Method for preventing disulfide bond reduction in protein and application thereof

Abstract

The invention relates to a method for preventing disulfide bond reduction in protein and application thereof, and the method for preventing disulfide bond reduction in protein comprises the following steps: introducing oxygen and nitrogen into a solution containing protein, and adjusting the dissolved oxygen level in the solution to reach a preset value. The concentration difference of oxygen on a gas-liquid interface is increased through the mixed gas of oxygen and nitrogen, so that the effect of quickly reaching a set DO level under the condition that KLa cannot be increased is achieved, and two specific control models are provided, so that the method is applied to determining a ventilation strategy for large-scale production of antibodies.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method for preventing disulfide bond reduction in proteins and an application thereof. Background Art

[0002] Chinese hamster ovary (CHO) cells are a commonly used mammalian cell expression system in biopharmaceutical production, particularly in the production of monoclonal antibodies (mAbs). Antibodies are composed of two light chains and two heavy chains, which are linked to each other by disulfide bonds. Under normal circumstances, these disulfide bonds are stable, maintaining the tetrameric structure of the antibody. However, during cell harvesting and / or Protein A chromatography steps, reduction of interchain disulfide bonds can occur.

[0003] Antibody disulfide bond reduction is essentially a reversible redox reaction initiated by intracellular enzymes. The thioredoxin (Trx) system and the glutathione (GSH) system are the two known enzyme systems involved in disulfide bond reduction. Their primary function is to maintain the intracellular redox environment and prevent oxidative stress damage. Figure 1 This is the mechanism by which the Trx system and GSH system regulate disulfide bond reduction.

[0004] In order to prevent antibody reduction, measures such as introducing a certain amount of air, cooling before harvesting, or adding EDTA / Cu are usually taken during the harvesting stage of antibody production. 2+ To prevent the reduction of antibody disulfide bonds from breaking, measures such as adding EDTA are used to prevent reduction, and additives need to be removed in the later purification. Therefore, the commonly used anti-reduction measure is to introduce a certain amount of air into the harvesting container during the harvesting process. Considering that the solubility of oxygen in water (about 0.25 mmol / L at 25°C) is very low, and the mass transfer coefficient (KLa) of the harvesting system (disposable mixing bag / stainless steel harvesting tank) is also very low, even under air-ventilated conditions, the dissolved oxygen (DO) level in the harvesting system often cannot quickly reach the lower limit of DO control requirements (>40%). In addition, due to the characteristics of some proteins that are easily reduced, and when the NADPH (reducing power) concentration in the harvesting fluid is high (over 0.5 mmol / L), the risk of protein reduction is higher, so the DO control requirements in the system are higher.

[0005] Existing methods for assessing protein reduction risk include adding varying concentrations of reducing agents to the target protein, adding varying concentrations of cell lysate to the target protein, and aerating the bottle containing clarified harvested target protein. These methods are qualitative and cannot provide production departments with quantitative anti-reduction strategies, such as the required aeration volume and the desired DO control level.

[0006] In summary, how to provide a method to quickly, comprehensively and accurately control the DO level in protein solutions, thereby preventing the reduction of disulfide bonds in proteins, has become one of the urgent problems to be solved in this field. Summary of the Invention

[0007] To solve the above technical problems, the present invention provides a method for preventing the reduction of disulfide bonds in proteins and its application. A combination of oxygen and nitrogen is used to control the dissolved oxygen level in the harvesting tank. Compared with using only air, the ratio of oxygen and nitrogen can be adjusted arbitrarily, and the dissolved oxygen level can be adjusted and controlled more accurately and quickly. A small-scale system for quickly and accurately controlling DO levels is established in the laboratory and applied to large-scale antibody production.

[0008] To achieve this object, the present invention adopts the following technical solutions:

[0009] In a first aspect, the present invention provides a method for preventing the reduction of disulfide bonds in proteins, the method comprising: introducing oxygen and nitrogen into a solution containing protein, and adjusting the dissolved oxygen level in the solution to a preset value, wherein the preset dissolved oxygen level is 10% to 120%.

[0010] Existing large-scale production harvesting typically uses stainless steel tanks or disposable liquid storage bags as harvesting tanks. These have the disadvantage of lacking DO feedback regulation and DO monitoring functions, and the KLa of the harvesting tank is relatively low. Therefore, using the air anti-reduction method, for certain harvesting liquids with high reducing power, it is impossible to quickly increase the dissolved oxygen concentration to the required DO level to meet the dissolved oxygen concentration required for certain molecular anti-reduction. As a result, even if air is ventilated during harvesting, it is impossible to prevent the reduction of recombinant proteins. The present invention uses a mixture of oxygen and nitrogen to increase the oxygen concentration difference at the gas-liquid interface, thereby achieving the desired DO level more quickly under conditions where KLa cannot be increased.

[0011] In addition to reduction, recombinant proteins may also undergo oxidation reactions. For example, the oxidation of histidine in antibodies can cause changes in the antibody charge, solubility, and immunogenicity. The oxidation of tryptophan causes an increase in antibody aggregates, etc. Therefore, when using ventilation to prevent reduction, the DO may be too high, causing protein oxidation. The present invention adopts a ventilation method that combines oxygen and nitrogen. By adjusting the ratio of oxygen and nitrogen, it ensures that the DO does not exceed a certain set limit, thereby avoiding protein oxidation caused by excessive DO during ventilation.

[0012] When the method of the present invention is applied to determine the aeration strategy for large-scale protein production, the optimal dissolved oxygen level in the solution is unknown. In this case, the preset value is 10% to 120% (for example, it can be 10%, 20%, 30%, 50%, 80%, 100%, or 120%, etc.), and the preset value is determined through multiple parallel experiments. When the method of the present invention is applied to a known optimal dissolved oxygen level, the dissolved oxygen level of the protein solution is controlled to prevent protein reduction or oxidation. In this case, the preset value is the optimal dissolved oxygen level.

[0013] Preferably, the protein is a monoclonal antibody.

[0014] Preferably, a stirring device is provided in the protein-containing solution.

[0015] Preferably, the rotation speed of the stirring device is 250-350 rpm.

[0016] Preferably, the method for preventing disulfide bond reduction in proteins specifically comprises the following steps:

[0017] (1) An oxygen injector with a first mass flow controller and a nitrogen injector with a second mass flow controller are respectively arranged at the bottom of the protein solution;

[0018] (2) providing a device for measuring the dissolved oxygen level of the protein solution, associated with the first mass flow controller and the second mass flow controller;

[0019] (3) measuring the dissolved oxygen level of the protein solution in real time, and adjusting the gas flow rate through the first mass flow controller and / or the second mass flow controller so that the dissolved oxygen level reaches a preset value.

[0020] Preferably, the associating with the first mass flow controller and the second mass flow controller in step (2) specifically comprises: associating the oxygen flow rate and the nitrogen flow rate with the dissolved oxygen level using Delta V software.

[0021] Preferably, the method for adjusting the gas flow rate in step (3) is: when the dissolved oxygen level is lower than the set value, the oxygen intake is increased; when the dissolved oxygen level is higher than the set value, the nitrogen intake is increased.

[0022] Preferably, the method for preventing disulfide bond reduction in proteins specifically comprises the following steps:

[0023] (I) connecting an oxygen injector and a nitrogen injector to form a mixed gas, and sequentially passing the mixed gas into a third mass flow controller and the bottom of the protein solution;

[0024] (II) providing a device for measuring the dissolved oxygen level of the protein solution, associated with the third mass flow controller;

[0025] (III) measuring the dissolved oxygen level of the protein solution in real time, and adjusting the mixed gas flow rate through a third mass flow controller so that the dissolved oxygen level reaches a preset value.

[0026] Preferably, step (I) comprises: connecting an oxygen injector with a first mass flow controller and a nitrogen injector with a second mass flow controller to a gas flow measuring device to form a mixed gas, and sequentially passing the mixed gas into a third mass flow controller and the bottom of the protein solution.

[0027] To expand the applicability of the specific control model provided by this invention, a gas flow rate control device is added before the mixed gas to adjust the volume ratio of oxygen and nitrogen, thereby adapting it to different proteins and different production conditions. In this control model, the volume ratio of oxygen and nitrogen was determined through preliminary experiments and ranged from 1:0 to 0:1 for O2:N2.

[0028] Preferably, the gas flow measurement device comprises a Venturi tube.

[0029] Preferably, the associating with the third mass flow controller in step (II) specifically comprises: associating the mixed gas flow rate with the dissolved oxygen level using Delta V software.

[0030] Preferably, the method for adjusting the mixed gas flow rate in step (III) is: when the dissolved oxygen level is lower than the set value, the amount of mixed gas introduced is increased; when the dissolved oxygen level is higher than the set value, the amount of mixed gas introduced is reduced.

[0031] In a second aspect, the present invention provides a device for preventing the reduction of disulfide bonds in proteins, wherein the device is used to perform the method for preventing the reduction of disulfide bonds in proteins described in the first aspect.

[0032] Preferably, the device comprises:

[0033] Sample module: used to store protein solution;

[0034] Dissolved oxygen measurement module: used for real-time measurement of dissolved oxygen levels in protein solutions;

[0035] Ventilation module: used to introduce oxygen and nitrogen into the protein solution;

[0036] Gas flow control module: used to control the amount of gas introduced into the ventilation module according to the dissolved oxygen level measured by the dissolved oxygen measurement module.

[0037] Preferably, the device further comprises:

[0038] Gas ratio control module: used to adjust the volume ratio of oxygen and nitrogen, mix oxygen and nitrogen to obtain mixed gas, and pass the mixed gas into the ventilation module.

[0039] Preferably, the ventilation module is used to introduce oxygen and nitrogen into the protein solution, and the gas flow control module is used to control the amount of oxygen and nitrogen introduced into the ventilation module according to the dissolved oxygen level measured by the dissolved oxygen measurement module.

[0040] Preferably, the ventilation module introduces a mixed gas of oxygen and nitrogen into the protein solution, and the gas flow control module controls the amount of the mixed gas introduced into the ventilation module according to the dissolved oxygen level measured by the dissolved oxygen measurement module.

[0041] In a third aspect, the present invention provides use of the method for preventing disulfide bond reduction in proteins as described in the first aspect and / or the device for preventing disulfide bond reduction in proteins as described in the second aspect in controlling the dissolved oxygen level of a protein solution.

[0042] In a fourth aspect, the present invention provides a method for determining a ventilation strategy for large-scale antibody production, the method comprising the following steps:

[0043] S1. Producing antibodies with tool cells, separating cells and antibody-containing supernatant by centrifugation, disrupting cells to prepare cell lysate, and mixing the cell lysate with the antibody-containing supernatant;

[0044] S2. Aeration is performed using the method of preventing disulfide bond reduction in proteins according to the first aspect to adjust the dissolved oxygen level in the solution;

[0045] S3. Check whether the protein is denatured. If it is denatured, repeat steps S1 and S2 until the protein is denatured. The ventilation method at this time is the ventilation strategy for antibody large-scale production. If it is not denatured, the ventilation method at this time is the ventilation strategy for antibody large-scale production.

[0046] Based on the oxygen consumption of small-scale models, the present invention proposes an aeration guidance strategy with equal KLa (mass transfer coefficient) and equal VVM (ventilation volume ratio) to guide the aeration process in large-scale production. This method helps to optimize the production process and improve the quality and stability of antibody products.

[0047] Preferably, after the cell lysate in step S1 is mixed with the supernatant containing the antibody, the ratio of lactate dehydrogenase to antibody is the same as that in large-scale antibody production.

[0048] Preferably, the ventilation time in step S2 is the same as the storage time of the antibody in the harvest tank during large-scale antibody production.

[0049] Other specific point values ​​within the above numerical ranges can be selected and will not be described in detail here.

[0050] Compared with the prior art, the present invention has the following beneficial effects:

[0051] (1) The present invention provides a method for preventing the reduction of disulfide bonds in proteins, which uses a combination of oxygen and nitrogen to control the dissolved oxygen level in the harvesting tank. Compared with using only air, the ratio of oxygen and nitrogen can be adjusted arbitrarily, and the dissolved oxygen level can be adjusted and controlled more accurately and quickly.

[0052] (2) Based on the mixed gas ventilation strategy, the present invention proposes two control models. One is to quickly achieve the DO level to the set value through direct feedback regulation of oxygen and nitrogen, which is the optimal condition for quantitative anti-reduction risk assessment; the other is to use a fixed oxygen partial pressure level to adjust the gas ventilation volume, and the DO reaches the set value relatively slowly, which can be used for the worst condition for quantitative anti-reduction risk assessment. These two models are used to establish a quantitative anti-reduction assessment model for recombinant proteins. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 Diagram of the antibody reduction mechanism.

[0054] Figure 2 Schematic diagram of the device for preventing disulfide bond reduction in proteins in Example 1.

[0055] Figure 3 Schematic diagram of the device for preventing disulfide bond reduction in proteins in Example 2.

[0056] Figure 4 This is the DO control curve of the method in Example 1.

[0057] Figure 5 This is the DO control curve of the method in Example 2.

[0058] Figure 6 This is the SDS-PAGE protein electrophoresis analysis result in Test Example 2. DETAILED DESCRIPTION

[0059] To further illustrate the technical means and effects of the present invention, the present invention is further described below with reference to the embodiments and drawings. It should be understood that the specific embodiments described herein are only used to explain the present invention, rather than to limit the present invention.

[0060] If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0061] Example 1

[0062] This embodiment provides a method and apparatus for preventing disulfide bond reduction in proteins, such as Figure 2As shown, the method for preventing the reduction of disulfide bonds in proteins comprises the following steps:

[0063] (1) An oxygen injector with a first mass flow controller (oxygen MFC) and a nitrogen injector with a second mass flow controller (nitrogen MFC) are respectively set at the bottom of the protein solution;

[0064] (2) setting up a device to measure the dissolved oxygen level of the protein solution and correlating the oxygen flow rate and nitrogen flow rate with the dissolved oxygen level using Delta V software;

[0065] (3) Real-time measurement of the dissolved oxygen level of the protein solution, adjusting the gas flow rate, and increasing the oxygen flow rate when the dissolved oxygen level is lower than the set value; and increasing the nitrogen flow rate when the dissolved oxygen level is higher than the set value, so that the dissolved oxygen level reaches the preset value.

[0066] This embodiment also provides a device for implementing the above method for preventing disulfide bond reduction in proteins, comprising the following modules:

[0067] Sample module: used to store protein solution;

[0068] Dissolved oxygen measurement module: used for real-time measurement of dissolved oxygen levels in protein solutions;

[0069] Ventilation module: used to introduce oxygen and nitrogen into the protein solution;

[0070] Gas flow control module: used to control the amount of oxygen and nitrogen introduced into the ventilation module according to the dissolved oxygen level measured by the dissolved oxygen measurement module.

[0071] Example 2

[0072] This embodiment provides a method and apparatus for preventing disulfide bond reduction in proteins, such as Figure 3 As shown, the method for preventing the reduction of disulfide bonds in proteins comprises the following steps:

[0073] (I) connecting an oxygen injector with a first mass flow controller (oxygen MFC) and a nitrogen injector with a second mass flow controller (nitrogen MFC) to a gas flow measurement device (Venturi tube) to form a mixed gas, and sequentially passing the mixed gas into a third mass flow controller (mixed gas MFC) and the bottom of the protein solution;

[0074] (II) setting up a device to measure the dissolved oxygen level of the protein solution and using Delta V software to correlate the mixed gas flow rate with the dissolved oxygen level;

[0075] (III) Real-time measurement of the dissolved oxygen level of the protein solution. When the dissolved oxygen level is lower than a set value, the amount of mixed gas introduced is increased. When the dissolved oxygen level is higher than the set value, the amount of mixed gas introduced is reduced to allow the dissolved oxygen level to reach the set value.

[0076] This embodiment also provides a device for implementing the above method for preventing disulfide bond reduction in proteins, comprising the following modules:

[0077] Sample module: used to store protein solution;

[0078] Dissolved oxygen measurement module: used for real-time measurement of dissolved oxygen levels in protein solutions;

[0079] Gas ratio control module: used to adjust the volume ratio of oxygen and nitrogen, mix oxygen and nitrogen to obtain mixed gas, and pass the mixed gas into the ventilation module;

[0080] Ventilation module: used to introduce a mixture of oxygen and nitrogen into the protein solution;

[0081] Gas flow control module: used to control the amount of mixed gas introduced into the ventilation module according to the dissolved oxygen level measured by the dissolved oxygen measurement module.

[0082] Example 3

[0083] This embodiment provides a method for determining the ventilation strategy for large-scale antibody production, comprising the following steps:

[0084] S1. Antibody production using tool cells: centrifuge to separate cells and antibody-containing supernatant, disrupt cells to prepare cell lysate, mix the cell lysate with the antibody-containing supernatant, and maintain the same ratio of lactate dehydrogenase to antibody as used in large-scale antibody production.

[0085] S2. Aeration is performed using the method for preventing disulfide bond reduction in proteins provided in Example 1 or Example 2. The aeration time is the same as the storage time of the antibody in the harvest tank during large-scale antibody production to adjust the dissolved oxygen level in the solution;

[0086] S3. Check whether the protein is denatured. If it is denatured, repeat steps S1 and S2 until the protein is denatured. The ventilation method at this time is the ventilation strategy for antibody large-scale production. If it is not denatured, the ventilation method at this time is the ventilation strategy for antibody large-scale production.

[0087] Test Example 1

[0088] This test example uses the methods provided in Example 1 and Example 2 to adjust the protein solution to a DO value of 50%. The DO control curve of Example 1 is as follows: Figure 4As shown, the speed of the stirring device is 250rpm, and the set DO value can be reached within 15 minutes. The DO value has an overcharge phenomenon, and the overcharge range is about 5%. The DO control curve of Example 2 is shown as follows: Figure 5 As shown, the rotation speed of the stirring device is 250 rpm, and the set DO value is reached in about 25 minutes, which is slightly slower than that in Example 1, but the DO control is more stable and there is no DO overcharge phenomenon.

[0089] Test Example 2

[0090] This test example uses the method provided in Example 3 to determine the ventilation strategy for antibody scale production based on the method for providing ventilation strategies in Example 1 and Example 2, thereby preventing disulfide bond reduction from occurring during antibody scale production. Different DO levels (0%, 10%, 30%) are simulated in a small-scale system, and samples are collected at different time points (0, 6h, 24h, 48h). Protein bands are observed by SDS-PAGE protein electrophoresis analysis to assess whether the antibody is reduced or oxidized. The test results are shown in FIG. Figure 6 shown.

[0091] Depend on Figure 6 It can be seen that the antibodies in the positive control, Example 1, and Example 2 did not reduce at the 0h sampling point. After 6h, the positive control (DO controlled at 0%) showed some reduction of the antibodies. However, after 6h, the antibodies did not reduce at the DO levels of Example 1 (10%) and Example 2 (30%), and this experimental result was maintained for 48h. Therefore, ≥10% can be selected as the ventilation strategy for large-scale antibody production.

[0092] In summary, the present invention provides a method for preventing the reduction of disulfide bonds in proteins. The method uses a combination of oxygen and nitrogen to control the dissolved oxygen level in the harvesting tank. Compared with using only air, the ratio of oxygen and nitrogen can be adjusted arbitrarily, and the dissolved oxygen level can be adjusted and controlled more accurately and quickly. A small-scale system for quickly and accurately controlling DO levels is established in the laboratory and applied to large-scale antibody production.

[0093] The applicant declares that the above is only a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention fall within the scope of protection and disclosure of the present invention.

Claims

1. A method for preventing disulfide bond reduction in proteins, characterized in that: The method for preventing disulfide bond reduction in protein comprises: introducing oxygen and nitrogen into a solution containing protein, and adjusting the dissolved oxygen level in the solution to reach a preset value, wherein the preset dissolved oxygen level is 10% to 120%.

2. The method for preventing disulfide bond reduction in proteins according to claim 1, wherein: The protein is a monoclonal antibody; Preferably, a stirring device is provided in the protein-containing solution; Preferably, the rotation speed of the stirring device is 250-350 rpm.

3. The method for preventing disulfide bond reduction in proteins according to claim 1 or 2, characterized in that: The method for preventing disulfide bond reduction in proteins specifically comprises the following steps: (1) An oxygen injector with a first mass flow controller and a nitrogen injector with a second mass flow controller are respectively arranged at the bottom of the protein solution; (2) providing a device for measuring the dissolved oxygen level of the protein solution, associated with the first mass flow controller and the second mass flow controller; (3) measuring the dissolved oxygen level of the protein solution in real time, and adjusting the gas flow rate through the first mass flow controller and / or the second mass flow controller so that the dissolved oxygen level reaches a preset value.

4. The method for preventing disulfide bond reduction in proteins according to claim 3, wherein: The associating with the first mass flow controller and the second mass flow controller in step (2) specifically includes: associating the oxygen flow rate and the nitrogen flow rate with the dissolved oxygen level using Delta V software; Preferably, the method for adjusting the gas flow rate in step (3) is: when the dissolved oxygen level is lower than the set value, the oxygen intake is increased; when the dissolved oxygen level is higher than the set value, the nitrogen intake is increased.

5. The method for preventing disulfide bond reduction in protein according to claim 1 or 2, characterized in that: The method for preventing disulfide bond reduction in proteins specifically comprises the following steps: (I) connecting an oxygen injector and a nitrogen injector to form a mixed gas, and sequentially passing the mixed gas into a third mass flow controller and the bottom of the protein solution; (II) providing a device for measuring the dissolved oxygen level of the protein solution, associated with the third mass flow controller; (III) measuring the dissolved oxygen level of the protein solution in real time, and adjusting the mixed gas flow rate through a third mass flow controller so that the dissolved oxygen level reaches a preset value.

6. The method for preventing disulfide bond reduction in proteins according to claim 5, characterized in that: Step (I) comprises: connecting an oxygen injector with a first mass flow controller and a nitrogen injector with a second mass flow controller to a gas flow measuring device to form a mixed gas, and sequentially passing the mixed gas into a third mass flow controller and the bottom of the protein solution; Preferably, the gas flow measurement device comprises a venturi tube; Preferably, the associating with the third mass flow controller in step (II) specifically comprises: associating the mixed gas flow rate with the dissolved oxygen level using Delta V software; Preferably, the method for adjusting the mixed gas flow rate in step (III) is: when the dissolved oxygen level is lower than the set value, the amount of mixed gas introduced is increased; when the dissolved oxygen level is higher than the set value, the amount of mixed gas introduced is reduced.

7. A device for preventing disulfide bond reduction in proteins, characterized in that: The device is used to perform the method for preventing disulfide bond reduction in proteins according to any one of claims 1 to 6.

8. The device according to claim 7, characterized in that The device comprises: Sample module: used to store protein solution; Dissolved oxygen measurement module: used for real-time measurement of dissolved oxygen levels in protein solutions; Ventilation module: used to introduce oxygen and nitrogen into the protein solution; Gas flow control module: used to control the amount of gas introduced into the ventilation module according to the dissolved oxygen level measured by the dissolved oxygen measurement module; Preferably, the device further comprises: Gas ratio control module: used to adjust the volume ratio of oxygen and nitrogen, mix oxygen and nitrogen to obtain mixed gas, and pass the mixed gas into the ventilation module; Preferably, the ventilation module is used to introduce oxygen and nitrogen into the protein solution, and the gas flow control module is used to control the amount of oxygen and nitrogen introduced into the ventilation module according to the dissolved oxygen level measured by the dissolved oxygen measurement module. Preferably, the ventilation module introduces a mixed gas of oxygen and nitrogen into the protein solution, and the gas flow control module controls the amount of the mixed gas introduced into the ventilation module according to the dissolved oxygen level measured by the dissolved oxygen measurement module.

9. Use of the method for preventing disulfide bond reduction in protein according to any one of claims 1 to 6 and / or the device for preventing disulfide bond reduction in protein according to claim 7 or 8 in controlling the dissolved oxygen level in a protein solution.

10. A method for determining ventilation strategy for large-scale antibody production, characterized in that: The method for determining the ventilation strategy for large-scale antibody production comprises the following steps: S1. Producing antibodies with tool cells, separating cells and antibody-containing supernatant by centrifugation, disrupting cells to prepare cell lysate, and mixing the cell lysate with the antibody-containing supernatant; S2. Aeration is performed using the method for preventing disulfide bond reduction in proteins according to any one of claims 1 to 6 to adjust the dissolved oxygen level in the solution; S3. Check whether the protein is denatured. If so, repeat steps S1 and S2 until the protein is not denatured. The ventilation method at this time is the ventilation strategy for antibody scale production; Preferably, after the cell lysate in step S1 is mixed with the supernatant containing the antibody, the ratio of lactate dehydrogenase to antibody is the same as that in large-scale antibody production; Preferably, the ventilation time in step S2 is the same as the storage time of the antibody in the harvest tank during large-scale antibody production.

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