Culture medium optimization method, system and application

Abstract

The invention provides a culture medium optimization method and system and application, and relates to the technical field of biology. The culture medium optimization method comprises the following steps: optimizing a to-be-optimized culture medium combination for at least one time, wherein the culture medium combination comprises a basic culture medium and at least one supplementary culture medium; the optimization comprises the steps of culturing cells by using the initial culture medium combination, obtaining to-be-optimized components in the initial culture medium combination, then optimizing the content of the to-be-optimized components to obtain an optimized culture medium combination, and the to-be-optimized components comprise components with the consumption rate greater than or equal to a first threshold value in the initial culture medium combination. According to the culture medium optimization method, the target protein expression quantity of the cultured cells can be remarkably improved.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a method, system, and application for optimizing culture media. Background Technology

[0002] The following statements are provided only as background information in relation to the present invention and do not necessarily constitute prior art.

[0003] Mammalian cells, particularly Chinese hamster ovary (CHO) cells, are the expression host for the commercial production of recombinant proteins. They are primarily cultured in serum-free, animal-derived, and chemically defined media, mainly using fed-batch culture, which includes basal and supplemental media. The main components of the media include water, carbohydrates, amino acids, vitamins, inorganic salts, trace metal ions, lipids, buffers, and other additives. The typical media development process involves first screening the basal medium, then adapting cells to the selected basal medium, followed by screening the supplemental medium, and finally optimizing it based on the quality requirements of the expressed protein and the production process. The final developed media meets the requirements for cell growth, protein expression, protein quality, and process control.

[0004] With the development of the biopharmaceutical industry and the increasingly widespread application of mammalian cells, especially CHO cells, in antibody-based protein drugs, the demand for cell culture media is growing, as is the demand for media quality. In the process development phase of a biopharmaceutical project, to accelerate progress, companies often directly use the culture media supplied by the purchased cell line company without much optimization, or simply select one general-purpose media from several media companies before starting subsequent process development. However, because different monoclonal cell lines express different exogenous genes, their growth and metabolism vary, as do their nutritional requirements. Using commercially available general-purpose media cannot fully realize the potential of the cell lines, thus creating a need to optimize the culture medium to improve cell growth and protein expression. Many cell culture-based drug development projects that have entered Phase II or III clinical trials or are already on the market exhibit low expression levels. To reduce the production cost of protein drugs, there is also a need to optimize culture medium formulations and improve protein expression levels after using the medium.

[0005] In view of this, the present invention is hereby proposed. Summary of the Invention

[0006] The purpose of this invention is to provide methods, systems, and applications for optimizing culture media, so as to optimize the means of culture media optimization.

[0007] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0008] In a first aspect, a method for optimizing culture media is provided, comprising optimizing a combination of culture media to be optimized at least once, the combination of culture media including a basal culture medium and at least one feed culture medium;

[0009] The optimization includes culturing cells using an initial culture medium combination, obtaining the component to be optimized in the initial culture medium combination, and then optimizing the content of the component to be optimized to obtain an optimized culture medium combination; the component to be optimized includes the component in the initial culture medium combination whose consumption rate is ≥ a first threshold.

[0010] The consumption rate of a specific component P in the initial culture medium composition is calculated according to formulas (a1) and (a2):

[0011] Consumption rate = (C (初始培养基组合) -C (上清) ) / C (初始培养基组合) ×100% (a1);

[0012]

[0013] C (上清) The concentration of component P in the supernatant of the culture medium after cell culture has ended;

[0014] C (基) The concentration of component P in the basal culture medium;

[0015] C (补，Feed i) Let P be the concentration of component P in the i-th type of feed medium, where i is a positive integer from 1 to n;

[0016] V (补，Feed i) The volume percentage is the sum of the volume percentages of the i-th type of feed medium added each time during the entire culture process, where the volume percentage is the percentage of the volume of the i-th type of feed medium added each time to the volume of the culture medium in the culture system after the addition; and the volume of the culture medium in the culture system remains unchanged after each addition of feed medium.

[0017] n represents the quantity of all types of feed media in the initial culture medium combination;

[0018] When optimizing the culture medium combination for the first time, the initial culture medium combination is the culture medium combination to be optimized.

[0019] In an optional embodiment, optimizing the content of the component to be optimized includes at least one of (I) to (III):

[0020] (I) Adjust the content of at least one component to be optimized in the basal culture medium;

[0021] (II) Adjust the content of the component to be optimized in at least one fed culture medium;

[0022] (III) At least one component to be optimized is added as a separate feed medium during the culture process, independent of the basal medium and the feed medium, wherein the content of the component to be optimized in the separate feed medium is entirely derived from all or part of the increase of the component to be optimized relative to the initial culture medium combination.

[0023] In an optional embodiment, the culture medium optimization method includes additive formulation design, and then increasing the content of the component to be optimized in the initial culture medium combination according to the additive formulation to obtain an optimized culture medium combination;

[0024] The additive formulation design includes determining the addition value ΔC for each of the components to be optimized and the distribution method of the addition value ΔC in each optimized culture medium; the addition value ΔC is the increment of the component to be optimized in the optimized culture medium combination relative to the initial culture medium combination; the distribution method is the proportion of the addition value ΔC in each optimized culture medium.

[0025] In an optional implementation, the additive formulation design meets at least one of the following criteria:

[0026] (i) First, design the increment of the component to be optimized in the basal culture medium, and allocate the remaining increment to at least one feed culture medium, wherein the increment of the component to be optimized in the basal culture medium is 0 to 100%;

[0027] (ii) The additive formulation shall not cause any of the components to be optimized to exceed the maximum solubility in the culture medium in which they are contained;

[0028] (iii) The osmotic pressure of the basal culture medium shall not exceed 330 mosm / kg.

[0029] In an optional implementation, the increase in concentration of the component P to be optimized in a specific optimized culture medium Q is calculated according to formulas (a3) ​​and (a4):

[0030] ΔC (增，P) =M Q ×ΔC / V (补，Q)… (a3);

[0031] ΔC=W×C (初始培养基组合) (a4);

[0032] 0 < W ≤ 1;

[0033] ΔC (增，P) This indicates the concentration increment of the component P to be optimized in the optimized culture medium Q;

[0034] When the optimized culture medium Q is the basal medium, V(补，Q) =1;

[0035] When the optimized culture medium Q is a fed culture medium, V (补，Q) The total volume percentage of the optimized culture medium Q added throughout the entire culture process is used; the volume percentage is the percentage of the volume of the optimized culture medium Q added each time to the volume of the culture medium in the culture system after the addition; and the volume of the culture medium in the culture system remains unchanged after each addition of culture medium.

[0036] M Q M is the partition coefficient of the component to be optimized, P, in the optimized culture medium, Q. It is a constant given by those skilled in the art when adding the expected amount of the component to be optimized to the optimized culture medium composition. Q The range is 0-100%, and the sum of the partition coefficients of the component P to be optimized in all optimized culture media is 100%.

[0037] W represents the added value ΔC relative to C. (初始培养基组合) The increase factor.

[0038] In an optional implementation, the effectiveness of the optimized culture medium combination is evaluated using culture data from cell culture of the optimized culture medium combination.

[0039] In an optional implementation, the culture medium optimization method includes the following steps:

[0040] (A1) Obtain the component to be optimized from the initial culture medium combination;

[0041] (A2) Increase the content of the component to be optimized in the initial culture medium combination in step (A1) in at least one manner to obtain at least one optimized culture medium combination;

[0042] (A3) Use the optimized culture medium combination obtained in step (A2) to culture cells, and obtain a number of culture data for each optimized culture medium combination. If the expected number of culture data reaches the corresponding threshold range, the optimized culture medium combination that best corresponds to the culture data is taken as the optimized culture medium combination and the optimization is completed; otherwise, proceed to (A4).

[0043] (A4) Take the optimal culture medium combination corresponding to the best culture data in step (A3) as the initial culture medium combination in step (A1), and repeat (A1) to (A4) until the expected number of culture data reaches the corresponding threshold range, and / or until the culture data obtained no longer reflects the culture effect. Take the optimal culture medium combination corresponding to the best culture data as the optimized culture medium combination, and complete the optimization.

[0044] When step (A1) is performed for the first time, the initial culture medium combination is the culture medium combination to be optimized.

[0045] In a second aspect, a culture medium optimization system is also provided, which is used in the culture medium optimization method described in the first aspect, and includes a data acquisition module and a first data analysis module;

[0046] The data acquisition module is used to acquire the following data: C (上清) C (基) C (补，Feed i) and V (补，Feed i) And, set at least one first threshold;

[0047] The first data analysis module is used to execute formulas (a1) and (a2), match the calculation results with the first threshold, and output the content of all components to be optimized corresponding to each first threshold.

[0048] Thirdly, it also provides the application of the culture medium optimization method of the first aspect, or the culture medium optimization system of the second aspect, in cell culture, preparation of culture medium, or preparation of protein.

[0049] Compared with the prior art, the present invention has the following beneficial effects:

[0050] The culture medium optimization method provided by this invention can improve the culture medium currently used by cell lines to a medium adapted to specific cells or to achieve the desired culture effect without rebuilding the culture process. This method can be improved for specific cells and specific uses of cells without changing the culture process. When this culture medium optimization method is used to optimize the culture medium of cells expressing proteins, it can increase the protein expression level. Attached Figure Description

[0051] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0052] Figure 1 This is a flowchart of the culture medium optimization process in Example 1;

[0053] Figure 2 The cell growth curves (viable cell density) for additive formulations 1, 2-2, and 3 in Example 1 are shown.

[0054] Figure 3 The cell growth curves (cell viability) are for additive formulations 1, 2-2, and 3 in Example 1.

[0055] Figure 4 The protein expression levels at harvest time on day 14 for additive formulations 1, 2-2, and 3 in Example 1;

[0056] Figure 5 The cell growth curves (viable cell density) for additive formulations 2-1, 2-2, and 2-3 in Example 1 are shown.

[0057] Figure 6 Cell growth curves (cell viability) for additive formulations 2-1, 2-2, and 2-3 in Example 1;

[0058] Figure 7 The protein expression levels at harvest time on day 14 for additive formulations 2-1, 2-2, and 2-3 in Example 1;

[0059] Figure 8 The cell growth curve (live cell density) for additive formulation 4 in Example 2;

[0060] Figure 9 The cell growth curve (cell viability) for additive formulation 4 in Example 2 is shown below.

[0061] Figure 10 The protein expression level at harvest on day 14 of additive formulation 4 in Example 2;

[0062] Figure 11 The cell growth curve (viable cell density) for additive formulation 2 in Example 3 is shown below when it is fed alone.

[0063] Figure 12 The cell growth curve (cell viability) for additive formulation 2 in Example 3 is shown below when the additive is fed alone.

[0064] Figure 13 The protein expression level at harvest on day 14 after supplementation with additive formulation 2 in Example 3 was performed.

[0065] Figure 14 The cell growth curves (viable cell density) for additive formulations 5 and 6 in Example 4 are shown.

[0066] Figure 15 The cell growth curves (cell viability) for additive formulations 5 and 6 in Example 4 are shown.

[0067] Figure 16 The protein expression levels at harvest time on day 14 for additive formulations 5 and 6 in Example 4;

[0068] Figure 17 The cell growth curve (live cell density) for additive formulation 7 in Example 5;

[0069] Figure 18 The cell growth curve (cell viability) for additive formulation 7 in Example 5;

[0070] Figure 19 The protein expression level at harvest on day 14 of additive formulation 7 in Example 5;

[0071] Figure 20 The cell growth curve (viable cell density) for additive formulation 8 in Example 6 is shown below when it is fed alone.

[0072] Figure 21 The cell growth curve (cell viability) for additive formulation 8 in Example 6 is shown below when the additive is fed alone.

[0073] Figure 22 The protein expression level at harvest on day 14 after supplementation with additive formulation 8 in Example 6 was performed.

[0074] Figure 23 The cell growth curves (viable cell density) for additive formulations 9 and 10 in Example 7 are shown.

[0075] Figure 24 The cell growth curves (cell viability) for additive formulations 9 and 10 in Example 7 are shown.

[0076] Figure 25 The protein expression levels at day 14 of the harvest for additive formulations 9 and 10 in Example 7;

[0077] Figure 26 The cell growth curve (live cell density) for additive formulation 11 in Example 8;

[0078] Figure 27 The cell growth curve (cell viability) for additive formulation 11 in Example 8;

[0079] Figure 28 The protein expression level at harvest on day 14 of additive formulation 11 in Example 8;

[0080] Figure 29 The cell growth curve (viable cell density) for additive formulation 12 in Example 9 is shown below when it is fed alone.

[0081] Figure 30 The cell growth curve (cell viability) for additive formulation 12 in Example 9 is shown below when it is fed alone.

[0082] Figure 31 The protein expression level at harvest on day 14 after supplementing with additive formulation 12 in Example 9. Detailed Implementation

[0083] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0084] Firstly, a method for optimizing culture media is provided. In this paper, "culture media" is interpreted in the broadest sense; any system that provides nutrients for cell culture can be used as a culture medium. This can be, but is not limited to, liquid or solid systems, and must contain at least one substance that functions in cell culture. This function includes, but is not limited to, providing nutrients to cells; maintaining the cell culture environment, such as maintaining osmotic pressure and pH; or inducing changes in cell state, such as secreting proteins or differentiating into a specific cell type. The components of the culture medium include, but are not limited to, one or more of the following: sugars, amino acids, organic acids, amines, vitamins, inorganic salts, metal ions, lipids, buffer reagents, and culture medium additives.

[0085] In this document, the culture medium combination includes a basal medium and at least one feed medium. The basal medium refers to the culture medium used in the cell culture system at the beginning of the culture. The feed medium refers to the culture medium added to the cell culture system during the culture process from the start to the end of the culture. The types of feed media may be, for example, but not limited to, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; optionally, the culture medium combination includes one feed medium; optionally, the culture medium combination includes two feed media.

[0086] In a first aspect, a method for optimizing a culture medium is provided, comprising optimizing a culture medium combination to be optimized at least once, the culture medium combination including a basal culture medium and at least one feed culture medium.

[0087] The optimization includes culturing cells using an initial culture medium combination, obtaining the component to be optimized in the initial culture medium combination, and then optimizing the content of the component to be optimized to obtain an optimized culture medium combination; the component to be optimized includes the component in the initial culture medium combination whose consumption rate is ≥ a first threshold.

[0088] The consumption rate of a specific component P in the initial culture medium composition is calculated according to formulas (a1) and (a2):

[0089] Consumption rate = (C (初始培养基组合) -C (上清) ) / C (初始培养基组合) ×100% (a1);

[0090]

[0091] C (上清) The concentration of component P in the supernatant of the culture medium after cell culture has ended;

[0092] C (基) The concentration of component P in the basal culture medium;

[0093] C (补，Feed i) Let P be the concentration of component P in the i-th type of feed medium, where i is a positive integer from 1 to n;

[0094] V (补，Feed i) The volume percentage is the sum of the volume percentages of the i-th type of feed medium added each time during the entire culture process, where the volume percentage is the percentage of the volume of the i-th type of feed medium added each time to the volume of the culture medium in the culture system after the addition; and the volume of the culture medium in the culture system remains unchanged after each addition of feed medium.

[0095] n represents the quantity of all types of supplemental culture media in the initial culture medium combination.

[0096] In this invention, the culture medium combination to be optimized refers to the original culture medium combination that has not undergone an optimization process and is expected to be improved. The initial culture medium combination refers to the culture medium combination for which the component to be optimized is to be screened. When the culture medium combination to be optimized is optimized for the first time, the initial culture medium combination is the culture medium combination to be optimized. If the first optimization of the culture medium combination to be optimized cannot achieve the culture effect expected by the art, those skilled in the art may optionally continue to optimize the culture medium. When the culture medium combination to be optimized is optimized more than once, it can be optimized based on the culture medium combination to be optimized, using the culture medium combination to be optimized as the basis for improving the optimized culture medium combination in another way; or it can be optimized by continuing to optimize several optimized culture medium combinations obtained in the previous time, using the several optimized culture medium combinations obtained in the previous time, or the optimized culture medium combination with the best effect among them, as the basis for calculation to obtain the component to be optimized, and continue to optimize. Therefore, the initial culture medium combination is the culture medium combination to be optimized when the culture medium combination to be optimized is optimized for the first time, while in the second or subsequent optimizations, the initial culture medium combination can be the culture medium combination to be optimized or the culture medium combination optimized in the previous or earlier time.

[0097] In this paper, optimized culture medium combination refers to a culture medium combination with increased content of the component to be optimized, based on the initial culture medium combination. Since the culture medium combination consists of a basal culture medium and at least one supplementary culture medium, the content of at least one of the components to be optimized in any one or more of the culture media can be adjusted during the optimization process, or a new supplementary culture medium containing at least one of the components to be optimized can be added. In an optional embodiment, optimizing the content of the component to be optimized includes at least one of (I) to (III):

[0098] (I) Adjust the content of at least one component to be optimized in the basal culture medium;

[0099] (II) Adjust the content of the component to be optimized in at least one fed culture medium;

[0100] (III) At least one component to be optimized is added as a separate feed medium during the culture process, independent of the basal medium and the feed medium, wherein the content of the component to be optimized in the separate feed medium is entirely derived from all or part of the increase of the component to be optimized relative to the initial culture medium combination.

[0101] The basal culture medium in the optimized culture medium combination can be the same as the culture medium in the initial culture medium combination before adjustment; at least one supplementary culture medium in the optimized culture medium combination can be the same as the corresponding supplementary culture medium in the initial culture medium combination before adjustment. Based on the adjustment method in (III) above, the quantity of supplementary culture medium in the optimized culture medium combination can be increased compared to the initial culture medium combination from which it originates.

[0102] In this document, unless otherwise specified, the culture medium in the initial culture medium combination is either basal medium or supplemental medium; the culture medium in the initial culture medium combination is also referred to as the initial culture medium. Similarly, unless otherwise specified, the culture medium in the optimized culture medium combination is either basal medium or supplemental medium; the culture medium in the optimized culture medium combination is also referred to as the optimized culture medium. In some embodiments, optimized basal medium and optimized supplemental medium are also referred to as complete culture media.

[0103] In formula (a2), n represents the number of all types of feed media in the current initial culture medium combination. When the optimization is the first optimization, n is the number of feed media in the culture medium combination to be optimized. When more optimizations occur, since individually fed media are added in some embodiments, all types of feed media in the initial culture medium combination also include individually fed media. If the culture medium combination to be optimized contains x types (x is an integer ≥ 1) of feed media, and the current initial culture medium combination also contains y types (y is an integer ≥ 0) of individually fed media, then n = x + y.

[0104] The specific value of the first threshold can be set by those skilled in the art based on general knowledge and the intended target of the culture medium, and the present invention does not limit this. In optional embodiments, the first threshold is at least 80%, and more preferably 80% to 95%, for example, but not limited to 80%, 82%, 85%, 87%, 88%, 90%, 92%, or 95%.

[0105] In an optional implementation, the optimization further includes sample collection to measure the desired concentrations in formulas (a1) and (a2). Sample collection includes collecting samples from: basal culture medium, fed culture medium, and culture supernatant from culture media at the end of cell culture; obtaining the types and concentrations of some or all components in the corresponding system by detecting the samples; culture supernatant from culture media at the end of cell culture refers to the supernatant collected from the culture system after centrifugation and removal of cells, according to known cell culture process specifications at the end time specified by the cell culture process.

[0106] In the culture medium optimization method, the concentration of any component in any step of any culture medium can be measured using any method known and acceptable in the art. Exemplary measurement methods include, but are not limited to, column chromatography (liquid chromatography, gas chromatography, mass spectrometry, or liquid chromatography-tandem mass spectrometry, etc.) and immunoassay methods (ELISA, immunoblotting, immunochromatography, and immunomagnetic bead assay, etc.).

[0107] In an optional implementation, at least one optimization includes additive formulation design, and then the content of the component to be optimized in the initial culture medium combination is increased according to the additive formulation to obtain an optimized culture medium combination;

[0108] The additive formulation refers to a scheme for increasing the content of the component to be optimized. This additive formulation does not necessarily need to form a specific, physical preparation. The additive formulation design includes determining the addition value ΔC for each of the components to be optimized and the distribution of the addition value ΔC in each optimized culture medium.

[0109] The added value ΔC is the increment of a specific component P to be optimized in the optimized culture medium combination relative to the initial culture medium combination. Those skilled in the art can set this increment according to actual adjustment needs and expectations.

[0110] In an optional implementation, the added value ΔC is C (初始培养基组合) The value is a multiple of the component to be optimized. This multiple is denoted as W, where W ≥ 0. For example, it can be, but is not limited to, 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.5, 1.8, 2, 2.1, 2.5, or 3, with 0 to 1 being the preferred value. The W value can be different for different components to be optimized.

[0111] The allocation method refers to the proportion of the added value ΔC in each optimized culture medium. Specifically, it determines in which optimized culture media the component P to be optimized is added, and the proportion of the addition in a specific optimized culture medium relative to the total addition in the entire optimized culture medium combination. Specific allocation methods include, but are not limited to, allocating the added value ΔC of the component P to the basal culture medium, or allocating it to at least one fed culture medium, or allocating it to at least one separately fed culture medium, or allocating it proportionally to the basal culture medium, at least one fed culture medium, and at least one separately fed culture medium.

[0112] In an optional implementation, the optimized culture medium can be prepared directly based on the final concentration of each component in the adjusted formula, or it can be obtained by adding an additive containing the component to be optimized to the initial culture medium.

[0113] In an optional implementation, at least two additive formulations are designed, with the addition value ΔC and / or the distribution method differing between any two additive formulations, and several optimized culture medium combinations are obtained.

[0114] In an optional implementation, according to C (初始培养基组合) The addition value ΔC for each additive formulation is determined by the multiple, and the addition value ΔC for different additive formulations increases in a gradient, with the multiple ranging from 0.1 to 1.

[0115] In an optional embodiment, the additive formulation design shall at least conform to any one of (i) to (iii):

[0116] (i) First, design the increment of the component to be optimized in the basal culture medium. The remaining increment is allocated to at least one supplemental culture medium. The increment of the component to be optimized in the basal culture medium ranges from 0% to 100%. An increment of 0% indicates that the content of the component to be optimized is not increased in the basal culture medium, while an increment of 100% indicates that the content of the component to be optimized is increased only in the basal culture medium. The supplemental culture medium in this step includes all types of supplemental culture media. Specifically, it includes supplemental culture media in which the content of the component to be optimized is increased or maintained by adjusting the supplemental culture medium in the combination of culture media to be optimized, and supplemental culture media supplemented separately.

[0117] (ii) The additive formulation shall not cause any of the components to be optimized to exceed the maximum solubility in the culture medium in which they are contained;

[0118] (iii) Optimize the osmotic pressure of the basal culture medium to not exceed 330 mosm / kg.

[0119] In an optional implementation, the increased concentration of the component P to be optimized in a specific optimized culture medium Q is calculated according to formulas (a3) ​​and (a4):

[0120] ΔC (增，P) =M Q ×ΔC / V (补，Q)… (a3);

[0121] ΔC=W×C (初始培养基组合) (a4);

[0122] 0 < W ≤ 1;

[0123] ΔC (增，P) This indicates the concentration increment of the component P to be optimized in the optimized culture medium Q;

[0124] When the optimized culture medium Q is the basal medium, V (补，Q) =1;

[0125] When the optimized culture medium Q is a fed culture medium, V (补，Q) The total volume percentage added to optimize culture medium Q throughout the entire culture process; the volume percentage is the percentage of the volume of the optimized culture medium Q added each time to the volume of the culture medium in the culture system after the addition; the feed medium includes feed mediums that are fed from the culture medium combination to be optimized by increasing or maintaining the original content of the component to be optimized, and feed mediums that are fed separately.

[0126] M Q M is the partition coefficient of the component to be optimized, P, in the optimized culture medium, Q. It is a constant given by those skilled in the art when adding the expected amount of the component to be optimized to the optimized culture medium composition. Q The value is 0–100%, and the sum of the partition coefficients of the component P to be optimized in all optimized culture media is 100%. That is, the ΔC of the component P to be optimized is allocated to different optimized culture media in a certain proportion, and the sum of the allocation proportions should be 100%. For example, when someone skilled in the art expects to add 20% of the component P to the basal culture medium with ΔC, M... Q If it is 20%, then the M of the remaining optimized component P in each culture medium is... Q The total is 80%.

[0127] 0 < W ≤ 1, where W represents the added value ΔC relative to C. (初始培养基组合) The multiplier, which may be, for example, but not limited to, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1.

[0128] In an optional implementation, among all the components to be optimized, at least one component has a different W value than the other components to be optimized; or, all the components to be optimized have the same W value.

[0129] In an optional implementation, the effectiveness of the optimized culture medium combination is evaluated using culture data from cell culture. Those skilled in the art can determine whether to continue optimization based on whether the culture data achieves the expected goals or whether the culture effect is improved compared to the initial culture medium combination. Culture data may include one or more cell culture-related data, including but not limited to cell viability, activity, number, density, or target protein expression levels.

[0130] In an optional implementation, the culture data includes physiological data of at least one cell to reflect the cell state.

[0131] In an optional implementation, the culture data includes the concentration of components in the culture supernatant at the end of cell culture, for use in calculating the consumption rate.

[0132] In an optional implementation, the culture data includes protein expression data.

[0133] In an optional implementation, a control group is also set up when using the optimized culture medium combination for cell culture. The culture medium combination used in the control group is the initial culture medium combination that forms the basis for the current optimization of the culture medium combination. In this document, "current" refers to a single process of optimizing the initial culture medium combination to obtain the optimized culture medium combination.

[0134] In an optional implementation, during an optimization process, the culture data includes a percentage increase in protein expression levels. This percentage increase is calculated as the difference between the protein expression level of the initial culture medium combination and the protein expression level of the optimized culture medium combination, expressed as a percentage of the protein expression level of the initial culture medium combination. The expression level of the cultured cells in the initial culture medium combination can be derived from the culture data of the control group or from its previous culture data when used as the optimized culture medium combination.

[0135] In an optional embodiment, the culture process of the optimized culture medium combination cell culture is the same as the culture process of the initial culture medium combination that serves as the basis for the current optimization of the optimized culture medium combination.

[0136] In an optional embodiment, the fed culture medium is used to culture cells in a fed-batch culture manner.

[0137] In an optional embodiment, when screening for the component to be optimized, one or more of the following are measured in the initial culture medium combination: including but not limited to sugars, amino acids, organic acids, amines, vitamins, inorganic salts, metal ions, lipids, buffer reagents and culture medium additives, preferably including at least one of amino acids, organic acids and amines.

[0138] In an optional implementation, this culture medium optimization method is used to optimize the culture medium for culturing mammalian cells.

[0139] In an optional embodiment, the mammalian cells include CHO cells, hybridoma cells, SH87 cells, BHK cells, COS cells, VERO cells, HeLa cells, 293 cells, PER-C6 cells, K562 cells, MOLT-4 cells, M1 cells, NS-1 cells, COS-7 cells, MDBK cells, MDCK cells, MRC-5 cells, WI-38 cells, WEHI cells, SP2 / 0 cells, CAP cells, AGE1.HN cells, or derived cells of any of these cells.

[0140] In an optional embodiment, the mammalian cells include CHO cells, which include any cell line derived from CHO cells, including but not limited to CHO-K1 cells, CHO-DG44 cells, CHO-S cells, CHO-DXB11 cells, or CHO-GS cells.

[0141] In an optional implementation, this culture medium optimization method is used to optimize the culture of cells expressing heterologous proteins.

[0142] In an optional implementation, the heterologous protein includes an antibody.

[0143] In an optional implementation, any step of cell culture may include one or more of the following stages: cell resuscitation, cell proliferation, cell passage, cell differentiation, and induction of cell expression of the target protein.

[0144] In an optional implementation, the culture medium optimization method includes the following steps:

[0145] (A1) Obtain the component to be optimized from the initial culture medium combination;

[0146] (A2) Increase the content of the component to be optimized in the initial culture medium combination in step (A1) in at least one manner to obtain at least one optimized culture medium combination;

[0147] (A3) Use the optimized culture medium combination obtained in step (A2) to culture cells, and obtain a number of culture data for each optimized culture medium combination. If the expected number of culture data reaches the corresponding threshold range, the optimized culture medium combination that best corresponds to the culture data is taken as the optimized culture medium combination and the optimization is completed; otherwise, proceed to (A4).

[0148] (A4) Take the optimal culture medium combination corresponding to the best culture data in step (A3) as the initial culture medium combination in step (A1), and repeat (A1) to (A4) until the expected number of culture data reaches the corresponding threshold range, and / or until the culture data obtained no longer reflects the culture effect. Take the optimal culture medium combination corresponding to the best culture data as the optimized culture medium combination, and complete the optimization.

[0149] When step (A1) is performed for the first time, the initial culture medium combination is the culture medium combination to be optimized. When step (A1) is performed for the second or subsequent times, the initial culture medium combination is the optimized culture medium combination finally obtained when steps (A1) to (A4) were performed in the previous time.

[0150] During the first execution of step (A1), the basal medium in the initial culture medium combination is the basal medium in the culture medium combination to be optimized; during the second or subsequent executions of step (A1), the basal medium of the initial culture medium combination is the basal medium in the optimized culture medium combination finally obtained during the previous executions of steps (A1) to (A4). Since increasing the content of the component to be optimized in the basal medium is not necessary for each execution of step (A2), the basal medium in the initial culture medium combination in the current step (A1) can be the same as the basal medium in the initial culture medium combination during at least one previous execution of step (A1).

[0151] When step (A1) is executed for the first time, the feed medium in the initial culture medium combination is the feed medium in the culture medium combination to be optimized; when step (A1) is executed for the second or more times, the feed medium in the initial culture medium combination is all the feed mediums in the optimized culture medium combination finally obtained when steps (A1) to (A4) were executed in the previous time.

[0152] Since increasing the content of the component to be optimized in each feeding medium is not necessary for each execution of step (A2), the feeding medium in at least one initial culture medium combination in the current step (A1) can be the same as the corresponding feeding medium in the initial culture medium combination during at least one previous execution of step (A1). Furthermore, since a new feeding medium, i.e., the separately fed feeding medium in (III), may be generated during the execution of step (A2), the number of feeding media in the initial culture medium combination can increase with the repetition of (A1) to (A4). For example, if the culture medium combination to be optimized includes two feeding media, and a new separately fed feeding medium is added during the execution of step (A2), then when step (A1) is executed a second time, the initial culture medium combination includes three feeding media.

[0153] In an optional embodiment, step (A2) includes additive formulation design, and then increasing the content of the component to be optimized in the initial culture medium combination according to the additive formulation to obtain an optimized culture medium combination.

[0154] In an optional implementation, the addition value ΔC and / or the distribution method of the additive formulation in step (A2) are different from the additive formulation in the previously performed step (A2).

[0155] In an optional implementation, during one execution (A1) to (A4), the additive obtained according to the additive formulation design is directly added to the initial culture medium combination in step (A1). After the additive formulation design is completed, each component of the additive is added according to its addition method and concentration when preparing the basal culture medium and / or the supplemental culture medium to obtain the optimized culture medium (complete culture medium) for that step (A2). This implementation can directly obtain the optimized culture medium based on the existing culture medium, reducing the cost of culture medium preparation and simplifying the optimization method.

[0156] In an optional implementation, the threshold in step (A3) can be a preset value set by the operator.

[0157] In an optional implementation, the culture data in step (A3) may include a variety of culture data, and the culture data may include physiological data of at least one cell, including but not limited to cell viability, activity, number, density, or expression level of the target protein. The culture data that needs to reach the threshold may be all the physiological data to be detected, or a portion of the culture data may meet the threshold set by the culture data to determine that the culture medium optimization is complete.

[0158] In an optional implementation, in a certain execution (A1) to (A4), a control group is further set in step (A3), which uses the initial culture medium combination of the current step (A1) for cell culture and obtains the culture data of the control group.

[0159] In an optional implementation, during a certain execution (A1) to (A4), the culture data obtained from culturing cells with the optimized culture medium combination in step (A3) is compared with the culture data of the control group, and the difference is used to determine whether the set threshold is met.

[0160] In an optional implementation, the culture data includes component concentration and protein expression data.

[0161] In an optional implementation, during a particular execution (A1) to (A4), the culture data includes a percentage increase in protein expression level. This percentage increase is calculated as the difference between the expression level of cells cultured in the initial culture medium combination in step (A1) and the expression level of cells cultured in the optimized culture medium combination in step (A2), expressed as a percentage of the expression level of cells cultured in the initial culture medium combination in step (A1). The expression level of the cells cultured in the initial culture medium combination can be derived from the protein expression level measured in step (A1) or from the expression level of the control group in step (A3).

[0162] In an optional implementation, the cell culture process in steps (A1) and (A3) is the same.

[0163] In some specific embodiments, the culture medium optimization method includes the following steps:

[0164] S1 sample collection:

[0165] The sample includes all initial culture medium and the supernatant from the culture medium after cell culture has ended. The types and concentrations of some or all components in the initial culture combination are obtained by detecting the initial culture medium (optionally by liquid chromatography, mass spectrometry, or a kit). Before use, 1 ml of the initial culture medium is transferred to a sample tube and stored at -20°C or below (preferably -65°C or below). 1–3 ml of the cell culture supernatant is collected and stored at -20°C or below (preferably -65°C or below). In some embodiments, the feed medium may consist of Feed A (neutral pH) and Feed B (alkaline pH).

[0166] In an optional implementation, fed-batch culture is used. Cells are first seeded into basal medium and cultured. Starting from a certain day, 2% to 7% of the current culture volume of feed medium is added daily or every other day until the end of the experiment. (One common fed-batch culture process is to seed cells at 0.5E+06 cells / ml and culture them in basal medium. Feed medium is added starting on the 3rd day. The feed medium accounts for 3% to 5% of the culture volume of the culture system at the time of addition. The entire cell culture process takes 14 days.)

[0167] The supernatant from the culture medium at the end of cell culture refers to the supernatant obtained after collecting a fixed volume of cell fluid (for subsequent protein purification) at the end of the cell culture process, centrifuging it, and removing the cells, according to known cell culture process specifications. The cell culture process is selectable and variable.

[0168] S2 metabolic data detection:

[0169] The components of the initial culture media and the supernatant obtained at the end of cell culture were analyzed in S1. The data revealed the types and concentrations of nutrients in these media. Metabolic data included the concentrations of each nutrient in the initial culture media and the supernatant at the end of cell culture. These nutrients included carbohydrates, amino acids, vitamins, metal ions, organic acids, nucleotides, and amines. Methods used for analyzing the metabolic data included liquid chromatography, mass spectrometry (LC / MS, ICP-MS), and other methods using reagent kits.

[0170] S3 metabolic data analysis:

[0171] Based on the nutrient concentration data of each initial culture medium and at the end of cell culture obtained in S2, the consumption rate of each known component type is calculated according to the aforementioned formulas (a1) and (a2).

[0172] The total percentage of feed is calculated by considering liquid evaporation and volume loss from liquid sampling during the culture process. This makes V t The percentage remains constant with respect to V1, where V1 is the volume of culture medium in the culture system after the first feeding. t Let t be the volume of culture medium in the culture system after the t-th feeding during the culture process, where t is a positive integer ≥2.

[0173] After calculating the consumption rate of each known component, components with a consumption rate greater than 80% are selected as components to be optimized. Preferably, components with a consumption rate greater than 85% are selected as components to be optimized. Preferably, components with a consumption rate greater than 87% are selected as components to be optimized. Preferably, components with a consumption rate greater than 95% are selected as components to be optimized.

[0174] S4 culture medium additive formulation design:

[0175] For the components to be optimized obtained in S3, according to the initial culture medium concentration C (初始培养基组合) A culture medium additive formulation is created by adding 0.1 times the initial culture medium concentration (W = 0.1) as the addition value ΔC; another culture medium additive formulation is created by adding 0.2 times the initial culture medium concentration (W = 0.2) as the addition value ΔC; different culture medium additive formulations are created by increasing the initial culture medium concentration by 0.1 times each time, up to a maximum of 1 times the initial concentration. Ultimately, 10 additive formulations can be designed.

[0176] S5 culture medium additive formulation and distribution design:

[0177] After obtaining the S4 culture medium additive formulation, the distribution design of the components to be optimized in each optimized culture medium is carried out according to the specific addition values ​​of each component, thus obtaining the actual culture medium additive formulation. The distribution ratio is based on the following principles:

[0178] 1) First, design the amount to be added to the basal culture medium, and then add the remaining amount to at least one supplemental culture medium;

[0179] 2) After allocation, the amount of the component to be optimized added should not exceed its maximum solubility in the optimized culture medium formed;

[0180] 3) The osmotic pressure of the optimized basal medium obtained after distribution should not exceed 330 mosm / kg;

[0181] 4) When distributing, the basal culture medium should be added at a ratio of 0% to 100% by mass.

[0182] Alternatively, follow these principles: the culture medium additive formulation is used as at least one separate feed medium, and is added to the cell culture medium during the cell culture process in a certain proportion together with the feed medium.

[0183] Preparation of S6 complete culture medium:

[0184] After completing the design of the S5 culture medium additive formulation, each component of the additive is added according to its addition method and concentration when preparing the basal culture medium and / or supplemental culture medium to obtain a complete culture medium.

[0185] S7 cell culture validation experiment:

[0186] Cells were revived and passaged. Using the complete culture medium obtained from S6, cells were cultured in a fed-batch manner according to the initial cell combination, and this process was kept constant during the validation experiment. The concentration and expression levels of the obtained components were compared with the control group data to obtain the optimized culture results. Based on the culture results, it was determined whether the optimization target was achieved.

[0187] The complete culture data includes component concentration and protein expression data.

[0188] The control group data refers to the culture data, such as growth and expression, obtained by using the initial culture medium combination in fed-batch experiments.

[0189] S8: Data Analysis

[0190] Analyzing the experimental data from S7, the percentage increase in protein expression is defined as the percentage of the difference between the expression levels of cells cultured in the initial culture medium combination and those cultured in the optimized culture medium combination, expressed as a fraction of the initial culture medium combination's expression level. Optimization is complete when the percentage increase in protein expression reaches the initially set threshold or when the expression level cannot be further increased using this optimization method. The initial target can be a preset value set by the operator. If the initial threshold is not reached but the optimization method still provides an increase (determined by the expression curve not reaching the peak), then steps S3 to S8 are repeated using the culture medium with the optimal expression level as the initial culture medium combination until the percentage increase in protein expression reaches the initial set threshold or when the optimization method can no longer provide an increase.

[0191] The culture medium optimization method provided by this invention can improve the culture medium currently used for cell lines to a medium adapted to specific cells or to achieve the desired culture effect without rebuilding the culture process. This method can be improved for specific cells and specific uses without changing the culture process. When this culture medium optimization method is used to optimize the culture medium for cells expressing proteins, it can increase the protein expression level. In a preferred embodiment, the medium is added as a culture medium additive, and the protein expression of cell lines cultured using this optimized medium is significantly improved without changing the culture process.

[0192] In a second aspect, a culture medium optimization system is also provided, which is used to implement the culture medium optimization method described in the first aspect, including a data acquisition module and a first data analysis module;

[0193] The data acquisition module is used to acquire the following data: C (上清) C (基) C (补，Feed i) and V (补，Feed i)， And, set at least one first threshold;

[0194] The first data analysis module is used to execute formulas (a1) and (a2), match the calculation results with the first threshold, and output the content of all components to be optimized corresponding to each first threshold.

[0195] In an optional embodiment, the culture medium optimization system further includes a second data analysis module, which performs the additive formulation design.

[0196] In an optional implementation, the second data analysis module designs the additive formulation according to formulas (a3) ​​and (a4).

[0197] In an optional implementation, the first data analysis module is further configured to obtain the multiple W and allocation coefficient M of each component to be optimized. Q .

[0198] In an optional implementation, the culture medium optimization system further includes a third data analysis module, which is used to match the several culture data obtained after culturing cells in the optimized culture medium with a threshold to determine whether the optimization is complete.

[0199] In an optional embodiment, the culture medium optimization system further includes a component detection device.

[0200] Thirdly, it also provides the application of the culture medium optimization method of the first aspect, or the culture medium optimization system of the second aspect, in cell culture, preparation of culture medium, or preparation of protein.

[0201] In an optional embodiment, the preparation of the protein includes the preparation of an antibody.

[0202] The present invention will be further illustrated below with specific embodiments. However, it should be understood that these embodiments are merely for the purpose of more detailed illustration and should not be construed as limiting the present invention in any way.

[0203] Example 1

[0204] Optimize the composition of ActiPro+CB7a / 7b medium with a consumption rate of 95% (first threshold). The basal medium is ActiPro, and the supplemental medium includes Cell Boost7a and Cell Boost7b. Design schemes to add all of them to the basal medium, add all of them to the supplemental medium, and add both the basal medium and the supplemental medium.

[0205] Specific equipment, reagents, and consumables involved include:

[0206] Equipment: CO2 incubator (Thermo 3951), cell culture shaker (Adolf KNEX ES-X SMX1300), stacked constant temperature shaker (Jingqi IS RDS6C5), clean bench (Sujing Antai), centrifuge (Sigma 3K30), counter (Countstar IC1000), biochemical analyzer (Roche Cedex), -80℃ deep cryogenic freezer (Haier), AB SCIEX4500 three-way quadrupole mass spectrometer, etc.

[0207] Consumables: SF125 shake flask (Corning 431943), 10ml pipette (Corning 4488), 50ml microreactor (Corning431720), counting plate (Ruiyu Biotechnology), biochemical analyzer reaction vessel (Roche), etc.

[0208] Reagents: Trypan Blue (Ruiyu Biotechnology), glucose, lactate, ammonia, IgG reagent kit (Roche); ActiPro basal medium (Hyclone), Cell Boost 7a / 7b supplemented medium (Hyclone) (abbreviated as CB7a, CB7b), Glutamax (Gibco35050061), etc.

[0209] Cell line: CHO-K1 monoclonal cell line (expressing plinomaclonazole).

[0210] The operation flow of this embodiment is as follows: Figure 1 As shown, the main steps include the following:

[0211] S1. Sample collection;

[0212] S2. Metabolic data detection;

[0213] S3. Metabolic data analysis;

[0214] S4. Culture medium additive formulation design;

[0215] S5. Culture medium additive formulation and distribution design;

[0216] S6. Culture medium preparation;

[0217] S7. Cell Culture Validation Assay

[0218] S8. Data Analysis.

[0219] Optimization methods:

[0220] (S1) Sample collection:

[0221] Resuscitation: Prepare ActiPro basal medium, Cell Boost7a supplemented medium, and Cell Boost7b supplemented medium according to the instructions of the commercially available product. Resuscitate one CHO-K1 monoclonal cell line expressing dupilumab from a liquid nitrogen tank and inoculate it into an SF125 shake flask for culture at a seeding density of 0.3 × 10⁻⁶. 6 Approximately 30 ml of culture medium was prepared and placed in a 37°C, 5% CO2 incubator on a shaker at a speed of 140 rpm and an amplitude of 50 mm for 3 days.

[0222] Subculturing: Take cell sap for counting, at a rate of 0.5 × 10⁻⁶. 6Calculate the required cell culture volume for seeding 30 ml of cells / ml. Transfer the corresponding volume of cell culture to a new SF125 shake flask, add culture medium and 1% culture volume of Glutamax, and place it in a 37°C, 5% CO2 incubator on a shaker at a speed of 140 rpm and an amplitude of 50 mm. Subculture every 3 days.

[0223] Feed-to-cell assay: After three passages, cells were ready for feeding (cell doubling time less than 24 hours, cell viability greater than 95%) and seeded into 50ml miniature reactors. Before seeding, the cell culture medium was centrifuged, resuspended in fresh ActiPro basal medium, and then seeded. After seeding, 1ml each of the ActiPro basal medium, Cell Boost7a feed-to-cell medium (abbreviated as CB7a), and Cell Boost7b feed-to-cell medium (abbreviated as CB7b) used in this experiment were labeled and stored at -80℃. Specific process parameters for the feed-to-cell batch assay are shown in Table 1 below.

[0224] Table 1. Experimental process parameters for Fed-batch feeding

[0225]

[0226] Note: The feed medium should be added according to the percentage of the culture volume after feeding compared to the culture volume after the first feeding.

[0227] During the experiment, starting from day 2, 0.6 ml of sample was taken before each feeding to count cell density and viability, and to detect glucose, lactate, and ammonia levels. Glucose was controlled to be above 2 g / L. On day 14, the last day of culture, the content of dupilumab IgG protein was detected.

[0228] On day 14 of culture, 1 ml of sample was taken, counted, and biochemical parameters were tested. The supernatant collected each time feed was added was labeled and stored in a -80°C cryogenic freezer.

[0229] (S2) Metabolic data detection:

[0230] Culture medium and supernatant samples were taken from a -80°C cryogenic freezer, and metabolic data, including amino acids, vitamins, metal ions, nucleotides, amines, organic acids, etc., were detected using mass spectrometry to obtain the content data of each original component in the culture medium that could be detected.

[0231] (S3) Metabolic data analysis:

[0232] The consumption rate formula is used to calculate the percentage of consumption of all nutrients. The nutrients are then sorted from highest to lowest consumption rate, and those with a consumption rate greater than 95% are selected.

[0233] Consumption rate = (C (初始培养基组合) -C (上清) ) / C (初始培养基组合) ×100%;

[0234] C (初始培养基组合) =C (基) +C (补料培养基A) ×28%+C (补料培养基B) ×2.8%.

[0235] As shown in Table 1, the process involves adding feed on day 2 at a rate of 3% for feed medium CB7a and 0.3% for feed medium CB7b after the initial feed medium addition. On days 4, 6, 8, 10, and 12, feed is then added at a rate of 5% for feed medium CB7a and 0.5% for feed medium CB7b after the initial feed medium addition. During this process, the total percentage of feed medium CB7a added is 28%, and the total percentage of feed medium CB7b added is 2.8%.

[0236] The following components were ultimately screened and require content optimization: pyridoxine hydrochloride, putrescine dihydrochloride, choline chloride, sodium pyruvate, and leucine. The initial values, residual values ​​after 14 days of culture, and consumption rates of these five components in basal and fed media are shown in Table 2 below.

[0237] Table 2 shows the initial values, residual values, and consumption rate data of the five components to be optimized.

[0238]

[0239] (S4) Culture medium additive formulation design;

[0240] The concentrations of the five components to be optimized in Table 2 are determined according to C. (初始培养基组合) Using values ​​of 0.1, 0.5, and 1, three additive formulations were designed, as detailed in Table 3 below:

[0241] Table 3. Required concentrations of the three additive formulations.

[0242]

[0243] Note: Concentration unit: mg / L.

[0244] (S5) Culture medium additive formulation and distribution design

[0245] Based on metabolic data analysis, the five components in Table 3 are only present in the basal medium (ActiPro) and the supplemental medium CB7a. The consumption rate of the components in CB7b does not meet the optimization requirements and does not require optimization; therefore, they are not involved in the distribution scheme. In all formulations, CB7b uses the initial CB7b medium by default.

[0246] Additive Formula 1:

[0247] All five components were allocated to the basal medium ActiPro, and the resulting new basal medium was named ActiPro-1. Since no components were added to CB7a and CB7b feed media, they were still fed using CB7a and CB7b feed media respectively, and the feed media names remained CB7a and CB7b.

[0248] Additive formulations 2-1 to 2-3:

[0249] The five components of additive formulation 1 were allocated to the basal medium ActiPro at ratios of 20%, 50%, and 80%, respectively, with the remainder allocated to the supplemental medium CB7a. Three experimental groups were conducted. The resulting new basal media were named ActiPro-2-1, ActiPro-2-2, and ActiPro-2-3, respectively. The specific concentrations of each component in the three formulations were calculated using the following formula:

[0250] C (ActiPro-2-1中的添加剂) =C (添加剂配方2中的添加剂) ×0.2;

[0251] C (ActiPro-2-2中的添加剂) =C (添加剂配方2中的添加剂) ×0.5;

[0252] C (ActiPro-2-3中的添加剂) =C (添加剂配方2中的添加剂) ×0.8.

[0253] The newly formed feed media were named CB7a-2-1, CB7a-2-2, and CB7a-2-3, respectively. The specific concentrations of each component in the three formulations were calculated according to the following formula:

[0254] C (CB7a-2-1中的添加剂) =C (添加剂配方2中的添加剂) ×0.8 / 0.28;

[0255] C (CB7a-2-2中的添加剂) =C (添加剂配方2中的添加剂) ×0.5 / 0.28;

[0256] C (CB7a-2-3中的添加剂) =C (添加剂配方2中的添加剂) ×0.2 / 0.28.

[0257] Additive formulation 3:

[0258] All five components were allocated to the supplemental feeding medium CB7a, and the resulting new supplemental feeding medium was named CB7a-3. The concentration of each component allocated to CB7a needs to be calculated based on the percentage added to CB7a, using the following formula:

[0259] C (CB7a-3中的添加剂) =C (添加配方3中的添加剂) / 0.28.

[0260] The final allocation results of the 5 additive formulations in ActiPro and CB7a in 5 experimental groups are shown in Table 4 below:

[0261] Table 4. Distribution of the three additive formulations in ActiPro and CB7a across five experimental groups.

[0262]

[0263] Note: 1. Unit of concentration of culture medium components: mg / L.

[0264] (S6) Preparation of complete culture medium

[0265] Prepare 100 ml each of ActiPro basal medium, CB7a supplemental medium, and CB7b supplemental medium according to the instructions. Also prepare 100 ml of each complete basal medium (ActiPro-1 to ActiPro-3) and complete supplemental medium (CB7a-1 to CB7a-3).

[0266] Because the components of the basal culture medium additives and supplemental culture medium additives are added at low concentrations, they are first prepared into high-concentration stock solutions. The volume of stock solution to be added is then calculated based on the prepared stock solution volume. The volume of stock solution to be added is calculated using the following formula:

[0267] Volume of each component mother liquor added = C (培养基中的添加剂) ×0.1 / mother liquor concentration, in ml.

[0268] The CB7a-3 additive formula contains a high concentration of leucine, which is added by weighing. The weighed mass = C (CB7a-3中的亮氨酸) ×0.1 / 1000, in grams.

[0269] The concentration, preparation method, and added volume or weighing mass of each component in the mother liquor are shown in Table 5 below:

[0270] Table 5. Concentration of each component in the mother liquor, preparation method, and volume or mass added.

[0271]

[0272] The CB7a-3 additive includes 0.036 mL of pyridoxine hydrochloride at a concentration of 0.5 g / L, 0.437 mL of putrescine dihydrochloride at a concentration of 5 g / L, 3.176 mL of choline chloride at a concentration of 50 g / L, 2.798 mL of sodium pyruvate at a concentration of 40 g / L, and 0.9468 g of leucine.

[0273] (S7) Cell culture assay verification:

[0274] Revive one cell line, and follow the same revival, passage, and fed culture conditions as described in step (S1) to complete the cell culture experiment. Cell growth and protein expression data for different additive formulations (data for additive formulation 2 are from a 50% partitioning scheme) are available in [link to relevant documentation]. Figures 2-4 Additive formulation 2 was distributed in different proportions in basal medium ActiPro and supplemented medium CB7a. Cell growth and protein expression data are shown in [link to data]. Figures 5-7 .

[0275] (S8) Data Analysis:

[0276] According to C (初始培养基组合) The additive formulations designed at 0.1x, 0.5x, and 1x yields showed a maximum increase of 8.2% in maximum cell density and a maximum increase of 13% in cell viability on day 14 after cell culture. Protein expression was also increased to varying degrees, with the formulations based on C... (初始培养基组合) The 0.5x formulation, when divided at 80% in ActiPro medium, showed the highest protein expression at 5.208 g / L, a 49.7% increase compared to the control group. Control group data refers to cell growth, viable cell density, cell viability, and protein expression levels obtained from fed-batch experiments using the original and fed-batch medium without additives.

[0277] Example 2

[0278] The optimized composition with a consumption rate of over 80% was added to both the basal and supplemental culture media, and the cell lines and products were the same as in Example 1.

[0279] The difference between this embodiment and Embodiment 1 lies in the optimization of the specific method and data analysis, as follows:

[0280] (S1)~(S2) are the same as in Example 1.

[0281] (S3) Metabolic data analysis:

[0282] The consumption rate formula is used to calculate the percentage of consumption of all nutrients. The nutrients are then sorted from highest to lowest consumption rate, and those with a consumption rate greater than 80% are selected.

[0283] Consumption rate = (C (初始培养基组合) -C (上清) ) / C (初始培养基组合) ×100%;

[0284] C (初始培养基组合) =C (基) +C (补料培养基A) ×28%+C (补料培养基B) ×2.8%.

[0285] According to Table 1, the process involves feeding the cultured material at a rate of 3% (CB7a) / 0.3% (CB7b) of the current culture volume on day 2, and at a rate of 5% (CB7a) / 0.5% (CB7b) of the current culture volume on days 4, 6, 8, 10, and 12. The total feeding percentage is 28% (CB7a) / 2.8% (CB7b).

[0286] Based on the above formula, the components with a consumption rate greater than 95% (first threshold) are: pyridoxine hydrochloride, putrescine dihydrochloride, choline chloride, sodium pyruvate, and leucine. The components with a consumption rate between 80% and 94% (first threshold) are lysine, valine, and succinic acid. The initial values, residual values ​​after 14 days of culture, and consumption rate data of these eight components in the basal and supplemented media are shown in Table 6 below:

[0287] Table 6 shows the initial values, residual values, and consumption rate data of the eight components to be optimized.

[0288]

[0289]

[0290] (S4) Culture medium additive formulation design:

[0291] For ingredients with a consumption rate greater than 95%, the design concentration is set at 0.5 times the initial addition amount; for ingredients with a consumption rate between 80% and 94%, the design concentration is set at 0.25 times the initial addition amount. One additive formulation is designed, as detailed in Table 7 below:

[0292] Table 7. Required Concentration of Additives in the Formulation

[0293]

[0294] Note: Concentration unit: mg / L.

[0295] (S5) Culture medium additive formulation and distribution design

[0296] The additive formulation was designed with a 50% allocation in the basal medium ActiPro and a 50% allocation in the supplemental medium CB7a (the component to be optimized is not in CB7b, therefore all components were added to CB7a). The concentration of the component allocated to CB7a needs to be converted based on the percentage added to CB7a. The concentration of each component in the basal and supplemental media was calculated using the following formula:

[0297] C (ActiPro-4中的添加剂) =C (添加剂配方4中的添加剂) ×0.5;

[0298] C (CB7a-4中的添加剂) =C (添加剂配方4中的添加剂) ×0.5 / 0.28.

[0299] The final allocation results of the additive formulations in ActiPro and CB7a are shown in Table 8 below:

[0300] Table 8. Distribution of additive formulations in ActiPro and CB7a

[0301]

[0302] Note: 1. Concentration unit: mg / L.

[0303] (S6) Preparation of complete culture medium

[0304] Prepare the complete basal medium (ActiPro-4) and complete supplemented medium (CB7a-4) according to Table 8. Simultaneously prepare the original basal medium ActiPro, and the original supplemented media CB7a and CB7b, 100 ml for each medium. Follow the instructions for each medium in the corresponding labeling. For additives, weigh them after adding them to the original basal or supplemented medium powder, or add them using the stock solution.

[0305] The lower concentration components of the basal culture medium additives and supplemental culture medium additives are first prepared into high-concentration stock solutions, and then the volume of stock solution added is calculated based on the prepared volume. The volume of stock solution added is calculated using the following formula: Volume of stock solution added for each component = C (培养基中的添加剂) ×0.1 / mother liquor concentration, in ml. The additives CB7a-4, which contain higher concentrations of leucine, succinic acid, valine, and lysine hydrochloride, are added by weighing. The weighed mass = C (培养基中的添加剂)×0.1 / 1000, in grams. CB7a-4 additives include 0.009 mL of pyridoxine hydrochloride at a mother liquor concentration of 0.5 g / L, 0.109 mL of putrescine dihydrochloride at a mother liquor concentration of 5 g / L, 0.794 mL of choline chloride at a mother liquor concentration of 50 g / L, 0.700 mL of sodium pyruvate at a mother liquor concentration of 40 g / L, 0.2367 g of leucine, 1.698 mL of succinic acid at a mother liquor concentration of 30 g / L, 4.869 mL of valine at a mother liquor concentration of 20 g / L, and 0.1244 g of lysine hydrochloride.

[0306] The concentration, preparation method, and added volume or weighing mass of each component in the mother liquor are shown in Table 9 below:

[0307] Table 9. Concentration of each component in the mother liquor, preparation method, and volume or mass added.

[0308]

[0309] (S7) Cell culture assay verification

[0310] Resuscitate one cell line, and follow the same resuscitation, passage, and fed culture conditions as described in step (S1) to complete the cell culture experiment. A control group (ActiPro+CB7a / 7b) was set up as a comparison. Cell growth and protein expression data for different additive formulations are shown below. Figures 8-10 .

[0311] (S8) Data Analysis: Components with a consumption rate greater than 80% are classified according to C... (初始培养基组合) The additive formulation, designed at 0.5 times the original level, was prepared into a complete culture medium with 50% allocated to the basal medium and 50% to the supplemental medium. After cell culture, the maximum cell density increased from 16.7E+06 cells / ml to 18.6E+06 cells / ml, an increase of 11.4%. Viability was slightly improved, and protein expression was 3.693 g / L, an increase of 18% compared to the control group. The control group data is the control group data from Example 1.

[0312] Example 3

[0313] Based on Example 1, the additive was added separately as another supplement, with the same cell line and product.

[0314] The difference between Example 3 and Example 1 is that the additive is added separately as a separate supplement. The specific differences are as follows:

[0315] (S1)~(S4) are the same as in Example 1.

[0316] (S5) Culture medium additive formulation design: Additive concentration confirmation: According to additive formulation 2 in Table 3, the five components are added at equal concentrations of 1% of the current culture volume on days 2, 4, 6, 8, 10, and 12, with a total addition volume of 6%. The formula for calculating the concentration of each component is as follows:

[0317] C (单独补料中的添加剂) =C (添加剂配方2中添加剂) / 0.06.

[0318] The concentrations of each component in separately fed culture media are shown in Table 10 below:

[0319] Table 10 Concentrations of each component in additive formulation 2 separately fed culture medium

[0320]

[0321] Note: Concentration unit: mg / L.

[0322] (S6) Preparation of culture medium for individual additives: First, prepare high-concentration stock solutions of the five components according to the concentrations and dissolution methods in Table 11 below. Then, calculate the volume of each component's stock solution to be added in 100 ml using the following formula (see Table 11 below). When preparing the medium, first add 20 ml of ultrapure water or water for injection, then add the stock solutions of the five components sequentially while stirring. Bring the volume to 100 ml, adjust the pH to 6.80–7.00, and filter for later use.

[0323] Volume of each component mother liquor added = C( 单独补料中的添加剂 )×0.1 / mother liquor concentration.

[0324] Table 11. Preparation methods of the five component mother liquors and the volume of each component mother liquor added in 100ml of individual feed.

[0325]

[0326] (S7) Cell culture assay verification

[0327] Resuscitate one cell line, and follow the same resuscitation, passage, and fed culture conditions as described in step (S1) to complete the cell culture experiment. A control group (ActiPro+CB7a / 7b) is set up as a comparison. The experimental protocol is shown in Table 12 below:

[0328] Table 12. Experimental process parameters for Fed-batch feeding

[0329]

[0330] Note: The feed medium should be added as a percentage of the culture volume before feeding.

[0331] The cell growth and protein expression data for additive formulation 2 alone are shown below. Figures 11-13 .

[0332] (S8) Data Analysis: Components with a consumption rate greater than 95% are classified according to C... (初始培养基组合) The additive formulation, designed at 0.5 times the original level, was added separately on days 2, 4, 6, 8, 10, and 12 at 1% of the current culture volume. After cell culture, the maximum cell density increased from 15.9E+06 cells / ml to 17.3E+06 cells / ml, an increase of 8.8%. Viability showed no significant change, and protein expression was 4.024 g / L, an increase of 21.8% compared to the control group. The control group data were the same as in Example 1.

[0333] Example 4

[0334] To optimize ActiPro+CB7a / 7b, the cell lines were changed (expressing another product), and the additive formulations were designed with only basal medium additives and only supplemental medium additives.

[0335] The difference between Example 4 and Example 1 is as follows:

[0336] Cell line: CHO-K1 monoclonal cell line (expressing PD-1 monoclonal antibody).

[0337] Optimization methods:

[0338] (S1) Sample collection: Resuscitation: Resuscitate a CHO-K1 monoclonal cell line expressing PD-1 monoclonal antibody, and follow the same steps as in Example 1.

[0339] (S2) Metabolic data detection: Same as in Example 1.

[0340] (S3) Metabolic data analysis: Use the consumption rate formula to calculate the consumption percentage of all nutrients, sort them from largest to smallest consumption rate, and screen out the components with a consumption rate greater than 95%.

[0341] Consumption rate = (C (初始培养基组合) -C (上清) ) / C (初始培养基组合) ×100%;

[0342] C (初始培养基组合) =C (基) +C (补料培养基A) ×28%+C (补料培养基B) ×2.8%.

[0343] According to Table 1, the process involves feeding the cultured material at a rate of 3% (CB7a) / 0.3% (CB7b) of the current culture volume on day 2, and at a rate of 5% (CB7a) / 0.5% (CB7b) of the current culture volume on days 4, 6, 8, 10, and 12. The total feeding percentage is 28% (CB7a) / 2.8% (CB7b).

[0344] The following components were ultimately screened and require optimization: pyridoxine hydrochloride, choline chloride, sodium pyruvate, and ornithine. The initial values, residual values ​​after 14 days of culture, and consumption rates of these four components in basal and fed media are shown in Table 13 below.

[0345] Table 13 Initial values, residual values, and consumption rate data of the four components to be optimized.

[0346]

[0347] (S4) Culture medium additive formulation design:

[0348] The concentrations of the four components to be optimized in Table 13 are determined according to C. (初始培养基组合) Using values ​​of 0.1 and 1, two additive formulations were designed, as detailed in Table 14 below:

[0349] Table 14 Concentration of each component added in the two additive formulations

[0350]

[0351] Note: Concentration unit: mg / L.

[0352] (S5) Culture medium additive formulation and distribution design

[0353] Additive formulation 5: All four components are allocated to the basal medium ActiPro, with the added concentration equal to that in additive formulation 5. To distinguish it from other examples, the medium in this example is named ActiPro-5. No components are added to CB7a and CB7b media; CB7a and CB7b feed media are still used for feeding, and the media names remain CB7a and CB7b.

[0354] Additive Formulation 6: All four components are allocated to the fed medium CB7a, forming a new fed medium CB7a named CB7a-6. No components are added to ActiPro and CB7b, and their names remain unchanged. The concentration of the components allocated to CB7a needs to be converted based on the percentage added to CB7a. The calculation formula is: C (CB7a-6中的添加剂) =C (添加剂配方6中的添加剂) / 0.28.

[0355] The final allocation results of the two additive formulations in ActiPro and CB7a are shown in Table 15 below:

[0356] Table 15. Distribution of the two additive formulations in ActiPro and CB7a

[0357]

[0358] Note: 1. Concentration unit: mg / L.

[0359] (S6) Preparation of complete culture medium

[0360] Prepare complete basal medium (ActiPro-5) and complete supplemented medium (CB7a-6) according to Table 15. Simultaneously prepare the original basal medium ActiPro, and the original supplemented media CB7a and CB7b, 100 ml for each medium. Follow the instructions for each medium in the corresponding packaging. For additives, add them to the original basal medium or supplemented medium powder first, then add them using the stock solution.

[0361] The components of the basal culture medium additives and supplemental culture medium additives are first prepared into high-concentration stock solutions, and then the volume of stock solution added is calculated based on the prepared volume. The volume of stock solution added is calculated using the following formula: Volume of each component added to the stock solution = C (培养基中的添加剂) ×0.1 / mother liquor concentration, in ml.

[0362] The concentration, preparation method, and addition volume of each component in the mother liquor are shown in Table 16 below:

[0363] Table 16. Concentration of each component in the mother liquor, preparation method, and volume or mass added.

[0364] Element Pyridoxine hydrochloride choline chloride Sodium pyruvate Pyridoxal hydrochloride Mother liquor concentration (g / L) 0.5 50 40 10 Dissolution method Dissolved in water Dissolved in water Dissolved in water Dissolved in water ActiPro-5 (ml) 0.001 0.089 0.078 0.004 CB7a-6 (ml) 0.036 3.176 2.798 0.126

[0365] (S7) Cell culture assay verification

[0366] Resuscitate one cell line, and follow the same resuscitation, passage, and fed culture conditions as described in step (1) above to complete the cell culture experiment. A control group (ActiPro+CB7a / 7b) was also set up. Cell growth and protein expression data for different additive formulations are shown below. Figures 14-16 .

[0367] (S8) Data Analysis: According to C (初始培养基组合) The additive formulations designed at 0.1x and 1x ratios increased the maximum cell density after cell culture from 17.7E+06 cells / ml to 19.8E+06 cells / ml and 22.0E+06 cells / ml, respectively, representing increases of 11.9% and 24.3%. Viability remained largely unchanged, but protein expression was increased to varying degrees in both formulations. Specifically, the formulations based on C... (初始培养基组合)The 1x design formulation showed the highest protein expression at 4.984 g / L, a 33.7% increase compared to the control group. Control group data refers to cell growth, cell density, cell viability, and protein expression levels obtained from fed-batch experiments using the original culture medium without additives and fed-batch culture media.

[0368] Example 5

[0369] Based on Example 4, 80% of the components were optimized, while the cell line and culture medium remained the same as in Example 4:

[0370] The difference between Example 5 and Example 1 is that the cell line is CHO-K1 monoclonal cell line (expressing PD-1 monoclonal antibody).

[0371] (S1) Sample collection: Resuscitation: Resuscitate a CHO-K1 monoclonal cell line expressing PD-1 monoclonal antibody, and follow the same steps as in Example 1.

[0372] (S2) Metabolic data detection: Same as in Example 1.

[0373] (S3) Metabolic data analysis: Use the consumption rate formula to calculate the consumption percentage of all nutrients, sort them from largest to smallest consumption rate, and screen out the components with a consumption rate greater than 80%.

[0374] Consumption rate = (C (初始培养基组合) -C (上清) ) / C (初始培养基组合) ×100%;

[0375] C (初始培养基组合) =C (基) +C (补料培养基A) ×28%+C (补料培养基B) ×2.8%.

[0376] According to Table 1, the process involves feeding the cultured material at a rate of 3% (CB7a) / 0.3% (CB7b) of the current culture volume on day 2, and at a rate of 5% (CB7a) / 0.5% (CB7b) of the current culture volume on days 4, 6, 8, 10, and 12. The total feeding percentage is 28% (CB7a) / 2.8% (CB7b).

[0377] Based on the above formula, the components with a consumption rate greater than 95% are: pyridoxine hydrochloride, choline chloride, sodium pyruvate, and pyridoxal hydrochloride. The components with a consumption rate between 80% and 94% are succinic acid, serine, and vitamin B2. The initial values, residual values ​​after 14 days of culture, and consumption rate data of these seven components in the basal and supplemented culture media are shown in Table 17 below:

[0378] Table 17 Initial values, residual values, and consumption rate data for the seven components to be optimized.

[0379]

[0380] (S4) Culture medium additive formulation design: For components with a consumption rate greater than 95%, the design concentration is set at 0.5 times the initial addition amount; for components with a consumption rate between 80% and 94%, the design concentration is set at 0.25 times the initial addition amount. One set of additive formulations is designed, as shown in Table 18 below:

[0381] Table 18 Required Concentration of Additives in the Formulation

[0382]

[0383] Note: Concentration unit: mg / L.

[0384] (S5) Culture medium additive formulation and distribution design

[0385] The additive formulation was designed with a 50 / 50 ratio allocated to the basal medium ActiPro and a 50 / 50 ratio allocated to the supplemental medium CB7a. The component consumption rate in CB7b did not meet the optimization requirements and was not optimized. The concentration calculation formula for the additive in the basal medium ActiPro is: C (ActiPro-7中的添加剂) =C (添加剂配方7中的添加剂) ×0.5, the concentration of the component allocated in CB7a needs to be converted according to the percentage of CB7a added. The calculation formula is: C (CB7a-7中的添加剂) =C (添加剂配方7中的添加剂) ×0.5 / 0.28. The final allocation results of the additive formulation in ActiPro and CB7a are shown in Table 19 below:

[0386] Table 19 Distribution of Additive Formulation 7 in ActiPro and CB7a

[0387]

[0388]

[0389] Note: 1. Concentration unit: mg / L.

[0390] (S6) Preparation of complete culture medium:

[0391] Prepare complete basal culture medium (ActiPro-7) and complete supplemented culture medium (CB7a-7) according to Table 19. Simultaneously prepare the original basal culture medium ActiPro, and the original supplemented culture media CB7a and CB7b, 100 ml for each medium. Follow the instructions for each culture medium. Prepare high-concentration stock solutions of the additives, adding them after the original basal and supplemented culture medium powders. The concentration of each component in the stock solution and the specific volume added are shown in Table 20 below.

[0392] Table 20: Concentration of each component in the mother liquor, preparation method, and volume or mass added.

[0393]

[0394] (S7) Cell culture assay verification

[0395] Resuscitate one cell line, and follow the same resuscitation, passage, and fed culture conditions as described in step (S1) to complete the cell culture experiment. A control group (ActiPro+CB7a / 7b) is also included. Cell growth and protein expression data for different additive formulations are shown below. Figures 17-19 .

[0396] (S8) Data Analysis: Components with a consumption rate greater than 80% are classified according to C... (初始培养基组合) The additive formulation, designed at 0.5 times the original level, was prepared as a complete medium, with 50% allocated to the basal medium and 50% to the fed medium. After cell culture, the maximum cell density increased from 17.9E+06 cells / ml to 21.6E+06 cells / ml, an increase of 20.7%. Viability decreased slightly, but this had no significant impact on the experimental results. Protein expression was 5.182 g / L, a 34.1% increase compared to the ActiPro group. The control group (ActiPro group) data refers to cell growth, viability, cell density, cell viability, and protein expression levels obtained from fed-batch experiments using the original basal and fed media without additives.

[0397] Example 6

[0398] The cell line and culture medium were the same as in Examples 4 and 5. The additives were added by preparing a separate feed culture medium and adding it at a ratio of 1%.

[0399] The difference between Example 6 and Example 1 is as follows:

[0400] Cell line: CHO-K1 monoclonal cell line (expressing PD-1 monoclonal antibody).

[0401] Optimization methods:

[0402] (S1) Sample collection: Resuscitation: Resuscitate a CHO-K1 monoclonal cell line expressing PD-1 monoclonal antibody, and follow the same steps as in Example 1.

[0403] (S2) Metabolic data detection: Same as in Example 1.

[0404] (S3) Metabolic data analysis: Use the consumption rate formula to calculate the consumption percentage of all nutrients, sort them from largest to smallest consumption rate, and screen out the components with a consumption rate greater than 95%.

[0405] Consumption rate = (C (初始培养基组合) -C (上清) ) / C (初始培养基组合) ×100%;

[0406] C (初始培养基组合) =C (基) +C (补料培养基A) ×28%+C (补料培养基B) ×2.8%.

[0407] According to Table 22, the process involves feeding the cultured material at a rate of 3% (CB7a) / 0.3% (CB7b) of the current culture volume on day 2, and at a rate of 5% (CB7a) / 0.5% (CB7b) of the current culture volume on days 4, 6, 8, 10, and 12. The total feeding percentage is 28% (CB7a) / 2.8% (CB7b).

[0408] The following components were ultimately selected for optimization: pyridoxine hydrochloride, choline chloride, sodium pyruvate, and pyridoxal hydrochloride. The initial values, residual values ​​after 14 days of culture, and consumption rates of these four components in basal and fed media are shown in Table 13 above.

[0409] (S4) Culture medium additive formulation design:

[0410] For ingredients with a consumption rate greater than 95%, a set of additive formulations was designed with a concentration of 0.5 times the initial addition amount, as shown in Table 21 below:

[0411] Table 21 Required Concentration of Additives in the Formulation

[0412] Additive formulation Culture medium name Pyridoxine hydrochloride choline chloride Sodium pyruvate Pyridoxal hydrochloride Additive Formula 8 <![CDATA[C (初始培养基组合) 0.5×]]> 0.025 222.335 156.71 1.762

[0413] Note: Concentration unit: mg / L.

[0414] (S5) Additive Formulation Design:

[0415] Additive concentration confirmation: According to additive formula 8 in Table 18, the four components were added at equal concentrations on days 2, 4, 6, 8, 10, and 12, each time at a rate of 1% of the current culture volume. The total feed volume was 6%. The formula for calculating the concentration of each component is as follows:

[0416] C (单独补料中的添加剂) =C (添加剂配方8中的添加剂) / 0.06.

[0417] The concentrations of each component in separately fed culture media are shown in Table 22 below:

[0418] Table 22 Concentrations of each component in additive formulation 8 separately fed culture medium

[0419]

[0420] Note: Concentration unit: mg / L.

[0421] (S6) Preparation of culture medium for individual additives: First, prepare high-concentration stock solutions of the four components according to the concentrations and dissolution methods in Table 23 below. Then, calculate the volume of each component's stock solution to be added in 100 ml using the following formula (see Table 23 below). When preparing the medium, first add 20 ml of ultrapure water or water for injection, then add the stock solutions of the four components sequentially while stirring. Make up to 100 ml, adjust the pH to 6.80–7.00, and filter for later use.

[0422] Volume of each component mother liquor added = C (单独补料中的添加剂) ×0.1 / Mother liquor concentration.

[0423] Table 23 Preparation methods of the four component mother liquors and the volume of each component mother liquor added in 100ml individual feed.

[0424] Element Pyridoxine hydrochloride choline chloride Sodium pyruvate Pyridoxal hydrochloride Mother liquor concentration (g / L) 5 50 40 10 Dissolution method Dissolved in water Dissolved in water Dissolved in water Dissolved in water Volume of mother liquor added (ml) 0.0084 7.41 6.53 0.294

[0425] (S7) Cell culture assay verification:

[0426] Resuscitate one cell line, and follow the same resuscitation, passage, and fed culture conditions as described in step (S1) to complete the cell culture experiment. A control group (ActiPro+CB7a / 7b) is also included. The experimental protocol is shown in Table 24 below.

[0427] Table 24. Experimental process parameters for Fed-batch feeding

[0428]

[0429] Note: The feed medium should be added as a percentage of the culture volume before feeding.

[0430] Cell growth and protein expression data for different additive formulations are shown in [link to relevant documentation]. Figures 20-22 .

[0431] (S8) Data Analysis: Components with a consumption rate greater than 95% are classified according to C... (初始培养基组合) The additive formulation, designed to be 0.5 times the original, was added separately on days 2, 4, 6, 8, 10, and 12 at 1% of the current culture volume. After cell culture, the maximum cell density increased from 18.4E+06 cells / ml to 20.9E+06 cells / ml, an increase of 13.6%. Viability decreased slightly, but had no significant impact. Protein expression was 4.777 g / L, an increase of 28.1% compared to the control group. The control group data refers to the cell growth, viability, cell density, cell viability, and protein expression levels obtained from fed-batch experiments using the original culture medium without additives and the fed-batch culture medium.

[0432] Example 7

[0433] Another culture medium was optimized on the cell line of Example 1: Advanced+CB7a / 7b medium, designed entirely on the basal level or entirely added in the feed.

[0434] The difference between Example 7 and Example 1 is as follows:

[0435] Optimization methods:

[0436] (S1) Collection of culture medium and cell culture supernatant on the last day:

[0437] Resuscitation: Prepare Advanced basal medium and Cell Boost 7a / 7b fed medium according to the instructions. Resuscitate one CHO-K1 monoclonal cell line expressing dupilumab from a liquid nitrogen tank and inoculate it into an SF125 shake flask for culture at a seeding density of 0.3 × 10⁻⁶. 6 The cells / ml were approximately 30ml in volume. The culture was placed in a 37℃, 5% CO2 incubator on a shaker at a speed of 140 rpm and an amplitude of 50 mm for 3 days.

[0438] Subculturing: Take cell sap for counting, at a rate of 0.5 × 10⁻⁶. 6 Calculate the required cell volume for seeding 30 ml of cells / ml. Transfer the corresponding volume of cell solution to a new SF125 shake flask, add culture medium and 1% (V / V) Glutamax, and incubate on a shaker in a 37°C, 5% CO2 incubator at 140 rpm and 50 mm amplitude. Subculture every 3 days.

[0439] Feed-to-cell assay: After three passages, cells were ready for feeding experiments once their doubling time was less than 24 hours and viability was greater than 95%. Cells were then seeded into 50 ml microreactors. The cell culture was centrifuged before seeding, then resuspended in fresh Advanced basal medium. After seeding, 1 ml each of Advanced, CB7a, and CB7b culture media were taken, labeled, and stored at -80°C. Specific process parameters for the fed-to-cell assay are shown in Table 25 below.

[0440] Table 25. Experimental process parameters for Fed-batch feeding

[0441]

[0442]

[0443] Note: The feed medium should be added as a percentage of the culture volume before feeding.

[0444] During the experiment, starting from day 3, 0.6 ml of sample was taken before each feeding to count cell density and viability, and to detect glucose, lactic acid, and ammonia. Glucose was controlled above 2 g / L, and IgG content was detected on day 15, the last day of culture.

[0445] On day 15 of culture, 1 ml of sample was taken, counted, and biochemical parameters were tested. The remaining supernatant was labeled and stored in a -80°C cryogenic freezer.

[0446] (S2) Metabolic data detection is the same as in Example 1.

[0447] (S3) Metabolic data analysis: Use the consumption rate formula to calculate the consumption percentage of all nutrients, sort them from largest to smallest consumption rate, and screen out the components with a consumption rate greater than 95%.

[0448] Consumption rate = (C (初始培养基组合) -C (上清) ) / C (初始培养基组合) ×100%;

[0449] C (初始培养基组合) =C (基) +C (补料培养基A) ×28%+C (补料培养基B) ×2.8%.

[0450] According to Table 25, the process involves feeding the culture at a rate of 3% (CB7a) / 0.3% (CB7b) of the current culture volume on day 3, and at a rate of 5% (CB7a) / 0.5% (CB7b) of the current culture volume on days 5 / 7 / 9 / 11 / 13, for a total feeding percentage of 28% (CB7a) / 2.8% (CB7b).

[0451] The following components were ultimately screened and require optimization: pyridoxine hydrochloride, asparagine, leucine, and taurine. The initial values, residual values ​​after 15 days of culture, and consumption rates of these four components in basal and fed media are shown in Table 26 below.

[0452] Table 26 Initial values, residual values, and consumption rate data for the four components to be optimized.

[0453]

[0454] (S4) Culture medium additive formulation design: The concentrations of the four components to be optimized in Table 26 are determined according to C... (初始培养基组合) Using values ​​of 0.1 and 1, two additive formulations were designed, as detailed in Table 27 below:

[0455] Table 27 Concentration of Additives to be Optimized in Additive Formulations 9 and 10

[0456]

[0457] Note: Concentration unit: mg / L.

[0458] (S5) Culture medium additive formulation and distribution design:

[0459] Additive formulation 9: All four components are allocated to the Advanced basal culture medium, calculated using the following formula: C (Advanced-9中的添加剂) =C (添加剂配方9中的添加剂) .

[0460] Additive formulation 10: All four components are allocated to the supplemental culture medium CB7a. The component consumption rate in CB7b does not meet the optimization requirements and will not be optimized. The component concentration allocated to CB7a needs to be converted based on the percentage added to CB7a, using the following formula: C (CB7a-10中的添加剂) =C (添加剂配方10中的添加剂) / 0.28. The final allocation results of the two additive formulations in Advanced and CB7a are shown in Table 28 below:

[0461] Table 28 Distribution of Additive Formulations 9 and 10 in Advanced and CB7a

[0462]

[0463] Note: 1. Concentration unit: mg / L.

[0464] (S6) Preparation of complete culture medium:

[0465] Prepare complete basal culture medium (Advanced-9) and complete supplemented culture medium (CB7a-10) according to Table 28. Simultaneously prepare the original basal culture medium (Advanced), and the original supplemented culture media (CB7a and CB7b), 100 ml for each medium. Follow the instructions for each culture medium during preparation. For additives, weigh them after adding them to the original basal or supplemented culture medium powder, or add them using the stock solution.

[0466] The lower concentration components of the basal culture medium additives and supplemental culture medium additives are first prepared into high-concentration stock solutions, and then the volume of stock solution added is calculated based on the prepared volume. The volume of stock solution added is calculated using the following formula: Volume of stock solution added for each component = C (添加剂配方4) ×0.1 / mother liquor concentration, in ml. For additives with higher concentrations in the formulation, add by weighing; the weighed mass = C (添加剂配方4) ×0.1 / 1000, in grams.

[0467] The concentration, preparation method, and added volume or weighing mass of each component in the mother liquor are shown in Table 29 below:

[0468] Table 29: Concentration of Mother Liquor for Each Component, Preparation Method, Volume Added or Mass Weighed

[0469]

[0470] (S7) Cell Culture Experiment Verification: Resuscitate one cell line. The resuscitation, passage, and fed culture conditions are the same as in step (1) above. Complete the cell culture experiment, and simultaneously set up a control group (Advanced+CB7a / 7b). Cell growth and protein expression data for additive formulations 9 and 10 are shown below. Figures 23-25 .

[0471] (S8) Data Analysis: According to C (初始培养基组合) The additive formulations designed at 0.1x and 1x increased the maximum cell density by 25.5% and 18.6% respectively after cell culture, with no significant difference in cell viability. Protein expression was increased to varying degrees in both formulations. Specifically, the formulations based on C... (初始培养基组合) The 0.1x design formulation showed the highest protein expression at 3.775 g / L, a 20.6% increase compared to the control group. Control group data refers to cell growth, cell density, cell viability, and protein expression levels obtained from fed-batch experiments using the original culture medium without additives.

[0472] Example 8

[0473] Based on Example 7, the components with a consumption rate of 80% to 94% were optimized, while the cell line and culture medium remained unchanged.

[0474] Optimization methods:

[0475] (S1) Sample collection:

[0476] Resuscitation: Prepare Advanced basal medium and CB7a / CB7b fed medium according to the instructions. Resuscitate one CHO-K1 monoclonal cell line expressing dupilumab from a liquid nitrogen tank and inoculate it into an SF125 shake flask for culture at a density of 0.3 × 10⁻⁶ cells / year. 6 The cells / ml were approximately 30ml in volume. The culture was placed in a 37℃, 5% CO2 incubator on a shaker at a speed of 140 rpm and an amplitude of 50 mm for 3 days.

[0477] Subculturing: Take cell sap for counting, at a rate of 0.5 × 10⁻⁶. 6Calculate the required cell volume for seeding 30 ml of cells / ml. Transfer the corresponding volume of cell solution to a new SF125 shake flask, add culture medium and 1% Glutamax, and incubate at 37°C in a 5% CO2 incubator on a shaker at 140 rpm and 50 mm amplitude. Subculture every 3 days.

[0478] Feed-to-cell assay: After three passages, cells were ready for feeding experiments once their doubling time was less than 24 hours and viability was greater than 95%. Cells were then seeded into 50 ml microreactors. Before seeding, the cell culture medium was centrifuged, resuspended in fresh Advanced basal medium, and then seeded. After seeding, 1 ml each of Advanced, CB7a, and CB7b culture media used in this experiment was taken, labeled, and stored at -80°C. Specific process parameters for the fed-to-cell assay are shown in Table 25 above.

[0479] During the experiment, starting from day 3, 0.6 ml of sample was taken before each feeding to count cell density and viability, and to detect glucose, lactic acid, and ammonia. Glucose was controlled above 2 g / L, and IgG content was detected on day 15, the last day of culture.

[0480] On day 15 of culture, 1 ml of sample was taken, counted, and biochemical parameters were tested. The remaining supernatant was labeled and stored in a -80°C cryogenic freezer.

[0481] (S2) Metabolic data detection: The culture medium and supernatant samples were taken out from the -80℃ deep cryogenic freezer and the metabolic data, including amino acids, vitamins, metal ions, nucleotides, amines and organic acids, were detected by mass spectrometry to obtain the content data of each nutrient.

[0482] (S3) Metabolic data analysis: Use the consumption rate formula to calculate the consumption percentage of all nutrients, sort them from largest to smallest consumption rate, and screen out the components with a consumption rate greater than 80%.

[0483] Consumption rate = (C (初始培养基组合) -C (上清) ) / C (初始培养基组合) ×100%;

[0484] C (初始培养基组合) =C (基) +C (补料培养基A) ×28%+C (补料培养基B) ×2.8%.

[0485] According to Table 25, the process involves feeding the culture at a rate of 3% (CB7a) / 0.3% (CB7b) of the current culture volume on day 3, and at a rate of 5% (CB7a) / 0.5% (CB7b) of the current culture volume on days 5 / 7 / 9 / 11 / 13, for a total feeding percentage of 28% (CB7a) / 2.8% (CB7b).

[0486] Based on the above formula, the components with a consumption rate greater than 95% are: pyridoxine hydrochloride, asparagine, leucine, and taurine. The components with a consumption rate between 80% and 94% are choline chloride and putrescine. The initial values ​​of these six components in the basal and supplemented culture media, the residual values ​​after 15 days of culture, and the consumption rate data are shown in Table 30 below:

[0487] Table 30 shows the initial values, residual values, and consumption rates of the six components to be optimized.

[0488]

[0489]

[0490] (S4) Culture medium additive formulation design:

[0491] For ingredients with a consumption rate greater than 95%, the design concentration is set at 0.5 times the initial addition amount; for ingredients with a consumption rate between 80% and 94%, the design concentration is set at 0.25 times the initial addition amount. One set of additive formulations is designed, as detailed in Table 31 below:

[0492] Table 31 Required Concentration of Additives in the Formulation

[0493]

[0494] Note: Concentration unit: mg / L.

[0495] (S5) Culture Medium Additive Formulation Design: The additive formulation was designed with a 50% allocation in the basal medium (Advanced) and a 50% allocation in the supplemental medium (CB7a). The component consumption rate in CB7b did not meet the optimization requirements and was not optimized. The concentration calculation formula for the additive in the basal medium (Advanced) is: C (Advdanced-11中的添加剂) =C (添加剂配方11中的添加剂) ×0.5, the concentration of the component allocated in CB7a needs to be converted according to the percentage of CB7a added. The calculation formula is: C (CB7a-11中的添加剂) =C (添加剂配方11中的添加剂) ×0.5 / 0.28. The final allocation results of the additive formulations in Advanced and CB7a are shown in Table 32 below:

[0496] Table 32 Distribution of additive formulations in Advanced and CB7a

[0497]

[0498] Note: 1. Concentration unit: mg / L.

[0499] (S6) Preparation of complete culture media: Prepare complete basal culture medium (Advanced-11) and complete supplemented culture medium (CB7a-11) according to Table 32. At the same time, prepare the original basal culture medium (Advanced), and the original supplemented culture media (CB7a and CB7b), 100 ml of each culture medium. The preparation process should be carried out according to the instructions of the corresponding culture medium. The ingredients in the additive formula should be weighed and added after adding the dry powder of the original basal culture medium or supplemented culture medium, or added using the stock solution.

[0500] The lower concentration components of the basal culture medium additives and supplemental culture medium additives are first prepared into high-concentration stock solutions, and then the volume of stock solution added is calculated based on the prepared volume. The volume of stock solution added is calculated using the following formula: Volume of stock solution added for each component = C (添加剂配方4) ×0.1 / mother liquor concentration, in ml. For additives with higher concentrations in the formulation, add by weighing; the weighed mass = C (添加剂配方4) ×0.1 / 1000, in grams.

[0501] The concentration, preparation method, and added volume or weighing mass of each component in the mother liquor are shown in Table 33 below:

[0502] Table 33 Concentration of each component in the mother liquor, preparation method, and volume or mass added.

[0503]

[0504]

[0505] (S7) Cell Culture Assay Verification: Resuscitate one cell line, and follow the same resuscitation, passage, and fed culture conditions as described in step (S1) to complete the cell culture experiment. A control group (Advanced+CB7a / 7b) is also included. Cell growth and protein expression data for additive formulation 11 are available in [link to additive formulation]. Figures 26-28 :

[0506] (S8) Data Analysis: Components with a consumption rate greater than 80% are classified according to C... (初始培养基组合)The additive formulation, designed to be 0.5 times the original, was prepared as a complete medium, with 50% allocated to the basal medium and 50% to the fed medium. After cell culture, the maximum density increased from 15.8E+06 cells / ml to 20.2E+06 cells / ml, an increase of 27.8%. Cell viability showed no significant difference. Protein expression was 4.045 g / L, an increase of 28.5% compared to the control group. The control group data refers to the cell growth, viability, cell density, cell viability, and protein expression levels obtained from fed-batch experiments using the original basal and fed media without additives.

[0507] Example 9

[0508] Based on Example 7, the additive is added as a separate supplement, not in the currently used basal or supplemental culture medium. The cell line and culture medium are the same as in Example 7. Example 9 differs from Example 1 in the following steps; the rest is the same as in Example 1:

[0509] Optimization methods:

[0510] (S1) Sample collection:

[0511] Resuscitation: Prepare Advanced basal medium and Cell Boost 7a / 7b fed medium according to the instructions. Resuscitate one CHO-K1 monoclonal cell line expressing dupilumab from a liquid nitrogen tank and inoculate it into an SF125 shake flask for culture at a seeding density of 0.3 × 10⁻⁶. 6 The cells / ml were approximately 30ml in volume. The culture was placed in a 37℃, 5% CO2 incubator on a shaker at a speed of 140 rpm and an amplitude of 50 mm for 3 days.

[0512] Subculturing: Take cell sap for counting, at a rate of 0.5 × 10⁻⁶. 6 Calculate the required cell volume for seeding 30 ml of cells / ml. Transfer the corresponding volume of cell solution to a new SF125 shake flask, add culture medium and 1% Glutamax, and incubate at 37°C in a 5% CO2 incubator on a shaker at 140 rpm and 50 mm amplitude. Subculture every 3 days.

[0513] Feed-to-catch assay: After three passages, cells were ready for feeding (doubling time less than 24 hours, viability greater than 95%) and seeded into 50ml microreactors. The cell culture was centrifuged before seeding, then resuspended in fresh Advanced basal medium. After seeding, 1ml each of Advanced, CB7a, and CB7b culture media were taken, labeled, and stored at -80℃. Specific process parameters for the feed-to-catch assay are shown in Table 25 above.

[0514] During the experiment, starting from day 3, 0.6 ml of sample was taken before each feeding to count cell density and viability, and to detect glucose, lactic acid, and ammonia. Glucose was controlled above 2 g / L, and IgG content was detected on day 15, the last day of culture.

[0515] On day 15 of culture, 1 ml of sample was taken, counted, and biochemical parameters were tested. The remaining supernatant was labeled and stored in a -80°C cryogenic freezer.

[0516] (S2) Metabolic data detection: The culture medium and supernatant samples were taken out from the -80℃ deep cryogenic freezer and the metabolic data, including amino acids, vitamins, metal ions, nucleotides, amines, organic acids and other components, were detected by mass spectrometry to obtain the content data of each nutrient.

[0517] (S3) Metabolic data analysis: Use the consumption rate formula to calculate the consumption percentage of all nutrients, sort them from largest to smallest consumption rate, and screen out the components with a consumption rate greater than 95%.

[0518] Consumption rate = (C (初始培养基组合) C (上清) ) / C (初始培养基组合) ×100%;

[0519] C (初始培养基组合) =C (基) +C (补料培养基A) ×28%+C (补料培养基B) ×2.8%.

[0520] According to Table 25, the process involves feeding the culture at a rate of 3% (CB7a) / 0.3% (CB7b) of the current culture volume on day 3, and at a rate of 5% (CB7a) / 0.5% (CB7b) of the current culture volume on days 5 / 7 / 9 / 11 / 13, for a total feeding percentage of 28% (CB7a) / 2.8% (CB7b).

[0521] The following components were ultimately screened and require optimization: pyridoxine hydrochloride, asparagine, leucine, and taurine. The initial values, residual values ​​after 15 days of culture, and consumption rates of these four components in basal and fed media are shown in Table 26 above.

[0522] (S4) Culture medium additive formulation design: Design one set of additive formulations for components with a consumption rate greater than 95% at a concentration of 0.25 times the initial addition amount, as shown in Table 34 below:

[0523] Table 34 Additive Formulation 12 Required Concentration

[0524] Additive formulation Culture medium name Pyridoxine hydrochloride Asparagine Leucine Taurine Additive Formulation 12 <![CDATA[C (初始培养基组合) 0.25×]]> 1.38 927.85 598.46 0.845

[0525] Note: Concentration unit: mg / L.

[0526] (S5) Culture medium additive formulation and distribution design:

[0527] Additive concentration confirmation: According to additive formula 12 in Table 34, the four components were added at equal concentrations on days 3, 5, 7, 9, 11, and 13, each time at a rate of 1% of the current culture volume. The total feed volume was 6%. The formula for calculating the concentration of each component is as follows:

[0528] C (单独补料中的添加剂) =C (添加剂配方12中的添加剂) / 0.06.

[0529] The concentrations of each component in separately fed culture media are shown in Table 35 below:

[0530] Table 35 Concentrations of each component in additive formulation 12 separately fed culture medium

[0531]

[0532] Note: Concentration unit: mg / L.

[0533] (S6) Preparation of culture medium for individual additives: First, prepare high-concentration stock solutions of the four components according to the concentrations and dissolution methods in Table 36 below. Then, calculate the volume of each component's stock solution to be added in 100 ml using the following formula (see Table 36 below). When preparing the medium, first add 20 ml of ultrapure water or water for injection, then add the stock solutions of the four components sequentially while stirring. Bring the volume to 100 ml, adjust the pH to 6.80-7.00, and filter for later use.

[0534] Volume of each component mother liquor added = C (单独补料中的添加剂) ×0.1 / Mother liquor concentration.

[0535] Mass of each component = C (单独补料中的添加剂) ×0.1 / 1000.

[0536] Table 36. Preparation methods of the four component mother liquors and the volume of each component mother liquor added in 100ml individual feed.

[0537] Element Pyridoxine hydrochloride Asparagine Leucine Taurine Mother liquor concentration (g / L) 5 - - 30 Dissolution method Dissolved in water - - Dissolved in water Mother liquor added volume (ml) 0.460 - - 0.047 Weigh the mass (g) - 1.5464 0.9974 -

[0538] (S7) Cell culture assay verification:

[0539] Resuscitate one cell line, and follow the same resuscitation, passage, and fed culture conditions as described in step (S1) to complete the cell culture experiment. A control group (Advanced+CB7a / 7b) is also included. The experimental protocol is shown in Table 37 below:

[0540] Table 37. Experimental process parameters for Fed-batch feeding

[0541]

[0542] Note: The feed medium should be added as a percentage of the culture volume before feeding.

[0543] Additive formulation 12 cell growth and protein expression data are shown in [link to data]. Figures 29-31 .

[0544] (S8) Data Analysis: Components with a consumption rate greater than 95% are classified according to C... (初始培养基组合) The additive formulation, designed to be 0.5 times the original, was added separately at 1% on days 3, 5, 7, 9, 11, and 13. After cell culture, there was no significant difference in maximum cell density and viability, and protein expression was 3.887 g / L, an increase of 24.2% compared to the control group. The control group data refers to the cell growth, cell density, cell viability, and protein expression data obtained from fed-batch experiments using the original culture medium without additives and the supplemented culture medium.

[0545] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for optimizing a culture medium, characterized in that, This includes optimizing the culture medium combination to be optimized at least once, the culture medium combination including a basal culture medium and at least one feed culture medium; The optimization includes culturing cells using an initial culture medium combination, obtaining the component to be optimized in the initial culture medium combination, and then optimizing the content of the component to be optimized to obtain an optimized culture medium combination; the component to be optimized includes the component in the initial culture medium combination whose consumption rate is ≥ a first threshold. The consumption rate of a specific component P in the initial culture medium composition is calculated according to formulas (a1) and (a2): Consumption rate = (C (初始培养基组合) -C (上清) ) / C (初始培养基组合) ×100% (a1); C (上清) The concentration of component P in the supernatant of the culture medium after cell culture has ended; C (基) The concentration of component P in the basal culture medium; C (补 , Feed i ) Let P be the concentration of component P in the i-th type of feed medium, where i is a positive integer from 1 to n; V (补 , Feed i ) The volume percentage is the sum of the volume percentages of the i-th type of feed medium added each time during the entire culture process, where the volume percentage is the percentage of the volume of the i-th type of feed medium added each time to the volume of the culture medium in the culture system after the addition; and the volume of the culture medium in the culture system remains unchanged after each addition of feed medium. n represents the quantity of all types of feed media in the initial culture medium combination; When optimizing the culture medium combination for the first time, the initial culture medium combination is the culture medium combination to be optimized.

2. The culture medium optimization method according to claim 1, characterized in that, The optimization of the content of the component to be optimized includes at least one of (I) to (III): (I) Adjust the content of at least one component to be optimized in the basal culture medium; (II) Adjust the content of the component to be optimized in at least one fed culture medium; (III) At least one component to be optimized is added as a separate feed medium during the culture process, independent of the basal medium and the feed medium, wherein the content of the component to be optimized in the separate feed medium is entirely derived from all or part of the increase of the component to be optimized relative to the initial culture medium combination.

3. The culture medium optimization method according to claim 2, characterized in that, This includes designing an additive formulation, and then increasing the content of the component to be optimized in the initial culture medium combination according to the additive formulation to obtain an optimized culture medium combination; The additive formulation design includes determining the addition value ΔC for each of the components to be optimized and the distribution method of the addition value ΔC in each optimized culture medium; The added value ΔC is the increment of the component to be optimized in the optimized culture medium combination relative to the initial culture medium combination; the allocation method is the proportion of the added value ΔC in each optimized culture medium; Optionally, at least two additive formulations are designed, with the addition value ΔC and / or the distribution method differing between any two additive formulations, and several optimized culture medium combinations are obtained; Optionally, according to C (初始培养基组合) The addition value ΔC for each additive formulation is determined by the multiple, and the addition value ΔC for different additive formulations increases in a gradient, with the multiple ranging from 0.1 to 1.

4. The culture medium optimization method according to claim 3, characterized in that, The additive formulation design must meet at least one of the following criteria: (i) First, design the increment of the component to be optimized in the basal culture medium, and allocate the remaining increment to at least one feed culture medium, wherein the increment of the component to be optimized in the basal culture medium is 0 to 100%; (ii) The additive formulation shall not cause any of the components to be optimized to exceed the maximum solubility in the culture medium in which they are contained; (iii) The osmotic pressure of the basal culture medium shall not exceed 330 mosm / kg.

5. The culture medium optimization method according to claim 3, characterized in that, The increase in concentration of the component P to be optimized in a specific optimized culture medium Q is calculated according to formulas (a3) ​​and (a4): ΔC (增，P) =M Q ×ΔC / V (补，Q)… (a3); ΔC=W×C (初始培养基组合) (a4); 0＜W≤1； ΔC (增，P) This indicates the concentration increment of the component P to be optimized in the optimized culture medium Q; When the optimized culture medium Q is the basal medium, V (补，Q) =1; When the optimized culture medium Q is a fed culture medium, V (补，Q) To optimize the total volume percentage of culture medium Q added throughout the entire culture process; The volume percentage is the percentage of the volume of the optimized culture medium Q added each time to the volume of the culture medium in the culture system after the addition; and the volume of the culture medium in the culture system remains unchanged after each addition of culture medium. M Q M represents the partition coefficient of the component P to be optimized in the optimized culture medium Q. Q The range is 0-100%, and the sum of the partition coefficients of the component P to be optimized in all optimized culture media is 100%. W represents the added value ΔC relative to C. (初始培养基组合) The increase factor.

6. The culture medium optimization method according to claim 1, characterized in that, The effectiveness of the optimized culture medium combination was evaluated using cell culture data from the optimized culture medium combination. Optionally, the culture data includes physiological data of at least one cell type; Optionally, the culture data includes component concentration and / or protein expression data; Optionally, during an optimization process, the culture data includes a percentage increase in protein expression level, which is the percentage of the difference between the protein expression level of the initial culture medium combination and the protein expression level of the optimized culture medium combination, expressed as a percentage of the protein expression level of the initial culture medium combination. Optionally, the culture process for the optimized culture medium combination cell culture is the same as the culture process for the initial culture medium that forms the basis for the current optimized culture medium combination.

7. The culture medium optimization method according to claim 1, characterized in that, The fed culture medium is used to culture cells in a fed-batch culture manner; Optionally, the first threshold is at least 80%; Optionally, the first threshold is 80% to 95%; Optionally, the ingredients include one or more of the following: sugars, amino acids, organic acids, amines, vitamins, inorganic salts, metal ions, lipids, buffer reagents, and culture medium additives; Optionally, the cells include mammalian cells; Optionally, the mammalian cells include CHO cells, hybridoma cells, SH87 cells, BHK cells, COS cells, VERO cells, HeLa cells, 293 cells, PER-C6 cells, K562 cells, MOLT-4 cells, M1 cells, NS-1 cells, COS-7 cells, MDBK cells, MDCK cells, MRC-5 cells, WI-38 cells, WEHI cells, SP2 / 0 cells, CAP cells, AGE1.HN cells, and their derivative cells; Optionally, the cells express heterologous proteins; Optionally, the heterologous protein includes an antibody.

8. The method for optimizing culture media according to any one of claims 1 to 7, characterized in that, Includes the following steps: (A1) Obtain the component to be optimized from the initial culture medium combination; (A2) Increase the content of the component to be optimized in the initial culture medium combination in step (A1) in at least one manner to obtain at least one optimized culture medium combination; (A3) Use the optimized culture medium combination obtained in step (A2) to culture cells, and obtain a number of culture data for each optimized culture medium combination. If the expected number of culture data reaches the corresponding threshold range, the optimized culture medium combination that best corresponds to the culture data is taken as the optimized culture medium combination and the optimization is completed; otherwise, proceed to (A4). (A4) Take the optimal culture medium combination corresponding to the best culture data in step (A3) as the initial culture medium combination in step (A1), and repeat (A1) to (A4) until the expected number of culture data reaches the corresponding threshold range, and / or until the culture data obtained no longer reflects the culture effect. Take the optimal culture medium combination corresponding to the best culture data as the optimized culture medium combination, and complete the optimization. When step (A1) is performed for the first time, the initial culture medium combination is the culture medium combination to be optimized; Optionally, in a certain execution (A1) to (A4), the additives obtained according to the additive formulation design are directly added to the initial culture medium combination in step (A1) to obtain the optimized culture medium for that step (A2); Optionally, the addition value ΔC and / or the distribution method of the additive formulation in step (A2) are different from the additive formulation in the previously performed step (A2).

9. A culture medium optimization system, characterized in that, The culture medium optimization system is used to implement the culture medium optimization method according to any one of claims 1 to 8, and includes a data acquisition module and a first data analysis module; The data acquisition module is used to acquire the following data: C (上清) C (基) C (补，Feedi) and V (补，Feedi) And, set at least one first threshold; The first data analysis module is used to execute formulas (a1) and (a2), match the calculation results with the first threshold, and output the content of all components to be optimized corresponding to each first threshold; Optionally, the culture medium optimization system further includes a second data analysis module, which performs the additive formulation design; Optionally, the culture medium optimization system further includes a third data analysis module, which is used to match the several culture data obtained after cell culture in the optimized culture medium with a threshold to determine whether the optimization is complete. Optionally, the culture medium optimization system also includes a component detection device.

10. The application of the culture medium optimization method according to any one of claims 1 to 8, or the culture medium optimization system according to claim 9, in cell culture, preparation of culture medium, or preparation of protein; Optionally, the preparation of the protein includes the preparation of an antibody.

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