Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/40—Immunoglobulins specific features characterized by post-translational modification
  • C07K2317/41—Glycosylation, sialylation, or fucosylation
• • 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
• • 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
  • C12N2500/30—Organic components
  • C12N2500/34—Sugars

Definitions

• the present invention relates to a method for producing a product, which includes culturing cells so as to satisfy predetermined conditions during the product production period after reaching a target viable cell density.
• the invention further relates to a product produced by the production method described above.
• Cell culture is carried out for the purpose of increasing the number of cells with useful properties, making the cells produce products, and so on.
• U.S. Pat. (iii) maintaining the perfusion culture at a second perfusion rate until the bispecific antibody product concentration in the bioreactor is , a method for producing a bispecific antibody product is described that includes a step of being kept below a threshold value.
• US Pat. No. 5,001,000 describes an integrated continuous method for manufacturing a therapeutic protein drug substance, comprising: (i) a liquid culture medium containing a substantially cell-free recombinant therapeutic protein; supplying a liquid culture medium to be fed into a first multi-column chromatography system (MCCS1); (ii) using MCCS1 to capture the recombinant therapeutic protein in the liquid culture medium; (iii) the eluate of MCCS1 containing MCCS2 is continuously fed into a second multi-column chromatography system (MCCS2); A method is described that includes purifying and polishing the protein to be a therapeutic protein drug substance.
• MCCS1 multi-column chromatography system
• US Pat. No. 5,001,002 describes a method for controlling the perfusion rate of a perfusion bioreactor, comprising: a) measuring the viable cell density of cells growing in culture using a biomass capacitance probe; and b) A method is described that includes dynamically adjusting a target perfusion rate to take into account viable cell density, wherein cells grow to high cell density during perfusion.
• An object of the present invention is to provide a method for producing a product by cell culture that can reduce the amount of mannose 5-modified antibody in the product. Furthermore, it is an object of the present invention to provide a product produced by the above method.
• the present inventors found that by culturing cells so as to satisfy predetermined conditions during the product production period after reaching the target viable cell density, The inventors have discovered that the above problems can be solved, and have completed the present invention.
• a method for producing a product comprising culturing cells in a culture container,
• the above culture is a perfusion culture method
• formulas (1) to (5) are satisfied,
• the variation in VCD during the production period is ⁇ 20% or less with respect to the average viable cell density during the production period
• the variation in VCV during the production period is ⁇ 20% or less with respect to the average value of VCV during the production period, How products are produced.
• Gln-C indicates the glutamine concentration [mmol/L] in the culture solution
• Gluc-C indicates the glucose concentration [g/L] in the culture solution
• NH4-C indicates the ammonia concentration [mmol/L] in the culture solution
• VCD indicates viable cell density [ ⁇ 10 6 cells/mL]
• VCV indicates VCD ⁇ /6 ⁇ Cd 3 [ ⁇ 10 6 ⁇ m 3 /mL]
• Cd indicates the average diameter [ ⁇ m] of living cells.
• ⁇ 2> Production of the product according to ⁇ 1>, wherein the culture solution is bled so that the variation in VCD during the production period is ⁇ 20% or less with respect to the average viable cell density during the production period.
• Method. ⁇ 3> The method for producing a product according to ⁇ 2>, wherein in the bleeding, the culture solution is bled while the capacitance of the culture solution is measured in-line.
• ⁇ 5> The production according to ⁇ 3> or ⁇ 4>, wherein the variation in the capacitance of the culture solution measured in-line is ⁇ 20% or less with respect to the average value of capacitance during the production period. How things are produced.
• the above bleeding is (i) Setting a target capacitance; (ii) setting a weight control value for the culture vessel; (iii) Measuring the capacitance of the culture medium at a cycle of 0.1 seconds or less; (iv) When the capacitance of the culture solution exceeds the above target, the pump that withdraws the culture solution should be operated at a speed of 0.3 vvd to 3 vvd (where vvd is the volume of the culture solution drawn/the volume of the culture solution).
• the method for producing the product according to any one of ⁇ 3> to ⁇ 5> comprising: ⁇ 7> The method for producing the product according to any one of ⁇ 1> to ⁇ 6>, which satisfies formula (6) during the production period.
• LDH indicates the concentration of lactate dehydrogenase in the culture solution [U/L]
• VCD indicates viable cell density [ ⁇ 10 6 cells/mL].
• ⁇ 8> The method for producing the product according to any one of ⁇ 1> to ⁇ 7>, wherein the cells are animal cells.
• ⁇ 9> The method for producing a product according to any one of ⁇ 1> to ⁇ 8>, wherein the cells are cells that produce proteins.
• the cell is a cell that produces an antibody.
• ⁇ 11> The method for producing a product according to any one of ⁇ 1> to ⁇ 10>, wherein the cells are CHO cells.
• ⁇ 12> A product produced by the method for producing a product according to any one of ⁇ 1> to ⁇ 11>.
• the amount of mannose 5 in the product can be reduced.
• FIG. 1 shows a cell culture device.
• FIG. 2 shows the results of measuring the change in VCV in Example 1.
• FIG. 3 shows the results of measuring changes in capacitance data (also referred to as Cp) in Example 1.
• FIG. 4 shows the results of measuring the change in the mannose 5 ratio in Example 1.
• 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 method for producing a product of the present invention includes culturing cells in a culture container, the culture is a perfusion culture method, and during the production period of the product after reaching a target viable cell density, the formula ( 1) to (5) are satisfied, the variation in VCD during the production period is ⁇ 20% or less with respect to the average viable cell density during the production period, and the variation in VCV during the production period is within the range of VCV during the production period.
• the method for producing a product of the present invention can reduce the amount of mannose 5-modified antibody in the product by satisfying the above conditions.
• Gln-C indicates the glutamine concentration [mmol/L] in the culture solution
• Gluc-C indicates the glucose concentration [g/L] in the culture solution
• NH4-C indicates the ammonia concentration [mmol/L] in the culture solution
• VCD indicates viable cell density [ ⁇ 10 6 cells/mL]
• VCV indicates VCD ⁇ /6 ⁇ Cd 3 [ ⁇ 10 6 ⁇ m 3 /mL]
• Cd indicates the average diameter [ ⁇ m] of living cells.
• Preferred embodiments of formula (1) are the following formulas (1A) and (1B). 0.1 ⁇ Gln-C ⁇ 2.0 Formula (1A) 0.2 ⁇ Gln-C ⁇ 1.5 Formula (1B)
• Preferred embodiments of formula (2) are the following formulas (2A) and (2B). 1.0 ⁇ Gluc-C ⁇ 6.0 Formula (2A) 1.5 ⁇ Gluc-C ⁇ 5.0 Formula (2B)
• Preferred embodiments of formula (3) are the following formulas (3A) and (3B). 1.5 ⁇ NH4-C ⁇ 7.0 Formula (3A) 2.0 ⁇ NH4-C ⁇ 6.0 Formula (3B)
• Preferred embodiments of formula (4) are the following formulas (4A) and (4B). 80 ⁇ VCD ⁇ 250 Formula (4A) 100 ⁇ VCD ⁇ 220 Formula (4B)
• Preferred embodiments of formula (5) are the following formulas (5A) and (5B). 0.8 ⁇ 10 5 ⁇ VCV ⁇ 3.0 ⁇ 10 5 formula (5A) 1.0 ⁇ 10 5 ⁇ VCV ⁇ 2.5 ⁇ 10 5 formula (5B)
• the variation in VCD during the production period is less than ⁇ 20% relative to the average viable cell density during the production period, but more preferably, the variation in VCD during the production period is less than ⁇ 20% relative to the average viable cell density during the production period.
• the variation in VCD during the production period is ⁇ 15% or less, more preferably the variation in VCD during the production period is ⁇ 12% or less with respect to the average viable cell density during the production period, and particularly preferably the variation in VCD during the production period is ⁇ 8% or less of the average viable cell density in the cell.
• the lower limit of the variation in VCD during the production period is not particularly limited and may be 0% or more, but when daily fluctuations in cell proliferation are taken into account, variations of 0.1% or more actually occur.
• the variation in VCV during the production period is ⁇ 20% or less with respect to the average value of VCV during the production period, more preferably ⁇ 15% or less with respect to the average value of VCV during the production period, More preferably, it is ⁇ 12% or less with respect to the average value of VCV during the production period, and particularly preferably, it is ⁇ 8% or less with respect to the average value of VCV during the production period.
• the lower limit of the variation in VCV during the production period is not particularly limited and may be 0% or more, but when daily fluctuations in cell proliferation are taken into account, variations of 0.1% or more actually occur.
• the concentration of glutamine in the culture solution [mmol/L], represented by Gln-C, can be measured using the filtrate after removing the culture solution from the culture container and removing cells by filtration. Measurement of glutamine concentration in the filtrate can be performed using a commercially available analyzer such as Nova biomedical's Bioprofile FLEX2.
• the frequency of measurement of glutamine concentration is not particularly limited, but can be performed, for example, once a day.
• the glucose concentration [g/L] in the culture medium, indicated by Gluc-C, can be measured by extracting the culture medium from the culture container and removing cells by filtration, using the filtrate. Measurement of glucose concentration in the filtrate can be performed using a commercially available analyzer such as Nova biomedical's Bioprofile FLEX2.
• the frequency of measurement of glucose concentration is not particularly limited, but can be performed, for example, once a day.
• the ammonia concentration [mmol/L] in the culture solution can be measured by removing the culture solution from the culture container and removing cells by filtration using the filtrate. Measuring the ammonia concentration in the filtrate can be performed using a commercially available analyzer such as Nova biomedical's Bioprofile FLEX2.
• the frequency of measurement of ammonia concentration is not particularly limited, but can be performed, for example, once a day.
• VCD viable cell density [ ⁇ 10 6 cells/mL] indicated by VCD can be measured by a conventional method by extracting the culture solution from the culture container.
• VCD ⁇ /6 ⁇ Cd 3 [ ⁇ 10 6 ⁇ m 3 /mL] is further calculated as VCV.
• Cd indicates the average diameter [ ⁇ m] of living cells.
• the density of living cells and the average diameter of living cells can be measured using a commercially available measurement device such as Beckman Coulter's Cell Viability Analyzer Vi-cell XR, and as an example, the Vi-cell software is Vi-cell XR2. 04, the parameters at the time of measurement are Min diameter set to 6 ⁇ m and Max diameter set to 50 ⁇ m. Furthermore, measurement parameters can be set as follows. Cell brightness: 85% Cell sharpness: 100 Viable cell spot brightness: 75% Viable cell spot area: 5% Minimum circularity: 0 Decluster degree: Medium
• sample used for measurement may be used without being diluted, or the sample may be used after dilution.
• the frequency of measuring the density of living cells and the average diameter of living cells is not particularly limited, but can be performed, for example, once a day.
• the variation in VCD during the production period is the variation rate (percentage) that indicates how much the VCD value measured during the production period fluctuated relative to the average value of VCD during the production period, that is, the average viable cell density.
• the average value of VCD during the production period is determined by extracting the culture solution from the culture tank every day during the production period and measuring the VCD value using Beckman Coulter's Cell Viability Analyzer Vi-cell XR etc. It can be determined by adding up the number of days and dividing it by the number of culture days during the production period.
• the fluctuation in VCV during the production period is a fluctuation rate (percentage) indicating how much the VCV value measured during the production period fluctuates with respect to the average value of VCV during the production period.
• the culture solution can be bled so that the variation in VCD during the production period is ⁇ 20% or less with respect to the average viable cell density during the production period.
• Bleeding means removing a portion of the culture medium along with the cells to reduce the density of living cells during culture so that the density of living cells does not become excessive.
• fresh medium can be added in the same amount as the removed culture medium.
• the variation in VCD during the production period is less than ⁇ 15% relative to the average viable cell density during the production period, and even more preferably, the variation in VCD during the production period is less than or equal to the average viable cell density during the production period.
• the variation in VCD during the production period is preferably ⁇ 8% or less with respect to the average viable cell density during the production period.
• the lower limit of the variation in VCD during the production period is not particularly limited and may be 0% or more, but when daily fluctuations in cell proliferation are taken into account, variations of 0.1% or more actually occur.
• Bleeding can be carried out as long as the culture solution containing cells can be removed from the culture container, and the method is not particularly limited.
• piping for draining the culture solution is installed in the culture solution.
• the culture solution can be inserted and drained from the culture vessel through the above-mentioned piping.
• the culture solution may be bled while the capacitance of the culture solution is measured in-line.
• In-line measurement refers to measuring some kind of measurement value that indicates the state of the culture solution while culturing cells.
• a capacitance sensor may be inserted into the culture solution, and the capacitance of the culture solution may be measured in-line while culturing.
• bleeding may be automatically controlled.
• Automatically controlling means automatically controlling the start and stop of bleeding based on some kind of measured value indicating the state of some kind of culture solution.
• the start and stop of bleeding can be automatically controlled based on the measured value of the capacitance of the culture solution.
• bleeding may be performed so that the variation in the capacitance of the culture solution measured in-line is within ⁇ 20% of the average value of capacitance during the production period.
• the fluctuation in the capacitance of the culture solution measured in-line is more preferably ⁇ 15% or less with respect to the average value of capacitance during the production period, and even more preferably, the variation in capacitance during the production period is It is ⁇ 12% or less with respect to the average value, particularly preferably ⁇ 8% or less with respect to the average value of capacitance during the production period.
• the lower limit of the variation in capacitance of the culture solution is not particularly limited and may be 0% or more, but when daily fluctuations in cell proliferation are taken into account, variations of 0.1% or more actually occur.
• An example of breeding is (i) Setting a target capacitance; (ii) setting a weight control value for the culture vessel; (iii) Measuring the capacitance of the culture medium at a cycle of 0.1 seconds or less; (iv) When the capacitance of the culture solution exceeds the above target, operate the pump that draws out the culture solution at a speed of 0.3 vvd to 3 vvd (where vvd is the volume of the culture solution drawn/the volume of the culture solution). (means volume/day); (v) Automatically supplying the culture medium to the culture container while measuring the mass of the culture solution so that the variation in the volume of the culture solution is within 10%; and (vi) The capacitance of the culture solution is adjusted to the above target. If it falls below 0.01% or more, stop the pump that draws out the culture solution. It can be carried out by a process including.
• formula (6) is satisfied during the production period.
• LDH indicates the concentration of lactate dehydrogenase in the culture solution [U/L]
• VCD indicates viable cell density [ ⁇ 10 6 cells/mL].
• formula (6) More preferred embodiments of formula (6) are the following formulas (6A) and (6B). 2 ⁇ LDH/VCD ⁇ 35 Formula (6A) 2 ⁇ LDH/VCD ⁇ 30 Formula (6B)
• the concentration of lactate dehydrogenase in the culture solution [U/L], represented by LDH, can be measured using the filtrate after removing the culture solution from the culture container and removing cells by filtration. Measuring the concentration of lactate dehydrogenase in the filtrate can be performed using a commercially available analyzer such as Roche's Cedex Bio. Although the frequency of measuring the concentration of lactate dehydrogenase is not particularly limited, it can be performed, for example, once a day.
• FIG. 1 shows an example of a cell culture device that can be used in culturing cells in the present invention.
• a culture container 10 is a container containing a culture solution containing cells. Cells are cultured in a culture solution inside the culture container 10.
• Oxygen and air are sent from the sparger air supply piping 1, and the oxygen and air are introduced into the culture solution through the sparger 11 with a pore diameter of 20 ⁇ m.
• the sparger 11 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 portion with an average pore diameter of 1 ⁇ m or more and 300 ⁇ m or less and that releases a gas containing 30 volume % or more of oxygen can be used.
• Air and carbon dioxide are introduced from the air supply pipe 2 to the top of the culture solution inside the culture container.
• a culture medium is supplied from the culture medium supply pipe 3 to the culture container.
• the culture medium supply pipe 3 is provided with a culture medium supply pump 15 .
• the exhaust pipe 4 is a pipe for exhausting air, and an exhaust filter 5 is connected to the end thereof.
• the sampling tube 6 is a pipe for collecting (sampling) the culture solution.
• the bleeding pipe 7 is a pipe for extracting (bleeding) the culture solution.
• a capacitance sensor 8 is mounted so as to be in contact with the culture solution in the culture container.
• a dissolved oxygen sensor 9 is mounted so as to be in contact with the culture solution in the culture container.
• a stirring member having stirring blades 12 may be provided inside the culture container 10. By rotating the stirring blade 12, the culture solution inside the culture container 10 is stirred, and the homogeneity of the culture solution is maintained. When the culture solution is stirred by the stirring blade 12, 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. Further, 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 12 be arranged at a position close to the sparger.
• a perfusion device 13 is connected to the bottom of the culture container 10. Further, the culture solution inside the culture container 10 passes through a perfusion device 13 , and the permeate containing cell products is taken out from the culture container 10 by a delivery pump 16 . The flow of permeate is indicated by arrow 14.
• the viable cell density of the culture solution is preferably 50 ⁇ 10 6 to 300 ⁇ 10 6 cells/mL, more preferably 80 ⁇ 10 6 to 250 ⁇ 10 6 cells/mL, and 100 ⁇ 10 6 to 250 ⁇ 10 6 cells/mL. More preferably, it is ⁇ 10 6 to 220 ⁇ 10 6 cells/mL. It is sometimes written as M instead of 106 .
• 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 perfusion device.
• the cell suspension extracted from the culture container is divided into a cell-containing liquid having a higher cell density than the cell suspension and a permeate liquid having a lower cell density than the cell suspension. Separated.
• Cell density can be measured using Vi-CELL XR, a live and dead cell analyzer manufactured by Beckman Coulter.
• the above membrane separation treatment step is preferably tangential filtration, more preferably Alternating tangential flow (ATF) or tangential flow, and most preferably Alternating tangential flow.
• ATF Alternating tangential flow
• filters that can perform alternating tangential flow include SuATF10-S02PES and F2 RF02PES 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 pH of the medium varies depending on the cells to be cultured, but is generally pH 6.0 to 8.0, preferably pH 6.4 to 7.6, and more preferably pH 6.7 to 7.4.
• 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 1 L or more, more preferably 50 L or more, and even more preferably 200 L or more.
• the culture period is not particularly limited, but is generally from 12 hours to 90 days, preferably from 1 day to 80 days, more preferably from 1 day to less than 70 days, and even more preferably from 5 days to 65 days. It is particularly preferably 7 days or more and less than 60 days. In the present invention, the culture period is preferably 14 days or longer.
• 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. is 30 to 100kW/ m3 .
• the dissolved oxygen concentration in the culture solution can be set as appropriate and is not particularly limited, but generally it is 20 to 150% when the saturated dissolved oxygen concentration in a 37°C solution in 1 atm air is taken as 100%. , preferably 30 to 120%, more preferably 50 to 100%.
• 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 cell culture device may be a single-use culture tank from the viewpoint of homogenizing the culture environment.
• the viscosity of the culture solution is preferably 1.2 mPa ⁇ s or more and less than 15 mPa ⁇ s, more preferably 1.4 mPa ⁇ s or more and less than 12 mPa ⁇ s, particularly preferably 1.6 mPa ⁇ s or more and less than 10 mPa ⁇ s. It is.
• a pH adjuster may be added during culturing.
• the daily amount of the pH adjuster added to the culture tank is preferably 8 mmol/day/L or less, more preferably 7 mmol/day/L or less.
• the pH adjuster is not particularly limited, but is preferably an aqueous Na 2 CO 3 solution, an aqueous NaOH solution or an aqueous NaHCO 3 solution, more preferably an aqueous NaHCO 3 solution.
• the pH adjuster may be added alone or mixed with the medium or antifoaming agent. In order to avoid local pH fluctuations in the culture medium due to the addition of the pH adjuster, it is preferable to add the pH adjuster mixed with the culture medium.
• the method of culturing cells is perfusion culture.
• Perfusion culture is a culture method in which fresh medium is added and used medium is removed at the same time. With perfusion culture, it is generally possible to achieve high viable cell densities.
• a typical perfusion culture begins with a batch culture startup lasting one or two days, followed by continuous, stepwise, and/or intermittent addition of fresh feed medium to the culture and simultaneous removal of spent medium. do.
• methods such as sedimentation, centrifugation or filtration can be used to 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 number of days for perfusion culture is preferably 5 days or more and 150 days or less, more preferably 10 days or more and 100 days or less, and even more preferably 20 days or more and 90 days or less. It is preferable to culture for 5 days or more because the number of cells obtained increases, and when the cells produce a product, the amount of product produced increases. It is preferable to limit the number of days of culture to 150 days or less from the viewpoint of preventing clogging of the filtration membrane used in perfusion culture and preventing contamination.
• the perfusion ratio is not particularly limited, but is generally 0.3vvd to 5.0vvd, preferably 0.5vvd to 3.0vvd, and more preferably 0.8vvd to 2.5vvd.
• VVD in the perfusion ratio means volume of culture solution withdrawn/volume of culture solution in culture container/day.
• the perfusion ratio referred to here is the same as "(iv) When the capacitance of the culture solution exceeds the above target, the pump that withdraws the culture solution should be operated at a speed of 0.3vvd to 3vvd" in an example of bleeding. are different parameters.
• the culture solution can usually be removed from the culture container using a pump, but other available liquid feeding means may also be used.
• the culture solution extracted from the culture container is subjected to treatments such as recovery of products and removal of dead cells.
• the culture fluid extracted from the culture container may be partially discarded or returned to the culture container after processing such as collecting products and removing dead cells. If medium loss occurs due to the above treatment, it can be compensated for by, for example, supplying fresh medium to the culture container.
• 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 80% or more, more preferably 85% or more, particularly preferably 90% or more, and most preferably 95% or more.
• 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, DHFR gene, puromycin resistance gene, blasticidin resistance gene, hygromycin resistance gene, cycloheximide resistance gene), or fluorescent genes (encoding green fluorescent protein GFP, etc.). Genes that can be used for this purpose can be used.
• 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 produced by the method for producing a product according to the present invention.
• the type of product is not particularly limited, but 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.
• a vector containing a nucleic acid sequence encoding IgG1 was constructed, and the constructed vector was introduced into CHO-DG44 cells to produce CHO-DG44 cells (IgG1 cells) that express IgG1. 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.
• 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 1.5vvd. Thereafter, when the cell density reached 120 ⁇ 10 6 cells/mL, cell bleeding was performed while withdrawing a portion of the culture medium so that the cell density was maintained at 120 ⁇ 10 6 cells/mL.
• the culture conditions (medium, cell bleed conditions, etc.) used at this time were set as shown in Table 1, and Examples 1 to 6 and Comparative Examples 1 and 2 were carried out.
• the media A to C listed in Table 1 are commercially available media to which glutamine and glucose are added in the amounts listed in Table 1. Thereafter, the culture was continued for 10 days or more, and the evaluation described below was performed.
• 500 L culture put 500 L of culture medium into the culture tank Hypeforma 500 manufactured by Themo Fisher, set the stirring rotation speed to 150 rpm, and set the upper surface air to 4.75 L/min + CO 2 0.25 L/min.
• the culture was carried out under the same conditions as the 1.3L culture except that ATF10 manufactured by Repligen was used.
• ⁇ Automatic cell bleed control method When cell bleed was performed under automatic control, cell bleed was continuously performed automatically while monitoring the capacitance of the culture solution using a FUTURA sensor manufactured by ABER. Specifically, the following method was used. (1) Set the target capacitance. (2) Set the weight control value of the culture tank. (3) Measure the capacitance of the culture solution at a cycle of 0.1 seconds or less. (4) When the capacitance of the culture solution exceeds the target, operate the pump that draws out the culture solution at a speed of 0.3vvd to 3vvd. (5) The culture medium is automatically supplied into the culture tank while measuring the weight of the culture tank so that the volume of the culture solution is kept approximately constant. (6) When the capacitance of the culture solution falls below the target by 0.01% or more, stop the pump that draws out the culture solution.
• ⁇ Measurement and evaluation method> Measurement of viable cell density, average diameter of living cells, 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 density was 100 ⁇ 10 5 cells/mL or less, the sample was not diluted, and the measurement was performed at a dilution of 1. When the cell density was 100 ⁇ 10 5 cells/mL or more, the sample was diluted 10 times, and the measurement was performed at a dilution of 10.
• Mannose 5 of antibodies was evaluated as an evaluation standard for Examples and Comparative Examples.
• the N-type sugar chain profile containing mannose 5 was measured by the following method.
• the antibody in the obtained antibody recovery solution was digested with peptide-N-glycosidase F to cut out the N-type sugar chain. Thereafter, the excised sugar chain was fluorescently labeled with 2-aminopyridine, and mannose 5 was quantitatively analyzed by reverse-phase HPLC (high performance liquid chromatography) to calculate the peak of mannose 5 relative to the total peak area.
• the case where the mannose 5 was less than 4% was evaluated as A
• the case where it was between 4 and 7% was evaluated as B
• the case where it exceeded 7% was evaluated as C.

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• 2023
  • 2023-03-27 CN CN202380031332.3A patent/CN118974267A/zh active Pending
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