Modeling of Xanthophyllomyces dendrorhous growth on glucose and overflow metabolism in batch and fed-batch cultures for astaxanthin production

An astaxanthin-producing yeast Xanthophyllomyces dendrorhous ENM5 was cultivated in a liquid medium containing 50 g/L glucose as the major carbon source in stirred fermentors (1.5-L working volume) in fully aerobic conditions. Ethanol was produced during the exponential growth phase as a result of overflow metabolism or fermentative catabolism of glucose by yeast cells. After accumulating to a peak of 3.5 g/L, the ethanol was consumed by yeast cells as a carbon source when glucose in the culture was nearly exhausted. High initial glucose concentrations and ethanol accumulation in the culture had inhibitory effects on cell growth. Astaxanthin production was partially associated with cell growth. Based on these culture characteristics, we constructed a modified Monod kinetic model incorporating substrate (glucose) and product (ethanol) inhibition to describe the relationship of cell growth rate with glucose and ethanol concentrations. This kinetic model, coupled with the Luedeking-Piret equation for the astaxanthin production, gave satisfactory prediction of the biomass production, glucose consumption, ethanol formation and consumption, and astaxanthin production in batch cultures over 25-75 g/L glucose concentration ranges. The model was also applied to fed-batch cultures to predict the optimum feeding scheme (feeding glucose and corn steep liquor) for astaxanthin production, leading to a high volumetric yield (28.6 mg/L) and a high productivity (5.36 mg/L/day).     

A robust fed-batch feeding strategy independent of the carbon source for optimal polyhydroxybutyrate production

A three-stage control strategy independent of the organic substrate was developed for automated substrate feeding in a two-phase fed-batch culture of Cupriavidus necator DSM 545 for the production of the biopolymer polyhydroxybutyrate (PHB). The optimal feeding strategy was determined using glucose as the substrate. A combined substrate feeding strategy consisting of exponential feeding and a novel method based on alkali-addition monitoring resulted in a maximal cell concentration in the biomass growth phase. In the PHB accumulation phase, a constant substrate feeding strategy based on the estimated amount of biomass produced in the first phase and a specific PHB accumulation rate was implemented to induce PHB under limiting nitrogen at different biomass concentrations. Maximal cell and PHB concentrations of 164 and 125 g/L were obtained when nitrogen feeding was stopped at 56 g/L of residual biomass; the glucose concentration was maintained within its optimal range. The developed feeding strategy was validated using waste glycerol as the sole carbon source for PHB production, and the three-stage control strategy resulted in a PHB concentration of 65.6 g/L and PHB content of 62.7% while keeping the glycerol concentration constant. It can thus be concluded that the developed feeding strategy is sensitive, robust, inexpensive, and applicable to fed-batch culture for PHB production independent of the carbon source.     

High density culture of insect cells using rational medium design and feeding strategy

The objective of this study is to achieve high density cell culture by a rational medium design and feeding strategy. Insect cell/baculovirus expression system is one of the widely used methods for the production of heterologous proteins in the cell culture domain. Insect cell Spodoptera frugiperda Sf-21 and a recombinant baculovirus with encoded gene for human interleukin-5 were chosen as the model system in this study. A stoichiometric model was established to study the demand of nutrients, including glucose, 20 amino acids, and yeastolate, for the synthesis of cell mass. The coefficients for individual nutrients in the stoichiometric equation governing insect cell growth were determined from the information of cell mass and compositions. Based on the stoichiometric coefficients, the initial and supplemental media for fed-batch cell cultures were designed. The experiments began with the inoculation of Sf-21 cells into a spinner flask with the initial medium, which provided a starting environment for achieving optimum cell growth. This was followed by the periodic feeding of supplemental medium designed by utilizing the stoichiometric equation that governs insect cell growth. With this strategy, it was demonstrated that the Sf-21 cell culture reached a cell density in excess of 1.9᎒⁷ cells/ml. During the cultivation process, the utilization of various nutrients and the production of metabolites were also monitored. Further experiments proved that high concentration of recombinant product (such as human interleukin-5) could be achieved by infecting the high density cells (resulting from the designed medium) with recombinant baculoviruses.     

Development of a fed-batch cultivation for antibody-producing cells based on combined feeding strategy of glucose and galactose

In order to achieve enhanced cell mass and productivity with less lactate accumulation, a fed-batch culture based on a combined feeding strategy of glucose and galactose was developed. Cell performance was first examined with feeding of galactose alone. While cell growth was improved compared with glucose-feeding culture, cell maintenance was inefficient with rapid lactate depletion and considerable ammonium accumulation. Subsequently, to improve cell maintenance, a combined feeding strategy of glucose and galactose was proposed focusing on optimizing the ratio of glucose to galactose and feeding time. In addition, the compositions of amino acids and vitamins in feeding medium were refined for balanced supply of nutrients. With the combined feeding strategy, the metabolic shift of lactate from production to consumption occurred, but not accompanied by rapid lactate depletion and ammonium production. Furthermore, energy metabolism was more efficient and better utilization of carbon sources was achieved. Compared with the glucose-feeding culture in bioreactor, maximum lactate concentration was reduced by 55%; IVCC and the specific production rate of antibody were increased by 45% and 143%, respectively.     

Development and optimization of two-stage cyclic fed-batch culture for hG-CSF production using L-arabinose promoter of Escherichia coli

The long-term process for producing human granulocyte-colony stimulating factor (hG-CSF) was developed using two-stage cyclic fed-batch culture, in which hG-CSF expressing-recombinant Escherichia coli was directed by an L-arabinose promoter system. For the optimization, the preinduction growth rate during the growth stage and the feeding strategy during the production stage were investigated. The maximum harvest volume during the production stage was predicted before long-term cyclic operation. Based on those optimized strategies, the two-stage cyclic fed-batch culture was performed for 12 cycles (86 h). The cell growths in both stages were maintained at 45-50 g/L and 71-77 g/L, respectively. hG-CSF was stably produced at a level of 8-9 g/L and the plasmid stability was maintained at more than 90%. Volumetric productivity by the two-stage cyclic fed-batch culture was 0.643 g/L/h, which was about 280% higher than that of conventional DO-stat fed-batch culture.     

Influences of yeast extract on specific cellular yield of Ovine growth hormone during fed-batch fermentation of E. coli

In most cases of E. coli high cell density fermentation process, maximizing cell concentration helps in increasing the volumetric productivity of recombinant proteins usually at the cost of lower specific cellular protein yield. In this report, we describe a process for maintaining the specific cellular yield of Ovine growth hormone (oGH) from E. coli by optimal feeding of yeast extract during high cell density fermentation process. Recombinant oGH was produced as inclusion bodies in Escherichia coli. Specific cellular yield of recombinant oGH was maintained by feeding yeast extract along with glucose during fed-batch fermentation. Glucose to yeast extract ratio of 0.75 was found to be optimum for maintaining the specific cellular oGH yield of 66 mg/g of E. coli cells. Continuous feeding of yeast extract along with glucose helped in reducing acetic acid secretion and promoted higher cell growth during fed-batch fermentation. High cell growth of E. coli and high specific yield of recombinant oGH thus helped in achieving high volumetric productivity of the expressed protein. A maximum of 2 g/l of ovine growth hormone was expressed as inclusion bodies in 12 h of fed-batch fermentation.     

Enhancement of poly-3-hydroxybutyrate (PHB) productivity by the two-stage supplementation of carbon sources and continuous feeding of NH4Cl

Gluconate and glucose were selected as the carbon substrates in the production of poly-3-hydroxybutyrate (PHB). Gluconate was utilized to maximize the specific growth rate during the first stage of cell growth, whereas glucose was used to maximize PHB biosynthesis during the second stage of PHB accumulation. The sequential feeding of gluconate and glucose resulted in a 50% enhancement of PHB productivity as compared to the cultures cultivated on glucose alone. In conjunction with secondary glucose uptake, the presence of a trace amount of ammonium increased the rate of PHB biosynthesis during the stage of PHB accumulation. Via the feeding of 0.03 mmol/h of NH₄Cl solution prior to the exhaustion of the initial amount of NH₄Cl, PHB productivity was significantly enhanced as compared to the cultures raised on glucose alone. The glucose-grown culture evidenced a higher level of NADPH during the NH₄Cl-exausted PHB accumulation stage than was observed in the gluconate-grown culture, which reflects that the reason of higher PHB production observed when glucose was used as a carbon source. NH₄Cl feeding following the depletion of initial NH₄Cl resulted in elevated levels of both ATP and NADPH, which increased the PHB biosynthesis rate, and also in a decrease in the level of NADH, which reflected the alleviation of the inhibitory effects on the cells caused by nitrogen depletion. Electronic Supplementary Material  Supplementary material is available for this article at and is accessible for authorized users.     

Feeding lactate for CHO cell culture processes: impact on culture metabolism and performance

Lactate has long been regarded as one of the key metabolites of mammalian cell cultures. High levels of lactate have clear negative impacts on cell culture processes, and therefore, a great amount of efforts have been made to reduce lactate accumulation and/or to induce lactate consumption in the later stage of cultures. However, there is virtually no report on the impact of lactate depletion after initial accumulation. In this work, we observed that glucose uptake rate dropped over 50% at the onset of lactate consumption, and that catabolism of alanine due to lactate depletion led to ammonium accumulation. We explored the impact of feeding lactate as well as pyruvate to the cultures. In particular, a strategy was employed where CO(2) was replaced by lactic acid for culture pH control, which enabled automatic lactate feeding. The results demonstrated that lactate or pyruvate can serve as an alternative or even preferred carbon source during certain stage of the culture in the presence of glucose, and that by feeding lactate or pyruvate, very low levels of ammonia can be achieved throughout the culture. In addition, low levels of pCO(2) were also maintained in these cultures. This was in strong contrast to the control cultures where lactate was depleted during the culture, and ammonia and pCO(2) build-up were significant. Culture growth and productivity were similar between the control and lactate-fed cultures, as well as various product quality attributes. To our knowledge, this work represents the first comprehensive study on lactate depletion and offers a simple yet effective strategy to overcome ammonia and pCO(2) accumulation that could arise in certain cultures due to early depletion of lactate.     

Effect of Corn steep liquor supplementation and scale up on Lactococcus starter production

Summary Three Lactococcus strains (Lactococcus ssp. lactis var. diacetylactis, Lactococcus ssp. lactis cremoris and Lactococcus ssp. lactis var. lactis) isolated from the Tunisian lben were grown at constant pH on CSL medium in stirred fermentors for lactic starters production. The agitation required to homogenate alkali used to pH control should be low because it affects the Lactococcus growth. Scale up from 20-liter fermentor to 400-liter fermentor was carried out at constant impeller tip speed below 150 cm s^у. The CSL supplementation and fed-batch with glucose increased the yield in the upper 10¹⁰ cfu/ml. The consumed glucose during fermentation was converted into lactic acid and cell. Before fed-batch, the maximum specific growth rate of Lactococcus ssp. lactis var. diacetylactis was around 1 h^у and the number of cells increased 20 to 40 times according to inoculum size. After fed-batch, the glucose consumption rate remains constant but specific growth rate decreased and number of cell trebled only.     

Alteration of mammalian cell metabolism by dynamic nutrient feeding

The metabolism of hybridoma cells was controlled to reduce metabolic formation in fed-batch cultures by dynamically feeding a salt-free nutrient concentrate. For this purpose, on-line oxygen uptake rate (OUR) measurement was used to estimate the metabolic demand of hybridoma cells and to determine the feeding rate of a concentrated solution of salt-free DMEM/F12 medium supplemented with other medium components. The ratios among glucose, glutamine and other medium components in the feeding nutrient concentrate were adjusted stoichiometrically to provide balanced nutrient conditions for cell growth. Through on-line control of the feeding rate of the nutrient concentrate, both glucose and glutamine concentrations were maintained at low levels of 0.5 and 0.2 mM respectively during the growth stage. The concentrations of the other essential amino acids were also maintained without large fluctuations. The cell metabolism was altered from that observed in batch cultures resulting in a significant reduction of lactate, ammonia and alanine production. Compared to a previously reported fed-batch culture in which only glucose was maintained at a low level and only a reduced lactate production was observed, this culture has also reduced the production of other metabolites, such as ammonium and alanine. As a result, a high viable cell concentration of more than 1.0 × 10⁷ cells/mL was achieved and sustained over an extended period. The results demonstrate an efficient nutrient feeding strategy for controlling cell metabolism to achieve and sustain a high viable cell concentration in fed-batch mammalian cell cultures in order to enhance the productivity. This revised version was published online in July 2006 with corrections to the Cover Date.     

Control of L-phenylalanine production by dual feeding of glucose and L-tyrosine

Control of L-phenylalanine production by a recombinant of Escherichia coli AT2471 by means of the dual feeding of glucose and L-tyrosine was investigated. A novel method was developed for on-line monitoring of the maximum glucose uptake rate (MGUR), in which the length of time required for the consumption of added glucose was measured. Accumulation of acetic acid was successfully prevented throughout the whole period of the culture when the glucose concentration was kept below 0.1 g/L by controlling the glucose feeding on the basis of on-line monitoring of the MGUR and the cell concentration with a laser sensor.In a batch culture with glucose feeding, after L-tyrosine was depleted cell growth and the L-phenylalanine production rate decreased along with decreases in the specific enzyme activities of chorismate mutase-p-prephenate dehydratase (CMP) and 3-deoxy-D-arabinoheputulosonate 7-phosphate synthase (DAHP), which are the key enzymes in the L-phenylalanine synthesis pathway. Increasing the L-tyrosine feed rate by an appropriate amount, but not so far as to cause L-tyrosine accumulation in the culture, increased the activities of the enzymes and the specific rates of growth and production while the product yield based on glucose consumption decreased.The average specific rates of growth, production, and MGUR could be expressed as functions of the specific L-tyrosine consumption rate during both the earlier and later periods of L-tyrosine feeding. Estimations of the amount of L-phenylalanine produced, the product yield, and the cost factor by using these functions with several different combinations of two specific L-tyrosine consumption rates for two 10-h periods resulted in a suggested optimum L-tyrosine feeding strategy giving a lower specific L-tyrosine consumption rate in the later period, to suppress cell growth, in comparison to that in the earlier period. During L-tyrosine feeding, the three specific rates (growth, production, and MGUR) could be successfully controlled by adjusting the specific L-tyrosine consumption rate to the predicted value. The cost factor was lowest in this controlled culture, demonstrating experimentally the effectiveness of the strategy. (c) 1996 John Wiley & Sons, Inc.     

Continuous optical in-line glucose monitoring and control in CHO cultures contributes to enhanced metabolic efficiency while maintaining darbepoetin alfa product quality

Great efforts are directed towards improving productivity, consistency and quality of biopharmaceutical processes and products. One particular area is the development of new sensors for continuous monitoring of critical bioprocess parameters by using online or in-line monitoring systems. Recently, we developed a glucose biosensor applicable in single-use, in-line and long-term glucose monitoring in mammalian cell bioreactors. Now, we integrated this sensor in an automated glucose monitoring and feeding system capable of maintaining stable glucose levels, even at very low concentrations. We compared this fed-batch feedback system at both low (< 1 mM) and high (40 mM) glucose levels with traditional batch culture methods, focusing on glycosylation and glycation of the recombinant protein darbepoetin alfa (DPO) produced by a CHO cell line. We evaluated cell growth, metabolite and product concentration under different glucose feeding strategies and show that continuous feeding, even at low glucose levels, has no harmful effects on DPO quantity and quality. We conclude that our system is capable of tight glucose level control throughout extended bioprocesses and has the potential to improve performance where constant maintenance of glucose levels is critical.     

Maintaining rapid growth in moderate-density Escherichia coli fermentations

A novel feeding strategy that prolongs rapid growth rates for Escherichia coli fermentations to moderately high cell density is presented. High-density fermentations are a common and successful means of producing biological products. However, acetate accumulation can be a substantial problem in these procedures. To avoid this problem, many feeding strategies and host modifications have been developed, but all result in relatively low growth rates. If a faster growth rate could be maintained, the growth phase of the process would be shortened, leading to increased productivity. It is also possible that the subsequent specific production rate could be enhanced by growing the early culture at a faster rate. We have developed a procedure to enable rapid growth to a cell density of 20 g/L and have used cell-free protein synthesis to evaluate the relative potential of the resulting cells for producing recombinant proteins. The method uses glucose pulses and the duration of the dissolved oxygen response to calculate the appropriate glucose feed rate based on the glucose demand of the culture. Amino acids and vitamins were supplied in the medium to increase the growth rate. We were able to sustain a growth rate of 0.8/h up to 20 g/L dry cell weight without significant acetate accumulation. Analysis of amino acid consumption indicates that cell composition is an accurate predictor of amino acid demand for most amino acids. Cell-free protein synthesis was used to compare the protein production potential of the high-density cultures with that of cells grown in complex medium and harvested at low cell density and maximum growth rate. Protein production for the extract from the controlled, high-density fermentations was 950 mg/L compared with 860 mg/L for the low-density control. Therefore, the new control procedure has promising potential for developing rapid and productive industrial fermentations.     

High viable cell concentration fed-batch cultures of hybridoma cells through on-line nutrient feeding

A hybridoma cell line was cultivated in fed-batch cultures using a low-protein, serum-free medium. On-line oxygen uptake rate (OUR) measurement was used to adjust the nutrient feeding rate based on glucose consumption, which was estimated on-line using the stoichiometric relations between glucose and oxygen consumption. Through on-line control of the nutrient feeding rate, not only sufficients were supplied for cell growth and antibody production, but also the concentrations of glucose and other important nutrients such as amino acids were maintained at low levels during the cell growth phase. During the cultivation, cell metabolism changed from high lactate production and low oxygen consumption to low lactate production and high oxygen consumption. As a result the accumulation of lactate was reduced and the growth phase was extended. In comparison with the batch cultures, in which cells reached a concentration of approximately 2 x 10(6) cells/mL, a very high concentration of 1.36 x 10(7) cells/mL with a high cell viability (>90%) was achieved in the fed-batch culture. By considering the consumption of glucose and amino acids, as well as the production of cell mass, metabolites, and antibodies, a well-closed material balance was established. Our results demonstrate the value of coupling on-line OUR measurement and the stoichiometric realations for dynamic nutrient feeding in high cell concentration fed batch cultures. (c) 1995 John Wiley & Sons, Inc.     

Optimization of Escherichia coli growth by controlled addition of glucose

During aerobic growth of Escherichia coli (recombinant K-12 and strain B) on protein hydrolysate (L-broth) and a carbon source (glucose), acetic acid is produced via glucose metabolism until the late log phase. At this point, the culture pH starts to increase and the growth rate decreases. In cultures without further glucose supplementation, these changes are associated with the accumulation of ammonia, the utilization of acetic acid, the depletion of amino acids, and the complete depletion of glucose. We hypothesize that, after depletion of the glucose, the bacteria catabolize amino acids for energy and carbon and give off the nitrogen as ammonia. Also contributing to the overall increase in pH is the depletion of the acetic acid produced earlier as it is metabolized upon exhaustion of glucose. However, there is a lag time of about 1 hour after the initial pH increase before the sustained accumulation of ammonia begins. This lag indicates that an unidentified factor, in addition to the increase in ammonia, contributes to the increase in pH. Advantage was taken of the turnaround from acid production to base production as reflected in the culture pH to implement the addition of glucose. In growth experiments during which the pH was controlled in the basic direction by glucose addition, the observed decrease in growth rate was significantly postponed and the pH change in the basic direction was reversed as a result of acid production by the cells from the newly added glucose. Furthermore, coll densities of twice that obtained without glucose feeding were demonstrated. Based on the media cost per unit cell density, the data indicate a 31% cost savings.     

Defining an optimal carbon source/methionine feed strategy for growth and cephalosporin C formation by Cephalosporium acremonium.

The effect of the method of methionine addition, growth-limiting carbon source (glucose vs sucrose), and culture growth rate on cephalosporin C production was investigated in a Cephalosporium acremonium defined medium fed batch fermentation. Batch addition of methionine, at a concentration of 3 g/L, prior to the start of a fed sucrose fermentation was found to interfere with the ability of the culture to utilize this sugar, thus limiting growth and decreasing cephalosporin C production. Batch methionine addition had no effect on glucose-limited cultures. Concurrent exponential feeding of methionine with sucrose improved both culture growth and productivity. Under the control of identical carbon source limiting feed profiles, sucrose was observed to support greater cephalosporin C production than glucose. Optimal cephalosporin C production in a C. acremonium defined medium fed batch fermentation was obtained through controlling culture growth during the rapid growth phase at a relatively low level with respect to mumax (mu approximately 0.036 h-1) until achieving a desired cell mass with a concurrent sucrose and methionine feed, followed by maintaining relatively vigorous growth (mu approximately 0.01 h-1) with sucrose for the duration of the fermentation.     

Feasibility of culturing C2C12 mouse myoblasts on glass microcarriers in a continuous stirred tank bioreactor

Commercial culturing of mammalian cell lines is increasing in importance as more biological products unique to mammals are being produced in genetically altered mammalian cells. Most mammalian cells are anchorage dependent, so they must be cultured on a support matrix. This limitation, along with the requirement of a low shear environment, severely effects the scale-up of bench-scale culture systems. The need to culture mammalian cells on a support matrix limits the increase in cell population to a factor of 10-20 before growth virtually stops due to contact inhibition. Commercial culturing systems for anchorage dependent cells are batch processes because of the combination of contact inhibition and support matrix requirements. Development of a continuous bioreactor system could allow both unlimited scale-up and continuous cell-mass production. To design a continuous reactor, a mathematical model to predict the reactor performance should be developed. This paper addresses the development of a mathematical model for predicting continuous bioreactor performance. It was found that anchorage dependent C2C12 mouse myoblast cells, a continuous cell line, followed Monod kinetics for glucose consumption and cell mass production in batch flask experiments, with w_max = 0.040 hr^у and K_m = 2.5 mM. Furthermore, it was found that these parameters could be used to predict the glucose consumption in a continuous bioreactor operated with constant feed of seeded microcarriers operated at two different residence times. The success of this model implies the possibility of developing a continuous cell harvesting and reinoculation system using a microcarrier bioreactor to produce cell mass.     

Optimization of fed-batch parameters and harvest time of CHO cell cultures for a glycosylated product with multiple mechanisms of inactivation

Optimization of fed-batch feeding parameters was explored for a system with multiple mechanisms of product inactivation. In particular, two separate mechanisms of inactivation were identified for the recombinant tissue-type activator (r-tPA) protein. Dynamic inactivation models were written to describe particular r-tPA glycoform inactivation in the presence and absence of free-glucose. A glucose-independent inactivation mechanism was identified, and inactivation rate constants were found dependent upon the presence of glycosylation of r-tPA at N184. Inactivation rate constants of the glucose-dependent mechanism were not affected by glycosylation at N184. Fed-batch optimization was performed for r-tPA production by CHO cell culture in a stirred-tank reactor with glucose, glutamine and asparagine feed. Feeding profiles in which culture supernatant concentrations of free-glucose and amino acids (combined glutamine and asparagine) were used as control variables, were evaluated for a wide variety of set points. Simulation results for a controlled feeding strategy yielded an optimum at set points of 1.51 g L(-1) glucose and 1.18 g L(-1) of amino acids. Optimization was also performed in absence of metabolite control using fixed feed-flow rates initiate during the exponential growth phase. Fixed feed-flow results displayed a family of optimum solutions along a mass flow rate ratio of 3.15 of glucose to amino acids. Comparison of the two feeding strategies showed a slight advantage of rapid feeding at a fixed flow rate as opposed to metabolite control for a product with multiple mechanisms of inactivation.     

Benzyl adenine restores anthocyanin pigmentation in suspension cultures of wild Vaccinium pahalae

The overriding influence of cytokinin source on flavonoid production in vitro was explored using a suspension culture system for Vaccinium pahalae. The substitution of kinetin by 20 μM benzyl adenine (BA) in the suspension culture media resulted in a three-fold increase in total anthocyanin yield, and a more rapid production during the cell culture cycle. Anthocyanin production reached a maximum after a 16–20 day interval in cultures containing an optimal kinetin concentration, but pigment accumulation peaked at only 12–16 days when BA was used as the sole cytokinin source. Unlike some other production systems which increase secondary metabolite production at the expense of cell growth, BA-supplementation promoted both increased growth and increased anthocyanin productivity. In BA-supplemented medium, cultures were not susceptible to typical osmotically-induced cell growth suppression. When, after multiple subcultures in kinetin-containing media, anthocyanin production capability was lost or diminished, productivity could be restored within 3 days after transfer of cells to a BA-supplemented medium. This revised version was published online in June 2006 with corrections to the Cover Date.     

High-level secretory production of human granulocyte-colony stimulating factor by fed-batch culture of recombinant Escherichia coli

Secretory production of human granulocyte colony-stimulating factor fusion protein (hG-CSF) by fed-batch culture of Escherichia coli was investigated in both 2.5-L and 30-L fermentors. To develop a fed-batch culture condition that allows efficient production of hG-CSF, different feeding strategies including pH-stat, exponential and constant feeding were examined. Among these, the constant feeding strategy (0.228 g glucose2min^-1) and the exponential feeding that supports a low specific growth rate (µ=0.116 h^-1) resulted in the best hG-CSF production. Under these conditions, 4.4 g2L^-1 of hG-CSF was produced. The effect of induction time on the protein production was also investigated. For the fed-batch cultures carried out with the pH-stat and exponential feeding strategies, induction at higher cell density (late-exponential phase) resulted in more hG-CSF production compared with induction at lower cell density (early to mid-exponential phase). The constant feeding strategy that supported best hG-CSF production was applied to the scale-up production of hG-CSF in 30 L of fermentor. The maximum dry cell weight and hG-CSF concentration of 51.7 and 4.2 g2L^-1, respectively, was obtained.