Method for producing product and product

Abstract

The present invention addresses the problem of providing: a method for producing a cell culture product, the method making it possible to reduce the amount of mannose 5 in the product; and a product produced by the method. The present invention provides a method for producing a product, the method comprising culturing cells in a culture vessel and being a perfusion culture method. In a product production period which comes after a target live cell density is achieved, formulas (1) to (5) are satisfied. The fluctuation in VCD during the production period is ±20% or less with respect to the average live cell density during the production period. The fluctuation in VCV during the production period is ±20% or less with respect to the average value of VCV during the production period. (1): 0 ≤ Gln-C ≤ 2.5; (2): 0.5 ≤ Gluc-C ≤ 8.0; (3): 1.0 ≤ NH4-C ≤ 8.0; (4): 50 ≤ VCD ≤ 300; (5): 0.6×105 ≤ VCV ≤ 4.0×105. The meanings of symbols used in formulas (1) to (5) are as defined in the description of the present application.

Description

Production method and product

[0001] The present invention relates to a method for producing a product, which comprises culturing cells under predetermined conditions during the production period of the product after a target viable cell density is reached.The present invention also relates to a product produced by the above-mentioned production method.

[0002] Cell culture is carried out for the purpose of increasing cells having useful properties, causing cells to produce products, etc. For example, Patent Document 1 describes a method for producing a bispecific antibody product, comprising the steps of (i) providing at least one mammalian cell capable of expressing a bispecific antibody product in a perfusion bioreactor, (ii) growing the mammalian cell culture at a first perfusion rate until a set viable cell density is reached, and (iii) maintaining the perfusion culture at a second perfusion rate, wherein the concentration of the bispecific antibody product in the bioreactor is maintained below a threshold value.

[0003] Patent Document 2 describes an integrated, continuous method for producing a therapeutic protein drug substance, the method including: (i) supplying a liquid culture medium containing a substantially cell-free recombinant therapeutic protein such that the liquid culture medium is fed into a first multi-column chromatography system (MCCS1); (ii) capturing the recombinant therapeutic protein in the liquid culture medium using MCCS1, and continuously feeding the MCCS1 eluate containing the recombinant therapeutic protein into a second multi-column chromatography system (MCCS2); and (iii) purifying and polishing the recombinant therapeutic protein using MCCS2 such that the eluate emerging from MCCS2 is the therapeutic protein drug substance.

[0004] US Patent No. 5,999,949 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) dynamically adjusting a target perfusion rate to take into account the viable cell density, wherein the cells grow to a high cell density during perfusion.

[0005] JP 2021-505182 A JP 2020-033364 A JP 2020-533983 A

[0006] Among antibodies produced by cell culture, antibodies whose sugar chain moieties are glycosylated with five mannoses (mannose 5-modified antibodies) have insufficient pharmacological efficacy, and therefore, from the viewpoint of enhancing the pharmacological efficacy of pharmaceuticals, it is necessary to reduce the amount of mannose 5. On the other hand, it is difficult to separate and remove mannose 5-modified antibodies by a purification step, and it is desirable to suppress the production of mannose 5-modified antibodies from cells in the culture step. An object of the present invention is to provide a method for producing a product by cell culture, which can reduce the amount of mannose 5-modified antibodies in the product. Another object of the present invention is to provide a product produced by the above method.

[0007] As a result of extensive research into solving the above problems, the inventors discovered that the above problems can be solved by culturing cells so as to satisfy specific conditions during the product production period after the target viable cell density is reached, and thus completed the present invention.

[0008] That is, the present invention provides the following inventions. <1> A method for producing a product, which includes culturing cells in a culture vessel, wherein the culturing is a perfusion culture method, and during a product production period after a target viable cell density is reached, formulas (1) to (5) are satisfied, and the fluctuation in VCD during the production period is ±20% or less relative to the average viable cell density during the production period, and the fluctuation in VCV during the production period is ±20% or less relative to the average VCV during the production period. 0≦Gln-C≦2.5 Formula (1) 0.5≦Gluc-C≦8.0 Formula (2) 1.0≦NH4-C≦8.0 Formula (3) 50≦VCD≦300 Formula (4) 0.6×10 ５ ≦VCV≦4.0×10 ５Formula (5) In the formula, Gln-C represents the glutamine concentration in the culture medium [mmol / L], Gluc-C represents the glucose concentration in the culture medium [g / L], NH4-C represents the ammonia concentration in the culture medium [mmol / L], and VCD represents the viable cell density [×10 ６ cells / mL], and VCV is VCD × π / 6 × Cd ３ [x10 ６ μm ３ / mL], and Cd represents the average diameter of viable cells [μm]. <2> The method for producing a product according to <1>, in which the culture medium is bled so that the fluctuation in VCD during the production period is ±20% or less of the average viable cell density during the production period. <3> The method for producing a product according to <2>, in which the culture medium is bled while measuring the capacitance of the culture medium in-line during the breeding. <4> The method for producing a product according to <3>, in which the bleeding is automatically controlled. <5> The method for producing a product according to <3> or <4>, in which the fluctuation in the capacitance of the culture medium measured in-line is ±20% or less of the average value of the capacitance during the production period. <6> The method for producing a product according to any one of <3> to <5>, wherein the bleeding includes: (i) setting a target capacitance; (ii) setting a weight control value for the culture vessel; (iii) measuring the capacitance of the culture solution at a period of 0.1 second or less; (iv) when the capacitance of the culture solution exceeds the target, operating a pump that extracts the culture solution at a speed of 0.3 vvd to 3 vvd (where vvd means volume of culture solution extracted / volume of culture solution / day); (v) automatically supplying medium to the culture vessel while measuring the mass of the culture solution so that the fluctuation in the liquid volume of the culture solution is within 10%; and (vi) stopping the pump that extracts the culture solution when the capacitance of the culture solution falls below the target by 0.01% or more. <7> The method for producing a product according to any one of <1> to <6>, wherein formula (6) is satisfied during the production period. 2≦LDH / VCD≦40 Equation (6) In the equation, LDH represents the concentration of lactate dehydrogenase in the culture medium [U / L], and VCD represents the viable cell density [×10 ６<8> The method for producing a 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 a protein. <10> The method for producing a product according to any one of <1> to <9>, wherein the cells are cells that produce 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>.

[0009] According to the present invention, in a method for producing a product by cell culture, the amount of mannose 5 in the product can be reduced.

[0010] Fig. 1 shows a cell culture device. Fig. 2 shows the results of measuring the transition of VCV in Example 1. Fig. 3 shows the results of measuring the transition of capacitance data (also referred to as Cp) in Example 1. Fig. 4 shows the results of measuring the transition of mannose 5 ratio in Example 1.

[0011] The present invention will be described in detail below. In this specification, a numerical range indicated using "to" means a range that includes the numerical values ​​before and after "to" as the minimum and maximum values, respectively.

[0012] The method for producing a product of the present invention includes culturing cells in a culture vessel, the culture being a perfusion culture method, and during the production period of the product after a target viable cell density is reached, the formulas (1) to (5) are satisfied, with the VCD fluctuating within the production period being ±20% or less relative to the average viable cell density during the production period, and the VCV fluctuating within the production period being ±20% or less relative to the average VCV during the production period. By satisfying the above conditions, the method for producing a product of the present invention can reduce the amount of mannose-5-modified antibody in the product. 0≦Gln-C≦2.5 Formula (1) 0.5≦Gluc-C≦8.0 Formula (2) 1.0≦NH4-C≦8.0 Formula (3) 50≦VCD≦300 Formula (4) 0.6×10 ５ ≦VCV≦4.0×10 ５Formula (5) In the formula, Gln-C represents the glutamine concentration in the culture medium [mmol / L], Gluc-C represents the glucose concentration in the culture medium [g / L], NH4-C represents the ammonia concentration in the culture medium [mmol / L], and VCD represents the viable cell density [×10 ６ cells / mL], and VCV is VCD × π / 6 × Cd ３ [x10 ６ μm ３ / mL], and Cd indicates the mean diameter of viable cells [μm].

[0013] Regarding formula (1), preferred embodiments are the following formulas (1A) and (1B): 0.1≦Gln-C≦2.0 formula (1A) 0.2≦Gln-C≦1.5 formula (1B)

[0014] Regarding formula (2), preferred embodiments are the following formulas (2A) and (2B): 1.0≦Gluc-C≦6.0 (formula (2A)) 1.5≦Gluc-C≦5.0 (formula (2B))

[0015] Regarding formula (3), preferred embodiments are the following formulas (3A) and (3B): 1.5≦NH4-C≦7.0 formula (3A) 2.0≦NH4-C≦6.0 formula (3B)

[0016] With respect to formula (4), preferred embodiments are the following formulas (4A) and (4B): 80≦VCD≦250 formula (4A) 100≦VCD≦220 formula (4B)

[0017] Regarding formula (5), preferred embodiments are the following formulas (5A) and (5B): ５ ≦VCV≦3.0×10 ５ Formula (5A) 1.0×10 ５ ≦VCV≦2.5×10 ５ Formula (5B)

[0018] The VCD fluctuates within the production period within ±20% of the average viable cell density within the production period, more preferably within ±15% of the average viable cell density within the production period, even more preferably within ±12% of the average viable cell density within the production period, and particularly preferably within ±8% of the average viable cell density within the production period. There is no particular lower limit for the VCD fluctuation within the production period, and it is sufficient if it is 0% or more, but considering daily fluctuations in cell growth, fluctuations of 0.1% or more will occur.

[0019] The fluctuation of VCV during the production period is ±20% or less from the average value of VCV during the production period, more preferably ±15% or less from the average value of VCV during the production period, even more preferably ±12% or less from the average value of VCV during the production period, and particularly preferably ±8% or less from the average value of VCV during the production period. There is no particular lower limit for the fluctuation of VCV during the production period, and it is sufficient as long as it is 0% or more, but considering daily fluctuations in cell proliferation, fluctuations of 0.1% or more will actually occur.

[0020] The glutamine concentration [mmol / L] in the culture medium, represented by Gln-C, can be measured using the filtrate obtained by removing the culture medium from the culture vessel during cultivation and removing the cells by filtration. The glutamine concentration in the filtrate can be measured using a commercially available analyzer such as Nova Biomedical's Bioprofile FLEX2. The frequency of measuring the glutamine concentration is not particularly limited, and can be, for example, once a day.

[0021] The glucose concentration [g / L] in the culture medium, indicated by Gluc-C, can be measured using the filtrate obtained by removing the culture medium from the culture vessel during cultivation and removing the cells by filtration. The glucose concentration in the filtrate can be measured using a commercially available analyzer such as Nova Biomedical's Bioprofile FLEX2. The frequency of measuring the glucose concentration is not particularly limited, and can be, for example, once a day.

[0022] The ammonia concentration [mmol / L] in the culture medium, represented by NH4-C, can be measured using the filtrate obtained by removing the culture medium from the culture vessel during cultivation and removing the cells by filtration. The ammonia concentration in the filtrate can be measured using a commercially available analyzer such as Nova Biomedical's Bioprofile FLEX2. The frequency of measurement of the ammonia concentration is not particularly limited, and can be measured, for example, once a day.

[0023] The viable cell density indicated by VCD [×10 ６ The VCV can be measured by extracting the culture medium from the culture vessel during the culture and measuring it by a conventional method. ３ [x10 ６ μm ３ / mL], where Cd represents the mean diameter of viable cells [μm].

[0024] Viable cell density and viable cell mean diameter can be measured using a commercially available measuring device such as Beckman Coulter's Cell Viability Analyzer Vi-cell XR. For example, the Vi-cell software Vi-cell XR2.04 can be used, and the measurement parameters can be set as follows: Min diameter 6 μm, Max diameter 50 μm. Cell brightness: 85% Cell sharpness: 100 Viable cell spot brightness: 75% Viable cell spot area: 5% Minimum circularity:0 Decluster degree:Medium

[0025] The sample may be used without dilution or after dilution. The frequency of measuring the viable cell density and the average diameter of the viable cells is not particularly limited, but may be, for example, once a day.

[0026] The fluctuation of VCD during the production period is a rate of fluctuation (percentage) that indicates how much the VCD value measured during the production period fluctuated relative to the average VCD value during the production period, i.e., the average viable cell density. The average VCD value during the production period can be determined by sampling the culture medium from the culture tank every day during the production period, measuring the VCD value using a Beckman Coulter Cell Viability Analyzer Vi-cell XR or the like, adding up the VCD values ​​for the number of culture days during the production period, and dividing the result by the number of culture days during the production period.

[0027] The fluctuation of VCV during the production period is the rate of fluctuation (percentage) that indicates how much the VCV value measured during the production period fluctuated relative to the average VCV value during the production period. The average VCV value during the production period was calculated by extracting the culture medium from the culture tank every day during the production period and measuring the VCD and average cell diameter Cd using a Beckman Coulter Cell Viability Analyzer Vi-cell XR or the like, and then calculating VCV = VCD x (π / 6 x Cd ３ ) by the formula VCV [× 10 ６ μm ３ / ml], add the VCV value to the number of days of culture during the production period, and divide the result by the number of days of culture during the production period.

[0028] In the present invention, the culture medium can be bled so that the VCD fluctuation during the production period is preferably within ±20% of the average viable cell density during the production period. Bleeding refers to removing a portion of the culture medium along with the cells to reduce the viable cell density so that the viable cell density during culture does not become excessive. To maintain the culture volume, fresh medium can be added in an amount equal to the amount of the removed culture medium.

[0029] More preferably, the VCD fluctuates within the production period within ±15% of the average viable cell density during the production period, even more preferably within ±12% of the average viable cell density during the production period, and particularly preferably within ±8% of the average viable cell density during the production period. There is no particular lower limit for the VCD fluctuation during the production period, and it is sufficient if it is 0% or more, but considering daily fluctuations in cell growth, fluctuations of 0.1% or more will occur.

[0030] Bleeding can be performed by any method as long as the culture solution containing the cells can be removed from the culture vessel. For example, as shown in Figure 1, a pipe for discharging the culture solution can be inserted into the culture solution, and the culture solution can be discharged from the culture vessel through the pipe.

[0031] Preferably, the culture medium may be bled while measuring the capacitance of the culture medium in-line. In-line measurement refers to measuring some measurement value that indicates the state of the culture medium while cell culture is being performed. To measure the capacitance in-line, for example, as shown in FIG. 1, a capacitance sensor may be inserted into the culture medium, and the capacitance of the culture medium may be measured in-line during culture.

[0032] Preferably, the bleeding may be automatically controlled. Automatic control means that the start and stop of bleeding are automatically controlled based on some measured value indicating the state of the culture solution. For example, the start and stop of bleeding can be automatically controlled based on a measured value of the capacitance of the culture solution.

[0033] Preferably, bleeding may be performed so that the variation in the capacitance of the culture medium measured inline is ±20% or less of the average capacitance during the production period. The variation in the capacitance of the culture medium measured inline is more preferably ±15% or less of the average capacitance during the production period, even more preferably ±12% or less of the average capacitance during the production period, and particularly preferably ±8% or less of the average capacitance during the production period. The lower limit of the variation in the capacitance of the culture medium is not particularly limited, and it is sufficient if it is 0% or more, but considering daily fluctuations in cell growth, variation of 0.1% or more will actually occur.

[0034] An example of bleeding can be performed by the steps of: (i) setting a target capacitance; (ii) setting a weight control value for the culture vessel; (iii) measuring the capacitance of the culture solution at a period of 0.1 seconds or less; (iv) when the capacitance of the culture solution exceeds the target, operating a pump that extracts the culture solution at a speed of 0.3 vvd to 3 vvd (where vvd means volume of culture solution extracted / volume of culture solution / day); (v) automatically supplying medium to the culture vessel while measuring the mass of the culture solution so that the fluctuation in the liquid volume of the culture solution is within 10%; and (vi) stopping the pump that extracts the culture solution when the capacitance of the culture solution falls below the target by 0.01% or more.

[0035] In the present invention, preferably, during the production period, the following formula (6) is satisfied: 2≦LDH / VCD≦40 formula (6), where LDH represents the concentration of lactate dehydrogenase in the culture medium [U / L], and VCD represents the viable cell density [×10 ６ cells / mL] are shown.

[0036] 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))

[0037] The lactate dehydrogenase concentration [U / L] in the culture medium, expressed as LDH, can be measured using the filtrate obtained by removing the culture medium from the culture vessel during cultivation and removing the cells by filtration. The lactate dehydrogenase concentration in the filtrate can be measured using a commercially available analyzer such as Roche's Cedex Bio. The frequency of measurement of the lactate dehydrogenase concentration is not particularly limited, and can be, for example, once a day.

[0038] An example of a cell culture device that can be used for culturing cells in the present invention is shown in Figure 1. In Figure 1, a culture vessel 10 is a vessel that contains a culture medium containing cells. Cells are cultured in the culture medium inside the culture vessel 10.

[0039] Oxygen and air are sent from the sparger air supply pipe 1 and introduced into the culture solution through a sparger 11 having a pore size of 20 μm. The dissolved oxygen concentration in the culture solution can be adjusted by the sparger 11. The sparger is not particularly limited, but for example, a sparger having an average pore size of 1 μm or more and 300 μm or less in the gas release section and releasing a gas containing 30% by volume or more of oxygen can be used.

[0040] Air and carbon dioxide are introduced from the air supply pipe 2 into the upper part of the culture solution inside the culture vessel. Culture medium is supplied to the culture vessel from the culture medium supply pipe 3. A culture medium supply pump 15 is provided on the culture medium supply pipe 3. The exhaust pipe 4 is a pipe for exhausting air, and an exhaust filter 5 is connected to its end. The sampling pipe 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 attached so as to come into contact with the culture solution in the culture vessel. A dissolved oxygen sensor 9 is attached so as to come into contact with the culture solution in the culture vessel.

[0041] A stirring member having a stirring blade 12 may be provided inside the culture vessel 10. By rotating the stirring blade 12, the culture solution inside the culture vessel 10 is stirred, maintaining the homogeneity of the culture solution. When the culture solution is stirred by the stirring blade 12, 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 may be designed depending on the cell type used, the amount of culture solution, the amount of oxygen supplied, the position, number, size, etc. of the sparger. Furthermore, in order to quickly stir bubbles coming out of the sparger and prevent the bubbles from coalescing, it is preferable to position the stirring blade 12 close to the sparger.

[0042] A perfusion device 13 is connected to the bottom of the culture vessel 10. The culture solution inside the culture vessel 10 passes through the perfusion device 13, and the permeate containing the cell product is removed from the culture vessel 10 by a delivery pump 16. The flow of the permeate is indicated by arrows 14.

[0043] In the present invention, the viable cell density of the culture medium is 50 × 10 ６ ~300 x 10 ６ cells / mL, preferably 80 x 10 ６ ~250 x 10 ６ More preferably, the concentration is 100×10 ６ ~220 x 10 ６ More preferably, the concentration is 10 cells / mL. ６ It is sometimes written as M instead of .

[0044] In the present invention, the cell suspension removed from the culture vessel may be passed through a separation membrane to separate it into a cell-containing solution and a permeate. This operation can be performed using a perfusion device. In this operation, the cell suspension removed from the culture vessel is separated into a cell-containing solution having a higher cell density than the cell suspension and a permeate having a lower cell density than the cell suspension. The cell density can be measured using a Vi-CELL XR viable cell analyzer manufactured by Beckman Coulter.

[0045] The membrane separation process is preferably tangential filtration, more preferably alternating tangential flow (ATF) or tangential flow, and most preferably alternating tangential flow. Filters capable of performing alternating tangential flow include SuATF10-S02PES and F2 RF02PES manufactured by Repligen.

[0046] As a medium for cell culture, a medium that is commonly used for culturing animal cells can be used. For example, CD OptiCHO (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, Iscove's Modified Dulbecco's Medium (IMDM), McCoy's 5A medium, Leibovitz's L-15 medium, and 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), etc. may be used. Alternatively, a self-made medium may be used.

[0047] The medium may be supplemented with serum, such as fetal calf serum (FCS), or serum-free. The medium may be supplemented with additional components, such as amino acids, salts, sugars, vitamins, hormones, growth factors, buffers, antibiotics, lipids, trace elements, plant protein hydrolysates, etc. Protein-free media may also be used.

[0048] 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, and more preferably 36°C to 38°C, and the culture temperature may be changed during culture. The culture is carried out under CO２ The process can be carried out in an atmosphere having a concentration of 0 to 40% by volume, preferably 2 to 25% by volume, and more preferably 3 to 20% by volume.

[0049] The amount of culture medium is preferably 1 L or more, more preferably 50 L or more, and even more preferably 200 L or more.

[0050] The culture period is not particularly limited, but is generally 12 hours to 90 days, preferably 1 day to 80 days, more preferably 1 day or more but less than 70 days, even more preferably 5 days or more but less than 65 days, and particularly preferably 7 days or more but less than 60 days. In the present invention, the culture period is preferably 14 days or more.

[0051] During the culture, the medium can be replaced, aerated, or stirred as necessary. When stirring is performed during the culture, the stirring speed is not particularly limited, but the stirring power per unit volume is generally 10 to 300 kW / m ３ and preferably 20 to 200 kW / m ３ and more preferably 30 to 100 kW / m ３ is.

[0052] The dissolved oxygen concentration in the culture solution can be set appropriately and is not particularly limited, but is generally 20 to 150%, preferably 30 to 120%, and more preferably 50 to 100%, when the saturated dissolved oxygen concentration in air at 1 atmosphere at 37°C is taken as 100%.

[0053] Cell culture can be carried out using a cell culture device having the configuration described above in this specification. The cell culture device may be any of a fermenter-type tank culture device, an airlift-type culture device, a culture flask-type culture device, a spinner flask-type culture device, a microcarrier-type culture device, a fluidized bed-type culture device, a hollow fiber-type culture device, a roller bottle-type culture device, or a packed tank-type culture device. From the viewpoint of homogenizing the culture environment, the culture vessel is preferably a single-use culture tank. From the viewpoint of homogenizing the culture environment, the cell culture device may also be a single-use culture tank.

[0054] The viscosity of the culture medium 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, and particularly preferably 1.6 mPa·s or more and less than 10 mPa·s.

[0055] In the present invention, a pH adjuster may be added during the culture. In view of clogging of the filtration channel, the amount of pH adjuster added to the culture tank per day is preferably 8 mmol / day / L or less, more preferably 7 mmol / day / L or less. However, it is not necessary to add a pH adjuster. The pH adjuster is not particularly limited, but examples thereof include Na ２ CO ３ aqueous solution, aqueous NaOH or NaHCO ３ An aqueous solution is preferred, more preferably NaHCO ３ The pH adjuster may be added alone or mixed with the medium or antifoaming agent. To avoid local pH fluctuations in the culture solution due to the addition of the pH adjuster, it is preferable to add the pH adjuster to the medium.

[0056] In the present invention, the cell culture method is perfusion culture. Perfusion culture is a culture method in which fresh medium is added and spent medium is simultaneously removed. Perfusion culture generally allows for a high viable cell density to be achieved. A typical perfusion culture begins with a batch culture start-up 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. In perfusion culture, spent medium can be removed while maintaining viable cell density using methods such as sedimentation, centrifugation, or filtration. An advantage of perfusion culture is that the culture in which the target protein is produced can be maintained for a longer period of time than batch or fed-batch culture.

[0057] Perfusion can be continuous, stepwise, intermittent, or a combination thereof, preferably continuous. The animal cells are maintained in culture, and the removed spent medium may be substantially cell-free or contain far fewer cells than the culture. The product expressed by the cell culture can be maintained in culture or recovered by selecting the membrane pore size.

[0058] The number of days for perfusion culture is preferably 5 to 150 days, more preferably 10 to 100 days, and even more preferably 20 to 90 days. A culture period of 5 days or more is preferred because it increases the number of cells obtained and, if the cells produce a product, increases the amount of product produced. A culture period of 150 days or less is preferred from the viewpoint of preventing clogging of the filtration membrane used in perfusion culture and preventing contamination.

[0059] The perfusion ratio is not particularly limited, but is generally 0.3 vvd to 5.0 vvd, preferably 0.5 vvd to 3.0 vvd, and more preferably 0.8 vvd to 2.5 vvd. The "vvd" in the perfusion ratio refers to the volume of culture medium extracted / volume of culture medium in the culture vessel / day. Note that the perfusion ratio referred to here is a different parameter from the example of bleeding, "(iv) When the capacitance of the culture medium exceeds the target, operate the pump extracting the culture medium at a speed of 0.3 vvd to 3 vvd."

[0060] The culture medium is usually removed from the culture vessel using a pump, but other available liquid-transporting means may also be used. The culture medium removed from the culture vessel is then subjected to treatments such as product recovery and dead cell removal. After product recovery and dead cell removal, the culture medium removed from the culture vessel may be partially discarded or returned to the culture vessel. If a loss of medium occurs due to the above treatments, it can be compensated for by, for example, supplying fresh medium to the culture vessel.

[0061] The type of cell used in the present invention is not particularly limited, and examples 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, and most preferably mammalian cells. The cells may be primary cells or established cell lines.

[0062] Examples of cells include Chinese hamster ovary (CHO) cells, HEK cells (human embryonic kidney-derived cells), BHK cells, 293 cells, C127 cells, myeloma cells (such as NS0 cells), PerC6 cells, SP2 / 0 cells, hybridoma cells, 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 cells), PC12 cells, and WI38 cells. The cells may also be stem cells such as embryonic stem cells (ES cells) or induced pluripotent stem cells (iPS cells). Among these, CHO cells, HEK cells, BHK cells, and hybridoma cells 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, coagulation factors, and antibodies. It is preferable to use dihydrofolate reductase (DHFR)-deficient CHO cells, and for example, CHO-DG44 can be used as the DHFR-deficient CHO cells.

[0063] The higher the cell viability, the better, with 80% or more being preferred, 85% or more being more preferred, 90% or more being particularly preferred, and 95% or more being most preferred.

[0064] These cells may be cells into which a foreign gene encoding a protein to be expressed (e.g., an antibody) has been introduced. Preferably, the cells are antibody-producing cells. An expression vector can be used to introduce a foreign gene encoding a protein to be expressed into a cell. By introducing an expression vector containing DNA encoding the protein to be expressed, an expression regulatory sequence (e.g., an enhancer, a promoter, a terminator, etc.), and optionally a selection marker gene into a cell, a cell into which a foreign gene encoding the protein to be expressed has been introduced can be produced. There are no particular limitations on the expression vector, and it can be selected and used appropriately depending on the type of cell, purpose, etc.

[0065] Any promoter can be used as long as it is functional in mammalian cells. Examples include the promoter of the cytomegalovirus (CMV) IE (immediate early) gene, the SV40 early promoter, retrovirus promoters, metallothionein promoters, heat shock promoters, SRα promoters, and promoters and enhancers of Moloney murine leukemia virus. Furthermore, an enhancer of the human CMV IE gene may also be used together with the promoter.

[0066] Examples of selectable marker genes that can be used include drug resistance genes (neomycin resistance gene, DHFR gene, puromycin resistance gene, blasticidin resistance gene, hygromycin resistance gene, cycloheximide resistance gene), and fluorescent genes (genes encoding green fluorescent protein (GFP) or the like).

[0067] The method for introducing an expression vector into cells is not particularly limited, and for example, the calcium phosphate method, electroporation, liposome method, gene gun method, lipofection method, etc. can be used.

[0068] The method for producing a product according to the present invention comprises culturing cells using a cell culture device and producing a product from the cells. The present invention also provides a product produced by the method for producing a product according to the present invention.

[0069] In the present invention, the type of product is not particularly limited, but is preferably a recombinant protein. Examples of the product include 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, fragmented immune immunoglobulins, single-chain antibodies (scFv), etc. In addition, adenoviruses, adeno-associated viruses, lentiviruses, etc. may also be used.

[0070] The product is preferably an antibody, more preferably a human antibody, a humanized antibody, a chimeric antibody, or a mouse antibody. Examples of fragmented immune immunoglobulins include Fab, F(ab')2, and Fv. The class of the antibody is not particularly limited, and may be any class, such as IgG (e.g., IgG1, IgG2, IgG3, and IgG4), IgA, IgD, IgE, or IgM, although IgG and IgM are preferred for pharmaceutical use.

[0071] Human antibodies include all antibodies that have one or more variable and constant regions derived from human immunoglobulin sequences, hi one embodiment, all of the variable and constant domains are derived from human immunoglobulin sequences (fully human antibodies).

[0072] A humanized antibody 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 humanized antibody is less likely to provoke an immune response and / or provokes a less severe immune response when administered to a human subject compared to the non-human species antibody. In one example, certain amino acids within the framework and constant domains of the heavy and / or light chains of the non-human species antibody are mutated to produce a humanized antibody. In another example, constant domains from a human antibody are fused to variable domains of the non-human species.

[0073] A chimeric antibody is an antibody in which variable and constant regions of different origins are linked. For example, an antibody consisting of the heavy 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 heterochimeric antibody. A recombinant vector that expresses a chimeric antibody can be produced by linking DNA encoding the variable region of a mouse antibody to DNA encoding the constant region of a human antibody and incorporating this into an expression vector. Recombinant cells transformed with the above vector are cultured to express the incorporated DNA, thereby obtaining the chimeric antibody produced during the culture.

[0074] Bispecific antibodies are antibodies that recognize two different antigen specificities. Various forms of bispecific antibodies exist. Reported methods for producing bispecific antibodies include linking two immunoglobulin molecules using crosslinkers such as N-succinimidyl 3-(2-pyridyldithiol) propionate or S-acetylmercaptosuccinic acid anhydride, or linking Fab fragments of immunoglobulin molecules. Bispecific antibodies can also be expressed by introducing genes encoding them into cells.

[0075] An Fc fusion protein refers to a protein having an Fc region, including antibodies. Fab is a monovalent fragment having the VL, VH, CL, and CH1 domains. F(ab')2 is a bivalent fragment having two Fab fragments linked by a disulfide bridge at the hinge region. An Fv fragment has the VL and VH domains of a single antibody arm. A single-chain antibody (scFv) is an antibody in which the VL and VH domains 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 allow the protein chain to fold back on itself and form a monovalent antigen-binding site.

[0076] The antibody is not particularly limited, but examples thereof include an anti-IL-6 receptor antibody, an anti-IL-6 antibody, an anti-glypican-3 antibody, an anti-CD3 antibody, an anti-CD20 antibody, an anti-GPIIb / IIIa antibody, an anti-TNF antibody, an anti-CD25 antibody, an anti-EGFR antibody, an anti-Her2 / neu antibody, an anti-RSV antibody, an anti-CD33 antibody, an anti-CD52 antibody, an anti-IgE antibody, an anti-CD11a antibody, an anti-VEGF antibody, and an anti-VLA4 antibody.

[0077] The product may be recovered by simply recovering the culture medium, or by recovering the liquid from which at least a portion of the cells have been removed using, for example, a filter or a centrifuge, and any known method can be used without particular limitation. If it is desired to improve the purity of the product, change the solvent, or change the form, for example, to make it into a powder, the culture medium or the liquid can be subjected to further treatment.

[0078] It is also possible to collect a portion of the culture medium during perfusion, or to use a filter or centrifuge on a portion of the culture medium during perfusion, and collect the liquid from which at least a portion of the cells have been removed.

[0079] The product can be purified by a purification treatment. The obtained product can be purified to a high purity. Separation and purification of the product can be performed using separation and purification methods commonly used for proteins. For example, the product can be separated and purified 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., but is not limited to these. The concentration of the product obtained above can be measured by absorbance measurement or enzyme-linked immunosorbent assay (ELISA), etc. Furthermore, when the product is an antibody, the antibody titer can also be measured using a commercially available analytical instrument such as Roche's Cedex Bio.

[0080] Examples of columns used in affinity chromatography include protein A columns and protein G columns. Chromatography other than affinity chromatography includes, for example, ion exchange chromatography, hydrophobic chromatography, gel filtration, reverse phase chromatography, and adsorption chromatography. These chromatographies can be performed using liquid phase chromatography such as HPLC (high performance liquid chromatography) or FPLC (fast protein liquid chromatography).

[0081] The product can be modified or partially depeptidized by treating it with an appropriate polypeptide-modifying enzyme before or after purification, such as trypsin, chymotrypsin, lysyl endopeptidase, protein kinase, or glucosidase.

[0082] The products produced by the present invention can be used in, for example, biopharmaceuticals and regenerative medicine.

[0083] The present invention will be explained in more detail with reference to the following examples, but the present invention is not limited to these examples.

[0084] <Establishment of antibody-producing cells> A vector containing a nucleic acid sequence encoding IgG1 was constructed, and the constructed vector was introduced into CHO-DG44 cells to generate CHO-DG44 cells expressing IgG1 (IgG1 cells). The vector was constructed and introduced into the cells in accordance with Example 2 of JP-A-2016-517691. As described above, CHO cells producing monoclonal antibodies were prepared and used in the following experiments.

[0085] <Cell Culture> In the examples and comparative examples, experiments were carried out using the cell culture device shown in Fig. 1. 1.3 L of medium was placed in a 3 L culture vessel (Biostat (registered trademark) manufactured by Sartorius). 0.5 x 10 ６CHO cells were seeded at 100 cells / mL. The agitation speed was 160 rpm, and air and CO were introduced from above at 38 mL / min. ２ The oxygen concentration in the culture solution was adjusted to 80% by adding O 2 from a sparger installed in the culture tank. ２ was automatically controlled and supplied.

[0086] After culturing for two days after seeding, the cell culture medium was continuously supplied at a perfusion ratio of 0.8 vvd, and the cell culture medium was continuously filtered using ATF2 (manufactured by Repligen) to collect the collected solution. On the fifth day, the perfusion ratio was changed to 1.5 vvd. After that, when the cell density reached 120 × 10 ６ When the cell density reaches 120 x 10 cells / mL, ６ Cell bleeding was performed by withdrawing a portion of the culture medium so that the cell density was maintained at 1000 cells / mL. The culture conditions (culture medium, cell bleeding 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. Note that media A to C shown in Table 1 are media prepared by adding glutamine and glucose to commercially available media in the amounts shown in Table 1. Thereafter, the culture was continued for 10 days or more, and the evaluation described below was carried out.

[0087] For 50 L cultivation, 50 L of medium was added to the Thermo Fisher Hypeforma 50 fermenter, the agitation speed was set to 250 rpm, and the upper air flow rate was set to 0.475 L / min + CO ２ The culture was carried out under the same conditions as the 1.3 L culture, except that the flow rate was 0.025 L / min and ATF4 manufactured by Repligen was used.

[0088] For 500L cultivation, 500L of medium was added to the Thermo Fisher Hypeforma 500 fermenter, the agitation speed was set to 150 rpm, and the upper air flow rate was set to 4.75L / min + CO ２ The culture was carried out under the same conditions as the 1.3 L culture, except that the flow rate was 0.25 L / min and ATF10 manufactured by Repligen was used.

[0089] <Method for Automatic Cell Bleed Control> When cell bleeding was performed automatically, a FUTURA sensor manufactured by ABER was used to monitor the capacitance of the culture solution while automatically and continuously performing cell bleeding. Specifically, this was performed using the following method. (1) A target capacitance was set. (2) A weight control value for the culture tank was set. (3) The capacitance of the culture solution was measured at intervals of 0.1 s or less. (4) When the capacitance of the culture solution exceeded the target, the pump that extracts the culture solution was operated at a speed of 0.3 vvd to 3 vvd. (5) The culture medium was automatically supplied into the culture tank while measuring the weight of the culture tank so that the volume of the culture solution remained roughly constant. (6) When the capacitance of the culture solution fell 0.01% or more below the target, the pump that extracts the culture solution was stopped.

[0090] <Measurement and evaluation methods> (1) Measurement of viable cell density, average diameter and viability of viable cells The culture medium in the culture vessel was extracted and measured using a Beckman Coulter Cell Viability Analyzer Vi-cell XR. The Vi-cell software used was Vi-cell XR2.04, and the measurement parameters were set as follows: Min diameter: 6 μm Max diameter: 50 μm Dilution: cell density 100 × 10 ５ When the cell density was 100 x 10 cells / mL or less, the sample was not diluted and the measurement was performed at a dilution of 1. ５ For concentrations of 0.1 to 1.5 cells / mL, the sample was diluted 10 times and measured at a dilution of 10. Cell brightness: 85% Cell sharpness: 100 Viable cell spot brightness: 75% Viable cell spot area: 5% Minimum circularity: 0 Decluster degree: Medium

[0091] Measurement was carried out once a day using the above method.

[0092] (2) Gln, Gluc, NH ４Measurement of Concentration The culture medium was removed, and the cells were filtered through Whatman (Pore Size 0.2 μm, 25 mm Diameter) manufactured by Cytiva, and the filtrate was then measured using Bioprofile FLEX2 manufactured by Nova Biomedical Co. Measurement was carried out once a day using the method described above.

[0093] (3) Measurement of LDH The culture medium was removed, and the cells were filtered through Whatman (Pore Size 0.2 μm, 25 mm Diameter) manufactured by Cytiva, and the filtrate was measured using Cedex Bio manufactured by Roche. Measurement was performed once a day using the method described above.

[0094] (4) Titer Measurement The culture medium was removed, and the cells were filtered through Whatman (Pore Size 0.2 μm, 25 mm Diameter) manufactured by Cytiva, and the filtrate was measured using Cedex Bio manufactured by Roche. The titer was measured once a day using the above method.

[0095] (5) Measurement of Mannose 5 (M5) Mannose 5 of the antibody was evaluated as the evaluation standard for the Examples and Comparative Examples. The N-glycan 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 excise the N-glycans. The excise sugar chains were then fluorescently labeled with 2-aminopyridine, and mannose 5 was quantitatively analyzed by reverse-phase HPLC (high performance liquid chromatography), and the mannose 5 peak relative to the total peak area was calculated. A case in which mannose 5 was less than 4% was rated A, 4 to 7% was rated B, and more than 7% was rated C.

[0096] The results of measuring the VCV, capacitance data, and changes in the mannose 5 ratio over time in Example 1 are shown in Figures 2 to 4. The measurement results for each Example and Comparative Example are shown in Tables 2 and 3.

[0097]

[0098]

[0099]

[0100] 1 Sparger air supply pipe 2 Air supply pipe 3 Culture medium supply pipe 4 Exhaust pipe 5 Exhaust filter 6 Sampling pipe 7 Bleeding pipe 8 Capacitance sensor 9 Dissolved oxygen sensor 10 Cultivation vessel 11 Sparger 12 Stirring blade 13 Perfusion device 14 Arrow 15 Culture medium supply pump 16 Delivery pump

Claims

1. A method for producing a product, comprising culturing cells in a culture vessel, The culture is a perfusion culture method, During the product production period after the target viable cell density is reached, formulas (1) to (5) are satisfied; the variation in VCD during said production period is no more than ±20% relative to the average viable cell density during said production period; The fluctuation of VCV during the production period is ±20% or less of the average value of VCV during the production period. The method of producing the product. 0≦Gln-C≦2.5 Formula (1) 0.5≦Gluc-C≦8.0 Formula (2) 1.0≦NH4-C≦8.0 Formula (3) 50≦VCD≦300 Formula (4) 0.6×10 5 ≦VCV≦4.0×10 5 Formula (5) During the ceremony, Gln-C indicates the glutamine concentration in the culture medium [mmol / L], Gluc-C represents the glucose concentration in the culture medium [g / L], NH4-C indicates the ammonia concentration in the culture solution [mmol / L], VCD is the viable cell density [×10 6 cells / mL], VCV is VCD x π / 6 x Cd 3 [x10 6 μm 3 / mL], Cd indicates the mean diameter of viable cells [μm].

2. 2. The method for producing a product according to claim 1, wherein the culture medium is bled so that the fluctuation of VCD during the production period is within ±20% of the average viable cell density during the production period.

3. The method for producing a product according to claim 2 , wherein the culture solution is bled while measuring the capacitance of the culture solution in-line in the breeding step.

4. 4. The method of claim 3, wherein the bleeding is automatically controlled.

5. 5. The method for producing a product according to claim 3 or 4, wherein the fluctuation in the capacitance of the culture solution measured in-line is within ±20% of the average value of the capacitance during the production period.

6. The bleeding (i) setting a target capacitance; (ii) setting a weight control value for the culture vessel; (iii) measuring the capacitance of the culture solution at a period of 0.1 seconds or less; (iv) when the capacitance of the culture medium exceeds the target, operating the pump for withdrawing the culture medium at a rate of 0.3 vvd to 3 vvd (where vvd means the volume of culture medium withdrawn / volume of culture medium / day); (v) automatically supplying the culture medium to the culture vessel while measuring the mass of the culture medium so that the fluctuation in the liquid volume of the culture medium is within 10%; and (vi) stopping the pump that draws the culture solution when the capacitance of the culture solution falls below the target by 0.01% or more; A method for producing the product according to claim 3 or 4, comprising:

7. The method for producing a product according to any one of claims 1 to 4, wherein, during the production period, formula (6) is satisfied. 2≦LDH / VCD≦40 Formula (6) In the formula, LDH represents the concentration of lactate dehydrogenase in the culture medium [U / L]; VCD is the viable cell density [×10 6 cells / mL] are shown.

8. 5. A method for producing a product according to any one of claims 1 to 4, wherein the cells are animal cells.

9. The method for producing a product according to any one of claims 1 to 4, wherein the cell is a cell that produces a protein.

10. The method for producing the product according to any one of claims 1 to 4, wherein the cell is an antibody-producing cell.

11. 5. The method for producing the product according to any one of claims 1 to 4, wherein the cells are CHO cells.

12. 5. A method for producing a product according to any one of claims 1 to 4, wherein formula (2B) is satisfied during the production period of the product after the target viable cell density is reached. 1.5≦Gluc-C≦5.0 Formula (2B) In the formula, Gluc-C represents the glucose concentration [g / L] in the culture medium.

13. A product produced by the method for producing a product according to any one of claims 1 to 4.

14. The product according to claim 13, wherein the proportion of mannose 5 in the total N-glycans contained in the product is 7% or less.