A bubble column bioreactor for cell suspension culture and a cell culture method

Abstract

The present application belongs to the field of animal cell culture bioreactor, and particularly relates to a reactor for animal cell suspension culture based on a bubbling bed and a culture method thereof, which comprises a reactor tank, a drainage pipe, a drainage stirring paddle, a microporous aeration device and an aeration disc. By respectively designing independent micro-bubble aeration devices and aeration discs, the supply of oxygen and the removal of carbon dioxide in the culture solution are performed in two parts, the mass transfer coefficients of oxygen and carbon dioxide are simultaneously improved, the demand of cell oxygen supply and carbon dioxide removal is met, the ventilation volume is reduced, and the cell damage caused by ventilation is reduced. Compared with the existing technology, for the same oxygen demand cells, the average ventilation volume is reduced by 63.6%, the maximum ventilation volume is reduced by 60.7%, and the damage of ventilation to cells is greatly reduced.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of animal cell culture bioreactor, in particular to a reactor based on bubble column for animal cell suspension culture and its culture method. BACKGROUND

[0002] Animal cells are cultured on a large scale for the production of various therapeutic proteins, vaccines and antibodies. A large amount of energy is required in the process of animal cell growth and reproduction and synthesis of target products. In this process, cells consume a large amount of oxygen and produce carbon dioxide. In CHO cell culture, the dissolved oxygen level is usually maintained at 10% to 80% of air saturation. Too high oxygen concentration will lead to the accumulation of reactive oxygen species (ROS), which in turn changes the mitochondrial respiratory chain and intracellular redox reactions, ultimately leading to cell growth inhibition and reduced specific productivity. Hypoxic conditions will induce ROS accumulation and also promote the increase of lactic acid. In mammalian cell culture, the dissolved carbon dioxide (pCO2) level is usually maintained at 4% to 10% CO2 saturation (30 to 70 mmHg). High concentration of dissolved carbon dioxide (150 to 200 mmHg) will have adverse effects on cell growth and product expression. In CHO cell culture, high pCO2 can inhibit cell growth, product expression and change product quality. Carbon dioxide dissolved in water forms carbonate by reacting with water, thereby causing acidification, and carbonate decomposes into carbonic acid. When carbon dioxide is higher than a certain level, the pH environment in the cell is affected, and cell growth and product yield will be inhibited, and the quality attributes of antibodies (such as N-glycosylation, antibody charge heterogeneity, molecular size) will also be affected. In addition, the acidification of the culture medium leads to the addition of alkali (usually sodium bicarbonate) to the culture medium for pH control, resulting in an increase in osmotic pressure. Compared with normal levels (260 to 320 mOsm / kg) of osmotic pressure, elevated osmotic pressure (460 to 500 mOsm / kg) will lead to reduced cell density and viability. Although carbon dioxide can have adverse effects on cell culture, it is still essential for nucleic acid metabolism and synthesis, so its concentration cannot be too low. Compared with normal pCO2 levels (28 to 54 mmHg), ultra-low pCO2 levels (12.5 to 24.5 mmHg) will reduce viable cell density and cell viability.

[0003] Large scale animal cell culture faces the problem of removing (or resolving) carbon dioxide produced by cells at approximately the same molar rate as oxygen is consumed. In large reactors, top aeration is often not sufficient to remove carbon dioxide. Therefore, the bottom aeration rate and agitation speed must be adjusted to ensure carbon dioxide resolution. However, the shear stress generated by bubble breakage at the gas-liquid interface during aeration damages a large number of cells. Since animal cells lack cell walls and are not tolerant to shear stress, low rotation speed and air flow rate are controlled during the culture process. Therefore, oxygen deficiency and carbon dioxide accumulation are prone to occur during scale-up culture. The industry has taken various ways to reduce the damage to cells caused by bubble breakage. For example, protective agents such as PF68 are used to reduce the cells around the bubbles during bubble breakage, semi-permeable membranes without bubbles are used for oxygen exchange, and surface aeration is used instead of deep aeration. However, these methods cannot completely solve the damage to cells during aeration. For specific shear-sensitive cells, the cell damage caused by aeration cannot be ignored.

[0004] Numerous studies have shown that small bubbles are beneficial to oxygen mass transfer, but not conducive to carbon dioxide removal; while large bubbles are more conducive to carbon dioxide removal, but not conducive to oxygen mass transfer. In addition, numerical simulation and experimental observation of bubble breakage process show that smaller diameter bubbles cause more serious damage to cells. The differences in oxygen supply, carbon dioxide removal, and cell damage caused by bubble breakage require different bubble sizes, which complicates the regulation strategy. How to avoid cell loss caused by bubble breakage while meeting the oxygen demand of cells and avoiding carbon dioxide accumulation becomes a problem to be solved. SUMMARY

[0005] The purpose of the present application is to provide a bubble column bioreactor for animal cell suspension culture and a cell culture method thereof to solve the problems raised in the above technical background.

[0006] To solve the above technical problems, the present application provides the following technical solutions:

[0007] The first aspect of the present application discloses a bubble column bioreactor, comprising a reactor tank, a draft tube, a draft stirring paddle, a microporous aeration device, and an aeration disc.

[0008] The reactor tank is used to contain the culture solution.

[0009] The draft tube is arranged in the reactor tank and is used to guide the flow direction of the culture solution in the reactor tank.

[0010] The draft stirring paddle is arranged in the draft tube and is used to provide the power for the downward flow of the culture solution along the draft tube.

[0011] The micro-porous aeration device is arranged in the draft tube and is located at the lower part of the draft tube, and is used for providing micro-bubbled pure oxygen bubbles.

[0012] The aeration disc is arranged below the inside of the reactor and is located outside the draft tube, and is used for providing large bubbles for facilitating carbon dioxide removal.

[0013] The reactor is further provided with a cylindrical agitator, which is located outside the draft tube and is composed of 3-6 solid cylinders, and is used for assisting the mixing of the culture solution.

[0014] The draft tube is located below the liquid level of the culture solution, and the cross-sectional area of the draft tube decreases from top to bottom.

[0015] The micro-porous aeration device and the draft agitator are arranged in the middle and lower part of the draft tube.

[0016] The second aspect of the present application discloses a specific method for using the above-mentioned reactor for cell culture, and the steps are as follows:

[0017] S1: adding the culture medium into the reactor, and passing the sterilized air or oxygen into the reactor through the aeration disc;

[0018] S2: introducing the seed cells into the reactor, turning on the micro-porous aeration device, generating micro-bubbled pure oxygen bubbles in the draft tube, controlling the rotation speed of the draft agitator, and controlling the pure oxygen bubbles in the draft tube;

[0019] S3: the dissolved oxygen content in the culture solution is associated with the aeration amount of the micro-porous aeration device, and the carbon dioxide partial pressure in the culture solution is associated with the aeration amount of the aeration disc, and in the culture process, the pure oxygen bubbles formed by the micro-porous aeration device supply oxygen, and the large bubbles formed by the aeration disc remove carbon dioxide.

[0020] Preferably, the reactor is subjected to steam sterilization before the culture solution is added in S1, and the culture solution is filtered and sterilized.

[0021] Preferably, the aeration disc in S1 provides large bubbles with a diameter of 0.5-1mm. The micro-porous aeration device in S2 provides pure oxygen bubbles with a diameter of 20-100μm. The dissolved oxygen in the culture solution in S3 is (40±20) % of the air-saturated dissolved oxygen. The carbon dioxide partial pressure in the culture solution in S3 is lower than 100mmHg.

[0022] The present application has the following beneficial effects:

[0023] 1. The bubble column bioreactor designed by the present application balances the buoyancy of the rising bubbles by the arrangement of the draft tube and the draft stirring paddle, retains the oxygen-rich micro-bubbles below the liquid surface, provides higher oxygen mass transfer efficiency, and avoids the damage of small bubble rupture to cells; pure oxygen is continuously or intermittently introduced through the microporous aeration device, and the stirring paddle below the draft tube promotes the downward flow of the culture medium. Due to the small cross-sectional area of the upper part of the draft tube, the liquid flow rate is faster, and the cross-sectional area of the lower part is large, so the fluid flow rate is slow. The resistance of the bubble in the upper part is greater than the liquid buoyancy, and the bubble cannot flow out of the draft tube or escape from the draft tube below the draft tube. Therefore, the bubble will not rupture at the gas-liquid interface. The bubble will form a region rich in small bubbles in the draft tube, and eventually the bubble will be completely dissolved in the culture medium.

[0024] 2. By separately designing independent micro-bubble aeration devices and aeration discs, the oxygen supply and carbon dioxide removal in the culture solution are performed in two parts, which simultaneously improves the mass transfer coefficients of oxygen and carbon dioxide, meets the needs of cell oxygen supply and carbon dioxide removal, reduces the aeration volume, and reduces the cell damage caused by aeration. Compared with the existing technology, the average aeration volume of cells with the same oxygen demand is reduced by 63.6%, and the maximum aeration volume is reduced by 60.7%, which greatly reduces the damage of aeration to cells.

[0025] 3. In the process of animal cell culture, by dividing the oxygen supply and carbon dioxide removal into two parts, the dissolved oxygen and carbon dioxide partial pressure in the culture solution are regulated respectively, which reduces the mutual interference of the two regulations and simplifies the regulation strategy. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 is a schematic diagram of the reactor;

[0027] Figure 2 is the oxygen demand in the process of cell culture;

[0028] Figure 3 is the gas input required to reach the oxygen supply in Figure 2 and control pCO2 less than 100 mmHg before and after the improvement of the method. DETAILED DESCRIPTION

[0029] In the following, the present application will be described in detail in conjunction with specific examples, and the examples given are only to illustrate the present application, but not to limit the scope of the present application.

[0030] Example 1:

[0031] In a 10L reactor, 7L of culture medium was added, and fed-batch culture was carried out according to 3% of the initial volume per day. The dissolved oxygen was controlled at (40±20)% of air-saturated dissolved oxygen during the culture period, and the volume power was 23W / m3 , pH 7.0±0.2, carbon dioxide partial pressure 100 mmHg, and the cell number and residual sugar were determined every day. The initial cell seeding density was (0.5-1.0) x 10 6 cells / mL. The culture time was 16 days.

[0032] In Example 1, the aeration amount associated with the dissolved oxygen was correlated with the micro-porous aeration device, 20-100 μm bubbles were provided by the micro-porous aeration device, the carbon dioxide partial pressure was correlated with the aeration disc aeration amount, 0.5-1 mm large bubbles were provided by the aeration disc, the flow stirring paddle speed was controlled to avoid micro-bubbles escaping from the flow pipe (so that pure oxygen micro-bubbles cannot rise to the liquid surface and are trapped below the liquid surface).

[0033] Comparative Example:

[0034] The conventional reactor was used, only including the reactor tank, flow pipe, flow stirring paddle and micro-porous aeration device, the dissolved oxygen was correlated with the bottom aeration, pure oxygen was introduced, and the rest of the conditions were the same as in Example 1.

[0035] In the cell culture process of Example 1 and the comparative example, under the condition of simultaneously meeting the same oxygen supply capacity and carbon dioxide resolution capacity, the required aeration amount is shown in Table 1. Figure 3 During the culture process, the cell oxygen demand was the most vigorous (D13), and the conventional aeration method required 0.154 vvm aeration amount; while the aeration amount of the reactor of the present application was only 0.060 vvm, and the aeration amount was reduced by 60.7%; during the entire culture period, the aeration amount of the conventional method was 0.073 vvm, and that of the new reactor was 0.027 vvm, and the new reactor was reduced by 63.6%.

[0036] The above examples are the preferred embodiments of the present application, but the embodiments of the present application are not limited by the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application are equivalent replacement methods, and are included in the protection scope of the present application.

Claims

1. A bubble column bioreactor, characterized in that, The reactor comprises a reactor tank, a flow guide pipe, a flow guide stirring paddle, a microporous aeration device, and an aeration disc. The reactor tank is used for containing the culture solution. The flow guide pipe is arranged in the reactor tank and used for guiding the flow direction of the culture solution in the reactor tank. The flow guide stirring paddle is arranged in the flow guide pipe and used for providing the power for the downward flow of the culture solution along the flow guide pipe. The microporous aeration device is arranged in the lower part of the flow guide pipe and used for providing microporous pure oxygen bubbles. The aeration disc is arranged below the reactor and outside the flow guide pipe and used for providing large bubbles for facilitating the removal of carbon dioxide. The flow guide pipe is below the liquid level of the culture solution, and the cross-sectional area of the flow guide pipe decreases from bottom to top.

2. A bubble column bioreactor according to claim 1, wherein The reactor further comprises a cylindrical stirrer which is arranged outside the flow guide pipe and comprises 3-6 solid cylinders.

3. A bubble column bioreactor according to claim 1, wherein The microporous aeration device and the flow guide stirring paddle are arranged in the middle part of the flow guide pipe.

4. The method for cell culture using the reactor according to any one of claims 1-3, and the specific steps are as follows: S1: adding the culture solution into the reactor and passing the sterilized air or oxygen into the reactor through the aeration disc; S2: adding the seed cells into the reactor and opening the microporous aeration device to generate microporous pure oxygen bubbles in the flow guide pipe and controlling the rotation speed of the flow guide stirring paddle to control the pure oxygen bubbles in the flow guide pipe; S3: correlating the oxygen saturation in the culture solution with the ventilation amount of the microporous aeration device and correlating the carbon dioxide partial pressure in the culture solution with the ventilation amount of the aeration disc, and in the culture process, the pure oxygen bubbles formed by the microporous aeration device supply oxygen and the large bubbles formed by the aeration disc remove carbon dioxide.

5. The method of culturing cells according to claim 4, wherein, S1: sterilizing the reactor by steam before adding the culture solution and filtering the culture solution.

6. The method of culturing cells according to claim 4, wherein, S1: the aeration disc provides 0.5-1mm large bubbles.

7. The method of culturing cells according to claim 6, wherein, S2: the microporous aeration device provides 20-100μm pure oxygen bubbles.

8. The method of culturing cells according to claim 7, wherein, S3: the oxygen saturation in the culture solution is 40±20% of the air saturated oxygen saturation.

9. The method of culturing cells according to claim 8, wherein, S3: the carbon dioxide partial pressure in the culture solution is lower than 100mmHg.

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