CN120366018A - Optimization method of cell culture medium preparation method and application thereof - Google Patents
Optimization method of cell culture medium preparation method and application thereofInfo
- Publication number
- CN120366018A CN120366018A CN202510441351.4A CN202510441351A CN120366018A CN 120366018 A CN120366018 A CN 120366018A CN 202510441351 A CN202510441351 A CN 202510441351A CN 120366018 A CN120366018 A CN 120366018A
- Authority
- CN
- China
- Prior art keywords
- culture medium
- preparation
- cell culture
- optimizing
- turbidity
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Apparatus Associated With Microorganisms And Enzymes (AREA)
Abstract
本发明提供了一种细胞培养基配制方法的优化方法及其应用，所述方法包括：(S1)基于培养基配制步骤中的可控参数设计DoE实验，进行培养基配制；记录配制参数和配制后溶液的浊度值和过滤压力值；(S2)对配制参数、配制后溶液的浊度值和过滤压力值进行回归分析，得到影响最终浊度值和过滤压力值的主要因素的控制参数范围；(S3)基于所述主要因素的控制参数范围设置细胞培养基配制方法，指导大规模培养基生产。所述方法基于对不同类型培养基配制的小规模配制过程进行汇总，通过统计分析建立不同类型培养基专属的操作或控制参数范围，进而更好的指导培养基在生物制药大规模生产中的使用。
The present invention provides an optimization method for a cell culture medium preparation method and its application, the method comprising: (S1) designing a DoE experiment based on controllable parameters in a culture medium preparation step to prepare the culture medium; recording the preparation parameters and the turbidity value and filtration pressure value of the prepared solution; (S2) performing regression analysis on the preparation parameters, the turbidity value and filtration pressure value of the prepared solution to obtain the control parameter range of the main factors affecting the final turbidity value and filtration pressure value; (S3) setting a cell culture medium preparation method based on the control parameter range of the main factors to guide large-scale culture medium production. The method is based on summarizing the small-scale preparation process of different types of culture media, establishing the operation or control parameter range exclusive to different types of culture media through statistical analysis, and thus better guiding the use of culture media in large-scale production of biopharmaceuticals.
Description
Technical Field
The invention belongs to the field of biological pharmacy, and particularly relates to an optimization method of a cell culture medium preparation method and application thereof.
Background
The cell, the culture medium and the culture process are used as three driving locomotives in the modern biomedical industry, and are driven by the same driver, so that the development of the biomedical industry is promoted.
Cells are increasingly being used in modern biomedical production as vectors for product expression. The mammalian cell has a complex expression system, can guide the correct folding of protein, provide complex N-glycosylation, accurate O-glycosylation and other post-translational modification functions, so that the target protein is close to the natural protein in the aspects of molecular structure, physical and chemical properties and biological functions, and has wide application in the research and development of recombinant protein medicines.
The culture medium is used as a nutrient source for cell growth, and contains carbohydrates, nitrogenous substances, inorganic salts (including trace elements), vitamins and the like which are required by the cell growth, and the elements provide a good basis for the cell growth and protein synthesis. With the development of science and technology, the development of cell culture media has undergone early formulations containing serum, to media containing complex hydrolysates, and to media with completely defined chemical compositions. The culture genes with completely determined chemical components have the characteristics of determined components, small batch difference and the like, and lay a foundation for steady large-scale commercial production.
The culture process is based on the characteristics of different cells and the requirements of expression products, and robust culture conditions suitable for cell growth expression are developed on the basis of corresponding hosts and culture media. This work is done first in the research and development sector and then scaled up to larger scale for production as needed. The main stream of culture media, especially in recent years, is free of animal-derived components and chemically defined media, which serve as a main nutrient source for cell growth, and which are formulated in research and development departments mainly with reference to formulation suggestions provided by suppliers, which are buried in the following risks for subsequent scale-up production.
(1) Mass transfer difference of preparation containers, namely, most of preparation containers in research and development departments are beakers/bottles (containing stirrers), stirring is carried out through a magnetic stirrer, and a mass-produced disposable/stainless steel mixing system realizes system mixing through a stirring paddle at the bottom, so that the mass transfer and other dimensions are greatly different. Generally, mass transfer capacity of a mass-produced liquid distribution system is inferior to that of a preparation container in a research and development department.
(2) The temperature control difference is that the small-scale preparation can not provide temperature control due to limited equipment capacity, the temperature of the initial solvent water can only be controlled, then the whole preparation process gradually dissipates heat due to the temperature difference with the environment, the temperature drops, and the large-scale preparation has relatively large preparation volume, and the heat dissipation is relatively small and can be slow and very much. The difference in formulation temperature potentially affects the dissolution of the powder.
(3) The main powder feeding speed difference is that the main powder prepared by large-scale production is one of the components with the largest feeding amount, the feeding amount is amplified in equal proportion compared with the research and development department, but the longer the storage time of the culture medium in an open container is, the higher the risk of microorganism breeding is, the longer the total preparation time is, and the feeding speed of the main powder is not too slow. This operation may have a large amount of powder that cannot be dissolved in time, and that floats on the surface of the liquid to form lumps, increasing the difficulty of the solution.
(4) The acid/alkali adding speed difference is that the acid/alkali solution is used as a cosolvent of the culture medium powder, and the proper pH value is provided for the dissolution of the culture medium powder. Because of the limitation of equipment (such as a large pump head) in the preparation of the mass production culture medium, the adding speed of acid/alkali can be relatively difficult to control, in addition, the small test of control precision does not have quantitative guidance, the difficulty is increased for the mass preparation of the practical operation level, and a certain risk is brought to the dissolution of main powder.
The poor mixing efficiency of the main powder in the mass production and preparation can affect the final sterilization, filtration and even release. As a matter of course, there is a need to develop a small-scale culture medium formulation model that provides guidance for parameter settings during the formulation of large-scale production culture media.
Disclosure of Invention
Aiming at the defects existing in the prior art, the invention aims to provide an optimization method of a cell culture medium preparation method and application thereof. The invention establishes a culture medium preparation model for the biopharmaceutical field, and also provides a scheme for testing the feeding speed of each main component of the culture medium, and the two schemes are combined to form a whole set of preparation parameter recommendation, so that the culture medium preparation model can be effectively applied to a disposable/stainless steel mixing system for mass production. Effectively avoiding the material loss and production scheduling effect caused by the problems generated in the preparation process of the culture medium.
In order to achieve the aim of the invention, the invention adopts the following technical scheme:
In a first aspect, the present invention provides a method of optimizing a method of preparing a cell culture medium, the method comprising:
(S1) designing a DoE experiment based on controllable parameters in a culture medium preparation step, and carrying out culture medium preparation, wherein the preparation parameters, the turbidity value and the filtration pressure value of the prepared solution are recorded;
(S2) carrying out regression analysis on the preparation parameters, the turbidity value and the filtration pressure value of the prepared solution to obtain a control parameter range of main factors influencing the final turbidity value and the filtration pressure value;
(S3) setting a cell culture medium preparation method based on the control parameter range of the main factors, and guiding the production of the large-scale culture medium.
The invention examines different key factors of preparation by means of a small-scale preparation device, and provides guidance for parameter setting of large-scale production and large-volume preparation by combining direct reference parameters of dissolution effect, namely turbidity of feed liquid and pressure change of filtration.
In the present invention, regression analysis may be performed by using statistical software such as JMP software and Minitab.
Preferably, the controllable parameters comprise any one or a combination of at least two of temperature, stirring rotation speed, main powder addition time or acid and/or alkali addition time.
In the invention, the above controllable parameters are found to be key factors influencing powder dissolution in the preparation process by examining the preparation process of various culture mediums. The main powder is usually added in a large amount, and the adding speed is the primary concern for the preparation of the culture medium. The acid/base generally provides an environment in which the primary powder is dissolved, the rate of addition of which potentially affects the pH uniformity of the formulation system, and in addition, both stirring and temperature control are aimed at the primary powder to aid in dissolution.
Preferably, the medium preparation device is a small-scale medium preparation device.
Preferably, the small-scale culture medium preparation device comprises a preparation reactor, a power control unit, a temperature control unit, a component adding unit, an off-line detection unit and a filtering unit;
the power control unit is used for controlling the stirring rotating speed of the culture medium preparation system;
the temperature control unit is used for controlling the temperature of the culture medium preparation system;
the component adding unit is used for controlling the adding time of the components of the culture medium;
the off-line detection unit is used for detecting turbidity, pH or conductivity of the solution in the preparation equipment off-line;
the filter unit is used for filtering the solution in the preparation equipment and detecting the filtering pressure.
In the invention, the control of the addition speed is realized by controlling the addition time of the components of the culture medium.
In the invention, the powder dissolution condition is judged by detecting the turbidity of the solution, and further, the filtering pressure is also detected, so that the powder dissolution condition is reflected from the side surface. The dissolution condition can be judged by adopting two standard instruments, so that the preparation effect can be judged more comprehensively.
Preferably, the formulation reactor is a 2-4L reactor, which may be, for example, 2L, 3L, 4L, or the like.
Preferably, the power control unit includes a motor and a stirring paddle.
Preferably, the temperature control unit includes a heating blanket and a temperature electrode.
Preferably, the component adding unit is a component adding device for component transfer.
In the present invention, the medium component adding apparatus is used for component addition, and for example, the liquid/powder transfer vessel may be a pipette and/or beaker, transferring the solution and powder required for the medium formulation into the preparation vessel. The component adding unit is used for adding substances such as main powder, acid and/or alkali, cell shearing resistant protective agent and the like.
Preferably, the off-line detection unit comprises a turbidity detector and a multi-parameter detector.
In the invention, the multi-parameter detector is used for detecting pH and/or conductivity.
In the present invention, the turbidity detector may be, for example, a turbidity meter.
Preferably, the filter unit comprises a filter and a pressure sensor.
In a second aspect, the present invention provides the use of the method for optimizing a method for preparing a cell culture medium according to the first aspect in the preparation of a culture medium.
The numerical ranges recited herein include not only the recited point values, but also any point values between the recited numerical ranges that are not recited, and are limited to, and for the sake of brevity, the invention is not intended to be exhaustive of the specific point values that the recited range includes.
Compared with the prior art, the invention has the following beneficial effects:
The invention is applied to the preparation field of cell culture media without animal source components and chemical limitation in the biopharmaceuticals, and overcomes the defect that the existing large-scale preparation is lack of effective guidance. The method has the advantages that a small-scale preparation device is established, the flow of preparation and amplification of the culture medium is perfected, precious guidance is provided for large-scale production and preparation through a series of DoE experimental designs such as process control or operation parameters of the specific culture medium, the preparation and release of the culture medium for producing pharmacy and the filtering operation are controlled, pollution/deviation and the like of large-scale production caused by the preparation of the culture medium are effectively avoided or reduced, and the method has great significance for stable production.
Drawings
FIG. 1 is a schematic diagram of a small-scale medium preparation apparatus.
FIG. 2 is a development flow of a method for preparing a culture medium.
FIG. 3 is a graph of turbidity change during formulation.
FIG. 4 is a graph of the slope of final solution turbidity versus sterile filtration pressure.
FIG. 5 is the effect of temperature and NaOH addition rate on turbidity.
FIG. 6 is a combination of conditions corresponding to the lowest turbidity exhibited by the predictive describer.
Detailed Description
The technical scheme of the invention is further described by the following specific embodiments. It will be apparent to those skilled in the art that the examples are merely to aid in understanding the invention and are not to be construed as a specific limitation thereof.
The specific techniques or conditions are not identified in the examples and are described in the literature in this field or are carried out in accordance with the product specifications. The reagents or apparatus used were conventional products commercially available through regular channels, with no manufacturer noted.
Example 1
The embodiment provides a small-scale culture medium preparation device which comprises a preparation reactor, a power control unit, a temperature control unit, a component adding unit, an off-line detection unit and a filtering unit. A schematic of a small-scale medium preparation apparatus is shown in FIG. 1.
The preparation reactor is a 3L reactor.
The power control unit is used for controlling the stirring rotating speed of the culture medium preparation system and comprises a motor and a stirring paddle.
The temperature control unit is used for controlling the temperature of the culture medium preparation system and comprises a heating blanket and a temperature electrode.
The component adding unit is used for controlling the adding time of the components of the culture medium and comprises a peristaltic pump and a pipettor.
The off-line detection unit is used for detecting turbidity, pH or conductivity of the solution in the preparation equipment in an off-line manner, and comprises a turbidity meter and a multi-parameter detector.
The filter unit is used for filtering the solution in the preparation equipment and detecting the filtering pressure, and comprises a filter and a pressure sensor.
The small-scale culture medium preparation device can realize investigation of key factors of various culture mediums and output key factor guidance influencing dissolution, provide guidance for parameter setting of large-scale preparation, and reduce the risk of large-scale preparation.
Example 2
The present example provides a method for optimizing a cell culture medium formulation method.
The development flow of the culture medium preparation method is shown in FIG. 2.
1. The DoE experiments were designed based on the controllable parameters in the medium preparation step, medium preparation was performed using the small-scale medium preparation apparatus described in example 1, and the preparation parameters and turbidity values and filtration pressure values of the prepared solutions were recorded.
(1) Basic formula of culture medium
This example is given as a commercial ActiPro medium (available from Cytiva). The basic formulation is shown in Table 1, actiPro ingredients Table (2.00 kg).
TABLE 1
| Composition of the components | Target value (g) | Minimum (g) | Maximum value (g) | Operational error |
| Purified water | 1800.00 | 1710.00 | 1890.00 | ±2% |
| ActiPro culture 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% |
| NaHCO3 | 3.60 | 3.53 | 3.67 | ±2% |
| 10N sodium hydroxide | NA | NA | NA | NA |
| 6N hydrochloric acid | NA | NA | NA | NA |
| Final volume to volume | 2000.00 | 1960.00 | 2040.00 | ±2% |
The table shows that pH adjustment was performed as needed.
(2) The preparation flow of the culture medium is as follows:
1) Selecting a container with proper size, weighing corresponding volume of purified water, adding the purified water into a preparation container, and controlling the water temperature at 18-30 ℃. The small scale formulation model used in this example is exemplified by a 3L glass reactor.
2) While stirring in step 1, slowly adding the weighed ActiPro culture medium powder into the preparation container in step 1) until the powder is completely mixed in the liquid, and stirring for 10min.
3) Slowly adding the weighed 10N NaOH solution into the container in the step 1), and stirring for 10min until the powder is dissolved.
4) L-glutamine, sodium inosinate, thymine and NaHCO 3 were added to the vessel of step 1) and stirred for 5min until dissolved.
5) Adjusting pH of the culture medium to 7.20-7.45 with 10N sodium hydroxide or 6N hydrochloric acid, adding purified water to a final volume, and mixing again for 5min;
6) The final pH was tested (standard: 6.90-7.55), turbidity (standard: 4.00 NTU), osmolality (standard: 300-340 mOsm/kg) and all were within the accepted range.
7) The formulation was completed using a 0.1 μm filter into a suitable sterile storage vessel.
(3) Design of DoE experiment
Based on the basic formula and the preparation flow, the selected controllable parameters comprise temperature, rotating speed, main powder adding speed, acid/alkali adding speed and shearing force protective agent adding speed.
Referring to the preparation details in the basic preparation flow, doE experiment design (including a center point) is carried out on two control parameters and two operation parameters, the design of the small-scale culture medium preparation parameters is shown in a table 2, the upper limit and the lower limit of a temperature stepping control range are shown in consideration of temperature electrode control precision and historical preparation experience, the rotation speed steps on the condition that the liquid level is just vortex, the rotation speed with high vortex in the equipment capacity is larger, actiPro and 10N NaOH are added, 1s represents a fast adding speed, and 10min represents a slow adding speed. A total of 9 conditions are specified in table 3 below.
TABLE 2
TABLE 3 Table 3
(3) Small model experiment
Experiments were performed in a 3L glass reactor according to the experimental design of Table 3, and turbidity data of the process were recorded after ActiPro and 1N NaOH additions, facilitating observation of the main influencing factors and variations of the turbidity of the formulation process.
(4) Recording the formulation parameters and the turbidity value and filtration pressure value of the solution after formulation
Turbidity data corresponding to each experimental condition and data of filtration pressure of the post-sterilization filter are shown in table 4.
TABLE 4 Table 4
2. And carrying out regression analysis on the preparation parameters, the turbidity value and the filtration pressure value of the prepared solution by JMP software to obtain control parameters of main factors influencing the final turbidity value and the filtration pressure value.
Turbidity changes during formulation as shown in fig. 3, it can be seen from fig. 3 that ActiPro powder is the main source of turbidity, and that turbidity drops significantly after NaOH addition, i.e. NaOH addition is critical for the formulation of the medium.
The turbidity data corresponding to each experimental condition and the data of the filtration pressure of the post-sterilization filter are shown in Table 4, wherein the highest temperature condition 29 ℃ and the lowest rotation speed of 230rpm, and the turbidity of the fastest 10N NaOH addition are relatively high. In addition, turbidity and the slope of the filtration pressure are in a relatively good linear relationship, with R 2 = 0.6883 (fig. 4), i.e., the higher the endpoint turbidity, the greater the filtration pressure.
Figure 5 presents that temperature and NaOH addition rate significantly affect turbidity. Fig. 6 is a graph showing that the combination of conditions corresponding to the lowest turbidity exhibited by the predictive markers is lower temperature control and slower NaOH addition rate.
By JMP software analysis, temperature and 10N NaOH addition rate are the main factors affecting the final turbidity, and the best combination is a slow addition of NaOH at a temperature of 21℃and 10N.
3. And setting a cell culture medium preparation method based on the control parameters of the main factors, and guiding the production of the large-scale culture medium.
(1) The temperature guidance is that the optimized temperature parameters of the small-scale model preparation can be directly applied to the parameter setting of the large-scale preparation.
(2) The optimized rotating speed parameter prepared by the small-scale model can be used for guiding large-scale preparation, and if the dissolution risk is low, special consideration is not needed. If there is a certain risk of dissolution, the rotational speed can be increased within the capacity of the device to a value near the upper limit of the capacity of the device.
(3) And guiding the adding time of main powder and the like, wherein the optimal time parameter of the small-scale model preparation can be used for referencing the large-scale preparation.
In summary, the invention provides a small-scale culture medium preparation device established according to a small-scale glass reactor, a method for preparing a large-scale culture medium is developed based on the model, and corresponding guidance is provided by combining limiting points of large-scale production.
The applicant declares that the above is only a specific embodiment of the present invention, but the scope of the present invention is not limited thereto, and it should be apparent to those skilled in the art that any changes or substitutions that are easily conceivable within the technical scope of the present invention disclosed by the present invention fall within the scope of the present invention and the disclosure.
Claims (10)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202510441351.4A CN120366018A (en) | 2025-04-09 | 2025-04-09 | Optimization method of cell culture medium preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202510441351.4A CN120366018A (en) | 2025-04-09 | 2025-04-09 | Optimization method of cell culture medium preparation method and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN120366018A true CN120366018A (en) | 2025-07-25 |
Family
ID=96450334
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202510441351.4A Pending CN120366018A (en) | 2025-04-09 | 2025-04-09 | Optimization method of cell culture medium preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN120366018A (en) |
-
2025
- 2025-04-09 CN CN202510441351.4A patent/CN120366018A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Junne et al. | Cultivation of cells and microorganisms in wave‐mixed disposable bag bioreactors at different scales | |
| Todaro et al. | Fermentation and biochemical engineering handbook | |
| EP2270129B1 (en) | Bioreactor systems and associated methods of processing bioreactor vessels | |
| CN101671712B (en) | Method and device for optimizing and amplifying abamectin fermenting process | |
| CN202030764U (en) | Fermentation bottle and bio-fermentation culture device provided with same | |
| EP1183326A1 (en) | U-shape and/or nozzle-u-loop fermentor and method of carrying out a fermentation process | |
| De Wilde et al. | Superior scalability of single-use bioreactors | |
| US20220380718A1 (en) | Continuous reconstitution of process materials from solids | |
| TWI230199B (en) | Optimisation of fermentation processes | |
| US20230313109A1 (en) | Device and process for cell culture media preparation and performing cell culture | |
| Apostolidis et al. | Investigation of low viability in sparged bioreactor CHO cell cultures points to variability in the Pluronic F-68 shear protecting component of cell culture media | |
| CN120366018A (en) | Optimization method of cell culture medium preparation method and application thereof | |
| Oosterhuis et al. | How multipurpose is a disposable bioreactor? | |
| Curtis | Application of bioreactor design principles to plant micropropagation | |
| Elias et al. | Recombinant protein production in large-scale agitated bioreactors using the baculovirus expression vector system | |
| TW201817868A (en) | Cell culture vessel adapter | |
| CN114075540B (en) | HEK293 cell culture medium with full chemical composition and application thereof | |
| WO2024193113A1 (en) | Continuous-culture turbidostat and use thereof in measuring bacterial growth and bacterial cell cycle | |
| Zeng et al. | Continuous culture | |
| Jones | Single-use processing for microbial fermentations | |
| CN111808822B (en) | Feed supplement liquid for cell culture and method for improving recombinant HEK293 cell protein expression quantity | |
| CN1320097C (en) | Culturing system of built-in light biological reactor of plant tissue cutter | |
| Harada et al. | Fermentation pilot plant | |
| Parekh et al. | Bioprocessing using novel cell culture systems | |
| CN209836154U (en) | Bioreactor |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |