Powder cell culture medium and preparation method thereof

Abstract

The invention discloses a powder cell culture medium and a preparation method thereof. The preparation method comprises the following steps: S1, dividing a culture medium base material into a first culture medium base material and a second culture medium base material according to the particle size of each component culture medium base material; s2, the first culture medium base materials of all the components are smashed into small-particle culture medium base materials; s3, weighing the small-particle culture medium base material of each component and the second culture medium base material of each component; and S4, mixing the small-particle culture medium base material of each component and the second culture medium base material of each component weighed in the S3 to prepare the powder cell culture medium. Due to the fact that the first culture medium base material is treated in advance, the second culture medium base material does not need to be smashed again, the large-particle material and the first culture medium base material can be treated to 100-200 meshes according to the properties of the materials by adopting suitable smashing parameters, and granularity controllability can be achieved more easily by adopting different smashing parameters for different materials. And the product can be basically kept at the optimal granularity of 100-200 meshes to the greatest extent, and the condition of excessive fine powder caused by grinding large particles is avoided.

Description

Technical Field

[0001] The invention relates to the field of biological cell culture, and in particular to a powdered cell culture medium and a preparation method thereof. Background Art

[0002] The current cell culture medium formula development is to weigh the powdered raw materials one by one, group them into liquids, mix the liquids evenly, adjust the pH and osmotic pressure, and then filter and sterilize them to form the finished product. Although the liquid preparation form has an accurate formula, when converted into a solid culture medium, the materials cannot be dissolved in advance by grouping, but all the materials are mixed together. Solid mixing cannot reproduce the microscopic chemical changes that occur during liquid mixing, which will cause the solid-liquid differences of insoluble and trace components to have a greater impact on cell growth.

[0003] In addition, the development of existing cell culture medium formulas must go through the following steps: DOE experimental design (component grouping, concentration grading, orthogonal experimental design) → prepare liquid culture media with different formulations according to the experimental design → culture cells to confirm the performance of each formula → use statistical analysis to analyze the best influencing factors and levels → form a liquid culture medium formula → prepare three batches of samples to verify the performance of the liquid culture medium formula.

[0004] After the liquid culture medium formula is developed, the liquid culture medium formula needs to be converted to a solid production process. The specific steps are: liquid culture medium formula → put all formula ingredients into a certain amount of aqueous solution to fully dissolve → adjust pH and osmotic pressure and record the dosage → culture cells after sterilization and filtration, consistent with the performance of group preparation → calculate the dosage of all materials and solidify it into a solid culture medium formula → use solid preparation technology (weighing, premixing, grinding, final mixing, packaging) to trial-produce small samples → dissolve the solid sample, fully sterilize and filter, and culture cells → consistent with the performance of liquid-prepared culture medium → complete the process transfer from liquid to solid.

[0005] In the process of solid production process expansion, the following steps are required: small-scale solid process test to determine the range of production process parameters (grinding parameters, mixing parameters, powder particle size requirements, dissolution time limit, etc.) → solid pilot scale-up to verify process parameters → commercial production scale-up.

[0006] Therefore, the current cell culture medium formula development method is lengthy and requires a long time of development and verification. How to reduce the steps and time from cell culture medium formula development to commercial production is still a technical problem that needs to be solved urgently. Summary of the invention

[0007] To overcome the above disadvantages, one of the objects of the present invention is to provide a method for preparing a powdered cell culture medium that can reduce the steps from cell culture medium formulation development to commercial production and shorten the development and verification time.

[0008] In order to achieve the above object, the technical solution adopted by the present invention is: a method for preparing a powdered cell culture medium, comprising the following steps:

[0009] S1. dividing the culture medium base into a first culture medium base and a second culture medium base according to the particle size of each component culture medium base;

[0010] S2, crushing each component of the first culture medium base material into small particles of the culture medium base material;

[0011] S3, weighing the small particle culture medium base material of each component and the second culture medium base material of each component;

[0012] S4. Mix the small-particle culture medium base materials of each component and the second culture medium base materials of each component weighed in S3 to prepare a powdered cell culture medium.

[0013] Furthermore, in S1, the particle size D90 of the first culture medium base material is greater than 200 μm, and the particle size D90 of the second culture medium base material is less than 200 μm.

[0014] Furthermore, in S1, the small particle culture medium base material includes at least one of the following technical features:

[0015] 1) The ratio of the weight of the small particle culture medium base material with a particle size in the range of 75 to 150 μm to the total weight of the small particle culture medium base material is greater than 50%;

[0016] 2) D90 ranges from 100 to 200 μm. Exemplarily, the D90 of the small particle culture medium base is 100 μm, 120 μm, 140 μm, 150 μm, 160 μm, 180 μm, 200 μm, or any two thereof.

[0017] Further, in S2, during the pulverizing process, the pulverizing speed is 9000-15000 rpm (the speed of the equipment with different processing amounts can be scaled proportionally). Exemplarily, the pulverizing speed is 9000 rpm, 10000 rpm, 11000 rpm, 12000 rpm, 13000 rpm, 14000 rpm, 15000 rpm or a range consisting of any two of them.

[0018] Furthermore, S2 also includes the following steps: adding auxiliary materials to the culture medium base material and then crushing, and the mass ratio of the culture medium base material to the auxiliary materials is 1: (99-999). Since the amount of some culture medium base materials in the formula is less than 10 mg, the balance cannot be accurately weighed, and concentrated preparation is required in the production process. The concentration of the concentrated preparation can be 10 times, 100 times, 1000 times, etc. of the original formula amount. The specific steps are: the culture medium base material with an amount of less than 10 mg is concentrated with auxiliary materials respectively, and the mass ratio of the culture medium base material to the auxiliary materials is 1: (9-999), that is, (9-999) g of auxiliary materials are added to 1 g of culture medium base material, and the concentration of the culture medium base material is 0.1%-1%.

[0019] During the feeding process, the amount of the culture medium base material after the concentration is calculated according to the amount of the culture medium base material and the concentration of the culture medium base material. The accuracy of weighing the culture medium base material is improved by pre-concentrating the culture medium base material with a small feeding amount.

[0020] The concentrated excipients are the excipients that already exist in the culture medium formula. During the addition of the formula excipients, the amount of excipients already contained in the concentrated culture medium base material is subtracted to ensure the accuracy of the amount of each component in the formula.

[0021] Furthermore, in S2, the auxiliary material is sodium chloride or glucose.

[0022] Furthermore, in S2, each component of the small particle culture medium base material is stored separately. During the development or production process, when multiple groups of parallel experiments are carried out, each group of experiments can be directly weighed and fed without re-crushing.

[0023] Furthermore, S3 also includes the following steps: placing each component culture medium base material in different funnels, weighing each component culture medium base material through the automatic weighing system and the receiving hopper, and realizing unmanned automatic weighing and batching operation according to the formula, reducing labor costs and improving batching efficiency. This set of automatic weighing and batching operation system can be divided into small-scale, pilot-scale, and commercial equipment systems according to different production capacity scales, realizing rapid process isotropic production, avoiding the lengthy steps of solid-liquid conversion in the existing process, and making it easier to go from small-scale to pilot-scale to commercial production.

[0024] Furthermore, in S4, each component culture medium base material is added into a mixer for mixing, the mixing frequency of the mixer is 30 to 40 Hz (10-25 rpm, the speed of equipment with different processing volumes can be scaled proportionally), and the mixing time is 25 to 45 min.

[0025] A powdered cell culture medium is prepared by the preparation method as described above.

[0026] The beneficial effects of the present invention are:

[0027] 1) Since the particles of the small-particle culture substrate and the second culture substrate both meet the requirements of the cell culture medium particles, they only need to be mixed and do not need to be crushed again. The cell culture medium formula has more than 60 materials, and each material has a different form. In the existing methods, it is very difficult to achieve the optimal particle size (100-200 mesh) to the greatest extent under the same crushing parameters. In this application, since the first culture medium base material has been processed in advance, the second culture medium base material does not need to be crushed again. The large-particle material, the first culture medium base material, can be processed to 100-200 mesh using applicable crushing parameters according to the properties of the material. Different materials use different crushing parameters to more easily achieve controllable particle size; the finished product in the later stage only needs to be mixed evenly to ensure that the product is basically at the optimal particle size of 100-200 mesh to the greatest extent, and will not cause excessive fine powder when large particles are ground.

[0028] 2) During the R&D process of orthogonal experiments, a large number of R&D experiments need to be carried out. Compared with the existing method in which each R&D experiment requires weighing the culture medium base material, mixing the materials and then crushing the materials at least once, the present application crushes each component separately in advance, and then weighs and mixes the components in turn according to the formula of each R&D experiment. During the R&D experiment, it is no longer necessary to crush each experiment, which can improve development efficiency and shorten the development cycle.

[0029] 3) Since the culture medium base material has been crushed in advance, it can be converted to develop new products at any time. Once the formula is determined, the ingredient experiment will be carried out immediately to prepare product samples to meet the development and customization needs of different customers. The development cycle is short. If the development sample meets the needs, it will be quickly scaled up and put into commercial production of the product, thereby improving the cycle of cell culture medium from development to commercial growth.

[0030] 4) By pre-crushing each large-particle material, and then weighing and mixing it, the material loss caused by the grinding step in the existing fixed culture medium preparation process is reduced; and the material loss caused by the overall material crushing link is avoided. The final finished product recovery rate can reach 99%, ensuring the accuracy of the formula.

[0031] 5) By pre-crushing each large-particle material and then mixing it, compared with the traditional process of mixing first and then crushing, the maximum crushing capacity of the crushing equipment can be reduced. The crushing requirements can be met by using a small-sized crushing equipment, which reduces the purchase cost of the crushing equipment and saves the floor space of the crushing equipment.

[0032] 6) During the mixing process, different types of culture medium base materials are placed in different funnels respectively, and the small particle culture medium base materials of each component are weighed through the automatic weighing system and the receiving hopper to realize unmanned automatic weighing and mixing operation according to the formula. This set of automatic weighing and mixing operations can also be divided into small-scale, pilot-scale, and commercial equipment systems according to different production capacity scales. In the small-scale development process, the small-scale equipment system is used. In the pilot-scale expansion process, the pilot-scale equipment system can be directly used. In the commercial production process, it can be proportionally enlarged and the commercial equipment system can be used. Since the same operating steps are implemented from small-scale research and development, pilot-scale expansion to commercial production, the same series of equipment does not need to be converted from liquid culture medium to solid production process, and rapid process proportional amplification production can be realized, which can save steps and time from development to scale-up production, and can ensure stability and uniformity during process amplification. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The drawings constituting a part of this application are used to provide a further understanding of the present invention. The illustrative embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0034] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0035] Figure 1 This is a particle size distribution diagram of the DMEM / F12 culture medium prepared in Example 2 of the present invention;

[0036] Figure 2 This is a particle size distribution diagram of the DMEM / F12 culture medium prepared in Example 3 of the present invention;

[0037] Figure 3 This is a particle size distribution diagram of the DMEM / F12 culture medium prepared in Example 4 of the present invention;

[0038] Figure 4 This is the particle size distribution diagram of the commercially available DMEM / F12 culture medium of the present invention;

[0039] Figure 5 This is another particle size distribution diagram of commercially available DMEM / F12 culture medium of the present invention;

[0040] Figure 6 The present invention is a flow chart of the preparation method of the powdered cell culture medium. DETAILED DESCRIPTION

[0041] In order to make the above-mentioned purpose, features and advantages of the present invention more obvious and easy to understand, the specific embodiments of the present invention are described in detail below. In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without violating the connotation of the present invention, so the present invention is not limited by the specific embodiments disclosed below.

[0042] Except as shown in the operating examples or otherwise indicated, all numbers used in the specification and claims to indicate the amount of ingredients, physicochemical properties, etc. are understood to be adjusted by the term "about" in all cases. Therefore, unless otherwise indicated, the numerical parameters listed in the above specification and the attached claims are approximate values, and those skilled in the art can use the teachings disclosed herein to seek to obtain the desired properties and appropriately change these approximate values. The use of numerical ranges expressed as endpoints includes all numbers within the range and any range within the range, for example, 1 to 5 includes 1, 1.1, 1.3, 1.5, 2, 2.75, 3, 3.80, 4 and 5, etc.

[0043] The applicant found that the current cell culture medium formula development is to weigh the powder raw materials one by one, group them into liquids, mix the liquids to adjust the pH and osmotic pressure, and then filter and sterilize them to form the finished product. Although the liquid preparation form has an accurate formula, when it is converted into a solid culture medium, the method steps are lengthy, and there is a lot of consumption in development time and experimental materials.

[0044] One embodiment of the present invention provides a method for preparing a powdered cell culture medium to solve the above-mentioned problem, which specifically comprises the following steps:

[0045] S1. Divide the culture medium base into a first culture medium base and a second culture medium base according to the particle size of each component culture medium base.

[0046] The first culture medium base material D90> 200 μm is a large particle culture medium base material, which needs to be crushed during the development of the cell culture medium formula.

[0047] The second culture medium base material has a D90 of ≤200 μm and a relatively small particle size, and does not need to be crushed in advance, and can be directly used to prepare a powdered cell culture medium.

[0048] S2, crushing each component of the first culture medium base material into small particles of the culture medium base material;

[0049] The first culture medium base material is crushed into small-particle culture medium base material that meets the requirements, and the crushed small-particle culture medium base material includes at least one of the following technical features:

[0050] The ratio of the weight of the small-particle culture medium base material with a particle size in the range of 75 to 150 μm to the total weight of the small-particle culture medium base material is greater than 50%; that is, after a first culture medium base material is crushed, the small-particle culture medium base material obtained, the weight of the small-particle culture medium base material with a particle size in the range of 75 to 150 μm accounts for more than 50% of the total weight of the small-particle culture medium base material.

[0051] The range of D90 is 100-200 μm. That is, after a first culture medium base material is crushed, the small-particle culture medium base material is obtained, and the particle size D90 of the small-particle culture medium base material is in the range of 100-200 μm.

[0052] During the pulverizing process, the pulverizing speed is 9000-15000rpm. It is specifically determined according to the particle size of the first culture substrate. A large pulverizing speed is required for the first culture substrate with a large particle size. For example, the D90 of sodium dihydrogen phosphate monohydrate is 774μm, and the pulverizing speed is 13000rpm. When pulverizing glucose with a smaller particle size (D90 is 247μm), the pulverizing speed is 9000rpm. Therefore, by pulverizing the first culture base materials with larger particles separately, the speed of the pulverizer can be adjusted according to the particle size of the first culture base materials. Different materials use different pulverizing parameters to more easily achieve controllable particle size, which will not cause large particles to be ground small, small particles to be crushed into fine powder with a smaller particle size, and excessive fine powder to occur, thereby ensuring that the particle size of the powdered cell culture medium prepared in the end is more concentrated.

[0053] When the amount of some culture medium base materials in the formula is less than 10 mg, since the balance cannot accurately weigh, concentrated preparation is required in the production process. The specific operation is: before crushing, the culture medium base materials with an amount less than 10 mg are concentrated with auxiliary materials, which are also auxiliary materials in the formula. Common auxiliary materials include sodium chloride. For example, 999g of sodium chloride is added to 1g of culture medium base materials and mixed and crushed. The mass ratio of culture medium base materials and sodium chloride is 1:999, and the concentration of culture medium base materials is 0.1%.

[0054] In the process of weighing materials in step S3, the concentrated culture medium base material is weighed according to the proportion, and in the process of weighing the auxiliary materials in the formula, the amount of auxiliary materials that have been added to the concentrated culture medium base material is subtracted to ensure the accuracy of the formula.

[0055] S3, weighing the small particle culture medium base material of each component and the second culture medium base material of each component;

[0056] During the mixing process, the small-particle culture medium base material of each component and the second culture medium base material of each component are mixed. Since the particles of the small-particle culture medium base material and the second culture medium base material meet the requirements of the cell culture medium particles, they only need to be mixed and do not need to be crushed again. The materials of the cell culture medium formula are as many as more than 60 materials, and each material has a different form. In the existing method, it is very difficult to achieve the optimal particle size (100-200 mesh) to the greatest extent under the same crushing parameters. In this application, since the first culture medium base material has been processed in advance, the second culture medium base material does not need to be crushed again. The large-particle material first culture medium base material can be processed to 100-200 mesh according to the properties of the material using applicable crushing parameters. Different materials use different crushing parameters to more easily achieve controllable particle size; the finished product in the later stage only needs to be mixed evenly, which can maximize the product. The optimal particle size of 100-200 mesh can be guaranteed, and there will be no excessive fine powder when the large particles are ground.

[0057] During the mixing process, different types of culture medium base materials are placed in different funnels respectively, and the small particle culture medium base materials of each component are weighed through the automatic weighing system and the receiving hopper to realize unmanned automatic weighing and mixing operation according to the formula. This set of automatic weighing and mixing operations can also be divided into small-scale, pilot-scale, and commercial equipment systems according to different production capacity scales. In the small-scale development process, the small-scale equipment system is used. In the pilot-scale expansion process, the pilot-scale equipment system can be directly used. In the commercial production process, it can be scaled up in proportion and the commercial equipment system can be used. Since the same operating steps are implemented from small-scale research and development, pilot-scale expansion to commercial production, the same series of equipment does not need to be converted from liquid culture medium to solid production process, and rapid process proportional scale production can be realized, which can save steps and time from development to scale-up production, and can ensure the stability and uniformity of the process scale-up process.

[0058] S4. Mix the small-particle culture medium base materials of each component and the second culture medium base materials of each component weighed in S3 to prepare a powdered cell culture medium.

[0059] In this scheme, the main process from development to commercial production is as follows: crushing large-particle culture medium base materials (100-200 mesh) → concentrating and preparing trace components → DOE experimental design (component grouping, concentration grading, orthogonal experimental design) → preparing powder culture medium samples of different formulations according to the experimental design → dissolving solid samples and culturing cells after full sterilization and filtration → culturing cells to confirm the performance of each formula → using statistical analysis to analyze the best influencing factors and levels → forming powder culture medium formula → preparing 3 batches of samples to verify the performance of powder culture medium formula → pilot scale-up of solids (only verifying mixing parameters) → commercial production scale-up. Compared with the existing development process, this process has simplified steps and simple operation, which can shorten the cycle from development to commercial production.

[0060] Example

[0061] The following examples describe the disclosure of the present invention in more detail, and these examples are intended for illustrative purposes only, as various modifications and variations within the scope of the disclosure of the present invention will be apparent to those skilled in the art. Unless otherwise stated, all parts, percentages, and ratios reported in the following examples are by weight, and all reagents used in the examples are commercially available or synthesized according to conventional methods and can be used directly without further processing, and the instruments used in the examples are commercially available.

[0062] Testing instrument: Topsizer laser particle size analyzer

[0063] System suitability test: Continuously test 5 working standards, and the RSD of the particle size value of D75 (75-150um) is ≤2%;

[0064] Sample testing: Each sample was tested twice and the average value was taken;

[0065] Example 1

[0066] S1. Divide the culture medium base into a first culture medium base and a second culture medium base according to the particle size of each component culture medium base.

[0067] Take the weighed component material and confirm and review the material type and weight shown on the weighing bag label according to the formula. The specific components are shown in Table 1. Sampling and testing the particle size of each component material. The testing instrument is Topsizer laser particle size analyzer. Each sample is tested twice and the average value is taken. According to the particle size, each component material is divided into the first culture medium base material (D90>200μm) and the second culture medium base material (D90≤200μm).

[0068] In this embodiment, the first culture medium base material that needs to be crushed includes sodium dihydrogen phosphate monohydrate, L-serine, L-leucine, sodium chloride and glucose. The second culture medium base material with a smaller particle size that does not need to be crushed includes L-tyrosine disodium salt dihydrate, glutamine and riboflavin.

[0069] S2, crushing each component of the first culture medium base material into small particles of the culture medium base material;

[0070] The first culture medium base material (D90>200 μm) that does not meet the particle size requirements of the cell culture medium base material is pulverized into small particles of culture medium base material, and the pulverizer speed is set to 9000-15000 rpm and the feeding frequency is 15±1 Hz. The particle sizes of the materials after pulverization are shown in Table 1.

[0071] Take the crushed samples for testing to ensure that the crushed first culture medium base material is crushed into small particle culture medium base materials respectively.

[0072] S3, weighing the small particle culture medium base material of each component and the second culture medium base material of each component;

[0073] After the crushing is completed, the first culture medium base material and the second culture medium base material that have been crushed in advance are respectively placed in different closed and dry hoppers. With the automatic weighing and batching system and the receiving hopper, unmanned automatic weighing and batching operation according to the formula can be realized.

[0074] S4. Mix the small-particle culture medium base materials of each component and the second culture medium base materials of each component weighed in S3 to prepare a powdered cell culture medium.

[0075] Add the small particle culture medium base material of each component and the first culture medium base material of each component into the 3L mixing hopper, set the mixing frequency of the small test mixer to 35±1HZ for mixing, and the mixing time is 35±10min. After the mixing is completed, observe whether there is obvious attachment on the inner surface of the mixing hopper. If there is, clean compressed air can be used to clean it and collect it together with the component materials.

[0076] After mixing, the particle size of the mixed product was tested. The specific particle size is shown in Table 1.

[0077] Table 1

[0078]

[0079]

[0080] Example 2

[0081] Prepare DMEM / F12 medium formula by following the steps below

[0082] S1. Divide the culture medium base into a first culture medium base and a second culture medium base according to the particle size of each component culture medium base.

[0083] S2. Crushing: Refer to Table 2 for the pilot development section. Collect each component material according to the formula. The amount of each material should be ≥1Kg (this is the minimum processing capacity of the pilot equipment). Each material is packaged independently and crushed according to the crusher parameters of each material. The speed is 9000-15000rpm and the feeding frequency is 15±1HZ.

[0084] Since the amount of some culture medium base materials in the formula is less than 10 mg, the balance cannot be accurately weighed, and concentrated preparation is required in the production process. The specific steps are: the culture medium base materials with an amount less than 10 mg are respectively concentrated with the auxiliary material sodium chloride, and the mass ratio of the culture medium base material to sodium chloride is 1:999, that is, 999g of sodium chloride is added to 1g of the culture medium base material, and the concentration of the culture medium base material is 0.1%. In this embodiment, the materials that need to be concentrated are: copper sulfate pentahydrate, D-biotin, pyridoxine hydrochloride, linoleic acid, ferric nitrate nonahydrate, putrescine dihydrochloride, and lipoic acid.

[0085] Packaging: After crushing, heat-seal each material with a double-layer weighing bag + tie + one layer of aluminum foil bag, calculate the yield, and label it. Store at 2-8℃ away from light.

[0086] S3. Weighing and feeding: Put different kinds of small particle culture medium base materials into different funnels respectively, weigh each component material according to the formula requirements through the automatic weighing system and the receiving hopper, and start feeding after confirming that it is correct.

[0087] S4. Final mixing: Add the materials into the 3L mixing hopper, set the mixing frequency of the pilot mixer to 35±1HZ for mixing, and mix for 35±10min. After mixing, observe whether there is obvious attachment on the inner surface of the mixing hopper. If there is, clean compressed air can be used to clean it and collect it together with the component materials.

[0088] After mixing, take samples for inspection; heat seal with double-layer weighing bag + tie + one layer of aluminum foil bag, calculate the yield, and label. Store at 2-8℃ away from light.

[0089] Table 2

[0090]

[0091]

[0092] Example 3

[0093] The components and preparation process steps of Example 3 and Example 2 are basically the same, the main difference is that the addition amount of each component is different. The specific addition amount is shown in the pilot scale part in Table 3.

[0094] Example 4

[0095] The components and preparation process steps of Example 4 and Example 2 are basically the same, the main difference is that the addition amount of each component is different. The specific addition amount is shown in Table 3 for commercial production.

[0096] Comparative Example 1

[0097] S1. Collect materials and confirm and review the material types and weights shown on the weighing bag labels according to the formula. The specific components are the same as those in Example 1, see Table 1. After confirmation, start feeding.

[0098] Weigh each component material and add it to a 3L mixing hopper. Set the mixing frequency of the small test mixer to 35±1HZ for mixing, and the mixing time is 35±10min. After mixing, observe whether there is obvious attachment on the inner surface of the mixing hopper. If there is, clean compressed air can be used to clean it and collect it together with the component materials. After mixing, test the particle size of the mixed product. The specific particle size parameters are shown in Table 3.

[0099] S2. After mixing, set the pulverizer speed to 12000±1000rpm and the feeding frequency to 15±1HZ to pulverize the mixed material.

[0100] S3. After the crushing is completed, the materials are added to the 3L mixing hopper, and the mixing frequency of the small test mixer is set to 35±1HZ for mixing, and the mixing time is 35±10min. After the mixing is completed, observe whether there is obvious attachment on the inner surface of the mixing hopper. If there is, clean compressed air can be used to clean it and collect it together with the component materials.

[0101] After mixing, the particle size of the mixed product was tested again. The particle size parameters are shown in Table 3.

[0102] Table 3

[0103]

[0104] From the particle size data of the mixture in Table 1 and Table 3, it can be seen that the range of D10 to D90 in Example 1 is 5.150-198.214 μm, and the range of D10 to D90 in Comparative Example 1 is 4.753-233.334 μm. Therefore, in Example 1, the particle size distribution of the mixture after the culture medium is crushed separately according to the particle size and then mixed is more concentrated.

[0105] Comparative Example 2

[0106] The DMEM / F12 culture medium formula was prepared by existing process steps, and the components and amounts of each component of the formula were the same as those in Example 2.

[0107] S1. Concentrated component preparation: Since the amount of some culture medium base materials in the formula is less than 10 mg, the balance cannot be accurately weighed, and concentrated preparation is required in the production process. The culture medium base materials with an amount less than 10 mg are concentrated with the auxiliary material sodium chloride. The mass ratio of the culture medium base material and sodium chloride is 1:999, that is, 999g of sodium chloride is added to 1g of the culture medium base material, and the concentration of the culture medium base material is 0.1%.

[0108] S2. Premixing: Add all the concentrated culture medium base materials into the mixing hopper, set the mixer mixing frequency to 35±1HZ for mixing, and mix for 35±10min; collect after mixing. The amount of each component is shown in Table 3.

[0109] S3. First crushing: After mixing, set the crusher speed to 12000±1000rpm and the feeding frequency to 15±1HZ for crushing. Crush all the concentrated culture medium base materials that have been mixed. After crushing, add the materials to the mixing hopper and set the mixer mixing frequency to 35±1HZ for mixing. The mixing time is 35±10min. Collect after mixing.

[0110] S4. Mixing: Add the concentrated component materials into the mixing hopper, add the prepared concentrated component at a ratio of 0.1%, weigh the basic component materials that do not need to be concentrated and have a usage of more than 10 mg, add them into the mixing hopper, set the pilot mixer mixing speed to 19±1rpm for mixing, and mix for 2100±600s; collect after mixing.

[0111] S5. Second crushing: After mixing, set the crusher speed to 10000±500rpm and the feeding frequency to 15±1HZ for crushing. After crushing, add the materials to the mixing hopper and set the mixer speed to 19±1rpm for mixing. The mixing time is 2100±600s. Collect after mixing. After mixing, add a layer of aluminum foil bag to the double-layer weighing bag and heat-seal it, and label it. Store at 2-8℃ away from light.

[0112] Experimental Example 1

[0113] The cells were cultured with the DMEM / F12 culture medium formula prepared in Example 2 and Comparative Example 2 and a commercially available DMEM / F12 culture medium formula, wherein the commercially available DMEM / F12 culture medium formula was purchased from ThermoFisher Scientific.

[0114] The specific steps are as follows:

[0115] 1) Usage

[0116] Add 90% of the final volume of ultrapure water or water for injection. The recommended water temperature is 25-35℃.

[0117] Slowly add DMEM / F12 culture medium (powder) at a ratio of 18.01 g / L.

[0118] Stir and mix for at least 40 minutes.

[0119] Using calibrated containers, make up to final preparation volume.

[0120] The mixture was stirred for an additional 20 minutes.

[0121] Adjust the pH to 7.0-7.4. Note that the pH of the culture medium will rise by 0.1-0.2 after filtration.

[0122] Immediately sterile filter with 0.22μm, 0.22 / 0.45μm combination or 0.45μm filter membrane. Once filtered, the product must be stored at 2-8℃ away from light and can be stored for more than 6 months.

[0123] 2) Culture conditions

[0124] Complete medium: DMEM / F12 basal medium + 10% fetal bovine serum.

[0125] Cell lines: MDCK, fibroblasts, endothelial cells, glial cells.

[0126] Culture container: cell culture flask

[0127] Temperature range: 37±0.5℃, temperature conversion according to cell culture conditions

[0128] Shaker settings: 5-10% CO2, ≥80% humidity

[0129] 3) Cell recovery

[0130] Thaw cells quickly in a 37°C water bath (<2 min);

[0131] Transfer the cell suspension to a 50 mL centrifuge tube and add 10 mL of preheated DMEM / F12 complete medium, centrifuge at 300g for 3 minutes, discard the supernatant, resuspend the cells in 10mL medium, take samples and count, and determine the cell density and viability. The specific test results are shown in Table 4;

[0132] Transfer the cells to a cell culture flask and wash with pre-warmed Dilute with DMEM / F12 complete medium to the desired cell density;

[0133] Place the culture bottle horizontally and gently shake the cell solution to make it evenly distributed in the culture bottle;

[0134] Culture in a humidified incubator with 5-10% carbon dioxide and 37°C;

[0135] If necessary, cells were cultured for 2 to 4 days after recovery and then passaged.

[0136] 4) Cell passaging

[0137] Will DMEM / F12 complete medium was preheated at 37°C for >30 min and protected from light;

[0138] For adherent cells, when the cell confluence reaches ≥80%, the cells can be digested and passaged according to the required seeding density;

[0139] For suspension cells, when the cell density is >10 6 cells / mL, the cells can be passaged according to the desired seeding density;

[0140] The recommended range of cell seeding density is 0.1 to 0.2 × 10 6 cells / mL;

[0141] Place at 37°C and 5-10% CO 2 Culture in a humidified incubator;

[0142] The next subculture can usually be performed after 2 to 4 days of culture.

[0143] 5) Cryopreservation

[0144] Prepare cells in logarithmic growth phase with viability >90% for freezing;

[0145] Prepare cell cryopreservation solution and store at 2-8°C until use;

[0146] The recommended final cell freezing density is 0.5~1×10 7 cells / mL;

[0147] The cells were harvested by centrifugation at 300 g for 5 min and suspended in a predetermined volume of freezing solution at 2 to 8 °C;

[0148] Immediately dispense the cell suspension into cryogenic vials according to the cryopreservation specifications;

[0149] Freeze to -80°C in an automatic or manually controlled rate cryopreservation device according to standard procedures (freezing rate of -1°C / min) and store at -80°C overnight;

[0150] Transfer the frozen cells to liquid nitrogen.

[0151] Table 4

[0152]

[0153] From the test data in Table 4, it can be seen that the viable cell concentration and cell viability of the DMEM / F12 cell culture medium prepared by this scheme are within the range. On the premise that the performance of the DMEM / F12 cell culture medium prepared by the traditional process and the commercially available DMEM / F12 cell culture medium is equivalent, the process development process of preparing the DMEM / F12 cell culture medium by this scheme is simpler, which can shorten the development cycle, improve the efficiency of formula development, and shorten the conversion cycle from development to pilot production.

[0154] Experimental Example 2

[0155] The DMEM / F12 cell culture medium prepared in Example 2-4 and the commercially available DMEM / F12 cell culture medium were tested for particle size. Figure 1-5 . Figure 4 The DMEM / F12 cell culture medium was purchased from Thermo Fisher Scientific. Figure 5 The DMEM / F12 cell culture medium was purchased from Shanghai Aupumai Biotechnology Co., Ltd.

[0156] When the particle size difference is large, the material with large particle size and the material with small particle size will be separated during transportation, resulting in poor material uniformity. Figure 1-5 It can be seen that the DMEM / F12 cell culture medium prepared by this process in the development stage, pilot scale-up stage and commercial production stage has a narrower particle size distribution range, the particle sizes of different materials are closer, and the stability and uniformity of the final product are better.

[0157] The present invention illustrates the detailed process equipment and process flow of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned detailed process equipment and process flow, that is, it does not mean that the present invention must rely on the above-mentioned detailed process equipment and process flow to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, change of structural morphology, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

[0158] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0159] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

[0160] In addition, various embodiments of the present invention may be arbitrarily combined, and as long as they do not violate the concept of the present invention, they should also be regarded as the contents disclosed by the present invention.

[0161] The above implementation modes are only for illustrating the technical concept and features of the present invention, and their purpose is to enable people familiar with this technology to understand the content of the present invention and implement it, and they cannot be used to limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a powdered cell culture medium, characterized in that: The steps include: S1. dividing the culture medium base into a first culture medium base and a second culture medium base according to the particle size of each component culture medium base; S2, crushing each component of the first culture medium base material into small particles of the culture medium base material; S3, weighing the small particle culture medium base material of each component and the second culture medium base material of each component; S4. Mix the small-particle culture medium base materials of each component and the second culture medium base materials of each component weighed in S3 to prepare a powdered cell culture medium.

2. The method for preparing the powdered cell culture medium according to claim 1, characterized in that: In S1, the particle size D90 of the first culture medium base material is greater than 200 μm, and the particle size D90 of the second culture medium base material is less than or equal to 200 μm.

3. The method for preparing the powdered cell culture medium according to claim 1, characterized in that: In S2, the small particle culture medium base material includes at least one of the following technical features: 1) The ratio of the weight of the small particle culture medium base material with a particle size in the range of 75 to 150 μm to the total weight of the small particle culture medium base material is greater than 50%; 2)D90 range is 100~200μm.

4. The method for preparing the powdered cell culture medium according to claim 1, characterized in that: In S2, during the pulverization process, the pulverization speed is 9000 to 15000 rpm.

5. The method for preparing the powdered cell culture medium according to claim 1, characterized in that: S2 also includes the following steps: adding auxiliary materials to the culture medium base material and then crushing them, and the mass ratio of the culture medium base material to the auxiliary materials is 1: (9-999).

6. The method for preparing the powdered cell culture medium according to claim 5, characterized in that: The auxiliary materials are sodium chloride or glucose.

7. The method for preparing the powdered cell culture medium according to claim 1, characterized in that: In S2, the small particle culture medium base materials of each component are stored separately.

8. The method for preparing the powdered cell culture medium according to claim 1, characterized in that: S3 also includes the following steps: placing each component culture medium base material in different closed hoppers respectively, weighing each component culture medium base material through an automatic weighing system and a receiving hopper, and realizing unmanned automatic weighing and mixing operation according to the formula.

9. The method for preparing the powdered cell culture medium according to claim 1, characterized in that: In S4, each component culture medium base material is added into a mixer for mixing. The mixing frequency of the mixer is 30 to 40 Hz and the mixing time is 25 to 45 minutes.

10. A powdered cell culture medium, characterized in that The composition is prepared by the preparation method according to any one of claims 1 to 9.

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