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Improvement of recombinant CHO cell growth, comparison of growth characteristics and Erythropoietin production.

Introduction

Chinese hamster ovary (CHO) cells are of great interest for bio processing and pharmaceutical research and development [1]. Maintenance of mammalian cell lines require the addition of serum to the medium, replacement of fetal calf serum is the most advancement, particularly if it can be achieved by media that are completely protein-free [2]. Various successful serum free media formulations have been developed are commercially available today. Unfortunately, many cell lines do not grow well under serum-free conditions, so it is necessary to determine which serum-free culture medium can lead to the optimal cell growth. Glutamine, a major nutrient in mammalian cell culture, is required for the protein and nucleotide synthesis [3]. Glutamine is hydrolyzed metabolically or degrades chemically to ammonia. It has poor thermal stability, glutamine break down and catabolism result in high ammonia accumulation. At the end of a recombinant protein production campaign, the ammonium concentration can reach 5-10 mM [4]. Achievement of high cell yields in such culture is often limited by an accumulation of ammonia [5]. Cells can grow well in a medium where glutamine is replaced by glutamate, which is metabolized more efficiently, reducing the accumulation of ammonia and allows the culture longevity [6].

Cell death is a major barrier to maintain high cell densities at high viability and often leads to lower protein yields and quality [7]. Death of mammalian cells [8], including Chinese hamster ovary cells[9] during cultivation proceeds mainly via apoptosis. Various strategies such as nutrient feeding, addition of antiapoptotic chemicals and genetic manipulation have been used to decrease apoptosis and extend culture viability [7]. The most common anti-apoptotic agent is insulin, a metabolic hormone can rescue many types of cells from apoptotic cell death. Anti-apoptotic effects of insulin are mediated by activation of the phosphatidylinositol-3 kinase signaling pathway [10].

Dissolved oxygen has significant impact on cell growth, metabolism and product synthesis [11]. Aeration and agitation variable from vessel to vessel, are the main factors which directly effect the dissolved oxygen. Hence it is essential to optimize the vessel conditions for the growth of mammalian cells. Spinner flasks cannot be used for liquid volumes greater than 1 litre because of insufficient oxygen transfer and roller bottles are suitable for the adherent cell lines [12]. Bioreactors are complex and expensive devices, yet they do not provide an ideal environment for cell growth due to high local fluid shear and bubble aeration. To avoid the problems associated with cell culture vessels, a new wave bioreactor was designed by Singh [12]. Wave bioreactors are an excellent choice for the elimination of need of cleaning, sterilization and associated validation requirements [11]. In this study we describe the serum free adaptation of CHO cells, selection of commercial serum-free medium from the pool of media library, replacement of glutamine by glutamate and glutamax, effect of insulin addition on cell growth and apoptosis. Studies were also conducted for selection of culture vessel conditions using simple batch mode to improve the cell growth and productivity.

Materials and Methods

Cell line and culture media

A cloned recombinant Chinese hamster ovary cell line (CHO-EPO) manipulated to secrete recombinant human erythropoietin was used in this work. These cells were kindly provided by Genomix Biotech Inc. (Atlanta, USA). Briefly, they were established by transfection of a vector containing dihydrofolate reductase (dhfr) and human EPO genes into dhfr- deficient CHO cells. Media used in this study are described in Table 1, additives were added in each medium following manufacturer instructions. All additions to the culture medium were cell culture grade and purchased from Sigma, Gibco- Invitrogen, if not specified.

Cell culture and maintenance

Several different serum-free media (Table:1) were used to compare their effectiveness on cell growth and protein expression. Serum free adapted CHO cells were inoculated at 2 x [10.sup.5] cells/ml in to 15 ml of all 7 different types of cell culture media including control medium (DMEM/F-12 + 5% FBS). All media supplemented with 4 mM glutamine and 10 [micro]g/ml bovine insulin. Cultures were incubated at 37[degrees]C and 5% C[O.sub.2] in the humidified incubator for 6 days with medium replacement for every 48 hours in all flasks. In the experiments with glutamine replacement, actively growing CHO cells (2 x [10.sup.5] cells/ml) were transferred in to the culture flasks containing Excell 325 SF medium with various concentrations (0, 2, 4, 8 and 12 mM) of glutamine, glutamate and glutamax. Medium was also supplemented with 10 [micro]g/ml bovine insulin. Cells were centrifuged before inoculation to remove the culture medium, particularly to ensure that remaining glutamine from inoculum preparation was not present.

For Insulin experiments, CHO cells growing at exponential phase were washed with PBS, and then placed in Excell 325 SF medium containing 4 mM glutamax. Insulin (10 [micro]g/ml) was added in the one flask at 0 hrs (single addition), second flask at 0 and 72 hrs (two additions) and one flask kept for control (without insulin) to see its effect on cell growth and apoptosis. Glucose (10 mM) and glutamax (4 mM) was added during the cultivation to avoid their depletion. To determine the growth characteristics of CHO cells in different culture vessel conditions, cells were grown in spinner flasks (1000ml, Wheaton), roller bottles (850[cm.sup.2], Corning) and wave 2-L perfusion cell bag (GE Health care). All vessels were inoculated at a density of 2 x [10.sup.5] cells/ml with 300 ml of Excell 325 medium containing 500 nM MTX, 4mM glutamax and 10 [micro] g/ml insulin (Insulin added at 1st and 3rd d of cultivation). Agitation speeds were set at 60 rpm (spinner), 20 rpm (roller) and 10 rocks/min (wave bioreactor). Parallel cultures in T-flask also performed for the comparison of static and agitated systems. Aeration was kept constant at 0.1 to 0.2lpm for wave cell bag and cultures were performed at batch mode.

Analytical Methods

Viable cell concentration and total cell concentration was determined by the trypan blue exclusion method using a haemocytometer[13]. Secreted

EPO concentrations were quantified by ELISA (EPO.96, MD Bioscience, USA) as for manufacturer's instructions. Glucose concentration was determined by enzymatic method (GOP/POD method, Excel diagnostics, India). Ammonia concentrations were measured using enzymatic UV method (Randox laboratories, UK).

DNA Fragmentation assay

CHO cell apoptosis was determined by the DNA fragmentation assay on agarose electrophoresis. Target cell samples were collected in 1.5 ml micro-centrifuge tube, spun down and resuspended with 0.5 ml 1X PBS, and added 55ul of Triton X-100 lysis buffer (40ml of 0.5M EDTA, 5ml of 1M Tris-HCl buffer (pH 8.0), 5ml of 100% Triton X-100 and made up to 100ml with MilliQ water), kept for 20 min on ice (4[degrees]C). Centrifuged the tube in cold at 10000 rpm for 15 minutes, transferred the sample to new micro-centrifuge tube and then extracted supernatant with 1:1 mixture of phenol: chloroform and precipitated in two equivalence of cold ethanol and one tenth equivalence of sodium acetate. The contents were spun down, decanted supernatant and resuspended the precipitate in 30[micro]l of RNase solution (0.4ml water + 5[micro]l of 50[micro]g/ml RNase) and 5[micro]l of loaded buffer was added. Samples were run on 1.5% agarose gel, the DNA fragments were observed under Bio-Rad gel doc with UV light at 254nm and images were captured.

Statistical Analysis

Results are expressed as mean [+ or -] SD, and the group means are compared with the Student t test. The accepted level of significance was set at P<0.05.

Results and Discussion Adaptation to serum free medium

In order to adapt the CHO cells to serum-free cultivation, cells cultured in serum containing medium were directly sub-cultured into serum-free medium. This direct adaptation process was performed with initial cell concentration of 2 x [10.sup.5] cells/ml. After growth for 7 days at 37[degrees]C, and 5% C[O.sub.2] with medium replacement every other day, no viable cells were visible in the flask (Fig:1) and no significant consumption of glucose was observed. CHO cells are usually grown in media containing fetal bovine serum (FBS) which contains key components required for cell growth [14]. Serumfree cultivation of mammalian cells requires many components, which were provided by serum [15], but these essential nutrients are not present in the DMEM/F-12 medium. It is concluded, this may be the reason for not growing cells in the DMEM/F-12 medium without serum.

Optimal cell concentration is essential when cells are growing in the serumfree conditions; Literature recommends a minimum of 2-5 x [10.sup.5] cells/ml for adaptation into a serum-free suspension culture. It was observed that a critical initial cell density was required for successful growth of chick embryo cells in tissue culture [16-17]. Hence, various initial cell concentrations (2, 4 & 6 x [10.sup.5] cells/ml) were tested (data not shown) but the same results were obtained. However, commercially available serum-free media generally include growth factors such as insulin and transferrin, substituting for mitogenic factors in serum[18]. It is decided to use commercial serum-free medium (Excell-302) for the direct adaptation, during this adaptation, small clumps appeared, but cells stop growing after third day of culture (Fig: 2). During the initial days of culture, cells utilized the glucose and then they entered into death phase resulting in the decision to stop further evaluation of this procedure.

Cells are conditioned to grow better in serum-free media by a gradual adaptation procedure [19]. Gradual reduction of the serum concentration increases the chance for successful adaptation of cells to serum-free environment [20]. For this reason, we decided to stepwise decrease the serum concentration in every other passage by 50% to wean cells off serum. The gradual adaptation to serum-free medium gave better results (Fig: 3). Cells were grown well (Fig: 4A) and viability (Fig: 4B) was maintained more than 84% during the adaptation. Glucose consumption was observed during the entire adaptation, as a result of active cell growth there were sharp decline in glucose concentration. Only a slight reduction in viable cell number occurred when the serum content was reduced from 5 to 1%. The cell viability rapidly decreased (Fig: 4B), however, from 1% to 0% FBS, indicating a serum component becoming growth limiting [21]. Compared to serum containing cultures, reduction of cell growth is generally observed when cells are transferred to serum-free media [22-24]. In most cases the proportion of suspended cells increased with decreasing serum concentration. Therefore, gradual adaptation of CHO cells were considered fully adapted to the serum-free medium.

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Serum-free medium (SFM) selection

One of the major problem associated with SFM, is that there is no one universal medium on which all cells will grow. It has been found that almost every cell line requires a different set of growth factors, hormones and attachment factors for the optimal cell growth and yield [15]. Although there is serum or protein-free media described for the culture of CHO cells [25], they are not really satisfactory, because they were developed for one special cell clone. In the present work, over seven different serum-free media from six vendors have been screened to determine the best medium for growth and productivity.

Cell growth rates were calculated for all the media tested. Cells showed inability to grow well in all the media (Fig: 5A). When all the media were inoculated with same no of cells, the highest cell densities were obtained on Excell 325 (1.56 [+ or -] 0.036 x [10.sup.6]) and Excell 302 (1.483 [+ or -] 0.016 x [10.sup.6] cells/ml) media. By using Invitrogen CD CHO and CHO S SFM medium, cell densities reached 0.48 [+ or -] 0.013 x [10.sup.6] and 0.79 [+ or -] 0.015 x [10.sup.6] cells/ml, whereas cell density level was 0.92 [+ or -] 0.025 x [10.sup.6] cells/ml in Sigma medium. 0.58 [+ or -] 0.014 x [10.sup.6] cells/ml & 0.89 [+ or -] 0.02 x [10.sup.6] cells/ml, cell densities were observed in Cellgro and Hyclone media. More or less similar growth support was observed in Excell 325 and Excell 302 media, when compared with control medium (1.5 [+ or -] 0.01 x [10.sup.6] cells/ml in DMEM/F-12 + 5% FBS).

CHO cell line demonstrated good viability in both Excell media during the entire culture period, with viabilities ranging from 87 - 90% (Fig: 5B). Protein production demonstrated variations in all the media tested during the entire cultivation (Fig: 5D). Higher consumption of glucose was observed in both Excell 302 and 325 media (Fig: 5C). These results show that Excell 325 and Excell 302 media are the most suitable for CHO cell growth and protein expression. Large variations were found in both growth and productivity for the choice of media. While the commonly used serumfree media no longer contains animal serum as an additive, however they contains animal derived components, particularly bovine proteins such as albumin, insulin, transferrin and lipoproteins. It is highly desirable to develop animal proteinfree media for cell culture processes [26], because any animal derived proteins could carry a theoretical risk of introducing prions [27] or other adventitious agents [28]. Although SFM is a step in the right direction it does not entirely alleviate all of the problems associated with serum, it merely reduces them, this has led to the development of protein-free media [29-30]. The main benefit of a protein-free media is in the downstream recovery of product. Hence, it was decided to use only Excell 325 medium (SF & PF) for the further developmental studies.

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Effect of L-glutamine, glutamate and glutamax on cell growth & protein production

In figure 6A it can be observed that the 4 mM glutamine gave highest cell growth, both the growth rate and final cell concentrations were low with the increase in glutamine concentration (8 and 12 mM). Cells did not grow at all in the medium without glutamine and cells grown at 2 mM glutamine were slightly higher than the higher glutamine concentration. At the end of the incubation, the highest cell density observed at 4 mM was 1.21 [+ or -] 0.04 x [10.sup.6]; this was almost 33.8-45.5% (Student's t-test p<0.001) higher than the other concentrations.

Accumulation of ammonia was significantly high (11 [+ or -] 0.24 & 11.9 [+ or -] 0.23mM, Student's t-test p<0.001) in 8 and 12 mM glutamine, when compared with other concentrations (Fig: 6C). Cells stop growing at the ammonia concentration higher than 8 mM. It can be observed that glucose is consumed less (8.8 [+ or -] 0.34, 8.2 [+ or -] 0.29 & 7.6 [+ or -] 0.31 mM) at the lower (2 mM) and higher concentrations (8 & 12 mM) of glutamine (Fig: 6B), where it was consumed more than 25.4-35.6% (Student's ttest p<0.002) higher in the medium containing 4 mM glutamine (11.8 [+ or -] 0.31 mM, 'starting medium glucose concentration was 20 mM and it was reached to 8.26 mM at the end of cultivation'). Figure 6D shows EPO concentration in the medium containing various concentrations of glutamine. The yield of EPO was significantly (3.55 [+ or -] 0.02 mg/l, Student's t-test p<0.001) higher in the culture containing 4 mM glutamine than other concentrations.

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In the series of experiments for glutamine replacement, same approach was followed to determine the effect of glutamate on CHO cell growth. Cell densities were more or less similar (1.45 [+ or -] 0.07 x [10.sup.6] & 1.39 [+ or -] 0.076 x [10.sup.6]) at concentrations of 4 and 8 mM, on the other hand at 12 mM, it was 1.21 [+ or -] 0.027 x [10.sup.6] cells/ml which was significantly (Student's t-test p<0.02) lower than the above concentrations (4 & 8 mM) (Fig: 7A). Interestingly, the maximum concentration of ammonia accumulation was only 3.6 [+ or -] 0.08 mM at the highest concentration of glutamate (12 mM) (Fig: 7C); which was 3 fold less when compared with 12 mM glutamine cultures (11.9 [+ or -] 0.23 mM). No ammonia build up was observed in 2 mM glutamate culture. The trends for the consumption of glucose can clearly be distinguished; it was less (8.1 [+ or -] 0.27 mM, Student's t-test p<0.001) at the low concentration (2 mM) of glutamate, when compared with other concentrations (4, 8 &12 mM) (Fig: 7B). Figure 7D shows the expression of EPO at various concentrations of glutamate, highest EPO production (4.2 [+ or -] 0.08 mg/l, Student's t-test p<0.002) was observed at the 8 mM glutamate concentration, but this was insignificant (4.09 [+ or -] 0.05 mg/l, Student's ttest p<0.09) with the 4 mM glutamate cultures.

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In the experiments with glutamax, maximum cell density was obtained at 4 mM (1.67 [+ or -] 0.022 x [10.sup.6], Student's t-test p<0.049), but this was insignificant (1.59 [+ or -] 0.08 x [10.sup.6], Student's t-test p<0.2) with the 8 mM glutamax cultures (Fig: 8A). However, cells at highest concentration of glutamax (12 mM) grew with very similar growth profiles to 4&8 mM glutamax. Glucose consumption was significantly (14.8 [+ or -] 0.26 mM, Student's t-test p<0.029) high with the culture containing 4 mM glutamax (Fig 8B). Near similar consumption rate was observed in 4, 8 & 12 mM, but in between these all, high consumption was observed at 4 mM glutamax. Very less accumulation of ammonia was observed in all concentrations of glutamax used. 3 [+ or -] 0.15 mM is the highest ammonia production observed at 12 mM glutamax cultures (Fig 8C). As seen in Fig 8D, highest EPO production (6.2 [+ or -] 0.2 mg/l, Student's t-test p<0.021) was observed with the culture containing 4 mM glutamax at the end of incubation (i.e., 170 hrs). Where as, 2.7 [+ or -] 0.18, 5.9 [+ or -] 0.13 & 4.79 [+ or -] 0.16 mg/l was obtained in the cultures with 2, 8 & 12 mM glutamax.

The only prior reports on glutamine replacement with glutamate have been studied on the human diploid cell lines[31], reported that the replacement of glutamine with glutamate will increase the cell growth and productivity. Altamirano et al., [32] improved the CHO cell culture media formulations by substituting the glutamine with glutamate. In those studies, they did not clearly state the effect of ammonia on cell growth and productivity of CHO cell line. In the present work, two different components were tested (glutamate and glutamax) with the main objective of obtaining a more efficient use of the nitrogen source, with a lower release of ammonium ions.

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Toxic levels of ammonia accumulation were crossed at day 4 in 8 & 12 mM glutamine cultures, cells stop growing in the same culture and the final level of ammonia production was reached to more than 11 mM in the same flasks (Fig: 6C). Cells stop growing at the level of 8 mM ammonia in the batch cultivation [33]. Where as in the experiments with glutamate and glutamax, maximum ammonia accumulations obtained were only 3.6 [+ or -] 0.08 & 3 [+ or -] 0.15 mM (Fig: 7C & 8C); this was too low to produce any inhibition [33]. The accumulation of ammonia was significantly very low in the media containing glutamine based dipeptide (Lglutamine and L-alanyl) [34]. However, the cell growth was inhibited at higher concentration of glutamate (1.21 [+ or -] 0.027 x [10.sup.6], Student's t-test p<0.021), there was no significant growth inhibition was observed at any concentrations of glutamax except 2 mM, this may be due to insufficient level of glutamine.

Cell viability and EPO concentrations were decreased with the increased accumulation ammonia. EPO content in the cultures grown at glutamate and glutamax was gradually increased up to the end of cultivation, except the low concentration (2 mM), where it was decreased at the day 5. More than 42% increase in maximum EPO concentration was achieved by replacing the glutamine with glutamax. Similarly, 15.5% higher EPO concentration was achieved by glutamate. When comparing the EPO expression in between glutamax and glutamate, there is a significant (32%, Student's t-test p<0.001) increase was observed with glutamax than glutamate. Similarly, cell growth rate was also increased (13.5%, Student's t-test p<0.007). Overall, the glutamax increased cell viability, utilized more glucose, produced less ammonia and yielded more EPO, when compared to glutamine & glutamate.

Effect of Insulin on rCHO cell growth and apoptosis

The effect of insulin addition (10 [micro]g/ml) at 0 & 72 hrs on the viable cell concentration and apoptosis was investigated during the serum-free cultivation. The addition of insulin at 0 h (1.6 [+ or -] 0.017 x [10.sup.6]/ml, Student's t-test p<0.001) and 0 & 72 hrs (1.82 [+ or -] 0.016 x [10.sup.6]/ml, Student's t-test p<0.001) resulted in a markedly higher viable cell concentration compared with the control (0.98 [+ or -] 0.01x106/ml) (Fig: 9A). While the viability decreased rapidly to 72 [+ or -] 1% from the initial viability of 94% at the end of the culture without insulin (Fig: 9B). A marked decrease of viability (89 [+ or -] 0.5%) was obtained in the cultures supplemented with insulin only at 0 h. CHO cells are characteristically begin to lose viability around days 4-5 of culture[35]. A significant increase of viability (96 [+ or -] 0.07%, Student's t-test p<0.001) was observed in cultures with double addition of insulin. However, in both cases upto 72 hrs the viability was super imposable.

In both cases (single & double), insulin addition increased the specific glucose consumption rate. Consequently, insulin added not only initially, but also during the period of cultivation increased the viable cell concentration together with the specific glucose consumption rate (Fig: 9C). It was examined whether insulin addition could rescue CHO cells from apoptosis by a DNA fragmentation assay. It was found that DNA from cells treated with insulin (single & double addition) was uncleaved until 3rd day of incubation but the DNA from untreated cells was partially cleaved at the same day. DNA from the control cells showed ladder formation at the 6th day of culture, whereas from insulin double addition it was still uncleaved. DNA from insulin single addition cells were cleaved partially and showed the ladder formation at the same day (Fig: 9D). These findings suggest the insulin can retard apoptosis of CHO cells.

Double addition of insulin apparently increased cell concentration (12% & 46%), viability (7.5% & 25%) and glucose uptake (26% & 44.7%) compared with single addition at 0 h and control cultures. The concentration of insulin added at 0 h rapidly decreased to approximately 0.07% within initial days of cultures. On the other hand, the insulin concentration was maintained at approximately 0.26% until 144 h after the 2nd addition at 24 h [36]. This may be the reason for not increasing the cell concentration and viability in cultures with insulin added at 0 h comparatively with repeated additions. Previous work has shown that withdrawal of insulin lead to a reduction in viable cell density and viability [37]. The addition of insulin also increased the production of EPO (Table: 2), this indicates that this effect was concomitant to the effect on viable cell maintenance (Fig: 9A). Viability loss due to apoptosis often limits recombinant protein production [7], based on the evidence that single addition of insulin to the serum-free culture had only a small effect on viable, total cell concentration and EPO production. The increase in EPO production by the insulin may be a result of the increased viability and total cell concentration. Insulin is well known antiapoptotic factor that regulates gene expression in various cells [38-39]. It is expected that the suppression of apoptosis during the production phase in serum-free or low serum media might increase protein production by mammalian cells such as CHO cells [40]. The production of EPO was 17.2% & 2.44 fold higher in double addition of insulin than in single addition and control cultures. Overcoming apoptosis, the major mode of cell death in many bioprocesses is desirable to enhance product yield and quality [41].

The concentration of EPO at the end of (day 7) culture, shown in Fig: 9, was determined.

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Culture vessel comparison for CHO cells grown in agitated systems

In an effort to increase CHO cell growth and protein production, the effect of different culture vessel conditions on cell growth and protein production in serum-free medium were investigated Cultures grown for 7 days in wave bioreactor achieved higher cell densities (3.1 [+ or -] 0.1 x [10.sup.6] cells/ml) than those grown in spinner flask (1.8 [+ or -] 0.03 x [10.sup.6] cells/ml), roller bottle (2.22 [+ or -] 0.12 x [10.sup.6] cells/ml) and T-flask (1.56 [+ or -] 0.11 x [10.sup.6] cells/ml) (Fig: 10A). Maximum cell density was obtained at 5th day of culture in wave bioreactor and viable cells started decrease after the same day, 49.67% (Student's t-test p<0.001) higher cell growth was observed in wave bioreactor compared to static culture; where as only 13% & 29.7% were seen in spinner and roller bottles, respectively. Significant decrease in viability (Fig: 10B) was observed with wave bioreactor after reaching to high cell density, this clearly indicates the depletion of nutrients in the medium. No significant decrease in viability was obtained in other cultures.

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Figure 10C shows the glucose consumption, highest consumption was observed in wave bioreactor (2.8 [+ or -] 0.055 g/l, 'starting medium glucose concentration was 3.2g/l and the glucose concentration at the end of cultivation was 0.4g/l'), whereas, 1.9 [+ or -] 0.048g/l, 2.2 [+ or -] 0.06g/l and 1.8 [+ or -] 0.045g/l were obtained in spinner, roller and T-flask. This correlates well with the cell growth and viability profiles, cell growth and viability were decreased when the glucose level falls below the 1g/l in wave bioreactor. EPO production also increased in wave bioreactor compared to other systems (Fig: 10D). The highest EPO production obtained at wave bioreactor was 42 [+ or -] 1.16mg/l, this was almost 31.6%, 21.2% and 40% (Student's t-test p<0.002) higher than spinner (28.7 [+ or -] 0.9mg/l), roller (33.1 [+ or -] 0.95mg/l) and static cultures (25.2 [+ or -] 0.65mg/l). The overall observation with different culture vessel conditions, cell growth and EPO production were highly increased in wave bioreactor, this may be due to the continuous aeration and good agitation. Hence, the wave bioreactor was chosen for the higher yields of cell growth and protein expression. These results indicate the continuous aeration and good agitation could increase CHO cell growth and EPO production significantly. Decrease in cell growth and protein production in T-flask (no agitation and only head space aeration), spinner flask and roller bottles (continuous agitation, but both systems are oxygenated by gas diffusion through the head space) may be due to limitation in aeration and agitation. Mammalian cells require dissolved oxygen for growth and metabolism, but due to the low solubility of [O.sub.2] in culture medium, a continuous supply of [O.sub.2] from the gas phase is required [42].

Cell growth and EPO production was significantly increased in roller bottles than in spinner flasks. Cultures in spinner flasks frequently show signs of oxygen limitation, especially at high cell densities[43]; this could be the one reason to decrease cell growth and EPO yields in spinner flasks, whereas in roller bottles, medium interact with head space oxygen properly due to the rotary motion of rollers, which can improve the dissolved oxygen level better than in spinner flask. Additionally, roller bottles were opened (up to one screw) for the better supply of atmospheric oxygen.

Conclusions

Gradual reduction of serum is essential to achieve fully adapted serum-free cultivation of CHO cells. Since all commercially available media will not support equally to all cell lines growth, it may be essential to select the best commercially available medium for growth and protein expression. Glutamine replacement with glutamine based dipeptide (glutamax) decreased ammonia production and enhanced cell growth, viability and EPO expression. Substitution of glutamine with glutamax is an added advantage for the commercial production of EPO by rCHO cells. Addition of insulin to serum-free cultivation of CHO cells improved the cell growth, viability, protein production and suppresses cell death. Appropriate timely repeat addition of insulin could suppress cell death more effectively and improve the protein production than single addition. CHO cell growth and EPO production was significantly increased in wave bioreactor by continuous supply of aeration and sufficient agitation than in static condition (T-flask), spinner flask and roller bottle. This work may be useful for further study on a large scale process for highly efficient production of proteins toward industrial applications. The information obtained in this work can be applied to other cell cultures, which have wide utilization and application.

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Rambabu Surabattula (1) *, Muthuvaduganathan Nagasundram (2), Ratnagiri Polavarapu (3) and K.R.S. Sambasiva Rao (4)

(1) Scientist, Research & Development, Genomix Molecular Diagnostics Pvt Ltd, 5-36/207, Prashanthnagar, Kukatpally, Hyderabad--72, AP, India E-Mail: ramsura@gmail.com

(2) Sr. Scientist, Nagarjuna Fertilizers and Chemicals Limited (NFLC), R&D Gowraram post, Village--warangal, Warangal X road, Medak Dist, Pin--502279, AP, India E-Mail: vaduga_2000@yahoo.com

(3) President & CEO, Genomix Molecular Diagnostics Pvt Ltd, 5-36/207, Prashanthnagar, Kukatpally, Hyderabad--72, AP, India E-Mail: giripolava@gmail.com

(4) Coordinator, Dept. of Biotechnology, Acharya Nagarjuna University, Nagarjuna Nagar, Guntur, Andhra Pradesh, India E-Mail: krssrao@yahoo.com
Table 1: Cell culture media used.

ID Medium Name

C Gibco- DMEM/F-12 + 5 %FBS
1 Sigma - CHO-DHFR Medium
2 JRH- Ex-Cell 302 SF Medium
3 JRH- Ex-Cell 325 SF & PF Medium
4 Invitrogen- CD CHO Medium
5 Invitrogen-CHO S SFM Medium
6 Cellgro- Cell gro free Medium
7 Hyclone- SFM4CHO Utility Medium

Table 2: Effect of the insulin addition on EPO production.

 Insulin addition EPO (mg/l)

 Control (without addition) 3.11 [+ or -] 0.12
 Single addition at 0 h 6.29 [+ or -] 0.16
 Two addition's at 0 & 72 hrs 7.6 [+ or -] 0.15

The concentration of EPO at the end of (day 7) culture,
shown in Fig: 9, was determined.
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Author:Surabattula, Rambabu; Nagasundram, Muthuvaduganathan; Polavarapu, Ratnagiri; Rao, K.R.S. Sambasiva
Publication:International Journal of Biotechnology & Biochemistry
Date:Feb 1, 2011
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