Cell Culture Medium Testing Methods and Their Applications in Biopharmaceuticals

Abstract

The present invention provides a method for testing cell culture media and its application in biopharmaceuticals. The present invention has established a method for testing culture media in the field of biopharmaceuticals, which can comprehensively and effectively detect culture media from different sources (such as commercial cell culture media or laboratory-prepared culture media, etc.), conduct quality analysis / stability analysis on them, effectively ensure that the performance of large-scale production processes and the consistency of product quality are not affected by differences between culture media batches, and increase the stability of biopharmaceutical production.

Description

Technical Field

[0001] The present invention belongs to the fields of detection technology and biopharmaceuticals, and particularly relates to a method for testing cell culture media and its application in biopharmaceuticals. Background Art

[0002] Cells, as carriers for product expression, are increasingly used in modern biopharmaceutical production. Among them, mammalian cells are the main hosts in industrial biopharmaceutical production. Mammalian cells can produce diverse, correctly folded, and glycosylated proteins. Complex proteins need to undergo correct post-translational modifications to have drug functions and non-immunogenicity. The growth and production of cells are inseparable from cell culture media, so the quality control of cell culture media is quite important for biopharmaceutical production.

[0003] In modern biopharmaceutical production, cell culture media that are animal-free and chemically defined are generally used to culture cells. The components of cell culture media are very complex, containing carbohydrates, nitrogenous substances, inorganic salts (including trace elements), vitamins, buffer systems, lipids, protective agents, etc. Although the components of commercialized culture media are generally formulated according to standards, differences between different batches can still lead to abnormal cell growth and even further affect the quality of biological products.

[0004] Compared with traditional adherent culture, cell suspension culture can obtain a higher cell density (an order of magnitude higher). The cell density in adherent culture is at the level of 10 5 cell / mL (cells per milliliter), and suspension culture can reach 10 6 cell / mL level, and even can reach 10 7 cells / mL or higher in fed-batch culture mode. Corresponding to single-batch production, more output can be obtained. However, a higher cell density means higher requirements for cell culture conditions, and more stable and sufficient nutrient components are needed to support the growth of high-density cells and the production of high protein amounts.

[0005] According to the "Cell Culture Medium Standards and Detection Methods" published by the Chinese Pharmaceutical Biotechnology Association in 2011, the detection items for detecting the quality standards of cell culture media include clarity, pH value, osmotic pressure, mass fraction of drying loss, bacterial endotoxin, biological limit, cell growth experiment, bovine serum albumin, and antibiotics. These detection items are all based on the detection methods of the Chinese Pharmacopoeia and the Chinese industry standard "Mammalian Cell Culture Medium". Due to the rather complex components of cell culture media, detecting chemical and physical properties such as clarity and pH cannot fully reflect the quality and stability of the culture medium. And in the industry standard, the cell growth experiment uses Vero cells, the culture method is adherent culture, and the required standard is that there are no abnormal morphological variations in the cells, and the cell density is not lower than 1×10 5cells / mL. After continuous culturing for 48 hours, the cell count is not less than 1×10 5 cells / mL (the ability to maintain cell growth is examined here). With the widespread use of high cell density culture in modern biopharmaceuticals, this method is no longer applicable. On the one hand, the cell line in this experiment is less representative. On the other hand, in actual production, the cell culture method is suspension culture, and the cell density is much higher than the requirements of traditional detection methods.

[0006] The test certificate of the culture medium provided by the culture medium supplier is similar to the standards in the "Cell Culture Medium Standards and Detection Methods". However, since the formula of the culture medium provided by the supplier often involves patent protection or technical secrets, for technicians who purchase the culture medium, the standards of specific nutrients cannot be provided. The growth promotion experiment provided by the supplier is also limited to the supplier's own cell line, and its acceptance standard also has a certain gap from the cell density requirements supported by the culture medium in actual large-scale production.

[0007] In view of the above, it is necessary in the art to study quality control methods for commercial culture media or prefabricated culture media to make the production based on the culture medium more stable and efficient, in order to reduce the occurrence of events with low production efficiency in culture / fermentation. Summary of the Invention

[0008] The object of the present invention is to provide a method for testing cell culture media and its application in biopharmaceuticals.

[0009] In a first aspect of the present invention, a method for analyzing the quality of a culture medium is provided, including: (1) analyzing the physical and chemical properties of the culture medium to be tested, and analyzing three physical and chemical property indicators thereof: pH value, turbidity, and osmotic pressure; determining the culture medium that meets the three physical and chemical property indicators and the culture medium that does not meet the three physical and chemical property indicators; (2) for the culture medium obtained in (1) that meets the three physical and chemical property indicators, analyzing four biochemical indicators and six metal element (trace metal ions) indicators; the four biochemical indicators are: glutamine, glucose, potassium ions, and sodium ions; the six metal element indicators are: calcium ions, copper ions, iron ions, magnesium ions, manganese ions, and zinc ions; determining the culture medium that meets the four biochemical indicators and the six metal element indicators, which is a qualified culture medium; determining the culture medium that does not meet the four biochemical indicators and the six metal element indicators, which is an unqualified culture medium; wherein, the qualified culture medium is a culture medium with stable function and suitable for cell growth promotion.

[0010] In another aspect of the present invention, a method for directionally selecting a medium with stable function and suitable for cell growth promotion is provided, including: (1') analyzing the physical and chemical properties of the medium to be tested, and analyzing three indicators of its physical and chemical properties: pH value, turbidity, and osmotic pressure; selecting a medium that meets the three indicators of physical and chemical properties; (2') for the medium obtained in (1') that meets the three indicators of physical and chemical properties, analyzing four biochemical indicators and six metal element indicators; the four biochemical indicators are: glutamine, glucose, potassium ion, and sodium ion; the six metal element indicators are: calcium ion, copper ion, iron ion, magnesium ion, manganese ion, and zinc ion; determining a medium that meets the four biochemical indicators and six metal element indicators, which is a medium that can stably and is suitable for cell growth promotion.

[0011] In one or more embodiments, in the medium, the three indicators of physical and chemical properties meet the following ranges:

[0012] pH: 6.90 - 7.55;

[0013] Turbidity: < 4.00 NTU;

[0014] Osmotic pressure: 300 - 340 mOsm / kg.

[0015] In one or more embodiments, in the medium, the four biochemical indicators meet the following ranges:

[0016]

[0017] In one or more embodiments, in the medium, the six metal element indicators meet the following ranges:

[0018]

[0019] In one or more embodiments, when performing the above analysis or the directional selection using a shake flask culture and a multi-stage seed medium subculture mode; preferably, the multi-stage seed culture and subculture include: a resuscitation stage and 1 - 5 times (such as 1 - 1 time, 1 - 2 times, 1 - 4 times, 1 - 6 times, 1 - 7 times) of subculture; preferably, the multi-stage is five-stage, including a resuscitation stage and 1 - 4 times of subculture; preferably, the shake flask culture includes a shake flask culture in a suspension oscillation form.

[0020] In another aspect of the present invention, a method for culturing cells or using the cells for protein production is provided, including: (a) directionally selecting a medium with stable function and suitable for cell growth promotion by any of the methods described above; (b) culturing cells using the medium in (a) or using the cells for protein production.

[0021] In one or more embodiments, the cells include: cells into which an exogenous gene has been introduced (expressing / producing a protein) or cells into which no exogenous gene has been introduced (undergoing cell proliferation).

[0022] In one or more embodiments, the cells include: CHO cells; for example, the cells are the CHO-K1 cell line.

[0023] In one or more embodiments, the culture medium includes: a serum-free and / or chemically defined culture medium.

[0024] In one or more embodiments, the culture medium includes: a commercial culture medium or a culture medium prepared in the laboratory.

[0025] In one or more embodiments, the culture medium includes: a CHO culture medium, such as but not limited to ActiPro medium.

[0026] In one or more embodiments, the biochemical parameters of the culture medium are detected by a blood gas analyzer (BGA) and / or a biochemical analyzer (Cedex); the biochemical parameters include four biochemical indicators.

[0027] In one or more embodiments, the metal elements (trace metal ions) in the culture medium are detected by an inductively coupled plasma mass spectrometer (ICP-MS); the metal elements include: calcium ions, copper ions, iron ions, magnesium ions, manganese ions, and zinc ions.

[0028] In another aspect of the present invention, there is provided the use of any of the methods described above for quality control of a culture medium; preferably, for quality analysis of a culture medium used for cell culture or protein drug production, excluding unqualified culture media and selecting qualified culture media.

[0029] In another aspect of the present invention, there is provided a system (such as a device) for quality control of a culture medium, which includes the following detection unit and data analysis unit:

[0030] (i) Detection unit 1 and data analysis unit 1; the detection unit 1 is provided with a component for measuring three physicochemical property indicators (such as an instrument or a device), and the three physicochemical property indicators are pH value, turbidity, and osmotic pressure; the data analysis unit 1 includes a processing unit for analyzing and processing the measurement results of the detection unit 1 and outputs a determination result of the three physicochemical property indicators;

[0031] (ii) Detection unit 2 and data analysis unit 2; the detection unit 2 is provided with a component for measuring four biochemical indicators, and the four biochemical indicators are glutamine, glucose, potassium ions, and sodium ions; the data analysis unit 2 includes a processing unit for analyzing and processing the measurement results of the detection unit 2 and outputs a determination result of the four biochemical indicators;

[0032] (iii) Detection unit 3 and data analysis unit 3; the detection unit 3 is provided with a six-index determination component for metal elements; wherein the six indexes of metal elements are calcium ions, copper ions, iron ions, magnesium ions, manganese ions and zinc ions; the data analysis unit 3 includes a processing unit for analyzing and processing the determination results of the detection unit 3, and outputs a determination result of the six indexes of metal elements.

[0033] In one or more embodiments, the three-index range values of physical and chemical properties are preset in (i). If it is within the range value, it is determined as qualified; if it is outside the range value, it is determined as unqualified:

[0034] pH: 6.90 - 7.55;

[0035] Turbidity: < 4.00 NTU;

[0036] Osmotic pressure: 300 - 340 mOsm / kg.

[0037] In one or more embodiments, the four-index range values of biochemical indexes are preset in (ii). If it is within the range value, it is determined as qualified; if it is outside the range value, it is determined as unqualified:

[0038]

[0039] In one or more embodiments, the six-index range values of metal elements are preset in (iii). If it is within the range value, it is determined as qualified; if it is outside the range value, it is determined as unqualified:

[0040]

[0041] In one or more embodiments, when (i) is determined as unqualified, (ii) and (iii) are not carried out; when (i) is determined as qualified, (ii) and (iii) are carried out; when either (ii) or (iii) is determined as unqualified, the culture medium is an unqualified culture medium; when both (ii) and (iii) are determined as qualified, the culture medium is a qualified culture medium;

[0042] In one or more embodiments, the system further includes: (iv) data analysis unit 4, which is used to summarize the determination results of (i), (ii) or (iii), and outputs a conclusion that the culture medium is an unqualified culture medium or a qualified culture medium.

[0043] Other aspects of the present invention will be obvious to those skilled in the art from the disclosure herein. Description of the Drawings

[0044] Figure 1, in the early stage, a large number of culture media were analyzed, long-term data was summarized and optimized, and a flowchart for establishing acceptance criteria for test results was formed, which was divided into detection of culture medium components, detection of culture medium preparation properties, and cell growth promotion experiment detection, and the acceptance criteria for each culture medium were formed based on long-term data accumulation.

[0045] Figure 2 , the measured contents of biochemical parameters and metal elements of multiple batches of culture media.

[0046] Figure 3 , a schematic flow chart of the cell small-scale model culture process (shake flask) in the cell growth promotion experiment. There are a total of five generations of seed chain stages and one generation of production stage as a scaled-down model corresponding to large-scale production.

[0047] Figure 4 , a graph showing the change of viable cell density (VCD) and cell viability over time in the N-2 to N-1 stages during growth promotion analysis.

[0048] Figure 5A -P, a graph showing the change of viable cell density over time in the production stage during cell screening. Through the selection process of Examples 2-3, the culture media confirmed to be qualified can all achieve a growth promotion effect close to or even exceeding that of the positive control. Detailed implementation mode

[0049] The inventor of the present invention, through in-depth preliminary analysis and research, has first established a method for testing culture media in the field of biopharmaceuticals. The culture media can be animal-free and chemically defined culture media. The technical solution of the present invention can comprehensively and effectively detect culture media from different sources (such as commercial cell culture media or laboratory-prepared culture media, etc.), conduct quality analysis / stability analysis on them, and can effectively ensure that the performance of large-scale production processes and the consistency of product quality are not affected by differences between culture medium batches, increasing the stability of biopharmaceutical production.

[0050] As used in the present invention, the "analysis" includes "detection", "measurement", "evaluation", "prediction" or "assessment", and these terms can be used interchangeably.

[0051] As used in the present invention, "cell culture" or "culture" refers to maintaining or proliferating cells in an artificial (e.g., in vitro) environment.

[0052] As used in the present invention, "cultivate / culture" refers to maintaining or proliferating cells in an artificial environment under conditions conducive to cell growth, differentiation or continuous survival. Therefore, "cultivate / culture" can be used interchangeably with "cell culture" or any of its above synonyms.

[0053] As used herein, "cell culture medium" or "culture medium" refers to a nutrient composition that supports the culture and / or growth of cells.

[0054] As used herein, the term "ingredient" refers to any substance that can be used in a cell culture medium to maintain or promote the growth or proliferation of cells, which can include substances of chemical or biological origin. The terms "constituent", "nutrient" and "ingredient" are used interchangeably and all refer to such compounds. Common ingredients that can be used in cell culture media include amino acids, salts, metals, sugars, carbohydrates, lipids, nucleic acids, hormones, vitamins, fatty acids, proteins, etc.

[0055] As used in the present invention, the terms "index" or "parameter" are used interchangeably, unless otherwise specified, and refer to the physical and chemical properties, biochemical indices, and metal element indices of interest pointed out in the present invention. In the present invention, their suitable ranges (values) are determined.

[0056] As used in the present invention, the "qualified medium" or "medium meeting the requirements" refers to a medium that can provide a stable environment for cell growth, give a stable nutrient supply, and there is no unstable situation in the level of nutrient components.

[0057] As used in the present invention, the "unqualified medium" or "medium not meeting the requirements" refers to a medium that is insufficient to provide a stable environment for cell growth, the nutrient supply is not stable, and there is an unstable situation in the level of nutrient components. This medium can also be a commercial medium or a pre-prepared medium. Those skilled in the art understand that due to different quality control standards or factors such as improper storage and transportation, commercial media also need to be further subjected to more precise quality control.

[0058] As used in the present invention, the setting of the "control" or "threshold" is easily set by those skilled in the art based on the gist of the present invention. Selecting a suitable "control" or "threshold" is a routine part of experimental design. For example, according to the research results of multiple batches, a medium with an ideal cell growth promoting effect is selected as the positive control.

[0059] Based on the accumulation and analysis of the results of a large number of cell culture experiments, the present inventor has established a method for testing culture media in the field of biopharmaceuticals. First, the present invention monitors the physical and chemical properties of the prepared culture media, including turbidity, pH value, and osmotic pressure; second, the present invention detects the nutrient components and concentrations in cell culture media, including biochemical parameters and the concentrations of trace metal ions, making up for the defect that conventional detection cannot monitor the nutrient components of commercial culture media; third, the present invention establishes a reasonable cell growth promotion experiment strategy according to actual production needs, which can solve the problem that the cells and culture processes used for growth testing in the release of supplier culture media are not representative. Using the established method, the present inventor has investigated multiple batches of commercial culture media and analyzed the impact of batch-to-batch differences on the stability of biopharmaceutical production. The results show that the method of the present invention can effectively avoid and solve the occurrence of such impacts.

[0060] Cell culture media contain many components, and such components can vary between different culture media. Although there are a large number of commercial culture media in the art, due to the fact that a large number of components are often integrated in the culture media, they still have instability. For example, when the present inventor conducts the culture of CHO cells and protein production, it is often found that there are nutritional shortfalls / defects in the culture media for promoting the growth of cell culture media.

[0061] Cell culture media components usually include carbohydrates, amino acids, salts, trace elements, and vitamins. For mammalian cells, the most common carbohydrate used in cell culture media is glucose. In addition to glucose, any hexose such as galactose, fructose, or mannose or a combination of such sugars can also be (but is not limited to) used.

[0062] The addition of amino acids in cell culture media is very important for maintaining the metabolic functions of cells. Cell culture media usually contain essential amino acids (i.e., those amino acids that usually cannot be synthesized by mammals in vivo) and certain non-essential amino acids. Non-essential amino acids are usually included in cell culture media if the cell line cannot synthesize the amino acid or if the cell line cannot produce sufficient amounts of the amino acid to support maximum growth.

[0063] The addition of salts in cell culture media can maintain isotonic conditions and prevent osmotic imbalance. The desired osmolarity of cell culture media for culturing specific cell types often needs to be determined by those skilled in the art based on experience.

[0064] Sometimes vitamins are used by cells as cofactors. Exemplary vitamins include biotin, choline chloride, folic acid, i-inositol, nicotinamide, pyridoxal, riboflavin, thiamine, pyridoxine, nicotinamide, A, B6, B12, C, D3, E, K, and p-aminobenzoic acid (PABA).

[0065] To promote cell growth in media lacking serum or with reduced serum, one or more growth factors are sometimes added to the media: for example, fibroblast growth factors (FGFs), including acidic FGF and basic FGF, insulin, insulin-like growth factors (IGFs), epidermal growth factor (EGF), nerve growth factor (NGF), platelet-derived growth factor (PDGF), and transforming growth factors (TGFs), including TGFα and TGFβ, any cytokine such as interleukin 1, 2, 6, granulocyte-stimulating factor, leukocyte inhibitory factor (LIF), etc. In certain embodiments, the cell culture media does not contain growth factors.

[0066] One or more lipids may also be added to the cell culture media, such as fatty acids, such as linoleic acid, linolenic acid, arachidonic acid, palmitoleic acid, oleic acid, polyunsaturated fatty acids, and / or fatty acids, phospholipids, lecithin, and cholesterol. In certain embodiments, the cell culture media does not contain lipids.

[0067] The cell culture media may optionally include one or more buffers. Suitable buffers are those that provide buffering capacity without significant cytotoxicity to the cultured cells.

[0068] The cell culture media can be serum-free media, and the serum can be replaced with defined hormones or a mixture of hormones such as HITES or ITES (which contains hydrocortisone, insulin, transferrin, ethanolamine, and selenite). Alternatively, the serum-free media may contain growth factor extracts from endocrine glands, such as epidermal growth factor or fibroblast growth factor. The serum-free media may also contain other components as substitutes for serum, including purified proteins (animal or recombinant), peptones, amino acids, inorganic salts, and animal or plant hydrolysates (or fractions thereof). The serum-free media can be chemically defined or undefined. In a chemically defined media, the identity and amount of the components are known, whereas for an undefined media, the opposite is true.

[0069] The serum-free media contains a reduced amount of protein compared to serum-containing cell culture media.

[0070] In a preferred embodiment of the present invention, the media is a media free of animal-derived components and / or chemically defined media. The media can include: commercially available media, or media prepared in the laboratory.

[0071] In a more preferred embodiment, the media includes: CHO media, such as but not limited to ActiPro media.

[0072] ActiPro medium is a chemically defined medium that contains no animal-derived components, no proteins, protein hydrolysates or peptides, and no insulin or growth factors. ActiPro medium is used for suspension culture of CHO cells at different scales and can be used for culturing at various culture scales, including large-scale production.

[0073] The cells using the medium described herein can be established cell lines and their strains, such as CHO cells, 293 embryonic kidney cells, BHK cells, HeLa cervical epithelial cells, MDCK cells, etc. In a preferred embodiment, the cells include: CHO cells; more preferably, the cell line is CHO-K1 cells.

[0074] The cells using the medium described herein can include: cells into which a foreign gene has been introduced (expressing / producing proteins) or cells into which no foreign gene has been introduced (for cell proliferation).

[0075] Except for the (interested) indicators / parameters specified in the present invention, other medium components or cell culture methods can be those known in the art. Conventional experiments can be used to determine the optimal seeding and culture conditions for a given animal cell type.

[0076] Organic substances and trace metal elements, such as copper ions, zinc ions, iron ions, etc., are closely related to the growth and metabolism of cells, but their contents in the medium are often low, for example, at the level of micrograms per liter (μg / L). Such organic substances and trace metal element contents are not emphasized in the conventional medium detection and release standards in the art. However, the present inventors have found that the detection and quality control of such organic substances and trace elements are necessary, and improving their quality control greatly helps large-scale cell culture or production.

[0077] In a preferred embodiment of the present invention, the data of the medium growth promotion experiment are summarized, and a cell growth promotion database and acceptance criteria exclusive to the medium are established through statistical analysis. Establishing a perfect database for medium testing, a standardized testing process and acceptance criteria to comprehensively monitor the quality and performance of the medium can better guide the use of commercial media in production.

[0078] In a preferred embodiment of the present invention, the present inventors comprehensively detect cell culture media for modern biopharmaceuticals from three aspects:

[0079] Detection method 1 - Physical and chemical properties of the medium: The present invention will conduct preliminary tests from the aspects of pH, osmotic pressure, turbidity, that is, the apparent physical and chemical properties of the medium, according to the production release standards of the medium formulation.

[0080] Detection method 2 - Analysis of the nutritional components of the culture medium: The biochemical parameters of the culture medium, including glutamine, glucose, sodium, and potassium ions, are detected by a blood gas analyzer (BGA) and a biochemical analyzer (Cedex); trace metal ions, including calcium ions, copper ions, iron ions, magnesium ions, manganese ions, and zinc ions, are detected by ICP-MS (inductively coupled plasma mass spectrometry). Since the contents of the biochemical parameters and trace metal ions can be regarded as the specific manifestations of all the nutritional components of the culture medium, method 2 reflects the quality attributes of the culture medium from the perspective of nutritional components. By using the above-mentioned highly sensitive and broad-spectrum detection means and combining statistical analysis methods (mean ± 3x standard deviation / mean ± 3SD or 95%-99% tolerance interval / 95-99% TI), method 2 establishes a database of the nutritional components of the culture medium and acceptance criteria. In addition, as a double verification of method 3, method 2 can deepen the understanding of the nutritional components of the culture medium and the causes of abnormalities.

[0081] Detection method 3 - Cell growth promotion experiment: Using this experiment, the inventor verified the growth promotion of the selected culture medium.

[0082] At the same time, the inventor also screened cell line A with stable growth and sensitive to batch differences of the culture medium from the CHO basic cell line library for commercial production (safe, clear genetic background, and gene stability) (derived from clone X cell line in the patent WO2020088180A1 "A Process for Producing Biologics from Cell Cultures and a System for Practicing" publicly reported by WuXi Biologics), and selected suspension shaking culture and five-stage seed passage process. Combining the requirements for important parameters of cell growth (cell density and viability) in commercial production, a scientific cell growth promotion detection system was established.

[0083] The present invention comprehensively detects the culture medium through the above three aspects, and establishes a database for the exclusive cell culture medium and acceptance criteria for the corresponding methods. This invention is closer to the actual needs in biopharmaceutical production and can prevent the impact of batch-to-batch fluctuations in the nutritional components of the culture medium on product quality and process performance.

[0084] In a preferred embodiment of the present invention, the technical solution of the present invention can be applied to the detection field of animal-free and chemically defined cell culture media in biopharmaceuticals. It not only improves the existing methods for detecting culture media, but also through comprehensive detection of the culture media, the quality of the culture media used in biopharmaceutical production is comprehensively and well controlled, effectively avoiding and reducing large-scale production deviations caused by the culture media, and making the production more stable. In the long run, the formed exclusive culture media control strategy can better identify the differences in components between specific culture media batches and guide the use of culture media in production, which is of great significance for production stability.

[0085] The following will further illustrate the present invention in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are usually carried out under conventional conditions or according to the conditions recommended by the manufacturer.

[0086] Biomaterials and culture reagents

[0087] The cell line used in the embodiments of the present invention is the CHO-K1 cell line (see the publicly disclosed patent WO2020088180A1; the clone X cell strain in "A Process for Producing Biologics from Cell Cultures and a System for Practicing").

[0088] In the embodiments of the present invention, the test culture medium is a commercial ActiPro culture medium, purchased from Cytiva.

[0089] Example 1: Establishment of a culture medium quality control scheme

[0090] Commercially purchased CHO culture media or pre-prepared culture media often deteriorate or denature when actually applied to cell culture due to various reasons such as improper storage, contamination, transportation, and deterioration of unstable biological / chemical substances, resulting in the inability to efficiently achieve cell growth or production, or even overall contamination or production failure. Since the cost of large-scale protein production is expensive and time-consuming, when there are unstable factors in the culture medium, it will cause greater losses, and the cleaning and washing are time-consuming and laborious when the whole tank is contaminated.

[0091] In order to avoid such problems from the source, for commercially purchased CHO culture media or pre-prepared culture media, the inventors have carried out quality analysis applicable to culture media through long-term quality observation, analysis, and experimental verification. The analysis process is at least as Figure 1 shown.

[0092] After in-depth comprehensive analysis and long-term accumulation of monitoring data, the inventor has obtained a reliable culture medium analysis scheme. Based on this analysis scheme, a culture medium with stable quality, capable of efficiently promoting cell growth and production can be obtained.

[0093] This scheme includes the following steps carried out in sequence:

[0094] (a) Analysis of the physical and chemical properties of the culture medium

[0095] The analysis indicators include: pH, turbidity, and osmotic pressure, and their quality control range values are shown in Table 1.

[0096] Table 1

[0097]

[0098]

[0099] (b) Analysis of the nutrient components of the culture medium

[0100] Through long-term accumulation of monitoring data, the inventor has established a database of the contents of organic substances and trace metal elements in ActiPrio culture medium, and analyzed the substances / elements that can reflect the overall quality of the culture medium. The biochemical parameters of the culture medium are detected by a blood gas analyzer (BGA) and a biochemical tester (Cedex); the trace metal ions are detected by ICP-MS (inductively coupled plasma mass spectrometer). During the analysis, the acceptance criteria are calculated by mean ± 3x standard deviation / mean ± 3SD or 95 - 99% tolerance interval / 95 - 99% TI.

[0101] Tables 2 and 3 show the determined nutrient components, which are the nutrient component indicators representing the overall quality of the culture medium. Monitoring only these indicators can reflect the overall quality of the culture medium, including: biochemical indicators: glucose, glutamine, sodium ions, and potassium ions; trace metal elements: calcium, copper, iron, magnesium, manganese, and zinc. Their quality control range values are listed in Tables 2 and 3 respectively.

[0102] Table 2

[0103] Biochemical index Range value Glutamine mmol / L 3.63-5.56 Glucose g / L 5.27-7.05 Potassium ion mmol / L 9.44-11.20 Sodium ion mmol / L 109-119

[0104] Table 3

[0105] Metal element index Range value Calcium ion mg / L 10.3-26.0 Copper ion μg / L 16.9-71.8 Iron ion mg / L 47.8-129.8 Magnesium ion mg / L 7.7-31.2 Manganese ion μg / L 17.1-69.0 Zinc ion μg / L 167.7-461.3

[0106] It is well-known to those skilled in the art that there are up to about 100 types of various nutrient components / elements in CHO culture medium. However, the inventor has found that the above indicators are sufficient to reflect the quality of the culture medium.

[0107] Example 2. Analysis of the physical and chemical properties of the culture medium

[0108] For the test medium, the inventors first analyzed the physical and chemical properties of the medium. The test medium was: preparation and solution property testing of 1 kg of ActiPro medium solution.

[0109] For the ActiPro powder of the first batch, the testing method was as follows:

[0110] (1) Weigh 901.80 g of purified water and add it to the preparation container;

[0111] (2) Weigh 22.37 g of the medium powder and add it to the preparation container in step (1);

[0112] (3) Mix for at least 30 min until completely dissolved. Confirm that there are no insoluble particles and impurities, measure the pH value, and according to the measured pH value, use 10 N sodium hydroxide or 6 N hydrochloric acid to adjust the pH value of the medium to 7.45 (measured with a pH meter), add purified water to make up to 1 kg, and mix again;

[0113] (4) Filter through a 0.1 μm filter into a suitable sterile storage container to prepare a liquid medium;

[0114] (5) Conduct solution property testing: The final measured pH was 7.50 (the pH changed slightly after volume fixation), the turbidity was 0.37 NTU, and the osmotic pressure value was 309 mOsm / kg, which was within the acceptable range. When the pH, turbidity, and osmotic pressure ranges in Table 1 above were met, the physical and chemical properties of the test medium were considered qualified.

[0115] For other multiple batches of ActiPro powder to be tested, repeat steps 1 - 5 to complete the preparation and performance testing of the corresponding batches of the medium. Among them, the range of purified water in step (1) can be 882.00 - 918.00 g; the weighed amount of the medium powder in step (2) can be 21.91 - 22.81 g; the pH adjustment range in step (3) can be 7.20 - 7.45.

[0116] Example 3. Analysis of the nutritional components of the medium

[0117] In this example, for the test medium identified as having qualified physical and chemical properties in the previous Example 1, nutritional component analysis was carried out.

[0118] Based on the medium solution prepared and analyzed in Example 1, two 5 - ml samples were taken from each batch of the solution to detect the biochemical parameters and the content of trace metal elements.

[0119] The biochemical parameters of the medium, including glutamine, glucose, sodium ions, and potassium ions, were detected by a blood gas analyzer (BGA) and a biochemical analyzer (Cedex).

[0120] Detect trace metal ions, including calcium ions, copper ions, iron ions, magnesium ions, manganese ions and zinc ions, by ICP-MS (Inductively Coupled Plasma Mass Spectrometry).

[0121] After analyzing the biochemical parameters and metal ions of multiple batches of the culture medium identified as qualified in terms of physical and chemical properties in Example 1, batches meeting the standards in Tables 2 and 3 (considered to have stable quality and capable of achieving high-density cell growth) and batches not meeting the standards (considered to have unstable quality and unable to support cell growth to high cell density) were distinguished.

[0122] As a result, the present inventors obtained 8 batches of culture media with biochemical parameter and metal element indicators meeting the standards in Tables 2 and 3 from multiple batches of test culture media ( Figure 2 , referred to as Test Batches 1-8), expecting them to have good culture effects and ideal growth-promoting effects (qualified); at the same time, excluding the culture media with unsatisfactory expected effects that do not meet Tables 2 and 3. Figure 2 Taking the example of the "negative batch" mentioned in

[0123] Furthermore, they were used for subsequent tests and verifications.

[0124] Example 4, Verification of Cell Growth-Promoting Analysis

[0125] In this example, the effects of the qualified culture media distinguished in Example 2 in cell culture were verified.

[0126] The experimental steps are as follows:

[0127] (1) Using the CHO-K1 cell line as the cells to be cultured, perform growth-promoting analysis on multiple batches of the test ActiPro culture medium preliminarily determined in Example 2 above. At the same time, set aside positive (culture media that have been experimentally verified to be able to effectively achieve the growth of CHO cells to high density, meeting the target values in Table 4) and negative batches (culture media that cannot support the growth of CHO cells to high cell density and do not meet the target values in Table 4) of culture media.

[0128] (2) The process flow diagrams of the seed chain and production stages are shown in Figure 3 and Table 4. Use a small-scale model in the form of a shake flask to simulate the large-scale production process, that is, simulate the actual production situation. There are a total of five generations of the seed chain stage (resuscitation and N-1 to N-4 stages) and one generation of the production stage as a scaled-down model corresponding to the large-scale production. Subculture is achieved by calculating the appropriate seeding volume and adding the seeds to fresh culture medium and culture containers.

[0129] Table 4, Model Culture Process Flow and Preset Target Values

[0130]

[0131]

[0132] Among them, CD (Chemical Defined) CHO is a kind of CHO culture medium used for cell resuscitation and early cell expansion. Its nutrient components are simpler than those of ActiPro, which is also part of simulating large-scale production.

[0133] (3) Passage according to the above passage process, and culture the CHO cell line using the above-mentioned multiple batches of culture media and the control culture medium respectively, where the negative batch (negative control) culture medium is used for the N-2 to N-1 stages.

[0134] (4) Collect data and conduct data analysis.

[0135] Compare the viable cell density at the N-2 to N-1 stages (3 days + 3 days respectively). The detection results of ActiPro test batches 1-4 are shown in Figure 4 , and the growth curves of ActiPro test batches 1-4 are very close to those of the positive control culture medium, but significantly different from those of the negative batch culture medium. It is proved that ActiPro test batches 1-4 culture media have good cell growth-promoting effects and are excellent culture media.

[0136] This shows that according to the selection processes of Example 1 and Example 2, culture media with stable quality and beneficial to cell growth can be analyzed and obtained therefrom; at the same time, culture media with poor quality and instability can be excluded.

[0137] According to Figure 4 The results can also show that there are significant differences between the positive control and the negative batch (negative control) of the CHO cell line. According to the comparison of the viable cell density at the N stage and the difference in the viable cell density of cell growth, it can be seen that the cell line has a high viable cell density. Therefore, the solution of the present invention is suitable for the quality analysis of the culture medium for CHO cells.

[0138] Taking the CHO cell line as the culture object, the viable cell density (taking the higher value of the two) and cell viability (taking the lower value of the two) of the qualified culture medium on the fifth or sixth day are shown in Table 5.

[0139] Table 5. Cell growth-promoting experiment

[0140]

[0141] Example 5. Analysis and verification of more batches of commercial ActiPro culture media

[0142] For other batches of commercial ActiPro culture media, the applicant also adopted the same scheme as in Examples 1-3 for analysis to obtain qualified culture media for CHO culture as in Example 4, and analyzed whether these qualified culture media were stable in promoting growth.

[0143] As Figure 5A -P, the results of multiple repeated experiments all showed that through the selection process of Examples 2-3, the culture media confirmed to be qualified had a viable cell density (VCD) that could increase significantly with the increase of the culture days and maintain excellent cell viability. And they could all achieve a growth-promoting effect close to or even exceeding that of the positive control. It can be seen that their nutrient components are stable, can provide a good nutrient environment for CHO, and effectively achieve growth promotion.

[0144] According to the above, the method of the present invention can comprehensively monitor the CHO culture medium, can better guide the use of the culture medium in production, and has important significance for stable production.

[0145] The above-described embodiments merely represent several implementation manners of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the patent of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the appended claims. At the same time, all the documents mentioned in the present invention are cited in this application as references, just as if each document is cited separately as a reference.

Claims

1. A method for analyzing the quality of a culture medium, comprising: (1) Analyze the physical and chemical properties of the culture medium to be tested, and analyze three physical and chemical property indicators thereof: pH value, turbidity, and osmotic pressure; determine the culture medium that meets the three physical and chemical property indicators and the culture medium that does not meet the three physical and chemical property indicators; the three physical and chemical property indicators meet the following ranges: pH: 6.90 - 7.55; Turbidity: < 4.00 NTU; Osmotic pressure: 300 - 340 mOsm / kg; (2) For the culture medium that meets the three physical and chemical property indicators obtained in (1), analyze four biochemical indicators and six metal element indicators; the four biochemical indicators are: glutamine, glucose, potassium ions, and sodium ions; the six metal element indicators are: calcium ions, copper ions, iron ions, magnesium ions, manganese ions, and zinc ions; the four biochemical indicators meet the following ranges: The six metal element indicators meet the following ranges: Determine the culture medium that meets the four biochemical indicators, six metal element indicators, and cell growth promotion test as a qualified culture medium; determine the culture medium that does not meet the four biochemical indicators, six metal element indicators, and cell growth promotion test as an unqualified culture medium; wherein, the qualified culture medium is a culture medium with stable function and suitable for cell growth promotion; the cell is a CHO cell.

2. A method for directionally selecting a culture medium with stable function and suitable for cell growth promotion, the cell being a CHO cell, the method comprising: (1’) Analyze the physical and chemical properties of the culture medium to be tested, and analyze three physical and chemical property indicators thereof: pH value, turbidity, and osmotic pressure; select the culture medium that meets the three physical and chemical property indicators; the three physical and chemical property indicators meet the following ranges: pH: 6.90 - 7.55; Turbidity: < 4.00 NTU; Osmotic pressure: 300 - 340 mOsm / kg; (2’) For the culture medium that meets the three physical and chemical property indicators obtained in (1’), analyze four biochemical indicators and six metal element indicators; the four biochemical indicators are: glutamine, glucose, potassium ions, and sodium ions; the six metal element indicators are: calcium ions, copper ions, iron ions, magnesium ions, manganese ions, and zinc ions; the four biochemical indicators meet the following ranges: The six metal element indicators meet the following ranges: Determine the culture medium that meets the four biochemical indicators and six metal element indicators as a culture medium that can stably and is suitable for cell growth promotion.

3. The method according to any one of claims 1 or 2, characterized in that, after determining that it meets the four biochemical indicators and six metal element indicators, it further includes: performing a cell growth promotion experiment, and analyzing the viable cell density and cell viability on the 5th or 6th day of culture: Viable cell density ≥ 14.3 x 10 6 cells / ml; Cell viability rate ≥ 93.8%.

4. The method according to any one of claims 1 or 2, characterized in that, Use the shake flask culture and multi-stage seed culture medium subculture mode for the said analysis or directional selection.

5. The method according to claim 4, characterized in that, The multi-stage seed culture and subculture include: a resuscitation stage and 1 - 5 subcultures.

6. The method according to claim 5, characterized in that, The multi-stage is five-stage, including a resuscitation stage and 1 - 4 subcultures.

7. The method according to claim 4, characterized in that, The shake flask culture includes shake flask culture in a suspension oscillation form.

8. The method according to any one of claims 1 or 2, characterized in that, the cells are CHO-K1 cell line.

9. The method according to any one of claims 1 or 2, characterized in that, the culture medium includes: a medium without animal-derived components and / or a chemically defined medium.

10. The method according to any one of claims 1 or 2, characterized in that, the culture medium includes: a commercial culture medium, or a culture medium prepared in the laboratory.

11. The method according to any one of claims 1 or 2, characterized in that, detecting the biochemical parameters of the culture medium with a blood gas analyzer and / or a biochemical analyzer; the biochemical parameters include four biochemical indicators.

12. The method according to any one of claims 1 or 2, characterized in that, detecting the metal elements in the culture medium with an inductively coupled plasma mass spectrometer; the metal elements include: calcium ions, copper ions, iron ions, magnesium ions, manganese ions and zinc ions.

13. The application of the method according to any one of claims 1-12, for quality control of the culture medium.

14. The application according to claim 13, characterized in that, conducting quality analysis on the culture medium for cell culture or protein drug production, excluding unqualified culture media, and selecting qualified culture media.

15. A system for quality control of the culture medium, which includes the following detection units and data analysis units: (i) Detection unit 1 and data analysis unit 1; the detection unit 1 is provided with a component for determining three physicochemical property indicators, and the three physicochemical property indicators are pH value, turbidity and osmotic pressure; the data analysis unit 1 includes a processing unit for analyzing and processing the measurement results of the detection unit 1, and outputs a determination result of the three physicochemical property indicators ； wherein the range values of the three physicochemical property indicators are preset, if within the range values, it is determined to be qualified, if outside the range values, it is determined to be unqualified: pH: 6.90 - 7.55; Turbidity: < 4.00 NTU; Osmotic pressure: 300 - 340 mOsm / kg; (ii) Detection unit 2 and data analysis unit 2; the detection unit 2 is provided with a component for determining four biochemical indicators, and the four biochemical indicators are glutamine, glucose, potassium ions and sodium ions; the data analysis unit 2 includes a processing unit for analyzing and processing the measurement results of the detection unit 2, and outputs a determination result of the four biochemical indicators; wherein the range values of the four biochemical indicators are preset, if within the range values, it is determined to be qualified, if outside the range values, it is determined to be unqualified: (iii) Detection unit 3 and data analysis unit 3; the detection unit 3 is provided with a component for determining six metal element indicators; wherein the six metal element indicators are calcium ions, copper ions, iron ions, magnesium ions, manganese ions and zinc ions; the data analysis unit 3 includes a processing unit for analyzing and processing the measurement results of the detection unit 3, and outputs a determination result of the six metal element indicators; wherein the range values of the six metal element indicators are preset, if within the range values, it is determined to be qualified, if outside the range values, it is determined to be unqualified:

16. The system according to claim 15, characterized in that, When (i) is determined to be unqualified, (ii) and (iii) are not carried out; when (i) is determined to be qualified, (ii) and (iii) are carried out; When either (ii) or (iii) is determined to be unqualified, the culture medium is an unqualified culture medium; when both (ii) and (iii) are determined to be qualified, the culture medium is a qualified culture medium.

17. The system according to claim 16, characterized in that the system further comprises: (iv) a data analysis unit 4, which is used to summarize the determination results of (i), (ii) or (iii) and output a conclusion that the culture medium is an unqualified culture medium or a qualified culture medium.

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