reduces apoptosis, and significantly improves productivity.• YAP5SA regulates genes involved in proliferation, survival, and mTOR activation.• YAP5SA increases productivity by improving cell cycle, c-MYC expression, and mTOR pathway.

Separation of a water/MIBK mixture by batch heteroazeotropic distillation: A self-entrained case

In this work, we present a study on the dehydration of methyl isobutyl ketone (MIBK) by batch distillation, using the dynamic module for batch distillation (BatchSep) provided by the commercial chemical process simulator Aspen Plus V11®. A 98 wt.% MIBK/water mixture was initially fed to the column. The scope of the separation was set at 99.8 wt.%, as a standard purity for solvent reuse. Taking advantage of the heteroazeotropic behaviour of the mixture water/MIBK (self-entrained), an alternative configuration with a decanter, which allowed selective reflux of the organic phase, was studied and compared with the conventional batch distillation configuration at 1 atm. This alternative configuration presented clear operating advantages compared to the conventional case, i.e. reduction of both operational time and energy demand by 50% and increase in specific production by 100%. Finally, the distillation-decanter configuration was studied at reduced pressures (50–400 mbar), registering further operating improvements.

РАЗВИТИЕ ОБОРУДОВАНИЯ ДЛЯ ТОНКОДИСПЕРСНОГО ИЗМЕЛЬЧЕНИЯ МИНЕРАЛЬНОГО СЫРЬЯ

Introduction. The huge energy costs of fine and ultrafine grinding operations have led to numerous studies aimed at developing new devices and methods for producing powdered materials. The first way leads to an increase in specific productivity while maintaining energy consumption at approximately the same level. The second way allows not only to increase the specific productivity, but also to reduce the specific energy consumption, since with a decrease in the exposure time, the body is more easily deformed and destroyed. The grinders in which the intensification of the destruction process is carried out in the first way can include planetary and vibrating mills. The second method of intensification is used in jet and various types of centrifugal mills. It is determined that vertical centrifugal mills are the equipment of a new type that can effectively perform grinding operations at high technological indicators and low energy consumption. The purpose of the research. Development of measures for obtaining a finely dispersed product by destroying mineral raw materials in vertical centrifugal mills by mechanical means. Research methodology. The paper uses a comprehensive research method: analysis of the state of grinding equipment according to literary and patent sources; development of mathematical models of the movement of bulk material in the working space of a vertical centrifugal mill based on the Navie-Stokes equations; computer modeling of the movement of the crushed material using the OpenFOAM package for solving hydrodynamic equations; a solver program was created to perform calculations, implementing the SIMPLER algorithm and using the MRF model; laboratory experiments; granulometric method for the analysis of grinding products; fractional analysis of crushed material with a grain size of 0.08 mm using an X-ray analytical centrifuge ВrооkhаvеnBI-ХDС. The development of measures for the effective evacuation of the crushed product from the working space of a centrifugal mill was evaluated by comparative tests of mills using various design solutions. Research results and discussion 1. It is established that vertical centrifugal mills are the devices of a new type capable of effectively performing grinding operations at high technological indicators and low energy consumption. 2. A mathematical model of the movement of bulk material in the working space of a vertical centrifugal mill has been developed based on the Navier-Stokes equations, which allows determining the speed and direction of movement of material layers at any point in the working space, the energy costs for their movement, the influence of the height of the material column on the nature of its movement in the space above the rotor. 3. The directions of increasing the efficiency of grinding mineral raw materials in a vertical centrifugal mill are determined by reducing the number of radial ribs installed in the cavity of the mill rotor; making cutouts in the radial ribs of the rotor, in the area near the rotor hub; installing coaxial rings in the working space of the mill above the rotor. 4. A method of effective evacuation of the crushed product from the mill body by installing additional screening surfaces in the peripheral part of the working space of the mill has been developed. 5. For the first time, the distribution of the granulometric composition of fine and medium - dispersed crushed product obtained as a result of processing lump dolomite in a centrifugal mill was obtained. It was found that particles with a size class of -0.08 + 0.03 mm are absent in the output of the crushed product from the mill. Conclusions. A mass-produced vertical centrifugal mill will find its effective application at enterprises of the Russian Federation for the preparation of powdered materials and will allow to obtain a significant effect by reducing the price of the final product, simplicity of design and maintenance of equipment, as well as small occupied production areas.

Застосування нанофільтрації для регулювання властивостей вторинних продуктів переробки тваринної сировини

This article focuses on the study of the membrane concentration process using buttermilk ultrafiltration nanofiltration membranes. Two types of Vladipor OPMN nanofiltration membranes are being investigated in order to identify the main filtration dependencies. For each type, a pressure in the range of 0.5 to 2.0 MPa and a temperature of 20 to 40 ° C are applied. The results indicate that both types of membranes effectively retain lactose at a pressure of 2.0 MPa, a temperature of 40 ° C and a concentration factor. about 2. Varying the flow velocity above the membrane from 0.5 m / s to 1.5 m / s allows obtaining an increase in specific productivity by 15% from the initial one. The influence of the effects of concentration polarization on the specific productivity of nanofiltration membranes is established. The limitation of the concentration factor due to the increase in the osmotic pressure of the solution is determined. The conclusion is made about the possibility of large-scale introduction of nanofiltration of secondary material resources of milk processing in the study of membrane regeneration processes. Key words: buttermilk, ultrafiltration, nanofiltration, lactose, membrane concentration.

Вплив функціональної годівлі молодняка курчат-бройлерів м’ясних кросів на якість і безпечність м’яса

This article focuses on the study of the membrane concentration process using buttermilk ultrafiltration nanofiltration membranes. Two types of Vladipor OPMN nanofiltration membranes are being investigated in order to identify the main filtration dependencies. For each type, a pressure in the range of 0.5 to 2.0 MPa and a temperature of 20 to 40 ° C are applied. The results indicate that both types of membranes effectively retain lactose at a pressure of 2.0 MPa, a temperature of 40 ° C and a concentration factor. about 2. Varying the flow velocity above the membrane from 0.5 m / s to 1.5 m / s allows obtaining an increase in specific productivity by 15% from the initial one. The influence of the effects of concentration polarization on the specific productivity of nanofiltration membranes is established. The limitation of the concentration factor due to the increase in the osmotic pressure of the solution is determined. The conclusion is made about the possibility of large-scale introduction of nanofiltration of secondary material resources of milk processing in the study of membrane regeneration processes. Key words: buttermilk, ultrafiltration, nanofiltration, lactose, membrane concentration.

Maximizing antibody production in a targeted integration host by optimization of subunit gene dosage and position.

Historically, therapeutic protein production in Chinese hamster ovary (CHO) cells has been accomplished by random integration (RI) of expression plasmids into the host cell genome. More recently, the development of targeted integration (TI) host cells has allowed for recombination of plasmid DNA into a predetermined genomic locus, eliminating one contributor to clone-to-clone variability. In this study, a TI host capable of simultaneously integrating two plasmids at the same genomic site was used to assess the effect of antibody heavy chain and light chain gene dosage on antibody productivity. Our results showed that increasing antibody gene copy number can increase specific productivity, but with diminishing returns as more antibody genes are added to the same TI locus. Random integration of additional antibody DNA copies in to a targeted integration cell line showed a further increase in specific productivity, suggesting that targeting additional genomic sites for gene integration may be beneficial. Additionally, the position of antibody genes in the two plasmids was observed to have a strong effect on antibody expression level. These findings shed light on vector design to maximize production of conventional antibodies or tune expression for proper assembly of complex or bispecific antibodies in a TI system.

Limiting the metabolic burden of recombinant protein expression during selection yields pools with higher expression levels.

In order to avoid the metabolic burden of protein expression during cell growth, and to avoid potential toxicity of recombinant proteins, microbial expression systems typically utilize regulated expression vectors. In contrast, constitutive expression vectors have usually been utilized for isolation of protein expressing mammalian cell lines. In mammalian systems, inducible expression vectors are typically utilized for only those proteins that are toxic when overexpressed. We developed a tetracycline regulated expression system in CHO cells, and show that cell pools selected in the uninduced state recover faster than those selected in the induced state even though the proteins showed no apparent toxicity or expression instability. Furthermore, cell pools selected in the uninduced state had higher expression levels when protein expression was turned on only in production cultures compared to pools that were selected and maintained in the induced state through production. We show a titer improvement of greater than twofold for an Fc-fusion protein and greater than 50% improvement for a recombinant antibody. The improvement is primarily due to an increase in specific productivity. Recombinant protein mRNA levels correlate strongly with protein expression levels and are highest in those cultures selected in the uninduced state and only induced during production. These data are consistent with a model where CHO cell lines with constitutive expression select for subclones with lower expression levels.

Engineering an Artificial Membrane Vesicle Trafficking System (AMVTS) for the Excretion of β-Carotene in Escherichia coli.

Large hydrophobic molecules, such as carotenoids, cannot be effectively excreted from cells by natural transportation systems. These products accumulate inside the cells and affect normal cellular physiological functions, which hinders further improvement of carotenoid production by microbial cell factories. In this study, we proposed to construct a novel artificial transport system utilizing membrane lipids to carry and transport hydrophobic molecules. Membrane lipids allow the physiological mechanism of membrane dispersion to be reconstructed and amplified to establish a novel artificial membrane vesicle transport system (AMVTS). Specifically, a few proteins in E.coli were reported or proposed to be related to the formation mechanism of outer membrane vesicles, and were individually knocked out or overexpressed to test their physiological functions. The effects on tolR and nlpI were the most significant. Knocking out both tolR and nlpI resulted in a 13.7% increase of secreted β-carotene with a 35.6% increase of specific production. To supplement the loss of membrane components of the cells due to the increased membrane vesicle dispersion, the synthesis pathway of phosphatidylethanolamine was engineered. While overexpression of AccABCD and PlsBC in TW-013 led to 15% and 17% increases of secreted β-carotene, respectively, the overexpression of both had a synergistic effect and caused a 53-fold increase of secreted β-carotene, from 0.2 to 10.7 mg/g dry cell weight (DCW). At the same time, the specific production of β-carotene increased from 6.9 to 21.9 mg/g DCW, a 3.2-fold increase. The AMVTS was also applied to a β-carotene hyperproducing strain, CAR025, which led to a 24-fold increase of secreted β-carotene, from 0.5 to 12.7 mg/g DCW, and a 61% increase of the specific production, from 27.7 to 44.8 mg/g DCW in shake flask fermentation. The AMVTS built in this study establishes a novel artificial transport mechanism different from natural protein-based cellular transport systems, which has great potential to be applied to various cell factories for the excretion of a wide range of hydrophobic compounds.

Estimating farm-level financing gap: a technical efficiency approach

PurposeMuch attention has been paid to farm credit access with less focus on determining the actual credit amount needed to bring about a specified increase in productivity relative to the finance being sought. The paper aims to discuss this issue.Design/methodology/approachUsing 2016 cross-sectional data of plantain farmers, the authors employ the Cobb–Douglas stochastic frontier production function to determine the technical efficiency (TE) of each farmer. Current plantain quantity produced by farmers and the TE are then used to estimate plantain quantity at a target efficiency. The finance needed to produce at the target efficiency is estimated using the Harrod–Domar (HD) growth equation and the authors then subtract the farmers’ savings from the estimated amount to determine the financing gap of the farmers.FindingsResults of this study show that the actual amount required to improve the productivity of farmers to target levels of TE can be estimated and that credit amount granted to farmers can be tied to a specific production efficiency. Credit schemes with interest rates below 9 per cent are more beneficial to farmers while access to credit is determined by interest rate, education, credit process duration, land ownership and asset value in the study area.Research limitations/implicationsThe implication of this research is that it opens up the possibility of further exploring the application of the HD theory at the micro level.Practical implicationsThe findings in this study have important implications on the provision of agricultural credit to small farmers. The first is that the TE of farmers plays a very critical role in determining the actual amount of credit needed to bridge the farm-level financing gap and impact positively on productivity. Second, while it is important to bridge the farm-level financing gap, this can only be beneficial to the farmers at single-digit interest rates below 7 per cent. Finally, granting of credit to farmers can be tied to specific production increase target to reduce indiscipline and mismanagement in credit use.Social implicationsThe findings of this study will go along in helping to prevent mismanagement and indiscipline in the use of scarce financial resources in agricultural production.Originality/valueThis study is the first of its kind, using TE and bringing the HD equation down to the farm level to estimate the exact amount required by farmers to bring about specific increase in production, determining the credit amount beyond which mismanagement may set in.

Estimation of application efficiency of “Flumag M” magnesia flux in agglomeration and BF production.

To decrease viscosity of BF slag and improve its desulfurization ability during hot metal production a magnesia oxide is used, which is introduced into a blast furnace, as a rule, within iron ore sinter, as well as in the form of a fluxing additive. Dolomite, sometimes iron ore materials with increased magnesia content (for example, Kovdor concentrate, raw or roasted Bakal siderite) as well as magnesia-contained wastes, most often BOF slag, are usually used as a main source of magnesia oxide during iron ore sinter production. Brucite, which is widely used abroad, mainly in Japan during iron ore sinter production, is a very prospective magnesia flux. However, brucite was never used in sinter production in Russia. Main parameters and efficiency of its application were obtained under Japan raw materials conditions. However sinter chemical and mineral compositions at Russian and Japanese sinter plants considerably differ. In this connection studies on influence of the magnesia flux “Flumag M”, which is identical by its composition to brucite, on the process parameters of sinter burden sintering and pellets production were carried out. The estimation of application efficiency of “Flumag M” magnesia flux was made during typical sintering of NLMK sinter burden. It was determined, that partial and complete substitution of dolomite by the “Flumag M” magnesia flux in the NLMK sinter burden results in an increase of specific productivity of sintering process by 10–20% (comparative) and the sinter strength by 3–5% (comparative) correspondently. Laboratory experiments on “Flumag M” magnesia flux application, carried out in STI NITU “MISiS”, showed, that raw pellets with magnesia flux additives have higher compressive strength comparing with the pellets having dolomite additives. Impact strength and abrasion strength of roasted pellets is higher, comparing with those with dolomite. Optimal content of “Flumag M” flux in the pellets burden is 2%. The application of “Flumag M” magnesia flux enables to remove burden from the burden and increase strength of roasted pellets.

A Pro-metastatic tRNA Pathway

tRNAs are best known as basic modules for global regulation of protein synthesis. Goodarzi etal. now show that two tRNAs upregulated in metastatic breast cancer cells enhance stability and translation of transcripts enriched with these codons, leading to specific increase in production of pro-metastatic proteins.

S-Sulfocysteine simplifies fed-batch processes and increases the CHO specific productivity via anti-oxidant activity

Industrial fed-batch cultivation of mammalian cells is used for the production of therapeutic proteins such as monoclonal antibodies. Besides medium ensuring initial growth, feeding is necessary to improve growth, viability and antibody production. Established processes include a slight acidic main feed and a separate alkaline feed containing l-tyrosine and l-cysteine. Since l-cysteine is not stable at neutral pH, a new derivative, S-sulfocysteine, was tested in neutral pH feeds. In small scale fed-batch processes, the S-sulfocysteine process yielded a comparable maximum viable cell density, prolonged viability and increased titer compared to the two feed system. Bioreactor experiments confirmed the increase in specific productivity. In depth characterization of the monoclonal antibody indicated no change in the glycosylation, or charge variant pattern whereas peptide mapping experiments were not able to detect any integration of the modified amino acid in the sequence of the monoclonal antibody. Finally, the mechanism of action of S-sulfocysteine was investigated, and results pointed out the anti-oxidative potential of the molecule, mediated through an increase in superoxide dismutase enzyme levels and in the total intracellular glutathione pool. Finally, we propose that the increase in specific productivity obtained in the S-sulfocysteine process results from the anti-oxidative properties of the molecule.

Intranasal vaccination with killed Leishmania amazonensis promastigotes antigen (LaAg) associated with CAF01 adjuvant induces partial protection in BALB/c mice challenged with Leishmania (infantum) chagasi.

The CAF01 adjuvant has previously been shown to be safe for human use and to be a potent adjuvant for several vaccine antigens. In the present work, we sought to optimize the Leishmania amazonensis antigens (LaAg) intranasal vaccine in an attempt to enhance the protective immune responses against Leishmania (infantum) chagasi by using the CAF01 association. LaAg/CAF01 vaccinated mice that were challenged 15 days after booster dose with L. (infantum) chagasi showed a significant reduction in their parasite burden in both the spleen and liver, which is associated with an increase in specific production of IFN-γ and nitrite, and a decrease in IL-4 production. In addition, LaAg/CAF01 intranasal delivery was able to increase lymphoproliferative immune responses after parasite antigen recall. These results suggest the feasibility of using the intranasal route for the delivery of crude antigens and of a human-compatible adjuvant against visceral leishmaniasis.

Chemical manipulation of the mTORC1 pathway in industrially relevant CHOK1 cells enhances production of therapeutic proteins.

The mammalian target of rapamycin complex 1 (mTORC1) is known as a central coordinator of protein synthesis and cell growth in response to the cellular environment. In this work, chemical manipulation of mTORC1 pathway was employed to enhance mAb production as well as increase understanding of intracellular pathways in GS-CHOK1 cells. Using the phosphorylation status of mTORC1 downstream targets, S6K1 and 4E-BP1, as read-outs of mTORC1 activity, we investigated the contribution of each target protein to growth and/or productivity. Inoculation of cultures in the presence of rapamycin, a specific inhibitor of mTORC1, increased viability and final titer. The initial increase in specific productivity and inhibition of growth by rapamycin correlated with diminished phospho-S6K1. However, inhibition was transient and cells recovered by unknown mechanisms. In contrast, phosphorylation of 4E-BP1 was preserved in response to rapamycin. Finally, we examined the activity of mTORC1 after addition of a custom-designed feed. Feeding led to substantial increase in growth and productivity and the phosphorylation of both targets was elevated. Though many details of mTORC1 signaling in CHO cells remain to be clarified, we have provided evidence that environmental manipulation of the mTORC1 pathway correlates with changes in cell growth and recombinant protein production.

Optimization of bioprocess conditions improves production of a CHO cell-derived, bioengineered heparin.

Heparin is the most widely used anticoagulant drug in the world today. Heparin is currently produced from animal tissues, primarily porcine intestines. A recent contamination crisis motivated development of a non-animal-derived source of this critical drug. We hypothesized that Chinese hamster ovary (CHO) cells could be metabolically engineered to produce a bioengineered heparin, equivalent to current pharmaceutical heparin. We previously engineered CHO-S cells to overexpress two exogenous enzymes from the heparin/heparan sulfate biosynthetic pathway, increasing the anticoagulant activity ∼100-fold and the heparin/heparan sulfate yield ∼10-fold. Here, we explored the effects of bioprocess parameters on the yield and anticoagulant activity of the bioengineered GAGs. Fed-batch shaker-flask studies using a proprietary, chemically-defined feed, resulted in ∼two-fold increase in integrated viable cell density and a 70% increase in specific productivity, resulting in nearly three-fold increase in product titer. Transferring the process to a stirred-tank bioreactor increased the productivity further, yielding a final product concentration of ∼90 μg/mL. Unfortunately, the product composition still differs from pharmaceutical heparin, suggesting that additional metabolic engineering will be required. However, these studies clearly demonstrate bioprocess optimization, in parallel with metabolic engineering refinements, will play a substantial role in developing a bioengineered heparin to replace the current animal-derived drug.

Stable overexpression of miR-17 enhances recombinant protein production of CHO cells

miRNAs negatively regulate gene expression at post-transcriptional level, and consequently play an important role in the control of many cellular pathways. The use of miRNAs to engineer Chinese hamster ovary (CHO) cells is an emerging strategy to improve recombinant protein production. Here, we describe the effect of transient and stable miRNA overexpression on CHO cell phenotype. Using an established transient miRNA screening protocol, the effects of miR-17, miR-92a and cluster miR17-92a on CHO growth and protein productivity were studied and followed by analysis of cell pools with stable overexpression of these miRNAs. CHO cells stably engineered with miR-17 exhibited both enhanced growth performance and a 2-fold increase in specific productivity, which resulted in a 3-fold overall increase in EpoFc titer. While further studies of miRNA–mRNA interactions will be necessary to understand the molecular basis of this effect, these data provide valuable evidence for miR-17 as a cell engineering target to enhance CHO cell productivity.

Inhibition of microRNA-let-7a Increases the Specific Productivity of Antibody-Producing CHO Cell Lines

Chinese hamster ovary cells (CHO) are the preferred cell line for the production of recombinant biopharmaceuticals, which constitutes a multi-billion dollar global market. Major challenges to improving protein productivity of CHO in large-scale production cultures include growth level, cellular stress, and translation rate. Because microRNA (miR, miRNA) can simultaneously perturb multiple pathways by inhibiting translation or destabilizing different mRNAs, we explored their utility to extend the cell growth phase or alter protein production per cell (specific productivity) with the goal of enhancing current optimization techniques to increase the production capacity of CHO cell cultures. To investigate the effect of altered microRNA expression on CHO cell viability and specific productivity, two clinically relevant antibody-producing CHO cell lines were stably transduced with lentiviral vectors encoding nine different miRNAs or anti-miRNAs based on their potential involvement in pathways critical for recombinant protein production. Inhibition of miR-let-7a led to a 50%~68% increase in specific productivity in two recombinant antibody-producing cell lines. Furthermore, following miR-let-7a inhibition, we identified increased expression of its targets HMGA2, MYC, NF2, NIRF, RAB40C, and eIF4a which are important mediators of apoptosis, protein translation, and cellular metabolism. Overall, this work provides proof of concept that exogenous microRNA modifications can positively affect specific productivity of CHO cell cultures and highlights the potential of miR-let-7a to have a broad impact on the complex biological functions necessary for improving the capabilities of CHO cell lines.

Protein folding and glycosylation process are influenced by mild hypothermia in batch culture and by specific growth rate in continuous cultures of CHO cells producing rht-PA

Background CHO cells are the primary host for the production of different biopharmaceuticals, including recombinant proteins, monoclonal antibodies, vaccines, etc. Primarily due to their ability to perform properly folding and glycosylation processes required for these proteins acquire adequate biological functionality. However, culturing of these cells in the bioreactor still presents a number of disadvantages, among which can be mention: nutrient depletion, toxic byproducts accumulation, limited oxygen transfer, etc. These issues limit the cell growth and early onset of programmed cell death, which restricts the longevity of cultures and jointly specific productivity of recombinant protein. To overcome these limitations, different approaches have been made to maximize the productivity of these cultures. One of these approaches, that has gained importance during the last 20 years is the use of mild hypothermic temperatures, within a range of 33°C to 30° C. This strategy has been demonstrated to reduce the rate of growth and metabolism of cells but in turn increases the longevity of cultures and increase in specific productivity of a wide range of recombinant proteins in batch cultures [1,2]. One possible cause involved in the increase of specific productivity of recombinant proteins, is the increase in folding capacity and expression of chaperones from endoplasmic reticulum [3,4]. However, the intracellular mechanisms underlying the effect of temperature on the stages of post-translational protein synthesis are still poorly understood. In this regard, the study of endoplasmic reticulum processes (folding, assembly and glycosylation of proteins, and degradation of misfolded proteins through ERAD pathway) has reached a high interest in recent years [4,5]. Reports show that the expression of several

Enhancement of Human Thyrotropin Synthesis by Sodium Butyrate Addition to Serum-Free CHO Cell Culture

The influence of sodium butyrate (NaBu) on the synthesis of recombinant human thyrotropin (r-hTSH) by CHO cells was investigated for the first time. A volumetric productivity of ~10 μg hTSH/mL was repeatedly obtained, with a 3.3-fold increase over a control culture carried out in the absence of NaBu. Since NaBu can induce CHO cell apoptosis and cell growth arrest, the increase in specific productivity was even higher, i.e., ca. 5-fold. Analysis of the N-glycan composition of r-hTSH obtained with the addition of NaBu to the culture medium showed an approximately 12 % increase in the amount of sialic acid, as well as in total carbohydrate, partly due to the increase in the site occupancy from 2.77 to 2.93 glycans per mole of hTSH. The two hormone preparations were characterized by N-glycan structural analysis, which showed that NaBu increased the bi-antennary structures by ca. 13 % while decreasing the tri-antennary structures by approximately the same amount. The in vivo biological activity and pharmacokinetic behavior (clearance) were found to be similar for the two hormone preparations.

Simultaneous environmental manipulations in semi-perfusion cultures of CHO cells producing rh-tPA

We evaluated the combined effect of decreasing the temperature to a mild hypothermia range (34 and 31oC) and switching to a slowly metabolizable carbon source (glucose substituted by galactose) on the growth and production of a recombinant human tissue plasminogen activator (rh-tPA) by Chinese hamster ovary cells in batch and semi-perfusion cultures. In batch cultures using glucose as a carbon source, decreasing the temperature caused a reduction in cell growth and an increase in specific productivity of rh-tPA of 32% at 34oC and 55% at 31oC, compared to cultures at 37oC. Similar behaviour was observed in cultures at 34oC using galactose as a carbon source. Nonetheless, at 31oC, the specific productivity of rh-tPA strongly decreased (about 58%) compared to the culture at 37oC. In semi-perfusion culture, the highest rh-tPA specific productivity was obtained at 34oC. Similarly, whether a decrease in the temperature is accompanied of the replacement of glucose by galactose, the rh-tPA specific productivity improved about 112% over that obtained in semi-perfusion culture carried out at 37oC with glucose as the carbon source. A semi-perfusion culture strategy was implemented based on the combined effect of the chosen carbon source and low temperatures, which was a useful approach for enhance the specific productivity of the recombinant protein.