A method for constructing a light-sensitive protein stability evaluation reduced model and application thereof

Abstract

The present application relates to a method for constructing a photosensitive protein stability evaluation scale-down model and application thereof, the method comprising the following steps: (1) collecting light information of a photosensitive protein scale production plant; (2) collecting container information of the scale production plant for storing the photosensitive protein in step (1); (3) calculating the light intensity and light time of the photosensitive protein solution stored in the scale-down model; (4) setting a light source consistent with the scale production plant to irradiate the photosensitive protein solution stored in the scale-down model, and setting the light intensity and light time according to the calculation result of step (3). The present application provides representative stability data for the scale commercial production of photosensitive proteins by introducing the scale-down model, and reduces the production risk.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a method for constructing a light-sensitive protein stability evaluation reduced model and application thereof. BACKGROUND

[0002] Light-sensitive proteins are a class of proteins sensitive to light in structure, conformation and activity. Light mainly affects the stability of proteins through photooxidation. The protein aromatic group mainly composed of tryptophan residues is particularly sensitive to light, which is prone to photooxidation to form free radicals, thereby causing fragmentation and crosslinking. In addition, light can also accelerate the degradation of proteins or cause crosslinking to form aggregates, thereby further affecting the physicochemical properties and stability of the proteins.

[0003] Currently, light-sensitive biological products need to be stored in the dark to reduce the impact of light on protein stability during large-scale production. For example, during large-scale production, an integrated stainless steel tank can provide some light protection. However, the viewing window of the tank can still allow light to pass through, thereby affecting the stability of the proteins. Since the closed stainless steel tank cannot obtain the light intensity transmitted through the viewing window, it is impossible to predict and estimate the possible impact on the proteins, which becomes a potential risk point in the production process. In addition, disposable bags are also used in large-scale production. Although the light-proof support of the bag has certain light protection function, a small amount of light may still pass through and affect the stability of the proteins.

[0004] Before entering the commercial large-scale preparation of biological products, the room temperature stability of proteins is evaluated at the laboratory scale during the laboratory process development stage to obtain the room temperature stability data of the intermediate products for reference in large-scale production. The current stability experiment is mainly carried out in a water bath under light or without light, but the existing technical methods cannot truly reflect the light intensity received by the proteins in the large-scale production process, thereby failing to provide representative stability data for the light-sensitive proteins in large-scale production.

[0005] In summary, how to simulate the light conditions in large-scale production in the laboratory and evaluate the stability of proteins under the light conditions to provide more reliable stability data for large-scale production and support commercial production has become one of the problems to be solved in the field. SUMMARY

[0006] To solve the above technical problems, the present application provides a method for constructing a light-sensitive protein stability evaluation reduced model and application thereof. The light conditions received by the light-sensitive proteins in a large-scale production plant are collected, and the light conditions are simulated in the laboratory for a short time, thereby evaluating the stability of the light-sensitive proteins to provide representative stability data for the light-sensitive proteins in large-scale commercial production and reduce the production risk.

[0007] To achieve the above object, the present application adopts the following technical solutions:

[0008] In a first aspect, the present application provides a method for constructing a reduced model for evaluating the stability of a light-sensitive protein, the method comprising the following steps:

[0009] (1) collecting light information of a light-sensitive protein production plant;

[0010] (2) collecting container information of the light-sensitive protein stored in the production plant in step (1);

[0011] (3) calculating the light intensity and light time of the light-sensitive protein solution stored in the reduced model according to the information collected in steps (1) and (2);

[0012] (4) setting a light source consistent with the production plant to irradiate the light-sensitive protein solution stored in the reduced model, and setting the light intensity and light time according to the calculation results of step (3).

[0013] The present application relates to the stability evaluation of light-sensitive proteins in the purification process of antibodies, FC fusion proteins, double antibodies and recombinant proteins. The volume of light-sensitive protein production is generally greater than 1L, while the reduced model provided by the present application has a volume of less than or equal to 5mL. The present application simulates the light irradiation conditions of large-scale production and evaluates the stability of the protein under the light irradiation conditions by reducing the time and increasing the light intensity in the laboratory.

[0014] Preferably, the light information in step (1) includes the type of lamp (such as incandescent lamp, fluorescent lamp or LED lamp, etc.), the spectral information of the lamp and the maximum light intensity value in the production plant.

[0015] Preferably, the storage container of the light-sensitive protein in the production plant includes a sealed cylinder or a disposable bag.

[0016] Preferably, the sealed cylinder includes a sealed cylinder or a sealed prism.

[0017] Preferably, a viewing window with light transmission is provided on the storage container.

[0018] The structure diagram of the sealed cylinder of the present application is shown in Figure 1 A viewing window is provided on the top of the sealed cylinder for observing the production situation.

[0019] Preferably, the container information in step (2) includes any one of the following container information:

[0020] (2-1) When the light-sensitive protein is stored in a sealed cylinder in the production plant, the liquid surface area of the light-sensitive protein and the area of the viewing window;

[0021] (2-2) When the light-sensitive protein is stored in a disposable bag in a large-scale production plant, the maximum light intensity received by the light-sensitive protein.

[0022] Preferably, the calculation of the light intensity and the light exposure time of the light-sensitive protein solution stored in the reduced model in step (3) is any one of the following calculation methods:

[0023] (3-1) When the light-sensitive protein is stored in a sealed cylinder in a large-scale production plant, the light intensity

[0024]

[0025] (3-2) When the light-sensitive protein is stored in a disposable bag in a large-scale production plant, the light intensity

[0026]

[0027] wherein A is the area of the window, in square meters; B is the maximum light intensity, in lux; C is the liquid surface area of the light-sensitive protein, in square meters; t is the light exposure time, in minutes (min), 0 < t < 1440; and I is the light intensity, in lux.

[0028] The value of t in the above 0 < t < 1440 can be 1, 5, 10, 13, 15, 17, 20, 50, 100, 200, 500, 750, 1000, 1200, or 1440, etc.

[0029] When the light-sensitive protein is stored in a sealed cylinder in a large-scale production plant, the sealed cylinder storing the light-sensitive protein has A x B lumens of light passing through the window, and the liquid surface will receive lux light intensity, and be exposed to 7 days at lux, which is equivalent to being exposed to 1 day (1440 minutes) at lux. Therefore, in order to reduce the light exposure time of the reduced model to 1 day, the light intensity For example, if the light exposure time is 15 minutes, the light intensity

[0030] When the light-sensitive protein is stored in a disposable bag in a large-scale production plant, it is exposed to 7 days at a light intensity of B lux, which is equivalent to being exposed to 1 day (1440 minutes) at 7 x B lux. Therefore, in order to reduce the light exposure time of the reduced model to 1 day, the light intensity For example, if the light exposure time is 15 minutes, the light intensity

[0031] In a second aspect, the present application provides a reduced model for photosensitive protein stability evaluation, which is obtained by the method for constructing a reduced model for photosensitive protein stability evaluation according to the first aspect.

[0032] Preferably, the reduced model comprises:

[0033] a sample module for storing photosensitive proteins to be evaluated for stability;

[0034] an illumination module for simulating the illumination conditions in a large-scale production plant, and the light source, illumination intensity and illumination time are set according to the method for constructing a reduced model for photosensitive protein stability evaluation according to the first aspect.

[0035] In a third aspect, the present application provides a method for evaluating the stability of photosensitive proteins, which comprises using the reduced model for photosensitive protein stability evaluation according to the second aspect.

[0036] Preferably, the method for evaluating the stability of photosensitive proteins specifically comprises the following steps:

[0037] (I) using the illumination module in the reduced model, irradiating the sample module according to the preset light source, illumination intensity and illumination time;

[0038] (II) determining the purity of the protein in the sample module to obtain the quality of the photosensitive protein after irradiation for 7 days in a large-scale production plant.

[0039] In a fourth aspect, the present application provides the use of any one of the method for constructing a reduced model for photosensitive protein stability evaluation according to the first aspect, the reduced model for photosensitive protein stability evaluation according to the second aspect or the method for evaluating the stability of photosensitive proteins according to the third aspect in the large-scale production of photosensitive proteins.

[0040] Other specific point values within the above-mentioned numerical ranges can also be selected, which will not be described here one by one.

[0041] Compared with the prior art, the present application has the following beneficial effects:

[0042] The present application provides a method for constructing a reduced model for photosensitive protein stability evaluation, and establishes a reduced model for photosensitive protein stability evaluation. Using the reduced model, the illumination conditions for 7 days of storage in a large-scale production plant can be simulated in less than 1 day, representative stability data for large-scale commercial production of photosensitive proteins is provided, the production risk is reduced, and the operation is simple and fast. BRIEF DESCRIPTION OF DRAWINGS

[0043] Figure 1 A schematic diagram of a sealed column structure in a large-scale production plant.

[0044] Figure 2 Stability curve of the reduced model of the stainless steel tank for Test Example 1.

[0045] Figure 3 Stability curve of the reduced model of the disposable bag for Test Example 2. DETAILED DESCRIPTION

[0046] In order to further clarify the technical means adopted by the present application and its effects, the present application will be further described in conjunction with the embodiments and drawings. It can be understood that the specific embodiments described herein are only used to explain the present application, but not to limit the present application.

[0047] Unless otherwise specified in the examples, the techniques or conditions are in accordance with the techniques or conditions described in the literature in the art, or in accordance with the product instructions. Unless otherwise specified, the reagents or instruments used are conventional products that can be commercially available through regular channels.

[0048] Example 1

[0049] The present embodiment provides a method for constructing a reduced model for evaluating the stability of a light-sensitive protein, wherein the storage container of the light-sensitive protein in a large-scale production plant comprises a sealed cylinder or a disposable bag, and a viewing window with light transmission is provided on the storage container. Different construction methods are provided according to different storage containers, including the following steps:

[0050] (1) Collecting light information of the light-sensitive protein in a large-scale production plant, wherein the light information includes the type of light in the large-scale production plant, the spectral information of the light, and the maximum light intensity value.

[0051] (2) Collecting container information for storing the light-sensitive protein in the large-scale production plant. When the light-sensitive protein is stored in a sealed cylinder in the large-scale production plant, the light-sensitive protein liquid surface area and the viewing window area information are collected; when the light-sensitive protein is stored in a disposable bag in the large-scale production plant, the maximum light intensity information received by the light-sensitive protein is collected.

[0052] (3) Calculating the light intensity and light time for the light-sensitive protein solution stored in the reduced model according to the information collected in steps (1) and (2);

[0053] When the light-sensitive protein is stored in a sealed cylinder in the large-scale production plant, the light intensity

[0054]

[0055] When the light-sensitive protein is stored in a disposable bag in the large-scale production plant, the light intensity

[0056]

[0057] wherein A is the area of the window, in square meters; B is the maximum light intensity, in lux; C is the area of the photosensitive protein liquid surface, in square meters; t is the light exposure time, in minutes, 0 < t < 1440; and I is the light intensity, in lux.

[0058] (4) Set up a light source to irradiate the photosensitive protein solution stored in the scaled-down model in accordance with the production plant, and set the light intensity and light exposure time according to the calculation results of step (3).

[0059] Example 2

[0060] This example provides a scaled-down model for photosensitive protein stability evaluation, which comprises:

[0061] a sample module for storing photosensitive proteins to be evaluated for stability;

[0062] a light exposure module for simulating the light exposure conditions in the production plant, and the light source, light intensity and light exposure time are calculated and set according to the method provided in Example 1.

[0063] Example 3

[0064] This example uses the method provided in Example 1 to construct a scaled-down model of a stainless steel tank for photosensitive protein stability evaluation.

[0065] According to the collected information, the type of light in the production plant is LED light, and the maximum brightness in the plant is 1116 lux. For the stainless steel tank, the area of the observation window on the top of the tank is about 0.00238 square meters (diameter 5.5 cm), and the minimum liquid surface area in the tank is about 1.5 square meters (diameter 1.3 meters). The light exposure of the photosensitive protein exposed to the current light intensity for 7 days is converted to a light intensity of 15 minutes in the scaled-down model, and the light intensity Take the light intensity of 2000 lux as the worst case.

[0066] Example 4

[0067] This example uses the method provided in Example 1 to construct a scaled-down model of a disposable bag for photosensitive protein stability evaluation.

[0068] According to the collected information, the type of light in the production plant is LED light, and the container for storing photosensitive protein is a disposable bag. When the window is not covered by the light shield, the light intensity inside the bracket where the disposable bag is placed is detected, and the maximum value is 100 lux. The light exposure of the photosensitive protein exposed to the current light intensity for 7 days is converted to a light intensity of 8 hours in the scaled-down model, and the light intensity about 2000 lux. When the window is covered by the shade, the maximum light intensity is measured to be 10 lux, and the light exposure of the light-sensitive protein at the present light intensity for 7 days is converted to the light intensity of 50 minutes in the reduced model, and the light intensity about 2000 lux.

[0069] Test Example 1

[0070] This test example uses the reduced model provided in Example 3, and the LED lamp is used to irradiate 5 mL of light-sensitive protein at a light intensity of 2000 lux for 15 minutes. The same kind of light-sensitive protein is used as a control group. The purity of the light-sensitive protein after light exposure is analyzed by capillary isoelectric focusing (CIEF). The sample and the amphoteric electrolyte are mixed, and after injection, the two electrode grooves are acid and alkali, respectively. After high voltage is applied, a pH gradient is generated in the capillary. The components of the sample migrate to their respective isoelectric points in the capillary to form bands, which are then detected. The results are shown in Table 1 and Figure 2 As shown in Table 1 and

[0071] Table 1

[0072]

[0073] Test Example 2

[0074] This test example uses the reduced model provided in Example 4, and the LED lamp is used to irradiate 5 mL of light-sensitive protein at a light intensity of 2000 lux for 8 hours and 50 minutes, respectively. The same kind of light-sensitive protein is used as a control group, and the purity of the light-sensitive protein after light exposure is analyzed by the method of Test Example 1. The results are shown in Table 2 and Figure 3 As shown in Table 2 and

[0075] Table 2

[0076]

[0077] In summary, the present application provides a method for constructing a reduced model for evaluating the stability of light-sensitive protein, and establishes a reduced model for evaluating the stability of light-sensitive protein. Using the reduced model, the light exposure of 7 days in a large-scale production plant can be simulated in less than 1 day, providing representative stability data for large-scale commercial production of light-sensitive protein, reducing production risk, and being simple and fast to operate.

[0078] The applicant states that the above description is only a specific embodiment of the present application, but the protection scope of the present application is not limited thereto, and it should be understood by those skilled in the art that any changes or replacements within the technical scope disclosed by the present application, which can be easily thought of by any person skilled in the art, falls within the protection scope and disclosure scope of the present application.

Claims

1. A method for constructing a scaled-down model for assessing the stability of photosensitive proteins, characterized in that, The method comprises the following steps: (1) collecting light information of a photosensitive protein large-scale production plant; (2) collecting container information of the photosensitive protein stored in the large-scale production plant in step (1); (3) calculating the light intensity and light time of the photosensitive protein solution stored in a reduced model according to the information collected in steps (1) and (2); (4) setting a light source consistent with the large-scale production plant to irradiate the photosensitive protein solution stored in the reduced model, and setting the light intensity and light time according to the calculation result of step (3); The storage container of the photosensitive protein in the large-scale production plant comprises a sealed cylinder or a disposable bag; The calculation method of the light intensity and light time of the photosensitive protein solution stored in the reduced model in step (3) is any one of the following calculation methods: (3-1) When the light-sensitive protein is stored in a sealed cylinder in a large-scale production plant, the light intensity ; (3-2) When the light-sensitive protein is stored in a disposable bag in a large-scale production plant, the light intensity ; Wherein, A is the area of the window, unit: square meter; B is the maximum light intensity, unit: lux; C is the photosensitive protein liquid surface area, unit: square meter; t is the light time, unit: minute, 0 < t ≤ 1440; I is the light intensity, unit: lux.

2. The method of claim 1, wherein the model is constructed by, The light information in step (1) includes the type of the light in the large-scale production plant, the spectral information of the light, and the maximum light intensity value.

3. The method of claim 1, wherein the model is constructed by, The sealed cylinder comprises a sealed cylinder or a sealed prism.

4. The method of claim 1, wherein the model is constructed by, The storage container is provided with a window with light transmission.

5. The method of claim 1, wherein the model is constructed by, The container information in step (2) includes any one of the following container information: (2-1) when the photosensitive protein is stored in a sealed cylinder in the large-scale production plant, the photosensitive protein liquid surface area and the window area; (2-2) when the photosensitive protein is stored in a disposable bag in the large-scale production plant, the maximum light intensity received by the photosensitive protein.

6. A reduced model for light-sensitive protein stability assessment, characterized in that, The reduced model is obtained by the method for constructing a photosensitive protein stability evaluation reduced model according to any one of claims 1-5.

7. The reduced model for light-sensitive protein stability evaluation according to claim 6, characterized in that, The reduced model comprises: a sample module for storing photosensitive proteins to be evaluated for stability; an irradiation module for simulating the light conditions of the large-scale production plant, and setting the light source, light intensity and light time according to the method for constructing a photosensitive protein stability evaluation reduced model according to any one of claims 1-5.

8. A method of assessing stability of a light-sensitive protein, characterized by, The method for evaluating the stability of the photosensitive protein comprises using the reduced model for photosensitive protein stability evaluation according to claim 6 or 7.

9. The method of assessing stability of a light-sensitive protein according to claim 8, wherein, The method for evaluating the stability of the photosensitive protein specifically comprises the following steps: (I) using the irradiation module in the reduced model, irradiating the sample module according to the preset light source, light intensity and light time; (II) determining the purity of the protein in the sample module to obtain the quality of the photosensitive protein after irradiation for 7 days in the large-scale production plant.

10. The application of any one of the method for constructing a photosensitive protein stability evaluation reduced model according to any one of claims 1-5, the reduced model for photosensitive protein stability evaluation according to claim 6 or 7, or the method for evaluating the stability of the photosensitive protein according to claim 8 or 9 in the large-scale production of photosensitive proteins.

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