Product manufacturing method and product

Abstract

The present invention addresses the problem of providing: a manufacturing method for a product of cell culturing, wherein membrane clogging is suppressed; and a product manufactured via said method. The present invention provides a product manufacturing method comprising cell culturing, wherein: perfusion culture is performed in which the cell density during production of the product is 80×106cells / mL to 300×106cells / mL; the period of the perfusion culture is 13-500 days; during a period of one or more days among the perfusion culture period, X[mol / L / day], which is the alkali addition speed per day, is controlled to the range 0≤X<0.029; and the pH of an added alkali aqueous solution is such that 7<pH<13.

Description

Manufacturing method of the product and the product

[0001] The present invention relates to a method for producing a product, which comprises culturing cells, and in which the rate of alkali addition and the pH of the aqueous alkaline solution are controlled.The present invention further 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 regulating the high mannose glycoform content of a recombinant protein containing an Fc region of immunoglobulin during a mammalian cell culture process, the method comprising establishing a mammalian cell culture that expresses the recombinant protein in a serum-free medium in a bioreactor, maintaining the mammalian cells during the production phase, and contacting the cell culture with monensin.

[0003] Furthermore, Patent Document 2 describes a fed-batch method for culturing mammalian cells, which includes controlling pH using a pH set point, comprising: a first culture stage including inoculating mammalian cells into a culture medium at a first pH and culturing the cells at the first pH, wherein the pH set point is maintained at the first pH; and a second culture stage including culturing the cells at a second pH higher than the first pH, wherein the set point is maintained at the second pH, wherein the second pH is at least 0.1 pH units higher than the first pH, and the second culture stage has a duration of at least 6 hours.

[0004] Furthermore, Patent Document 3 describes a method for culturing a suspension of immortalized human blood cells, preferably cells originating from or derived from myeloid leukemia, which method provides high productivity, high cell viability and growth rate, and high batch-to-batch consistency, and can be scaled up without changing these parameters.

[0005] Special table 2016-534732 publication Special table 2021-503916 publication Special table 2014-500032 publication

[0006] The ATF (Alternating Tangential Flow Filtration) method commonly used in perfusion culture is less prone to membrane clogging than the TFF (Tangential Flow Filtration) method due to the backwash effect of the liquid flowing back from the secondary side of the membrane to the primary side. However, as developments in productivity improvement progress, cell density has become increasingly high, and membrane clogging has become an issue even in the ATF method. A common measure to suppress membrane clogging is to increase the membrane area, but there are problems with the area of ​​commercially available membranes being small and the device becoming larger.

[0007] An object of the present invention is to provide a method for producing a product by cell culture that can suppress membrane clogging. Another object of the present invention is to provide a product produced by the method.

[0008] As a result of intensive research to solve the above problems, the inventors discovered that membrane clogging can be suppressed by controlling the pH and addition rate of the alkaline aqueous solution used for pH control, and thus completed the present invention.

[0009] That is, the present invention provides the following inventions: <1> A method for producing a product, which includes culturing cells, wherein the cell density during production of the product is 80 x 10 ６ cells / mL or more 300×10 ６ <2> A method for producing a product, wherein the perfusion culture is performed at a cell density of 100 x 10 cells / mL, the perfusion culture period is 13 days or more and 500 days or less, and during at least one day of the perfusion culture period, the daily alkali addition rate X [mol / L / day] is controlled in the range of 0≦X<0.029, and the pH of the added aqueous alkaline solution is 7<pH<13. ６ cells / mL or more 300×10 ６<1> A method for producing the product according to <1>, wherein the cell count is 1000 cells / mL or less. <2> A method for producing the product according to <1> or <2>, wherein the perfusion culture period is 18 days or more and 500 days or less. <3> A method for producing the product according to <1> or <2>, wherein the perfusion culture period is 18 days or more and 500 days or less. <4> A method for producing the product according to any one of <1> to <3>, wherein the culture period for product production is 5 days or more and 450 days or less. <5> A method for producing the product according to any one of <1> to <4>, wherein the daily alkali addition rate X [mol / L / day] is controlled in the range of 0≦X<0.008 for at least 13 days or more of the perfusion culture period. <6> A method for producing the product according to any one of <1> to <5>, wherein the daily alkali addition rate X [mol / L / day] is 0 for at least 13 days or more of the perfusion culture period. <7> The method for producing a product according to any one of <1> to <6>, wherein the daily alkali addition rate X [mol / L / day] is controlled within the range of 0≦X<0.029 for at least 50% of the period of the perfusion culture. <8> The method for producing a product according to any one of <1> to <7>, wherein the method for continuous separation of the cell culture solution in the perfusion culture is membrane filtration. <9> The method for producing a product according to any one of <1> to <8>, wherein the membrane filtration is alternating tangential flow filtration (ATF). <10> The method for producing a product according to any one of <1> to <8>, wherein the flux during filtration Y [L / m ２ / hour] satisfies 0<Y≦10. <11> A method for producing the product according to any one of <1> to <10>, wherein the average pH of the culture solution during the culture is 6.7 to 7.2. <12> A method for producing the product according to any one of <1> to <11>, wherein the minimum pH of the culture solution during the culture is 6.6 or higher. <13> A method for producing the product according to any one of <1> to <12>, wherein the pH of the culture solution during the culture is controlled by automatically adding an alkaline aqueous solution while measuring the pH in the culture solution in-line. <14> A method for producing the product according to any one of <1> to <12>, wherein the cell density during the perfusion culture is 80×10 ６<15> The method for producing a product according to any one of <1> to <13>, wherein after the cell density reaches a target cell density of 80×10 cells / mL or more, the cell density is maintained within ±40% of the target cell density by removing the culture medium containing the cells, and the product is recovered during the period in which the cell density is maintained within ±40% of the target cell density. ６ <16> The method for producing the product according to any one of <1> to <13>, comprising, after the target cell density of 80 x 10 cells / mL or more has been reached, adjusting the cell density to within ±10% of the target cell density by removing the cell-containing culture medium at least once per day. ６ Dissolved CO2 during the period when it is maintained at or above 100 cells / mL ２ <17> The method for producing the product according to any one of <1> to <15>, wherein the concentration is 60 to 180 mmHg. <17> The alkali is NaHCO ３ and / or Na ２ CO ３ <18> The method for producing the product according to any one of <1> to <16>, wherein the alkali is NaHCO ３ <19> A method for producing the product according to any one of <1> to <18>, wherein the concentration Z [mol / L] of the alkaline aqueous solution is 0<Z<5. <20> A method for producing the product according to any one of <1> to <19>, wherein the pH of the medium added in perfusion culture is 7.0 to 8.0. <21> A method for producing the product according to any one of <1> to <20>, wherein, when the culture is performed without adding alkali and the pH of the culture solution drops, the pH of the culture solution is controlled by supplying a medium to which alkali has been added. <22> A method for producing the product according to any one of <1> to <20>, wherein, when the pH of the culture solution falls below 6.85, the pH of the culture solution is controlled by supplying a medium to which alkali has been added. －３<23> A method for producing the product according to any one of <1> to <22>, comprising supplying a medium supplemented with an alkali of up to 2.0 mol / L. <24> A method for producing the product according to any one of <1> to <23>, wherein an antifoaming agent containing dimethicone is added during the perfusion culture, and the alkali addition rate X [mol / L / day] and the dimethicone addition rate B [g / L / day] satisfy B<-0.79X+0.0228. <25> A method for producing the product according to any one of <1> to <24>, wherein no alkali is added throughout the perfusion culture. <26> A method for producing the product according to any one of <1> to <25>, wherein the cells are CHO cells. <27> A method for producing a product according to any one of <1> to <26>, wherein the product is an antibody. <28> A product produced by the method for producing a product according to any one of <1> to <27>.

[0010] According to the present invention, membrane clogging during perfusion culture can be suppressed.

[0011] Figure 1 shows a cell culture apparatus. The entire apparatus shown in Figure 1 is the cell culture apparatus. Figure 2 shows the relationship between the alkali addition rate and the number of days from the start of culture until membrane clogging. Figure 3 shows the relationship between the alkali addition rate and microparticle density. Figure 4 shows the relationship between the alkali addition rate and LDH. Figure 5 shows the relationship between the alkali addition rate, dimethicone addition rate, and the number of days from the start of culture until membrane clogging.

[0012] 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.

[0013] The present invention relates to a method for producing a product, which comprises culturing cells, wherein the cell density at the time of producing the product is 80×10 ６ cells / mL or more 300×10 ６The present invention relates to a method for producing a product, wherein the perfusion culture is performed at a cell density of 1000 cells / mL or less, the perfusion culture period being 13 to 500 days, and the daily alkali addition rate, X [mol / L / day], is controlled within the range of 0≦X<0.029 for at least one day of the perfusion culture period, and the pH of the added aqueous alkali solution is 7<pH<13. According to the present invention, membrane clogging during perfusion culture can be suppressed. According to the present invention, productivity can be improved, particularly in perfusion culture using a membrane such as ATF / TFF. Here, the alkali addition per day refers to alkali addition added directly to the culture solution as an aqueous alkali solution separately from the medium. Even if the medium added to the culture tank during perfusion culture contains alkali, this does not fall under the alkali addition referred to here. Furthermore, unless there is a description such as "addition to the medium," alkali addition refers to addition directly to the culture solution.

[0014] In the present invention, the cell density during product production is 80 x 10 ６ cells / mL or more, and ６ ~300 x 10 ６ cells / mL, preferably 90 x 10 ６ ~300 x 10 ６ More preferably, the concentration is 100×10 ６ ~300 x 10 ６ More preferably, the concentration is 110×10 ６ ~300 x 10 ６ More preferably, the concentration is 120 x 10 cells / mL. ６ ~300 x 10 ６ More preferably, the concentration is 10 cells / mL. ６ In particular, the cell density is sometimes expressed as 120 × 10 ６ ~300 x 10 ６ By setting the concentration to 1000 cells / mL, membrane clogging becomes more pronounced, and the effects of the present invention are more pronounced.

[0015] In the present invention, preferably, in the perfusion culture described below, the cell density is 80 × 10６ After the target cell density of 80×10 cells / mL or more is reached, the culture medium containing the cells is removed, thereby maintaining the cell density within ±40% of the target cell density, and the product can be recovered during the period when the cell density is maintained within ±40% of the target cell density. ６ After the target cell density of 1000 cells / mL or more is reached, the cell density can be adjusted to within ±10% of the target cell density by removing the cell-containing culture medium at least once per day.

[0016] In the present invention, the method for culturing cells is perfusion culture. Perfusion culture is a culture method in which fresh medium is supplied to a cell culture solution and a portion of the medium in which the cells are cultured is removed. By performing this perfusion culture, waste products excreted from the cells can be removed from the culture vessel. In perfusion culture, the medium is continuously supplied to the culture vessel, and the liquid from which the cells in the culture solution are continuously separated can be collected.

[0017] Perfusion culture generally allows for the achievement of high viable cell densities. A typical perfusion culture begins with a batch culture lasting one or two days, after which fresh feed medium is added to the culture continuously, stepwise, and / or intermittently, with simultaneous removal of spent medium. In perfusion culture, cells can also be separated using methods such as sedimentation, centrifugation, or filtration to remove spent medium while maintaining viable cell density. 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 in batch or fed-batch culture.

[0018] 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.

[0019] The method for continuous separation of cell culture medium in perfusion culture is preferably membrane filtration, more preferably alternating tangential flow filtration (ATF).

[0020] The flux during filtration is Y [L / m ２ / hour] is preferably 0<Y≦10, more preferably 0<Y≦5, and even more preferably 0<Y≦2.

[0021] The filtration flux is the amount of culture solution that permeates the membrane per unit time and per unit filtration area, and is defined by the following formula:

[0022]

[0023] The perfusion rate is not particularly limited, but is generally 0.3 vvd to 5.0 vvd, preferably 0.5 vvd to 2.0 vvd, and more preferably 0.5 vvd to 1.4 vvd. vvd refers to the amount of cell culture medium exchanged with fresh medium per volume of cell culture medium per day, i.e., volume of supply medium / volume of culture medium / Day.

[0024] The product can be extracted from the culture medium by a pump from the secondary side of the filtration membrane, but other available liquid transport means may also be used. The extracted culture medium is then subjected to processes such as product recovery and dead cell removal. After product recovery and dead cell removal, the extracted culture medium may be partially discarded or returned to the culture vessel. If a loss of culture medium occurs due to the above processes, it can be compensated for by, for example, supplying fresh medium to the culture vessel.

[0025] In perfusion culture, the cell density was 80 × 10 ６After reaching a target cell density of 1000 cells / mL or more, the cell density can be adjusted to within ±10% of the target cell density by removing the cell-containing culture medium at least once per day. Reducing the viable cell density by removing a portion of the culture medium along with the cells to prevent excessive viable cell density during culture is called cell bleeding, and the amount of culture medium can be maintained by adding an equal amount of fresh medium to the removed culture medium. "At least once per day" also includes continuous, automatic cell bleeding.

[0026] The period of perfusion culture (this period is the culture period from the start of perfusion and includes the period before the cells reach high density) is not particularly limited, but is generally from 1 day to 1000 days, preferably from 7 days to 1000 days, more preferably from 18 days to 500 days, even more preferably from 25 days to 200 days, and even more preferably from 30 days to 100 days. Perfusion may be initiated 2 days after seeding, which is considered to be day 0.

[0027] The cell density is 80 x 10 ６ The culture period for producing a product having a cell count of 1000 cells / mL or more is preferably 5 days or more and 990 days or less, and may be 5 days or more and 450 days or less, more preferably 10 days or more and 450 days or less, even more preferably 15 days or more and 190 days or less, and even more preferably 20 days or more and 90 days or less.

[0028] In the present invention, during a period of at least 13 days or more during the perfusion culture period, the daily alkali addition rate, X [mol / L / day], is controlled within the range of 0≦X<0.029, preferably 0≦X<0.015, more preferably 0≦X<0.0127, and more preferably 0≦X<0.008.

[0029] In the present invention, during at least 13 days or more of the perfusion culture period, the daily alkali addition rate X [mol / L / day] may be 0. When X is 0, this means that alkali is not added from a line separate from the medium supply line.

[0030] In the present invention, the daily alkali addition rate X [mol / L / day] is preferably controlled within the range of 0≦X<0.029 for 50% or more (preferably 70% or more, more preferably 80% or more, even more preferably 90% or more, and most preferably 100%) of the perfusion culture period.

[0031] The alkali is not particularly limited, but is preferably Na ２ CO ３ , NaOH or NaHCO ３ and more preferably NaHCO ３ and / or Na ２ CO ３ and particularly preferably NaHCO ３ The alkali is an aqueous solution (e.g., Na ２ CO ３ aqueous solution, aqueous NaOH or NaHCO ３ It can be added as an aqueous solution.

[0032] Na ２ CO ３ or NaHCO ３ When each of these is added to the cell culture medium, NaHCO ３ The viscosity of the cell culture medium containing NaHCO tends to be lower. ３ Na is better ２ CO ３ It can be said that NaHCO has a weaker effect of gelling the culture medium than NaHCO and is more likely to prevent membrane clogging. ３ It is particularly preferred to use

[0033] The addition of alkali should be started at least 7 days after the start of the culture, preferably at least 6 days, and more preferably at least 5 days.

[0034] The pH of the aqueous alkaline solution to be added is 7<pH<13, preferably 7.5<pH<12, more preferably 7.5<pH<10, and even more preferably 8<pH<10.

[0035] The pH of an alkaline aqueous solution is measured when the alkali is dissolved in water. The pH can be measured using a commercially available pH sensor. For example, there are pH meters (Seven Excellence, Seven Direct, Five Easy) from Mettler TOLEDO.

[0036] The concentration Z [mol / L] of the aqueous alkaline solution to be added is preferably 0<Z<5, more preferably 0<Z<3, and even more preferably 0<Z<2.

[0037] It is possible to culture the cells without adding any alkali throughout the entire perfusion culture period. However, not adding alkali can lower the pH of the culture medium, which can lead to a deterioration in the quality of the product (Table 3). In such cases, a method is needed to maintain the quality of the product while preventing membrane clogging.

[0038] When the culture solution pH falls below a certain value M, adding alkali to the medium at N [mol / L] can suppress membrane clogging and improve antibody quality. That is, if the culture solution pH drops when culturing without adding alkali, the pH of the culture solution can be controlled by supplying a medium with added alkali. The denominator of N is the medium volume after preparation. At this time, a new medium with increased alkali is prepared, and the container containing the old medium (the medium being supplied to the culture tank) and the tubing connecting to the culture tank are aseptically connected to a container containing the new medium using a thermal welder, and supply begins. Alternatively, alkali may be aseptically added to a container containing the medium connected to the culture tank. The certain value M of the culture solution pH is 6.85, preferably 6.80, and more preferably 6.75.

[0039] The amount of alkali added to the medium, N [mol / L], is 1.0 × 10 －３ up to 2.0 mol / L, preferably 2.0×10 －３ to 1.0 mol / L, and more preferably 1.0 × 10 －２ ~2.0 x 10 －１The alkali added to the medium here does not mean alkali added directly to the culture solution, but alkali added to the medium before it is supplied to the culture tank. For example, when the pH of the culture solution falls below 6.85, 1.0 × 10 －３ It is possible to supply a medium supplemented with an alkali of up to 2.0 mol / L. In this way, when culturing is performed without directly adding alkali to the culture solution, if the pH of the culture solution decreases, the pH of the culture solution can be controlled by supplying a medium with an increased pH. This medium with an increased pH may be prepared in advance, or preparation can be started when a change in the pH of the culture solution is observed. Within 24 hours after the pH begins to fall below M, the supply medium can be switched to a medium with an increased pH, and perfusion culture can be performed. Preferably, this is within 12 hours, more preferably within 6 hours, even more preferably within 3 hours, even more preferably within 1 hour, even more preferably within 30 minutes, and even more preferably within 5 minutes.

[0040] The amount of the solution to be prepared is preferably 0.5 to 10 times the amount of the culture solution. The alkali to be added to the medium is not particularly limited, but is preferably Na ２ CO ３ , NaOH or NaHCO ３ and more preferably NaHCO ３ and / or Na ２ CO ３ and particularly preferably NaHCO ３ is.

[0041] The pH of the medium added during perfusion culture is preferably 7.0 to 8.0, more preferably 7.0 to 7.8, and even more preferably 7.0 to 7.6. The pH of the medium can be measured using a commercially available pH sensor after incubation at 37°C in 5% CO for one day. Examples of such sensors include pH meters from Mettler TOLEDO (Seven Excellence, Seven Direct, and Five Easy).

[0042] Preferably, the average pH of the culture medium during cultivation is 6.7 to 7.2, more preferably 6.8 to 7.0.

[0043] The minimum pH of the culture medium during the culture is preferably 6.6 or higher, more preferably 6.7 or higher, and even more preferably 6.8 or higher.

[0044] Preferably, the pH of the culture medium during cultivation can be controlled by automatically adding an alkaline aqueous solution while measuring the pH of the culture medium in-line.

[0045] Preferably, an antifoaming agent can be added in perfusion culture. Silicon-based antifoaming agents are preferred as the antifoaming component, with dimethicone being particularly preferred. The antifoaming component of the antifoaming agent is preferably one containing polydimethylsiloxane, more preferably one containing polydimethylsiloxane and finely powdered silica. For example, Cytiva's HyClone ADCF Antifoam Agent can be used as the antifoaming agent. More preferably, the alkali addition rate X [mol / L / day] and the dimethicone addition rate B [g / L / day] per day preferably satisfy the relationship B<-0.79X+0.0228. In this case, B is not a negative value. Therefore, X≦0.0288. The denominator of B is the volume of culture medium contained in the culture tank and ATF. By satisfying this relationship, membrane clogging can be more effectively suppressed.

[0046] 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 14 is a vessel that contains a culture medium containing cells. Cells are cultured in the culture medium inside the culture vessel 14.

[0047] A culture medium is supplied to the culture vessel through a culture medium supply pipe 1. An antifoaming agent for suppressing foaming is supplied to the culture vessel through an antifoaming agent supply pipe 2. An alkali is supplied to the culture vessel through an alkali supply pipe 3. If no alkali is added, the alkali supply pipe 3 may be omitted. Carbon dioxide (CO ) is supplied through an air supply pipe 4. ２ ) and air are introduced into the upper part of the culture solution inside the culture vessel. Oxygen (O ２) and / or air is fed into the culture solution through a sparger 15 having a pore size of 20 μm. The dissolved oxygen concentration in the culture solution can be adjusted by the sparger 15. 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 (for example, a pore size of 20 μm) in the gas release section and releasing a gas containing 30% by volume or more of oxygen can be used.

[0048] The exhaust pipe 6 is a pipe for exhausting air, and an exhaust filter (not shown) may be connected to the end thereof. The sampling pipe 7 is a pipe for collecting (sampling) the culture medium or extracting (cell bleeding) the culture medium. In the case of automatic and continuous cell bleeding, a separate pipe may be installed (not shown). A pH sensor 8 is attached so as to come into contact with the culture medium. A dissolved oxygen sensor 9 is attached so as to come into contact with the culture medium. The cell culture device may be provided with pressure sensors 10, 11, and 13. A hollow fiber membrane 12 is attached to the cell culture device.

[0049] A stirring member having a stirring blade 16 may be provided inside the culture vessel 14. By rotating the stirring blade 16, the culture solution inside the culture vessel 14 is stirred, maintaining the homogeneity of the culture solution. When the culture solution is stirred by the stirring blade 16, 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 16 close to the sparger.

[0050] In the present invention, the cell suspension extracted 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 cell culture device. In this operation, the cell suspension extracted from the culture vessel is separated into a cell-containing solution having a higher cell concentration than the cell suspension and a permeate having a lower cell concentration than the cell suspension. The cell concentration can be measured using a Vi-CELL XR viable cell analyzer manufactured by Beckman Coulter.

[0051] The membrane separation process is preferably tangential filtration, more preferably alternating tangential flow (ATF) filtration or tangential flow filtration, and most preferably alternating tangential flow filtration. Filters capable of performing alternating tangential flow filtration include SuATF10-S02PES and F2 RF02PES manufactured by Repligen.

[0052] 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.

[0053] The medium may contain serum such as fetal calf serum (FCS), or may not contain serum. The medium may be supplemented with additional components such as amino acids, salts, sugars, vitamins, hormones, growth factors, buffers, antibiotics, lipids, trace elements, and plant protein hydrolysates. Protein-free media may also be used. 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 in a CO ２ 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.

[0054] The amount of culture medium is preferably 0.5 L or more, more preferably 50 L or more, and even more preferably 200 L or more.

[0055] During the culture, the medium can be replaced, aerated, or stirred as necessary. When stirring the culture solution, 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.

[0056] The dissolved oxygen concentration in the culture medium can be appropriately set and is not particularly limited, but is generally 10 to 100%, preferably 30 to 90%. ６ Dissolved CO2 during the period when it is maintained at or above 100 cells / mL ２ The concentration is preferably 60 to 180 mmHg, more preferably 80 to 160 mmHg, and even more preferably 100 to 140 mmHg.

[0057] 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, and a packed tank-type culture device. From the viewpoint of homogenizing the culture environment, the culture vessel is preferably a single-use culture tank.

[0058] The cell density is 80 x 10 ６ The viscosity of the culture medium for producing a product having a cell density of 1.2 mPa·s or more and less than 15 mPa·s 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, even more preferably 1.6 mPa·s or more and less than 10 mPa·s, particularly preferably 1.6 mPa·s or more and less than 5 mPa·s, and most preferably 1.6 mPa·s or more and less than 3 mPa·s.

[0059] 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.

[0060] 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.

[0061] The higher the cell viability, the better, with 85% or more being preferred, 90% or more being more preferred, 95% or more being particularly preferred, and 99% or more being most preferred.

[0062] 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.

[0063] 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.

[0064] Examples of selectable marker genes that can be used include drug resistance genes (neomycin resistance gene, dihydrofolate reductase (DHFR) gene, puromycin resistance gene, blasticidin resistance gene, hygromycin resistance gene, cycloheximide resistance gene, etc.) and fluorescent genes (genes encoding green fluorescent protein (GFP), etc.).

[0065] 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.

[0066] 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.

[0067] In the present invention, the type of product is not particularly limited, but is preferably a protein, more 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.

[0068] 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.

[0069] 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).

[0070] 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.

[0071] 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.

[0072] 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.

[0073] 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.

[0074] 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.

[0075] 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.

[0076] 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.

[0077] 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.

[0078] 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).

[0079] 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.

[0080] The products produced by the present invention can be used, for example, in biopharmaceuticals and regenerative medicine.

[0081] 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.

[0082] <Establishment of antibody-producing cells> Vectors containing nucleic acid sequences encoding IgG1 and IgG4 were constructed, and the constructed vectors were introduced into CHO-DG44 cells to generate CHO-DG44 cells expressing IgG1 (IgG1 cells) and CHO-DG44 cells expressing IgG4 (IgG4 cells). The vectors were 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.

[0083] <Cell Culture> In the examples and comparative examples, experiments were carried out using the cell culture device shown in Figure 1. Briefly, 120 x 10 CHO cells producing antibodies were cultured by perfusion culture. ６ cells / mL and grown to 120 x 10 ６ When the cell density reached 1000 cells / mL, cell bleeding was started, and the number of days until the hollow fiber membrane became clogged was evaluated. Details are as follows.

[0084] One liter of medium was placed in a 2-liter glass culture vessel (product name, manufactured by ABLE). The medium was CD OptiCHO (manufactured by ThermoFisher Scientific), and 0.9% by mass of poloxamer (poloxamer-188) was added to the medium. On day 0, 0.5 × 10 cells were cultured. ６ The stirring speed was 150 rpm, and air was supplied from above at 38 mL / min + CO ２The oxygen concentration in the culture medium was adjusted to 80% by a sparger installed in the culture tank. ２ The pH in the fermenter was read to ensure that the pH in the fermenter was 7.0, and when the pH fell below 7.0, 1.0 mol / L NaHCO was automatically supplied. ３ An aqueous solution was added, and pH control was initiated. After culturing on the second day after seeding, the cell culture solution was continuously filtered using ATF2 manufactured by Repligen while continuously supplying the medium at a perfusion ratio of 0.8 vvd, and the recovery solution was collected. On the fifth day, the perfusion ratio was changed to 1.6 vvd. On the sixth day, the addition rate of simethicone in the antifoaming agent was changed to 7.2 mg / day / L. On the seventh day, the pH of the culture solution decreased, and an alkaline aqueous solution began to be automatically added to the culture solution. On the ninth to eleventh days, the cell density reached 120 x 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 100 cells / mL. The culture was continued until the membrane became clogged and the ATF could no longer function.

[0085] Several experiments were carried out by changing the medium used, the rate of addition of simethicone, pH control, etc. as shown in the table below.

[0086] <Evaluation Method> (1) Measurement of Cell Density and Viability The culture medium was extracted from the culture vessel 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:

[0087] Min diameter: 6 μm, Max diameter: 50 μm Dilution: Cell concentration 10 × 10 ６ When the cell concentration was 10 × 10 cells / mL or less, the sample was not diluted and the dilution was set to 1. ６When the concentration was higher than 100 cells / mL, the sample was diluted 10 times to make the dilution 10. Cell brightness: 75%, Cell sharpness: 100, Viable cell spot brightness: 75%, Viable cell spot area: 5%, Minimum circularity: 0, Decluster degree: Medium

[0088] (2) pH, dissolved CO ２ The culture medium in the culture tank was extracted and measured using a RAPIDLab 348EX manufactured by SIEMENS. ２ The carbon dioxide (CO ２ ) The flow rate was adjusted.

[0089] (3) Measurement of particle (debris) density The culture medium in the culture tank was extracted and diluted 10,000 times with ISOTON manufactured by Beckman Coulter. The particle size distribution of the diluted culture medium from 1.46 to 20 μm was measured using a precision particle size distribution measuring device, Multisizer 4e manufactured by Beckman Coulter.

[0090] (4) Measurement of Lactate Dehydrogenase (LDH) The culture medium was removed, and the cells were separated from the supernatant at 300 G for 5 minutes. The supernatant was measured using Roche's Cedex Bio.

[0091] (5) Measurement of antibody quality The culture medium was removed from the culture vessel, and the cells were filtered using Whatman (Pore Size 0.2 μm, 25 mm Diameter) manufactured by Cytiva. The culture supernatant was loaded onto a MabSelect Sure column equilibrated at pH 6.0, and the antibody was eluted at pH 3.2.

[0092] The antibody charge was evaluated by measuring the antibody recovery solution using cation exchange high-performance liquid chromatography (HPLC). Mannose 5 (M5) was evaluated by digesting the antibody in the antibody recovery solution with peptide-N-glycosidase F to remove the N-glycans, which were then fluorescently labeled with 2-aminobenzamide. The M5 of the treated antibody was measured using a C18 (ODS) reverse-phase HPLC column.

[0093] (6) Evaluation criteria The evaluation criteria for each category are as follows. Evaluation of membrane clogging: S: When the number of days from the start of culture to clogging is 30 days or more, the number of days from the start of perfusion to clogging is 28 days or more, and the number of days from the start of cell bleeding to clogging is 20 days or more. A: When the number of days from the start of culture to clogging is 20 to 29 days, the number of days from the start of perfusion to clogging is 18 to 27 days, and the number of days from the start of cell bleeding to clogging is 10 to 19 days. B: When the number of days from the start of culture to clogging is 15 to 19 days, the number of days from the start of perfusion to clogging is 13 to 17 days, and the number of days from the start of cell bleeding to clogging is 5 to 9 days. C: When the number of days from the start of culture to clogging is 14 days or less, the number of days from the start of perfusion to clogging is 12 days or less, and the number of days from the start of cell bleeding to clogging is 4 days or less.

[0094] Evaluation of antibody charge: A: The area percentage X of the acidic peak satisfies both 14%≦X≦22% and the area percentage X of the basic peak satisfies 11%≦X≦19%. B: The area percentage X of the acidic peak satisfies one or more of 10%≦X<14% or 22%<X≦26%, and the area percentage X of the basic peak satisfies one or more of 6%≦X<11% or 19%<X≦23%. C: The area percentage X of the acidic peak satisfies one or more of X<10% or 26%<X, and the area percentage X of the basic peak satisfies X<6% or 23%<X. The peak area percentage refers to the ratio of the peak area of ​​the chromatography of the acidic component or the peak area of ​​the basic component to the total peak area of ​​all chromatography.

[0095] Evaluation of antibody M5: A: M5 peak area ratio X is 0%≦X≦5% B: M5 peak area ratio X is 5%<X≦6% C: M5 peak area ratio X is 6%<X The peak area ratio refers to the ratio of the chromatographic peak area of ​​the M5 (Mannose 5) component to the total peak area of ​​all chromatography.

[0096] <Measurement and Evaluation Results> The measurement and evaluation results are shown in Tables 2 and 3. As shown in Table 3, it was confirmed that the antibodies produced in Examples 5, 10, and 11 were of good quality.

[0097] Figure 2 shows the relationship between the alkali addition rate and the number of days until membrane clogging in Examples 1 to 3, 5, and 7 to 9. The results in Figure 2 show that membrane clogging can be suppressed by lowering the alkali addition rate.

[0098] The relationship between the alkali addition rate and the microparticle density in Examples 1 to 3, 5, and 7 to 9 is shown in Figure 3. The microparticle density was defined as the cumulative number of cells with a size of 1 to 6 μm. The microparticles are debris / fragments derived from cells (14 μm) and are an indicator of cell damage. The results in Figure 3 show that cell damage can be suppressed by lowering the alkali addition rate.

[0099] The relationship between the alkali addition rate and LDH in Examples 1 to 3, 5, and 7 to 9 is shown in Figure 4. LDH is an enzyme component that leaks from damaged cells and is generally used as an indicator of cell damage. The results in Figure 4 show that cell damage can be suppressed by lowering the alkali addition rate.

[0100] Furthermore, the relationship between the alkali addition rate, the antifoaming agent addition rate, and the number of days until film clogging in Examples 1 to 9 and 11 and Comparative Example 1 is shown in Figure 5. It can be seen that the higher the antifoaming agent addition rate and the alkali addition rate, the more likely film clogging occurs. The region where the alkali addition rate X [mol / L / day] and the antifoaming agent dimethicone addition rate B [g / L / day] satisfy B < -0.79X + 0.0228 is the region where the film clogging evaluation is S.

[0101]

[0102]

[0103]

[0104] 1 Culture medium supply pipe 2 Antifoam agent supply pipe 3 Alkali supply pipe 4 Air supply pipe 5 Sparger air supply pipe 6 Exhaust pipe 7 Sampling pipe 8 pH sensor 9 Dissolved oxygen sensor 10 Pressure sensor 11 Pressure sensor 12 Hollow fiber membrane 13 Pressure sensor 14 Culture vessel 15 Sparger 16 Stirring blade

Claims

1. 1. A method for producing a product, comprising culturing a cell, the method comprising: The cell density at the time of product production is 80×10 6 cells / mL or more 300×10 6 The perfusion culture is performed at 1000 μg / mL for 13 days or more and 500 days or less. An alkali is added for at least one day during the perfusion culture period; During at least one day of the perfusion culture period, the alkali addition rate per day, X [mol / L / day], is controlled in the range of 0<X<0.029, and the pH of the aqueous alkali solution added is 7<pH<13. The method of producing the product.

2. The cell density in the perfusion culture was 100×10 6 cells / mL or more 300×10 6 The method for producing the product according to claim 1, wherein the concentration of the product is less than 1000 cells / mL.

3. The method for producing a product according to claim 1 or 2, wherein the period of the perfusion culture is from 18 days to 500 days.

4. The method for producing a product according to claim 1 or 2, wherein the culture period for producing the product is from 5 days to 450 days.

5. 3. The method for producing a product according to claim 1 or 2, wherein the daily alkali addition rate X [mol / L / day] is controlled in the range of 0≦X<0.008 during at least 13 days or more of the perfusion culture period.

6. 3. The method for producing a product according to claim 1 or 2, wherein the daily alkali addition rate X [mol / L / day] is 0 during at least 13 days or more of the perfusion culture period.

7. 3. The method for producing a product according to claim 1 or 2, wherein the daily alkali addition rate X [mol / L / day] is controlled in the range of 0≦X<0.029 during 50% or more of the period of the perfusion culture.

8. The method for producing a product according to claim 1 or 2, wherein the method for continuous separation of the cell culture solution in the perfusion culture is membrane filtration.

9. 3. The method for producing a product according to claim 1 or 2, wherein the membrane filtration is an alternating tangential flow filtration, also called ATF.

10. The flux during filtration is Y [L / m 2 9. The method for producing the product according to claim 8, wherein Y is 0<Y≦10.

11. The method for producing a product according to claim 1 or 2, wherein the average pH of the culture medium during the culture is 6.7 to 7.

2.

12. The method for producing a product according to claim 1 or 2, wherein the minimum pH of the culture medium during the culture is 6.6 or higher.

13. 3. The method for producing a product according to claim 1 or 2, wherein the pH of the culture solution during the culture is controlled by automatically adding an aqueous alkaline solution while measuring the pH of the culture solution in-line.

14. In the perfusion culture, the cell density was 80×10 6 3. The method for producing a product according to claim 1 or 2, wherein the cell density is maintained within ±40% of the target cell density by removing the culture medium containing the cells after the target cell density of not less than ±40% has been reached, and the product is recovered during a period during which the cell density is maintained within ±40% of the target cell density.

15. In the perfusion culture, the cell density was 80×10 6 3. The method for producing the product according to claim 1 or 2, comprising adjusting the cell density to within ±10% of the target cell density by removing the culture medium containing the cells at least once per day after the target cell density of at least 100,000 cells / mL has been reached.

16. The cell density is 80×10 6 Dissolved CO during the period when it is maintained at or above cells / mL 2 The method for producing the product according to claim 1 or 2, wherein the concentration is 60 to 180 mmHg.

17. The alkali is NaHCO 3 And / or Na 2 CO 3 The method for producing the product according to claim 1 or 2,

18. The alkali is NaHCO 3 The method for producing the product according to claim 1 or 2,

19. The method for producing a product according to claim 1 or 2, wherein the concentration Z [mol / L] of the alkaline aqueous solution is 0<Z<5.

20. The method for producing the product according to claim 1 or 2, wherein the pH of the medium added in the perfusion culture is 7.0 to 8.

0.

21. 3. The method for producing a product according to claim 1 or 2, wherein when the culture is performed without adding alkali and the pH of the culture solution decreases, the pH of the culture solution is controlled by supplying a medium containing added alkali.

22. When the pH of the culture medium falls below 6.85, 1.0 × 10 -3 3. A method for producing the product according to claim 1 or 2, comprising feeding a medium supplemented with up to 2.0 mol / L of alkali.

23. An antifoaming agent containing dimethicone is added to the perfusion culture, The method for producing the product according to claim 1 or 2, wherein the addition rate of the alkali, X [mol / L / day], and the addition rate of the dimethicone, B [g / L / day], satisfy B<-0.79X+0.0228.

24. 1. A method for producing a product comprising culturing a cell, the method comprising: The cell density at the time of product production is 80×10 6 cells / mL or more 300×10 6 The perfusion culture is performed at 1000 μg / mL for 13 days or more and 500 days or less. During at least one day of the perfusion culture period, the alkali addition rate per day, X [mol / L / day], is controlled in the range of 0≦X<0.029, and the pH of the aqueous alkali solution added is 7<pH<13; When the pH of the culture solution drops when the culture is performed without adding alkali, the pH of the culture solution is controlled by supplying a medium to which alkali has been added. When the pH of the culture medium falls below 6.85, 1.0 × 10 -3 providing a medium supplemented with 2.0 mol / L of alkali; The method of producing the product.

25. The method for producing a product according to claim 1 or 2, wherein the cells are animal cells.

26. The method for producing a product according to claim 1 or 2, wherein the cells are CHO cells.

27. The method for producing a product according to claim 1 or 2, wherein the product is an antibody.

28. A product produced by the method for producing a product according to claim 1 or 2.