Glucose And Glutamine Metabolism Optimized Serum Free Suspension Fed-batch Culture And Integrated Multiply Parameters Control

With increasing demand of recombinant protein and expanding scale of animal cell culture, the need for overcome and optimiazation of defiency in culture performance is evident. Recenty, the worldwide research groups mainly focused on the improvement of cell perforamance, the control of cell metabolism, the maintaining of product quality while maximizaing productivity. Newly arising issues in large-scale culture, including suspension culture, serum-free medium, the conception of development of product yield and productivity simultaneously, have obtained great achievements. But thses research lacked technology integration relating to products, and neglected systematic characteristic of culture process. Thus the integrated and systematic research of serum-fre medium development, suspension cultrure adaptation, glucose and glutamine metabolism optimzed fed-batch, and process parameters control would provide mode and basis for establishment of large-scale culture technology platform in China.[ Objectives ] 1. To develop serum-free medium, and to adapt cell to serum-free suspension culture; 2. To screen optimized initial glucose and glutamine concentration, and to coduct glucose and glutamine metabolism optimized serum-free suspension fed-batch culture; 3. To define the process parameters ranges of seeding cell precultivation stages, and to control the stability of the seeding cells' physiological status.[ Methods]1. HPLC analysis and orthogonal design were used to screen serum-free medium(1) HPLC was used to analyze the amino acids exhaustion in basic medium; (2) Orthogonal design was used to screen serum-free medium supplement ingredients and their concentrations; (3) Serum-free adaptation culture was conducted by gradual serum weaning; (4) Cell density and viability were determined by cytometry; (5) Antibody production was detected Sandwiwh ELISA; (6) Antigen-binding activity was mearsured by indirect ELISA and FCM; (7) P and q MAb were calculated by equation: μ; (8) Cell growth, antibody productionand antigen-binding activity under serum-free cultrure condition were evaluated by cytometry, Sandwich ELISA, indirect ELISA, FCM, μ and qMAb, repectively.2. Serum-free suspension adaptaion of CHO-TS28 cells by using heparin(1) The influence of heparin on cell adhesion was studied by cell adhesion experiments; (2) The influence of heparin on cell growth and antibody production was analyzed by cell density, viability, antibody production, p and qMAb; (3) The influence of heparin on cell cycle and apoptosis was analyzed FCM; (4) Serum-free suspension adaptation was conducted by combining gradual serum weaning, heparin supplement, and stepwise increasing of agitation speed; (5) Cell growth, antibody production and antigen-binding activity under serum-free cultrure condition were evaluated by cytometry, Sandwich ELISA, indirect ELISA, FCM, μ and qMAb, repectively; (6) E-cadherin and integrin expression were detected by indirect immunofluorescence and FCM after cells culitivated with heparin for 72 hours.3. Dynamic parameter analysis was used to optimize the metabolism of glucose and glutamine(1) HAbl8 cells were inoculated in serum-free medium containingdifferent levels of glucose and glutamine and cultivated for 144 hours, then cell density, antibody yied, glucose and glutamine concentration, lactate and ammonia concentration were detectd, P , qMAb, tfoiuc, qain, tfLac, * Ammonia/Glutamine; * Cell/Glucose? JCell/Glutamines ^Ah/Glucose and^Ab/Giutamine were calculated; (2) HAbl8 cells were inoculated in 5Lbioreator, initial glucose and glutamine were controlled at low level. When glucose and glutamine concentration below the setpoint, condensed glucose and amino acids solution were fed according to stoichiometric ratio between glutamine and amino acids and the relationship Vin = ff'K^*)?+i +(L)J(Li~t?X^)B4. Integrating multiply process control parameters was used to explore stability of seeding cells' physiological status at the start of the production phase stable(1) HAbl8 cells were continuously cultivated for 1 to 6 months, and cell density, antibody yield, positivity rate of antigen-binding and cloning were detected regularly,- (2) HAbl8 cells from 9 groups ranging from 0.075><106 cells/ml to 0.5xlO6 cells/ml were cultivated for 72 hours, then cell density and antibody yield were mearsured; (3) HAbl8 cells were inoculated at 9 timeposts from 8h to 36h after passage and were cultured for 24 hours, then cell density and cell cycle distrubtion were determined; (4) HAbl8 cells were inoculated at 1L, 5L and 75L bioreator by selected inoculated condition for fed-batch culture, cell density, antibody yield, positivity rate of antigen-binding were detected, P and quAb were calculated; (5) Ion exchange chromatography was used to purify IgG in supernatant; (6) SDS-PAGE and FACE were conducted to observe band shift of protein and band pattern of oligosaccharide prepared at different culture condition. [Results]1. Serum-free medium SFM18 and SFM28 were developed through HPLC analysis and orthogonal design(1) Hiss Pro> Vak Leu> Ile> Phe and Met in HAbl 8 low/serim-free culture, and His> Pkk Cys^ Arg in CHO-TS28 low/serim-free culture were exhausted above 50%;(2) SFM18 and SFM28 for HAbl8 and CHO-TS28 serum-free culturerespectively were screened by orthogonal test. The two screened medium had simple and defined components, with protein at a concentration of 20-25mg/L;(3) Cell growth restored, u returned to 0.03/h and cell aggregation reduced after 2 months serum-free adaptaion;(4) Camparing with 10%FBS, p was decreased slightly while qMAb significantly increased (0.0207/h vs. 0.0218/h, 0.387 vs. 0.218 pg/cell/h, P <0.01) in SFM18. In SFM18 culture, the metabolism pathway of glutcose and glutamine was the same as those in 10%FBS; and lactate was reduced while ammonia was increased. The positivity rate of FHCC98 cell-binding activity and the intensity of fluorescence, the HAbl8G/CD147 antigen-binding activity in SFM18 was higher than in 10%FBS.2. CHO-TS28 cells serum-free suspension culture was achieved by using heparin through inhibiting E-cadherin expresion(1) Heparin showed a dose depended inhibition of intracellular adhesion in CHO-TS28, with the highest inhibition occurring at the concentration above 250 u g/ml;(2) Heparin increased the viable cell density and viability (70% vs. 93%), with the highest cell density reaching at a concentration of 125 n g/ml [ (1.205 ±0.035)xl06cells/ml] and maximum antibody yield at 250 u g/ml (lOmg/L);(3) FCM indicated that heparin reduced the apoptosis both in early phase (20.7 vs. 10.8-13.4%) and in late phase (8.9 vs. 2.1-3.8 %). While the cell number in S phase was not affected by heparin;(4) Heparin effectively dispersed cell aggregates and single-cell suspension culture could be achieved. And u restored to 0.02-0.03/h while maintaining the viability above 85% after continuous passage of 11 generations in suspension culture;(5) Camparing with 10%FBS, u was significantly decreased in static serum-free medium; while restored to 0.0298 ± 0.01/h after suspension adaptation. qMAb stayed at similar level among conditions of 10%FBS, SFM28 and suspension culture. The utilization of glutcose and glutamine was increased in serum-free culture. The positivity rate of FHCC98 cell-bindingactivity and the intensity of fluorescence, the HAbl8G/CD147 antigen-binding activity in SFM28 were higher than in 10%FBS;(6) CHO-TS28 expressed E-cadherin and 250 u g/ml heparin could reduced the positive rate of its expression from 51.5% to 26.4%.3. Cell density and antibody yield were both enhanced in glucose and glutamine concentration optimized fed-batch culture(1) When glucose concentration decreased below 0.5mM, glutamine concentration below 0.3mM , #Lac > qAmm and Jlatatec/Giucose decreased significantly, while Fceii/Giucos and Fceii/Giutamine increased about 2~3folds. #Gin showed a trend of increase at lower glucose level; amino acid (Lys, Thr, Val, Leu, lie, Phe and Met) consumption increased by 30-50% at low glutamine concentration. Additionly, qrowth limitations existed at glucose level <0.5 mM and glutamine level <0.3 raM; while identical growth rates were obtained for different nutrients levels when glucose was above 0.5mM and glutamine above 0.3mM. The different effects of glucose and glutamine on specific antibody productivity were demonstrated by the results that <7MAb increased to 2 folds of original media at 0.5mM glucose, and decreased to 37.6% of original media at 0.3mM glutamine.(2) Lactate and ammonia production reduced greatly at low glucose and glutamine level. The maximum lactate and ammonia concentration were 2.982mM and 1.537mM, respectively. Compared with high-level glucose and glutamine fed-batch culture, low-level glucose and glutamine led to higher cell density (1.0 vs. 0.3 X 107 cells/ml), longer culture span (14 vs. 8 days) and higher antibody yield (250 vs. 150mg/L).4. Stability of seeding cells' physiological status at the start of the production phase were achieved by integrating multiply process control parameters in fed-batch culture(1) When cells were subcultured within 3 months after revival, ^MAb, binding activity for targeted cell FHCC98 and targeted antigen HAbl8G, positivity rate of cloning could be maintained above 85% of initial culture;(2) In a range of inoculation density from 0.1 to 0.3 xlO6 cells/ml, the obtained maximum cell density was enhanced with inoculation density...