full issue - Association of Biotechnology and Pharmacy

More documents

Recommendations

Info

Current Trends in Biotechnology and Pharmacy Vol. 5 (3) 1298 -1308 July 2011, ISSN 0973-8916 (Print), 2230-7303 (Online) 1299 therefore, large amounts of culture are needed per clinical dose of the final product. High cell density cultures can overcome this problem by increasing the volumetric productivity, which is directly related to both cell density and specific productivity (2,3). Packed-bed bioreactors enable the achievement of high-volume cell density and productivity under low-volume media conditions. Cong et al. (2001) managed to achieve a CHO cell density of 2 × 10 7 cells/ml after fifteen days cultivation in a packed-bed perfusion bioreactor (4). Culture compositions have a great impact on cell viability, density and productivity, and also influence cell attachment to microcarriers. Reduction in glutamine concentrations decreases cell growth and viability (5,6). Whereas glucose and glutamine are vital for rCHO cells, glutamine cannot support cell maintenance in the absence of glucose. Meanwhile, glutamine metabolism represents the main source of ammonium as a toxic by-product (5,7,8). Elevated ammonium concentrations significantly inhibit cell growth and final cell density and consequently decrease volumetric productivity of recombinant proteins. The adverse effects of ammonium on gene expression and protein glycosylation in rCHO cells have also been previously reported (9-12). Lactate is another toxic by-product, which is formed in the presence of excess glucose. Glutamine metabolism and ammonium buildup increase intracellular pH, while lactate formation decreases intracellular pH in mammalian cell cultures. Various strategies have been developed to alleviate the detrimental effects of both byproducts in CHO cells (13). Addition of higher concentrations of certain amino acids has been reported to diminish the negative effects of ammonium, especially cell growth and productivity in CHO cells (14). Besides, certain amino acids can protect rCHO cells from elevated osmolality and carbon dioxide pressure which inhibit both cell growth and recombinant protein production (15,16). Shift of cultivation temperature from 37 o C to 30 o C has been shown to cause a growth arrest and improvement in the productivity of rCHO cells producing alkaline phosphatase. Since temperature shifts have been found to have different effects on mammalian cell growth and recombinant protein production that is dependent on the cell line and expression system, optimization of temperature has been proposed for each cell line (17,18). Human Coagulation factor VIII is a large plasma glycoprotein that is necessary for prophylactic treatment of hemophilia A patients. Increasing demand for rhFVIII has resulted in the development and improvement of rhFVIII production, in order to decrease the cost of rhFVIII (19,20). However, there are many problems with regard to rhFVIII production, such as interaction with protein chaperones and its stability in medium (21,22). Secretion of rhFVIII has been found to increase in culture medium supplemented with different concentrations of propionic and butyric acids (23). Implementing different cell lines and improving glycosylation sites have also been effective methods with regard to increasing rhFVIII production (24,25). It is commonly accepted that all mammalian cell lines have the same basic nutrient requirements; however their individual metabolisms, e.g. specific nutrient consumption and metabolite production rates often differ based on the cell line and expression system. Consequently, the main objective of this study was to investigate the physico-chemical culture conditions of the rCHO cells that affect their growth behaviors, in order to improve the cell density and recombinant protein production in a packed-bed bioreactor. Materials and methods Materials: Dulbecco’s modified Eagle’s medium (DMEM), Ham’s F12, fetal bovine serum (FBS), Shakibaie et al
Current Trends in Biotechnology and Pharmacy Vol. 5 (3) 1298 -1308 July 2011, ISSN 0973-8916 (Print), 2230-7303 (Online) 1300 and trypsin were purchased from Gibco. All chemicals were of analytical grade, and were purchased from Sigma-Aldrich (St. Louis, MO). Cell line: The rhFVIII expressing CHO cell line obtained from the National Institute of Genetic Engineering and Biotechnology (26) was used as the rCHO cell line. Culture conditions: The rCHO cells were cultured in a basal medium containing DMEM and Ham’s F12 nutrient mixture at a ratio of 1:1 (v/v), supplemented with 10% FBS. The rCHO cells were grown at different concentrations of glucose (1.2, 3.5, 7 g/l) in a T-75 flask so as to obtain an appropriate glucose concentration in the basal medium, for the purpose of obtaining higher amounts of cell density. The basal medium was prepared at different concentrations of NH 4 Cl (2, 4, 6, 8, 10, 12 mM) to determine the effect of ammonium stress on rCHO cell growth and rhFVIII production. The control flask contained basal medium without excess ammonium. The amino acid-supplemented T-flasks contained basal medium plus 20 mM of each of the appropriate amino acids and 10 mM NH 4 Cl. The negative control flask contained the basal medium plus 10 mM NH 4 Cl, and the positive control flask contained only basal medium. This experiment was carried out at low and high glucose concentrations (1.2, 3.5 g/l). The precursor-supplemented T-flasks contained basal medium plus 10 -3 M of each of the dNTPs (dATP, dCTP, dTTP, dGTP). The control flask did not contain any precursors. For studying effects of temperature on cell growth and protein production, the rCHO cells were cultivated at 37 o C until the culture reached the exponential phase of growth. The temperature was then reduced to 30 o C., but the control flask was maintained at 37 o C. The osmolality of the medium supplemented with 10% FBS was 320 mOsmol/kg. To determine the effects of osmolality on cell growth and protein production, NaCl stock solution was added to the basal medium increasing osmolality by up to 410 mOsmol/kg (1 mg NaCl/ml = 32 mOsm increase). The control flask had no excess sodium chloride. In order to investigate the effects of certain additives on cell growth, the rCHO cells were cultivated in basal medium with 10% FBS. After 48 h of cultivation, the medium was replaced with fresh medium without FBS. Appropriate amino acids and biotin were added to the serumfree medium. Basal medium was supplemented with 10 mg/l biotin. The serum-free control flask had no additives. Bioreactor: The batch bioreactor experiment was performed in a 5 l (3.5 l working volume) Celligen bioreactor (New Brunswick Scientific, USA) maintained at 37 o C, pH 7.2, and stirred at 50-100 rpm. The pH level was controlled by acidic and basic solutions (2N HCl and 80g/l NaHCO 3 ). Dissolved oxygen (DO) was adjusted to 50% of air saturation, as manipulated by the bioreactor control program. The polyester disks (134 g) purchased from New Brunswick Scientific Co., were used as microcarriers for the attachment of the rCHO cells in the packed-bed bioreactor. The appropriate conditions obtained in the T-flask were also employed in packed-bed bioreactor. Glutamine was added after 48 h of cultivation at 20 mM and other additives (proline, glycine, threonine, glutamine, biotin, dNTP and glucose) were prepared in the basal medium. The concentrations of the additives were; amino acids (proline, glycine, threonine) 20 mM, biotin 2 mg/ l, dNTP 10 -3 M, and glucose 3.5 g/l. After 120 h of cultivation, the polyester microcarriers were removed from the bioreactor and the rCHO cells were separated by the EDTA-Trypsin solution. Effect of culture conditions on rCHO cell growth
Page 3 and 4:
ISSN 0973-8916 Current Trends in Bi
Page 5 and 6:
Current Trends in Biotechnology and
Page 7 and 8:
Page 9 and 10:
Page 11 and 12:
Page 13 and 14:
Page 15 and 16:
Page 17 and 18:
Page 19 and 20:
Page 21 and 22:
Page 23 and 24:
Page 25 and 26:
Page 27 and 28:
Page 29 and 30:
Page 31 and 32:
Page 33 and 34:
Page 35 and 36:
Page 37 and 38:
Page 39 and 40:
Page 41 and 42:
Page 43 and 44:
Page 45 and 46:
Page 47 and 48:
Page 49 and 50: Current Trends in Biotechnology and
Page 61 and 62: β Current Trends in Biotechnology
Page 99: Current Trends in Biotechnology and
Page 151 and 152:
Page 153 and 154:
Page 155 and 156:
Page 157 and 158:
Page 159 and 160:
Page 161 and 162:
Page 163 and 164:
Page 165 and 166:
Page 167 and 168:
Page 169 and 170:
Page 171 and 172:
Page 173 and 174:
Page 175:
(December 7-9, 2011) Venue: Karunya
show all

full issue - Association of Biotechnology and Pharmacy

Create successful ePaper yourself

Delete template?

Save as template?