Abstract:
A cell culture vessel comprising a housing chamber which has an inverted frusto-conical bottom having a vertical axis. The culture vessel further comprises an upper and lower section which are concentric. The vessel requires little or no shear-force and less stress by shaking or impellor action.

Description:
RELATED APPLICATION 
       [0001]    This application claims priority to U.S. provisional Application Serial No. US60/690,587 entitled “suspension culture vessels,” filed on Jun. 15, 2005, the content of which is incorporated by reference in its entirety. 
     
    
     FIELD OF THE INVENTION 
       [0002]    The present invention relates to a wide-body mammalian cell culture vessel with an inversed frusto-conical or inverted frustum bottom for reduced seed volume and better mixing with less hydro-mechanical stress and greater aeration. 
       BACKGROUND OF THE INVENTION 
       [0003]    High-density suspension culture of mammalian cells is a useful tool for protein drug or vaccine development. It often requires small-volume cell culture vessels for production of animal testing materials, and large-volume cell culture vessels for production of clinical trial material. Most cell biologists prefer simplified small volume suspension culture equipments, while scale-up professionals enjoy “easy to optimize” large-volume cell culture systems. 
         [0004]    Tall cylinder is a typical shape for current bioreactor culture vessels and spinner bottles. Such a vessel or bottle has a height:diameter ratio of larger than 1/1 of at work volume. The surface area of the culture medium in the vessel is not enough for effective O2 uptake. Air or O2 sparging has been used to address this problem. However, overdose of air sparging often causes cell damage by foaming and bubble burst. Also, overdose of pure O2 sparging is toxic to cells. Thus, a sophisticated control tower and related dissolved oxygen (DO) probes are required to monitor and control air or pure O2 sparging. Nonetheless, it is tedious and costly to optimize the control tower or probe. Thus, there is a need for small volume culture vessels, particularly disposable vessels, without control tower and related DO probe, and for “easy to optimize” large volume culture vessels. 
         [0005]    This design of this invention allows one to culture large volume of cells without using sophisticated control tower and related probes, Also, disposable or autoclavable small-volume shaker-based suspension culture systems have been developed. In addition, a prototype for an “easy to optimize” impellor-based large volume industrial inverted frusto-conical bioreactor system has also been studied. 
       SUMMARY OF INVENTION 
       [0006]    This invention features a wide-body culture vessel with an inverted frusto-conical bottom. Comparing with traditional flat-bottom bioreactor vessels, the vessel of this invention has a significant larger culture medium surface area for O2 uptake and CO2 stripping. The inverted frusto-conical bottom provides significant seed volume reduction for initiation of a cell culture process. The inverted frusto-conical bottom makes a low-positioned air sparging possible for extended air bubble traveling time course. Meanwhile, orbital shaking can be used so that the culture medium climb up onto the wall of the vessel easily with no share-force and less hydro-mechanical stress. This creates a broad thin medium layer for extended surface and greater aeration and better mixing. By using this design, plastic “single use” or glass autoclavable small-volume shaker-based suspension culture systems with no sophisticated control tower and related probes have been developed. In addition, a prototype for an “easy to optimize” impellor-based large volume industrial bioreactor system has been studied. 
     
    
     
       BRIEF DESCRIPTION OF DRAWINGS 
         [0007]      FIG. 1   a  is a drawing of 3-liter wide-body vessel with an inverted frusto-conical bottom; work volume=3 L; culture medium surface area/culture medium volume &gt;0.143 cm2/cm 3 ; minimum seeding volume=150 ml, 1/20 of marked 3-liter work volume. 
           [0008]      FIG. 1   b  is a photograph of a 3-liter wide-body shaker vessel with an inverted frusto-conical bottom and a traditional flat-bottom Applikon bioreactor vessel on the background. 
           [0009]      FIG. 2  is a photograph of a 150 ml wide-body shaker bottle with an inverted frusto-conical bottom; work volume=150 ml culture medium; total free air space=500 ml; minimum seeding volume=7.5 ml, 1/20 of the marked work volume. A shaker platform, a flow meter, a low-positioned air bubbling device and an air pump are required to support this system. This system is mainly designed for screening production cell line candidates for their robustness and productivity, as well as seed train support. 
           [0010]      FIG. 3  is a series of drawings of wide-body suspension culture vessels with inverted frusto-conical bottoms providing a simplified seed train process. 
           [0011]      FIG. 4   a  is a diagram showing scale up issues and factors that negatively affect scale-up optimization related to mixing, shear force, hydro-mechanical stress, aeration, and CO2 stripping, of industrial tall-cylinder stir-tank bioreactors. 
           [0012]      FIG. 4   b  is a diagram showing advantages of wide-body bioreactor vessels with inverted frusto-conical bottom for large-scale industrial cell culture. 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0013]    This invention is based, at least in part, on the discovery that, without using sophisticated control tower and related DO and pH probes, suspension adapted mammalian cells grew well when culture medium surface area/culture medium volume is above 0.14 cm2/cm 3 . This culture was conducted in a shaker bottle with a bottom air sparging or overlay pure O2 supply at 37° C. using a serum-free suspension medium supplemented with 20-25 mM HEPES. The above discovery indicated that wide-body culture vessels have advantage for medium surface aeration over traditional tall cylinder stir-tank culture vessels. 
         [0014]    Many robust mammalian cell lines have been developed for serum-free and animal component-free suspension culture. These include CHOD (B11 and DG44), CHOK, and NS0 (serum-free adapted) cells. Also, powerful animal component-free suspension culture media (basal growth and feed) have also been developed. By adding a non-CO2 dependent buffer such as 20 mM HEPES buffer and sufficient surface aeration, sophisticated control tower and related dissolved oxygen (DO) and pH probes become not essential for small volume suspension cultures when wide body culture vessels are employed. The above developments enabled us to freely test and develop simplified and “easy to optimize” bioreactor vessels for serum-free suspension culture of mammalian cells. 
         [0015]    In one example, we have designed and made 3-liter ( FIG. 1   a,b ) and 150 ml ( FIG. 2 ) work volume wide-body culture vessels with inverted frusto-conical bottom. These vessels were first used as shaker culture vessels for mammalian cell culture. Surprisingly, they worked much better than conventional Applikon flat-bottom bioreactor vessels of similar volume in term of cell density, recombinant protein productivity, reliability, and user-friendlyness. Clearly, combination of orbital shaking, frusto-conically shaped bottom, wide-body and pH stable culture medium worked in favor of mammalian cell culture and recombinant protein production. 
         [0016]    In the first part of this invention, the focus was on a small-scale 150 ml wide-body shaker bottle with an inverted frusto-conical bottom for robustness screening of production cell line candidates. The important invention for this part was the inverted frusto-conical bottom for reduced seed volume for initiation of a cell culture process. 
         [0017]    In this study, a wide-body vessel with an inverted frusto-conical bottom ( FIG. 2 ) was studied first side by side with a wide-body shaker vessel with a “sharp-point” conical bottom (Corning, cat #431123). Each e vessel has a total volume of 500 ml and a designated 150 ml work volume. CHO cells expressing recombinant protein VEGFR1-Fc-IL-1ra were used for this study. Fed-batch results in Table 1 indicate that these 150 ml shaker vessels were good for small-scale suspension fed-batch study. A small seed volume 7.5-15 ml ( 1/20- 1/10 of the 150 ml work volume) was achieved for successful initiation of a cell culture process. The shaker vessel with “sharp-point” conical bottom caused cells precipitation particularly cell clusters (Table 1) at the top of the bottom, indicating poor mixing at the region. Thus it was abandoned. 
         [0018]    The second part of this invention related to effective cone angles for the culture vessels with conical bottom. Influence of the cone angle on orbital shaking, culture medium mixing, and seed volume were studied by using plastic conical centrifugation tubes with different cone angles (Table 2). The orbital shaking may not be able to make the culture medium climbing up on the wall easily, if the angle is too narrow (such as &lt;30 degrees) or too wide (such as &gt;70 degrees). 
         [0019]    In the third part of this invention, the focus was on 3-liter suspension culture vessels with no sophisticated control tower and related DO, pH probes. Aeration was through either air sparging at the lowest point of the inverted frusto-conical bottom for extended air bubble travel time course or pure O2 overlay over large medium surface area. 
         [0020]    First, we found that orbital shaking was able to make the culture medium easily climb up onto the wall of the culture vessel, thereby achieving low hydro-mechanical stress and creating a broad thin medium layer for extended surface, greater aeration and better mixing. 
         [0021]    In this study, a 3-liter wide-body shaker vessel with an inverted frusto-conical bottom ( FIG. 1   a, b ) was constructed and tested with no control tower or related DO probe. Filtered air (containing about 21% of O2) was pumped into this shaker vessel for air sparging at low-flow rate without causing significant cell damage. Alternatively very low flow-rate of pure O2 was used through a tube device at bottom of the vessel (serves as a flow-rate monitor) for generation of larger bubbles or direct O2 overlay. 
         [0022]    CHO cells expressing TNFR1-Fc-IL-1ra, IL-18 bp-Fc-IL-1ra, VEGFR1-Fc-IL-1ra, and Tie2-Fc-IL-1ra were used for this study. Applikon 2 liter flat-bottom stir-tank bioreactor ( FIG. 1   b  background) and 20 liter coy-boy vessel served as flat bottom shaker bottles. Each had a 3 liter work volume culture and was employed as a control. Table 3, 4, 5 summarized the results from the inverted frusto-conical bottom vessels compared with results from Applikon bioreactor vessels and flat-bottom shaker vessels. 
         [0023]    Surprisingly, the wide-body shaker vessel (Table 3) has worked significantly better than flat-bottom Applikon stir-tank (Table 4) and flat-bottom shaker vessels (Table 5). Clearly, orbital shaking motion is able to push culture medium up onto the vessel wall easily with no shear force and less hydro-mechanical stress, and create a broad thin medium layer for greater aeration. 
         [0024]    The final part of this invention focused on design, analysis and prototype study of a large volume impellor-based industrial stir-tank wide-body bioreactor with an inverted frusto-conical bottom. Application of the inverted frusto-conical bottom contributes significantly to reduced seed volume ( FIG. 3 ) as well as better mixing with less hydro-mechanical stress and possible low shear force. In addition, the larger surface area of the design certainly contributes to better aeration and more effective CO2 stripping ( FIG. 4   a, b ). Our goal was to make larger industrial bioreactors stable, easier to optimize and use of significantly reduced seed volume.  FIG. 4   a  shows scale up issues and factors that negatively affect scale up optimization related to mixing, shear force, hydro-mechanical stress, aeration, and CO2 stripping. These factors make scale up optimization more difficult particularly for large volume industrial bioreactors. After the analysis, we have concluded that, at least in theory, a wide-body vessel with a conical bottom not only improves scale up optimization (such as better mixing with less hydro-mechanical stress and possible less shear force)( FIG. 4   b ), but also reduces feed volume of impellor driven stir-tank bioreactor vessels ( FIG. 3 ). At a given agitation speed, the inverted frusto-conical bottom of the vessel and the increased surface area together improve scale up optimization process. 
       EXAMPLE 1 
     Study of 150 ml Wide-Body Shaker Vessel with Inverted Frusto-Conical Bottom 
       [0025]    Use of small-scale shaker vessels for fed-batch culture of CHO production cell line candidates is an important approach to screen production cell lines for robustness and high productivity. In this study, 150 ml wide-body vessels with the inverted frusto-conical bottom ( FIG. 2 ) were tested side by side with wide-body shaker bottles with inverted “sharp-point” conical bottom (Corning, Cat #431123). The fed-batch results (Table 1) indicated that these 150 ml shaker bottles were suitable for small-scale suspension fed-batch study. Small seed volume ( 1/20 of the work volume) was achieved. Expression titers were comparable to the 2-liter bioreactor. The shaker vessel with the inverted “sharp-point” conical bottom caused obvious cells precipitation particularly large size cell clusters showing poor mixing in the bottom tip area. For this reason, we abandoned use of the inverted “sharp-point” conical bottom and adapted the inverted frusto-conical bottom. 
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 The culture vessels were placed on a shaker platform at speed 100 rpm. Feed 
               
               
                 (4.5 ml) started at day 7 when batch culture volume reached 90 ml for two days. 
               
             
          
           
               
                   
                   
                   
                   
                   
                   
                   
                   
                 Cells or cell 
               
               
                   
                   
                   
                   
                   
                   
                 End of 
                   
                 cluster 
               
               
                 Vessel 
                   
                 Seed 
                 Seed 
                 Before 
                 After feed at 
                 culture at 
                 Expression 
                 precipitation 
               
               
                 bottom 
                 Cell line 
                 volume 
                 density 
                 feed at Day 7 
                 day 9 
                 day 11 
                 titer (mg/L) 
                 at bottom 
               
               
                   
               
               
                 Frusto- 
                 CHO 
                 15 ml 
                 1 ± 0.2 × 10-6 
                 6.0 ± 0.8 × 10-6 
                 10.2 ± 1.5 × 10-6 cells/ml 
                 7.1 ± 0.9 × 10-6 cells/ml 
                 128 ± 8.0 
                 No 
               
               
                 conical 
                 expressing 
                   
                 cells/ml 
                 cells/ml 
               
               
                 bottom 
                 VEGFR1- 
               
               
                   
                 Fc-IL-1ra 
               
               
                 Sharp- 
                 CHO 
                 15 ml 
                 1 ± 0.2 × 10-6 
                 6.5 ± 0.7 × 10-6 
                  9.9 ± 1.3 × 10-6 cells/ml 
                    6.8 × 10-6 cells/ml 
                 118 ± 0.6 
                 Yes 
               
               
                 point 
                 expressing 
                   
                 cells/ml 
                 cells/ml 
               
               
                 conical 
                 VEGFR1- 
               
               
                 bottom 
                 Fc-IL-1ra 
               
               
                   
               
             
          
         
       
     
       EXAMPLE 2 
     Study of Culture Vessels with the Inverted Conical Bottom by Different Cone Angles 
       [0026]    Influence of cone angle of the conical bottom on orbital shaking, medium mixing, and seed volume were studied by using plastic centrifugation tubes with different cone angles on shaker platform (Table 2). The orbital shaking might not be able to make the culture volume climbing up on vessel wall easily and forming a broad thin layer with extended medium surface area, if the angle was too narrow or too wide. For example, it was not significantly different from the flat bottom vessels if the angle was too wide (such as &gt;70 degrees). It was also not much different from flat bottom vessels if the angle was too narrow (such as &lt;30 degrees). Results in Table 3 suggested that 30 degree of the angle from the cylinder wall perhaps was probably the minimum angle for effective orbital shaking and mixing. According to calculation, &gt;70 degrees of cone angle, there was no significant reduced seed volume was achieved from that of regular round bottom culture vessels, thus being abandoned. 
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 Study of vessels with the conical bottom by different angles on effective orbital 
               
               
                 shaking and mixing, and seed volume. 
               
             
          
           
               
                   
                   
                   
                   
                 Effect on 
                   
                 Effect on 
               
               
                 Brand and 
                   
                 Cone angle 
                 Total 
                 orbital 
                 Effect on 
                 seed 
               
               
                 Cat# 
                 Measured angle 
                 description 
                 volume 
                 shaking 
                 mixing 
                 volume 
               
               
                   
               
               
                 Corning 
                 Conical bottom 
                 Narrow 
                 250 ml 
                 Not 
                 Precipitation at 
                 No 
               
               
                 Cat#43776 
                 is 30 degree 
                   
                   
                 shaking 
                 the apex 
                 problem 
               
               
                   
                 angle from the 
                   
                   
                 well 
               
               
                   
                 cylinder wall 
               
               
                 Kendall 
                 Frusto-conical 
                 Wide 
                  50 ml 
                 Shaking 
                 No 
                 No 
               
               
                 Cat#20820 
                 bottom is 40 
                   
                   
                 well 
                 precipitation at 
                 problem 
               
               
                   
                 degree angle 
                   
                   
                   
                 both low and 
               
               
                   
                 from the 
                   
                   
                   
                 high speed 
               
               
                   
                 cylinder wall 
               
               
                 Corning 
                 Conical bottom 
                 Wide 
                 500 ml 
                 Shaking 
                 Precipitation at 
                 No 
               
               
                 Cat# 
                 is 40 degree 
                   
                   
                 great 
                 the apex at low 
                 problem 
               
               
                 431123 
                 angle from the 
                   
                   
                   
                 speed 
               
               
                   
                 cylinder wall 
               
               
                 Custom- 
                 Frusto-conical 
                 Wide 
                 500 ml 
                 Shaking 
                 No 
                 No 
               
               
                 made 
                 bottom is 40 
                   
                   
                 great 
                 precipitation at 
                 problem 
               
               
                   
                 degree angle 
                   
                   
                   
                 both low and 
               
               
                   
                 from the 
                   
                   
                   
                 high speed 
               
               
                   
                 cylinder wall 
               
               
                 Custom- 
                 Frusto-conical 
                 Wide 
                 3000 ml  
                 Shaking 
                 No 
                 No 
               
               
                 made 
                 bottom is 45 
                   
                   
                 great 
                 precipitation at 
                 problem 
               
               
                   
                 degree angle 
                   
                   
                   
                 both low and 
               
               
                   
                 from the 
                   
                   
                   
                 high speed 
               
               
                   
                 cylinder wall 
               
               
                   
               
               
                 Note: 
               
               
                 CHO cells expressing TNFR2-Fc-IL-1ra were used for this study. 
               
             
          
         
       
     
       EXAMPLE 3 
       [0027]    Study of 3-liter wide-body shaker vessel with inverted frusto-conical bottom 3-liter wide-body shaker vessel with inverted frusto-conical bottom together with O2 tube bubbling device or air sparging was assembled ( FIG. 1   a, b ). The culture medium at work volume has larger surface area for medium O2 uptake (&gt;0.143 cm2 surface area per cm 3  medium volume). The inverted frusto-conical bottom makes a low-positioned air sparging device possible for extended air (containing about 21% of O2) traveling time course (Figure a, b). The O2 bubble tube device at the bottom serves as a low flow meter monitor for very low flow rate of pure O2 medium surface supply. The orbital shaking motion is able to make the culture volume climbing up on the conical bottom wall easily and creating a broad thin medium layer for extended medium surface area and better aeration. 
         [0028]    We have been routinely using CHO cell lines expressing TNFR1-Fc-IL-lra, IL-18 bp-Fc-IL-lra, VEGFR1-Fc-IL-lra, and Tie2-Fc-IL-lra for production of animal testing materials. The above 3-liter wide-body shaker vessel with inverted frusto-conical bottom, Applikon 2 liter flat-bottom stir-tank bioreactor and 20 liter shaker-based coy-boy culture vessel were employed for the routine production. Table 3, 4, 5 summarized all the results from the inverted frusto-conical bottom vessels comparing with results from flat-bottom Applikon bioreactor vessel and flat-bottom shaker vessel. 
         [0029]    Surprisingly, the wide-body shaker vessel with inverted frusto-conical bottom (Table 3) worked significantly better than flat-bottom Applikon stir-tank (Table 4) and shaker vessel (Table 5). Clearly, orbital shaking motion is able to push culture medium climbing up on the vessel wall easily with no shear force and low hydro-mechanical stress, and creating a broad thin medium layer for greater aeration. 
         [0030]    The wide-body shaker culture vessel with inverted frusto-conical bottom was designed to be better than recently developed “Wave” plastic bag bioreactor. Our analysis showed that it is similar to “Wave” with large surface area for O2 uptake and CO2 remival. However, the orbital shaking together with frusto-conical bottom of the vessel makes the culture medium climbing up on the vessel wall easily and creating a broad thin layer of culture medium for extended medium surface aeration. Similar to “Wave”, it is “single use” plastic vessels. However, it is better than “Wave” due to significantly reduced seed volume for initiating a given cell culture process. It is also better than “Wave” that an air sparging device is placed at the frusto-conical bottom for significantly extend air bubble travel time course. Although we did not compare “Wave” directly with the wide-body culture vessel with inverted frusto-conical bottom in terms of cell density, cost-effectiveness and user-friendlyness, all the indirect data pointed that the wide-body culture vessel with inverted frusto-conical bottom is much better than “Wave” in all the aspects mentioned. 
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 3-liter wide-body shaker vessel with inverted frusto-conical bottom 
               
               
                 together with O2 tube bubbling device or air sparging was used for routine fed-batch 
               
               
                 production of animal testing materials by using CHO cell lines expressing TNFR1-Fc-IL- 
               
               
                 1ra, IL-18bp-Fc-IL-1ra, VEGFR1-Fc-IL-1ra, and Tie2-Fc-IL-1ra. 
               
             
          
           
               
                   
                   
                   
                   
                   
                   
                 Final dot 
                   
               
               
                 Gas 
                   
                   
                   
                 Total 
                 Packed cell 
                 blot titer 
                 Final 
               
               
                 supply 
                 CHO cells 
                 Seed 
                 The highest 
                 culture 
                 volume 
                 (+after 
                 purification 
               
               
                 mode 
                 expressing 
                 volume 
                 cell density 
                 length 
                 (pcv) % 
                 dilution) 
                 yield 
               
               
                   
               
               
                 Low- 
                 TNFR1- 
                 300 ml 
                 20.4 ± 6.0 × 10-6 cells/ml 
                 13 
                 4.2% 
                 518 mg/ml 
                 77 mg/L 
               
               
                 positioned 
                 Fc-IL-1ra 
                   
                   
                 days 
               
               
                 air 
               
               
                 sparging 
               
               
                 Low- 
                 IL-18bp- 
                 300 ml 
                 18.8 ± 0.6 × 10-6 cells/ml 
                 14 
                 4.0% 
                 518 mg/ml 
                 65 mg/L 
               
               
                 positioned 
                 Fc-IL-1ra 
                   
                   
                 days 
               
               
                 air 
               
               
                 sparging 
               
               
                 Low- 
                 VEFR1- 
                 300 ml 
                 15.0 ± 0.3 × 10-6 cells/ml 
                 14 
                 3.5% 
                 256 mg/ml 
                 66 mg/L 
               
               
                 positioned 
                 Fc-IL-1ra 
                   
                   
                 days 
               
               
                 air 
               
               
                 sparging 
               
               
                 Low- 
                 Tie2-Fc- 
                 300 ml 
                 10.0 ± 0.3 × 10-6 cells/ml 
                 15 
                 2.2% 
                 128 mg/ml 
                 42 mg/L 
               
               
                 positioned 
                 IL-1ra 
               
               
                 air 
               
               
                 sparging 
               
               
                 O2 
                 TNFR1- 
                 300 ml 1 
                 15.0 ± 0.3 × 10-6 cells/ml 
                 14 
                 3.4% 
                 518 mg/ml 
                 73 mg/L 
               
               
                 Overlay 
                 Fc-IL-1ra 
                   
                   
                 days 
               
               
                 O2 
                 IL-18bp- 
                 300 ml 
                  9.0 ± 0.9 × 10-6 cells/ml 
                 15 
                 2.3% 
                 518 mg/ml 
                 66 mg/L 
               
               
                 Overlay 
                 Fc-IL-1ra 
                   
                   
                 days 
               
               
                 O2 
                 VEFR1- 
                 300 ml 
                 18.0 ± 0.7 × 10-6 cells/ml 
                 13 
                 3.8% 
                 256 mg/ml 
                 67 mg/L 
               
               
                 Overlay 
                 Fc-IL-1ra 
                   
                   
                 days 
               
               
                 O2 
                 Tie2-Fc- 
                 300 ml 
                 12.0 ± 0.5 × 10-6 cells/ml 
                 14 
                 2.8% 
                 128 mg/ml 
                 39 mg/L 
               
               
                 Overlay 
                 IL-1ra 
                   
                   
                 days 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 4 
               
             
             
               
                   
               
               
                 Applikon 2 liter flat-bottom stir-tank bioreactor vessel was routinely 
               
               
                 employed for routine Fed-batch production of animal testing materials by using CHO cell 
               
               
                 lines expressing TNFR1-Fc-IL-1ra, IL-18bp-Fc-IL-1ra, VEGFR1-Fc-IL-1ra, and Tie2- 
               
               
                 Fc-IL-1ra. Some of the results summarized here as controls. 
               
             
          
           
               
                   
                   
                   
                   
                   
                   
                 Final dot 
                   
               
               
                 Gas 
                   
                   
                   
                 Total 
                   
                 blot titer 
                 Final 
               
               
                 supply 
                 CHO cells 
                 Seed 
                 The highest 
                 culture 
                 Packed cell 
                 (+after 
                 purification 
               
               
                 mode 
                 expressing 
                 volume 
                 cell density 
                 length 
                 volume (pcv) % 
                 dilution) 
                 yield 
               
               
                   
               
               
                 Low- 
                 TNFR1- 
                 800 ml 
                 9.4 ± 6.0 × 10-6 cells/ml 
                 13 
                 2.2% 
                 256 mg/ml 
                 44 mg/L 
               
               
                 positioned 
                 Fc-IL-1ra 
                   
                   
                 days 
               
               
                 air 
               
               
                 sparging 
               
               
                 Low- 
                 IL-18bp- 
                 800 ml 
                 8.8 ± 0.7 × 10-6 cells/ml 
                 12 
                 2.0% 
                 256 mg/ml 
                 41 mg/L 
               
               
                 positioned 
                 Fc-IL-1ra 
                   
                   
                 days 
               
               
                 air 
               
               
                 sparging 
               
               
                 Low- 
                 VEFR1- 
                 800 ml 
                 10.0 ± 0.7 × 10-6 cells/ml  
                 12 
                 2.3 
                 256 mg/L 
                 42 mg/L 
               
               
                 positioned 
                 Fc-IL-1ra 
                   
                   
                 days 
               
               
                 air 
               
               
                 sparging 
               
               
                 Low- 
                 Tie2-Fc- 
                 800 ml 
                 N/A 
                 N/A 
                 Did not grow 
                 N/A 
                 N/A 
               
               
                 positioned 
                 IL-1ra 
               
               
                 air 
               
               
                 sparging 
               
               
                 Low- 
                 TNFR1- 
                 800 ml 
                 8.0 ± 0.3 × 10-6 cells/ml 
                 13 
                 1.8% 
                 256 mg/ml 
                 46 mg/L 
               
               
                 positioned 
                 Fc-IL-1ra 
                   
                   
                 days 
               
               
                 pure O2 
               
               
                 sparging 
               
               
                 Low- 
                 IL-18bp- 
                 800 ml 
                 9.0 ± 0.9 × 10-6 cells/ml 
                 12 
                 2.0% 
                 256 mg/ml 
                 42 mg/L 
               
               
                 positioned 
                 Fc-IL-1ra 
                   
                   
                 days 
               
               
                 pure O2 
               
               
                 sparging 
               
               
                 Low- 
                 VEFR1- 
                 800 ml 
                 N/A 
                 N/A 
                 contaminated 
                 N/A 
                 N/A 
               
               
                 positioned 
                 Fc-IL-1ra 
               
               
                 pure O2 
               
               
                 sparging 
               
               
                 Low- 
                 Tie2-Fc- 
                   
                   9.1 ± 0.310-6 cells/ml 
                 13 
                 2.2% 
                 128 mg/L 
                 27 mg/L 
               
               
                 positioned 
                 IL-1ra 
                   
                   
                 days 
               
               
                 pure O2 
               
               
                 sparging 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 5 
               
             
             
               
                   
               
               
                 20 liter shaker-based coy-boy culture vessel with O2 tube bubbling 
               
               
                 device or air sparging was also routinely employed for routine Fed-batch production of 
               
               
                 animal testing materials by using CHO cell lines expressing TNFR1-Fc-IL-1ra, IL-18bp- 
               
               
                 Fc-IL-1ra, VEGFR1-Fc-IL-1ra, and Tie2-Fc-IL-1ra. Some of the results summarized here 
               
               
                 as controls. 
               
             
          
           
               
                   
                   
                   
                   
                   
                   
                 Final dot 
                   
               
               
                 Gas 
                   
                   
                   
                 Total 
                 Packed cell 
                 blot titer 
                 Final 
               
               
                 supply 
                 CHO cells 
                 Seed 
                 The highest 
                 culture 
                 volume 
                 (+after 
                 purification 
               
               
                 mode 
                 expressing 
                 volume 
                 cell density 
                 length 
                 (pcv) % 
                 dilution) 
                 yield 
               
               
                   
               
               
                 Low- 
                 TNFR1- 
                 800 ml 
                  9.0 ± 3.0 × 10-6 cells/ml 
                 9 days 
                 2.0% 
                 256 mg/ml 
                 40 mg/L 
               
               
                 positioned 
                 Fc-IL-1ra 
               
               
                 air 
               
               
                 sparging 
               
               
                 Low- 
                 IL-18bp- 
                 800 ml 
                  8.8 ± 0.6 × 10-6 cells/ml 
                 8 days 
                 2.2% 
                 256 mg/ml 
                 42 mg/L 
               
               
                 positioned 
                 Fc-IL-1ra 
               
               
                 air 
               
               
                 sparging 
               
               
                 Low- 
                 VEFR1- 
                 800 ml 
                 10.0 ± 0.3 × 10-6 cells/ml 
                 8 days 
                 2.2% 
                 256 mg/ml 
                 38 mg/L 
               
               
                 positioned 
                 Fc-IL-1ra 
               
               
                 air 
               
               
                 sparging 
               
               
                 Low- 
                 Tie2-Fc- 
                 800 ml 
                  9.0 ± 0.3 × 10-6 cells/ml 
                 9 days 
                 2.0% 
                 128 mg/ml 
                 26 mg/L 
               
               
                 positioned 
                 IL-1ra 
               
               
                 air 
               
               
                 sparging 
               
               
                 O2 
                 TNFR1- 
                 800 ml 
                 10.0 ± 0.3 × 10-6 cells/ml 
                 9 days 
                 2.4% 
                 256 mg/ml 
                 41 mg/L 
               
               
                 overlay 
                 Fc-IL-1ra 
               
               
                 O2 
                 IL-18bp- 
                 800 ml 
                  9.0 ± 0.9 × 10-6 cells/ml 
                 9 days 
                 2.3% 
                 256 mg/ml 
                 39 mg/l 
               
               
                 overlay 
                 Fc-IL-1ra 
               
               
                 O2 
                 VEFR1- 
                 800 ml 
                  8.9 ± 0.7 × 10-6 cells/ml 
                 10 days  
                 2.2% 
                 256 mg/ml 
                 40 mg/L 
               
               
                 overlay 
                 Fc-IL-1ra 
               
               
                 O2 
                 Tie2-Fc- 
                 800 ml 
                 10.0 ± 0.5 × 10-6 cells/ml 
                 10 days  
                 2.2% 
                  64 mg/ml 
                 27 mg/L 
               
               
                 overlay 
                 IL-1ra 
               
               
                   
               
             
          
         
       
     
       EXAMPLE 4 
     Two Detailed Examples Fed-Batch Runs of TNFR1-Fc-IL-1ra by Using 3-Liter Wide-Body Shaker Vessel with Inverted Frusto-Conical Bottom and 2-Liter Applikon Flat-Bottom Bioreactor 
       [0031]    3-liter wide-body shaker vessel with inverted frusto-conical bottom together with O2 tube bubbling device, and 2-liter flat-bottom Applikon bioreactor was used to produce TNFR1-Fc-IL-1ra in serum-suspension medium. Fed-batch mode was used. Table 6 shows the results. Note: 2% pcv=9.6×10 −6  cells/ml. Clear advantage of 3-liter wide-body shaker vessel with inverted frusto-conical bottom was demonstrated over 2-liter Applikon flat-bottom bioreactor. 
         [0000]    
       
         
               
             
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 6 
               
             
             
               
                   
               
               
                 3-liter wide-body shaker vessel with inverted frusto-conical bottom together 
               
               
                 with O2 tube bubbling device, and 2-liter flat-bottom Applikon bioreactor was used to 
               
               
                 produce TNFR1-Fc-IL-1ra in serum-suspension medium. Fed-batch mode was used. 
               
               
                 Table 6 shows the results. 
               
             
          
           
               
                   
                 Day 
               
             
          
           
               
                   
                 1 
                 2 
                 3 
                 4 
                 5 
                 6 
                 7 
                 8 
                 9 
                 10 
                 11 
                 12 
               
               
                   
                   
               
             
          
           
               
                 frusto- 
                 0.2% 
                 0.2% 
                 0.5% 
                 1.0% 
                 1.8% 
                 2.2% 
                 2.2% 
                 2.3% 
                 2.8% 
                 3.0% 
                 2.6% 
                 2.2% 
               
               
                 conical 
                 pcv 
                 pcv 
                 pcv 
                 pcv 
                 pcv 
                 pcv 
                 pcv 
                 pcv 
                 pcv 
                 pcv 
                 pcv 
                 pcv 
               
               
                 bottom 
                 seeded 
                   
                   
                   
                   
                 5 
               
               
                 Applikon 
                 0.2% pcv 
                 0.2% 
                 0.4% 
                 0.9% 
                 1.6% 
                 2.0% 
                 2.1% 
                 2.0% 
                 1.8% 
                 1.6% 
               
               
                 flat- 
                 seeded 
                 pcv 
                 pcv 
                 pcv 
                 pcv 
                 pcv 
                 pcv 
                 pcv 
                 pcv 
                 pcv 
               
               
                 bottom 
               
               
                   
               
               
                 Note: 
               
               
                 2% pcv (packed cell volume) = 9.6 × 10-6 cells/ml. Clear advantage of 3-liter wide-body shaker vessel with inverted frusto-conical bottom was demonstrated over 2-liter Applikon flat-bottom bioreactor.