Classifications

• • 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
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P21/00—Preparation of peptides or proteins
  • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
  • C07K2317/14—Specific host cells or culture conditions, e.g. components, pH or temperature
• • 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
• • 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
  • C12N2511/00—Cells for large scale production

Definitions

• the present invention relates to a method for producing a product, which involves culturing cells, and in which the rate of alkali addition and the pH of the aqueous alkali solution are controlled.
• the invention further relates to a product manufactured by the above-described manufacturing method.
• Cell culture is carried out for the purpose of increasing the number of cells with useful properties, making the cells produce products, etc.
• U.S. Pat. A method is described that includes establishing a mammalian cell culture expressing a modified protein, maintaining the mammalian cell during a production phase, and contacting the cell culture with monensin.
• US Pat. No. 5,002,302 describes a fed-batch method for culturing mammalian cells that includes controlling pH using a pH set point, in which mammalian cells are seeded in a culture medium at a first pH.
• a first culture step comprising culturing the cells at a first pH, wherein the pH set point is maintained at the first pH; and culturing the cells at a second pH that is higher than the first pH.
• a second culture stage comprising: a second culture stage in which the set point is maintained at a second pH; the second culture stage is at least 0.1 pH unit higher than the first pH;
• a method is described having a duration of at least 6 hours.
• Patent Document 3 describes a method for culturing immortalized human blood cells, preferably cells originating from myeloid leukemia or a suspension of cells derived therefrom, which provides high productivity and high cell survival. A method is described that provides rate and growth rate and high batch-to-batch consistency and can be scaled up without changing these parameters.
• the ATF (Alternating tangential flow filtration) method which is commonly used in perfusion culture, has a backwashing effect in which the liquid flows back from the secondary side of the membrane to the primary side, resulting in less membrane clogging compared to the TFF (Tangential flow filtration) method. Rishi Hateful. However, as development to improve productivity progresses, cell density increases, and membrane clogging has become a problem even in the ATF method. A common measure to suppress membrane clogging is to increase the membrane area, but there are problems in that the area of commercially available membranes is small and the equipment becomes large.
• An object of the present invention is to provide a method that can suppress membrane clogging in a method for producing products by cell culture. Furthermore, it is an object of the present invention to provide a product manufactured by the above method.
• a method for producing a product comprising culturing cells,
• the cell density during production of the product is perfusion culture of 80 x 10 6 cells/mL or more and 300 x 10 6 cells/mL, and the period of perfusion culture is 13 days or more and 500 days or less,
• the daily alkali addition rate X [mol/L/day] is controlled within the range of 0 ⁇ X ⁇ 0.029.
• the pH of the alkaline aqueous solution is 7 ⁇ pH ⁇ 13. The method of manufacturing the product.
• ⁇ 2> The method for producing the product according to ⁇ 1>, wherein the cell density in the perfusion culture is 100 ⁇ 10 6 cells/mL or more and 300 ⁇ 10 6 cells/mL or less.
• ⁇ 3> The method for producing a product according to ⁇ 1> or ⁇ 2>, wherein the period of the perfusion culture is 18 days or more and 500 days or less.
• ⁇ 4> The method for producing a product according to any one of ⁇ 1> to ⁇ 3>, wherein the culture period for producing the product is 5 days or more and 450 days or less.
• ⁇ 5> During at least 13 days or more of the above perfusion culture period, the daily alkali addition rate X [mol/L/day] is controlled within the range of 0 ⁇ X ⁇ 0.008.
• the method for producing the product according to any one of ⁇ 1> to ⁇ 4> is any one of ⁇ 1> to ⁇ 4>.
• ⁇ 6> Any one of ⁇ 1> to ⁇ 5>, in which the daily alkali addition rate X [mol/L/day] is 0 during at least 13 days or more of the perfusion culture period.
• ⁇ 7> During 50% or more of the perfusion culture period, the daily alkali addition rate X [mol/L/day] is controlled within the range of 0 ⁇ X ⁇ 0.029.
• ⁇ 8> The method for producing a product according to any one of ⁇ 1> to ⁇ 7>, wherein the continuous separation method of the cell culture solution in the perfusion culture is membrane filtration.
• the membrane filtration is alternating tangential flow filtration called ATF.
• Y [L/m 2 /hour] which is the flux during filtration, satisfies 0 ⁇ Y ⁇ 10.
• ⁇ 11> The method for producing the product according to any one of ⁇ 1> to ⁇ 10>, wherein the average pH of the culture solution during the culture is 6.7 to 7.2.
• ⁇ 12> The method for producing a product according to any one of ⁇ 1> to ⁇ 11>, wherein the minimum pH of the culture solution during the culture is 6.6 or higher.
• ⁇ 13> Any one of ⁇ 1> to ⁇ 12>, wherein the pH of the culture solution during the culture is controlled by automatically adding an alkaline aqueous solution while measuring the pH in the culture solution in-line. Method of manufacturing the product described.
• ⁇ 14> In the above perfusion culture, after the cell density reaches the target cell density of 80 ⁇ 10 6 cells/mL or more, the culture solution containing the cells is taken out, so that the cell density is adjusted to the above target cell density ⁇ 40%.
• the method for producing a product according to any one of ⁇ 1> to ⁇ 13> wherein the product is collected during a period in which the cell density is maintained within ⁇ 40% of the target cell density.
• the cell density is reduced by removing the culture medium containing cells at least once per day.
• the method for producing the product according to any one of ⁇ 1> to ⁇ 13> which comprises adjusting the target cell density to within ⁇ 10%.
• ⁇ 16> Production of the product according to any one of ⁇ 1> to ⁇ 15>, wherein the dissolved CO 2 concentration during the period when the cell density is maintained at 80 ⁇ 10 6 cells/mL or more is 60 to 180 mmHg.
• Method. ⁇ 17> The method for producing a product according to any one of ⁇ 1> to ⁇ 16>, wherein the alkali is NaHCO 3 and/or Na 2 CO 3 .
• ⁇ 18> The method for producing a product according to any one of ⁇ 1> to ⁇ 17>, wherein the alkali is NaHCO 3 .
• ⁇ 19> The method for producing a product according to any one of ⁇ 1> to ⁇ 18>, wherein the concentration Z [mol/L] of the alkaline aqueous solution is 0 ⁇ Z ⁇ 5.
• ⁇ 20> The method for producing the product according to any one of ⁇ 1> to ⁇ 19>, wherein the pH of the medium added in perfusion culture is 7.0 to 8.0.
• the pH of the culture solution decreases when culturing without adding an alkali, control the pH of the culture solution by supplying a medium to which an alkali has been added, ⁇ 1> to ⁇ 20 >The method for manufacturing the product described in any one of >.
• ⁇ 22> ⁇ 1> to ⁇ 21 including supplying a medium to which 1.0 ⁇ 10 ⁇ 3 to 2.0 mol/L of alkali has been added when the pH of the culture solution is below 6.85.
• an antifoaming agent containing dimethicone is added, From ⁇ 1>, where X [mol/L/day], which is the addition rate of the alkali, and B [g/L/day], which is the addition rate of the dimethicone, satisfy B ⁇ -0.79X+0.0228.
• ⁇ 24> The method for producing the product according to any one of ⁇ 1> to ⁇ 23>, wherein alkali addition is not performed during the entire period of the perfusion culture.
• ⁇ 25> The method for producing a product according to any one of ⁇ 1> to ⁇ 24>, wherein the cells are animal cells.
• ⁇ 26> The method for producing a product according to any one of ⁇ 1> to ⁇ 25>, wherein the cells are CHO cells.
• ⁇ 27> The method for producing the product according to any one of ⁇ 1> to ⁇ 26>, wherein the product is an antibody.
• ⁇ 28> A product manufactured by the method for manufacturing a product according to any one of ⁇ 1> to ⁇ 27>.
• membrane clogging in perfusion culture can be suppressed.
• FIG. 1 shows a cell culture device.
• the entire apparatus shown in FIG. 1 is a cell culture apparatus.
• FIG. 2 shows the relationship between the alkali addition rate and the number of days from the start of culture until membrane clogging.
• FIG. 3 shows the relationship between alkali addition rate and fine particle density.
• FIG. 4 shows the relationship between alkali addition rate and LDH.
• FIG. 5 shows the relationship between the alkali addition rate, dimethicone addition rate, and the number of days from the start of culture until membrane clogging.
• a numerical range indicated using “ ⁇ ” means a range that includes the numerical values listed before and after “ ⁇ " as the minimum and maximum values, respectively.
• the present invention is a method for producing a product, the method comprising culturing cells,
• the cell density during production of the product is perfusion culture of 80 x 10 6 cells/mL or more and 300 x 10 6 cells/mL or less, and the period of perfusion culture is 13 days or more and 500 days or less,
• the daily alkali addition rate X [mol/L/day] is controlled within the range of 0 ⁇ X ⁇ 0.029.
• the pH of the alkaline aqueous solution is 7 ⁇ pH ⁇ 13. It relates to a method for manufacturing products. According to the present invention, membrane clogging in perfusion culture can be suppressed.
• productivity can be improved particularly in perfusion culture using membranes such as ATF/TFF.
• the addition of alkali per day refers to the addition of alkali that is directly added to the culture solution as an aqueous alkali solution separately from the culture medium, and does not include alkali in the culture medium added to the culture tank during perfusion culture. Even if it were, it does not fall under the category of alkali addition here.
• addition of an alkali is intended to be added directly to the culture solution.
• the cell density during production of the product is 80 ⁇ 10 6 cells/mL or more, preferably 80 ⁇ 10 6 to 300 ⁇ 10 6 cells/mL, and 90 ⁇ 10 6 to 300 It is more preferably ⁇ 10 6 cells/mL, even more preferably 100 ⁇ 10 6 to 300 ⁇ 10 6 cells/mL, even more preferably 110 ⁇ 10 6 to 300 ⁇ 10 6 cells/mL. , more preferably 120 ⁇ 10 6 to 300 ⁇ 10 6 cells/mL. It is sometimes written as M instead of 106 . Particularly, when the cell density is set to 120 ⁇ 10 6 to 300 ⁇ 10 6 cells/mL, membrane clogging becomes noticeable, and the effects of the present invention are significantly obtained.
• the culture solution containing the cells is taken out, so that the cell density can be adjusted to the above-mentioned level.
• the target cell density is maintained within ⁇ 40%, and the product can be collected during the period in which the cell density is maintained within ⁇ 40% of the target cell density.
• the cell density is reduced by removing the culture medium containing cells at least once per day. The above target cell density can be adjusted within ⁇ 10%.
• the method for culturing cells is perfusion culture.
• Perfusion culture is a culture method in which fresh medium is supplied into a cell culture medium and a portion of the medium in which the cells are cultured is removed. By performing this perfusion culture, waste products discharged from the cells can be removed from the culture tank.
• perfusion culture while continuously supplying a culture medium to a culture tank, cells in the culture solution can be continuously separated and a solution can be collected.
• a typical perfusion culture begins with a batch culture lasting one or two days, after which fresh feed medium is continuously, stepwise, and/or intermittently added to the culture, and spent medium is simultaneously removed. .
• methods such as sedimentation, centrifugation or filtration can also be used to separate cells and remove spent medium while maintaining viable cell density.
• the advantage of perfusion culture is that the culture in which the protein of interest is produced is maintained for a longer period of time than in batch or fed-batch culture methods.
• Perfusion may be continuous, stepwise, intermittent, or a combination thereof. Preferably, it has a continuous form.
• the animal cells may be maintained in culture and the spent medium that is removed may be substantially free of cells or have far fewer cells than the culture. Products expressed by cell culture can be retained or recovered in culture by selection of membrane pore size.
• the method for continuous separation of cell culture fluid in perfusion culture is preferably membrane filtration, more preferably alternating tangential flow filtration (ATF).
• Y [L/m 2 /hour], which is the flux during filtration, preferably satisfies 0 ⁇ Y ⁇ 10, more preferably 0 ⁇ Y ⁇ 5, and still more preferably 0 ⁇ Y ⁇ 2.
• the flux during filtration is the amount of culture solution that permeates the membrane per unit time and unit filtration area, and is defined by the following formula.
• the perfusion ratio is not particularly limited, but is generally 0.3vvd to 5.0vvd, preferably 0.5vvd to 2.0vvd, and more preferably 0.5vvd to 1.4vvd.
• vvd means the amount of cell culture solution to be replaced with fresh medium per volume of cell culture solution for one day, that is, volume of supply medium/volume of culture solution/Day.
• the product can be removed from the culture solution by using a pump from the secondary side of the filtration membrane, but other available liquid delivery means may also be used.
• the extracted culture solution is subjected to treatments such as recovery of products and removal of dead cells. Further, the extracted culture solution may be partially discarded after processing such as product recovery and removal of dead cells, or may be returned to the culture container. If a loss of culture solution occurs due to the above treatment, it can be compensated for by, for example, supplying a fresh medium to the culture container.
• the culture medium containing cells is removed at least once per day to adjust the cell density to the target cell density. It can be adjusted within ⁇ 10%.
• a portion of the culture solution is removed together with the cells to reduce the density of living cells. This is called cell bleeding.
• the volume of the culture solution can be maintained by adding a medium. Once a day or more includes cases where cell bleeding is performed continuously and automatically.
• the period of perfusion culture (this period is the culture period from the start of perfusion and includes the period before cells reach high density) is not particularly limited, but is generally one day. 1000 days or more, preferably 7 days or more and 1000 days or less, more preferably 18 days or more and 500 days, still more preferably 25 days or more and 200 days, even more preferably 30 days or more and 100 days. It is as follows. Perfusion may be started 2 days after seeding on day 0.
• the culture period for producing a product with a cell density of 80 ⁇ 10 6 cells/mL or more is preferably 5 days or more and 990 days or less, and may be 5 days or more and 450 days or less, and more preferably 10 days.
• the period is not less than 450 days, more preferably not less than 15 days and not more than 190 days, and even more preferably not less than 20 days and not more than 90 days.
• the daily alkali addition rate X is controlled within the range of 0 ⁇ X ⁇ 0.029 during at least 13 days or more of the perfusion culture period. , preferably within the range of 0 ⁇ X ⁇ 0.015, more preferably within the range of 0 ⁇ X ⁇ 0.0127, and even more preferably within the range of 0 ⁇ X ⁇ 0.008.
• the daily alkali addition rate X may be 0 during at least 13 days or more of the perfusion culture period.
• X is 0, it means that alkali is not added from a line separate from the medium supply.
• the alkali addition rate X [mol/L/day] is controlled within the range of 0 ⁇ X ⁇ 0.029.
• the alkali is not particularly limited, but is preferably Na 2 CO 3 , NaOH or NaHCO 3 , more preferably NaHCO 3 and/or Na 2 CO 3 , particularly preferably NaHCO 3 .
• the alkali can be added as an aqueous solution (eg, an aqueous Na2CO3 , NaOH or NaHCO3 aqueous solution).
• the cell culture medium to which NaHCO 3 is added tends to have a lower viscosity. That is, it is particularly preferable to use NaHCO 3 because it can be said that NaHCO 3 has a weaker effect of gelling the culture solution than Na 2 CO 3 and can easily suppress membrane clogging.
• the time to start adding alkali is after 7 days from the start of culture, preferably after 6 days, and more preferably after 5 days.
• the pH of the alkaline aqueous solution to be added is 7 ⁇ pH ⁇ 13, preferably 7.5 ⁇ pH ⁇ 12, more preferably 7.5 ⁇ pH ⁇ 10, and even more preferably 8 ⁇ pH ⁇ 10. It is.
• pH of an aqueous alkali solution is measured at the point when the alkali is dissolved in water. pH can be measured using a commonly available pH sensor. For example, there are pH meters (Seven Excellence, Seven Direct, Five Easy) manufactured by Mettler TOLEDO.
• the concentration Z [mol/L] of the aqueous alkali solution to be added is preferably 0 ⁇ Z ⁇ 5, more preferably 0 ⁇ Z ⁇ 3, and even more preferably 0 ⁇ Z ⁇ 2.
• N [mol/L] of alkali By adding N [mol/L] of alkali to the culture medium when the culture solution pH falls below a certain value M, membrane clogging can be suppressed and antibody quality can be improved. That is, if the pH of the culture solution decreases when culturing is performed without adding an alkali, the pH of the culture solution can be controlled by supplying a medium to which an alkali has been added.
• the denominator of N is the volume of the medium after preparation. At this time, prepare a new medium with increased alkali, and use a heat welder to connect the container containing the old medium (the medium being supplied to the culture tank) and the tube connected to the culture tank, and connect it to the container containing the new medium. Reconnect aseptically and start supplying.
• an alkali may be added aseptically to a container containing a medium connected to a culture tank.
• a certain value M of the culture solution pH is 6.85, preferably 6.80, and more preferably 6.75.
• the amount of alkali added to the medium, N [mol/L], is 1.0 ⁇ 10 ⁇ 3 to 2.0 mol/L, preferably 2.0 ⁇ 10 ⁇ 3 to 1.0 mol/L. , more preferably 1.0 ⁇ 10 ⁇ 2 to 2.0 ⁇ 10 ⁇ 1 mol/L.
• the alkali added to the culture medium here does not mean the alkali added directly to the culture solution, but refers to the alkali added to the culture medium before being supplied to the culture tank. For example, when the pH of the culture solution falls below 6.85, a medium supplemented with 1.0 ⁇ 10 ⁇ 3 to 2.0 mol/L of alkali can be supplied.
• the pH of the culture solution decreases when culturing without directly adding alkali to the culture solution, it is possible to control the pH of the culture solution by supplying a medium with increased pH. can.
• This pH-enhanced medium may be prepared in advance, or preparation may begin when a change in the pH of the culture solution is observed.
• the feeding medium can be switched to a medium with increased pH and perfusion culture can be performed. Preferably within 12 hours, more preferably within 6 hours, even more preferably within 3 hours, even more preferably within 1 hour, even more preferably within 30 minutes, even more preferably 5 minutes.
• the amount of the solution is preferably 0.5 to 10 times the amount of the culture solution.
• the alkali added to the medium is not particularly limited, but is preferably Na 2 CO 3 , NaOH or NaHCO 3 , more preferably NaHCO 3 and/or Na 2 CO 3 , particularly preferably NaHCO 3 .
• the pH of the medium added in perfusion culture is preferably 7.0 to 8.0, more preferably 7.0 to 7.8, and even more preferably 7.0 to 7.6. .
• the pH of the culture medium can be measured using a commonly available pH sensor after being stored for one day under 5% CO2 and 37°C incubation. For example, there are pH meters (Seven Excellence, Seven Direct, Five Easy) manufactured by Mettler TOLEDO.
• the average pH of the culture solution during cultivation is 6.7 to 7.2, more preferably 6.8 to 7.0.
• the minimum pH of the culture solution during cultivation is 6.6 or higher, more preferably 6.7 or higher, and even more preferably 6.8 or higher.
• the pH of the culture solution during cultivation can be controlled by automatically adding an alkaline aqueous solution while measuring the pH in the culture solution in-line.
• an antifoaming agent can be added in perfusion culture.
• silicone type is preferable, and dimethicone is particularly preferable.
• the antifoaming component of the antifoaming agent preferably contains polydimethylsiloxane, and more preferably contains finely powdered silica in polydimethylsiloxane.
• the antifoaming agent for example, HyClone ADCF Antifoam Agent manufactured by Cytiva can be used.
• X [mol/L/day] which is the addition rate of alkali and B [g/L/day] which is the addition rate of dimethicone per day are preferably B ⁇ -0.79X+0 Satisfies .0228. At this time, B does not take a negative value. Therefore, X ⁇ 0.0288.
• the denominator of B is the volume of the culture solution contained in the culture tank and ATF. By satisfying this relationship, membrane clogging can be suppressed more effectively.
• FIG. 1 shows an example of a cell culture device that can be used in culturing cells in the present invention.
• the culture container 14 is a container containing a culture solution containing cells. Cells are cultured in a culture solution inside the culture container 14.
• a culture medium is supplied from the culture medium supply pipe 1 to the culture container.
• An antifoaming agent for suppressing foaming is supplied from the antifoaming agent supply pipe 2 to the culture container.
• Alkali is supplied from the alkali supply pipe 3 to the culture container. If alkali is not added, the alkali supply pipe 3 may be omitted.
• Carbon dioxide (CO 2 ) and air are introduced from the air supply pipe 4 to the top of the culture solution inside the culture container.
• Oxygen (O 2 ) and/or air is sent from the sparger air supply piping 5, and the oxygen and/or air is introduced into the culture solution through the sparger 15 with a pore diameter of 20 ⁇ m.
• the sparger 15 allows the dissolved oxygen concentration in the culture solution to be adjusted.
• the sparger is not particularly limited, but for example, a sparger that has a gas release part with an average pore diameter of 1 ⁇ m or more and 300 ⁇ m or less (an example is a pore diameter of 20 ⁇ m) and that releases a gas containing 30% by volume or more of oxygen may be used. I can do it.
• the exhaust pipe 6 is a pipe for exhausting air, and an exhaust filter (not shown) may be connected to the end thereof.
• the sampling tube 7 is a pipe for collecting the culture solution (sampling) or extracting the culture solution (cell bleed). If cell bleed is performed automatically and continuously, separate piping may be installed (not shown).
• a pH sensor 8 is mounted so as to be in contact with the culture solution.
• a dissolved oxygen sensor 9 is mounted so as to be in contact with the culture solution.
• Pressure sensors 10, 11 and 13 can be provided in the cell culture device.
• a hollow fiber membrane 12 is installed in the cell culture device.
• a stirring member having stirring blades 16 may be provided inside the culture container 14. By rotating the stirring blade 16, the culture solution inside the culture container 14 is stirred, and the homogeneity of the culture solution is maintained. When the culture solution is stirred by the stirring blade 16, the bubbles released by the sparger are also stirred.
• the position of the stirring member having the stirring blade, the size of the stirring blade, etc. are not particularly limited, and are designed depending on the cell type used, the amount of culture solution, the amount of oxygen to be supplied, the position, number, size of the sparger, etc. do it. Furthermore, in order to quickly stir the bubbles coming out of the sparger and to suppress coalescence of the bubbles, it is preferable that the stirring blade 16 be arranged at a position close to the sparger.
• the cell suspension extracted from the culture container may be passed through a separation membrane to be separated into a cell-containing liquid and a permeated liquid.
• This operation can be performed using a cell culture device.
• the cell suspension extracted from the culture container is divided into a cell-containing liquid having a higher cell concentration than the cell suspension and a permeate liquid having a lower cell concentration than the cell suspension. Separated.
• Cell concentration can be measured using Vi-CELL XR, a live/dead cell analyzer manufactured by Beckman Coulter.
• the membrane separation treatment step described above is preferably tangential filtration, more preferably Alternating tangential flow (ATF) filtration or tangential flow filtration. Most preferably, it is an initial flow filtration.
• filters that can perform alternating tangential flow filtration include SuATF10-S02PES and F2RF02PES manufactured by Repligen.
• a medium used for normal animal cell culture a medium used for normal animal cell culture can be used.
• CD OptiCHO manufactured by ThermoFisher
• Dulbecco's modified Eagle's medium DMEM
• Eagle's minimum essential medium MEM
• RPMI-1640 medium RPMI-1641 medium
• F-12K medium Ham's F12 medium
• Iscobb's modified Dulbecco's medium a medium used for normal animal cell culture.
• IMDM IMDM
• McCoy 5A medium McCoy 5A medium
• Leibovitz L-15 medium EX-CELL 300 series
• EX-CELL 300 series JRH Biosciences
• CHO-S-SFMII Invitrogen
• CHO-SF Sigma-Aldrich
• CD-CHO Invitrogen
• IS CHO-V Irvine Scientific
• PF-ACF-CHO Sigma-Aldrich
• Serum such as fetal calf serum (FCS) may or may not be added to the medium.
• the medium may be supplemented with additional components such as amino acids, salts, sugars, vitamins, hormones, growth factors, buffers, antibiotics, lipids, trace elements, hydrolysates of plant proteins, etc. Protein-free media can also be used.
• the culture temperature is generally 30°C to 40°C, preferably 32°C to 39°C, more preferably 36°C to 38°C, and the culture temperature may be changed during culture. Cultivation can be carried out in an atmosphere with a CO 2 concentration of 0 to 40% by volume, preferably 2 to 25% by volume, and more preferably 3 to 20% by volume.
• the amount of culture solution is preferably 0.5 L or more, more preferably 50 L or more, and still more preferably 200 L or more.
• the stirring rotation speed is not particularly limited, but the stirring power per unit volume is generally 10 to 300 kW/m 3 , preferably 20 to 200 kW/m 3 , and more Preferably it is 30 to 100 kW/m 3 .
• the dissolved oxygen concentration in the culture solution can be set appropriately and is not particularly limited, but is generally 10 to 100%, preferably 30 to 90%. Further, the dissolved CO 2 concentration during the period when the cell density is maintained at 80 ⁇ 10 6 cells/mL or more is preferably 60 to 180 mmHg, more preferably 80 to 160 mmHg, and still more preferably 100 to 140 mmHg. It is.
• Cell culture can be performed using a cell culture device having the configuration described above in this specification.
• Cell culture devices include fermenter type tank culture device, air lift type culture device, culture flask type culture device, spinner flask type culture device, microcarrier type culture device, fluidized bed type culture device, holofiber type culture device, roller Either a bottle type culture device or a filled tank type culture device may be used.
• the culture container is preferably a single-use culture tank from the viewpoint of homogenizing the culture environment.
• the viscosity of the culture solution for producing a product with a cell density of 80 ⁇ 10 6 cells/mL or more is preferably 1.2 mPa ⁇ s or more and less than 15 mPa ⁇ s, more preferably 1.4 mPa ⁇ s or more. less than 12 mPa ⁇ s, more preferably 1.6 mPa ⁇ s or more and less than 10 mPa ⁇ s, particularly preferably 1.6 mPa ⁇ s or more and less than 5 mPa ⁇ s, and most preferably 1.6 mPa ⁇ s or more and less than 3 mPa ⁇ s. less than s.
• the types of cells in the present invention are not particularly limited, but include animal cells, plant cells, eukaryotic cells such as yeast, prokaryotic cells such as Bacillus subtilis, and Escherichia coli.
• the cells are preferably animal cells (more preferably mammalian cells) or insect cells, most preferably mammalian cells.
• the cells may be primary cells or established cell lines.
• Examples of cells include Chinese hamster ovary (CHO) cells, HEK cells (Human Embryonic Kidney-derived cells), BHK cells, 293 cells, C127 cells, myeloma cells (NS0 cells, etc.), PerC6 cells, SP2/0 cells, hybridoma cells, Examples include COS cells (African green monkey kidney-derived cells), 3T3 cells, HeLa cells, Vero cells (African green monkey kidney epithelial cells), MDCK cells (canine kidney tubular epithelial cell-derived cells), PC12 cells, WI38 cells, etc. .
• the cells may be stem cells, such as embryonic stem cells (ES cells) or induced pluripotent stem cells (iPS cells).
• ES cells embryonic stem cells
• iPS cells induced pluripotent stem cells
• CHO cells HEK cells, BHK cells, and hybridomas are preferred, CHO cells and HEK cells are more preferred, and CHO cells are most preferred.
• CHO cells are widely used for the production of recombinant proteins such as cytokines, clotting factors, and antibodies. It is preferable to use CHO cells deficient in dihydrofolate reductase (DHFR), and as the DHFR deficient CHO cells, for example, CHO-DG44 can be used.
• DHFR dihydrofolate reductase
• the cell survival rate is preferably higher, preferably 85% or higher, more preferably 90% or higher, particularly preferably 95% or higher, and most preferably 99% or higher.
• These cells may be cells into which a foreign gene encoding a protein (for example, an antibody, etc.) to be expressed is introduced.
• the cells are preferably cells that produce antibodies.
• Expression vectors can be used to introduce foreign genes encoding proteins to be expressed into cells.
• the protein to be expressed is encoded by introducing into cells an expression vector containing DNA encoding the protein to be expressed, an expression control sequence (e.g., enhancer, promoter, terminator, etc.), and optionally a selection marker gene. Cells into which foreign genes have been introduced can be produced. There are no particular limitations on the expression vector, and it can be appropriately selected and used depending on the cell type, purpose, etc.
• any promoter can be used as long as it can function in mammalian cells.
• CMV cytomegalovirus
• SV40 early promoter SV40 early promoter
• retrovirus promoter metallothionein promoter
• heat shock promoter SR ⁇ promoter
• Moloney murine leukemia virus promoter SR ⁇ promoter
• Moloney murine leukemia virus promoter SR ⁇ promoter
• Moloney murine leukemia virus promoter Moloney murine leukemia virus promoter.
• enhancer of the human CMV IE gene may be used together with the promoter.
• selectable marker genes include drug resistance genes (neomycin resistance gene, dihydrofolate reductor (DHFR) gene, puromycin resistance gene, blasticidin resistance gene, hygromycin resistance gene, cycloheximide resistance gene, etc.), or fluorescent genes ( A gene encoding green fluorescent protein GFP, etc.) can be used.
• drug resistance genes neomycin resistance gene, dihydrofolate reductor (DHFR) gene, puromycin resistance gene, blasticidin resistance gene, hygromycin resistance gene, cycloheximide resistance gene, etc.
• fluorescent genes A gene encoding green fluorescent protein GFP, etc.
• the method for introducing the expression vector into cells is not particularly limited, and for example, the calcium phosphate method, electroporation method, liposome method, gene gun method, lipofection method, etc. can be used.
• the method for producing a product according to the present invention includes culturing cells using a cell culture device and producing a product from the cells. According to the present invention, there is provided a product manufactured by the method for manufacturing a product according to the present invention.
• the type of product is not particularly limited, but it is preferably a protein, and more preferably a recombinant protein.
• Products include, for example, recombinant polypeptide chains, recombinant secreted polypeptide chains, antigen-binding proteins, antibodies (e.g., human antibodies, humanized antibodies, chimeric antibodies, mouse antibodies, bispecific antibodies, etc.), Fc fusion proteins, fragments. Examples include immunized immunoglobulins, single chain antibodies (scFv), and the like.
• adenovirus, adeno-associated virus, lentivirus, etc. may also be used.
• the product is preferably an antibody, more preferably a human antibody, humanized antibody, chimeric antibody, or mouse antibody.
• Fragmented immune immunoglobulins include Fab, F(ab')2, Fv, and the like.
• the class of the antibody is also not particularly limited, and may be any class such as IgG such as IgG1, IgG2, IgG3, and IgG4, IgA, IgD, IgE, and IgM, but IgG and IgM are preferred when used as a medicine.
• Human antibodies include all antibodies that have one or more variable and constant regions derived from human immunoglobulin sequences. In one embodiment, all variable and constant domains are derived from human immunoglobulin sequences (fully human antibody).
• Humanized antibodies are designed such that when administered to a human subject, humanized antibodies are less likely to elicit an immune response and/or induce a severe immune response compared to non-human species antibodies. has a sequence that differs from that of an antibody derived from a non-human species by one or more amino acid substitutions, deletions, and/or additions such that the antibody has a lower sequence.
• certain amino acids within the heavy and/or light chain framework and constant domains of a non-human species antibody are mutated to produce a humanized antibody.
• a constant domain from a human antibody is fused to a variable domain of a non-human species.
• a chimeric antibody is an antibody in which a variable region and a constant region of different origins are linked.
• an antibody consisting of the heavy chain and light chain variable regions of a mouse antibody and the heavy and light chain constant regions of a human antibody is a mouse-human heterologous chimeric antibody.
• a recombinant vector that expresses a chimeric antibody can be created by linking the DNA encoding the variable region of a mouse antibody with the DNA encoding the constant region of a human antibody and incorporating this into an expression vector. By culturing recombinant cells transformed with the above vector and expressing the integrated DNA, chimeric antibodies produced during the culture can be obtained.
• Bispecific antibodies are antibodies that recognize two different antigen specificities. Bispecific antibodies exist in various forms. Bispecific antibodies can be produced by linking two immunoglobulin molecules using a cross-linking agent such as N-succinimidyl 3-(2-pyridyldithiol) propionate or S-acetylmercaptosuccinic acid anhydride. Several methods have been reported, including a method in which Fab fragments of immunoglobulin molecules are linked together. Furthermore, expression can also be achieved by introducing a gene encoding a bispecific antibody into cells.
• a cross-linking agent such as N-succinimidyl 3-(2-pyridyldithiol) propionate or S-acetylmercaptosuccinic acid anhydride.
• Fc fusion protein refers to a protein having an Fc region, and includes antibodies.
• Fabs are monovalent fragments with VL, VH, CL and CH1 domains.
• F(ab')2 is a bivalent fragment with two Fab fragments connected by a disulfide bridge in the hinge region.
• Fv fragments have the single-armed VL and VH domains of an antibody.
• a single chain antibody (scFv) is an antibody in which the VL and VH regions are joined via a linker (e.g., a synthetic sequence of amino acid residues) to form a continuous protein chain, where the linker is long enough to fold onto itself to form a monovalent antigen binding site.
• Antibodies include, but are not particularly limited to, anti-IL-6 receptor antibodies, anti-IL-6 antibodies, anti-glypican-3 antibodies, anti-CD3 antibodies, anti-CD20 antibodies, anti-GPIIb/IIIa antibodies, anti-TNF antibodies, and anti-CD25 antibodies.
• Antibodies include anti-EGFR antibodies, anti-Her2/neu antibodies, anti-RSV antibodies, anti-CD33 antibodies, anti-CD52 antibodies, anti-IgE antibodies, anti-CD11a antibodies, anti-VEGF antibodies, and anti-VLA4 antibodies.
• the product may be collected by simply collecting the culture solution, or by collecting a liquid obtained by removing at least a portion of the cells from the culture solution using, for example, a filter or a centrifuge, and known methods may be used. Can be used without any particular restrictions. If it is desired to improve the purity of the product, change the solvent or change the form, for example into a powder, the culture solution or the liquid described above can be subjected to further processing.
• a portion of the culture solution may be collected during perfusion, or a filter or centrifuge may be used to remove at least a portion of the cells from the culture solution during perfusion. It is also possible to collect
• the product can be purified by purification treatment.
• the resulting product can be purified to high purity.
• the product may be separated and purified using conventional separation and purification methods used for proteins. For example, by appropriately selecting and combining chromatography columns such as affinity chromatography, filters, ultrafiltration, salting out, dialysis, sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, isoelectric focusing, etc. can be separated and purified, but is not limited to these.
• the concentration of the product obtained above can be measured by absorbance measurement, enzyme-linked immunosorbent assay (ELISA), or the like.
• ELISA enzyme-linked immunosorbent assay
• the product is an antibody
• the antibody titer can also be measured with a commercially available analytical instrument such as Roche's Cedex Bio.
• chromatography examples include protein A columns and protein G columns.
• chromatography other than affinity chromatography examples include ion exchange chromatography, hydrophobic chromatography, gel filtration, reversed phase chromatography, and adsorption chromatography. These chromatography can be performed using liquid phase chromatography such as HPLC (high performance liquid chromatography) or FPLC (fast protein liquid chromatography). I can.
• the product can be modified or peptides can be partially removed by treating the product with an appropriate polypeptide modification enzyme before or after purification.
• polypeptide-modifying enzymes used include trypsin, chymotrypsin, lysyl endopeptidase, protein kinase, and glucosidase.
• the products produced according to the present invention can be used, for example, in biopharmaceuticals, regenerative medicine, and the like.
• CHO-DG44 cells IgG1 cells
• IgG4 cells CHO-DG44 cells that express IgG4 are obtained.
• Construction of the vector and introduction into cells were performed according to Example 2 of Japanese Translated Patent Publication No. 2016-517691.
• CHO cells producing monoclonal antibodies were prepared as described above and used in the following experiments.
• CHO cells that produce antibodies are grown by perfusion culture to 120 x 10 6 cells/mL, cell bleeding is started when the cell density of 120 x 10 6 cells/mL is reached, and the hollow fiber membrane is grown. The number of days of clogging was evaluated. Details are as follows.
• the pH in the culture tank was read so that the pH in the culture tank became 7.0, and when the pH fell below 7.0, 1.0 mol/L NaHCO 3 aqueous solution was automatically added to start pH control. .
• the cell culture solution was continuously filtered using ATF2 manufactured by Repligen while the culture medium was continuously supplied at a perfusion ratio of 0.8 vvd, and a recovered solution was collected. Furthermore, on the 5th day, the perfusion ratio was changed to 1.6 vvd. Furthermore, on the 6th day, the addition rate of simethicone in the antifoaming agent was set to 7.2 mg/day/L.
• the pH of the culture solution decreases, and an aqueous alkaline solution begins to be automatically added to the culture solution.
• days 9 to 11 when the cell density reached 120 x 10 6 cells/mL, cell bleeding was performed while withdrawing a portion of the culture medium to maintain the cell density at 120 x 10 6 cells/mL. Ta. Culture was continued until membrane clogging occurred and ATF could no longer operate.
• ⁇ Evaluation method> Measurement of Cell Density and Viability The culture solution in the culture tank was taken out and measured using Cell Viability Analyzer Vi-cell XR manufactured by Beckman Coulter. Note that the Vi-cell software used was Vi-cell XR2.04, and the parameters at the time of measurement were set as follows.
• Min diameter 6 ⁇ m
• Max diameter 50 ⁇ m
• Dilution When the cell concentration was 10 ⁇ 10 6 cells/mL or less, the sample was not diluted and the dilution was set to 1. When the cell concentration was higher than 10 ⁇ 10 6 cells/mL, the sample was diluted 10 times and the dilution was set to 10.
• Cell sharpness 100
• Minimum circularity 0 Decluster degree: Medium
• the culture solution in the culture tank was extracted and diluted 10,000 times with ISOTON from Beckman Coulter.
• the particle size distribution of the diluted culture solution from 1.46 to 20 ⁇ m was measured using a precision particle size distribution analyzer Multisizer 4e manufactured by Beckman Coulter.
• LDH Lactate dehydrogenase
• Antibody charge was evaluated by measuring the above antibody recovery solution using cation exchange high performance liquid chromatography (HPLC).
• Mannose 5 (M5) was evaluated by digesting the antibody in the obtained antibody recovery solution with peptide-N-glycosidase F to cut out the N-glycan chain, which was fluorescently labeled with 2-aminobenzamide.
• the M5 of the treated antibody was measured using a C18 (ODS) reverse phase HPLC column.
• Evaluation of membrane clogging S: When the number of days from the start of culture until clogging is 30 days or more, the number of days from the start of perfusion until clogging is 28 days or more, and the number of days from the start of cell bleeding until clogging is 20 days or more.
• A When the number of days from the start of culture until clogging is 20 to 29 days, the number of days from the start of perfusion until clogging is 18 to 27 days, and the number of days from the start of cell bleeding until clogging is 10 to 19 days. .
• B When the number of days from the start of culture until clogging is 15 to 19 days, the number of days from the start of perfusion until clogging is 13 to 17 days, and the number of days from the start of cell bleeding until clogging is 5 to 9 days.
• C The number of days from the start of culture until clogging is 14 days or less, the number of days from the start of perfusion until clogging is 12 days or less, and the number of days from the start of cell bleeding until clogging is 4 days or less.
• the peak area ratio is the ratio of the chromatographic peak area of the Acidic component or the chromatographic peak area of the Basic component to the total peak area of all chromatographic peaks.
• M5 peak area ratio X is 0% ⁇ X ⁇ 5%
• B: M5 peak area ratio X is 5% ⁇ X ⁇ 6%
• C: M5 peak area ratio X is 6% ⁇ X
• the peak area ratio refers to the ratio of the chromatographic peak area of the M5 (Mannose 5) component to the total chromatographic peak area.
• FIG. 2 shows the relationship between the alkali addition rate and the number of days until membrane clogging in Examples 1 to 3, 5, and 7 to 9. The results in FIG. 2 show that membrane clogging can be suppressed by lowering the alkali addition rate.
• FIG. 3 shows the relationship between the alkali addition rate and the fine particle density in Examples 1 to 3, 5, and 7 to 9.
• the particle density was defined as the cumulative number of cells with a density of 1 to 6 ⁇ m.
• Microparticles are dregs/debris derived from cells (14 ⁇ m) and are an indicator of cell damage. The results in FIG. 3 show that cell damage can be suppressed by lowering the alkali addition rate.
• FIG. 4 shows the relationship between the alkali addition rate and LDH in Examples 1 to 3, 5, and 7 to 9.
• LDH is an enzyme component that leaks from damaged cells and is commonly used as an indicator of cell damage. The results in FIG. 4 show that cell damage can be suppressed by lowering the alkali addition rate.
• FIG. 5 shows the relationship between the alkali addition rate, antifoaming agent addition rate, and number of days until membrane clogging in Examples 1 to 9 and 11 and Comparative Example 1.

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• 2023
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