A culture method for improving cell resistance to high pCO2 environmental pressure

Abstract

The present invention relates to a culture method for improving cell resistance to high pCO2 environmental pressure, which specifically comprises the following steps: a) inoculating cells and culturing at 36-37°C; b) culturing cells at a temperature of 8-30×10 6 cells / ml, lowering the culture temperature to 30-32°C, introducing CO2 to lower the pH from a first pH in the range of 7.00±0.20 to a pH in the range of 6.7-6.90, wherein the first pH is greater than the second pH, and maintaining the pH until the cells are harvested; c) in step b), simultaneously with lowering the temperature, adding acetazolamide at a concentration of 50 μM-150 μM; and d) harvesting the cells and collecting the antibody protein product. Also provided are alternative methods for increasing the CO2 partial pressure in step b), and the corresponding use of acetazolamide for reducing lactic acid accumulation in cell culture and reducing the pentamethylene glycol ratio of the antibody protein produced after cell culture.

Description

Technical Field

[0001] The present invention belongs to the field of biopharmaceuticals, in particular to the technical field of mammalian cell batch fed-batch culture, and relates to a method for reducing the modification ratio of pentamethylene glycol (Man5) in antibody proteins under low pH culture conditions by resisting high carbon dioxide partial pressure. Background Art

[0002] In the biopharmaceutical field, antibody drugs are widely used in disease treatment. High-mannosylation is a common immature glycoform in antibody N-glycosylation modifications. The high-mannosylation form of antibodies expressed in CHO cells is primarily Man 5 (pentamannose). A high proportion of Man 5 increases the clearance efficiency of antibodies in the human body, thereby affecting the pharmacokinetic and pharmacodynamic parameters of antibody drugs. Therefore, Man 5 levels are often an important quality parameter for antibody drugs, and reducing Man 5 levels is often a goal pursued during the development of new drugs or biosimilars. Currently, the relationship between pH and Man 5 levels and how to control them are unclear in this field.

[0003] On the contrary, during the development or production of cell culture processes, it is often unavoidable that the pCO2 in the culture system is too high. During scale-up production, due to the enhanced O2 mass transfer capacity of large-scale bioreactors, the difficulty of CO2 overflow increases, pCO2 increases, and the pH of the culture medium decreases. Therefore, a large amount of alkaline solution Na2CO3 needs to be added, which exacerbates the increase in pCO2, thus forming a vicious cycle. During process development, in order to meet the needs of regulating the quality of certain product proteins, such as regulating the proportion of sugar forms, traditional low-pH processes need to be adopted, and CO2 will also be introduced in large quantities, thus generating higher pCO2. A large amount of research data shows that cells are prone to abnormal aerobic metabolism in a high pCO2 environment, and lactic acid continues to accumulate, accompanied by a decrease in cell viability, reduced protein production and changes in protein quality. In severe cases, it may lead to the failure of the entire batch production or process application [1]. Although this problem has caused industry practitioners and scholars to conduct extensive research on why high pCO2 has such a serious negative impact and how to reduce pCO2 in the culture system, there is currently no effective method to improve cell resistance to high pCO2 environmental pressure [2].

[0004] Some studies have suggested that excessive CO2 produces a large amount of H+ and HCO3- after intracellular hydration, resulting in excessive acidification of the intracellular environment. At the same time, HCO3- may form peroxide HCO4-. These changes may cause irreversible damage to mitochondrial function, thereby leading to deterioration of cell performance in a high pCO2 environment [3].

[0005] In fact, the key hydration reaction of CO2 in cells does not occur naturally after dissolution in the cytoplasm, but is a rapid reaction catalyzed by carbonic anhydrase (CA, EC 4.2.1.1) to quickly meet the needs of the substrate HCO3- in the glycolysis and aerobic metabolic pathways. Mammalian cells have a total of 14 carbonic anhydrase isozymes, of which CA II located in the cytoplasm and CA V located on the mitochondrial membrane are mainly responsible for the reversible hydration reaction of CO2. CA V is the only carbonic anhydrase located in the mitochondria and is also one of the key enzymes to maintain the normal operation of the mitochondrial TCA cycle. Its catalytic conversion constant (kcat) for CO2 is as high as 3×105s-1[4]. In a high pCO2 environment, its catalysis of excessive hydration of CO2 may be the most direct cause of mitochondrial damage. According to previous studies, sulfonamides have a strong inhibitory effect on CA enzymes, among which acetazolamide is the most effective, with an inhibition constant of 58 nM. It also has the same level of inhibitory effect on CA II (Ki = 10 nM), which has the same function, while its inhibitory effect on other isoenzymes is weaker [5].

[0006] However, the prior art does not provide any method for protecting cells from environmental stress under high pCO2 while avoiding the production of antibodies with high Man5 glycoform modification. Summary of the Invention

[0007] To this end, an object of the present invention is to provide a method for effectively maintaining low pH culture conditions and reducing Man5 levels during antibody production, which method comprises adding acetazolamide during the culture of cells expressing the antibody.

[0008] Another object of the present invention is to provide the use of acetazolamide in effectively maintaining low pH culture conditions and reducing Man5 levels during antibody production.

[0009] In one aspect of the present invention, a method for reducing pentamethylene glycol in an antibody protein produced by cell culture using acetazolamide is provided, the method comprising the following steps:

[0010] a) inoculating cells and culturing at 36-37° C., preferably 36.5° C.;

[0011] b) When the cells grow to 8-30x10 6 The cell density of cells / ml is preferably 15.0x10 6 cells / ml, lowering the culture temperature to 30-32°C, preferably 31°C, and introducing CO2 on the cooling day to lower the pH from a first pH in the range of 7.00±0.20 to a second pH in the range of 6.7-6.90, preferably 6.80, wherein the first pH is greater than the second pH, and maintaining the pH until the cells are harvested;

[0012] c) in step b), adding acetazolamide while lowering the temperature, at a concentration of 50 μM to 150 μM, preferably 150 μM; and

[0013] d) Harvesting the cells and collecting the antibody protein product.

[0014] In another aspect of the present invention, a method for producing an antibody is provided, the method comprising the steps of:

[0015] a) inoculating cells and culturing at 36-37° C., preferably 36.5° C.;

[0016] b) When the cells grow to 8-30x10 6 The cell density of cells / ml is preferably 15.0x10 6 cells / ml, lowering the culture temperature to 30-32° C., preferably 31° C., and increasing the pCO2 from a first pCO2 of 20 mmHg-60 mmHg to a second pCO2 of greater than 80 mmHg, preferably 80 mmHg-200 mmHg, on the cooling day, wherein the second pCO2 is higher than the first pCO2, and the pCO2 is maintained until the cells are harvested, optionally by increasing the carbon dioxide ventilation ratio from less than 6% to 8-30%, preferably 15% CO2;

[0017] c) in step b), adding acetazolamide while lowering the temperature, at a concentration of 50 μM to 150 μM, preferably 150 μM; and

[0018] d) Harvesting the cells and collecting the antibody protein product.

[0019] In a preferred embodiment of the above aspects of the present invention, in step (a), the pH is maintained in the range of 7.0±0.20.

[0020] In another preferred embodiment of the above aspect of the present invention, in step (a), pCO2 is maintained at 6% or below.

[0021] In another preferred embodiment of the above aspect of the invention, the cells are cultured in step (a) with a pCO2 partial pressure of 20 mmHg-60 mmHg, preferably 30 mmHg. Optionally, the pCO2 partial pressure is achieved by introducing carbon dioxide with a ventilation ratio of less than 6%.

[0022] In one embodiment of the present invention, the cell is a mammalian cell, which can be derived from mammals, such as humans and non-human primates, as well as rabbits, rats, mice, goats, pigs and other mammalian species. In a preferred embodiment, the mammal is a human. In another embodiment, the cell is preferably a CHO cell.

[0023] In one embodiment of the present invention, the antibody protein is a monoclonal antibody or a polyclonal antibody, or an antigen-binding fragment thereof. In a preferred embodiment, the antibody is an IgG antibody selected from IgG1, IgG2, IgG3, IgG4, and a fusion protein having an IgG Fc-terminal structure. In a more preferred embodiment, the antibody protein is selected from IgG1 and a fusion protein having an IgG Fc-terminal structure.

[0024] In another aspect of the present invention, there is provided a use of acetazolamide for reducing lactic acid accumulation in cell culture, characterized in that 50-150 μM acetazolamide is added during cell culture at a pH lower than 7.0, preferably pH 6.8, or a CO2 partial pressure greater than 80 mmHg, or a CO2 ventilation ratio greater than 6%, preferably 8-30%.

[0025] In another aspect of the present invention, there is provided the use of acetazolamide for reducing the pentamethylene glycol ratio of an antibody protein produced after cell culture, characterized in that 50-150 μM acetazolamide is added during cell culture at a pH lower than 7.0, preferably pH 6.8, or a CO2 partial pressure greater than 80 mmHg or a CO2 ventilation ratio greater than 6%, preferably 8-30%.

[0026] The advantages of the present invention are that acetazolamide is used to eliminate high pCO2 pressure during cell culture for antibody production, improving lactic acid accumulation during low pH and high pCO2 culture, maintaining ideal cell viability and production, and thereby improving the cells' ability to withstand high CO2 partial pressures. Furthermore, pentamerin levels are kept low during cell culture, providing a stabilizing effect on maintaining ideal antibody pharmacokinetics and pharmacodynamics.

[0027] Other features and advantages of various embodiments will be described in part in the following description, and in part will be apparent from the description, or can be learned through the practice of various embodiments. The objectives and other advantages of various embodiments will be realized and achieved through the elements and combinations particularly pointed out in the description and the appended claims.

[0028] Unless otherwise indicated, the reagents, cells, and instruments used in the present invention are commonly commercially available and publicly available. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The graph shows the change of viable cell density over time in the experimental group after relieving high pCO2 pressure. It can be seen that the viable cell density in the control group under high pCO2 decreases more dramatically over time than that in the group treated with acetazolamide.

[0030] Figure 2The graph shows the changes in cell viability over time in the experimental group that was relieved of high pCO2 pressure. It can be seen that under high CO2 partial pressure, the cell viability of the control group decreased by 10% on day 14, while that of the acetazolamide group decreased by less than 5%.

[0031] Figure 3 The graph shows the change in lactate concentration over time in the high pCO2 pressure experimental group. It can be seen that after day 10, lactate accumulation in the control group increased significantly, reaching 2-3 times that of the two different acetazolamide concentration groups.

[0032] Figure 4 The offline pCO2 time curve of the experimental group that relieved high pCO2 pressure is shown. It can be seen that acetazolamide does not lead to a substantial reduction in the CO2 pressure in the environment, but maintains high CO2 pressure, consistent with the control group. Acetazolamide improves other physiological parameters and metabolic rate under this pressure, as shown by Figure 2 and Figure 3 .

[0033] Figure 5 The graph shows the change in viable cell density over time for the acetazolamide-low pH process. It shows that the viable cell density at low pH after cooling without acetazolamide was significantly lower than that with acetazolamide; the latter was essentially equivalent to the viable cell density of cells cultured at high pH.

[0034] Figure 6 The graph shows the change in cell viability over time in the acetazolamide-low pH process combination group. In both the high pH (7.2) control group and the pH 6.8±0.1 group without acetazolamide, cell viability dropped sharply after 12 days. Under the pressure of low pH and high CO2, a large number of cells died, with a mortality rate of nearly 30% at harvest (nearly 14 days). However, the group with acetazolamide significantly alleviated this stress, with less than 10% cell death.

[0035] Figure 7 A graph showing the change in lactate concentration over time in the acetazolamide-low pH process combination group shows similar lactate concentration trends around day 14 in the low pH + acetazolamide group and the high pH control group, while lactate concentration increased dramatically in the low pH group without acetazolamide.

[0036] Figure 8The online pH curve over time for the acetazolamide combined with low pH process is shown. It can be seen that in the experimental group (pH 6.8 ± 0.1), the pH strategy was adjusted to 6.8 ± 0.1 one day after the cooling day. The pH then dropped significantly to 6.90 and remained at this level until the end of the culture. In the control group (pH 7.0 ± 0.2), the pH gradually recovered to 7.20 after cooling. A slight fluctuation occurred on the 12th day (dropping to 7.05 and then rebounding to 7.20), which may be caused by the slight fluctuation in lactate on that day. The pH of the experimental group remained stable after the addition of acetazolamide.

[0037] Figure 9 The offline pCO2 profile for the acetazolamide combined with low pH is shown. This graph shows that the pCO2 partial pressure remains stable at high pH, ​​while at low pH and with high pCO2 addition, the CO2 partial pressure remains above 80 mmHg, causing stress to the culture.

[0038] Figure 10 The figure shows the protein yield and quality results for the acetazolamide-low pH process. As shown in the table below the x-axis, the addition of acetazolamide increased protein yield by approximately 23.5% per liter compared to the low pH process without acetazolamide, while maintaining the Man5 ratio, effectively reducing the negative effects of the low pH process. Furthermore, the Man5 level was reduced by approximately 1.1% compared to the high pH process. DETAILED DESCRIPTION

[0039] Reference will now be made in detail to certain embodiments of the present invention, examples of which are illustrated in the accompanying drawings. Although the present invention will be described in conjunction with the illustrated embodiments, it will be understood that they are not intended to limit the invention to those embodiments. On the contrary, the present invention is intended to cover all alternatives, modifications, and equivalents thereof as defined by the appended claims.

[0040] It should be understood that as used herein throughout the specification and claims, the meanings of "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0041] Unless otherwise apparent from the context, when a value is expressed as "about" X or "approximately" X, the stated value of X is to be understood to be accurate to ±10%.

[0042] In this disclosure, when a time period or duration or length of an interval is expressed in days or a time point is expressed in days, this means that the time or timing is calculated or identified in days, wherein the numbers do not necessarily represent exact multiples of 24 hours.

[0043] The core technical solution of the present invention is to add acetazolamide during the cell culture process on the cooling day of batch fed-batch culture or at any time point when the high pCO2 environmental pressure needs to be relieved.

[0044] The “cooling day” mentioned herein refers to the day when cells reach a high cell density (e.g., 15.0×10 6 cells / ml) and then lower the temperature to control cell proliferation and maintain high viability (productivity) for a long time.

[0045] Conventional culture conditions can be used in the cell culture step before the cooling day, such as culturing cells at 36-37°C, preferably 36.5°C, and a low pCO2 environment pressure, for example, culturing cells under a carbon dioxide partial pressure of 20-60 mmHg, preferably 20-50 mmHg.

[0046] As used herein, "high pCO2 ambient pressure" refers to a pCO2 partial pressure greater than 80 mmHg, preferably 80-200 mmHg, and more preferably 80 mmHg, 100 mmHg, 120 mmHg, or 150 mmHg. "Normal pCO2 conditions" are conditions with a pCO2 of less than 60 mmHg. High pCO2 conditions can lead to severe accumulation of cellular lactate, and decreased cell viability and protein production. Those skilled in the art can determine the pCO2 pressure and its impact on cell culture and production by monitoring the CO2 partial pressure in real time and combining these findings with lactate accumulation and decreased viability. The CO2 partial pressure described herein can optionally be achieved by conventional methods, such as by varying the CO2 aeration ratio. For example, introducing a CO2 aeration ratio of 8-30% can achieve an increase in the CO2 partial pressure relative to a conventional aeration ratio of less than 6%.

[0047] As used herein, "antibodies" or "antibody proteins" refer to immunoglobulins that are produced by the body in response to the presence of an antigen and that bind to the antigen, as well as antigen-binding fragments and engineered variants thereof. Thus, the term "antibody" includes, for example, complete monoclonal antibodies (e.g., antibodies produced using hybridoma technology) and antigen-binding antibody fragments, such as F(ab')2 and Fab fragments. Also included are genetically engineered complete antibodies and fragments, such as chimeric antibodies, humanized antibodies, single-chain Fv fragments, single-chain antibodies, bifunctional antibodies, minibodies, linear antibodies, multivalent or multispecific (e.g., bispecific) hybrid antibodies, and the like. Thus, the term "antibody" is used expansively to include any protein that contains an antibody's antigen-binding site and is capable of specifically binding to its antigen. The term "antibody" also includes antibodies themselves ("naked antibodies") or antibodies bound to cytostatic or cytotoxic drugs.

[0048] The core technical solution of the present invention is to add acetazolamide under pressure when culturing cells under high CO2 partial pressure and low pH pressure, thereby unexpectedly improving the effect of cell culture and reversing the adverse effects of high pressure. These pressures are used to enable cells to better produce the required antibody protein and limit the proportion of its Man5 glycoform to a low proportion required for antibody preparation.

[0049] The experimental data presented in the examples of the present invention demonstrate that, first, under high pCO₂ conditions simulating large-scale production, at least two clones exhibited the following performance: compared to conventional pCO₂ conditions (pCO₂ < 60 mmHg), under high pCO₂ conditions (pCO₂ > 80 mmHg), cellular lactate accumulated significantly, and both cell viability and protein yield decreased. Furthermore, the addition of acetazolamide significantly improved this situation, with lactate levels decreasing by up to 90%, viability increasing by 15%-25%, and protein yield maintained at a higher level. This result fully demonstrates that the addition of acetazolamide can significantly enhance the ability of cells to withstand the stress of a high pCO₂ environment.

[0050] Furthermore, compared to the traditional low-pH process, the addition of acetazolamide not only maintained pentamethylenetetramine levels at a low level, but also improved overall cell culture performance, including lactate metabolism, cell viability, and protein yield. This suggests that the addition of acetazolamide further optimizes the Man5-low pH process.

[0051] Having now generally described the present invention, the same may be understood more readily by reference to the following description of the following examples which are provided by way of illustration and are not intended to be limiting of the present invention unless expressly stated.

[0052] Unless otherwise specifically specified, any feature, step, element, embodiment or aspect of the present invention may be used in combination with any other feature, step, element, embodiment or aspect. Although the present invention has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be apparent that certain changes and modifications may be practiced within the scope of the appended claims.

[0053] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications and patents specifically mentioned herein are incorporated by reference for all purposes. All references cited in this specification should be considered as an indication of the level of skill in the art, and this should not be construed as an admission that the present invention is not entitled to antedate disclosure by virtue of prior invention.

[0054] Example 1 Effect of Acetazolamide on Cell Fermentation under High pCO2 Environmental Pressure

[0055] 1. Equipment and Reagents:

[0056] 1.1 Device Information

[0057]

[0058]

[0059] 1.2 Reagent Information

[0060] Material abbreviation Manufacturer Item No. <h2 style=";text-align:left;direction:ltr"><![CDATA[Cell Boost <h2 style=";text-align:left;direction:ltr"> TM <h2 style=";text-align:left;direction:ltr"> 7a]]><h2 style=";text-align:left;direction:ltr"> NA <![CDATA[HyClone TM ]]> SH31026.04 <h2 style=";text-align:left;direction:ltr"><![CDATA[Cell Boost <h2 style=";text-align:left;direction:ltr"> TM <h2 style=";text-align:left;direction:ltr"> 7b]]><h2 style=";text-align:left;direction:ltr"> NA <![CDATA[HyClone TM ]]> SH31027.02CN ActiProTM NA <![CDATA[HyClone TM ]]> SH31037.01 Acetazolamide NA McCaw 59-66-5 L-Glutamine Gln JTBaker 2078-06 Anhydrous dextran Glucose JT Baker 1919-09 HT additives HT Gibco 11067030 Blasticidin S HCl BS Gibco A11139 Bleomycin selection antibiotics Zeocin Invitrogen R25001 Sodium bicarbonate NaHCO3 Merck 1.37013.2500 Hydrochloric acid, 6.0N solution HCl JT.Baker 0327-02 Sodium bicarbonate NaHCO3 Merck 1.37013.2500 sodium carbonate <![CDATA[Na2CO3]]> Merck 1.06398.5000 Poloxamer 188 NA Merck 1.37065.1000 defoaming agent Antifoam <![CDATA[HyClone TM ]]> SH30897.01

[0061] 2. Test method:

[0062] 2.1 Preparation of 3mM acetazolamide stock solution. The preparation steps are as follows (taking 1L aqueous solution as an example):

[0063] a. Record the weight of the liquid preparation container used

[0064] b. Record the temperature of ultrapure water used

[0065] c. Add 0.99 kg of ultrapure water to the liquid preparation container, heat and maintain the temperature at 70°C

[0066] d. Weigh 0.67g of acetazolamide, add it to ultrapure water, and stir until completely dissolved

[0067] e. Add ultrapure water to make up the volume and record the final weight

[0068] f. Mix for 15-30 minutes

[0069] 2.2 Method of adding acetazolamide (taking 150uM concentration as an example)

[0070] When added once to the culture medium as an additive, the volume to be added is calculated as follows:

[0071] If the cell culture volume is V, then the added volume of 3mM acetazolamide is Vi=(0.15×V) / 3

[0072] 2.3 Cell culture steps:

[0073] 2.3.1 General batch fed-batch culture process steps:

[0074] 2.3.1.1 Inoculation: CHO cells were plated at 0.4 x 10 6 Cells / mL density was inoculated into the basic medium Actipro.

[0075] 2.3.1.2 Feeding: Feeding was carried out on days 3, 5, 7, 10 and 12 from the date of inoculation. The feeding amount was: CB7a7b (CB7a:CB7b=10:1) 3%, 3%, 3%, 3%, 2%.

[0076] 2.3.1.3 Temperature: The initial culture temperature is 36.5°C. When the viable cell density is >15.0x10 6 cells / mL and then cooled to 31.0°C.

[0077] 2.3.1.4 pH: 7.0 ± 0.2

[0078] 2.3.1.5 Harvest: On the 14th day of culture, collect the culture supernatant by centrifugation.

[0079] 2.3.1.6 Analysis and testing: The harvested supernatant was filtered through a 0.22 μm filter membrane and then subjected to antibody expression and glycosylation analysis.

[0080] 2.3.2 Process steps of high pCO2 pressure experimental group

[0081] Carry out inoculation as described in 2.3.1, except that the CO2 ventilation ratio is controlled at 6% from the day of inoculation to before cooling, and the CO2 ventilation ratio is increased to 15% after cooling to the day of harvest.

[0082] 2.3.3 Process steps of low pH process experimental group

[0083] The inoculation was carried out as described in 2.3.1. The pH was controlled at 7.0±0.2 from the inoculation day to the cooling day. The pH of the control condition was kept constant at 7.0±0.2 from the cooling day to the harvest day. The pH control strategy of the experimental conditions was adjusted to 6.8±0.1 (the upper limit of pH was controlled by the introduction of CO2, and the lower limit of pH was controlled by the addition of 1M Na2CO3).

[0084] 2.3.4 Process steps for combining acetazolamide with low pH or high pCO2 processes

[0085] Carry out inoculation as described in 2.3.2 or 2.3.3. From the date of inoculation to before cooling, the pH is controlled at 7.0±0.2 or the CO2 ventilation ratio is controlled at 6%. From cooling to the day of harvest, the pH control strategy is adjusted to 6.8±0.1 (the upper limit of pH is controlled by introducing CO2, and the lower limit of pH is controlled by adding 1M Na2CO3). The CO2 ventilation ratio is maintained at 6%, or the CO2 ventilation ratio is adjusted to 15%.

[0086] Add acetazolamide stock solution: on the cooling day or by real-time monitoring of CO2 partial pressure, and in combination with lactic acid accumulation and activity decline, adjust the pCO2 concentration at any time point when the high pCO2 environmental pressure needs to be relieved. After the adjustment, add acetazolamide stock solution.

[0087] 2.4 Detection method:

[0088] The viable cell density was counted using Vi-cell, the lactate concentration was measured using a Cedex automated multifunctional biochemical analyzer, and the offline pH and pCO2 were measured using a BGA blood gas analyzer. All detection methods were as specified in the manufacturer's instructions.

[0089] Protein expression and glycosylation analysis: The cell culture supernatant was filtered through a 0.22 μm filter membrane, and the antibody expression level was determined by protein-A affinity high-performance liquid chromatography. The glycosylation analysis method was HILIC (HALOPenta-HILIC, 2.1×150 mm, 2.7 μm).

[0090] 3. Test results:

[0091] The experimental effects of the present invention are mainly compared through two groups of experiments. The first group is the high pCO2 pressure relief experimental group, which shows that the addition of acetazolamide can effectively improve the ability of cells to resist the high pCO2 environmental pressure; the second group is the acetazolamide and low pH process combined regulation group, which shows that the addition of acetazolamide in the low pH process can effectively correct the negative impact of high pCO2 caused by low pH on cell culture performance, while not affecting its ability to reduce the level of pentamethylenetetramine.

[0092] Figures 1 to 4 Shows the comparison of the effects of relieving high pCO2 pressure in the experimental groups.

[0093] Under high pCO2 culture conditions, the addition of acetazolamide significantly reduced lactate levels in the late culture period and increased cell harvest viability. Furthermore, the protein yield was higher after the addition of acetazolamide. Furthermore, the effect of the high addition concentration (150uM) was more pronounced than that of the low addition concentration (50uM) (see Table 1).

[0094] Table 1 Protein yield in the high pCO2 pressure experimental group

[0095]

[0096] Figures 5 to 10 The results of the combined acetazolamide and low pH process are shown. Compared with the control pH (7.0±0.2), the low pH process (6.8±0.1) can significantly reduce the level of pentamethylenetetramine. However, under this process condition, the pCO2 level can reach up to 200mmHg. Under this high pCO2 environment, the viable cell density and cell viability decreased rapidly. The lactic acid level continued to increase in the late stage of culture, and the final protein yield decreased by more than 25% (see Figure 10 ); while the addition of acetazolamide can effectively reduce the negative effects of the low pH process while maintaining the low pentamethylenetetramine level unchanged ( Figure 10 ).

[0097] In summary, under high pCO2 conditions simulating large-scale production, compared with conventional pCO2 conditions (pCO2 < 60 mmHg), high pCO2 conditions (pCO2 > 80 mmHg) resulted in significant accumulation of lactate, reduced cell viability, and decreased protein production. Adding acetazolamide significantly improved this situation, with lactate levels decreasing by up to 90%, viability increasing by 15%-25%, and protein production maintained at a higher level. These results clearly demonstrate that the addition of acetazolamide significantly enhances the cell's ability to withstand the stress of a high pCO2 environment.

[0098] Furthermore, compared to the traditional low-pH process, the addition of acetazolamide not only maintained low pentamethylene glycol levels but also improved overall cell culture performance, including lactate metabolism, cell viability, and protein yield. This suggests that the addition of acetazolamide further optimizes the low-pH process for pentamethylene glycol reduction.

[0099] References

[0100] [1]XU S, JIANG R, MUELLER R, et al. Probing lactate metabolism variations in large-scale bioreactors[J / OL]. Biotechnology Progress, 2018, 34(3):756-766. https: / / doi.org / 10.1002 / btpr.2620.

[0101] [2]ZHANG

[0102] [3]BRUNNER M, DOPPLER P, KLEIN T, et al. Elevated pCO2 affects the lactate metabolic shift in CHO cell culture processes[J / OL]. Engineering in Life Sciences, 2018, 18(3): 204 - 214. https: / / doi.org / 10.1002 / elsc.201700131.

[0103] [4]HECK R W, TANHAUSER S M, MANDA R, et al. Catalytic properties of mouse carbonic anhydrase V.[J / OL]. Journal of Biological Chemistry, 1994, 269(40): 24742 - 24746. https: / / doi.org / 10.1016 / S0021-9258(17)31454-0.

[0104] [5]FRANCHI M, VULLO D, GALLORI E, et al. Carbonic anhydrase inhibitors: Inhibition of human and murine mitochondrial isozymes V with anions[J / OL]. Bioorganic & Medicinal Chemistry Letters, 2003, 13(17): 2857 - 2861. https: / / doi.org / 10.1016 / S0960-894X(03)00581-X.

Claims

1. A method for producing an IgG antibody, comprising the following steps: a) inoculating cells and culturing them at 36-37° C., maintaining the pH at 7.0±0.20, and using 20-60 mmHg of CO 2 ; b) When the cells grow to 8-30×10 6 cells / ml, lowering the culture temperature to 30-32°C, increasing the pCO2 from a first pCO2 of 20 mmHg-60 mmHg to a second pCO2 of 80 mmHg-200 mmHg on the day of cooling, wherein the second pCO2 is higher than the first pCO2, and maintaining the pCO2 until the cells are harvested; c) in step b), while lowering the temperature, adding acetazolamide at a concentration of 50 μM to 150 μM; and d) harvesting the cells and collecting the antibody protein product, wherein the cells are IgG antibody-producing CHO cells.

2. The method of claim 1, wherein in step a) the cells are cultured at 36.5°C.

3. The method of claim 1, wherein the cell density is 1.5×10 7 cells / ml and lower the culture temperature.

4. The method of claim 1, wherein the temperature is lowered to 31°C in step b).

5. The method of claim 1, wherein the IgG antibody is selected from IgG1, IgG2, IgG3, IgG4, and a fusion protein having an IgG Fc-terminal structure. The method according to claim 5 , wherein the antibody protein is selected from IgG1 and a fusion protein having an IgG1 Fc terminal structure.

7. The method of claim 1, wherein the cells are cultured with 30 mmHg of CO2 during the culture in step a).

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