Cell culture method and method for producing useful substances

Abstract

The present invention addresses the problem of providing: a cell culturing method whereby it becomes possible to prevent oxygen deficiency in cells when the cells are cultured at a high density; and a method for producing a useful substance utilizing the cell culturing method. According to the present invention, a cell culturing method is provided, in which the relationship among an agitation power per unit volume P / V, a dissolved oxygen concentration CL0 and a viable cell density VCD satisfies the requirement represented by formula 1 in cell culturing at a viable cell density of 60×106 cell / mL or more. Formula 1: 0.0059×exp[0.0243×(P / V)]×CL0 / VCD ≧ 1.10×10-17 In the formula, CL0 represents a dissolved oxygen concentration [mol / mL]; VCD represents a viable cell density [cell / mL]: and P / V represents an agitation power per unit volume [W / m3] which is defined by a specified formula.

Description

Technical Field

[0001] The present invention relates to a cell culture method for suppressing oxygen deficiency in cells. The present invention further relates to a method for producing a useful substance using the above-described cell culture method.

Background Art

[0002] In cell culture, oxygen supply to cells is very important. When the amount of supplied oxygen is insufficient, the cells enter an oxygen-deficient state, which causes growth stagnation and a decrease in the productivity of useful substances. Generally, the oxygen supply capacity of a cell culture device is indicated by kLa (gas-liquid oxygen transfer volume coefficient), which is a parameter representing the rate (size) of oxygen dissolution from bubbles into the culture solution. Conventionally, in response to an increase in the required oxygen amount accompanying cell densification, studies have been made to obtain a sufficient dissolved oxygen concentration by increasing kLa (increasing the rate of dissolution from bubble oxygen into the medium).

[0003] Patent Document 1 describes a method for culturing living cells, characterized in that the dissolved oxygen concentration in the culture solution in the culture tank is measured at a plurality of locations in the space in the culture tank, and the stirring speed of the culture solution in the culture tank is determined based on the difference in the measured dissolved oxygen concentrations at the plurality of locations. The culture method described in Patent Document 1 is a method for continuously culturing living cells over a long period by precisely controlling the stirring speed of the culture solution, particularly when culturing living cells using microcarriers.

Prior Art Documents

Patent Documents

[0004] <从这里开始,原文中没有对应的翻译内容,按照要求保留原文标签即可

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0005] However, it has been found that when cells are cultured at high densities, a problem arises where cells become oxygen-deprived even if the dissolved oxygen concentration in the culture medium is sufficient. The problem that this invention aims to solve is to provide a cell culture method that can suppress oxygen deprivation in cells when culturing cells at high densities. Furthermore, the problem that this invention aims to solve is to provide a method for producing useful substances using the cell culture method described above. [Means for solving the problem]

[0006] The inventors investigated the cause of the problem that cells become oxygen-deprived when the cell density is high, even though the dissolved oxygen concentration in the culture medium is sufficient. As a result, they found that when the cell density is high, the rate of oxygen supply from the culture medium to the cells decreases, leading to oxygen deprivation. The inventors diligently investigated to solve the above problem and found that when the relationship between the stirring power per unit volume P / V, the dissolved oxygen concentration CL0, and the viable cell density VCD satisfies predetermined conditions, oxygen deprivation in high-density culture can be improved and stable culture can be achieved. Furthermore, the inventors found that when the relationship between the newly defined oxygen transfer capacity coefficient (kcella) from the culture medium to the cells, the dissolved oxygen concentration CL0, and the viable cell density VCD satisfies predetermined conditions, oxygen deprivation in high-density culture can also be improved and stable culture can be achieved. The present invention was completed based on the above findings.

[0007] In other words, the present invention provides the following invention. <1> The density of living cells is 60 × 10 6 A cell culture method in which, in cell cultures with a density of cells / mL or higher, the relationship between the stirring power per unit volume P / V, the dissolved oxygen concentration CL0, and the viable cell density VCD satisfies the conditions of Equation 1 below. Equation 1: 0.0059×exp[0.0243×(P / V)]×CL0 / VCD≧1.10×10 -17 During the ceremony, CL0 indicates the dissolved oxygen concentration [mol / mL]; VCD indicates the density of living cells [cells / mL]: P / V represents the stirring power per unit volume [W / m 3 defined by the following formula: P / V = ρ × (n / 60) 3 × Di 5 / V × Np wherein, ρ represents the density of the culture solution [kg / m 3 ; n represents the stirring rotation speed [rpm]; Di represents the diameter of the stirring blade [m]; V represents the amount of the culture solution [m 3 ; Np represents the stirring power coefficient. <2> The cell culture method according to <1>, wherein the stirring power P / V per unit volume satisfies 60 ≤ P / V ≤ 2000. <3> The cell culture method according to <1> or <2>, wherein the stirring rotation speed n satisfies 120 ≤ n ≤ 600. <4> The relationship among the stirring power P / V per unit volume, the dissolved oxygen concentration CL0, and the viable cell density VCD satisfies 0.0059 × exp[0.0243 × (P / V)] × CL0 / VCD ≤ 4.00 × 10 -17 The cell culture method according to any one of <1> to <3>. <5> The cell culture method according to any one of <1> to <4>, wherein the cell culture period satisfying the conditions of Formula 1 is 20 days or more. <6> The cell culture method according to any one of <1> to <5>, wherein the conditions of Formula 1 are satisfied throughout the entire cell culture period. <7> In cell culture where the viable cell density is 60 × 10 6 cell / mL or more, the relationship among kcella defined below, the dissolved oxygen concentration CL0 in the steady state, and the viable cell density VCD satisfies the conditions of the following Formula 2. The cell culture method. Formula 2: kcella × CL0 / VCD ≥ 1.00 × 10 -17 [mol / s / cell] wherein, CL0 represents the dissolved oxygen concentration [mol / mL]; VCD indicates the density of living cells [cells / mL]: kcella is Ln(CL / CL0) = -kcella × t This indicates the total oxygen transfer capacity coefficient [ / s] from the culture medium to the cells, as defined by: During the ceremony, The steady state is defined as the state in which the dissolved oxygen concentration in the culture medium is maintained at a predetermined set value, CL0, for 24 hours or more by continuously stirring the culture medium and supplying oxygen. The moment when the oxygen supply is stopped while stirring continues from the steady state is defined as the temporal starting point. The dissolved oxygen concentration CL0 at the above starting point and the change in the dissolved oxygen concentration CL in the liquid over 60 seconds from the above starting point are measured, and the slope obtained by linearly approximating the change in Ln(CL / CL0) with respect to elapsed time t using the least squares method is defined as -kcella. <8> kcella satisfies 0.03 ≤ kcella ≤ 0.39. <7> The cell culture method described above. <9> The relationship between kcella, steady-state dissolved oxygen concentration (CL0), and viable cell density (VCD) is as follows: kcella × CL0 / VCD ≤ 4.00 × 10 -17 [mol / s / cell] The conditions that satisfy, <7> or <8> The cell culture method described above. <10> The correlation between the calculated turbulent energy dissipation rate ε of the culture medium and the measured kcella value is determined in advance for the target culture vessel and liquid volume, and the kcella value obtained from the above correlation is increased by increasing the stirring power as the cells proliferate, thereby controlling the culture to satisfy the conditions of Equation 2. <7> from <9> A cell culture method as described in any one of the following. <11> As VCD increases, the culture is controlled to satisfy the conditions of Equation 2 by increasing at least one of kcella and CL0. <7> from <9> A cell culture method as described in any one of the following. <12> The cell culture period that satisfies the conditions of Equation 2 is 20 days or longer. <7> from <11> A cell culture method as described in any one of the following. <13> The conditions in Equation 2 are satisfied throughout the entire cell culture period. <7> from <12> A cell culture method as described in any one of the following. <14> CL0 is 0.45 × 10 -8 ≤CL0 ≤ 6.75 × 10 -8 That is, <1> from <13> A cell culture method as described in any one of the following. <15> The density of living cells is 80 × 10 6 cells / mL or more <1> from <14> A cell culture method as described in any one of the following. <16> The culture medium volume is 50L or more. <1> from <15> A cell culture method as described in any one of the following. <17> It is a perfusion culture. <1> from <16> A cell culture method as described in any one of the following. <18> The cells are mammalian cells. <1> from <17> A cell culture method as described in any one of the following. <19> Mammalian cells are Chinese hamster ovary cells. <18> The cell culture method described above. <20> The average cell diameter of the cells is 12 μm or more. <1> from <19> A cell culture method as described in any one of the following. <21> During cell culture, the lactate concentration in the culture medium is less than 1.2 g / L. <1> from <20> A cell culture method as described in any one of the following. <22> During cell culture, the concentration of lactate dehydrogenase in the culture medium is less than 2000 U / L. <1> from <21> A cell culture method as described in any one of the following. <23> <1> from <22> A method for producing a useful substance, comprising culturing cells by any one of the cell culture methods described in the following, thereby causing cells to produce a useful substance. <24> The useful substance is recombinant protein. <23> A method for producing the useful substances described above. [Effects of the Invention]

[0008] According to the cell culture method of the present invention, oxygen deficiency in cells can be suppressed even when culturing cells at high density. The cell culture method of the present invention is useful in the production of useful substances. [Brief explanation of the drawing]

[0009] [Figure 1] Figure 1 shows the relationship between the rate of molecular diffusion of oxygen transport, the overall oxygen transport capacity coefficient (defined as kcella), and the concentration difference between the oxygen concentration in the culture vessel and the oxygen concentration on the cell surface (CL0-Ccell). [Figure 2] Figure 2 shows a schematic diagram of the case where the density of living cells is low. [Figure 3] Figure 3 shows a schematic diagram of a case with a high density of living cells. [Figure 4] Figure 4 shows a schematic diagram of the increase in kcella when the density of living cells is high. [Figure 5] Figure 5 shows the measurement method for kcella. [Figure 6] Figure 6 shows the cell proliferation phase and antibody production phase in the example. [Figure 7] Figure 7 shows the relationship between kcella and 0.0059 × exp[0.0243 × (P / V)] × CL0 / VCD × 10¹⁷. [Figure 8] Figure 8 shows the relationship between kcella and kcella×CL0 / VCD×10¹⁷. [Figure 9] Figure 9 shows the relationship between the turbulent energy dissipation rate ε and kcella. [Modes for carrying out the invention]

[0010] The details of the present invention will be described below. In this specification, numerical ranges indicated using "~" mean a range that includes the numbers before and after "~" as the minimum and maximum values, respectively. In this specification, s, which indicates a unit, refers to seconds.

[0011] This invention provides a living cell density of 60 × 10 6This invention relates to a cell culture method in which, in cell cultures with a cell density of cells / mL or higher, the relationship between the stirring power per unit volume P / V, the dissolved oxygen concentration CL0, and the viable cell density VCD satisfies a predetermined condition (the condition of Equation 1 described below), or the relationship between kcella as defined below, the steady-state dissolved oxygen concentration CL0, and the viable cell density VCD satisfies a predetermined condition (the condition of Equation 2 described below).

[0012] In Patent Document 1, the range of viable cell density is given as an example of 7 × 10 6 While it is described that the oxygen concentration distribution during proliferation (see Figures 3 and 4) is controlled by the stirring speed to a level of cells / mL or less, the range of viable cell densities described in Patent Document 1 is different from the viable cell density defined in the present invention. Furthermore, in adherent cultures including microcarriers described in Patent Document 1, the viable cell density is typically 10 × 10 6 The cell density is less than or equal to cells / mL, which differs from the live cell density defined in this invention.

[0013] <Oxygen supply rate between culture medium and cells and the mechanism of oxygen deficiency> Even with dynamic culture methods, it is thought that a layer of culture medium (boundary layer) exists around the cells where the culture medium accumulates.

[0014] In the oxygen supply system for cell culture, within the boundary layer where the culture medium stagnates around the cells, the amount of oxygen supplied from the culture medium to the cells is proportional to (1) and (2) below (Figure 1). (1) Total oxygen transport capacity coefficient (defined as kcella). The thickness of the boundary layer depends on the flow state of the surrounding culture medium. (2) The difference in oxygen concentration between the surrounding culture vessel and the cell surface (CL0-Ccell)

[0015] When the density of living cells is low, the distance between cells is sufficiently large, and the kLa, which is the transfer volume coefficient of oxygen dissolution from air bubbles into the culture medium, is sufficiently high, i.e., the dissolved oxygen concentration CL0 is sufficiently high. In such cases, oxygen is supplied from the culture medium to the cells, expressed as kcella × (CL0 - Ccell), and oxygen deficiency does not occur (Figure 2).

[0016] We discovered that even when the dissolved oxygen concentration is sufficiently high in proportion to the cell density, oxygen deficiency occurs when the cell density is high. When the cell density is high, the relative distance between cells in the culture vessel decreases. Even though the distance between cells decreases, adjacent cells do not directly overlap, but the boundary layers of adjacent cells overlap. When areas where the boundary layers of adjacent cells overlap occur, the boundary layers of other cells are present around each cell, so the apparent boundary layer thickness increases, and kcella, which is inversely proportional to the boundary layer thickness, decreases. In addition, the surface area effective for oxygen transport decreases, and kcella decreases. As a result, even when the dissolved oxygen concentration (≒kLa) is sufficiently high, the amount (rate) of oxygen transport expressed as kcella × (CL0 - Ccell) decreases, and oxygen deficiency occurs (Figure 3).

[0017] In situations with such high cell density, increasing kcella levels can eliminate boundary layer overlap even when the distance between cells is small, making it possible to achieve sufficient oxygen supply (Figure 4).

[0018] <First aspect of the present invention> In the cell culture method of the first aspect of the present invention, the relationship between the stirring power per unit volume P / V, the dissolved oxygen concentration CL0, and the viable cell density VCD satisfies the following condition of Equation 1. Equation 1: 0.0059×exp[0.0243×(P / V)]×CL0 / VCD≧1.10×10 -17 During the ceremony, CL0 indicates the dissolved oxygen concentration [mol / mL]; VCD indicates the density of living cells [cells / mL]: P / V is the stirring power per unit volume [W / m²] defined by the following formula. 3 ] indicates: P / V = ρ × (n / 60) 3 × Di 5 / V × Np During the ceremony, ρ is the density of the culture medium [kg / m³] 3 ] indicates; n represents the stirring speed [rpm]; Di indicates the diameter of the stirring blade [m]; V is the volume of culture medium [m³] 3 ] indicates; Np represents the stirring power coefficient.

[0019] The stirring power coefficient can be calculated based on the physical properties of the liquid (viscosity, density), information about the culture tank and agitator (number of agitators, agitator width, agitator diameter, culture tank inner diameter), and information about the stirring rotation speed. Specifically, the stirring power coefficient can be determined by the following formula. Power coefficient Np = A / Re + B × {(10 3 +1.2×Re 0.66 ) / (10 3 +3.2×Re 0.66 )} p ×(H / D) (0.35+a) a = np × h / 2 / D b = d / D A = 14 + a × {670 × (b - 0.6)} 2 +185} B = 10 {1.3-4×(a-0.5)2-1.14×b} p = 1.1 + 4 × a - 2.5 × (b - 0.5) 2 -7 × a 4 Re = n / 60 × d 2 ×ρ / μ np: Number of stirring blades [pieces] h: Stirring blade width [m] d: Stirring blade diameter [m] n: Stirring speed [rpm] D: Culture tank inner diameter [m] H: Depth of the liquid [m] ρ: liquid density [kg / m 3 ] μ: Liquid viscosity [Pa s]

[0020] The stirring power P / V per unit volume preferably satisfies 60 ≤ P / V ≤ 2000, more preferably 60 ≤ P / V ≤ 1500, even more preferably 60 ≤ P / V ≤ 1000, particularly preferably 60 ≤ P / V ≤ 500, and most preferably 60 ≤ P / V ≤ 200.

[0021] The stirring rotation speed n preferably satisfies 100 ≤ n ≤ 600, more preferably 110 ≤ n ≤ 400, and even more preferably 120 ≤ n ≤ 300.

[0022] The relationship between the stirring power per unit volume P / V, the dissolved oxygen concentration CL0, and the viable cell density VCD is preferably 0.0059 × exp[0.0243 × (P / V)] × CL0 / VCD ≤ 4.00 × 10 -17 The conditions are met. The lower limit of 0.0059 × exp[0.0243 × (P / V)] × CL0 / VCD is 1.20 × 10 -17 The above is 1.30 × 10 -17 The above is 1.40 × 10 -17 The above, or 1.50 × 10 -17 That's fine too. The upper limit of 0.0059 × exp[0.0243 × (P / V)] × CL0 / VCD is 3.50 × 10 -17 Below, 3.00 × 10 -17 The following is also acceptable.

[0023] The cell culture period that satisfies the conditions of Formula 1 is preferably 10 days or more, more preferably 20 days or more, and even more preferably 30 days or more. It is particularly preferable that the conditions of Formula 1 are satisfied throughout the entire cell culture period.

[0024] <Second aspect of the present invention> In the cell culture method of the second aspect of the present invention, the relationship between kcella as defined below, the dissolved oxygen concentration CL0 in a steady state, and the viable cell density VCD satisfies the conditions of the following equation 2. Formula 2: kcella×CL0 / VCD≧1.00×10 -17 [mol / s / cell] During the ceremony, CL0 indicates the dissolved oxygen concentration [mol / mL]; VCD indicates the density of living cells [cells / mL]: kcella is Ln(CL / CL0) = -kcella × t This indicates the total oxygen transfer capacity coefficient [ / s] from the culture medium to the cells, as defined by: In the formula, the steady state is defined as the state in which the dissolved oxygen concentration in the culture medium is maintained at a predetermined set value, CL0, for 24 hours or more by continuously stirring the culture medium and supplying oxygen. The moment when the oxygen supply is stopped from the steady state is defined as the temporal starting point. The dissolved oxygen concentration CL0 at the above starting point and the change in the dissolved oxygen concentration CL in the liquid over 60 seconds from the above starting point are measured, and the slope obtained by linearly approximating the change in Ln(CL / CL0) with respect to elapsed time t using the least squares method is defined as -kcella.

[0025] The kcella during steady-state cultivation can be calculated from the rate of decrease in dissolved oxygen concentration in the culture medium when only the oxygen supply is stopped while maintaining the stirring conditions and culture temperature from the steady state. Oxygen supply from the culture medium to the cells is thought to be limited by oxygen transport in the boundary layer around the cells and by cell respiration. However, when measuring the dissolved oxygen concentration in the culture medium after stopping oxygen supply from a steady-state culture state, the rate of oxygen depletion in the culture medium also changes depending on the stirring speed during measurement, indicating that boundary layer transport is the limiting factor. Furthermore, the fluidity of the culture medium changes with the stirring speed, and the thickness of the boundary layer around the cells also changes. In the boundary layer transport-limited stage, the amount of oxygen required by the cells is greater than the oxygen transport rate, so the oxygen consumption rate = oxygen transport rate for the entire system (if the oxygen concentration on the cell surface is Ccell [mol / mL], then CL - Ccell ≈ CL). The equation for oxygen transfer in the culture medium at this time is as follows:

[0026] dCL / dt=-kcella(CL-Ccell)=-kcella×CL

[0027] Ln(CL / CL0) = -kcella × t

[0028] In other words, the slope of the linearly approximated curve of the dissolved oxygen concentration Ln(CL / CL0) in the culture medium plotted against elapsed time t from t=0 to 60[s] is -kcella (Figure 5).

[0029] kcella preferably satisfies the condition 0.03 ≤ kcella ≤ 0.39. The lower limit of kcella may be 0.04 or greater, 0.05 or greater, or 0.10 or greater. The upper limit of kcella may be 0.35 or less, 0.30 or less, or 0.26 or less.

[0030] The relationship between kcella, steady-state dissolved oxygen concentration (CL0), and viable cell density (VCD) is preferably kcella × CL0 / VCD ≤ 4.00 × 10 -17 It satisfies the condition [mol / s / cell].

[0031] The lower limit of kcella×CL0 / VCD is 1.10×10 -17 The above is 1.20 × 10 -17 The above is 1.30 × 10 -17 The above is 1.40 × 10 -17 The above, or 1.50 × 10 -17 That's fine too. The upper limit of kcella×CL0 / VCD is 3.50×10 -17 Below, 3.00 × 10 -17 The following, or 2.50 × 10 -17 The following is also acceptable.

[0032] In the present invention, the correlation between the calculated value of the turbulent energy dissipation rate ε of the culture medium and the measured value of kcella when the stirring speed is changed for the target culture vessel and liquid volume may be determined in advance, and the culture may be controlled to satisfy the conditions of Equation 2 by increasing the stirring power (i.e., increasing the stirring speed) as the cells proliferate, thereby increasing the kcella obtained from the above correlation.

[0033] The turbulent energy dissipation rate ε can be calculated using commercially available CFD (Computational Fluid Dynamics) software (e.g., Fluent from ANSIS). The turbulent energy dissipation rate ε can be calculated, for example, by combining the mass conservation equation, the momentum conservation equation, the k-ε model transport equation, and the modified wall-treated ε equation. The turbulent energy dissipation rate can be calculated by creating a mesh that simulates the culture tank shape, impeller shape, and predetermined liquid volume, setting parameters such as fluid viscosity, fluid density, stirring speed, rotation angle, gravity direction, and liquid surface boundary conditions, and then performing a convergence calculation.

[0034] In the present invention, the culture may be controlled to satisfy the conditions of Formula 2 by increasing at least one of kcella and CL0 as VCD increases.

[0035] The cell culture period that satisfies the conditions of Equation 2 is preferably 10 days or more, more preferably 20 days or more, and even more preferably 30 days or more. It is particularly preferable that the conditions of Equation 2 are satisfied throughout the entire cell culture period.

[0036] <About the cell culture method of the present invention> For cell culture, you can use the same culture media that are commonly used for animal cell culture. For example, you can use CD OptiCHO (ThermoFisher), Dulbecco's modified Eagle medium (DMEM), Eagle's minimal essential medium (MEM), RPMI-1640 medium, RPMI-1641 medium, F-12K medium, Ham F12 medium, Iscob's modified Dulbecco medium (IMDM), McCoy's 5A medium, Leibowitz 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. Alternatively, you may use your own homemade culture media.

[0037] Serum, such as fetal bovine serum (FCS), may or may not be added to the culture medium. Additional components such as amino acids, salts, sugars, vitamins, hormones, growth factors, buffers, antibiotics, lipids, trace elements, and hydrolyzed plant proteins may be added to the culture medium. Protein-free medium can also be used.

[0038] The pH of the culture medium varies depending on the cells being cultured, but is generally between pH 6.0 and 8.0, preferably between pH 6.8 and 7.6, and more preferably between pH 7.0 and 7.4. The culture temperature is generally 30°C to 40°C, preferably 32°C to 37°C, and more preferably 36°C to 37°C, and the culture temperature may be changed during cultivation. The culture is preferably carried out in an atmosphere with a CO2 concentration of 0-40%, preferably 2-10%.

[0039] The incubation period is not particularly limited, but is generally 12 hours to 90 days, preferably 24 hours to 60 days, and more preferably 24 hours to 30 days. During culture, the culture medium can be replaced, aerated, and stirred as needed.

[0040] The cell culture method of the present invention can be carried out in a culture device (also called a bioreactor) or in any other suitable container. Suitable culture devices include fermentation tank type culture devices, air-lift type culture devices, culture flask type culture devices, spinner flask type culture devices, microcarrier type culture devices, fluidized bed type culture devices, hollow fiber type culture devices, roller bottle type culture devices, and packed tank type culture devices.

[0041] In the cell culture method of the present invention, the volume of culture medium is generally 1 L to 20,000 L, preferably 50 L or more, for example, 50 L to 2,000 L, or 500 L to 2,000 L.

[0042] In the cell culture method of the present invention, the dissolved oxygen concentration CL0 (mol / mL) is preferably 0.45 × 10⁻⁶. -8 ≤CL0 ≤ 6.75 × 10 -8 Therefore, the lower limit of CL0 is 0.50 × 10 -8 mol / mL or more, 0.60×10 -8 mol / mL or more, 0.70×10 -8 mol / mL or more, 0.80×10 -8 mol / mL or higher, or 0.90 × 10⁻⁶ -8 It is acceptable to have concentrations of mol / mL or higher. The upper limit of CL0 is 6.50 × 10⁻⁶. -8 mol / mL or less, 6.00×10 -8 mol / mL or less, 5.50×10 -8 mol / mL or less, or 5.00 × 10⁻⁶ -8 A concentration of mol / mL or less is also acceptable.

[0043] In the cell culture method of the present invention, the viable cell density is 60 × 10 6 The number of cells / mL should be greater than or equal to 70 × 10⁻¹⁰. 6 More than 80 × 10 6 The cell / mL is greater than or equal to 100 × 10⁻¹⁰. 6 The cell density is 120 × 10⁻¹⁴ or higher. 6 cell / mL or more, 150×10 6 cell / mL or more, 200×10 6 cell / mL or higher, or 300 × 10 6 The cell density may be higher than or equal to cells / mL. There is no particular upper limit to the viable cell density, but generally it is 500 × 10⁻⁶. 6 The value is less than or equal to cells / mL.

[0044] The method of cell culture according to the present invention is not particularly limited and may be any of perfusion culture, batch culture, or fed-batch culture, but perfusion culture is preferred.

[0045] Batch culture is a discontinuous method in which cells are grown for a short period in a fixed volume of culture medium and then completely harvested.

[0046] Fed-batch culture is a culture method that improves the batch process by supplying culture medium in bolus or continuous manner to replenish consumed medium components.

[0047] Perfusion culture is a culture method that involves adding fresh culture medium and simultaneously removing used medium, and it has the potential to further improve batch culture and fed-batch culture. Perfusion culture generally makes it possible to achieve high viable cell densities. A typical perfusion culture begins with a batch culture startup lasting one or two days, after which fresh supply medium is continuously, stepwise, and / or intermittently added to the culture while simultaneously removing used medium. In perfusion culture, used medium can be removed while maintaining viable cell density using methods such as sedimentation, centrifugation, or filtration. The advantage of perfusion culture is that the culture producing the target protein can be maintained for a longer period than with batch culture or fed-batch culture.

[0048] Perfusion may be continuous, stepwise, intermittent, or a combination thereof. Animal cells are retained in the culture, and the used medium that is removed may contain substantially no cells or far fewer cells than the culture. Useful substances expressed by cell culture can be retained or recovered in the culture by selecting the pore size. To prevent excessive cell density during culture, a portion of the culture medium, including the cells, may be removed and an equal amount of fresh medium added to reduce the cell density (cell bleeding).

[0049] The type of cell used in this invention is not particularly limited, but is preferably an animal cell, and more preferably a mammalian cell. The cell may be a primary cell or a cell line.

[0050] Examples of suitable cells include Chinese hamster ovary (CHO) cells, BHK cells, 293 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-derived cells), PC12 cells, and WI38 cells. Among these, CHO cells, BHK cells, 293 cells, myeloma cells (such as NS0 cells), PerC6 cells, SP2 / 0 cells, and hybridoma cells are particularly preferred, with CHO cells being even more preferred. CHO cells are widely used for the production of recombinant proteins, such as cytokines, coagulation factors, and antibodies. It is preferable to use CHO cells lacking dihydrofolate reductase (DHFR), and for example, CHO-DG44 can be used as a DHFR-deficient CHO cell.

[0051] These cells may also be cells into which an exogenous gene encoding the protein to be expressed has been introduced. An expression vector can be used to introduce a foreign gene encoding the protein to be expressed into cells. By introducing an expression vector containing the DNA encoding the protein to be expressed, an expression regulatory sequence (e.g., enhancer, promoter, and terminator), and optionally a selected marker gene into cells, cells into which the foreign gene encoding the protein to be expressed can be created. There are no particular limitations on the expression vector; it can be appropriately selected and used depending on the type of cell and its intended use.

[0052] Any promoter capable of functioning in mammalian cells can be used. Examples include the promoter of the cytomegalovirus (CMV) IE (immediate early) gene, the early promoter of SV40, retrovirus promoters, metallothionein promoters, heat shock promoters, SRα promoters, and the promoter and enhancer of Moloney murine leukemia virus. Additionally, the enhancer of the human CMV IE gene may be used in conjunction with the promoter.

[0053] Examples of selectable marker genes include drug resistance genes (neomycin resistance gene, DHFR gene, puromycin resistance gene, blastosidine resistance gene, hygromycin resistance gene, cycloheximide resistance gene), or fluorescent genes (genes encoding green fluorescent protein GFP, etc.).

[0054] There are no particular limitations on the method for introducing expression vectors into cells; for example, calcium phosphate methods, electroporation, liposome methods, gene gun methods, and lipofection methods can be used.

[0055] In the present invention, the average cell diameter of the cells is preferably 12 μm or more. When cells have an average cell diameter of 12 μm or more, oxygen deficiency due to the overlapping of the culture medium layer (boundary layer) around the cells tends to become significant. Therefore, the cell culture method of the present invention is particularly useful for culturing cells with an average cell diameter of 12 μm or more.

[0056] In the present invention, during cell culture, the lactate concentration in the culture medium is preferably less than 1.2 g / L, more preferably less than 1.0 g / L, even more preferably less than 0.8 g / L, and particularly preferably less than 0.6 g / L. The lactate concentration in the culture medium can be measured using a commercially available product such as the BioProfile400 from Nova Biomedical.

[0057] In the present invention, during cell culture, the concentration of lactate dehydrogenase in the culture medium is preferably less than 2000 U / L, more preferably less than 1800 U / L, even more preferably less than 1500 U / L, and particularly preferably 1200 U / L or less. The concentration of lactate dehydrogenase in the culture medium can be measured using a commercially available product such as Cedex Bio from Roche Diagnostics.

[0058] <Method for producing useful substances> The present invention relates to a method for producing useful substances, which includes causing cells to produce useful substances by culturing cells using the cell culture method of the present invention described above.

[0059] In the present invention, the type of useful substance is not particularly limited, but recombinant proteins are preferred. Examples of useful substances include recombinant polypeptide chains, recombinant secretory polypeptide chains, antigen-binding proteins, human antibodies, humanized antibodies, chimeric antibodies, mouse antibodies, bispecific antibodies, Fc fusion proteins, fragmented immunoglobulins, and single-chain antibodies (scFv).

[0060] The useful substances are preferably human antibodies, humanized antibodies, chimeric antibodies, or mouse antibodies. Examples of fragmented immunoglobulins include Fab, F(ab')2, and Fv. The antibody class is not particularly limited and can be any class such as IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, or IgM, but IgG and IgM are preferred when used as pharmaceuticals.

[0061] Human antibodies include all antibodies having one or more variable and constant regions derived from human immunoglobulin sequences. In one embodiment, all variable and constant domains are derived from human immunoglobulin sequences (full human antibody).

[0062] Humanized antibodies have sequences that differ from those of antibodies derived from non-human species by one or more amino acid substitutions, deletions, and / or additions, so that when administered to human subjects, the humanized antibody is less likely to induce an immune response and / or induce a less severe immune response compared to non-human species antibodies. In one example, certain amino acids in the heavy and / or light chain framework and constant domain of the non-human species antibody are mutated to produce a humanized antibody. In another example, the constant domain from a human antibody is fused to the variable domain of a non-human species.

[0063] A chimeric antibody is an antibody in which variable and constant regions of different origins are linked. For example, an antibody consisting of the variable regions of the heavy and light chains of a mouse antibody and the constant regions of the heavy and light chains of a human antibody is a mouse-human heterologous chimeric antibody. By linking the DNA encoding the variable region of a mouse antibody with the DNA encoding the constant region of a human antibody and incorporating this into an expression vector, a recombinant vector that expresses a chimeric antibody can be created. By culturing recombinant cells transformed with the above vector and expressing the incorporated DNA, the chimeric antibody produced during culture can be obtained.

[0064] Bispecific antibodies are antibodies produced by chemical methods or cell fusion that recognize two different antigen specificities. Methods for producing bispecific antibodies include linking two immunoglobulin molecules using crosslinking agents such as N-succinimidyl 3-(2-pyridyldithiol) propionate or S-acetylmercaptosuccinic acid anhydride, and linking Fab fragments of immunoglobulin molecules.

[0065] An Fc fusion protein refers to a protein that contains an Fc region, and includes antibodies. Fab is a monovalent fragment having VL, VH, CL, and CH1 domains. F(ab')2 is a divalent fragment having two Fab fragments linked by disulfide bridges in the hinge region. The Fv fragment contains the VL and VH domains of the single arm of the antibody. A single-chain antibody (scFv) is an antibody in which the VL and VH regions are joined via a linker (e.g., a synthetic sequence of amino acid residues) to form a continuous protein chain, where the linker is long enough to fold the protein chain over itself and form a monovalent antigen-binding site.

[0066] The useful substances produced by the culture described above can be purified. The separation and purification of these useful substances can be carried out using the same separation and purification methods used for ordinary proteins. For example, useful substances 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 are not limited to these methods. The concentration of the useful substances obtained above can be measured by absorbance measurement or enzyme-linked immunosorbent assay (ELISA), etc.

[0067] Examples of columns used in affinity chromatography include protein A columns and protein G columns. Other types of chromatography include ion exchange chromatography, hydrophobic chromatography, gel filtration, reversed-phase chromatography, and adsorption chromatography. These chromatography methods can be performed using liquid-phase chromatography such as HPLC (high-performance liquid chromatography) or FPLC (fast protein liquid chromatography).

[0068] Furthermore, useful substances can be modified or partially peptide-removed by treating them with an appropriate polypeptide-modifying enzyme before or after purification. Examples of polypeptide-modifying enzymes include trypsin, chymotrypsin, lysyl endopeptidase, protein kinase, and glucosidase.

[0069] The present invention will be described in more detail by the following examples, but the present invention is not limited to these examples. [Examples]

[0070] <Establishment of antibody-producing cells> Vectors containing nucleic acid sequences encoding IgG1 and IgG4 were constructed, and these vectors were introduced into CHO-DG44 cells to produce CHO-DG44 cells expressing IgG1 (IgG1 cells) and CHO-DG44 cells expressing IgG4 (IgG4 cells). The construction of the vectors and their introduction into the cells were carried out in accordance with Example 2 of Japanese Patent Publication No. 2016-517691. As a result, CHO cells that produce monoclonal antibodies were prepared.

[0071] <Cell culture method> Cell culture was performed using the above-mentioned CHO cells via perfusion culture. CD OptiCHO (ThermoFisher) was used as the culture medium, and the cell seeding density was set to 5 × 10⁻⁶. 5 The culture medium was set to cells / mL. The culture environment was maintained with a pH of 6.7 to 7.4, a culture temperature of 36°C to 38°C, and a CO2 concentration of 25% or less. Fresh culture medium was supplied and filtered culture solution was removed from the culture vessel from the second day onward.

[0072] For culture volumes of 1 L or less, a glass container with a diameter of 114 mm was used as the culture vessel, equipped with a paddle-shaped stirring blade with a diameter of 85 mm, and the predetermined amount of culture medium was filled and cultured. Filtration was performed using a Repligen ATF2 system with an RF02PES membrane.

[0073] For culture volumes greater than 1L but less than or equal to 3L, a glass container with a diameter of 143mm was used as the culture vessel, equipped with a propeller-type stirring blade with a diameter of 48mm, and filtration was performed using a Repligen ATF2 system with RF02PES membrane.

[0074] For culture volumes greater than 3L but less than or equal to 50L, a 350mm diameter plastic single-use bag was used as the culture vessel, equipped with a 116mm diameter propeller-type stirring blade, and a Repligen ATF4 single-use system was used for filtration.

[0075] For culture volumes greater than 50L but less than or equal to 500L, a 756mm diameter plastic single-use bag was used as the culture vessel, equipped with a 251mm diameter propeller-type stirring blade, and a Repligen ATF10 single-use system was used for filtration.

[0076] After reaching the target viable cell density, the cell suspension was removed from the culture vessel at least once a day, or continuously (cell bleeding), and the same amount of fresh medium was replenished to maintain the target viable cell density while keeping the predetermined volume of culture medium. The period from the start of culture until the first cell bleeding was started was defined as the cell proliferation phase, and the period from the first cell bleeding onward was defined as the antibody production phase (Figure 6). The culture medium was sampled daily, and the viable cell density, lactate concentration in the culture medium, and bleb formation rate were measured using the method described below. In addition, filtered culture permeate was sampled, and the concentration and quality of antibodies contained in the culture permeate were measured and evaluated.

[0077] <Evaluation Method and Judgment> The viable cell density was measured using Beckman Coulter's Vi-CELL XR. Cell proliferation was determined as follows: in the proliferation phase, a ratio of 1.25 times or more to the previous day's viable cell density was classified as A, a ratio of 1.1 times or more but less than 1.25 times was classified as B, and a ratio of less than 1.1 times was classified as C. In the antibody production phase, a ratio of 1.15 times or more to the previous day's viable cell density was classified as A, a ratio of 1.05 times or more but less than 1.15 times was classified as B, and a ratio of less than 1.05 times was classified as C.

[0078] In cell culture, lactate concentration is generally considered one of the key indicators of oxygen deficiency. The lactate concentration in the culture medium was measured using Nova Biomedical's BioProfile400. During the cell proliferation phase, lactate concentration was not used to determine the presence or absence of oxygen deficiency. During the antibody production phase, a lactate concentration of less than 0.6 g / L was classified as A, a concentration of 0.6 g / L or more but less than 1.2 g / L was classified as B, and a concentration of 1.2 g / L or more was classified as C.

[0079] Bleb formation is a known characteristic of apoptosis, where blister-like projections form on the cell membrane surface. Previous studies have shown that bleb formation rates increase when cultured cells are deprived of oxygen. The bleb formation rate was calculated by counting the total number of cells and the number of cells with blister formation on their surface in 10 or more randomly taken microscopic images of cell suspensions with a field of view of 0.3 mm to less than 3 mm, and then determining the ratio of blister-forming cells to the total number of cells. Cases with a bleb formation rate of less than 5% were classified as A, cases with a rate of 5% or more but less than 20% as B, and cases with a rate of 20% or more as C.

[0080] The presence or absence of oxygen deficiency was determined by comprehensively evaluating the viable cell density, lactate concentration, and bleb formation rate. Oxygen deficiency was determined to have occurred if one or more cells with B and C were present, or if two or more cells with C were present.

[0081] <Measurement and evaluation of antibody concentration and antibody quality in culture permeate> Antibody concentrations in the culture permeate were measured using Cedex Bio from Roche Diagnostics.

[0082] The quality of the antibody in the culture supernatant was evaluated by measuring and evaluating the purity, sugar chains, and charge of the antibody.

[0083] The purity was evaluated by two methods. The first was performed using a column of Tosoh's TSKgel G3000WXL (7.8 mm × 300 mm). The ratio of the maximum peak area to the total peak area at 4 to 11 minutes of the chromatogram was evaluated as the monomer.

[0084] The second was performed using SCIEX's CE PA800 plus. The ratio of the heavy chain partial area to the total area was calculated from the results of the non-reducing electrophoregram.

[0085] The sugar chains were evaluated by G0F, G1F, and Man5, which are indicators of antibody maturity. The measurement was performed using a column of Thermo Fisher Scientific's ODS HYPERSIL 3μm (2.1 mm × 150 mm). The ratio of the peak area of each of G0F, G1F, and Man5 to the total peak area at 22 to 46 minutes of the chromatogram was calculated.

[0086] The charge measurement was performed using a column of Thermo Fisher Scientific's ProPac WCX 5μm (4 mm × 250 mm). The maximum peak at 10 to 60 minutes of the chromatogram was defined as Neutral, the peak that appeared before Neutral was defined as Acidic, and the peak that appeared after Neutral was defined as Basic, and the ratio of the peak area of each to the total peak area was calculated.

[0087] <Measurement method of <kcella>> With continuous stirring of the culture medium and oxygen supply, in a steady culture state (steady state) where the dissolved oxygen concentration in the culture medium maintained a predetermined set value (CL0) for 24 hours or more, the oxygen supply was stopped, and this moment was set as the temporal starting point (t = 0). The dissolved oxygen concentration CL0 at the starting point and the change in the dissolved oxygen concentration CL in the liquid over 60 seconds at 3-second intervals from the starting point were measured, and Ln(CL / CL0) was calculated. Ln(CL / CL0) was plotted against the elapsed time t, and the slope obtained by linear approximation was defined as -kcella.

[0088] <Method for calculating the turbulent energy dissipation rate ε> The turbulent energy dissipation rate ε can be calculated using commercially available CFD (Computational Fluid Dynamics) software. In this embodiment, it was calculated using Fluent from ANSIS. The software's standard mass conservation equation, momentum conservation equation, k-ε model transport equation, and modified wall-treated ε equation were combined. A mesh was created that simulated the culture tank shape, impeller shape, and predetermined liquid volume. Fluid viscosity, fluid density, stirring speed, rotation angle, gravity direction, and liquid surface boundary conditions were set as parameters, and the turbulent energy dissipation rate was calculated by performing a convergence calculation. Note that "creating a mesh" generally means dividing the object to be analyzed into simple-shaped elements in the software when performing numerical analysis, applying defined models and equations to each element to solve the calculations, and then combining the calculation results of each element to obtain the analysis result for the entire object.

[0089] <Methods for evaluating cell damage> Cell damage was evaluated by measuring LDH (lactate dehydrogenase) in the culture medium. The sampled culture medium was centrifuged at 300G for 5 minutes, and the supernatant was measured using a Cedex Bio analyzer from Roche Diagnostics. LDH levels below 2000 U / L were classified as A (no problem), levels between 2000 U / L and 3000 U / L were classified as B (not practically desirable), and levels above 3000 U / L were classified as C (difficult to continue culturing).

[0090] The results of the above measurements and evaluations are shown in Tables 1-4 and Figures 7-9. As shown in Table 2, when the conditions of Formula 1 or Formula 2 of the present invention were met (Examples 1 to 7), oxygen deficiency did not occur. However, when the conditions of Formula 1 and Formula 2 were not met (Comparative Examples 1 to 4), oxygen deficiency occurred.

[0091] [Table 1]

[0092] [Table 2]

[0093] [Table 3]

[0094] [Table 4]

Claims

1. The density of living cells is 60 x 10 ６ A cell culture method in which, in a cell culture with cells / mL or higher, the relationship between the stirring power per unit volume P / V, the dissolved oxygen concentration CL0, and the viable cell density VCD satisfies the conditions of Equation 1 below, wherein the cells are Chinese hamster ovary cells. Equation 1: 0.0059 × exp [0.0243 × (P / V)] × CL0 / VCD ≥ 1.10 × 10 －１７ During the ceremony, CL0 indicates the dissolved oxygen concentration [mol / mL]; VCD indicates the live cell density [cells / mL]: P / V is the stirring power per unit volume [W / m²] defined by the following formula. ３ [Indicates:] P / V=ρ × (n / 60) ３ × Di ５ / V × Np During the ceremony, ρ is the density of the culture medium [kg / m³] ３ ] indicates; n represents the stirring speed [rpm]; Di represents the diameter of the stirring blade [m]; V is the volume of culture medium [m³] ３ ] indicates; Np represents the stirring power coefficient.

2. The cell culture method according to claim 1, wherein the stirring power P / V per unit volume satisfies 60 ≤ P / V ≤ 2000.

3. The cell culture method according to claim 1 or 2, wherein the stirring rotation speed n satisfies 120 ≤ n ≤ 600.

4. The relationship between stirring power per unit volume P / V, dissolved oxygen concentration CL0, and living cell density VCD is, 0.0059×exp[0243×(P / V)]×CL0 / V4≦4.00×10 －１７ A cell culture method according to any one of claims 1 to 3, which satisfies the conditions.

5. A cell culture method according to any one of claims 1 to 4, wherein the cell culture period that satisfies the conditions of formula 1 is 20 days or longer.

6. A cell culture method according to any one of claims 1 to 5, wherein the condition of formula 1 is satisfied throughout the entire cell culture period.

7. The viable cell density is 60×10 ６ In cell culture where the viable cell density is 60×10 ６ cells / mL or more, a cell culture method in which the relationship between kcell a defined below, the dissolved oxygen concentration CL0 in the steady state, and the viable cell density VCD satisfies the conditions of the following formula 2, and the cells are Chinese hamster ovary cells. Equation 2: kcella×CL0 / VCD ≥ 1.00×10 －１７ [mol / s / cell] During the ceremony, CL0 indicates the dissolved oxygen concentration [mol / mL]; VCD indicates the live cell density [cells / mL]: kcella is, Ln(CL / CL0)=-kcella×t This indicates the total oxygen transfer capacity coefficient [ / s] from the culture medium to the cells, as defined by: During the ceremony, The steady state is defined as the moment when the dissolved oxygen concentration in the culture medium is maintained at a predetermined set value, CL0, for 24 hours or more by continuously stirring the culture medium and supplying oxygen. The moment when the oxygen supply is stopped while stirring continues from the steady state is defined as the temporal starting point. The dissolved oxygen concentration CL0 at the starting point and the change in the dissolved oxygen concentration CL in the liquid over 60 seconds from the starting point are measured, and the slope obtained by linearly approximating the change in Ln(CL / CL0) with respect to elapsed time t using the least squares method is defined as -kcella.

8. The cell culture method according to claim 7, wherein kcella satisfies 0.03 ≤ kcella ≤ 0.

39.

9. The relationship between kcella, the steady-state dissolved oxygen concentration CL0, and the viable cell density VCD is as follows: kcella×CL0 / VCD ≦ 4.00×10 －１７ [mol / s / cell] A cell culture method according to claim 7 or 8 that satisfies the conditions.

10. A cell culture method according to any one of claims 7 to 9, wherein, for a target culture vessel and liquid volume, the correlation between the calculated value of the turbulent energy dissipation rate ε of the culture medium and the measured value of kcella when the stirring speed is changed is determined in advance, and the culture is controlled to satisfy the conditions of Equation 2 by increasing the stirring power as the cells proliferate, thereby increasing the kcella obtained from the above correlation.

11. A cell culture method according to any one of claims 7 to 9, wherein the culture is controlled to satisfy the conditions of formula 2 by increasing at least one of kcella and CL0 in conjunction with the increase in VCD.

12. A cell culture method according to any one of claims 7 to 11, wherein the cell culture period that satisfies the conditions of formula 2 is 20 days or longer.

13. A cell culture method according to any one of claims 7 to 12, wherein the condition of formula 2 is satisfied throughout the entire cell culture period.

14. CL0 is 0.45 × 10 －８ ≤CL0 ≤ 6.75 × 10 －８ The cell culture method according to any one of claims 1 to 13.

15. The density of living cells is 80 x 10 ６ A cell culture method according to any one of claims 1 to 14, wherein the cell concentration is cell / mL or higher.

16. A cell culture method according to any one of claims 1 to 15, wherein the amount of culture medium is 50 L or more.

17. A cell culture method according to any one of claims 1 to 16, wherein the method is perfusion culture.

18. A cell culture method according to any one of claims 1 to 17, wherein the average cell diameter of the cells is 12 μm or more.

19. A cell culture method according to any one of claims 1 to 18, wherein the lactic acid concentration in the culture medium is less than 1.2 g / L during cell culture.

20. A cell culture method according to any one of claims 1 to 19, wherein the concentration of lactate dehydrogenase in the culture medium is less than 2000 U / L during cell culture.

21. A method for producing a useful substance, comprising culturing cells by a cell culture method described in any one of claims 1 to 20 to cause cells to produce a useful substance.

22. A method for producing a useful substance according to claim 21, wherein the useful substance is a recombinant protein.

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