Improved method for producing cell culture medium lyophilized powder
By grouping trace elements according to their mass percentage, solubility, and pH level after dissolution and then freeze-drying them independently, the problem of insufficient dissolution and precipitation of trace elements during freeze-drying in existing technologies has been solved. This method achieves uniformity and accuracy in the detection of freeze-dried powder, ensuring batch stability and cell culture performance of the finished culture medium.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- 无锡多宁生物科技有限公司
- Filing Date
- 2026-03-24
- Publication Date
- 2026-06-09
AI Technical Summary
The one-pot production process in the existing technology is prone to insufficient dissolution, mutual reaction and precipitation or chemical state change of trace elements during high-concentration preparation and freeze-drying. This results in uneven composition of freeze-dried powder, poor resolubility and fluctuation in detection accuracy, affecting the batch-to-batch quality stability of the finished culture medium.
Trace elements were grouped according to their mass percentage, solubility, and pH after dissolution, and then mixed separately with a freeze-drying carrier in purified water to prepare a homogeneous freeze-drying solution. The solution was then freeze-dried under vacuum to obtain trace element freeze-dried powder, which was then mixed with other components to form the finished cell culture medium powder.
This method achieves uniform composition of lyophilized powder, good solubility, and high batch-to-batch consistency, ensuring the quality stability of the finished culture medium and the reliability of cell culture performance. It also reduces the deviation of quality control testing and improves the accuracy of test results and the stability of cell growth.
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Figure CN122168508A_ABST
Abstract
Description
Technical Field
[0001] This application relates to the field of cell culture medium production technology, and more specifically, it relates to an improved method for producing freeze-dried cell culture medium powder. Background Technology
[0002] Cell culture media are raw materials for biopharmaceuticals, cell therapy, and other fields. Their quality stability and component precision are increasingly important as the demand for large-scale production of biopharmaceuticals grows. Culture media formulations contain dozens to hundreds of components. Although trace elements account for a low proportion, they are crucial for cell growth, metabolism, and product expression. Fluctuations in their content affect the quality of biopharmaceuticals. Because trace elements account for a very low proportion, the industry uses inorganic salt carriers to first prepare lyophilized trace element powders, and then processes them with other components to form finished products. The current mainstream production process is the one-pot method: all trace elements and carriers are mixed at once, dissolved at high concentrations, and then lyophilized.
[0003] The related one-pot production process mixes all trace elements with the carrier in one go, dissolves them at a high concentration, and then freezes them. However, this method ignores the differences in the physicochemical properties of different trace elements. As a result, during the high-concentration preparation and freeze-drying process, some components are prone to insufficient dissolution, mutual reaction to produce precipitation, or changes in chemical state. This leads to uneven composition of the freeze-dried powder, poor resolubility, and fluctuations in detection accuracy, ultimately affecting the batch-to-batch quality stability of the finished culture medium. Summary of the Invention
[0004] To address the issue of batch-to-batch quality stability of the finished culture medium caused by the one-pot production process of mixing all trace elements with the carrier at once, dissolving them at high concentrations, and then freeze-drying them, this application provides an improved method for producing freeze-dried cell culture medium powder.
[0005] This application provides an improved method for producing freeze-dried cell culture medium powder, employing the following technical solution: An improved method for producing lyophilized cell culture medium powder includes the following steps: S1. The trace elements are grouped according to their mass percentage in the cell culture medium formulation, their solubility, and their pH after dissolution. S2. For each group of trace elements, mix them separately with the lyophilization carrier in purified water to prepare a homogeneous lyophilized solution. S3. The freeze-dried liquid obtained in S2 is subjected to vacuum freeze-drying to obtain trace element freeze-dried powder; S4. Mix the trace element freeze-dried powder obtained in S3 with other components of the cell culture medium to obtain the finished cell culture medium powder.
[0006] By adopting the above technical solution, and by grouping trace elements according to the principles of similar mass proportions, similar solubility, and consistent pH after dissolution, and then freeze-drying them independently, this method avoids signal interference from high-proportion components to low-proportion components during detection, ensuring the accuracy of quality control testing. At the same time, the grouping preparation allows each group of trace elements to completely dissolve under conditions suitable for its physicochemical properties, avoiding uneven mixing and precipitation. This results in freeze-dried trace element powders with uniform composition, good solubility, and high batch-to-batch consistency, thereby ensuring the quality stability of the finished culture medium and the reliability of cell culture performance.
[0007] Preferably, in step S1, the principle for grouping by mass percentage is as follows: trace elements with a mass percentage difference of less than 10 times are grouped into the same group, and trace elements with a mass percentage difference of more than 10 times are grouped into different groups.
[0008] By adopting the above technical solution, and by using a grouping principle that controls the mass ratio difference to within 10 times, this method ensures that the content of each trace element in the same group is at the same order of magnitude. This measure reduces the signal suppression or enhancement effect of high-concentration components on low-concentration components when using instruments such as inductively coupled plasma mass spectrometry for quality control detection in the subsequent process, thereby obtaining accurate detection results, controlling the detection deviation of individual elements within a certain range, and realizing the control of trace element content.
[0009] Preferably, based on the principle of grouping by mass proportion, trace elements with similar solubility are grouped into the same group; wherein, the criterion for judging similar solubility is: at 25°C and normal pressure, the solubility of each trace element in purified water does not differ by more than 10 times.
[0010] By adopting the above technical solution, and by grouping trace elements with solubility differences within 10 times into a group based on similar solubility, this method ensures that the dissolution conditions set for each group of lyophilized liquid can simultaneously meet the dissolution requirements of all trace elements within the group. This operation eliminates local insolubility or precipitation caused by differences in the solubility of different components, thus obtaining a clear, uniform, high-concentration lyophilized liquid, laying the foundation for subsequent lyophilization processes to prepare lyophilized powder with uniform component distribution.
[0011] Preferably, based on the principle of grouping by mass percentage and similar solubility, trace elements with consistent pH after dissolution are grouped into the same group; wherein, the criterion for consistent pH is: at 25°C, the pH value of each trace element after dissolving in purified water is acidic, alkaline or neutral.
[0012] By adopting the above technical solution, and by grouping trace elements with consistent pH levels after dissolution into a single group, this method ensures that no acid-base neutralization reaction will occur between the components in the group due to pH differences when preparing high-concentration freeze-drying solutions. This process avoids the precipitation or decomposition of trace elements caused by local pH fluctuations, thereby obtaining a freeze-drying solution with stable chemical properties and uniform ionic state, ensuring the stability and biological activity of trace elements during the freeze-drying process.
[0013] Preferably, in step S2, when preparing the lyophilized solution, first take purified water, place it in a clean beaker, control the water temperature at 20~25℃, and the stirring speed at 750~850r / min; then weigh each group of trace elements, slowly add them to the purified water, and continue stirring for 15~20min; then weigh the lyophilized carrier, add it and continue stirring for 10~15min to obtain a uniform lyophilized solution.
[0014] By adopting the above technical solution, the operation of controlling the water temperature between 20 and 25°C and maintaining a stirring speed of 800 revolutions per minute provides a suitable environment for the dissolution of trace elements. The stepwise feeding sequence, that is, adding trace elements first to ensure their dispersion and dissolution, and then adding a large amount of freeze-drying carrier, avoids the influence of the high ionic strength environment on the dissolution of certain trace elements. Therefore, a freeze-dried liquid with uniform dissolution and mixing of each component is obtained, which provides raw materials for the subsequent freeze-drying preparation of freeze-dried powder with loose structure and consistent water content.
[0015] Preferably, in step S2, the freeze-drying carrier is sodium chloride.
[0016] By adopting the above technical solution, sodium chloride is used as the freeze-drying carrier. Sodium chloride has the characteristics of rapid dissolution in purified water, chemical stability, no decomposition during freeze-drying, non-toxicity to cell growth, and low cost. During freeze-drying, this carrier can form a solid framework, effectively carrying and fixing distributed trace elements. At the same time, it has good compatibility with other components in the culture medium. Therefore, a freeze-dried powder of trace elements that can be stably stored, reconstituted rapidly, and does not adversely affect cells is obtained.
[0017] Preferably, in step S3, the vacuum freeze-drying includes the following steps: S31. Pre-freezing: The trace freeze-dried liquid obtained in S2 is placed into freeze-drying trays and placed in a freezer at -90~-70℃ for 11~13h to obtain frozen solids. S32, Sublimation Drying: The temperature of the frozen solid obtained in S31 is lowered to -55~-50℃, while controlling the vacuum degree to 10~15Pa, and freeze-drying is carried out for 72 hours to ensure that the moisture content of the freeze-dried powder is ≤3.0%; S33. After freeze-drying is complete, quickly remove each group of trace element freeze-dried powders and seal them in packaging.
[0018] By adopting the above technical solution, the deep pre-freezing operation at -80℃ for 12 hours can freeze the freeze-dried liquid into a solid and form an ice crystal structure, thereby preventing foaming or component migration during subsequent sublimation drying. The sublimation drying process using a condenser temperature of -53℃ and a vacuum of 12 Pascals for 72 hours can sublimate the ice crystals into water vapor at low temperature and remove them, thus protecting the activity of heat-sensitive trace elements. Therefore, a trace element freeze-dried powder with a water content of less than 3%, a porous structure, good resolubility, and preserved biological activity is obtained.
[0019] Preferably, in step S4, other components include amino acids, vitamins, carbohydrates, or inorganic salts.
[0020] By adopting the above technical solution, other culture medium components such as amino acids, vitamins, carbohydrates, and inorganic salts are added in the final mixing step. These substances, together with the grouped lyophilized trace element powder, constitute a complete cell culture medium formula. This operation physically mixes the trace elements, which have undergone quality control and homogenization, with other nutrients, thus obtaining a finished cell culture medium powder with comprehensive components and balanced nutrition, which can provide a material basis for cell growth and metabolism.
[0021] Preferably, in step S4, the mixing is carried out using a ball mill with a rotation speed of 260–290 r / min and a mixing time of 2–3 h.
[0022] By adopting the above technical solution, the mechanical mixing method, which uses a ball mill and continuously mixes at a speed of 280 rpm for 2.5 hours, provides shear force and tumbling action. This process can disperse and interpenetrate trace element freeze-dried powders with different specific gravities, particle sizes and physical forms with other culture medium component powders, thus obtaining finished culture medium powders with uniformity at both the macroscopic and microscopic scales, ensuring that the content of each component in each sample is consistent with the formulation design.
[0023] Preferably, in step S4, the finished cell culture medium powder is passed through a 50-mesh sieve and then sealed and packaged.
[0024] By adopting the above technical solution, the step of passing the mixture through a 50-mesh sieve after mixing is added. This operation removes a small number of large particles or lumps formed during the ball milling process. Combined with subsequent sealed packaging, this process ensures that the physical state of the finished powder is uniform, which is conducive to its rapid dissolution. On the other hand, it effectively isolates air and moisture, preventing the finished product from absorbing moisture and deteriorating. Therefore, a final product with consistent physical properties, good stability, and easy storage and use is obtained.
[0025] In summary, this application has the following beneficial effects: 1. This application employs a process of grouping trace elements according to their mass percentage, solubility, and pH level after dissolution, and then freeze-drying them independently. The mass percentage grouping ensures that the concentration of elements within the same group is at a similar level, while the solubility and pH consistency grouping sets process conditions for each group. This allows the preparation of high-concentration freeze-drying solutions to achieve the dissolution and chemical stability of all components, thereby obtaining freeze-dried powder with uniform components and good solubility. This avoids precipitation and detection deviations caused by property conflicts and improves the batch stability of the finished culture medium.
[0026] 2. In this application, the preferred approach is to adopt a grouping principle that controls the difference in mass percentage within a specific multiple. Since this principle groups trace elements with similar concentrations into the same unit, when used with precision instruments for detection, this measure reduces the interference effect of high-concentration components on the signals of low-concentration components, so that the detection can more accurately reflect the content of each element, thus obtaining accurate detection results and providing a basis for the control of the formula content.
[0027] 3. The method of this application groups trace elements with similar solubility and consistent pH after dissolution into a group, and adopts a process of first dissolving the trace elements and then adding the freeze-drying carrier to obtain a chemically stable and homogeneous freeze-dried solution. The similarity of solubility ensures that all components in the group can dissolve synchronously under the set suitable conditions; the consistency of pH avoids precipitation caused by local acid-base reactions during the preparation process; and the stepwise addition of materials ensures that the trace elements dissolve in a suitable environment before the freeze-drying carrier is introduced to avoid the salting-out effect. This process, together with the coordination of the grouped chemical components, provides a qualified raw material solution for subsequent freeze-drying.
[0028] 4. Since this application uses inorganic salts as a carrier and performs pre-freezing and sublimation drying processes, this process works synergistically with the lyophilized liquid prepared in groups; the solid framework formed by the carrier during lyophilization can fix trace elements; deep pre-freezing allows the solution to freeze rapidly, forming uniform ice crystals, ensuring the uniformity of the solution and preventing component migration during subsequent drying; long-term sublimation drying under low temperature vacuum removes the ice crystals, protecting the activity of heat-sensitive elements, thereby obtaining lyophilized powder with low water content, good resolubility and retained activity. Attached Figure Description
[0029] Figure 1 This is a flowchart of an improved method for producing freeze-dried cell culture medium powder proposed in this application. Detailed Implementation
[0030] The present application will be further described in detail below with reference to the accompanying drawings and embodiments.
[0031] Technical concept: The related one-pot production process mixes all trace elements with the carrier in one go, dissolves them at a high concentration, and then freezes them. However, this method ignores the differences in the physicochemical properties of different trace elements. As a result, during the high-concentration preparation and freeze-drying process, some components are prone to insufficient dissolution, mutual reaction to produce precipitation, or changes in chemical state. This leads to uneven composition of the freeze-dried powder, poor resolubility, and fluctuations in detection accuracy, ultimately affecting the batch-to-batch quality stability of the finished culture medium.
[0032] This application discloses an improved method for producing lyophilized cell culture medium powder. The method includes the following steps: S1, grouping the trace elements according to their mass percentage in the cell culture medium formulation, solubility, and pH level after dissolution; S2, mixing each group of trace elements with a lyophilization carrier in purified water to prepare a homogeneous lyophilized solution; S3, subjecting the lyophilized solution obtained in S2 to vacuum freeze-drying to obtain lyophilized trace element powder; S4, mixing the lyophilized trace element powder obtained in S3 with other components of the cell culture medium to obtain the finished cell culture medium powder.
[0033] This application employs a process of grouping trace elements according to their mass percentage, solubility, and pH level after dissolution, and then freeze-drying them independently. The mass percentage grouping ensures that the concentration of elements within the same group is at a similar level, while the solubility and pH consistency grouping sets process conditions for each group. This allows the preparation of high-concentration freeze-drying solutions to achieve the dissolution and chemical stability of all components, thereby obtaining freeze-dried powders with uniform components and good solubility. This avoids precipitation and detection deviations caused by conflicting properties and improves the batch stability of the finished culture medium.
[0034] Example 1: This example provides an improved method for producing lyophilized cell culture medium powder, comprising the following steps: S1. Based on the mass percentage, solubility, and pH level of the trace elements in the cell culture medium formulation, the trace elements are grouped.
[0035] When grouping trace elements by mass percentage, trace elements whose mass percentage differs by less than 10 times within the same group belong to different groups.
[0036] S2. For each group of trace elements, mix them separately with the lyophilization carrier in purified water to prepare a homogeneous lyophilized solution.
[0037] In the preparation process, first, purified water was placed in a clean beaker, and the water temperature was controlled at 20℃ while the stirring speed was 750 r / min. Then, each group of trace elements was weighed and slowly added to the purified water, with stirring continued for 15 min. Next, the lyophilization carrier was weighed, added, and stirring continued for 10 min to obtain a homogeneous lyophilized solution. The lyophilization carrier was sodium chloride.
[0038] S3. The freeze-dried liquid obtained in S2 is subjected to vacuum freeze-drying to obtain trace element freeze-dried powder.
[0039] The vacuum freeze-drying process includes the following steps: S31, Pre-freezing: The freeze-dried liquid is placed into freeze-drying trays and placed in a -90℃ freezer for 11 hours to obtain frozen solids; S32, Sublimation drying: The temperature of the frozen solids is lowered to -55℃, while the vacuum degree is controlled at 10Pa, and freeze-drying is carried out for 72 hours to ensure that the water content of the freeze-dried powder is ≤3.0%; S33, After freeze-drying is completed, each group of trace element freeze-dried powders is quickly taken out and sealed in packaging.
[0040] S4. Mix the trace element freeze-dried powder obtained in S3 with other components of the cell culture medium to obtain the finished cell culture medium powder.
[0041] Other components include amino acids, vitamins, carbohydrates, and inorganic salts. Mixing was carried out using a ball mill at 260 rpm for 2 hours. The finished cell culture medium powder was passed through a 50-mesh sieve and then sealed in packaging.
[0042] Example 2: This example provides an improved method for producing lyophilized cell culture medium powder, comprising the following steps: S1. Based on the mass percentage, solubility, and pH level of the trace elements in the cell culture medium formulation, the trace elements are grouped.
[0043] Based on the principle of grouping by mass percentage, trace elements with similar solubility are grouped together. The criterion for judging similar solubility is that, at 25℃ and normal pressure, the solubility of each trace element in purified water does not differ by more than 10 times.
[0044] S2. For each group of trace elements, mix them separately with the lyophilization carrier in purified water to prepare a homogeneous lyophilized solution.
[0045] In the preparation process, firstly, purified water was placed in a clean beaker, and the water temperature was controlled at 22.5℃ with a stirring speed of 800 r / min. Then, each group of trace elements was weighed and slowly added to the purified water, with stirring continued for 17.5 min. Next, the lyophilization carrier was weighed, added, and stirring continued for 12.5 min to obtain a homogeneous lyophilized solution. The lyophilization carrier was sodium chloride.
[0046] S3. The freeze-dried liquid obtained in S2 is subjected to vacuum freeze-drying to obtain trace element freeze-dried powder.
[0047] The vacuum freeze-drying process includes the following steps: S31, Pre-freezing: The freeze-drying liquid is placed into freeze-drying trays and placed in a -80℃ freezer for 12 hours to obtain frozen solids; S32, Sublimation drying: The temperature of the frozen solids is lowered to -52.5℃, while the vacuum degree is controlled at 12.5Pa, and freeze-drying is carried out for 72 hours to ensure that the water content of the freeze-dried powder is ≤3.0%; S33, After freeze-drying is completed, each group of trace element freeze-dried powders is quickly taken out and sealed in packaging.
[0048] S4. Mix the trace element freeze-dried powder obtained in S3 with other components of the cell culture medium to obtain the finished cell culture medium powder.
[0049] Other components include amino acids, vitamins, carbohydrates, and inorganic salts. Mixing was carried out using a ball mill at 275 rpm for 2.5 hours. The finished cell culture medium powder was passed through a 50-mesh sieve and then sealed in packaging.
[0050] Example 3: This example provides an improved method for producing lyophilized cell culture medium powder, comprising the following steps: S1. Based on the mass percentage, solubility, and pH level of the trace elements in the cell culture medium formulation, the trace elements are grouped.
[0051] Among them, based on the principles of mass proportion grouping and similar solubility, trace elements with consistent pH values after dissolution are grouped together. The criterion for consistent pH is: at 25℃, the pH value of each trace element after dissolving in purified water is acidic, alkaline, or neutral.
[0052] S2. For each group of trace elements, mix them separately with the lyophilization carrier in purified water to prepare a homogeneous lyophilized solution.
[0053] In the preparation process, firstly, purified water was placed in a clean beaker, and the water temperature was controlled at 25℃ while the stirring speed was 850 r / min. Then, each group of trace elements was weighed and slowly added to the purified water, with stirring continued for 20 min. Next, the lyophilization carrier was weighed, added, and stirring continued for 15 min to obtain a homogeneous lyophilized solution. The lyophilization carrier was sodium chloride.
[0054] S3. The freeze-dried liquid obtained in S2 is subjected to vacuum freeze-drying to obtain trace element freeze-dried powder.
[0055] The vacuum freeze-drying process includes the following steps: S31, Pre-freezing: The freeze-dried liquid is placed into freeze-drying trays and placed in a -70℃ freezer for 13 hours to obtain frozen solids; S32, Sublimation drying: The temperature of the frozen solids is lowered to -50℃, while the vacuum degree is controlled at 15Pa, and freeze-drying is carried out for 72 hours to ensure that the water content of the freeze-dried powder is ≤3.0%; S33, After freeze-drying is completed, each group of trace element freeze-dried powders is quickly taken out and sealed in packaging.
[0056] S4. Mix the trace element freeze-dried powder obtained in S3 with other components of the cell culture medium to obtain the finished cell culture medium powder.
[0057] Other components include amino acids, vitamins, carbohydrates, and inorganic salts. Mixing was carried out using a ball mill at 290 rpm for 3 hours. The finished lyophilized cell culture medium powder was passed through a 50-mesh sieve and then sealed in packaging.
[0058] Example 4: In this example, the cell culture medium used is a chemically defined medium suitable for CHOK1 cell culture, in which trace elements include copper sulfate pentahydrate, zinc sulfate heptahydrate, manganese chloride tetrahydrate, aluminum chloride hexahydrate, sodium selenite, sodium molybdate dihydrate, nickel(II) sulfate hexahydrate, and tin(II) chloride dihydrate. Sodium chloride is used as the lyophilization carrier. Quality control is performed using ICP-MS (inductively coupled plasma mass spectrometry). Cell culture is performed in batch culture mode. The detection index is the viable cell density (VCDmax). The RSD value (relative standard deviation) is used to evaluate batch stability. The smaller the RSD value, the better the batch stability.
[0059] Step 1: Trace element classification Taking the eight trace elements in my DM20 cell culture medium formula as an example, the concentrations of each component are as follows: copper sulfate pentahydrate 0.00027 g / L, zinc sulfate heptahydrate 0.0019 g / L, manganese chloride tetrahydrate 0.0000089 g / L, aluminum chloride hexahydrate 0.0000013 g / L, sodium selenite 0.000028 g / L, sodium molybdate dihydrate 0.0000042 g / L, nickel(II) sulfate hexahydrate 0.000000664 g / L, and tin(II) chloride dihydrate 0.00000061 g / L.
[0060] Based on the principles of "mass percentage difference ≤ 10 times, similar solubility ≤ 10 times, and consistent pH value after dissolution," the above eight trace elements were grouped, and the specific grouping results are as follows: Group 1 of lyophilized solutions: copper sulfate pentahydrate (0.00027 g / L) and zinc sulfate heptahydrate (0.0019 g / L); the mass ratio of the two solutions differs by approximately 7 times. The difference in solubility between the two is less than 10 times; and both are sulfates, with a difference of 57.5 ÷ 25.0 = 2.3 times in purified water at 25℃, which is less than 10 times, indicating similar solubility; both have acidic pH values in their saturated aqueous solutions at 25℃, so they are grouped together. Two lyophilized solutions were prepared: manganese chloride tetrahydrate (0.0000089 g / L) and aluminum chloride hexahydrate (0.0000013 g / L); the difference in their mass percentages was approximately 6.8 times. The difference in solubility between the two compounds is less than 10 times; both are chlorides, and their solubility in purified water at 25℃ differs by approximately 4.667 times (210÷45≈4.667), which is less than 10 times, indicating similar solubility; both have acidic pH values in their saturated aqueous solutions at 25℃, so they are grouped together. Three lyophilized solutions were prepared: sodium selenite (0.000028 g / L) and sodium molybdate dihydrate (0.0000042 g / L), with a mass ratio difference of approximately 6.7 times. The difference in solubility in purified water at 25℃ is less than 10 times; the difference in solubility is 66.1÷43.2≈1.53 times, which is less than 10 times, indicating similar solubility; the pH value of the saturated aqueous solution at 25℃ is alkaline, so they are grouped together. Four lyophilized solutions were prepared: nickel(II) sulfate hexahydrate (0.000000664 g / L) and tin(II) chloride dihydrate (0.00000061 g / L); the mass ratio of the two solutions differed by approximately 1.1 times. The difference in solubility between the two in purified water at 25℃ is less than 10 times; and the difference in solubility between the two in purified water at 25℃ is 118.7÷60.83≈1.95 times, which is less than 10 times, indicating similar solubility; the pH value of their saturated aqueous solutions at 25℃ is acidic, so they are grouped together. Step 2: Freeze-drying the carrier Sodium chloride was selected as the freeze-drying carrier.
[0061] Step 3: Prepare high-concentration lyophilization solutions in groups Each group was prepared with 3000× high-concentration lyophilized solution. The specific components and masses are shown in Table 1 below: Table 1: Summary Table of Components and Mass of Lyophilized Liquid ; Preparation method: Place 100 mL of purified water in a beaker, control the water temperature at 25 ± 2 ℃, and stir at 800 r / min; accurately weigh each group of trace elements, slowly add them to the purified water, and stir for 20 min until completely dissolved; then add the weighed sodium chloride, and continue stirring for 10 min until completely dissolved to obtain a uniform, precipitate-free, high-concentration lyophilized solution of trace elements.
[0062] Step 4: Group freeze-drying Four groups of 3000× high-concentration trace element lyophilized solutions were placed into lyophilization trays and lyophilized according to the following process parameters: (1) Pre-freezing: Each group of lyophilized liquids is placed into a lyophilization tray and placed in a -80℃ refrigerator for 12 hours to allow the lyophilized liquids to be completely frozen into solids, thus avoiding problems such as bubbles and component separation during the lyophilization process; (2) Sublimation drying: The temperature of the condenser of the vacuum freeze dryer is reduced to -53℃, the freeze drying chamber is evacuated and the vacuum degree is controlled at 12Pa. The freeze drying is carried out for 72 hours so that the water in the frozen freeze-dried liquid is removed in the form of sublimation, ensuring that the water content of the freeze-dried powder is ≤3.0%, improving the stability of the freeze-dried powder and extending the storage period. (3) After freeze-drying is completed, quickly take out each group of trace element freeze-dried powders, seal them in packaging and keep them for later use.
[0063] This experiment produced a total of 3 batches (batch 1, batch 2, and batch 3), and each batch prepared 4 groups of trace element freeze-dried powders according to the above steps.
[0064] Step 5: Preparation of finished culture medium dry powder According to the CHOK1 cell culture medium formula, accurately weigh 4 groups of trace element lyophilized powders from 3 batches, as well as 3 batches of amino acids, vitamins, glucose and other components, and put them into a ball mill jar. Grind at 280 r / min for 2.5 h; pulverize and pass through a 50 mesh sieve to obtain the finished cell culture medium dry powder; seal and package.
[0065] Example 1: Detection Results and Analysis 1. Status detection of lyophilized liquid The state of the three batches of four groups of 3000× high-concentration trace element freeze-dried solutions was observed. All of them were uniform and transparent solutions without any precipitation or turbidity. This indicates that the trace elements in each group were completely dissolved and met the freeze-drying requirements. This verifies the rationality of the "similar solubility" principle in the grouping principle of this invention and avoids the occurrence of non-dissolution in traditional methods.
[0066] 2. ICP-MS Detection Results of Trace Element Lyophilized Powder The actual content of trace elements in each group of lyophilized powders from three batches was determined by ICP-MS, and the deviation from the theoretical value was calculated. The results are shown in Table 2 below (deviation calculation formula: deviation% = |actual value - theoretical value| / theoretical value × 100%). Table 2: Summary Table of Trace Element Deviation Results ; Analysis: As shown in Table 2, the deviations of the ICP-MS test results of all three batches of trace element freeze-dried powder in Example 1 from the theoretical values were all within 15%, with the maximum deviation being 13.9% for sodium molybdate dihydrate, far lower than the deviation of over 30% for traditional methods. Among them, the deviations for manganese chloride tetrahydrate, sodium selenite, and nickel(II) hexahydrate were relatively small, with maximum deviations of 7.2%, 10.8%, and 11.8%, respectively; the deviations for copper sulfate pentahydrate, zinc sulfate heptahydrate, aluminum chloride hexahydrate, and tin(II) chloride dihydrate were all controlled within 13.4%. This indicates that the present invention, through group freeze-drying, effectively avoids the interference of high-proportion trace elements on the detection of low-proportion trace elements, significantly improving the accuracy of quality control detection and enabling precise control of trace element content, achieving the goal of quality control friendliness. Furthermore, the small fluctuations in deviation among the three batches indicate that the production method of the present invention has good repeatability and can stably produce trace element freeze-dried powder that meets the formulation requirements.
[0067] 3. Cell culture performance testing of finished culture medium Three batches of the finished culture medium powder were prepared according to the instructions and used for batch culture of CHOK1 cells. Culture conditions: 37℃. The shaking speed was 130 r / min, and the cells were cultured for 7 days. The maximum viable cell density (VCDmax) was measured, and the RSD value was calculated. The results are shown in Table 3 below. Table 3: Summary of Maximum Viable Cell Density for Each Batch ; Analysis: As shown in Table 3, when the three batches of finished culture medium in Example 1 were used for CHOK1 cell batch culture, the VCDmax were 243E5 cells / mL, 231E5 cells / mL, and 226E5 cells / mL, respectively, with an average of 233.3E5 cells / mL and an RSD of 3.6%, which is much lower than the RSD of the traditional method (around 10%). This indicates that the finished culture medium prepared by this invention has good batch uniformity, can provide a stable nutritional environment for CHOK1 cells, and exhibits stable cell growth performance without significant fluctuations, effectively solving the problem of poor batch stability of finished culture medium in the traditional method. Simultaneously, the cell growth density is at a high level, indicating that the method of this invention does not affect the biological activity of trace elements and can meet the nutritional requirements for cell growth.
[0068] Comparative Example 1: Preparation of cell culture medium lyophilized powder using the traditional "one-pot method" Using the traditional "one-pot method," all trace elements were mixed to form a freeze-dried powder. The other steps (freeze-drying carrier pretreatment, freeze-drying process parameters, and finished product preparation) were completely consistent with Example 1, as detailed below: Step 1: Preparation of lyophilization solution All eight trace elements from Example 1 were mixed together with sodium chloride carrier to prepare a 3000× high-concentration lyophilization solution. The composition and concentration of the lyophilization solution are shown in Table 4 below. Table 4: Summary Table of Components and Concentrations of Lyophilized Liquid ; The preparation method is the same as in Example 1: Take 100 mL of purified water, control the water temperature at 25 ± 2 °C, and stir at 800 r / min. Add the trace elements one by one to the purified water and stir for 20 min. A small amount of precipitate is observed in the solution, which cannot be completely dissolved. Finally, add sodium chloride and stir for 10 min, but the precipitate still does not disappear. A high-concentration lyophilized suspension is obtained.
[0069] Step 2: Freeze-drying The turbid freeze-drying liquid was loaded into a freeze-drying tray and freeze-dried according to the freeze-drying process parameters of Example 1. Three batches (batch 1, batch 2, and batch 3) were produced. After freeze-drying, the trace element freeze-dried powder was obtained.
[0070] Step 3: Preparation of finished culture medium dry powder Consistent with step 5 of Example 1, three batches of trace element lyophilized powder were mixed with three batches of other components to prepare the finished cell culture medium dry powder.
[0071] Comparative Example 1: Detection Results and Analysis 1. Status detection of lyophilized liquid The three batches of freeze-dried solutions were observed to be turbid solutions with a small amount of precipitate that could not be completely dissolved, which confirmed that the traditional "one-pot method" has the problem of poor solubility.
[0072] 2. ICP-MS Detection Results of Trace Element Lyophilized Powder The actual content of trace elements in the lyophilized powder of three batches was determined by ICP-MS, and the deviation from the theoretical value was calculated. The results are shown in Table 5 below: Table 5: Summary of Deviations Between Actual and Theoretical Values of Trace Elements ; Analysis: As shown in Table 5, the ICP-MS detection results of the three batches of trace element lyophilized powder in Comparative Example 1 generally deviated significantly from the theoretical values, with the largest deviation reaching 26.7% (nickel(II) hexahydrate group 2). The deviations for several trace elements exceeded 20%, such as manganese chloride tetrahydrate (24.1%), sodium molybdate dihydrate (24.2%), sodium selenite (21.5%), and tin(II) chloride dihydrate (25.7%), all near the critical deviation value of 30%. This is because some trace elements were not completely dissolved, resulting in uneven component distribution after lyophilization. Furthermore, the high proportion of trace elements interfered with the detection of low proportion trace elements, further exacerbating the detection deviation.
[0073] 3. Cell culture performance testing of finished culture medium Three batches of the finished culture medium powder were prepared according to the instructions and used for batch culture of CHOK1 cells. The culture conditions were the same as in Example 1. VCDmax was measured and RSD value was calculated. The results are shown in Table 6 below: Table 6: Summary Table of VCDmax and RSD Results for Each Batch ; Analysis: Table 6 shows that when the three batches of finished culture medium from Comparative Example 1 were used for CHOK1 cell batch culture, the VCDmax were 259E5 cells / mL, 217E5 cells / mL, and 209E5 cells / mL, respectively, with an average of 228.3E5 cells / mL. The RSD value was 10.8%, significantly higher than the 3.6% in Example 1, indicating that the batch stability of the finished culture medium prepared by the traditional "one-pot method" is poor, and the cell growth performance fluctuates greatly. This is because the trace element lyophilized powder prepared by the traditional method has uneven composition and large content deviations, resulting in an unstable nutrient environment in the finished culture medium. The trace element content in some batches is too high or too low, affecting cell growth and leading to large fluctuations in cell growth density, which will have a certain impact on industrial production.
[0074] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.
Claims
1. An improved method for producing freeze-dried cell culture medium powder, characterized in that, Includes the following steps: S1. The trace elements are grouped according to their mass percentage in the cell culture medium formulation, their solubility, and their pH after dissolution. S2. For each group of trace elements, mix them separately with the lyophilization carrier in purified water to prepare a homogeneous lyophilized solution. S3. The freeze-dried liquid obtained in S2 is subjected to vacuum freeze-drying to obtain trace element freeze-dried powder; S4. Mix the trace element freeze-dried powder obtained in S3 with other components of the cell culture medium to obtain the finished cell culture medium powder.
2. The improved method for producing freeze-dried cell culture medium powder according to claim 1, characterized in that, In step S1, the principle of grouping by mass percentage is as follows: trace elements with a mass percentage difference of less than 10 times are grouped into the same group, and trace elements with a mass percentage difference of more than 10 times are grouped into different groups.
3. The improved method for producing freeze-dried cell culture medium powder according to claim 2, characterized in that, Based on the principle of grouping by mass proportion, trace elements with similar solubility are grouped together; the criterion for judging similar solubility is that the solubility of each trace element in purified water at 25℃ and normal pressure does not differ by more than 10 times.
4. The improved method for producing freeze-dried cell culture medium powder according to claim 3, characterized in that, Based on the principles of mass proportion grouping and similar solubility, trace elements with consistent pH after dissolution are grouped together. The criterion for consistent pH is that, at 25°C, the pH value of each trace element after dissolving in purified water is acidic, alkaline, or neutral.
5. The improved method for producing freeze-dried cell culture medium powder according to claim 1, characterized in that, In step S2, when preparing the lyophilized solution, first take purified water and place it in a clean beaker, control the water temperature at 20~25℃, and the stirring speed at 750~850r / min; then weigh each group of trace elements, slowly add them to the purified water, and continue stirring for 15~20min. Then weigh the freeze-drying carrier, add it and continue stirring for 10-15 minutes to obtain a uniform freeze-dried liquid.
6. The improved method for producing freeze-dried cell culture medium powder according to claim 1, characterized in that, In step S2, the freeze-drying carrier is sodium chloride.
7. The improved method for producing freeze-dried cell culture medium powder according to claim 1, characterized in that, In step S3, the vacuum freeze-drying includes the following steps: S31. Pre-freezing: The trace freeze-dried liquid obtained in S2 is placed into freeze-drying trays and placed in a freezer at -90~-70℃ for 11~13h to obtain frozen solids. S32, Sublimation Drying: The temperature of the frozen solid obtained in S31 is lowered to -55~-50℃, while controlling the vacuum degree to 10~15Pa, and freeze-drying is carried out for 72 hours to ensure that the moisture content of the freeze-dried powder is ≤3.0%; S33. After freeze-drying is complete, quickly remove each group of trace element freeze-dried powders and seal them in packaging.
8. The improved method for producing freeze-dried cell culture medium powder according to claim 1, characterized in that, In step S4, other components include amino acids, vitamins, carbohydrates, or inorganic salts.
9. The improved method for producing freeze-dried cell culture medium powder according to claim 1, characterized in that, In step S4, the mixing is carried out using a ball mill with a rotation speed of 260–290 r / min and a mixing time of 2–3 h.
10. The improved method for producing freeze-dried cell culture medium powder according to claim 1, characterized in that, In step S4, the finished cell culture medium powder is passed through a 50-mesh sieve and then sealed and packaged.