Characteristics of CHO-K1 cell culture producing two types of recombinant soluble thrombomodulin

Recombinant CHO-K1 cells, expressing human soluble thrombomodulin, were cultured in a serum-free medium and characteristics of the culture associated with glucose and lactate were investigated. In 3 L fermentor (3LFM) cultures, the cell density was found to have a proportional relationship with the volumetric glucose consumption rate, and the specific glucose consumption rates were constant at about 0.2 mg/(10⁶ cells·d) despite many differences in the culture conditions. Thus, it was concluded that the glucose consumption rate is little influenced by the condition of the cells or the culture conditions, and that the cell density can be estimated by the glucose consumption rate calculated from glucose measurement. Two types of thrombomodulin (rsTMα and rsTMβ) were produced, in which rsTMβ possesses chondroitin-4-sulfate and has greater anticoagulant activities than rsTMα. Therefore, it is important to investigate the rsTMα and rsTMβ production properties, and to determine the optimal culture conditions for high rsTMβ production. The most important factor to increase the production of rsTMβ relative to rsTMα (the β/α ratio) was effective aeration. Moreover, a lower ratio of lactate production/glucose consumption (the L/G ratio) with sufficient oxygen, high glucose concentration, and a longer medium exchange interval contributed to a higher specific rsTMβ production rate. Since there was a linear relationship between the production rate of each type of rsTM and the overall rsTM production rate per liter, it is expected that the rsTMα and rsTMβ production rates may be able to be estimated from the overall rate and the rsTMβ production increased by increasing the overall rsTM production with a lower L/G ratio.     

Continuous culture of CHO-K1 cells producing thrombomodulin and estimation of culture conditions

Recombinant Chinese hamster ovary (CHO-K1) cells expressing human soluble thrombomodulin (rsTM) were cultured in a continuous culture system with a fluidized-bed reactor. Cells were grown in a medium containing 1% serum for 10 d, and then cultured in a serum-free medium. The protein production rate increased remarkably in the serum-free culture, with a decrease in the lactate production rate. This suggests that CHO-K1 cells exhibit different physiological characteristics in response to serum removal from the medium, which resulted in a higher rsTM concentration (about 60 mg/l). A procedure for estimating protein productivity was developed using experimental glucose and lactate measurements. In this procedure, cell density was estimated from the glucose consumption rate, and the specific protein (rsTM) production rate was obtained from the ratio of lactate production/glucose consumption (ΔL/ΔG). Since the cell density and protein productivity in repeated batch culture were well estimated, the procedure was applied to continuous culture in a fluidized-bed bioreactor culture. The estimation procedure was also found to be effective in this continuous culture using the models derived from the repeated batch culture.     

Comparison of manufacturing techniques for adenovirus production

We have compared three different production methods, which may be suitable for the large scale production of adenovirus vectors for human clinical trials. The procedures compared 293 cells adapted to suspension growth in serum-free medium in a stirred tank bioreactor, 293 cells on microcarriers in serum-containing medium in a stirred tank bioreactor, and 293 cells grown in standard tissue culture plasticware. With a given virus, yields varied between 2000 and 10,000 infectious units/cell. The stirred tank bioreactor routinely produced between 4000 and 7000 infectious units/cell when 293 cells were grown on microcarriers. The 293 cells adapted to suspension growth in serum-free medium in the same stirred tank bioreactor yielded between 2000 and 7000 infectious units/cell. Yields obtained from standard tissue culture plasticware were up to 10,000 infectious units/cell. Cell culture conditions were monitored for glucose consumption, lactate production, and ammonia accumulation. Glucose consumption and lactate accumulation correlated well with the cell growth parameters. Ammonia production does not appear to be significant. Based on virus yields, ease of operation and linear scalability, large-scale adenovirus production seems feasible using 293 cells (adapted to suspension/serum free medium or on microcarriers in serum containing medium) in a stirred tank bioreactor. This revised version was published online in July 2006 with corrections to the Cover Date.     

表达抗-CD25单克隆抗体的GS-NS0骨髓瘤细胞无血清培养及代谢特性

抗-CD25单克隆抗体作为免疫抑制剂拥有广阔的市场前景和巨大的经济价值。本实验以表达抗?CD25单克隆抗体的GS-NS0细胞为研究对象,开发了支持其大规模培养和抗体表达的无血清低蛋白培养基,批培养最大活细胞密度和最大抗体浓度分别达3×106cells/mL和300mg/L以上,比商业无血清培养基(Excell 620+0.2% primatone)分别提高了100%和46%。通过批培养实验,研究了细胞的生长、葡萄糖和氨基酸代谢、以及产物表达特点,并揭示了批培养过程中初始葡萄糖浓度对GS-NS0细胞生长与代谢的影响规律。为优化GS-NS0细胞培养过程和抗CD25单抗成功迈向产业化提供了重要的科学依据。     

Chinese hamster ovary cell growth and interferon production kinetics in stirred batch culture

Summary Recombinant human interferon- production by Chinese hamster ovary cells was restricted to the growth phase of batch cultures in serum-free medium. The specific interferon production rate was highest during the initial period of exponential growth but declined subsequently in parallel with specific growth rate. This decline in specific growth rate and interferon productivity was associated with a decline in specific metabolic activity as determined by the rate of glucose uptake and the rates of lactate and ammonia production. The ammonia and lactate concentrations that had accumulated by the end of the batch culture were not inhibitory to growth. Glucose was exhausted by the end of the growth phase but increased glucose concentrations did not improve the cell yield or interferon production kinetics. Analysis of amino acid metabolism showed that glutamine and asparagine were exhausted by the end of the growth phase, but supplementation of these amino acids did not improve either cell or product yields. When glutamine was omitted from the growth medium there was no cell proliferation but interferon production occurred, suggesting that recombinant protein production can be uncoupled from cell proliferation. Offprint requests to: P. M. Hayter     

Hybridoma cell growth and anti-neuroblastoma monoclonal antibody production in spinner flasks using a protein-free medium with microcarriers

The main disadvantages of foetal calf serum as the world-wide common serum supplement for cell growth are its content of various proteins of variable concentrations between batches as well as its high cost. The use of serum-free and protein-free media is gradually becoming one of the goals of cell culture especially for standardizing culture conditions or for simple purification of cell products like monoclonal antibodies. The mouse hybridoma cells 14/2/1 were cultivated either in protein-free UltraDOMA medium or in serum-containing RPMI medium with and without microcarriers to generate high quantities of monoclonal antibodies against neuroblastoma tumour cells. Cell growth rate, IgG production, viability, glucose and lactate concentrations, attachment rate and doubling time have been used as investigation criteria. Modifications of culture procedures (static or stirred), inoculum density, and microcarrier concentration caused an improvement of monoclonal antibody production. The kinetics of antibody synthesis was best in spinner culture with 2 ml of microcarriers in protein-free medium. These results of short-term microcarrier culture in stirred spinner flasks indicate that IgG yields in protein-free medium 2.5-fold higher to those in serum-supplemented medium can be achieved.     

Increased production of succinic acid in Escherichia coli by overexpression of malate dehydrogenase

Escherichia coli NZN111 is a double mutant with inactivated lactate dehydrogenase and pyruvate formate-lyase. It cannot utilize glucose anaerobically because of its unusually high intracellular NADH/NAD(+) ratio. We have now constructed a recombinant strain, E. coli NZN111/pTrc99a-mdh, which, during anaerobic fermentation, produced 4.3 g succinic acid l(-1) from 13.5 g glucose l(-1). The NADH/NAD(+) ratio decreased from 0.64 to 0.26. Furthermore, dual-phase fermentation (aerobic growth followed by anaerobic phase) resulted in enhanced succinic acid production and reduced byproduct formation. The yield of succinic acid from glucose during the anaerobic phase was 0.72 g g(-1), and the productivity was 1.01 g l(-1) h(-1).     

Physiological changes during the adaptation of hybridoma cells to low serum and serum-free media

Two murine hybridoma cell lines (167.4G5.3 and S3H5/gamma2bA2) were adapted to grow in low-serum and serum-free media by a weaning procedure. The changes in cell growth, metabolic, and antibody production rates with adaptation were examined using biochemical and flow cytometric analyses. After adaptation to a particular serum level, the short-term serum response of the cells was experimentally determined. Specific growth rates, glucose and glutamine uptake and lactate and ammonia production rates, and specific antibody production rates were evaluated from the data. For both cell lines, an improvement in cell growth was observed after adaptation, and both higher growth rates and higher cell concentrations were obtained. The specific glucose and glutamine uptake rates and the lactate and ammonia production rates changed insignificantly with adaptation. Conversely, changes in the specific antibody production rate of the two cell lines differed. Cell line 167.4G5.3 showed a loss in antibody productivity at low serum levels, while the S3H5/gamma2bA2 kept its original productivity in low-serum-containing media. The intracellular antibody content for S3H5/gamma2bA2 cells remained unaltered by adaptation, but a low antibody containing cell population appeared in the 167.4G5.3 culture. The loss of specific antibody productivity in this cell line was due to the appearance of this population.     

重组CHO-GS细胞降低氨毒副作用的代谢研究

在重组CHOGS细胞无血清批培养过程中,由于GS系统的引入,使氨对细胞的毒副作用显著降低,从而引起细胞生长和代谢途径发生变化。当起始氨浓度为1.42mmolL时,细胞最高密度可达到15.6×105cellsmL,随着氨浓度的增加,尽管细胞生长受到一定的抑制,但在氨浓度为12.65mmolL时,细胞密度仍可达到8.9×105cellsmL。当起始氨浓度从0.36mmolL增加到12.65mmolL时,细胞对葡萄糖的得率系数和乳酸对葡萄糖的得率系数降低,己糖激酶(HK)、丙酮酸激酶(PK)和乳酸脱氢酶(LDH)酶活分别提高了43%、140%和25%,表明细胞对葡萄糖的利用增加,糖代谢更倾向于高能量生成途径。在谷氨酰胺代谢途径中,氨促进了谷丙转氨酶(GPT)酶活,谷氨酸到α酮戊二酸的转化逐渐倾向于谷丙转氨途径,谷氨酸脱氢酶(GDH)酶活降低,脱氨途径相应受到抑制。此外,氨浓度的增加使细胞群体处于G0G1期的比例逐渐升高,当氨浓度为12.65mmolL时,重组蛋白比生产速率比氨浓度为0.36mmolL时提高了2.1倍。     

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.     

A novel serum-free medium for the cultivation of Vero cells on microcarriers

Summary A novel serum-free medium for the cultivation of Vero cells on microcarriers was developed,which composed of the 1:1 mixture of Dubecco's Modified Eagle Medium: Nutrient Mixture F12, bovine serum albumin(BSA) or human serum albumin(HSA), epidermal growth factor(EGF), gelatin and Dbiotin. Both BSA and EGF were effective on cell growth, adhesion and spreading. Further addition of gelatin and biotin led to the enhanced cell adhesion and spreading without growth promoting activity. The serum-free medium was suitable for the cultivation of vero cells on several different microcarriers with cell density reached over 3×l0⁶cells/ml.     

Continuous production and recovery of recombinant Ca2+ binding receptor from HEK 293 cells using perfusion through a packed bed bioreactor

The extracellular domain of human parathyroid Ca²⁺ receptor was needed in order to study itsstructure and clinical application. The Ca²⁺receptor is a unique member of the G protein-coupledreceptor super-family, expressed in parathyroid andkidney cells where it has been shown to play acritical role in extracellular calcium homeostasis.The desired protein was produced by immobilizing thetransformed HEK 293 cells in a packed-bedconfiguration using a 1.6 l (working volume)bioreactor equipped with a vertical mixing impellerassembly and an internal basket. The process includeda propagation phase followed by a production phase. Inthe propagation phase, lasting approximately 160 h, the bed was perfused with a serum-containingmedium, allowing the cells to grow at a constantgrowth rate to approximately 3 × 10¹⁰. At this point the production phase was begun, replacing themedium with serum-free medium and continuing theperfusion process for additional 350 h. Duringthis phase, the medium was pumped through the packedbed at a rate of 4–6 l per day, keeping theresidual glucose concentration around 1 g l^-1 andcollecting and processing approximately 80 l ofspent medium. This continuous perfusion method of thepacked-bed bioreactor was compared to a repeated batchmethod in which existing medium was replenished whenthe glucose concentration was down to 1 g l^-1. Using this method, serum-free medium was replaced withserum containing medium a few times when a decline inthe glucose consumption was observed. Though mediumconsumption and protein yield are similar in bothmethods (roughly 10 mg l^-1), there aredifferences related to the ease of operation andprocessing of the produced protein. The continuousperfusion operation was found to be preferable and waschosen as the production strategy.     

Vero细胞微载体培养的化学成分明确无血清培养基的设计

目的：设计适用于Vero细胞微载体培养的化学成分明确无血清培养基。方法：以商品化的DMEM／F12合成培养基为基础培养基,应用Plackett—Burman实验设计和响应面分析法设计支持Vero细胞微载体培养的化学成分明确无血清培养基。结果：以细胞密度为评价指标,在单因素实验的基础上采用Plackett-Burman实验设计考察10种培养基添加成分对Vero细胞生长的影响,确定了3种对Vero细胞生长起明显促进作用的培养基添加成分,为胰岛素、血清素和腐胺。继而利用响应面法分析了这3种添加成分的最佳水平范围,设计了一种支持Vero细胞贴附培养的无血清培养基（VERO—SFM—A）。在Bellco搅拌式培养瓶中采用VERO-SFM．A和Cytodex1微载体培养Vero细胞,细胞密度由接种时的4×10^5cells／ml增加到培养6d后的22．3×10^cells／ml,细胞活力保持在96％以上。结论：VERO—SFM—A能够有效地支持Vero细胞在微载体表面固定化生长并达到较高的细胞密度,具有实际应用于Vero细胞微载体规模化培养的应用潜力。     

Perfusion-microcarrier cultivation of rCHO-cells in serum-free medium for production of human renin

A recombinant CHO cell line producing human prorenin was cultivated on microcarriers in serum-free medium. Best growth was obtained when the cells were cultivated on a collagen coated microcarrier (Cytodex 3) using a serum-free medium (SF-02) supplemented with fibronectin. It was possible to reduce the necessary concentration of fibronectin in the feed medium from 10 g/cm³ to 2 g/cm³ during perfusion cultures in a spinner reactor equipped with an UF-membrane. Also in this system, the prorenin concentration increased up to 8 times higher compared to that in a conventional repeated-batch culture. The cells grew in multilayers on the microcarriers during the perfusion culture. The specific prorenin productivity was not significantly affected by the cell growth rate, and the secretion of prorenin continued even after the cells had ceased to grow.     

Repeated batch cultivation of rBHK cells on Cytodex 3 microcarriers: antithrombin III,amino acid,and fatty acid metabolic quotients

Anchorage-dependent human antithrombin III-producing recombinant baby hamster kidney (rBHK) cells were cultivated on Cytodex 3 microcarriers in repeated batch mode. During a 3-month experiment four different low-serum (0.025% fetal bovine serum) or serum-free medium formulations were evaluated for (a) the initial growth phase of cells and (b) the subsequent production phase, whereby two free fatty acid (FFA) supplements were examined with respect to their growth-promoting and product-formation-enhancing properties. Selected nutrient and (by)product consumption and production rates (including those for antithrombin III, amino acids, and fatty acids) are reported. The calculated metabolic quotients reflect the prevailing slow growth conditions ( approx. 0.06 day^–1) associated with microcarrier cultures. Specific antithrombin III productivities vary singificantly as a function of the feed medium supplementation with FFA. Correspondence to: G. Schmid     

多孔微载体无血清培养rCHO细胞生产u-PA

在30L搅拌式反应器中无血清培养分泌尿激酶型纤溶酶原激活剂(u-PA)的DNA重组CHO细胞,定期部分更换Cytopore多孔微载体,使生长在多孔微载体中的细胞不断更新繁殖,解决大规模细胞培养中的细胞凋亡问题。在91d连接换液培养过程中,细胞密度可维持在(1.3～2.6)×107／mL,活细胞比率维持在90％以上。在7.5L搅拌罐中培养细胞,利用外部周期性压力振荡刺激并结合载体更新技术,可减轻密度效应对细胞生长和表达的影响,在一定程度上提高细胞在高密度培养条件下的表达水平。在67d连续换液培养中,细胞最高密度为2.64×107／mL,活细胞比率维持在95％以上。与稳压操作相比,利用周期变压刺激技术可提高产量10％～20％,且可降低葡萄糖厌氧代谢生成乳酸的转化率,利用4步纯化工艺,从含u-PA约135g的2100L上清中获得约80gu-PA(单链比例约为90％)。     

Protein-free fed-batch culture of non-GS NS0 cell lines for production of recombinant antibodies

Presented is a novel antibody production platform based on the fed-batch culture of recombinant, NS0-derived cell lines. A standardized fed-batch cell culture process was developed for five non-GS NS0 cell lines using enriched and optimized protein-free, cholesterol-free, and chemically defined basal and feed media. The process performed reproducibly and scaled faithfully from the 2-L to the 100-L bioreactor scale achieving a volumetric productivity of > 120 mg/L per day. Fed-batch cultures for all five cell lines exhibited significant lactate consumption when the cells entered the stationary or death phase. Peak and final lactate concentrations were low relative to a previously developed fed-batch process (FBP). Such low lactate production and high lactate consumption rates were unanticipated considering the fed-batch culture basal medium has an unconventionally high initial glucose concentration of 15 g/L, and an overall glucose consumption in excess of 17 g/L. The potential of this process platform was further demonstrated through additional media optimization, which has resulted in a final antibody concentration of 2.64 +/- 0.19 g/L and volumetric productivity of > 200 mg/L per day in a 13-day FBP for one of the five production cell lines. Use of this standardized protein-free, cholesterol-free NS0 FBP platform enables consistency in development time and cost effectiveness for manufacturing of therapeutic antibodies.     

Low-cost propionate salt as road deicer: evaluation of cheese whey and other media constituents

Propionate and acetate salts are environmentally friendly, effective road deicer substitutes for widely used sodium chloride. A low-cost medium, using raw cheese whey and hydrolyzed whey permeate/whey permeate powder as substrates, and corn-steep liquor as a nutrient supplement, was studied for lactic acid production, replacing synthetic lactose and other high-cost nutrients. A non-sterile stage-I fermentation process for improved lactate productivity using an inexpensive commercial medium was performed at a 20-L fermenter level. A lactate yield of 0.98 g/g lactose and a productivity of 1.1 g/L/h was obtained with complete lactose utilization. When synthetic lactate and glucose were used as substrates in propionate and acetate fermentation, a total acid yield of 0.55 g/g glucose and lactate consumed and a batch productivity of 0.22 g/L/h was obtained. A stage-II fermentation process to produce propionate and acetate salts from cheese whey-derived lactate (stage-I fermentation broth) resulted in 1.6%( w/v) propionate after a total of 161 h (stages I and II).     

Enhancement of tissue plasminogen activator production from human normal fibroblast IMR-90 cells by limitation of cell growth

Summary Tissue plasminogen activator (t-PA) production induced by proteose peptone from IMR-90 cells was investigated. Cells monolayered on plastic surfaces had a higher ability to produce t-PA per unit cell compared to those grown tri-dimensionally on ceramic pieces. Furthermore, confluent monolayers of the cells, which suffered contact inhibition and resulted in limited growth, were available for t-PA production. Repeated batch production with microcarriers, on which the cells were almost confluent monolayers similar to those in T-flasks, was performed. Utilization of the cells, which had limited serum in the growth phase, resulted in an increase in production. Moreover, dilution of the basal components of the medium at initiation of the production phase markedly promoted t-PA production. The volumetric productivity was stable for 30 days at 100 IU/cm³ per day. The cells were then mostly retained on microcarriers. Thus, an effective and scalable method of t-PA production by normal fibroblast cells was developed. Offprint requests to: S. Mitsuda     

Culture of insect cells in helical ribbon impeller bioreactor

An 11-L helical ribbon impeller (HRI) bioreactor was tested for the culture of Spodoptera frugiperda (Sf-9) cells. This impeller and surface baffling ensured homogeneous mixing and high oxygen transfer through surface aeration and surface-induced babble generation. Serum-supplemented and serum-free cultures, using TNMFH and IPL/41 media, respectively, grew a similar specific growth rates(0.031 and 0.028 h(-1)) to maximum cell densities of 5.5 x 10(6)-6.0 x 10(6) cells. mL(-1) with viability exceeding 98% during exponential growth phase. Growth limitation coincided with glucose and glutamine depletion and production of significant amounts of alanine. The bioreactor was further tested under more stringent conditions by infecting a serum-free medium culture with a recombinant baculovirus. Heterologous protein production of approximately 35 mug per 10(6) cells was comparable to yields obtained in serum-free cultures grown in spinner flasks and petri dishes. Average specific oxygen up-take and carbon dioxide production rates of the serum-free culture prior to infection as measured by on-line mass spectroscopy were 0.20 mumol O(2)mu.(10(6) cells)(-1) h(-1) and 0.22 mumol CO(2) . (10(6) cells)(-1)h(-1) and increased by 30-40% during infection. Therefore, the mixing and oxygenation conditions of this bioreactor were suitable for insect cell culture and recombinant protein production, with limitation being mainly attributed to nutrient depletion and toxic by-product generation.