High-yield CHO-K1 instantaneous transfection method with linearized PEI as transfection reagent

Abstract

The invention discloses an instantaneous transfection method for high-yield CHO-K1 (CHO-K1) by taking linearized PEI (Polyetherimide) as a transfection reagent. The antibody titer reaches about 1g / L within 10 days by replacing a culture medium, zero incubation of a compound, a specific DNA proportion and temperature regulation, the dosage of an expression vector is remarkably reduced, the research and development period is shortened, and the method is suitable for early research and development of antibody drugs and sample preparation.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a high-yield transient transfection method for CHO-K1 using linearized PEI as the transfection reagent. Background Technology

[0002] Mammalian cell expression systems, especially Chinese hamster ovary cells (CHO), have become the preferred choice for the industrial production of recombinant protein drugs. The CHO-K1 cell line is widely used due to its rapid growth, ease of suspension culture, and excellent protein folding and post-modification capabilities. Traditionally, the research and development phase requires months to construct stable cell lines to obtain small quantities of test samples, severely slowing down the evaluation of candidate drugs. Transient transfection can yield milligrams to grams of protein within days, but the transient transfection efficiency of CHO-K1 has long been lower than that of sublines such as CHO-S, resulting in limited yield.

[0003] Linearized polyethyleneimine (PEI) is one of the most commonly used non-liposome transfection reagents, offering advantages such as low cost, easy scale-up, and absence of animal-derived components. However, in current technologies, CHO-K1 cells exhibit low transfection efficiency under pristine conditioned media, and the formation and incubation methods of the PEI-DNA complex have not been systematically optimized. While reports indicate that exogenous "carrier DNA" can improve transfection efficiency in certain cell lines and reduce the amount of expensive expression vectors used, no literature has specifically utilized herring sperm DNA for CHO-K1 suspension transient transfection systems.

[0004] The GS (glutamine synthase) expression system can achieve stable and high yields through MSX pressure selection, but there are no reports on whether introducing the GS gene in the transient transformation stage can increase yield. Summary of the Invention

[0005] The purpose of this invention is to provide a high-yield transient transfection method for CHO-K1 using linearized PEI as the transfection reagent.

[0006] Technical solution: A high-yield transient transfection method for CHO-K1 using linearized PEI as the transfection reagent, comprising the following steps: (a) Centrifuge CHO-K1 suspension cells and completely replace them with culture medium for chemical transfection; (b) Adjust the cell density to 5 × 10^6–2.5 × 10^7 live cells / mL; (c) Prepare a DNA solution in the transfection medium by diluting the DNA with 8-16% of the transfection system volume of the medium. The total amount of DNA consists of GS expression vector DNA, expression vector DNA and herring sperm DNA, and the mass ratio of GS expression vector DNA: expression vector DNA: herring sperm DNA is 1:2-3:6-10. (d) To prepare the DNA-PEI complex, linearized polyethyleneimine (PEI) is added to the diluted DNA solution at a mass ratio of 2:1–2.5:1 and mixed immediately, and the mixture is inverted for 10 seconds to obtain the DNA-PEI complex. (e) Immediately add the DNA-PEI complex prepared in step (d) to the cell suspension and mix well before transfection culture; (f) Add Titer enhancer and culture 5-10 min after transfection; (g) Add nutrients: Add CHO TransFeed and glucose 1 day / 4 days after transfection; (h) Recombinant protein was harvested between days 5 and 14.

[0007] Preferably, the centrifugation parameters in step (a) are 400 g × 5 min, and the culture medium for chemical transfection is MetaCell CHO-320 or CHO-320 Plus.

[0008] Preferably, in step (c), the expression vector DNA is 2.5-7.5 μg / mL; the herring sperm DNA is 5-15 μg / mL; and the GS expression vector DNA is 0.5-2 μg / mL, wherein the herring sperm DNA fragment is 500 bp-1 kbp in length, and the expression vector DNA is an expression system carrying the CMV promoter.

[0009] Preferably, the linearized polyethyleneimine (PEI) in step (d) is commercially available PEI 40K.

[0010] Preferably, the culture temperature after transfection in step (e) is 37 ℃ when the live cell density is ≤ 7.5 × 10^6 cells / mL, and 32 ℃ when the live cell density is > 7.5 × 10^6 cells / mL.

[0011] Preferably, in step (f), the amount of MetaCell titer enhancer added is 1-3% of the volume of the transfection complex.

[0012] Preferably, in step (g), the amount of CHO TransFeed added is 4-12% of the transfection system volume and the total amount is less than 16% of the transfection system volume, and the amount of glucose added is maintained above 5 g / L.

[0013] Preferably, the recombinant protein expression peaks 5-14 days post-transfection in step (h). The human antibody is detected using the Cedex Bio IGHB model (immunoturbidimetric assay).

[0014] The beneficial effects of this invention are as follows: 1. By using a synergistic process of "complete replacement of culture medium and instant mixing of DNA-PEI complex", the inhibitory components of conditioned medium are eliminated, and the efficiency decay caused by long-term incubation of the complex is avoided. This improves the CHO-K1 transfection efficiency by more than 80% and the antibody expression level exceeds 1 g / L, solving the industry problem of low efficiency of PEI transfection of CHO-K1.

[0015] 2. By partially replacing the expression vector with herring sperm DNA, the amount of vector DNA used is reduced by 50% at a mass ratio (vector DNA: herring sperm DNA = 1:2-3), and the cost of herring sperm DNA is only 1 / 10 of that of vector DNA, which greatly reduces the cost of transfection reagents.

[0016] 3. By using the GS expression vector and gradient temperature control, the GS system (0.5-2 μg / mL) improves antibody expression efficiency; by switching the culture temperature according to the live cell density (≤7.5×10^6 cells / mL: 37℃; >7.5×10^6 cells / mL: 32℃), the high viability production period is extended to 14 days, and the cumulative expression level continues to increase.

[0017] 4. The entire process uses commercially available components: culture medium (MetaCell CHO-320 / Plus); enhancer (MetaCell Titer Enhancer, 1-3%); and feed (CHO TransFeed 4-12% + glucose), ensuring batch-to-batch consistency (experimental data CV < 15%), and can be directly scaled up to a bioreactor.

[0018] 5. The preparation of DNA-PEI complex without incubation shortens the transfection operation to within 1 minute; protein expression can be quickly detected 3 days after transfection, and harvesting can be flexibly carried out in 5-14 days, which greatly accelerates the research and development cycle. Attached Figure Description

[0019] Figure 1 This is a gradient data graph showing antibody expression levels at different cell transfection densities under the same conditions.

[0020] Figure 2A graph showing the impact of GS carrier addition amount on yield.

[0021] Figure 3 This is a graph showing the impact of herring sperm DNA on transfection efficiency and the amount of expression vector required.

[0022] Figure 4 The cumulative expression data of Example 2 and Example 3 were detected on the 7th, 10th and 14th days of culture, respectively; 1-1 / 1-2 are parallel data of the same experiment in Example 3, and 2-1 / 2-2 are parallel data of the same experiment in Example 2.

[0023] Figure 5 The graph shows the transfection efficiency data for CHO-K1 transfection with and without complete replacement of the culture medium.

[0024] Figure 6 Graph showing the effect of not incubating or adding the DNA-PEI complex independently on transfection efficiency.

[0025] Figure 7 The graph shows that herring sperm DNA can replace expression vectors and improve transfection expression efficiency.

[0026] Figure 8 Graph showing the effect of optimized PEI-mediated CHO-K1 transfection on antibody expression.

[0027] Figure 9 A graph showing data on enhancing antibody expression in a traditional dual-vector system using GS vector DNA. Detailed Implementation

[0028] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. 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. Example 1

[0029] (a) Centrifuge CHO-K1 cells (400 g × 5 min) and replace with MetaCell CHO-320 medium; (b) Adjust the live cell density to 5 × 10^6 cells / mL; (c) DNA solution preparation (8% volume dilution): GS vector DNA 0.5 μg / mL, target vector DNA 1.0 μg / mL (GS:target vector = 1:2), herring sperm DNA 5 μg / mL (target vector:herring sperm = 1:5); (d) DNA-PEI complex: PEI 40K was added at a ratio of DNA:PEI = 1:2.5 and mixed for 10 seconds; (e) The complex was immediately added to cells (viable cell density ≤ 7.5 × 10^6 cells / mL) and incubated at 37°C; (f) Add 1% MetaCell Titer Enhancer 5 min after transfection; (g) Feeding: Add 12 / 4% CHO TransFeed + glucose 1 day / 4 after transfection; (h) Detection 3 days post-transfection: Antibody expression level 305 mg / L (Cedex Bio assay); (i) Day 10 results: cumulative expression level 899 mg / L, viability 85%. Example 2

[0030] (a) Centrifuge CHO-K1 cells (400 g × 5 min) and replace with MetaCell CHO-320 medium; (b) The viable cell density was adjusted to 1.25 × 10^7 cells / mL; (c) DNA solution preparation (12% volume dilution): GS vector DNA 1.0 μg / mL, target vector DNA 2.5 μg / mL (GS:target vector = 1:2.5), herring sperm DNA 7.5 μg / mL (target vector:herring sperm = 1:3); (d) DNA-PEI complex: PEI 40K was added at a ratio of DNA:PEI = 1:2.5 and mixed for 10 seconds; (e) The complex was immediately added to cells (viable cell density > 7.5 × 10^6 cells / mL) and incubated at 32°C; (f) Add 2% MetaCell Titer Enhancer 5 min after transfection; (g) Feeding: Add 8% CHO TransFeed + glucose on days 1 and 4 after transfection; (h) Detection 3 days post-transfection: Antibody expression level 348 mg / L; (i) Day 10 results: cumulative expression level 943 mg / L, viability 88%. Example 3

[0031] (a) Centrifuge CHO-K1 cells (400 g × 5 min) and replace with MetaCell CHO-320 medium; (b) The viable cell density was adjusted to 2.5 × 10^7 cells / mL; (c) DNA solution preparation (16% volume dilution): GS vector DNA 2.0 μg / mL, target vector DNA 6.0 μg / mL (GS:target vector = 1:3), herring sperm DNA 15 μg / mL (target vector:herring sperm = 1:2.5); (d) DNA-PEI complex: Add PEI 40K at a ratio of DNA:PEI = 1:2 and mix for 10 seconds; (e) The complex was immediately added to cells (viable cell density > 7.5 × 10^6 cells / mL) and incubated at 32°C; (f) Add 3% MetaCell Titer Enhancer 5 min after transfection; (g) Feeding: Add 12 / 4% CHO TransFeed + glucose 1 day / 4 after transfection; (h) Detection 3 days post-transfection: Antibody expression level 479 mg / L; (i) Harvest on day 14: cumulative expression level 1440 mg / L, viability 82%.

[0032] Comparative Example 1: Same as Example 1, except that (b) the live cell density was changed to 5.0 × 10^6 cells / mL.

[0033] Comparative Example 2: Compared with Example 2, only (a) was modified to retain the original culture medium (without centrifugation and replacement).

[0034] Comparative Example 3: Compared with Example 2, the only modification was that (d) DNA-PEI mixture was incubated at room temperature for 30 minutes before transfection.

[0035] Comparative Example 4: Compared with Example 2, only (c) the target vector DNA was increased to 10 μg / mL (herring sperm DNA was deleted).

[0036] Comparative Example 5: Compared with Example 2, only the DNA solution preparation (12% volume dilution) was modified: GS vector DNA 0 μg / mL, target vector DNA 3.0 μg / mL, and herring sperm DNA 7.5 μg / mL (target vector: herring sperm = 1:2.5).

[0037] Comparative Example 6: Compared with Example 2, only (f) and (g) were modified by not adding Titer Enhancer and TransFeed / glucose.

[0038] The statistical results of the experiment are shown in the table below: Table 1 Comparative Examples and Implementation Examples Test Table sample Expression level over 3 days (mg / L) Peak expression level (mg / L) Survival rate (%) Cost Index Example 1 305 899(D10) 85 35 Example 2 348 726(D10) 88 42 Example 3 479 1012 (D10) 82 78 Comparative Example 1 121 441(D10) 70 100 Comparative Example 2 119 389(D10) 81 95 Comparative Example 3 256 620(D10) 83 90 Comparative Example 4 278 654(D10) 84 150 Comparative Example 5 293 678(D10) 76 42 Comparative Example 6 156 410(D10) 80 30 Cost index explanation (benchmark comparison 1=100).

[0039] Examples 1, 2, and 3, and Comparative Example 1, show that different cell transfection densities are closely related to antibody expression levels. The gradient data of antibody expression levels at different cell transfection densities under the same conditions are shown in the attached figure. Figure 1 The cumulative expression levels in Examples 2 and 3, measured on days 7, 10, and 14 of culture, are shown in the attached figures. Figure 4 The synergistic process of "replacement culture medium and instantaneous PEI complex" (claims 1a, 1d, 1e) solves the problem of low transfection efficiency of traditional PEI. The expression level of Comparative Example 2 (without replacement) is only 34% of that of Example 2. The transfection efficiency data of CHO-K1 before complete and incomplete replacement of culture medium are shown in the attached figure. Figure 5 Comparative Example 3 (incubation for 30 min) showed a 26% efficiency decrease, demonstrating the necessity of incubation-free immediate transfection. The data on the effect of no incubation or independent addition of the DNA-PEI complex on transfection efficiency are shown in the attached figure. Figure 6 The data on the effect of optimized PEI-mediated CHO-K1 transfection on antibody expression are shown in the attached figure. Figure 8 Herring sperm DNA partially replaces the expression vector (claim 1c), achieving cost reduction and efficiency improvement. In Example 2, 726 mg / L was achieved by adding 7.5 μg / mL herring sperm to 2.5 μg / mL of the target vector, while in Comparative Example 4, only 654 mg / L was achieved with 10 μg / mL of the target vector (without herring sperm), resulting in a lower vector cost (Comparative Example 4, cost index 150). The impact of herring sperm DNA on transfection efficiency and the required amount of expression vector is shown in the attached graph. Figure 3 Herring sperm DNA can replace expression vectors and improve transfection expression efficiency. (See attached data). Figure 7 Comparative Example 5 shows that the addition of GS vector DNA has a significant impact on antibody yield. The data on the effect of the amount of GS vector DNA added on the yield is shown in the attached figure. Figure 2 The attached graph shows the data on how GS vector DNA enhances antibody expression in traditional dual-vector systems. Figure 9 Simultaneously, the feeding strategy enabled the expression window to cover D7-D14 (Example 1 D10: 899 mg / L; Example 3 D14: 1440 mg / L; Comparative Example 6 D10: 410 mg / L).

[0040] 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 high-yield transient transfection method for CHO-K1 using linearized PEI as the transfection reagent, characterized in that, Includes the following steps: (a) Centrifuge CHO-K1 suspension cells and completely replace them with culture medium for chemical transfection; (b) Adjust the cell density to 5 × 10^6–2.5 × 10^7 live cells / mL; (c) Prepare a DNA solution in the transfection medium by diluting the DNA with 8-16% of the transfection system volume of the medium. The total amount of DNA consists of GS expression vector DNA, expression vector DNA and herring sperm DNA, and the mass ratio of GS expression vector DNA: expression vector DNA: herring sperm DNA is 1:2-3:6-10. (d) To prepare the DNA-PEI complex, linearized polyethyleneimine (PEI) is added to the diluted DNA solution at a mass ratio of 2:1–2.5:1 and mixed immediately, and the mixture is inverted for 10 seconds to obtain the DNA-PEI complex. (e) Immediately add the DNA-PEI complex prepared in step (d) to the cell suspension and mix well before transfection culture; (f) Add Titer enhancer and culture 5-10 min after transfection; (g) Add nutrients: Add CHO TransFeed and glucose 1 day / 4 days after transfection; (h) Recombinant protein was harvested between days 5 and 14.

2. The high-yield transient CHO-K1 transfection method using linearized PEI as the transfection reagent according to claim 1, characterized in that, In step (a), the centrifugation parameters are 400 g × 5 min, and the culture medium used for chemical transfection is MetaCell CHO-320 or CHO-320 Plus.

3. The high-yield transient CHO-K1 transfection method using linearized PEI as the transfection reagent according to claim 1, characterized in that, In step (c), the expression vector DNA is 2.5–7.5 μg / mL; the herring sperm DNA is 5–15 μg / mL; and the GS expression vector DNA is 0.5–2 μg / mL, wherein the herring sperm DNA fragment is 500 bp–1 kbp in length, and the expression vector DNA is an expression system carrying the CMV promoter.

4. The high-yield transient CHO-K1 transfection method using linearized PEI as the transfection reagent according to claim 1, characterized in that, In step (d), the linearized polyethyleneimine (PEI) is commercially available PEI 40K.

5. The high-yield transient CHO-K1 transfection method using linearized PEI as the transfection reagent according to claim 1, characterized in that, In step (e), the culture temperature after transfection is 37 ℃ when the viable cell density is ≤ 7.5 × 10^6 cells / mL, and 32 ℃ when the viable cell density is > 7.5 × 10^6 cells / mL.

6. The high-yield transient CHO-K1 transfection method using linearized PEI as the transfection reagent according to claim 1, characterized in that, In step (f), the amount of MetaCell titer enhancer added is 1-3% of the volume of the transfection complex.

7. The high-yield transient CHO-K1 transfection method using linearized PEI as the transfection reagent according to claim 1, characterized in that, In step (g), the amount of CHO TransFeed added is 4-12% of the transfection system volume and the total amount is less than 16% of the transfection system volume, and the amount of glucose added is kept above 5 g / L.

8. The high-yield transient CHO-K1 transfection method using linearized PEI as the transfection reagent according to claim 1, characterized in that, In step (h), the recombinant protein expression is highest between 5 and 14 days post-transfection.

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