Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M27/00—Means for mixing, agitating or circulating fluids in the vessel
  • C12M27/10—Rotating vessel
  • C12M27/12—Roller bottles; Roller tubes
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M27/00—Means for mixing, agitating or circulating fluids in the vessel
  • C12M27/16—Vibrating; Shaking; Tilting

Definitions

• roller bottle systems still prevail in research and industry. Additionally, for industrial scale production of cell culture products (i.e. vaccines), cells are frequently passaged in roller bottles prior to transfer to microcarrier cultures for the final growth phase even when unit operation based systems are utilized [V. G. Edy, Adv. Exp. Med. Biol. 172, 169 (1984)].
• roller bottle Widespread use of the roller bottle is due to several reasons. Most notably, the process relies on very simple technology: a horizontal cylindrical vessel is filled approximately one-third full and axially rotated. Thus, scale-up development is not required, resulting in reduced developmental timelines for industry and faster introduction to market for new products.
• the system allows constant fluid-gas contact, and easy addition of nutrients without interruption of the process.
• the process is capable of maintaining sterile conditions for prolonged times, contamination of one or more roller bottles does not result in the contamination of an entire lot, precise control of nutrient and waste-product levels is possible, and the direct monitoring of the cells is relatively simple [E. I. Tsao, M. A. Bohn, D. R. Omstead, and M. J. Munster. Annals N.Y. Acad. Sci. 665, 127 (1992)].
• roller bottles are limited in surface area available for growth and in the volume of harvest fluid obtained.
• the instant application describes detail mathematical and experimental characterizations of the fluid flow profiles within rotating roller bottles, including particle trajectories, fluid mixing patterns, and unsteady-state flow strategies.
• the results point to ways in which cell proliferation and infection can be optimized by simple modifications to the roller bottle's rotation.
• the instant invention describes an improved method for mixing in roller bottles that introduces cross- sectional and axial flow perturbations. These perturbations disrupt the closed orbits that cells experience during conventional mixing and facilitate particle settling.
• this invention relates to a mixing process that ensures that cells come in contact with adequate amounts of nutrient-rich medium and by increasing the contact between the cells and the roller bottle wall or cell sheet and thereby enhances productivity.
• This invention relates to a method for enhanced mixing in a roller bottle by the introduction of controlled axial and cross-sectional flow perturbations.
• the effectiveness of the method is demonstrated by achieving higher efficiency in cell culturing and virus propagation in roller bottles.
• Figures 4a shows the initial condition
• Fig. 4b through 4e represent the initial condition and the line after 4, 8, 16, and 32 bottle revolutions, respectively.
• the colors in this figure range from dark blue to red, and correspond to zero residence time to infinite residence time.
• FIG. 20 Experimental apparatus used to investigate mixing enhancement via introduction of rocking motion.
• Figure 21(a)-21(c) demonstrate axial mixing with no rocking after 0, 32 and 64 revolutions, respectively.
• Roller machine capable of introducing a rocking motion.
• Figure 23 Protuberances on (a) the roller bottle and (b) rollers which introduce a rocking motion.
• the invention relates to a method for enhancing the mixing of materials in a roller bottle comprising controlled axial and cross- sectional flow perturbations.
• material refers to the a solution of or suspension of liquid and solid materials, such as a cell culture and the media needed to sustain that culture, the virus infected cells and the media needed to sustain these cells.
• the axis of rotation of the roller bottle is the axis defined by the line connecting the center of the roller bottle and running the length of the roller bottle.
• the rocking motion is defined as the angle swept by the roller bottle about an axis that is perpendicular to the axis of rotation of the roller bottle, that angle of about 0 degrees to about + 20 degrees or -20 degrees, a speed of rotation of about 0 to about 50 rpm, and a rock to roll frequency of about 0 to about 31.4.
• the preferred conditions for the combined rotation of the bottle with rocking motion are defined by a rocking angle of about +10 degrees to about -10 degrees, and a rock to roll frequency of about 1.8.
• Figure 21 (a)-(c) shows the control experiment where there is no rocking.
• Figure 21 (d)-(i) shows two different rocking frequencies, 1.6 and 3.2 revolutions per rocking cycle. Without rocking, half of the bottle appears yellow after 32 revolutions, and after 64 revolutions the blue yellow interface has barely moved. On the other hand, when rocking is implemented, after 64 revolutions the fluid is completely yellow for both rocking frequencies, indicating that complete mixing has been achieved throughout the system.
• the rocking motion can be introduced in a number of ways, including but not limited to, introducing a rocking motion to a roller machine (Figure 22), or by introducing protuberances to the rollers or the roller bottles (Figure 23).
• the roller machine rack as depicted in Figure 22, provides the most flexibility in varying the rock to roll frequency, rocking angle, frequency of bottle rotation and the speed of rotation. Additionally, a single level roller machine is also contemplated by the instant invention.
• Another way of disturbing periodic cell trajectories is to operate the bottle in a stop-go mode.
• the bottle motion consists of two alternating parts.
• the bottle is rotated at constant velocity U for a time t ls and then it is kept stationary for a time t 2 .
• cells undergo settling at terminal speed.
• the purpose of using this mode is to try to break the periodic cell orbit and let cells settle.
• Another approach is to vary the speed of rotation as a function of time. This approach has been applied to the varicella vaccine production process. Previously, the method had utilized a rotation rate of 0.25 rpm throughout. Experiments were conducted with faster rotation rates for the cell expansion phase and slower rotation rates for the first 6 hours of the infection phase. As described below, slower rotation rates during the virus propagation phase enabled more varicella-infected cells to reach the inside surfaces of the roller bottle sooner, resulting in higher levels of virus production. In addition, rotation faster than 0.25 rpm during the cell expansion phase resulted in more efficient cell growth.
• the method for enhancing the mixing in a roller bottle wherein the controlled axial flow perturbation is introduced by combining a time-dependent rotation speed of the roller bottle with a rocking motion, which is further defined by a speed of rotation of about 0 to about 50 rpm, a frequency of rotation rate changes per revolution of about 0 to about 1, a rocking angle of about 0° to about +10 degrees or -10 degrees, and a rock to roll frequency of about 0 to about 31.4.
• the method for mixing in a roller bottle wherein the controlled axial flow perturbation is introduced by combining a time-dependent rotation direction of the roller bottle with a rocking motion, which is further defined by a speed of rotation (roll frequency) of about 0 to about 50 rpm, and a frequency of rotation direction changes per revolution of about 0 to about 1, a rocking angle of about 0° to about +10 degrees or -10 degrees, and a rock to roll frequency of about 0 to about 31.4.
• the method for mixing in a roller bottle wherein the controlled axial flow perturbation is introduced by combining a time- dependent rotation direction of the roller bottle with a rocking motion and a time- dependent speed of rotation, which is further defined by a speed of rotation of about 0 to about 50 rpm, and a frequency of rotation direction changes per revolution of about 0 to about 1, rocking angle of about 0° to about +10 degrees or -10 degrees, and a rock to roll frequency of about 0 to about 31.4.
• Typical parameters and preferred values are the following: for rotation frequency, 0 to 50 rpm, 0.1 to 5 rpm preferred; for rocking angle, 0 to ⁇ 20 degrees, 0 to ⁇ 10 degrees preferred; for the magnitude of rotation speed variations, the low speed is 0% to 100% of the high speed, preferred value is when the low speed is less than or equal to 50% of the high speed; for the frequency of rotation speed variations, from 0 to 10 times the frequency of rotation, from 0.05 to 0.5 times the frequency of rotation is preferred; and for the frequency of rotation direction variations, from 0 to 2 times the frequency of rotation, 0.5 is the preferred value
• FLUENT The use of FLUENT and similar software packages has been used by several researchers in the past to solve fluid dynamics problems [A. K. Majumdar, R. H. Whitesides, S. L. Jenkins, and D. L. Bacchus, J. Prop. 6, 5 (1990); V. Dupont, M. Pourkashanian, A. Williams, and R. Wooley, Fuel 72, 497 (1993); J. Seth and W. R. Wilcox, J. Crystal Growth 114: 357 (1991); D. Y. Tang, J. J. Ou, R. H. Heist, S. H. Chen and A. J. Dukat, Ind Eng. Chem. Res. 32, 1727 (1993); P. D. Swanson, F. J.
• the physical domain was discretized using a curvilinear mesh; while the bottle has a total length to diameter ratio of 2:1, due to the symmetry of the roller bottle, only one-half of the system needed to be simulated, giving a length to diameter ratio of 1:1.
• the liquid height in the bottle was one-third the total diameter, which was consistent with industrial practice.
• a 80 x 20 x 94 structured computation grid is used;
• Figure lb represents a schematic of the z-plane grid (for the computation a higher node density was used).
• An acceptable node density for the computational mesh was found by creating several meshes with different node densities. The number of nodes in the mesh was successively increased by 25% until an average velocity difference of less than 3% was achieved for two successive meshes. The lower node density was then used to generate the computational mesh.
• Velocity and pressure fields were obtained via iteration using a Sun SPARC 20 workstation. Approximately 140 Mb of RAM and 60 hours of CPU time were need for solution convergence. In order to determine convergence, a criterion of 10"° was used for each normalized residual velocity and pressure component. Residuals for each iteration were normalized versus the residual values obtained after the second iteration of the solver. In order to test the sufficiency of the convergence criterion, the simulation was run using convergence criteria ranging from 10 ⁇ 5 to 10" ⁇ with no significant change in the velocity field when the convergence criteria was varied over this range ( ⁇ 1.0% change in velocity magnitude, on average). Sensitivity analysis (i.e., trajectory closing) showed that such a mesh is sufficiently accurate. This flow data was then used for all subsequent particle tracking simulations.
• the flow was seeded with 12 ⁇ m diameter silver-coated hollow glass spheres (Potter's Industries) which have a specific gravity of 1.17.
• a 10 mJ New Wave Research pulsed mini-YAG laser was used as the illumination source.
• the laser beam passed through a series of optical components to produce a sheet of light 1 mm thick (at a focal distance of lm).
• a 32x32 grid was analyzed using cross-correlation data acquisition, with 10,000 ⁇ s between laser pulses. Resultant data from Pr was in the form of velocity direction and magnitude at the center of each interrogation area.
• the experimental velocity field was measured at the vertical plane in the middle of the bottle.
• Figure 2a,b respectively show the experimental (2a) and computational (2b) velocity vector fields.
• the length and color of the arrows represent the velocity magnitude in the plane, ranging from low (dark blue) to high (red).
• the velocity fields are qualitatively identical, with the exception of some small experimental scattering near the free surface of the liquid.
• the computational velocity field was further validated by comparing computational and experimental mixing patterns obtained for passive tracers at the center plane of the bottle. This first step for simulating mixing patterns is to determine the trajectories followed by point particles as a result of the equation of motion
• Figure 7f corresponds to the center of the bottle and is therefore identical to Fig. 2b Moreover, Fig. 7d and 7e are also identical to Fig. 7f, indicating that end effects play little or no role in the fluid flow past 5 cm from the end of the bottle.
• the velocity field shows both especular symmetry relative to the center plane (expected from the symmetry of the boundary) and antisymmetry between the left and right sides of the bottle (expected from the creeping flow condition and the rotational symmetry of the bottle). As discussed below, this observation can have important implications, because the flow induced by the ends of the bottle is the only means of axial mixing.
• Figure 7a represents the velocity field at the end of the roller bottle. Due to the no-slip condition at the bottle wall, all fluid follows the bottle wall in a counter-clockwise direction. When the fluid reaches the free surface (at the right side), it detaches from the wall and flows outward in the z-direction. At the left side, the fluid immediately adjacent to the wall is drawn downward; this motion is "fed” by an inward flow from the z-direction.
• Figures 7b and 7c show the presence of a high velocity component near the surface of the liquid, caused by the end effects. However, detailed examination of the velocity vectors displayed in Fig. 7b-c indicate that the x and y components of the velocity field are largely unaffected by end effects; such effects appear primarily in the axial (z) component of the velocity field.
• Figure 8 shows a velocity contour plot just below the liquid free surface.
• the end of the bottle is at the bottom of this figure, and the symmetry plane is at the top.
• Flow in the axial (z) direction is clearly visible in this figure.
• the fluid flows upward from the end wall in the lower right-hand corner travels in a counter-clockwise loop, then flows downward at the lower right-hand corner due to the end-wall flow.
• FIG. 9 shows six pathlines for fluid particles initially placed 0.2 cm from the bottom of the bottle.
• the center of the bottle is at the left- hand side of the figure, and the end wall is at the right. Looking at the pathlines from left to right, we see the effect that the end wall has on the flow field.
• the pathline at the center plane is a two-dimensional vertical loop identical to the pathlines observed in Fig 3. As we move toward the end of the bottle, the pathlines begin to bend near the free surface toward the center of the bottle. All the pathlines shown are closed loops.
• the pathlines at the center plane of the bottle are two-dimensional loops, and the pathlines away from the symmetry plane are three-dimensional loops.
• u is the fluid velocity vector
• v is the cell velocity vector
• a is the cell radius
• v is the fluid kinematic viscosity
• m p and mp respectively correspond to the weigh of the cell and that of the fluid displaced by the cell.
• the five terms in the right hand side of the equation correspond to buoyancy forces, pressure forces, flow history effects, added mass effect, and Stokes drag forces. This equation can be significantly simplified for the roller bottle problem. Since u is known from the numerical solution of the flow, the relative magnitude of each term can be evaluated. Analysis has shown that the leading order terms are inertia, buoyancy, and drag forces, thus the equation becomes:
• v the total particle velocity
• V s the particle terminal settling velocity
• FIG. 11 closely resembles the pattern shown in Figure 10, which is a simulation for particles of roughly similar settling velocity.
• Figure 10 is a simulation for particles of roughly similar settling velocity.
• the particle was originally placed in a different location, closer to the bottle center, the particle followed a different closed pathline.
• the particle When placed close enough to the upper surface, the particle eventually settled and remain attached to the bottle wall.
• V s 0 (Fig. 12a)
• the structure of the particle positions is identical to those of the flow streamlines since such particles are in fact fluid particles.
• Particle positions form closed orbits, indicating that all particles are confined inside closed orbits.
• no settling occurs in the flow.
• V s 0.02U
• V s 0.02U
• 0.2U, Fig. 12e are most particles able to reach the bottle walls.
• the periodic flow can be simulated with two identical steady flows that are mirror images of each other, each one acting for a predetermined amount of time in an alternating fashion. While mixing of passive tracers will not improve at all in such periodic flows because the particles would simply move back-and- forth along the same streamlines, settling of heavy particles is tremendously enhanced by periodic reversal of the direction of rotation.
• T ⁇ 25 the bottle does not complete a rotation between reversals, and therefore some regions of the bottle wall never touch the liquid. Such a condition would lead to starvation and death of cells in such regions of the bottle wall, and therefore to reduced process efficiency.
• Figures 2 ld-f represent the effect of rocking on mixing in the system.
• the roller bottle was rocked approximately 12 degrees at a rocking rate of 1.6 bottle revolutions per rock. It can be seen that mixing is improved substantially in the system as a result of the rocking.
• Figures 21g-i represent mixing for a rocking rate of 3.2 bottle revolutions per rock.
• Time Dependent Speed of Rotation Frozen MRC-5 cells were thawed, and the cells were cultivated in either T-flasks or Nunc Cell Factories, which are flat, stationary growth surfaces. The cells attached to and propagated on the lower surfaces of these containers, where they were submerged under a nutrient growth medium. Every 6 days, the cells were removed from the surface by enzymatic means, known as trypsinization. The resulting cell suspension was then diluted and placed into a larger number of vessels for further propagation.
• the freshly trypsinized, concentrated cell suspension was then placed in the roller bottles along with 125 mL of growth medium.
• the bottles were rotated at 0.25 rpm or 0.5 rpm and incubated at 37 °C.
• An additional 300 mL of growth medium was added to the bottles on day 33.
• the figures represent the average number of cells yielded per bottle on day 35 of the process. This represents a 23% ⁇ 10% increase in cell number over the 0.25 rpm rate of rotation.
• the higher cell yield is obtained by a prolongation of the exponential growth phase. See Figure 18, which depicts the daily growth curves for two of the five trials. The figure illustrates that the higher cell numbers are not due to a higher growth rate, rather the exponential growth phase of the culture has been extended. This may be attributable to improved transport of nutrients, gases, and waste products resulting from the faster rotation.
• Roller bottle cultures from Trials 4 and 5 above were then infected by the addition of freshly trypsinized, virus-infected MRC-5 "working seed” cells.
• the virus infected cells were added to the liquid nutrient medium in the roller bottle, where they attached to the uninfected cell monolayer which had been established on the inside surfaces of the roller bottle. Once the bottles were inoculated with the "working seed", they were rotated at alternate rates.
• the potency was evaluated by five replicate plaque assays for each of the four experimental groups, for each of the two experiments.
• the combination strategy of the faster rotation for cell growth and slower rotation for the initial period of infection resulted in 32 % more product, with a 95% confidence interval of 15%, indicating a statistically significant result.
• the rotating bottle creates circular liquid flows, in which the varicella-infected cells can become "trapped.” As these infected cells circulate in the liquid phase, they gradually lose their infectivity (the 1/2-life of the infectivity is less than 5 hours). Although slowing the rotation rate of the bottles does not break up the circular flows, modeling had shown that it would allow more of the trapped cells to reach the surface sooner via settling, as borne out by these experiments.
• Figures 15 through 17 illustrate that a slower rotation rate results in faster transport of infected cells to the cell culture which has been established on the inner surfaces of the roller bottle. It is important to minimize the length of time which the infected cells spend in the culture supernatant for two reasons. First, time in the supernatant is process time during which a given virus-infected particle is not infecting the cell monolayer, and thus, generating product. Second, the half life of the virus activity less than 5 hrs. Time spent in the supernatant translates directly into degraded viral material. Figures 15 and 17 show the numbers of infected cells present in the supernatant through time for several different rotation rates.
• Figure 15 shows that at a faster rotation rate of 1/2 rpm, more infected cells are left in the supernatant relative to the regular production (1/4 rpm) and T-flask stationary controls.
• Figure 16 confirms that the disappearance of infected cells from the supernatant correlates with the appearance of infectious foci on the cell monolayer. For all cases, the faster rpm resulted in fewer infectious foci per unit area.
• Figure 17 shows similar data for an experiment which tested 1/4, and 1/8 rpm, where the slower rpm again resulted in enhanced transport of infected cells to the surface.
• the technique used for cell enumeration in Figure 17 was much more sensitive than the technique used for cell enumeration in Figure 15, so the 1/4 rpm control cases for the two experiments differ.
• the Oka strain of varicella was obtained from the Biken Institute, Japan.
• MRC-5 cells were obtained from ATCC or NIBSC and used to generate a Manufacturer's Working Cell Bank (MWCB) at Merck.
• MEM was manufactured by Merck and supplemented at the time of use with fetal bovine serum (FBS), neomycin and glutamine.
• FBS fetal bovine serum
• HBME was purchased from GIBCO.
• Horseradish peroxidase- conjugated donkey anti-goat antibody (affinity purified F(ab')2 fragment) was obtained from Jackson Immunoresearch Labs, Inc., West Grove, PA, (Cat# 705-036-147).
• the goat anti-varicella antiserum was obtained from Lampire, Inc. (Lot 2200).
• DAB 3,3-diaminobenzidine
• MRC-5 ampules (ATCC No. CCL 171) were thawed, and the cells were cultivated in either T-flasks or Nunc Cell Factories, which are flat, stationary growth surfaces.
• the cells were added to BME medium supplemented with 10% FBS, 50 ⁇ g/mL neomycin, and 2 mM glutamine; and were incubated at 37°C. Every 3 to 9 days (depending upon the exact process), the cells were removed from the surface by enzymatic means using trypsin. The resulting cell suspension was then diluted and placed into a larger number of vessels for further propagation.
• roller Bottle Cell Expansion Freshly trypsinized, concentrated cell suspension was placed in the roller bottles with MEM growth medium supplemented with 10% FBS, 50 ⁇ g/mL neomycin, and 2 mM glutamine. The bottles were incubated at 37 °C and rotated at the desired rate. After 4-6 days (depending upon the process used), the roller bottles were refed with fresh nutrient medium.
• Roller bottle cultures with an established cell monolayer on the roller bottle surface were infected by the addition of freshly trypsinized, virus-infected MRC-5 cells.
• the virus infected cells were added to the liquid nutrient medium in the roller bottle 6-8 days after the initial roller bottle cell plant.
• Samples were thawed in 20-30 °C water with frequent swirling.
• the sonicator was calibrated so that when the sonicator tip was immersed in 20 mL of room temperature water and energized for 1 minute, the temperature of the water would be increased by 5 °C.
• the sonicator tip was then sterilized by the application of 90% isopropyl alcohol.
• the thawed samples were each sonicated for 1 minute in 20 mL aliquots. Sonicated samples were then frozen at -70 °C for potency testing.
• MRC-5 cells were grown in 60 mm plates with 5 ml/plate of BME with 10% FBS, 0.2 % glutamine, and 0.05% neomycin. 10 plates were prepared per sample with 5 plates per dilution. The plates were then incubated at 35.5°C with a 3% CO 2 gas environment for 2 days. Prior to addition of the samples, the cell growth medium was removed and 5 ml/plate of MEM with 2% FBS, 0.2% glutamine, and 0.05% neomycin was added to each plate. Diluted virus samples of 0.1ml were added to each plate. Samples were diluted to ensure a plaque counting range of 15-80 plaques per plate. The plates were mixed well and incubated for 7 days at 35.5°C with a 3% CO 2 gas environment.
• Particle counting and size analysis were performed using an Elzone Particle size analyzer model with a 48 ⁇ m orifice. Samples were diluted to 20ml in PBS. The diluted sample was placed in the analyzer sample holder following calibration with particles of a known diameter (10 and 20 ⁇ m microspheres).
• the medium was aspirated from the culture vessel (T-flask or roller bottle) using vacuum.
• the culture vessel was then rinsed three times with PBS with 1% BSA.
• a fixing solution of 5% acetic acid, 5% water, and 90% methanol was added to the culture vessel and exposed to all surfaces for 15-30 minutes.
• the methanol was then aspirated from the culture vessel with vacuum.
• the culture vessels were then rinsed three more times with PBS, and stored with a PBS overlay at 4-8 °C until stained. Immunostaining of the fixed bottles was carried out by a dual antibody method.

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