A HEK 293 cell culture additive, culture medium and its application

Abstract

The present application relates to the technical field of HEK 293 cell culture, in particular to a HEK 293 cell culture additive, a culture medium and application thereof.The additive is composed of HEK 293 cell culture additive A and HEK 293 cell culture additive B.The present application proposes an additive A for improving the density of HEK 293 cells, which is matched with commercial HEK 293 basic culture medium HE02, further matched with commercial HEK 293 feeding culture medium HA02, and the protein yield is further improved through additive B.

Description

Technical Field

[0001] This invention relates to the field of HEK 293 cell culture technology, specifically to an HEK 293 cell culture additive, culture medium, and their applications. Background Technology

[0002] Polyethyleneimine (PEI)-mediated transient gene expression (TGE) is a core technology in the biopharmaceutical field for producing recombinant proteins and gene therapy vectors (such as rAAV and VLP). It introduces exogenous genetic material (such as plasmid DNA) into eukaryotic cells, enabling rapid and high-level expression of the target protein within a short period (usually 3-7 days) through the cell's own transcription and translation mechanisms. However, its industrial application has long been limited by the cell density effect (CDE), meaning that transfection efficiency drops sharply with increasing HEK 293 cell density. Research indicates that at high cell densities, the bottleneck in transfection lies in the uptake and intracellular transport of the DNA / PEI polymeric complex, rather than plasmid expression. Furthermore, after cell culture, metabolites lead to Na+... + / Cl - Increased mediated ionic strength leads to decreased transfection efficiency, resulting in reduced yield.

[0003] Overcoming this bottleneck can be achieved through the following approaches: 1. Culture medium replacement: Replacing the culture medium with fresh medium before transfection is the most direct and effective method, quickly removing waste culture medium containing inhibitors. 2. Enhanced culture medium: Developing specialized culture medium formulations with stronger buffering capacity that can effectively resist changes in ionic strength during culture. 3. Transfection reagent development: Developing interference-resistant, highly penetrating molecules, cationic polymers, or cell-penetrating peptides to resist non-specific binding in the culture environment while enhancing membrane permeability, thereby bypassing inhibitory barriers and improving delivery efficiency.

[0004] There is currently no better way than the above methods. Culture medium replacement is the most effective, but it is not very practical. Centrifugation cannot be scaled up. Summary of the Invention

[0005] To address the technical problems mentioned in the background section, this invention provides an HEK 293 cell culture additive and its application. The technical solution is as follows: An HEK 293 cell culture additive A is composed of the following components: asparagine 80~800mg / L, lysine 30~300mg / L, acetylcysteine ​​15~150mg / L, insulin 0.1~1mg / L, retinoic acid 1~10mg / L, resveratrol 0.05~0.5mg / L, edaravone 2~20mg / L, hydrocortisone 0.001~0.01mg / L, and CHIR-99021 0.1~1mg / L.

[0006] A HEK 293 cell culture additive B is composed of the following components: putrescine dihydrochloride 2~20 mg / L, ethanolamine 20~200 mg / L, spermine 0.1~1 mg / L, curculigoside 0.05~0.5 mg / L, transferrin 10~100 mg / L, cholesterol 0.1~1 mg / L, and β-mercaptoethanol 0.1~1 mg / L.

[0007] Preferably, the HEK 293 cell culture additive A contains asparagine 400 mg / L, lysine 150 mg / L, acetylcysteine ​​75 mg / L, insulin 0.5 mg / L, retinoic acid 5 mg / L, resveratrol 0.25 mg / L, edaravone 10 mg / L, hydrocortisone 0.005 mg / L, and CHIR-99021 0.5 mg / L.

[0008] Preferably, the HEK 293 cell culture additive B contains 10 mg / L putrescine dihydrochloride, 100 mg / L ethanolamine, 1 mg / L spermine, 0.5 mg / L curculigoside, 100 mg / L transferrin, 1 mg / L cholesterol, and 1 mg / L β-mercaptoethanol.

[0009] A HEK 293 cell culture medium, comprising HEK 293 serum-free basal medium HE02 and HEK 293 serum-free supplemental medium HA02; The HEK 293 serum-free basal medium HE02 is supplemented with one of the aforementioned HEK 293 cell culture additives A, with an addition volume ratio of 10%.

[0010] The HEK 293 serum-free supplemented culture medium HA02 is supplemented with one of the aforementioned HEK 293 cell culture additives B, at a volume ratio of 10%.

[0011] According to any of the preceding descriptions, the HEK 293 cell culture is used for the culture of HEK293 cells.

[0012] HEK 293 cells were prepared and cultured in HEK 293 serum-free basal medium HE02 under the following shaker conditions: 37℃, 110 rpm, 8% CO2, 80% humidity, at a cell density of 0.6 × 10⁶ cells / year. 6 Cells were passaged at a density of 100 cells / mL every three days for three passages until they were stable. The cells were then cultured at a concentration of 0.6 × 10⁶ cells / mL. 6 Cells were seeded at a density of 1000 cells / mL in HEK293 serum-free basal medium HE02, with HEK 293 cell culture additive A added, and cultured in a shaker. The basal culture process for HEK 293 cells is as follows: S1 and HEK 293 cells were cultured at 1.1 × 10⁻⁶. 6 Cells per mL were seeded into 125 mL shake flasks, and cell count and viability were recorded on day 00, day 3 (D03), and day 5 (D05) after seeding.

[0013] The transfection process of HEK 293 cells is as follows: S2. The day before the transfection experiment, the cell density was controlled at 1.7 × 10⁻⁶. 6 Cell count / mL, cell count on the day of the experiment for future reference; S3. On the day of transfection, following the 20 mL shake flask culture system, adjust the volume of cells to 18 mL with fresh HEK 293 serum-free protein expression medium HE02, resulting in a total cell count of 60 × 10⁶ cells / mL. 6 Cell count / mL, cell density at transfection was 3.3 × 10⁻⁶. 6 about.

[0014] Preparation of PEI / DNA complex: In this protocol, the transfection system is 20 mL, and the final cell density is 3.0 × 10⁻⁶ cells / mL. 6 Cell count / mL, final DNA concentration 1.5 μg / mL, DNA:PEI = 1.5:4. The specific steps are as follows: PEI MAX dilution: Dilute 80 μg PEI MAX (Polysciences brand PEIMAX, catalog number 24765-1 used in this protocol) to 1 mL with HEK 293 serum-free protein expression medium HE02 and incubate at room temperature for 5 min.

[0015] DNA dilution: Dilute 30 μg of DNA to 1 mL with HEK 293 serum-free protein expression medium HE02 and incubate at room temperature for 5 min.

[0016] Mixing PEI with DNA: Add PEI MAX diluent to DNA diluent to form a PEI / DNA complex, mix well, and incubate at room temperature for 10 min.

[0017] S4. Slowly add the prepared 2 mL PEI / DNA complex to the culture system, and then transfer it to an incubator for further culture.

[0018] S5. About 16-24 hours after transfection, add 5% volume of HEK 293 serum-free feed medium HA02 (1 mL feed for every 20 mL of the system), add HEK 293 cell culture additive B, and supplement with 6 g / L glucose.

[0019] S6. On day 3 (D03) and day 5 (D05) after transfection, record the number of cells and their viability. On day 5, harvest the supernatant and detect the protein content.

[0020] The main beneficial effects of this invention are as follows: This invention proposes an additive A to increase the density of HEK 293 cells, which, when combined with commercial HEK 293 basal culture medium HE02 and further combined with commercial HEK 293 supplemental culture medium HA02, and through additive B, further increases the yield of protein (rabbit-derived IgG, which is a reference antibody protein and serves as a control positive plasmid).

[0021] Industrial customers are limited by the cell density effect (CDE). However, HEK 293 cell culture additive A, used in combination with retinoic acid, resveratrol, and curculigoside, can promote HEK 293 cell proliferation. During cell transfection, specifically in step S2, one day before the transfection experiment, the cell density is controlled at 1.7 × 10⁻⁶ cells / year. 6 Cell count / mL: Higher cell density on the day of the experiment allows for an increased proportion of fresh culture medium to be added, which can clear some inhibitors, reduce the cell density effect, and improve transfection efficiency. HEK 293 cell culture additive B promotes HEK 293 cell protein expression. The combination of curculigoside, transferrin, and β-mercaptoethanol, when added 16-24 hours after transfection (hereinafter referred to as D01), will not affect transfection efficiency, but can improve cell viability and provide a favorable environment.

[0022] Mechanism of action of the present invention: This application describes the development of a culture medium that can increase the density of HEK 293 cells by enhancing the culture medium. Increased cell density allows for the addition of more fresh culture medium during transfection, achieving a balance between the cell density effect and medium replacement. This application optimizes the culture medium for this purpose and further enhances yield by adding substances that promote protein expression in the later stages of transfection.

[0023] The transfection efficiency of HEK 293 cells is limited by the cell density effect (CDE), which is the phenomenon that transfection efficiency decreases sharply as HEK 293 cell density increases. The key to the cell density effect is the aggregation and fusion of membrane heparan sulfate proteoglycans (HSPGs) such as Glypican-4, which triggers the internalization of DNA / PEI polymeric complexes. However, the accumulation of extracellular free proteoglycans / glycosaminoglycans (GAGs) competitively inhibits CDE during transfection. Therefore, transfection can be promoted by adding more fresh culture medium. To add more fresh culture medium, a higher cell density needs to be achieved at the cell seeding stage.

[0024] HEK 293 cell culture additive A contains asparagine, lysine, acetylcysteine, and insulin, all key substances for cell growth. Asparagine, in particular, is not only a metabolic substrate but also an essential activator of mTORC1, a crucial node connecting nitrogen metabolism, nucleotide synthesis, protein translation, and cell signaling. However, its content in general culture media is slightly low, only enough to maintain the basic needs of cells. Supplementation with HEK 293 cell culture additive A, as a substrate, provides more energy substances. In addition, retinoic acid can bind to RAR / RXR nuclear receptors to regulate gene expression, promote proliferation and protein synthesis. Resveratrol can protect cells from oxidative stress through the SIRT1 / AMPK pathway while eliminating abnormal cells, exhibiting a bidirectional regulatory characteristic on cells, protecting normal cells while inducing apoptosis in abnormal cells. CHIR-99021 is an aminopyrimidine derivative and a GSK-3β inhibitor that activates Wnt / β-catenin to promote proliferation. The overall cell culture environment is better, enhancing cell viability and achieving higher cell density, thus breaking through the bottleneck of cell density effect.

[0025] HEK 293 cell culture additive B contains putrescine dihydrochloride, ethanolamine, and spermine, polyamines that play important roles in cell growth, proliferation, differentiation, migration, gene regulation, and protein and nucleic acid synthesis. However, their levels are generally low in common culture media. Transferrin provides iron. 3+ Supporting cell survival and proliferation, it is an essential component of DNA synthesis (ribonucleotide reductase) and energy metabolism (cytochrome). β-mercaptoethanol can be converted into cysteine ​​in the culture medium, serving as a precursor for glutathione (GSH) synthesis, increasing antioxidant capacity, and maintaining the correct disulfide bond pairing between antibody light and heavy chains. Curculigo glycoside can improve cell viability, upregulate the expression of target gene SOD, enhance antioxidant capacity, and reduce ROS levels. As a bidirectional regulator, when used in combination with transferrin and β-mercaptoethanol, it maintains enzyme activity and the correct folding of membrane proteins, thus promoting protein expression. Detailed Implementation

[0026] The technical solutions of the present invention will be further described below with reference to specific embodiments. The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. 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.

[0027] I. Experimental Materials: The amino acid components and reagents described in the following examples were all purchased from Sigma-Aldrich; the commercial culture media: HEK 293 serum-free basal medium HE02 (catalog number: HE000~N052) and HEK 293 serum-free supplemental medium HA02 (catalog number: HA000~N012) were all purchased from Suzhou Ekosei Biotechnology Co., Ltd.; Expi293 cells and plasmids (catalog number: EA40002926) were purchased from Thermo Fisher Scientific; the other methods and reagents used in this example, unless otherwise stated, are conventional methods and reagents in the art.

[0028] Live cell density and cell viability were measured using the following instrument: Name: Countstar BioTech Automated Cell Counter, Manufacturer: Countstar, Shanghai Ruiyu Biotechnology Co., Ltd., Model: ic1000.

[0029] II. Experimental Methods: This application discloses an additive suitable for HEK 293 cell culture. HEK 293 cell culture additive A increases the growth density of HEK 293 cells. Example 8 is preferred. Subsequent transfection tests all used Example 8 as the basal culture medium for cell transfection. Protein yield was increased by adding HEK 293 additive B to HEK 293 serum-free feed HA02.

[0030] Specific implementation scheme: HEK 293 Cell Culture Additive A Example 1: Asparagine 400 mg / L, lysine 150 mg / L, acetylcysteine ​​75 mg / L, insulin 0.5 mg / L; Example 2: Asparagine 400 mg / L, Lysine 150 mg / L, Acetylcysteine ​​75 mg / L, Insulin 0.5 mg / L, CHIR-99021 0.5 mg / L; Example 3: Asparagine 400 mg / L, Lysine 150 mg / L, Acetylcysteine ​​75 mg / L, Insulin 0.5 mg / L, Retinoic acid 5 mg / L, Resveratrol 0.25 mg / L, CHIR-99021 0.5 mg / L; Example 4: Asparagine 400 mg / L, Lysine 150 mg / L, Acetylcysteine ​​75 mg / L, Insulin 0.5 mg / L, Edaravone 10 mg / L, Hydrocortisone 0.005 mg / L, CHIR-99021 0.5 mg / L; Example 5: Asparagine 400 mg / L, lysine 150 mg / L, acetylcysteine ​​75 mg / L, insulin 0.5 mg / L, retinoic acid 5 mg / L, resveratrol 0.25 mg / L; Example 6: Asparagine 400 mg / L, lysine 150 mg / L, acetylcysteine ​​75 mg / L, insulin 0.5 mg / L, edaravone 10 mg / L, hydrocortisone 0.005 mg / L; Example 7: Asparagine 80 mg / L, Lysine 30 mg / L, Acetylcysteine ​​15 mg / L, Insulin 0.1 mg / L, Retinoic acid 1 mg / L, Resveratrol 0.05 mg / L, Edaravone 2 mg / L, Hydrocortisone 0.001 mg / L, CHIR-99021 0.1 mg / L; Example 8: Asparagine 400 mg / L, Lysine 150 mg / L, Acetylcysteine ​​75 mg / L, Insulin 0.5 mg / L, Retinoic acid 5 mg / L, Resveratrol 0.25 mg / L, Edaravone 10 mg / L, Hydrocortisone 0.005 mg / L, CHIR-99021 0.5 mg / L; Example 9: Asparagine 800 mg / L, Lysine 300 mg / L, Acetylcysteine ​​150 mg / L, Insulin 1 mg / L, Retinoic acid 10 mg / L, Resveratrol 0.5 mg / L, Edaravone 20 mg / L, Hydrocortisone 0.01 mg / L, CHIR-99021: 1 mg / L; Control group 1: HEK 293 serum-free basal medium HE02 conventional cell culture.

[0031] HEK 293 Cell Culture Additive B Example 10: Putrescine dihydrochloride 2 mg / L, ethanolamine 20 mg / L, spermine 0.1 mg / L, curculigoside 0.05 mg / L, transferrin 10 mg / L, cholesterol 0.1 mg / L, β-mercaptoethanol 0.1 mg / L; Example 11: Putrescine dihydrochloride 20 mg / L, ethanolamine 200 mg / L, spermine 2 mg / L, curculigoside 1 mg / L, transferrin 200 mg / L, cholesterol 2 mg / L, β-mercaptoethanol 2 mg / L; Example 12: Putrescine dihydrochloride 10 mg / L, ethanolamine 100 mg / L, spermine 1 mg / L; Example 13: Curculigoside 1 mg / L, transferrin 200 mg / L, cholesterol 2 mg / L, β-mercaptoethanol 2 mg / L; Example 14: Putrescine dihydrochloride 10 mg / L, ethanolamine 100 mg / L, spermine 1 mg / L, curculigoside 0.5 mg / L, transferrin 100 mg / L, cholesterol 1 mg / L; Example 15: Putrescine dihydrochloride 10 mg / L, ethanolamine 100 mg / L, spermine 1 mg / L, curculigoside 0.5 mg / L, transferrin 100 mg / L, β-mercaptoethanol 1 mg / L; Example 16: Putrescine dihydrochloride 10 mg / L, ethanolamine 100 mg / L, spermine 1 mg / L, transferrin 100 mg / L, cholesterol 1 mg / L, β-mercaptoethanol 1 mg / L; Example 17: Putrescine dihydrochloride 10 mg / L, ethanolamine 100 mg / L, spermine 1 mg / L, curculigoside 0.5 mg / L, cholesterol 1 mg / L, β-mercaptoethanol 1 mg / L; Example 18: Putrescine dihydrochloride 10 mg / L, ethanolamine 100 mg / L, spermine 1 mg / L, curculigoside 0.5 mg / L, transferrin 100 mg / L, cholesterol 1 mg / L, β-mercaptoethanol 1 mg / L; Control group 2: routine cell culture using HEK 293 serum-free basal medium HE02 combined with HEK 293 serum-free supplemented medium HA02.

[0032] The test results are as follows: 1. Table 1 shows the cell viable cell density and cell growth in different embodiments. Different concentrations of HEK 293 cell culture additive A were added, compared to the control group (which grew to 7.32 × 10⁻⁶ cells on day 5). 6 (cell count / mL), Examples 7, 8, and 9 show cell densities reaching 14 × 10⁻⁶. 6 The cell count was approximately [number] / mL, significantly higher than the cell growth density of control group 1.

[0033] Table 1

[0034] 2. Table 2 shows the cell viability in different examples. Examples 7, 8, and 9 show a cell growth trend that is slightly higher than that of control group 1.

[0035] Table 2

[0036] 3. Table 3 shows the cell viability density and cell growth in different examples after transfection with HEK 293 basal medium HE02 and HEK 293 cell culture additive A (i.e., Example 8). Different concentrations of HEK 293 cell culture additive B were added, compared to the control group (which grew to 11.4 × 10⁻⁶ cells on day 3). 6 (cell count / mL), Examples 15, 17, and 18 show that the cell density is significantly higher than that of the control group 2.

[0037] Table 3

[0038] 4. Table 4 shows the cell viability after transfection with HEK 293 basal medium HE02 and HEK 293 cell culture additive A (i.e., Example 8). The cell viability in Example 18 is the highest. The viability of other examples is not significantly different from that of control group 2.

[0039] Table 4

[0040] 5. Table 5 shows the protein yield in different examples based on HEK 293 basal medium HE02 with HEK 293 cell culture additive A (i.e., Example 8). The yields in Examples 15 and 18 were higher than those in the control group. In Example 15, the yield was 52.1% higher than that in the control group, indicating that HEK 293 additive B can significantly enhance protein expression.

[0041] Table 5

[0042] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A HEK 293 cell culture medium, characterized in that, It consists of component one and component two; Component 1 consists of HEK 293 serum-free basal medium HE02 (product number HE000~N052) with HEK 293 cell culture additive A added at a volume ratio of 10%. Component 2 consists of HEK 293 serum-free fed culture medium HA02 (product number HA000~N012) with HEK 293 cell culture additive B added at a volume ratio of 10%. HEK 293 cell culture additive A consists of the following components: asparagine 400 mg / L, lysine 150 mg / L, acetylcysteine ​​75 mg / L, insulin 0.5 mg / L, retinoic acid 5 mg / L, resveratrol 0.25 mg / L, edaravone 10 mg / L, hydrocortisone 0.005 mg / L, and CHIR-99021 0.5 mg / L; HEK 293 cell culture additive B consists of the following components: putrescine dihydrochloride 10 mg / L, ethanolamine 100 mg / L, spermine 1 mg / L, curculigoside 0.5 mg / L, transferrin 100 mg / L, cholesterol 1 mg / L, and β-mercaptoethanol 1 mg / L.

2. The application of the HEK 293 cell culture medium as described in claim 1, characterized in that, This HEK 293 cell culture medium is used for the culture of HEK 293 cells.

3. The application of the HEK 293 cell culture medium as described in claim 2, characterized in that, The HEK 293 cell culture process is as follows: S1. Prepare HEK 293 cells and culture them in the serum-free basal medium HE02 (product number HE000~N052) as described above. The culture conditions on a shaker are: 37℃, 110 rpm, 8% CO2, 80% humidity, and a cell density of 0.6 × 10⁶ cells / year. 6 Cells were passaged at a density of 100 cells / mL every three days for three passages until they were stable. The cells were then cultured at a concentration of 0.6 × 10⁶ cells / mL. 6 Cells were seeded at a density of 100 cells / mL in HEK 293 serum-free basal medium HE02 (product number HE000~N052), HEK 293 cell culture additive A was added, and the cells were cultured in a shaker. S2. The day before the transfection experiment, the cell density was controlled at 1.7 × 10⁻⁶. 6 Cell count / mL, cell count on the day of the experiment for future reference; S3. On the day of transfection, following the 20 mL shake flask culture system, adjust the cells to 18 mL with fresh HEK 293 serum-free protein expression medium (HE02, catalog number HE000~N052), resulting in a total cell count of 60 × 10⁶ cells / mL. 6 Cell count / mL, cell density at transfection was 3.3 × 10⁻⁶. 6 about; Preparation of PEI / DNA complex: The transfection volume was 20 mL, and the final cell density was 3.0 × 10⁻⁶. 6 Cell count / mL, final DNA concentration 1.5 μg / mL, DNA:PEI = 1.5:

4. The specific steps are as follows: PEI MAX dilution: Dilute 80 μg PEI MAX to 1 mL with HEK 293 serum-free protein expression medium HE02 (product number HE000~N052) and incubate at room temperature for 5 min; DNA dilution: Dilute 30 μg of DNA to 1 mL with HEK 293 serum-free protein expression medium HE02 (product number HE000~N052) and incubate at room temperature for 5 min; Mixing PEI with DNA: Add PEI MAX dilution to DNA dilution to form a PEI / DNA complex, mix well, and incubate at room temperature for 10 min; S4. Slowly add the prepared 2 mL PEI / DNA complex to the culture system, and then transfer it to an incubator for further culture. S5. About 16-24 hours after transfection, add 5% volume of HEK 293 serum-free feed medium HA02 (product number HA000~N012), add 1 mL of feed to a 20 mL system, add HEK 293 cell culture additive B, and supplement with 6 g / L glucose. S6. On the 5th day after transfection, the supernatant was harvested.

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