WO2010017338A1 - Procédé de commande de ph, d'osmolalité et de niveau de dioxyde de carbone dissous dans un processus de culture cellulaire de mammifère pour améliorer une viabilité de cellules et un rendement de produit biologique - Google Patents

Procédé de commande de ph, d'osmolalité et de niveau de dioxyde de carbone dissous dans un processus de culture cellulaire de mammifère pour améliorer une viabilité de cellules et un rendement de produit biologique Download PDF

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WO2010017338A1
WO2010017338A1 PCT/US2009/052912 US2009052912W WO2010017338A1 WO 2010017338 A1 WO2010017338 A1 WO 2010017338A1 US 2009052912 W US2009052912 W US 2009052912W WO 2010017338 A1 WO2010017338 A1 WO 2010017338A1
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carbon dioxide
cell culture
dissolved carbon
osmolality
dissolved
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PCT/US2009/052912
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English (en)
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Alan T. Y. Cheng
Ying Zhou
Amitabh Gupta
Balazs Hunek
Nigel Grinter
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Praxair Technology, Inc.
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Priority to EP09791207A priority Critical patent/EP2310493A1/fr
Priority to CN2009801393185A priority patent/CN102171331A/zh
Publication of WO2010017338A1 publication Critical patent/WO2010017338A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0018—Culture media for cell or tissue culture
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/60—Buffer, e.g. pH regulation, osmotic pressure

Definitions

  • the present invention relates to mammalian cell culture processes, and more particularly to methods for enhancing cell growth, cell density, cell viability, product concentration and product yield through improved control of process parameters including pH, osmolality and dissolved carbon dioxide level of the cell culture medium.
  • Bioreactors adapted for culturing suspensions of genetically optimized mammalian, insect or other cell types.
  • Mammalian cell culture bioreactors typically have several hundred to several thousand liters in working volume.
  • Most common full scale manufacturing plants have bioreactors with working volumes ranging from approximately 1,000 liters up to 25,000 liters.
  • Drug candidates for clinical trials are produced in laboratory scale bioreactors having five (5) liters to several hundred liters of working volume.
  • a seed culture inoculum is typically prepared. This involves culturing production cells in a series of flasks in incubators and/or smaller bioreactors of increasing volume until enough cells are available for inoculation into the production bioreactor. The process involves transferring a ceil population from one culture vessel to a larger one. Generally, a 20% dilution of the cell population is used for each transfer or subculture. In the incubator, the flasks with culture medium are clamped to a rotating platform to swirl the culture and facilitate gas transfer between the culture medium and the atmosphere in the incubators.
  • the incubator for a mammalian cell culture process is set at 37 0 C with 5% carbon dioxide (CO 2 ) and a humidity level higher than about 80%. Similar temperatures and CO 2 levels are used for seed cultures grown in bioreactors. When the seed culture reaches a sufficient volume and cell density, it is inoculated into the production bioreactor.
  • CO 2 carbon dioxide
  • pH is typically controlled by adding basic or acidic solutions when necessary during the process.
  • Commonly used base solutions include sodium bicarbonate, sodium carbonate and sodium hydroxide solutions.
  • Dissolution of carbon dioxide (CO 2 ) is commonly used to achieve a more acidic pH.
  • CO 2 carbon dioxide
  • the preferred temperature of the culture medium or solution for mammalian cell cultivation processes is about 37 0 C.
  • the desired level of dissolved oxygen in the culture medium or solution is typically achieved through air sparging using sparger installed on the bottom of the bioreactor, along with agitation of the culture medium or solution using impellers which breakup the large air/oxygen bubbles to enhance the transfer of oxygen to the cell medium from the sparged air bubbles.
  • Purging the bioreactor headspace with a cover gas provides a limited degree of surface gas exchange.
  • air-sparging and agitation of the culture medium or solution may result in foaming and shear damage to the mammalian cells which adversely impacts cell viability. Accumulations of foam on the surface of the culture medium also serve to further limit surface gas exchange and to reduce the available working volume of the bioreactor.
  • Temperature probes, pH detectors, dissolved oxygen probes and dissolved CO 2 probes or sensors are used to monitor the temperature, pH, dissolved oxygen and dissolved CO 2 levels of the cell medium or solution in real time.
  • cell culture medium or solution samples can be withdrawn from the bioreactor at selected intervals to determine cell density and cell viability, as well as to analyze other characteristics such as metabolites and osmolality. Based on such analytical results, additional feed or other additives can be added to the cell culture medium or solution in an effort to prolong the cell viability and increase production of biological products.
  • a prescribed lower threshold is often determined empirically based on the results of down-stream recovery and purification of the harvested biological products.
  • the mammalian cells exhibit three phases, namely the lag phase, the exponential growth phase, and the stationary or production phase.
  • the lag phase occurs immediately after inoculation and is generally a period of physiological adaptation of mammalian cells to the new environment.
  • the mammalian cells are considered in the exponential growth phase.
  • the exponential growth phase the mammalian cells multiply and cell density increases exponentially with time. Many cells actually start to produce the desired protein, antibody or biological product during some point in the exponential growth phase.
  • Cell density refers to the total number of cells in culture, usually indicated in the density of viable and non-viable cells.
  • Mammalian cells are known to be sensitive to the amount of dissolved carbon dioxide in the cell culture media or solution. Mammalian cell cultures exposed to excess carbon dioxide levels during the exponential growth phase may demonstrate reduced production of monoclonal antibodies or other desired biological products. Before inoculation, the pH of the slightly alkaline culture media has to be lowered with carbon dioxide adjusted to an optimum value. This often leads to elevated levels of dissolved carbon dioxide at the beginning of the lag phase of many mammalian cell culture processes. [0010] Dissolved carbon dioxide in mammalian cell culture bioreactors originates from chemical and biological sources.
  • the chemical source of carbon dioxide is equilibrium chemical reactions occurring within the cell culture medium or solution that includes a selected amount of a buffer solution containing sodium bicarbonate and/or sodium carbonate. Additionally, carbon dioxide may be directly sparged into the slightly alkaline culture medium or solution to reduce the pH of the broth to a prescribed level, usually around 7.0, resulting in more dissolved carbon dioxide.
  • the biological source of carbon dioxide is a product of the respiration of the mammalian cells within the bioreactor. This biological source of carbon dioxide increases with cell density and generally reaches its maximum value at about the same time that cell density within the bioreactor is maximized.
  • the conventional method of removing or stripping dissolved carbon dioxide from a mammalian cell culture solution is by sparging the cell culture solution with air or a gas mixture of air/oxygen/nitrogen in agitated tanks.
  • gas sparging in agitated tanks results in adverse effects to the cell culture process.
  • the gas-bubble breakage at the tip of the rotating agitator is a source of high shear rate that damages mammalian cell membranes, often sufficiently to cause cell death. Even when damage is sub-lethal, cell productivity is compromised in the period that the damaged membrane is repaired.
  • sparging air or nitrogen into the bioreactor creates gas bubbles rising to the surface of the solution within the bioreactor where the gas is released into the headspace.
  • Gas bubble breakage at the top surface of the cell culture solution is often more damaging to the mammalian cells than the damage caused by the agitator. Restraining the agitator speed and limiting the gas sparging rate are currently viewed as the best means to avoid such damage and increase cell viability. However, these measures reduce the amount of carbon dioxide that can be removed and the excess that cannot be removed also inhibits cell growth and viability. These disadvantages are particularly challenging to overcome in large, commercial-scale bioreactors where the shear rate goes up substantially with the diameter of the impellers. Also, the greater hydrostatic head of large scale bioreactors tends to increase the solubility of carbon dioxide, meaning that more needs to be removed to maintain dissolved CO 2 levels within an optimal range.
  • the present invention may be characterized as a method for enhancing cell growth, cell viability, cell density, product yield and product concentration in a mammalian cell culture process comprising the steps of: (a) maintaining the dissolved carbon dioxide in a cell culture medium at a generally stable level of less than 10% concentration of dissolved carbon dioxide during a growth phase or production phase of the mammalian cell culture process; and maintaining the osmolality in the cell culture medium at a value of between about 300 mOsmol/kg and 700 mOsmol/kg during the growth phase of the mammalian cell culture process.
  • Fig. 1 is a graph that depicts percentage of dissolved carbon dioxide for three different runs of a mammalian cell line in a process having a moderate level of osmolality, wherein the three runs include one having a high peak level of dissolved carbon dioxide, another having moderate peak level of dissolved carbon dioxide, and the third having a low peak level of dissolved carbon dioxide; [0018] Fig.
  • FIG. 2A is a graph that depicts viable cell density in a mammalian cell culture process as a function of time in days for the three different runs of a mammalian cell line in the process from Fig. 1 having a moderate osmolality;
  • Fig. 2B is a graph that depicts cell viability as a percentage as a function of time in days for the three different runs of a mammalian cell line in the process from Fig. 1 having a moderate osmolality;
  • Fig. 2C is a graph that depicts total cell density cell in a mammalian cell culture process as a function of time in days for the three different runs of a mammalian cell line in the process from Fig. 1 having a moderate osmolality
  • Fig. 3 is a graph that depicts biologic product concentration in a mammalian cell culture process as a function of time in days for the three different runs of a mammalian cell line in the process from Fig. 1 having a moderate osmolality;
  • Fig. 4 is a graph that depicts percentage of dissolved carbon dioxide in a mammalian cell culture process as a function of time in days for two different runs of a mammalian cell line in a process having generally constant or stable osmolalities wherein the first run includes a low peak level of dissolved carbon dioxide and the second run includes a moderate overall peak level of dissolved carbon dioxide;
  • Fig. 5 is a graph that depicts viable cell density in a mammalian cell culture process as a function of time in days for the two different runs of a mammalian cell line in the process from Fig. 4 having a moderate osmolality and generally constant or stable levels of dissolved carbon dioxide;
  • Fig. 6 is a graph that depicts biologic product titer or concentration in a mammalian cell culture process as a function of time in days for the two different runs of a mammalian cell line in the process from Fig. 4 having a moderate osmolality and generally constant or stable levels of dissolved carbon dioxide;
  • Fig. 7 is a graph that depicts the dissolved carbon dioxide profile during the growth and production phases of a mammalian cell culture process
  • Fig. 8 is a graph that depicts viable cell density in a mammalian cell culture process as a function of time in days for yet another two different runs of a mammalian cell line in which different but generally constant levels of dissolved carbon dioxide are maintained;
  • Fig. 9 is a graph that depicts osmolality in a mammalian cell culture process as a function of time in days for the two different runs of the mammalian cell line in the process from Fig. 8 having different but generally constant levels of dissolved carbon dioxide;
  • Fig. 10 is a graph that depicts percentage of viable cells in a mammalian cell culture process as a function of time in days for the two different runs of the mammalian cell line in the process from Fig. 8 having different but generally constant levels of dissolved carbon dioxide;
  • Fig. 11 is a graph that depicts biologic product yield or titer in a mammalian cell culture process as a function of time in days for the two different runs of a mammalian cell line in the process from Fig. 8 having different but generally constant levels of dissolved carbon dioxide;
  • Fig. 12 is a graph that depicts dissolved carbon dioxide in a mammalian cell culture process as a function of time in days for the two different runs using the present Dynamic Gas Control (DGC) process compared against a standard run;
  • DGC Dynamic Gas Control
  • Fig. 13 is a graph that depicts cell viability in a mammalian cell culture process as a function of time in days for the two different runs using the present Dynamic Gas Control (DGC) process compared against a standard run;
  • DGC Dynamic Gas Control
  • Fig. 14 is a graph that depicts viable cell density in a mammalian cell culture process as a function of time in days for the two different runs using the present Dynamic Gas Control (DGC) process compared against a standard run;
  • DGC Dynamic Gas Control
  • Fig. 15 is a graph that depicts biologic product yield or titer in a mammalian cell culture process as a function of time in days for the two different runs using the present Dynamic Gas Control (DGC) process compared against a standard run;
  • DGC Dynamic Gas Control
  • Fig. 16 is a chart that depicts the trend of IgG titer versus peaked dCO 2 in the cell culture process with varying levels of osmolality;
  • Fig. 17 is a chart that depicts the trend of IgG titer versus maximum osmolality in the DGC type cell culture process with low levels of dissolved carbon dioxide;
  • Fig. 18 is a table that provides the data collected during various cell culture process runs at various combinations of osmolality and dissolved carbon dioxide.
  • HC(V has a fairly low dissociation constant, producing only low concentrations of hydrogen ions and achieving only a moderate lowering the solution pH.
  • the net result of increasing atmospheric carbon dioxide is to depress pH by shifting the series of equilibria shown in (1) above to the right.
  • an alkali such as sodium bicarbonate is used to neutralize the effect of elevated carbon dioxide tension:
  • the cells cultured in a batch process reach the exponential growth phase, they become maximally metabolically active and each cell produces its maximum carbon dioxide output.
  • the cell density is low, most of carbon dioxide can be removed by sparging the broth with air or sweeping the headspace of the bioreactor with a cover gas or air. A few days into the batch cycle, however, the carbon dioxide generation will exceed the normal carbon dioxide removal capacity of a typical bioreactor.
  • the excess carbon dioxide generated by the cells will increase the dissolved carbon dioxide level and decrease the solution pH. In order to maintain the preferred pH, additional base has to be added, resulting in excessive dissolved carbon dioxide and undesirably high osmolality in the bioreactor broth.
  • the pH set-point in a mammalian cell culture process can significantly affect the cell-culture performance.
  • Cell culture medium pH is known to affect intracellular enzymatic activity of many mammalian cell types. Lowering pH reduces specific glucose consumption and lactate production rates, reducing the risk of glucose depletion or toxic levels of lactate.
  • the lower pH set point in typical mammalian cell cultures is about 7.0; a pH below about 6.8 is known to inhibit cell growth.
  • Medium or moderate pH values also are known to affect the specific growth rate and specific production rate of mammalian cells, which ultimately affects the overall culture productivity. Excessively low or high pH can kill the cells.
  • a pH range of about 7.0 to 7.4 is commonly used in mammalian cell culture processes.
  • the wide fluctuations in pH that often occur during the process as, for example, when medium is replenished have an adverse effect on the cells.
  • Controlling pH in mammalian cell culture processes is particularly important nowadays because high cell densities (>lxl ⁇ 6 cells/ml) are routinely achieved. Without proper pH control, the cell culture broth can rapidly become acidic when cells are so concentrated.
  • human fibroblasts are grown at a higher pH (7.6-7.8) than established cells (pH 7.0-7.4), and it is usual to culture primary cells at a pH of 1.2-1 A.
  • the optimum pH for growth of human foreskin fibroblasts (e.g. FS-4) at low culture densities is more alkaline than the optimum pH for growth of human lung fibroblasts (e.g. MRC-5).
  • the pH should be about 7.7 to 7.8 for FS-4 cells and about 7.5 to 7.6 for MRC-5 cells.
  • equation (1) Since only the dissolved carbon dioxide molecule is transported across the gas- liquid interface, the bicarbonate must be re-associated to form carbon dioxide molecules. Separating equation (1) above into its two sections, it is noted that the reverse reaction set forth below as equations (3) and (4) is generally fast, whereas the first part of the reaction, represented by equation (5), is much slower.
  • Control of p ⁇ is a key operating condition as many types of mammalian cells die when the p ⁇ is substantially outside the range between p ⁇ 7.0 and pH7.4.
  • the primary target is pH regulation, with dissolved carbon dioxide/bicarbonate levels and osmolality largely uncontrolled and varying significantly during the culture cycle. Few data are available demonstrating the benefits of simultaneously maintaining constant pH, dissolved carbon dioxide and osmolality.
  • Culture media must be buffered under two sets of cell growth conditions: (1) in small open containers (e.g., inside an incubator), wherein the carbon dioxide can be lost to the atmosphere, causing the pH to rise, and (2) in a bioreactor when maximal production of carbon dioxide and lactic acid by high cell concentrations causes pH to fall.
  • a buffer may be incorporated into the medium to stabilize the pH, but additional gaseous carbon dioxide is still required by some cell lines, particularly at low cell concentrations, to prevent the total loss of dissolved carbon dioxide and bicarbonate from the medium.
  • bicarbonate buffer is still used more frequently than any other buffer because of its low toxicity, low cost, and nutritional benefits to the culture.
  • Leibovitz L- 15 cell culture medium that does not utilize carbon dioxide for buffering or to control pH.
  • Leibovitz L- 15 cell culture medium is preferably used when low tensions of carbon dioxide are required.
  • Leibovitz L- 15 contains a higher concentration of sodium pyruvate (550 mg/L) but lacks NaHC(V and does not require carbon dioxide in the gas phase.
  • the inclusion of pyruvate in the medium enables mammalian cells to increase their production of carbon dioxide, making them independent of external supplied carbon dioxide, as well as HC(V. Buffering in the Leibovitz L- 15 cell culture medium is achieved via the relatively high amino acid concentrations.
  • Increased medium osmolality has been shown to decrease specific cell-growth rate and increase specific production rate.
  • the initial medium osmolality can be predicted from the medium formulation.
  • the amount of interaction between medium components typically does not make the osmolality significantly different from the sum of each component's contribution.
  • Individual osmolalities for components of a typical medium are shown in the following table.
  • the growth and function of cells in culture depends on maintaining an appropriate osmolality in the medium. Some cells (e.g. HeLa and other established cell lines) can tolerate wide fluctuations in osmolality. In contrast, primary cells and normal diploid strains are very sensitive to changes in osmolality, and high yields can only be obtained if it is kept within a narrow range. [0064] Controlling osmolality is reported to give more reproducible cultures.
  • Osmolality of cell culture media produced by commercial suppliers may differ, probably because of differences in interpretation of original formulations.
  • high-yield cultures often require various additions to the medium during the culture cycle. These can include buffers (HEPES), acid (HCl), base (NaOH), growth hormone and nutrients.
  • HEPES buffers
  • HCl acid
  • NaOH base
  • growth hormone growth hormone
  • NaCl NaCl
  • M osm measured osmolality (mOsm).
  • X ml of stock of NaCl (mOsm) to be added per milliliter of medium.
  • NaCl (1 mg/ml) that must be added to achieve the desired osmolality is calculated.
  • Measuring osmolality by freezing point depression is the most practical method, since it does not require diluting the nutrients in the medium or adding large volumes of buffers or saline solutions.
  • Vapor pressure depression is another popular method of measuring osmolality.
  • the system and method disclosed herein for controlling pH in a mammalian cell culture process comprises ascertaining the desired pH range and desired level of dissolved carbon dioxide for the selected cell culture medium; providing an initial minimum amount of bicarbonate to adjust the pH of the cell culture medium to fall within the desired pH range and produce the desired level of dissolved carbon dioxide within the cell culture media. It was found that this initial equilibrium between dissolved carbon dioxide level and bicarbonate level has a significant impact on final cell viability and product level and yield.
  • Enough sodium bicarbonate is added into the medium before inoculation sufficient to allow an equilibrium of dissolved carbon dioxide to attain only a low level, less than 10% and more preferably about 5%.
  • pH is maintained by adding sodium hydroxide as required to maintain pH within the desired range to avoid further increase in bicarbonate and an associated increase in dissolved carbon dioxide.
  • the sodium hydroxide - a strong base - also maintains pH within the desired range without significantly increasing the osmolality and maintains the levels of dissolved carbon dioxide relatively stably at or near the desired levels. Controlling Dissolved Carbon Dioxide Levels to Enhance Cell Culture Process
  • the present system and method provides for tight control of the dissolved carbon dioxide level in the cell culture media both at start-up and during the exponential growth phase which provides a beneficial effect on cell viability during the production phase.
  • the accumulated product yield is also influenced by the exposure of the cells to prescribed levels of dissolved carbon dioxide during the growth phase.
  • various test runs or test batches demonstrate that tightly controlling the level of dissolved carbon dioxide during the exponential growth phase yields higher accumulated product yield during production phase and also results in a slower degradation or reduction in cell viability during the production phase.
  • the present method of controlling the dissolved CO 2 removal employs a bioreactor system having an upward flow impeller disposed within a draft tube disposed in the bioreactor vessel.
  • the upward pumping impeller is driven via shaft by a motor outside the bioreactor vessel.
  • the upward flow of the impeller provides a top surface renewal method that enhances surface gas exchange in a highly controllable manner.
  • the upward pumping impeller moves cell culture medium and suspended mammalian cells from the bottom of the bioreactor vessel toward the liquid/headspace gas interface in the upper part of the reactor. In doing so, dissolved carbon dioxide in the cell culture solution or medium is continuously and rapidly brought to the surface of the liquid in the bioreactor where gas-liquid exchange is occurring.
  • a high turnover in the surface liquid allows rapid removal of dissolved carbon dioxide to the headspace.
  • the upward flow impeller allows a higher pumping velocity without creating sufficient shear to damage or kill the mammalian cells.
  • a sweeping gas consisting of oxygen, nitrogen, air, carbon dioxide or other suitable gases and mixtures thereof that is introduced to the headspace in the bioreactor vessel, where it interacts with the top surface of the solution to achieve the desired liquid gas exchange, and is subsequently exhausted from the headspace in the bioreactor vessel.
  • the preferred bioreactor system also may include a plurality of sensors and analyzers including a pH sensor, a dCO 2 sensor, a temperature indicator, a dissolved oxygen analyzer, and a vent gas analyzer. Such sensors and analyzers are coupled as inputs to a system controller (not shown) that controls or adjusts the gas supply of oxygen, nitrogen, and carbon dioxide to the bioreactor vessel.
  • the system may also include an exhaust subsystem, a plurality of biological filters as well as a means for sterilizing the bioreactor vessel with water and steam, as needed. .
  • the upward pumping impeller is preferably located near the middle of the main bioreactor vessel so that the impeller is submerged for low liquid medium or solution starting levels.
  • the impeller speed is adjustable and may be varied throughout the cell culture process to maintain the desired level of dissolved carbon dioxide at all times for the particular mammalian cell culture process.
  • the impeller speed is maintained at very low speeds when the liquid or solution level within the bioreactor vessel is low and should be increased as the liquid or solution level rises.
  • a draft tube is to be added to increase the upward flowing velocity, resulting in a higher gas exchange rate.
  • the impeller speed is preferably highest during the end of the exponential growth phase of the cell culture process, when the liquid or solution level in the bioreactor vessel is also highest.
  • the liquid or solution in the bottom of a large bioreactor vessel is exposed to significant hydrostatic pressures, and the dissolved carbon dioxide trapped inside the mammalian cells will be slow to equilibrate.
  • the presently disclosed upward pumping impeller mitigates this problem.
  • the mammalian cells are exposed to a lower overall average hydrostatic pressure regime and thus achieve a better equilibrium level of dissolved carbon dioxide.
  • the continuous axial or upward recirculating of the cell culture medium or solution provides a varying level hydrostatic pressure on the mammalian cells which is believed to enhance the ability of the cells to expel excess dissolved carbon dioxide deep inside the plasma of the cells
  • the upward flowing liquid can reach the top surface very rapidly before rolling outward towards the bioreactor wall. This provides a very rapid renewal of the liquid surface which promotes rapid removal of dissolved carbon dioxide.
  • impellers can be used to provide the upward recirculating flow with or without the draft tube.
  • the upward pumping impeller is a screw impeller or propeller.
  • propellers may also be used so long as the lateral or radial flow from the propeller is minimized which, in turn reduces shearing and other damage to the mammalian cells.
  • Rapid gas-liquid surface renewal is also useful for dissolving gases into the liquid.
  • the presently disclosed gas-liquid surface renewal method can be used to dissolve the prescribed amount of oxygen needed for the growing cells.
  • the oxygen composition in the sweeping gas in the headspace is increased, resulting in increased transfer of oxygen to the top surface of the recirculating liquid.
  • the oxygen dissolution requirement is low, the oxygen composition in the sweeping gas in the headspace is reduced and replaced with air or nitrogen.
  • the variation in oxygen composition of the sweeping gas has little or no impact on the carbon dioxide removal rate.
  • the dissolved oxygen concentration is preferably maintained at about 50% in many mammalian cell culture processes.
  • very low oxygen concentrations are used in the cell culture solution to enhance protein production by the cells.
  • the dissolved carbon dioxide level can be adjusted or maintained at any desirable level. To decrease the dissolved carbon dioxide level at any time during the cell culture process, the flow rate of the sweeping gas going into the headspace of the bioreactor can be increased to more rapidly eliminate CO 2 from the liquid near the surface. The impeller rotational speed can also be increased to speed up the surface liquid renewal rate. To increase the dissolved carbon dioxide level, one would reduce the sweeping gas flow rate and/or decrease rotational speed of the upward pumping impeller.
  • additional carbon dioxide is needed as, for example, may be the case in the earliest stages of the process shortly after inoculation of the production bioreactor, it can be added to the sweeping gas mixture in the headspace as required.
  • the dissolved oxygen requirement increases as the batch proceeds from the initial lag phase to the end of the exponential growth phase, while the dissolved carbon dioxide concentration increases due to cell respiration, reaches a maximum concentration towards the end of the exponential growth phase, and then is gradually reduced during the production phase. Therefore, gaseous carbon dioxide is added mostly during the lag phase to regulate and maintain pH. Also, some prescribed level of dissolved oxygen needs to be maintained during the cell production phase.
  • the gas supply of nitrogen, oxygen and carbon dioxide to the bioreactor vessel is introduced above the top surface of the liquid in the headspace and preferably closely adjacent to the rolling surface of the liquid solution in the bioreactor vessel.
  • Such gas introduction can be achieved by making the gas injectors movable so as to always inject the gases at or near the top surface as the liquid level in the bioreactor vessel rises. Impingement of the gas at the rolling top surface reduces the momentum boundary layer on the gas side and improves the total mass transfer rate between the liquid and gas.
  • the gas supply may be delivered using fixed gas injectors disposed so as to introduce the gas at a location near the maximum liquid height that will be attained in the bioreactor vessel.
  • controlled introduction of the gas supply of nitrogen, air, oxygen and carbon dioxide to the bioreactor vessel may be done by sparging the gases within the solution using one or more spargers disposed within the bioreactor vessel.
  • the sparger used to dissolve oxygen can have finer nozzles (or holes) to generate small oxygen bubbles that dissolve or are absorbed before breaking the liquid surface.
  • the sparger for the stripping gas typically introduced at considerably higher flow rates, can have much larger nozzles to provide large diameter gas bubbles. Large gas bubbles are less damaging when they break at the surface of the liquid and have less tendency to produce foam.
  • Such submerged gas spargers can assist with the independent control of both oxygen and dissolved carbon dioxide levels in combination with the headspace gas exchange method.
  • the gas spargers are preferably located apart from the upward flow impeller to maximize their residence time in the cell culture medium. With this method, the stripping gas bubbles are much bigger than those injected into axial flow impellers and the potential for foaming is greatly diminished. Gas exchange now occurs both on the surface and in the bulk of the liquid. Sparging small volumes of gases intermittently for short periods of time allows oxygen uptake and carbon dioxide removal to be maximized without resorting to very high flows of sweeping gas or employing the fastest impeller speeds. It is important that such sparging be done only at peak demand for oxygen dissolution and carbon dioxide removal in order to minimize cell damage.
  • the preferred upward pumping device is a helical impeller that can move large volumes of liquid upward with minimal radial flow.
  • carbon dioxide removal rate was measured from a simulated broth and reported as Volumetric Mass Coefficient. The higher the mass transfer coefficient, the better the gas exchange efficiency.
  • Even with an upward pumping impeller the moving liquid stream is going to be rotated by the rotation of the agitator. As a result, the surface liquid is going to swirl, greatly reducing liquid surface renewal as the surface liquid rotates in the plane of the surface.
  • a vertical baffle system is also used on top of the impeller to break the rotation of the liquid and redirect the flow straight to the surface.
  • DGC Dynamic Gas Control
  • a third sample (identified as Run 40) has a starting level of dissolved carbon dioxide of about 6% to 10% during the lag phase and a high level of variability in dissolved carbon dioxide level ranging from about 5% to about 44% throughout days 4 through 11 of the cell culture process.
  • DGC 8 maintained a higher product yield (mg/1) of IgG than the product yield of Run 30 and corresponding product yield of Run 40. Also, the specific productivity (pg/viable cell .day) in DGC 8 with low dCO2 was increased significantly. Specific productivity for the sample processes were about 40 pg/viable cell. day (DGC 8) , 20 pg/viable cell. day (Run 32) and 16 pg/viable cell. day (Run 40), respectively. As evidenced by the DGC 8 data in Figs. 1, 2A, 2B, 2C, and 3, maintaining a stable and low level of dissolved carbon dioxide throughout the cell culture process can enhance cell viability, increase product yield and specific productivity.
  • Figs. 4-6 there are shown graphs depicting the characteristics and results of two additional test runs of a mammalian cell culture process.
  • the sample runs maintained a generally constant or stable level of dissolved carbon dioxide and moderate osmolality of the cell culture medium.
  • Run 50 maintained a moderate level of dissolved carbon dioxide between about 13% and 18% during the exponential growth phase and production phase of the cell culture process
  • Run 55 maintained a low level of dissolved carbon dioxide between about 2% and 6% during the exponential growth phase and production phase of the cell culture process.
  • Fig. 4 As seen in Fig. 4, Run 50 maintained a moderate level of dissolved carbon dioxide between about 13% and 18% during the exponential growth phase and production phase of the cell culture process whereas Run 55 maintained a low level of dissolved carbon dioxide between about 2% and 6% during the exponential growth phase and production phase of the cell culture process.
  • the present system and method preferably maintains a generally constant or stable and low level of dissolved carbon dioxide of less than 10% during the lag and exponential growth phase, and more preferably around 3% to 5% while maintaining a moderate osmolality of between about 300 and 700 mmole/kg, and more preferably between about 350 and 560 mmole/kg during the lag phase and exponential growth phase (See Fig. 1, Fig 4 and Fig. 9).
  • This combined dissolved carbon dioxide level and osmolality process condition provides longer cell viability and highest biological product yield during the production phase for given mammalian cell culture processes (see Figs 2, 3, 5, 6, 8, 10 and 11).
  • FIG. 7 depicts the dissolved carbon dioxide levels during the growth and production phases of Run 62 which has a low level of dissolved carbon dioxide of about 5% during the lag and exponential growth phases and Run 63 which has a moderate level of dissolved carbon dioxide of about 10% during the lag and exponential growth phases of the cell culture process.
  • Fig. 8 further shows that the viable cell density for sample Run 62, with the low level of dissolved carbon dioxide demonstrated a higher degree of cell viability during the production phase than Run 63 having a moderate level of dissolved carbon dioxide.
  • Figs. 7 depicts the dissolved carbon dioxide levels during the growth and production phases of Run 62 which has a low level of dissolved carbon dioxide of about 5% during the lag and exponential growth phases and Run 63 which has a moderate level of dissolved carbon dioxide of about 10% during the lag and exponential growth phases of the cell culture process.
  • Fig. 8 further shows that the viable cell density for sample Run 62, with the low level of dissolved carbon dioxide demonstrated a higher degree of cell viability during the production phase than
  • FIG. 10 and 11 shows that sample Run 62, with the low level of dissolved carbon dioxide demonstrated a higher percentage of cell viability and higher product yield during the production phase that Run 63 having a moderate level of dissolved carbon dioxide.
  • the present system and method also provides for higher osmolality and higher levels of dissolved carbon dioxide in the production phase (See Figs. 7 and 9). Such higher osmolalities and higher levels of dissolved carbon dioxide in the production phase may actually enhance the overall bioreactor productivity. Specifically, Fig.
  • Fig. 9 shows the osmolalities for Run 62 and Run 63 during the exponential growth phase and production phase of the cell culture process which were maintained in a moderate range of about 350 m ⁇ smol/kg to about 400 m ⁇ smol/kg during the growth phase and between about 400 m ⁇ smol/kg to about 700 m ⁇ smol/kg during the production phase.
  • Fig. 7 shows the dissolved carbon dioxide levels during production phase of between about 20% to 50% for Run 62 and between about 20% and 30% for Run 63.
  • FIGs. 12 through 17 there are shown charts containing data comparing the cell culture process using Dynamic Gas Control (DGC) compared to a cell culture process without employing the Dynamic Gas Control (DGC).
  • DGC Dynamic Gas Control
  • the data on the illustrated charts suggest that sample runs employing the Dynamic Gas Control (DGC) process at moderate osmolality, namely samples DGC2 and DGC3,
  • the dissolved carbon dioxide was started at about 8.45%, and was subsequently maintained in a range between about 7.0% to 7.5% throughout the remaining cell culture process.
  • the dissolved carbon dioxide was started at about 5.5%, and was maintained in a range between about 5.5% to 6.3% for Day 1 and Day 2, and subsequently decreased to about 4.5% at Day 3 and Day 4, and further reduced to about 4.0% from Day 4 to Day 15.
  • Run 32 had a dissolved carbon dioxide profile very typical cell culture process where the average dCO2 was maintained about 6% in the growth phase, followed with increasing dCO2 to about 15%, then gradually lowered to about 10% in the production phase.
  • Figs 12-17 The data contained in Figs 12-17 shown that the dissolved carbon dioxide levels can be well maintained at desired low level through the process with Dynamic gas Control (DGC) process Both the DGC2 and DGC3 sample runs had higher viable cell density and viability during later stages of protein production. Sample run DGC3 had the highest product titer among these three runs, and reached maximum product titer much earlier than either DGC2 or Run 32.
  • Fig. 18 is a table that provides the cell culture process data collected during various sample runs at various combinations of osmolality and dissolved carbon dioxide.
  • the present system and method also provides for a low level of dissolved carbon dioxide of less than 10%, and more preferably around 5% or less while diluting the mammalian cell culture batch with water during the in the production phase while adding additional nutrient and cell booster during the production phase.
  • This dilution and nutrient supplementation procedure provides higher mammalian cell culture bioreactor product yields and appears to dilute some of the critical toxic waste buildup.
  • the present cell culture process optimization and control method comprises: (1) process optimization phase; and (2) active control phase.
  • the process optimization phase involves empirically determining the desired pH, osmolality and dissolved carbon dioxide levels for a given mammalian cell culture process, cell line and bioreactor configuration.
  • a microprocessor-based controller is then programmed to establish the initial settings as well as permissible values or ranges for overlay gas composition, overlay gas flow rate, sparged gas composition, sparged gas flow rate, acid addition, base addition, nutrient addition, media harvest, etc. to achieve the desired dissolved carbon dioxide and osmolality in the bioreactor while maintaining pH within the desired range and maintaining one or more of the other process parameters such as dissolved oxygen level, agitator speed, temperature, pressure, nutrient content, waste product content, etc. within specifications
  • Individual gases or gas mixtures relevant for cell culture bioreactor sparging and overlaying may include the addition and removal of oxygen, nitrogen, air, argon, carbon dioxide, or combinations thereof.
  • the empirical determination of desired pH, osmolality and dissolved carbon dioxide level for a given mammalian cell culture process is preferably accomplished in laboratory scale bioreactors running scaled-down process conditions and may be supplemented with appropriate model-based studies.
  • the active control phase involves monitoring or measuring a plurality of parameters to be used as inputs to the microprocessor-based controller.
  • Such inputs include the dissolved carbon dioxide level, osmolality and pH, as well as optional inputs of dissolved oxygen level, temperature, nutrient and waste product concentration, agitation, gas sparging or overlay gas flow rate and composition, nutrient feed volume and composition and product harvest volume and composition.
  • Such inputs are fed to the controller at a regular interval or a continuous basis throughout the production and growth phase of the cell culture process.
  • the microprocessor based controller receives the inputs and produces one or more output signals representing the value and setting of at least one parameter selected from the group of overlay gas composition, overlay gas flow rate, sparged gas composition, sparged gas flow rate, agitator speed, acid addition, base addition, nutrient addition, media harvest, etc.
  • the output signals are used to control or adjust the overlay gas composition, overlay gas flow rate, sparged gas composition, sparged gas flow rate, agitator speed, acid addition, base addition, nutrient addition, media harvest, etc which maintains the dissolved carbon dioxide level, osmolality, or pH at the desired values or ranges.
  • This proposed process control scheme is applicable for nearly constant physiological temperature and also hypothermic cell culture processes. Hypothermic cell culture processes run at least part of the time at less than the typical approx. 37°C process temperature. This proposed process control scheme is also applicable to nearly any configuration of bioreactor and operating in any mode, including batch mode, fed-batch mode, or a continuous mode of operation. [0104] From the foregoing, it should be appreciated that the present invention thus provides various methods and systems for controlling the dissolved carbon dioxide level, pH and osmolality during a mammalian cell culture process to enhance cell viability and biologic product yield. Numerous modifications, changes, and variations of the present methods and systems will be apparent to a person skilled in the art and it is to be understood that such modifications, changes, and variations are to be included within the purview of this application.

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Abstract

L'invention concerne des procédés pour commander le niveau de dioxyde de carbone dissous et limiter l'osmolalité dans un processus de culture cellulaire de mammifère, afin d'améliorer une croissance cellulaire, une viabilité et une densité, et d'augmenter une concentration et un rendement de produits biologiques. Une telle commande du niveau du dioxyde de carbone dissous et du pH de même que l'aptitude résultante à limiter l'osmolalité dans un processus de culture cellulaire de mammifère, sont obtenues en adoptant des stratégies de contrôle de pH en variante et des techniques de lavage de CO2 en variante pendant un processus de culture cellulaire de mammifère. De telles techniques de commande de pH et de stockage de dioxyde de carbone surviennent avec peu ou pas de détérioration des cellules de mammifère.
PCT/US2009/052912 2008-08-06 2009-08-06 Procédé de commande de ph, d'osmolalité et de niveau de dioxyde de carbone dissous dans un processus de culture cellulaire de mammifère pour améliorer une viabilité de cellules et un rendement de produit biologique WO2010017338A1 (fr)

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CN2009801393185A CN102171331A (zh) 2008-08-06 2009-08-06 控制哺乳动物细胞培养过程中pH值、重量克分子渗透压浓度和溶解二氧化碳水平来增强细胞生存力和生物产物产量的方法

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WO2013041487A1 (fr) * 2011-09-21 2013-03-28 F. Hoffmann-La Roche Ag Culture à profil de co2
WO2014004791A1 (fr) * 2012-06-29 2014-01-03 Stroot Peter Méthodes et systèmes de contrôle des vitesses de croissance de cultures de microbes autotrophes
US20140199729A1 (en) * 2011-04-29 2014-07-17 Biocon Research Limited Method for Reducing Heterogeneity of Antibodies and a Process of Producing the Antibodies Thereof
US9515780B2 (en) 2011-12-23 2016-12-06 Nokia Technologies Oy Shifting HARQ feedback for cognitive-radio-based TD-LTE systems
EP2697251B1 (fr) 2011-04-13 2019-02-27 LEK Pharmaceuticals d.d. Procédé de contrôle des structures principales complexes des n-glycanes et des variants acides ainsi que de la variabilité des bioprocessus produisant des protéines recombinantes
US11459538B2 (en) 2012-10-26 2022-10-04 Sanofi Humidity control in chemical reactors
WO2023114949A1 (fr) 2021-12-16 2023-06-22 Sana Biotechnology, Inc. Procédés et systèmes de production de particules
US11827871B2 (en) 2013-08-23 2023-11-28 Massachusetts Institute Of Technology Small volume bioreactors with substantially constant working volumes and associated systems and methods

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CN109517738A (zh) * 2018-12-14 2019-03-26 杭州奕安济世生物药业有限公司 一种调控生物反应器中二氧化碳含量的方法

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US8178318B2 (en) 2008-08-06 2012-05-15 Praxair Technology, Inc. Method for controlling pH, osmolality and dissolved carbon dioxide levels in a mammalian cell culture process to enhance cell viability and biologic product yield
WO2011073377A1 (fr) * 2009-12-18 2011-06-23 Boehringer Ingelheim International Gmbh Méthode pour optimiser un processus de production biopharmaceutique
US8822198B2 (en) 2009-12-18 2014-09-02 Boehringer Ingelheim International Gmbh Method for optimizing a biopharmaceutical production process
EP2365060A1 (fr) * 2010-02-09 2011-09-14 Praxair Technology, Inc. Procédé de contrôle du pH, de l'osmolalité et des taux de dioxyde de carbone dissout dans un procédé de culture de cellules de mammifère pour améliorer la viabilité des cellules et le rendement en produit biologique
CN102191275A (zh) * 2010-02-09 2011-09-21 普莱克斯技术有限公司 增强细胞生存力和生物产物产量的方法
EP2697251B1 (fr) 2011-04-13 2019-02-27 LEK Pharmaceuticals d.d. Procédé de contrôle des structures principales complexes des n-glycanes et des variants acides ainsi que de la variabilité des bioprocessus produisant des protéines recombinantes
US9631216B2 (en) * 2011-04-29 2017-04-25 Biocon Research Limited Method for reducing heterogeneity of antibodies and a process of producing the antibodies thereof
US20140199729A1 (en) * 2011-04-29 2014-07-17 Biocon Research Limited Method for Reducing Heterogeneity of Antibodies and a Process of Producing the Antibodies Thereof
EP2702164B1 (fr) 2011-04-29 2015-11-25 Biocon Research Limited Procédé permettant de réduire l'hétérogénéité des anticorps et procédé de production desdits anticorps
WO2013041487A1 (fr) * 2011-09-21 2013-03-28 F. Hoffmann-La Roche Ag Culture à profil de co2
US10513697B2 (en) 2011-09-21 2019-12-24 Hoffmann-La Roche Inc. CO2 profile cultivation
US9515780B2 (en) 2011-12-23 2016-12-06 Nokia Technologies Oy Shifting HARQ feedback for cognitive-radio-based TD-LTE systems
WO2014004791A1 (fr) * 2012-06-29 2014-01-03 Stroot Peter Méthodes et systèmes de contrôle des vitesses de croissance de cultures de microbes autotrophes
US11459538B2 (en) 2012-10-26 2022-10-04 Sanofi Humidity control in chemical reactors
US11725176B2 (en) 2012-10-26 2023-08-15 Sanofi Humidity control in chemical reactors
US11827871B2 (en) 2013-08-23 2023-11-28 Massachusetts Institute Of Technology Small volume bioreactors with substantially constant working volumes and associated systems and methods
WO2023114949A1 (fr) 2021-12-16 2023-06-22 Sana Biotechnology, Inc. Procédés et systèmes de production de particules

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