Method for constructing photosensitive protein stability assessment narrowing model and application of photosensitive protein stability assessment narrowing model

Abstract

The invention relates to a method for constructing a photosensitive protein stability evaluation reduction model and application thereof. The method comprises the following steps: (1) collecting light information of a photosensitive protein large-scale production plant; (2) collecting the container information of the photosensitive protein stored in the large-scale production plant in the step (1); (3) calculating the illumination intensity and illumination time of the photosensitive protein solution stored in the reduced model; and (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 illumination intensity and illumination time according to a calculation result in the step (3). By introducing the reduction model, representative stability data is provided for large-scale commercial production of the photosensitive protein, and the production risk is reduced.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method for constructing a reduced model for evaluating the stability of a photosensitive protein and an application thereof. Background Art

[0002] Photosensitive proteins are a type of protein whose structure, conformation and activity are sensitive to light. Light mainly affects the stability of proteins through photooxidation. Among them, aromatic proteins mainly composed of tryptophan residues are particularly sensitive to light and are prone to photooxidation to form free radicals, which leads to breakage and cross-linking. In addition, light can accelerate protein degradation or cross-link to form aggregates, further affecting the physical and chemical properties and stability of proteins.

[0003] At present, photosensitive biological products need to be stored away from light during large-scale production to reduce the impact of light on protein stability. For example, in large-scale production, one-piece stainless steel tanks can play a certain role in shielding from light. However, the window of the tank may still allow light to pass through, thus affecting the stability of the protein. Since the closed stainless steel tank cannot obtain the light intensity that penetrates through the window, it is impossible to predict and estimate the possible impact on the protein, which becomes a potential risk point in the production process. In addition, disposable bags are also used in large-scale production. Although the opaque bracket has a certain light-shielding function, a small amount of light may still pass through and affect the stability of the protein.

[0004] Before entering the commercial scale preparation of biological products, the room temperature stability of proteins will be evaluated at the laboratory scale during the laboratory process development stage to obtain room temperature stability data of intermediates for reference in large-scale production. The current stability experiments are mainly conducted in a water bath in a light-proof or non-light-proof manner, but the existing technical methods cannot truly feedback the light intensity received by the protein during the large-scale production process, and thus there is no way to provide representative stability data for the large-scale production of photosensitive proteins.

[0005] In summary, how to simulate the lighting conditions for large-scale production in the laboratory and evaluate the stability of proteins under such lighting conditions to provide more reliable stability data for large-scale production and support commercial production has become one of the urgent issues to be solved in this field. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides a method for constructing a scaled-down model for evaluating the stability of photosensitive proteins and its application. By collecting the light conditions to which the photosensitive proteins are exposed in large-scale production plants and simulating such light conditions in the laboratory for a short time, the stability of the photosensitive proteins can be evaluated, thereby providing representative stability data for the large-scale commercial production of photosensitive proteins and reducing production risks.

[0007] To achieve this object, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention 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 from a large-scale production plant for photosensitive proteins;

[0010] (2) collecting information on containers storing the photosensitive protein in the large-scale production plant described in step (1);

[0011] (3) calculating the illumination intensity and illumination time of the photosensitive protein solution stored in the reduced model based on the information collected in step (1) and step (2);

[0012] (4) Setting a light source consistent with that in a large-scale production plant to illuminate the photosensitive protein solution stored in the scaled-down model, and setting the light intensity and illumination time according to the calculation results of step (3).

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

[0014] Preferably, the light information in step (1) includes the type of lamps in a large-scale production plant (such as incandescent lamps, fluorescent lamps or LED lamps, etc.), the spectrum information of the lamps and the maximum light intensity value.

[0015] Preferably, the storage container of the photosensitive protein in a large-scale production plant includes a sealed column or a disposable bag.

[0016] Preferably, the sealing column comprises a sealing cylinder or a sealing prism.

[0017] Preferably, a light-transmitting window is provided on the storage container.

[0018] The structural schematic diagram of the sealing column of the present invention is as follows Figure 1 As shown, a window for observing the production situation is arranged above the sealing column.

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

[0020] (2-1) When the photosensitive protein is stored in a sealed column in a large-scale production plant, the liquid surface area and window area of ​​the photosensitive protein;

[0021] (2-2) The maximum light intensity that the photosensitive protein receives when it is stored in disposable bags in a large-scale production plant.

[0022] Preferably, the method for calculating the illumination intensity and illumination time of the photosensitive protein solution stored in the reduced model in step (3) is any one of the following calculation methods:

[0023] (3-1) When photosensitive proteins are stored in sealed columns in large-scale production plants, the light intensity

[0024]

[0025] (3-2) When photosensitive proteins are stored in disposable bags in large-scale production plants, the light intensity

[0026]

[0027] Among them, A is the window area, in square meters; B is the maximum light intensity, in lux; C is the photosensitive protein liquid surface area, in square meters; t is the illumination time, in minutes (min), 0<t≤1440; I is the light intensity, in lux.

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

[0029] When the photosensitive protein is stored in a sealed column in a large-scale production plant, the sealed column storing the photosensitive protein has A×B lumens of light passing through the window, and the liquid surface will receive Lux light intensity, in Exposure to lux for 7 days is equivalent to Lux exposure for 1 day (1440 minutes). Therefore, in order to reduce the illumination time of the reduced model to 1 day, the light intensity For example, if the illumination time is 15 minutes, the illumination intensity

[0030] When light-sensitive proteins are stored in disposable bags in a large-scale production plant, exposure to a light intensity of B lux for 7 days is equivalent to exposure to 7×B lux for 1 day (1440 minutes). Therefore, in order to reduce the illumination time of the scaled-down model to 1 day, the light intensity For example, if the illumination time is 15 minutes, the illumination intensity

[0031] In a second aspect, the present invention provides a reduced model for evaluating the stability of a light-sensitive protein, wherein the reduced model is constructed by the method for constructing a reduced model for evaluating the stability of a light-sensitive protein described in the first aspect.

[0032] Preferably, the reduced model comprises:

[0033] Sample module: used to store photosensitive proteins to be evaluated for stability;

[0034] Lighting module: used to simulate the lighting conditions of a large-scale production plant, and to set the light source, light intensity and lighting time according to the method for constructing a scaled-down model for evaluating the stability of a photosensitive protein as described in the first aspect.

[0035] In a third aspect, the present invention provides a method for evaluating the stability of a light-sensitive protein, wherein the method for evaluating the stability of a light-sensitive protein comprises using the reduced model for evaluating the stability of a light-sensitive protein described in the second aspect for evaluation.

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

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

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

[0039] In a fourth aspect, the present invention provides the use of any one of the method for constructing a scaled-down model for evaluating the stability of a photosensitive protein as described in the first aspect, the scaled-down model for evaluating the stability of a photosensitive protein as described in the second aspect, or the method for evaluating the stability of a photosensitive protein as described in the third aspect in the large-scale production of photosensitive proteins.

[0040] Other specific point values ​​within the above numerical ranges can be selected and will not be described in detail here.

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

[0042] The present invention provides a method for constructing a scaled-down model for evaluating the stability of a photosensitive protein, and establishes a scaled-down model for evaluating the stability of a photosensitive protein. The scaled-down model can be used to simulate the illumination conditions of 7 days of storage in a large-scale production plant in less than 1 day, thereby providing representative stability data for the large-scale commercial production of photosensitive proteins, reducing production risks, and being simple and fast to operate. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 This is a schematic diagram of the sealing column structure in a large-scale production plant.

[0044] Figure 2 This is the stability curve of the reduced model of the stainless steel tank in Test Example 1.

[0045] Figure 3 This is the stability curve of the disposable bag reduction model of Test Example 2. DETAILED DESCRIPTION

[0046] To further illustrate the technical means and effects of the present invention, the present invention is further described below in conjunction with the embodiments and drawings. It should be understood that the specific implementation methods described herein are only used to explain the present invention, rather than to limit the present invention.

[0047] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0048] Example 1

[0049] This embodiment provides a method for constructing a reduced model for evaluating the stability of a photosensitive protein. The storage container of the photosensitive protein in a large-scale production plant includes a sealed column or a disposable bag. A light-transmitting window 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 a large-scale production plant of a photosensitive protein, wherein the light information includes the type of lamps in the large-scale production plant, the spectrum information of the lamps, and the maximum light intensity value.

[0051] (2) Collecting information about containers for storing the photosensitive protein in large-scale production plants; when the photosensitive protein is stored in a sealed column in a large-scale production plant, collecting information about the liquid surface area and window area of ​​the photosensitive protein; and when the photosensitive protein is stored in a disposable bag in a large-scale production plant, collecting information about the maximum light intensity received by the photosensitive protein.

[0052] (3) calculating the illumination intensity and illumination time of the photosensitive protein solution stored in the reduced model based on the information collected in step (1) and step (2);

[0053] When light-sensitive proteins are stored in sealed columns in large-scale production plants, the light intensity

[0054]

[0055] When light-sensitive proteins are stored in disposable bags in large-scale production plants, the light intensity

[0056]

[0057] Among them, A is the window area, in square meters; B is the maximum light intensity, in lux; C is the photosensitive protein liquid surface area, in square meters; t is the illumination time, in minutes, 0<t≤1440; I is the light intensity, in lux.

[0058] (4) Setting a light source consistent with that in a large-scale production plant to illuminate the photosensitive protein solution stored in the scaled-down model, and setting the light intensity and illumination time according to the calculation results of step (3).

[0059] Example 2

[0060] This embodiment provides a scaled-down model for evaluating the stability of a light-sensitive protein, the scaled-down model comprising:

[0061] Sample module: used to store photosensitive proteins to be evaluated for stability;

[0062] Lighting module: used to simulate the lighting conditions of a large-scale production plant, and calculate and set the light source, lighting intensity and lighting time according to the method provided in Example 1.

[0063] Example 3

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

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

[0066] Example 4

[0067] This example uses the method for constructing a reduced model for evaluating the stability of a photosensitive protein provided in Example 1 to construct a reduced model of a disposable bag for evaluating the stability of a photosensitive protein.

[0068] The collected information shows that the type of lights in the large-scale production plant is LED lights, and the container for storing light-sensitive proteins is a disposable bag. When the window is not covered by the light shield, the light intensity in the bracket where the disposable bag is placed is detected, and the maximum value is 100 lux. The light intensity received by the light-sensitive protein under the current light intensity for 7 days is converted into the light intensity of 8 hours in the reduced model. When the window is covered by a sunshade, the maximum light intensity is measured to be 10 lux. The light intensity of the photosensitive protein exposed to the current light intensity for 7 days is converted to the light intensity of 50 minutes in the reduced model. About 2000 lux.

[0069] Test Example 1

[0070] This test example uses the scaled-down model provided in Example 3, and uses an LED lamp to irradiate 5 mL of photosensitive protein for 15 minutes at a light intensity of 2000 lux. A control group is a photosensitive protein of the same type that is protected from light. The purity of the photosensitive protein after illumination is analyzed using the capillary isoelectric focusing (CIEF) method. The sample and amphoteric electrolytes are mixed, and after injection, the two electrode slots contain acid and alkali, respectively. After high voltage is applied, a pH gradient is generated in the capillary, and the components of the sample migrate to their respective isoelectric points in the capillary to form zones, which are then tested. The results are shown in Tables 1 and Figure 2 As shown, the photosensitive protein can be stable for 7 days in both light and light-protection experiments. Therefore, it is recommended that the storage time of this photosensitive protein produced in this stainless steel tank be less than or equal to 7 days.

[0071] Table 1

[0072]

[0073] Test Example 2

[0074] This test example uses the scaled-down model provided in Example 4, and uses an LED lamp to irradiate 5 mL of photosensitive protein at a light intensity of 2000 lux for 8 hours and 50 minutes, respectively. The same type of photosensitive protein is protected from light as a control group, and the purity analysis method of the photosensitive protein after illumination is carried out in accordance with Test Example 1. The results are shown in Tables 2 and Figure 3 As shown in the figure, when placed at room temperature for 7 days in the dark, the quality of the protein did not change significantly. However, the light experiment showed that the quality of the protein decreased significantly with the extension of the light exposure time, and it could only be stable for 7 days when the bracket was covered with a light shield. Therefore, it is recommended that if you want to store it at room temperature for 7 days during the large-scale production stage, you need to cover the disposable bag bracket with an additional light shield.

[0075] Table 2

[0076]

[0077] In summary, the present invention provides a method for constructing a scaled-down model for evaluating the stability of a photosensitive protein, and establishes a scaled-down model for evaluating the stability of a photosensitive protein. The scaled-down model can be used to simulate the illumination conditions of 7 days of storage in a large-scale production plant in less than 1 day, thereby providing representative stability data for the large-scale commercial production of photosensitive proteins, reducing production risks, and being simple and fast to operate.

[0078] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope 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 shall fall within the protection scope and disclosure scope of the present invention.

Claims

1. A method for constructing a reduced model for evaluating the stability of a light-sensitive protein, characterized in that: The method comprises the following steps: (1) Collecting light information from a large-scale production plant for photosensitive proteins; (2) collecting information on containers storing the photosensitive protein in the large-scale production plant described in step (1); (3) calculating the illumination intensity and illumination time of the photosensitive protein solution stored in the reduced model based on the information collected in step (1) and step (2); (4) Setting a light source consistent with that in a large-scale production plant to illuminate the photosensitive protein solution stored in the scaled-down model, and setting the light intensity and illumination time according to the calculation results of step (3).

2. The method for constructing a reduced model for evaluating the stability of a light-sensitive protein according to claim 1, characterized in that: The light information in step (1) includes the type of lamps in the large-scale production plant, the spectrum information of the lamps and the maximum light intensity value.

3. The method for constructing a reduced model for evaluating the stability of a light-sensitive protein according to claim 1 or 2, characterized in that: The storage container of the photosensitive protein in the large-scale production plant includes a sealed column or a disposable bag; Preferably, the sealing column comprises a sealing cylinder or a sealing prism; Preferably, a light-transmitting window is provided on the storage container.

4. The method for constructing a reduced model for evaluating the stability of a light-sensitive protein according to any one of claims 1 to 3, characterized in that: 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 column in a large-scale production plant, the liquid surface area and window area of ​​the photosensitive protein; (2-2) The maximum light intensity that the photosensitive protein receives when it is stored in disposable bags in a large-scale production plant.

5. The method for constructing a reduced model for evaluating the stability of a light-sensitive protein according to any one of claims 1 to 4, characterized in that: The method for calculating the illumination intensity and illumination 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 photosensitive proteins are stored in sealed columns in large-scale production plants, the light intensity (3-2) When photosensitive proteins are stored in disposable bags in large-scale production plants, the light intensity Among them, A is the window area, in square meters; B is the maximum light intensity, in lux; C is the photosensitive protein liquid surface area, in square meters; t is the illumination time, in minutes, 0<t≤1440; I is the light intensity, in lux.

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

7. The scaled-down model for evaluating the stability of a light-sensitive protein according to claim 6, characterized in that: The reduced model includes: Sample module: used to store photosensitive proteins to be evaluated for stability; Lighting module: used to simulate the lighting conditions of a large-scale production plant, and to set the light source, light intensity and lighting time according to the method for constructing a scaled-down model for evaluating the stability of a photosensitive protein according to any one of claims 1 to 5.

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

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

10. Use of any one of the method for constructing a reduced model for evaluating the stability of a light-sensitive protein as described in any one of claims 1 to 5, the reduced model for evaluating the stability of a light-sensitive protein as described in claim 6 or 7, or the method for evaluating the stability of a light-sensitive protein as described in claim 8 or 9 in the large-scale production of light-sensitive proteins.

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