Elucidating the effects of postinduction glutamine feeding on the growth and productivity of CHO cells

Inducible mammalian expression systems are increasingly being used for the production of valuable therapeutics. In such system, maximizing the product yield is achieved by carefully balancing the biomass concentration during the production phase and the specific productivity of the cells. These two factors are largely determined by the availability of nutrients and/or the presence of toxic waste metabolites in the culture environment. Glutamine is one of the most important components of cell culture medium, since this substrate is an important building block and source of energy for biomass and recombinant protein production. Its metabolism, however, ultimately leads to the formation of ammonia, a well known inhibitor of cellular growth and productivity. In this work, we show that nutrient feeding post‐induction can greatly enhance the product yield by alleviating early limitations encountered in batch. Moreover, varying the amount of glutamine in the feed yielded two distinct culture behaviors post‐induction; whereas excess glutamine allowed to reach greater cell concentrations, glutamine‐limited fed‐batch led to increased cell specific productivity. These two conditions also showed distinctive lactate metabolism. To further assess the physiological impact of glutamine levels on the cells, a comparative ¹³C‐metabolic flux analysis was conducted and a number of key intracellular fluxes were found to be affected by the amount of glutamine present in the feed during the production phase. Such information may provide useful clues for the identification of physiological markers of cell growth and productivity that could further guide the optimization of inducible expression systems. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:535–546, 2014     

CHO cells engineered for fluorescence read out of cell cycle and growth rate in real time

For efficient production of recombinant proteins by mammalian cells in a bioreactor, optimal growth rates are required and represent the most important process parameter. We present the first successful attempt to monitor the growth behavior and cell cycle state of a mammalian production relevant cell line under bioreactor cultivation conditions up to 1.2 l, utilizing a fluorescent read‐out without the need of additional staining or marking. For this purpose, we developed two new production relevant cell line derivatives (CHO‐K1 FUCCI CM & CHO‐K1 FUCCI CN) and corresponding analytical methods. The approach is easily scalable, applicable to mammalian recombinant protein production cell lines, and it allows for real‐time monitoring using appropriate fluorescence probes. It is based on the Ubiquitination‐based Cell Cycle Indicator (FUCCI) system developed by Miyawaki et al. CHO‐K1 was chosen as a model cell line due to its close relationship to several production cell lines.¹ We defined a new process parameter i_red, a quantitative and numerically robust representation of the cell cycle distribution, and demonstrate it to be linearly correlated with the cell cycle state and inversely related to the real time growth rate. Detection of growth rate limitations is possible earlier than using cell‐count‐based approaches. Analytics were compatible with bulk fluorescence methods, using a plate reader as well as a flow cytometer. For future real time applications in industry scale bioreactors we recommend the use of on‐line or at‐line fluorescence probes. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1408–1417, 2017     

CHO工程细胞无血清悬浮分批培养的生长代谢特征及动力学模型

以悬浮适应的表达尿激酶原CHO工程细胞为研究对象,在100mL的摇瓶中进行无血清悬浮培养,以细胞密度、细胞活力、Pro-UK活性、葡萄糖比消耗速率(qglc)、乳酸比生产速率(qlac)、乳酸对葡萄糖的得率系数(Ylac/glc)为观察指标,同时以细胞有血清悬浮培养作为参照,考察CHO工程细胞无血清悬浮培养生长和代谢特征。观察结果表明,CHO工程细胞在无血清及有血清悬浮培养条件下表现为大致相似的细胞生长和代谢特征。在此基础上,依据实际检测的数据,应用MATLAB软件对细胞对数生长期的细胞生长、乳酸生成及葡萄糖消耗的模型参数进行非线性规划,获得全局性收敛的最优参数估计值,建立了细胞在无血清培养条件下的生长及代谢动力学模型。     

On-line calibration of a computerized biosensor system for continuous measurements of glucose and lactate

An on-line calibration procedure for application in continuous monitoring systems has been developed. Control of the calibration value and recalibration on-line during monitoring is possible without having to disrupt the sample withdrawal. The calibration procedure has been applied and evaluated in a continuous biosensor system based on the detection of oxygen depletion during enzymatic substrate conversion by immobilized oxidases. Evaluation included on-line calibration during continuous measurements of glucose and lactate in bovine blood samples. Calibration of the complete system consisting of a sampling device, a sample handling step, a biocatalytic step, a detection step, and a data processing unit is performed by the on-line addition of a calibration solution to a blank sample which is fed through the system. The calibration cycle is completed within 5.5 min. When recalibration is carried out during monitoring, the calibration solution is added to the sample, instead of to a blank sample, and the increase in outlet singl is registered. The major advantage of this internal standard principle is that the calibration solution is fed through the whole system according to the same path as the sample solution and thus takes into account all parameters influencing the sample. (c) 1995 John Wiley & Sons, Inc.     

Design of serum-free medium for suspension culture of CHO cells on the basis of general commercial media

The design of serum-free media for suspension culture of genetically engineered Chinese hamster ovary (CHO) cells using general commercial media as a basis was investigated. Subcultivation using a commercial serum-free medium containing insulin-like growth factor (IGF)-1 with or without FCS necessitated additives other than IGF-1 to compensate for the lack of FCS and improve cell growth. Suspension culture with media containing several combinations of growth factors suggested the effectiveness of addition of both IGF-1 and the lipid signaling molecule lysophosphatidic acid (LPA) for promoting cell growth. Subcultivation of CHO cells in suspension culture using the commercial serum-free medium EX-CELL™302, which contained an IGF-1 analog, supplemented with LPA resulted in gradually increasing specific growth rate comparable to the serum-containing medium and in almost the same high antibody production regardless of the number of generations. The culture with EX-CELL™302 supplemented with LPA in a jar fermentor with pH control at 6.9 showed an apparently higher cell growth rate than the cultures without pH control and with pH control at 6.8. The cell growth in the medium supplemented with aurintricarboxylic acid (ATA), which was much cheaper than IGF-1, in combination with LPA was synergistically promoted similarly to that in the medium supplemented with IGF-1 and LPA. In conclusion, the serum-free medium designed on the basis of general commercial media could support the growth of CHO cells and antibody production comparable to serum-containing medium in suspension culture. Moreover, the possibility of cost reduction by the substitution of IGF-1 with ATA was also shown.     

Chemically defined medium for the production of biologically active substances of CHO cells

A recombinant CHO cell line (GT19) secreting a high level of human growth hormone (hGH) was constructed with amplification of the introduced hGH gene. The cells grew well in the alpha MEM medium supplemented with 5% dialyzed fetal calf serum (dFCS), but not with less than 1% dFCS. Therefore we examined various medium components and obtained an improved medium which supported cell growth at low serum concentrations. The production of hGH by the cells was also enhanced in this medium.Abbreviations CHO Chinese hamster ovary - hGH human growth hormone - dFCS dialyzed fetal calf serum - dhfr dihydroforate reductase - MTX methotrexate     

Estimation of surface heat flux and temperature distributions in a multilayer tissue based on the hyperbolic model of heat conduction

In this study, an inverse algorithm based on the conjugate gradient method and the discrepancy principle is applied to solve the inverse hyperbolic heat conduction problem in estimating the unknown time-dependent surface heat flux in a skin tissue, which is stratified into epidermis, dermis, and subcutaneous layers, from the temperature measurements taken within the medium. Subsequently, the temperature distributions in the tissue can be calculated as well. The concept of finite heat propagation velocity is applied to the modeling of the bioheat transfer problem. The inverse solutions will be justified based on the numerical experiments in which two different heat flux distributions are to be determined. The temperature data obtained from the direct problem are used to simulate the temperature measurements. The influence of measurement errors on the precision of the estimated results is also investigated. Results show that an excellent estimation on the time-dependent surface heat flux can be obtained for the test cases considered in this study.     

Enhancement of immunoregulatory effects of Lactobacillus gasseri TMC0356 by heat treatment and culture medium

Aims: The aim of this study was to investigate the influence of heat treatment and culture media on the immunoregulatory effects of a probiotic strain, Lactobacillus gasseri TMC0356 (TMC0356). Methods and Results: TMC0356 cultured in deMan–Rogosa–Sharpe and same food grade (FG) media were inactivated with the heat treatment at 70 and 90°C. Viable and heat‐killed TMC0356 were tested for their ability to induce interleukin (IL)‐12 production in the murine macrophage cell line J774.1. These TMC0356 were examined for their resistance to N‐acetylmuramidase. Their morphology was observed by scanning electron microscopy. The heat‐killed TMC0356 significantly induced IL‐12 production in J774.1 cells and exhibited enhanced resistance to N‐acetylmuramidase compared with viable TMC0356. Morphological changes were observed in TMC0356 when cultured in FG medium. Cell morphology and induction of IL‐12 production in J774.1 cells were also associated. Conclusions: These results suggest that heat treatment and culture medium composition modified the immunoregulatory effects of TMC0356 to induce IL‐12 production in macrophages. Significance and Impact of the Study: These results demonstrate that probiotic immunoregulatory effects may be modified by the processing technology of cell preparation.     

Uncoupling of cell growth and proliferation results in enhancement of productivity in p21CIP1-arrested CHO cells

Chinese hamster ovary cells have been engineered to inducibly over-express the p21(CIP1) cyclin-dependent kinase inhibitor, to achieve cell cycle arrest and increase cell productivity. In p21(CIP1)-arrested cells production of antibody from a stably integrated lgG4 gene, was enhanced approximately fourfold. The underlying physiological basis for enhanced productivity was investigated by measuring a range of cellular and metabolic parameters. Interestingly, the average cell volume of arrested cells was approximately fourfold greater than that of proliferating cells. This was accompanied by significant increases in mitochondrial mass, mitochondrial activity, and ribosomal protein S6 levels. Our results suggest that p21(CIP1)-induced cell cycle arrest uncouples cell growth from cell-cycle progression, and provides new insight into how improved productivity can be achieved in a cell line commonly used for large-scale production of pharmaceutical proteins.     

Role of iron and sodium citrate in animal protein-free CHO cell culture medium on cell growth and monoclonal antibody production

Chemically defined iron compounds were investigated for the development of animal protein-free cell culture media to support growth of CHO cells and production of monoclonal antibodies (mAb). Using a multivessel approach of 96-well plates, shake flasks, and bioreactors, we identified iron and its chemical partner citrate as critical components for maintenance of continuous cell growth and mAb production. The optimized iron concentration range was determined to be 0.1-0.5 mM and that for citrate 0.125-1 mM. This complete formulation is able to maintain cell growth to similar levels as those supplemented with iron compounds alone; however, mAb productivity was enhanced by 30-40% when citrate was present. The addition of sodium citrate (SC) did not affect product quality as determined by size exclusion chromatography, ion exchange chromatography, reversed phase and normal phase-HPLC. No significant changes in glucose and lactate profiles, amino acid utilization, or mAb heavy and light chain expression ratios were observed. Cellular ATP level was ～30% higher when SC was included suggesting that SC may have a role in enhancing cellular energy content. When cell lysates were analyzed by LC-MS to assess the overall cellular protein profile, we identified that in the SC-containing sample, proteins involved in ribosome formation and protein folding were upregulated, and those functions in protein degradation were downregulated. Taken together, this data demonstrated that iron and citrate combination significantly enhanced mAb production without altering product quality and suggested these compounds had a role in upregulating the protein synthetic machinery to promote protein production.     

Development of a new medium for the culture of agallian leafhopper cells

Summary The optimal composition of a medium for tissue culture of cells from the leafhopperAgallia constricta was estimated from experiments in which the rate of growth of the cells was measured at different concentrations of each component. Wound tumor virus and theconstricta variety of potato yellow dwarf virus multiply, inA. constricta when these viruses are transmitted from one plant to another. Differences weredetected in the suitablility of different batches of fetal bovine serum (after heating at 56°C for 30 min), and histidine as components in the tissue culture medium. Without heat treatment even thebest fetal bovine serum was not suitable. Estimated optimal concentrations of fetal bovine serum, histidine, salts, dextrose, lactalbumin hydrolysate and yeast autolysate were determined. Fungizone (amphotericin B) at 50 or 100 mg per 1 caused harmful effects; effective concentrations of penicillin, neomycin and steptomycin did not. Combinations of histidine-HCl and histidine (free-base) made it possible to prepare buffered medium at my pH between 6.0 and 7.0. Optimal growth ofA. constricta cells occurred at pH 6.43, and ofAceratagallia sanguinolenta at pH 6.30. Osmotic pressures of the new medium between 360 and 405 mOSM were better than lower osmotic pressures. The new medium was still suitable for growth of the cells after, storage for 6 months at 4°C. Portion of a thesis submitted for the Ph.D. degree by the senior author to the Graduate College of the University of Illinois. This research was supported in part by Grant GB 20915 from the National Science Foundation and Grant AI 6392 from the National Institutes of Health.     

Metabolic flux analysis of CHO cells at growth and non-growth phases using isotopic tracers and mass spectrometry

Chinese hamster ovary (CHO) cells are the main platform for production of biotherapeutics in the biopharmaceutical industry. However, relatively little is known about the metabolism of CHO cells in cell culture. In this work, metabolism of CHO cells was studied at the growth phase and early stationary phase using isotopic tracers and mass spectrometry. CHO cells were grown in fed-batch culture over a period of six days. On days 2 and 4, [1,2-¹³C] glucose was introduced and the labeling of intracellular metabolites was measured by gas chromatography-mass spectrometry (GC–MS) at 6, 12 and 24 h following the introduction of tracer. Intracellular metabolic fluxes were quantified from measured extracellular rates and ¹³C-labeling dynamics of intracellular metabolites using non-stationary ¹³C-metabolic flux analysis (¹³C-MFA). The flux results revealed significant rewiring of intracellular metabolic fluxes in the transition from growth to non-growth, including changes in energy metabolism, redox metabolism, oxidative pentose phosphate pathway and anaplerosis. At the exponential phase, CHO cell metabolism was characterized by a high flux of glycolysis from glucose to lactate, anaplerosis from pyruvate to oxaloacetate and from glutamate to α-ketoglutarate, and cataplerosis though malic enzyme. At the stationary phase, the flux map was characterized by a reduced flux of glycolysis, net lactate uptake, oxidative pentose phosphate pathway flux, and reduced rate of anaplerosis. The fluxes of pyruvate dehydrogenase and TCA cycle were similar at the exponential and stationary phase. The results presented here provide a solid foundation for future studies of CHO cell metabolism for applications such as cell line development and medium optimization for high-titer production of recombinant proteins.     

A simple medium for the study of hepatocyte growth in culture under defined conditions

Summary The combination (1∶1) of Dulbecco's modified Eagle's medium and Waymouth's medium MAB 87/3 was found to provide favorable conditions for serum-free culture and growth of adult rat hepatocytes. In this simple medium, a majority of hepatocytes stimulated by epidermal growth factor plus insulin entered S phase and divided, with a normal (13 h) interval between DNA synthesis and cell division. The proliferative response did not require extra substratum or the presence of serum, even during cell isolation and plating. This work was supported by the Norwegian Cancer Society.     

Evaluation of the serum-free medium MDSS2 for the production of poliovirus on vero cells in bioreactors

The serum-free medium MDSS2 (Merten et al., 1994), was used for cultivating Vero cells as well as for producing poliovirus (Sabin type 1) in static and in perfused micro-carrier cultures. At slightly different growth rates of 0.0120/h and 0.0106/h, respectively, static cultures in serum-containing (SCM) and serum-free (SFM) medium produced titers of ^106.75 and 10^6.67 TCID50 per 50 μl; signifying a specific productivity of 0.89 and 1.07 TCID50/c. Serum-free bioreactor cultures of Vero cells on DEAE-dextran microcarriers at 6.25 g/l produced cell densities of about 1.5×10⁶c/ml. After infection with virus (multiplicity of infection (MOI) 0.1–0.3) titers of about 6.3×10⁸ TCID50/ml were obtained, signifying an average specific productivity of 7.1 TCID50/c.h. Although these values were 4 and 2 fold, respectively, higher than in classical resum-based production processes (Montagnon et al. Dev. biol. Stand. 1981, 47, 55), a reference culture, for which cell growth was done in SCM and only virus production was done in SFM, produced 2×10⁹ TCID/ml with an average specific virus production rate of 18.9 TCID50/c.h. The differences between the fully serum-free and our reference process were mainly due to physiological differences of cells grown in SCM and SFM and also due to strongly modified consumption kinetics after virus infection leading to limitations of one or several essential medium compounds, like glucose and amino acids. Avoiding these limitations by increasing the residual concentration of glucose, glutamine, histidine, and SH-amino acids, led to specific virus production rates (of about 17.9 TCID59/c.h.) comparable to those found in the reference virus production process. The optimisation of the production of the poliovirus (Sabin 1) will be described with respect to the modification of the medium composition. This revised version was published online in June 2006 with corrections to the Cover Date.     

Addressing the influence of instrument surface heat exchange on the measurements of CO2 flux from open-path gas analyzers

There is a growing concern in the flux community that using the eddy covariance method with open‐path CO₂ analyzers often leads to measurements of an apparent ecosystem CO₂ uptake during off‐season periods, especially in cold climates. Such uptake has not been observed when measurements were made with closed‐path analyzers, chambers, or profile methods, suggesting it is an artifact due in some way to the use of open‐path analyzers. In this study, a series of laboratory tests and field experiments were conducted to determine the magnitude of the instrument surface heat exchange in the open path and its relationship with the measured CO₂ flux. Results showed that (1) the surface of an open‐path instrument became substantially warmer than ambient due to electronics and radiation load during daytime, while at night, radiative cooling moderated temperature increases in the path; (2) high‐frequency temperature measurements inside the path were correlated with vertical wind speed producing sensible heat flux inside the instrument path exceeding the ambient heat flux by up to 14%; (3) enclosing the open‐path instrument eliminated the sensible heat flux in the path, and caused measured CO₂ flux to match a closed‐path reference; (4) using sensible heat flux measured directly inside the open path in the WPL term instead of the ambient sensible heat flux also led to a match in CO₂ flux between open‐path instrument and closed‐path reference; and (5) correcting previously collected open‐path CO₂ flux data was possible by estimating the instrument heating effect with a semi‐empirical model using standard weather variables. Results showed that all proposed techniques led to a significant reduction in apparent CO₂ uptake during off‐season periods and to a reduction of the underestimation of CO₂ release in other periods. Close agreement between the open‐path measurements and closed‐path references was achieved in all cases.     

Clonal growth and culture life span of bovine adrenocortical cells in a serum-free medium

Summary The life span and growth from clonal density of bovine adrenocortical cell cultures were studied in serum-supplemented medium and a serum-free defined medium, which supported sustained cell proliferation and steroid production. The total culture life span was 79 population doublings in serum-supplemented medium with fibroblast growth factor (FGF) and 36 population doublings in the defined medium without serum. Older passage cell cultures grown in the defined medium progressively lost the ability to produce 11β- and 21-hydroxylated steroids, which was observed previously for cultures in serum-supplemented medium, and also had a decline of 17α-hydroxylated steroid production. The cloning efficiency in the defined medium was 12.2% as compared to 24% in serum-supplemented medium with FGF. Five isolated clonal cell lines grown in the defined medium were characterized for steroid function in response to steroidogenic agents. All five clonal cell lines had stimulated steroid production with 8-bromo-cAMP, but only four of the clonal lines were stimulated also by adrenocorticotropin. None of the clonal cell lines produced 11β-, 21- or 17α-hydroxylated steroids in response to treatment with either steroidogenic agent, results that were similar to data obtained from older mass cultures. The apparent deficiency of the defined medium as compared to serum-supplemented medium for maximum support of the culture life span and cloning efficiency may be useful in studies of cellular aging and its relation to differentiated function for this cell culture system. This study was supported by the Iowa Diabetes and Endocrinology Research Center (grant AM25295 from the National Institutes of Health, Bethesda, MD). D.A.F. was supported by a National Research Service Award from the National Institutes of Health (grant HL07485).     

A serum-free medium for the culture of insect cells and production of recombinant proteins

Summary A low protein aqueous lipid supplement (Ex-Cyte VLE), in combination with pluronic polyol, is an effective replacement for fetal bovine serum for insect Sf-9 cells. Serum-free medium with lipid supplement and pluronic (SFM-LP) supported higher cell viability and maximum cell populations than serum-supplemented medium. No adaptation procedures are required when switching cells from serum-containing medium to SFM-LP, and growth rates remain constant during continued passages in SFM-LP. The amounts of recombinant proteins produced, which is the major use for the Sf-9 cells, are better or equal in SFM-LP compared to serum-supplemented medium. SFM-LP also supports growth of the TN-368 cell line but IPLB-SF-21AE or IZD-Mb0503 lines grow poorly in this medium.     

Serum-free culture conditions for the growth of normal rat mammary epithelial cells in primary culture

Summary A monolayer culture system has recently been developed for the extended growth and serial passage of normal rat mammary epithelial (RME) cells. In this system the cells undergo greater than 20 population doublings when grown on type I collagen-coated tissue culture dishes in Ham's F12 medium supplemented with insulin, hydrocortisone, epidermal growth factor, prolactin, progesterone, cholera toxin, and 5% fetal bovine serum (FBS). The purpose of the present studies was to define additional growth factors that would allow equivalent RME cell proliferation in serum-free medium. Ethanolamine (EA) was effective at reducing the FBS requirements for RME cell proliferation and at its optimum concentration did so by greater than 20-fold. Even with optimum levels of EA there was essentially no cell proliferation in the absence of FBS. However, addition of bovine serum albumin (BSA) to the hormone, growth factor, and EA-supplemented medium resulted in substantial proliferation in the absence of serum, and the further addition of transferrin (T) potentiated this effect. Thus, in this culture system, replacement of FBS with EA, BSA, and T resulted in RME cell proliferation in primary culture which was equivalent to that obtained in the 5% FBS-containing medium. This work was supported by grant RR-05529 from the Division of Research Resources, National Institutes of Health, Bethesda, MD, and by Public Health Service grant CA40064-01 from the National Cancer Institute, Bethesda, MD.     

A protein-free medium for the growth of hybridomas and other cells of the immune system

Summary A protein-free medium, termed ABC, has been developed which essentially eliminates the need for serum proteins. ABC supports the long-term growth of murine hybridomas as well as other transformed cells of the immune system. The requirement of hybridoma growth for transferrin has been met by substituting the soluble organo-iron compound, sodium nitroprusside. Substantial improvement in the growth of hybridomas was afforded by the inclusion of 18 trace elements complexed to disodium ethylene diaminetetraacetate (EDTA). The medium was further improved by the inclusion of components not found in Ham's F12 medium or by raising the concentrations of existing low molecular weight components. Murine hybridomas can be cultured routinely in this protein-free medium in an anchorage-independent manner with doubling times generally under 24 h. Visualized on electrophoretic gels, levels of monoclonal antibody taken from those cultures often exceeded 80% of the total protein. The medium was also able to support the growth of HuT 78 and H9 cells as well as certain other transformed cells of the immune system. In addition, normal human peripheral blood lymphocytes, activated with phytohemagglutinin and cultured with 50 U/ml recombinant interleukin 2, could be grown for 2 wk with a 50-fold expansion over input cell number.     

Development and manufacturability assessment of chemically‐defined medium for the production of protein therapeutics in CHO cells

Advantages of using internally developed chemically‐defined (CD) media for cell culture‐based therapeutic protein production over commercial media include better raw material control and medium vendor options, and most importantly, flexibility for process development and subsequent optimization needed for therapeutic protein production. Through several rounds of design of experiment (DOE) screening, and medium component supplementation and optimization studies, we successfully developed a CD basal medium (CDM) for CHO cell culture. The internally prepared liquid CDM demonstrated comparable cell culture performance to that from a commercially available control medium. However, when the same CDM formulation was transferred to two major commercial medium suppliers for manufacturing, cell culture performance utilizing these newly prepared media was significantly reduced compared with the in‐house prepared counterpart. An investigation was launched to assess whether key medium components were sensitive to large‐scale preparation of the final bulk media by the vendors. Further work necessitated the reformulation of the original CDM formulation into a core medium that was suitable for large‐scale media manufacturing. The modified preparation of the core medium with two separate supplements to generate the final CDM was able to recover the expected cell culture performance and monoclonal antibody (mAb) productivity. Confirmation of cell culture robustness in cell growth and production was corroborated in two additional mAb‐expressing cell lines. This work demonstrates that a robust CD medium is not only one that performs during the development stage, but also one that must be reproducible by commercial media vendors. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1163–1171, 2015