Optimization method and application of cell culture medium preparation method

Abstract

The invention provides an optimization method and application of a cell culture medium preparation method, and the method comprises the following steps: (S1) designing a DoE experiment based on controllable parameters in a culture medium preparation step, and carrying out culture medium preparation; recording preparation parameters and a turbidity value and a filtering pressure value of the prepared solution; (S2) performing regression analysis on the preparation parameters and the turbidity value and the filtering pressure value of the prepared solution to obtain a control parameter range of main factors influencing the final turbidity value and the filtering pressure value; and (S3) setting a cell culture medium preparation method based on the control parameter range of the main factors, and guiding large-scale culture medium production. According to the method, on the basis of summarizing small-scale preparation processes of different types of culture media, special operation or control parameter ranges of the different types of culture media are established through statistical analysis, and then the culture media are better guided to be used in large-scale production of biopharmacy.

Description

Technical Field

[0001] The present invention belongs to the field of biopharmaceuticals, and particularly relates to an optimization method for preparing a cell culture medium and its application. Background Art

[0002] Cells, culture media, and culture processes, as the three main components in the modern biopharmaceutical industry, work together to promote the development of the biopharmaceutical industry.

[0003] Cells, as the carriers for product expression, are increasingly used in modern biopharmaceutical production. Mammalian cells, due to their complex expression systems, can guide the correct folding of proteins, provide complex N-glycosylation and accurate O-glycosylation, and other post-translational modification functions, making the target protein close to the natural protein in terms of molecular structure, physical and chemical properties, and biological functions. Therefore, they are widely used in the research and production of recombinant protein drugs.

[0004] As the nutrient source for cell growth, the culture medium contains carbohydrates, nitrogen-containing substances, inorganic salts (including trace elements), vitamins, etc. required for cell growth, which provide a good basis for cell growth and protein synthesis. With the development of science and technology, the development of cell culture media has gone through early formulations containing serum, to media containing complex hydrolysates, and then to completely chemically defined media. The completely chemically defined culture medium, due to its characteristics such as definite composition and small batch differences, has laid a foundation for robust large-scale commercial production.

[0005] The culture process is a robust culture condition developed based on the characteristics of different cells and the requirements of the expressed product, on the basis of the corresponding host and culture medium, suitable for cell growth and expression. This work is first completed in the R & D department and then scaled up to a larger scale for production as needed. Culture media, especially the mainstream animal-free and chemically defined media in recent years, as the main nutrient source for cell growth, are mainly prepared in the R & D department with reference to the preparation suggestions provided by suppliers, which poses the following several potential problems for subsequent large-scale production.

[0006] (1) Mass transfer differences in preparation containers: The preparation containers in the R & D department are mostly beakers / flasks (with magnetic stirrers), and stirring is carried out through a magnetic stirrer, while the disposable / stainless steel mixing systems for large-scale production achieve system mixing through bottom stirrers. There are significant differences in mass transfer and other dimensions between the two. Generally, the mass transfer capacity of the large-scale production liquid preparation system is worse than that of the preparation containers in the R & D department.

[0007] (2) Temperature control differences: Due to limited equipment capabilities in small-scale preparation, temperature control cannot be provided. Only the temperature of the initial solvent "water" can be controlled. Subsequently, during the entire preparation process, due to the temperature difference from the environment, heat dissipation occurs gradually, and the temperature drops. In large-scale preparation, because the preparation volume is relatively large, heat dissipation is much slower compared to small-scale trials. The difference in preparation temperature may potentially affect the dissolution of the powder.

[0008] (3) Differences in the feeding speed of the main powder: The main powder in large-scale production preparation is one of the components with the largest feeding amount, and the feeding amount will be proportionally enlarged compared to that of the R & D department. However, considering that the culture medium is a nutrient-rich component, the longer it is stored in an open container, the higher the risk of microbial growth. The total preparation time should not be too long, and the feeding speed of the main powder should not be too slow. This operation may result in a large amount of powder not being dissolved in time, floating on the liquid surface and caking, increasing the difficulty of the solution.

[0009] (4) Differences in the addition speed of acid / alkali: Acid / alkali solutions, as co-solvents for the culture medium powder, provide an appropriate pH value for the dissolution of the culture medium powder. Due to equipment limitations in large-scale production of culture medium preparation (for example: larger pump heads), the addition speed of acid / alkali is relatively difficult to control. In addition, there is no quantitative guidance for small-scale trials in terms of control accuracy, which further increases the difficulty in actual large-scale preparation operations and poses a certain risk to the dissolution of the main powder.

[0010] In large-scale production preparation, the mixing efficiency of the main powder is poor, which will affect the final sterile filtration and even release. Therefore, it is urgent to develop a small-scale culture medium preparation model to provide guidance for parameter setting in large-scale production of culture medium preparation. Summary of the Invention

[0011] Aiming at the deficiencies of the existing technology, the purpose of the present invention is to provide an optimization method for a cell culture medium preparation method and its application. The present invention establishes a culture medium preparation model for the biopharmaceutical field, and also provides a test scheme for the feeding speed of each main component in the scale-up of the culture medium. The combination of the two forms a complete set of recommended preparation parameters, enabling it to be effectively applied to disposable / stainless steel mixing systems in large-scale production. Effectively avoid material losses and production scheduling impacts caused by problems during the culture medium preparation process.

[0012] To achieve the purpose of this invention, the present invention adopts the following technical solutions:

[0013] In the first aspect, the present invention provides an optimization method for a cell culture medium preparation method, and the method includes:

[0014] (S1) Design a DoE experiment based on the controllable parameters in the culture medium preparation steps, and conduct culture medium preparation; record the preparation parameters and the turbidity value and filtration pressure value of the solution after preparation;

[0015] (S2) Perform a regression analysis on the formulation parameters, the turbidity value of the solution after formulation, and the filtration pressure value to obtain the control parameter range of the main factors affecting the final turbidity value and the filtration pressure value;

[0016] (S3) Set the cell culture medium formulation method based on the control parameter range of the main factors to guide large-scale culture medium production.

[0017] The present invention examines different key formulation factors by means of a small-scale formulation device, and combines the dissolution effect to directly refer to the parameters - the turbidity of the feed liquid and the change in filtration pressure, providing guidance for setting the parameters of large-scale large-volume formulation.

[0018] In the present invention, for example, statistical software such as JMP software and Minitab can be used for the regression analysis.

[0019] Preferably, the controllable parameters include: any one or a combination of at least two of temperature, stirring speed, main powder addition time, or acid and / or base addition time.

[0020] In the present invention, by examining the formulation processes of various culture media, it is found that the above-mentioned controllable parameters are key factors affecting the dissolution of powders during the formulation process. The main powder usually has a large addition amount, and the addition speed is the primary concern in culture medium formulation. Acids / bases usually provide the environment for dissolving the main powder, and their addition speed potentially affects the pH uniformity of the formulation system. In addition, the purposes of stirring and temperature control are also to assist in dissolving the main powder.

[0021] Preferably, the device used for the culture medium formulation is a small-scale culture medium formulation device.

[0022] Preferably, the small-scale culture medium formulation device includes: a formulation reactor, a power control unit, a temperature control unit, a component addition unit, an off-line detection unit, and a filtration unit;

[0023] The power control unit is used to control the stirring speed of the culture medium formulation system;

[0024] The temperature control unit is used to control the temperature of the culture medium formulation system;

[0025] The component addition unit is used to control the addition time of the culture medium components;

[0026] The off-line detection unit is used to off-line detect the turbidity, pH, or conductivity of the solution in the formulation equipment;

[0027] The filtration unit is used to filter the solution in the formulation equipment and detect the filtration pressure.

[0028] In the present invention, the control of the addition speed is achieved by controlling the addition time of the culture medium components.

[0029] In the present invention, the turbidity of the solution is detected to judge the dissolution of the powder. Further, the filtration pressure is also detected to reflect the dissolution of the powder from the side. Using two standard instruments to judge the dissolution can more comprehensively judge the preparation effect.

[0030] Preferably, the preparation reactor is a 2 - 4L reactor, for example, it can be 2L, 3L, or 4L, etc.

[0031] Preferably, the power control unit includes a motor and a stirring paddle.

[0032] Preferably, the temperature control unit includes a heating blanket and a temperature electrode.

[0033] Preferably, the component addition unit is a component addition device for component transfer.

[0034] In the present invention, the use of the medium component addition device is for component addition; for example, the liquid / powder transfer container can be a pipette and / or a beaker to transfer the solution and powder required for the medium formulation into the preparation container. The component addition unit is used for the addition of substances such as main powder, acid and / or base, and cell anti - shear protectant.

[0035] Preferably, the off - line detection unit includes a turbidity detector and a multi - parameter detector.

[0036] In the present invention, the multi - parameter detector is used for the detection of pH and / or conductivity.

[0037] In the present invention, the turbidity detector can be a turbidimeter, for example.

[0038] Preferably, the filtration unit includes a filter and a pressure sensor.

[0039] In a second aspect, the present invention provides the application of the optimization method of the cell culture medium preparation method described in the first aspect in medium preparation.

[0040] The numerical ranges described in the present invention not only include the above - listed point values, but also include any point values between the above - mentioned numerical ranges that are not listed. Due to space limitations and for the sake of brevity, the present invention does not exhaustively list the specific point values included in the range.

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

[0042] The present invention is applied to the field of preparing animal-free and chemically defined cell culture media in biopharmaceuticals, making up for the deficiency of the existing large-scale preparation lacking effective guidance. A small-scale preparation device is established to improve the process of medium preparation scale-up. Through a series of DoE experimental designs on specific medium process control or operating parameters, etc., valuable guidance is provided for large-scale production preparation, enabling the release and filtration operations of medium preparation for pharmaceutical production to be controlled, effectively avoiding or reducing pollution / deviations in large-scale production caused by medium preparation, which is of great significance for stable production. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 It is a schematic diagram of a small-scale medium preparation device.

[0044] Figure 2 It is the development process of the medium preparation method.

[0045] Figure 3 It is the turbidity change diagram during the preparation process.

[0046] Figure 4 It is the slope relationship diagram between the turbidity of the final solution and the sterilizing filtration pressure.

[0047] Figure 5 It is the influence of temperature and NaOH addition rate on turbidity.

[0048] Figure 6 It is to show the combination of conditions corresponding to the lowest turbidity using a prediction profiler. DETAILED DESCRIPTION OF THE INVENTION

[0049] The technical solution of the present invention will be further described below through specific embodiments. Those skilled in the art should understand that the embodiments are only for helping to understand the present invention and should not be regarded as specific limitations to the present invention.

[0050] For those technical or conditions not specified in the embodiments, they shall be carried out according to the techniques or conditions described in the literature in this field or according to the product specifications. For the reagents or instruments whose manufacturers are not indicated, they are all conventional products that can be obtained through regular commercial channels.

[0051] Example 1

[0052] This example provides a small-scale medium preparation device, which includes: a preparation reactor, a power control unit, a temperature control unit, a component addition unit, an off-line detection unit, and a filtration unit. The schematic diagram of the small-scale medium preparation device is as Figure 1 shown.

[0053] The preparation reactor is a 3L reactor.

[0054] The power control unit is used to control the stirring speed of the culture medium preparation system; the power control unit includes a motor and a stirring paddle.

[0055] The temperature control unit is used to control the temperature of the culture medium preparation system; the temperature control unit includes a heating blanket and a temperature electrode.

[0056] The component addition unit is used to control the addition time of the culture medium components; the component addition unit includes a peristaltic pump and a pipettor.

[0057] The off-line detection unit is used to off-line detect the turbidity, pH or conductivity of the solution in the preparation equipment; the off-line detection unit includes a turbidimeter and a multi-parameter detector.

[0058] The filtration unit is used to filter the solution in the preparation equipment and detect the filtration pressure; the filtration unit includes a filter and a pressure sensor.

[0059] The small-scale culture medium preparation device can investigate various key factors of the culture medium and output the key factors affecting dissolution to guide the parameter setting of large-volume preparation, reducing the risk of large-scale preparation.

[0060] Example 2

[0061] This example provides an optimization method for a cell culture medium preparation method.

[0062] The development process of the culture medium preparation method is as Figure 2 shown.

[0063] 1. Design a DoE experiment based on the controllable parameters in the culture medium preparation steps, and use the small-scale culture medium preparation device described in Example 1 to prepare the culture medium; record the preparation parameters and the turbidity value and filtration pressure value of the solution after preparation.

[0064] (1) Basic formula of the culture medium

[0065] This example takes the commercial ActiPro culture medium (purchased from Cytiva) as an example. The basic formula is shown in Table 1, ActiPro ingredient list (2.00 kg).

[0066] Table 1

[0067] Component Target value (g) Minimum value (g) Maximum value (g) Operating error Purified water 1800.00 1710.00 1890.00 ±2％ ActiPro medium main powder 44.72 43.83 45.61 ±2％ 10N Sodium hydroxide 8.58 8.41 8.75 ±2％ L-Glutamine powder 1.1692 1.1458 1.1926 ±2％ Sodium hypoxanthine 31.62 (mg) 30.99 (mg) 32.25 (mg) ±2％ Thymine 7.75 (mg) 7.60 (mg) 7.91 (mg) ±2％ <![CDATA[NaHCO3]]> 3.60 3.53 3.67 ±2％ 10N Sodium hydroxide * NA NA NA NA 6N Hydrochloric acid * NA NA NA NA Final volume made up 2000.00 1960.00 2040.00 ±2％

[0068] In the table, * indicates: adjust the pH as required.

[0069] (2) The preparation process of the culture medium is as follows:

[0070] 1) Select a container of appropriate size, weigh out the corresponding volume of purified water and add it to the preparation container, with the water temperature controlled at 18 - 30 °C. In this example, the small-scale preparation model used takes a 3L glass reactor as an example.

[0071] 2) While stirring in step 1, slowly add the weighed ActiPro medium powder to the preparation container in step 1) until it is completely mixed in the liquid, and stir for 10 min.

[0072] 3) Slowly add the weighed 10N NaOH solution to the container in step 1), and stir for 10 min until the powder is dissolved.

[0073] 4) Add L-glutamine, sodium hypoxanthine, thymine, and NaHCO3 to the container in step 1), and stir for 5 min until dissolved.

[0074] 5) Use 10N sodium hydroxide or 6N hydrochloric acid to adjust the pH value of the medium to 7.20 - 7.45, add purified water to make up the volume to the final volume, and mix well again for 5 min;

[0075] 6) Test the final pH (standard: 6.90 - 7.55), turbidity (standard: < 4.00 NTU), and osmotic pressure value (standard: 300 - 340 mOsm / kg), and all are within the acceptable range.

[0076] 7) Filter through a 0.1μm filter into a suitable sterile storage container to complete the preparation.

[0077] (3) DoE experimental design

[0078] Based on the above basic formula and preparation process, the controllable parameters selected include: temperature, rotation speed, main powder addition speed, acid / alkali addition speed, and shear force protector addition speed.

[0079] Referring to the preparation details in the basic preparation process, conduct a DoE experimental design (including the central point) for two control parameters and two operating parameters. The small-scale medium preparation parameter design is shown in Table 2. Set the temperature at the upper and lower limits of the control range, and consider the control accuracy of the temperature electrode and historical preparation experience; set the rotation speed at two conditions, where the low speed is when there is just a vortex on the liquid surface, and the high speed is the rotation speed with a larger vortex within the equipment capacity; for the addition of ActiPro and 10N NaOH, 1 s represents the fast addition speed, and 10 min represents the slow addition speed. There are a total of 9 conditions, as shown in Table 3 below.

[0080] Table 2

[0081]

[0082]

[0083] Table 3

[0084]

[0085] (3) Small-scale model experiment

[0086] According to the experimental design in Table 3, experiments were carried out in a 3L glass reactor, and the turbidity data of the process were recorded after the addition of ActiPro and 1N NaOH to facilitate the observation of the main influencing factors and changes in turbidity during the preparation process.

[0087] (4) Record the formulation parameters and the turbidity value and filtration pressure value of the solution after formulation

[0088] The turbidity data corresponding to each experimental condition and the data of the filtration pressure of the subsequent sterilizing filter are shown in Table 4.

[0089] Table 4

[0090]

[0091]

[0092] 2. Use JMP software to perform regression analysis on the formulation parameters and the turbidity value and filtration pressure value of the solution after formulation to obtain the control parameters of the main factors affecting the final turbidity value and filtration pressure value.

[0093] The change in turbidity during the formulation process is as Figure 3 shown. It can be seen from Figure 3 that ActiPro powder is the main source of turbidity. After the addition of NaOH, the turbidity decreases significantly, indicating that the addition of NaOH is very crucial for the preparation of this medium.

[0094] The turbidity data corresponding to each experimental condition and the data of the filtration pressure of the subsequent sterilizing filter are shown in Table 4. Among them, the turbidity is relatively high under the highest temperature condition of 29°C, the lowest rotation speed of 230 rpm, and the fastest addition of 10N NaOH. In addition, the slope of turbidity and filtration pressure shows a relatively good linear relationship, R 2 = 0.6883( Figure 4 ), that is, the higher the final turbidity, the greater the filtration pressure.

[0095] Figure 5 It shows that temperature and the addition rate of NaOH significantly affect turbidity. Figure 6 It is shown by the prediction profiler that the combination of conditions corresponding to the lowest turbidity is lower temperature control and slower addition rate of NaOH.

[0096] Through JMP software analysis, temperature and the addition rate of 10N NaOH are the main factors affecting the final turbidity. The optimal combination is a temperature of 21 °C and a slow addition of 10N NaOH.

[0097] 3. Set the control parameters for the cell culture medium preparation method based on the above main factors to guide large-scale medium production.

[0098] (1) Guidance for temperature: The optimized temperature parameters for small-scale model preparation can be directly applied to the parameter settings for large-scale preparation.

[0099] (2) Guidance for rotation speed: The optimized rotation speed parameters for small-scale model preparation can guide large-scale preparation. If the dissolution risk is low, no special consideration is required. If there is a certain dissolution risk, the rotation speed can be increased to near the upper limit of the equipment capacity within the equipment's capabilities.

[0100] (3) Guidance for the addition duration of the main powder, etc.: The optimal duration parameters for small-scale model preparation can be used as a reference for large-scale preparation.

[0101] In summary, the present invention provides a small-scale culture medium preparation device established based on a small-scale glass reactor, develops a method for large-scale culture medium preparation based on the model, and provides corresponding guidance in combination with the limiting points of large-scale production.

[0102] The applicant declares that the above description is only the specific implementation manner 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 fall within the protection scope and the disclosure scope of the present invention.

Claims

1. An optimization method for a method of preparing a cell culture medium, characterized in that, The method includes: (S1) Design a DoE experiment based on the controllable parameters in the culture medium preparation step, and conduct the culture medium preparation; record the preparation parameters, the turbidity value and the filtration pressure value of the solution after preparation. (S2) Conduct a regression analysis on the preparation parameters, the turbidity value and the filtration pressure value of the solution after preparation to obtain the control parameter ranges of the main factors affecting the final turbidity value and the filtration pressure value. (S3) Set the cell culture medium preparation method based on the control parameter ranges of the main factors to guide large-scale culture medium production.

2. The optimization method of the cell culture medium preparation method according to claim 1, characterized in that The controllable parameters include any one or a combination of at least two of: temperature, stirring speed, main powder addition time, acid and / or alkali addition time, or cell anti-shear protection agent addition time.

3. The optimization method of the cell culture medium preparation method according to claim 1 or 2, characterized in that The device used for the culture medium preparation is a small-scale culture medium preparation device. The small-scale culture medium preparation device includes: a preparation reactor, a power control unit, a temperature control unit, a component addition unit, an off-line detection unit, and a filtration unit. The power control unit is used to control the stirring speed of the culture medium preparation system. The temperature control unit is used to control the temperature of the culture medium preparation system. The component addition unit is used to control the addition time of the culture medium components. The off-line detection unit is used to off-line detect the turbidity, pH or conductivity of the solution in the preparation equipment. The filtration unit is used to filter the solution in the preparation equipment and detect the filtration pressure.

4. The optimization method of the cell culture medium preparation method according to claim 3, wherein The preparation reactor is a 2-4L reactor.

5. The optimization method of the cell culture medium preparation method according to claim 3 or 4, characterized in that The power control unit includes a motor and a stirring paddle.

6. The optimization method of the cell culture medium preparation method according to any one of claims 3-5, characterized in that, The temperature control unit includes a heating blanket and a temperature electrode.

7. The optimization method of the cell culture medium preparation method according to any one of claims 3-6, characterized in that, The component addition unit is a culture medium component addition device for component transfer.

8. The optimization method of the cell culture medium preparation method according to any one of claims 3-7, characterized in that, The off-line detection unit includes a turbidity detector and a multi-parameter detector.

9. The optimization method of the cell culture medium preparation method according to any one of claims 3-8, characterized in that, The filtration unit includes a filter and a pressure sensor.

10. Application of the optimization method of the cell culture medium preparation method according to any one of claims 1-9 in culture medium preparation.

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