Application of Palbociclib in the preparation of serum-free culture medium for CHO cells

Abstract

This invention relates to the field of serum-free culture medium technology. Specifically, it provides the application of Palbociclib in the preparation of serum-free culture medium for CHO cells. A serum-free culture medium for CHO cells is prepared by adding Palbociclib, wherein the final concentration of Palbociclib in this serum-free CHO cell culture medium is 2-50 μM. The serum-free CHO cell culture medium of this invention also includes vitamin C, which, when used in combination with Palbociclib, can better improve the expression level of the target protein in CHO cells, increase cell-specific productivity, promote CHO cell growth, improve cell viability and yield, and simultaneously reduce production costs.

Description

Technical Field

[0001] This invention relates to the field of serum-free culture medium technology, and more particularly to the application of Palbociclib in the preparation of serum-free culture medium for CHO cells. Background Technology

[0002] In recent years, biotherapy and diagnostics have experienced rapid growth in the pharmaceutical market. Over the past two decades, more than 300 biopharmaceuticals have been approved by the U.S. Food and Drug Administration (FDA), and this number continues to grow, with an increasing number of biological products (proteins, nucleic acids, sugars, and complexes) entering the diagnostic and therapeutic fields. As a crucial component of biopharmaceuticals, recombinant protein drugs such as monoclonal antibodies possess immense scientific and commercial value. CHO cells are one of the most important platforms for producing recombinant proteins and monoclonal antibodies; over 80% of approved monoclonal antibodies are produced in CHO cells. The culture medium plays a vital role in cell growth and productivity. Conventional cell culture requires the addition of serum, but serum has complex compositions, batch-to-batch variations, and drawbacks such as contamination by exogenous viral and pathogenic factors, allergic reactions caused by serum residues, and difficulties in standardizing cell culture and purifying the final product, making it unsuitable for the production of recombinant protein drugs. Serum-free culture media have a clearly defined composition, avoiding the influence of different batches or unknown components of serum on cell culture. They largely avoid or improve upon the aforementioned defects of serum-containing culture media and are currently widely used for CHO cell culture in the production of recombinant protein drugs.

[0003] In recent years, to meet the growing demand for monoclonal antibodies, a series of measures have been taken in the industrial production of recombinant protein drugs from CHO cells to increase recombinant protein expression levels and reduce production costs. Early efforts to improve CHO cell recombinant protein expression focused on optimizing expression vectors, modifying host cells and culture medium composition, and employing fed-batch strategies to increase integrated viable cell density (IVCD) and specific cell productivity (Qp) over the cell culture period. Later, it was discovered that optimizing culture processes (such as low temperature and high osmotic pressure environments) and adding small molecule reagents could improve protein specific productivity. Among these, optimizing serum-free culture media by adding small molecule additives has become an effective strategy for promoting CHO cell protein expression, as exemplified by the serum-free culture medium for CHO cells disclosed in CN116478913A. Therefore, developing a simple, low-cost formulation that can better improve recombinant protein expression levels in CHO cell culture has become a pressing technical challenge. Summary of the Invention

[0004] The purpose of this invention is to provide the application of Palbociclib in the preparation of serum-free culture medium for CHO cells. By adding Palbociclib and using it in combination with vitamin C, the expression level of the target protein in CHO cells is improved, cell-specific productivity is increased, and CHO cell growth, cell viability and yield are promoted.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides the application of Palbociclib in the preparation of serum-free culture medium for CHO cells.

[0007] Preferably, the CHO cells include CHO-K1, CHO-S, or CHO-DG44 cells.

[0008] The present invention also provides a serum-free culture medium for CHO cells, comprising a basic serum-free culture medium and Palbociclib.

[0009] Preferably, the final concentration of Palbociclib is 2-50 μM.

[0010] Preferably, the final concentration of Palbociclib is 20-45 μM.

[0011] Preferably, the serum-free culture medium for CHO cells also includes vitamin C.

[0012] Preferably, the concentration ratio of Palbociclib to vitamin C in the serum-free culture medium for CHO cells is (2-50) μM:15 μM.

[0013] By adopting the above technical solution, the present invention has the following beneficial effects:

[0014] The technical solution of this invention can improve the expression level of the target protein in CHO cells to a certain extent by adding palbociclib to the serum-free culture medium. Furthermore, combining palbociclib and vitamin C at a reasonable concentration ratio can further enhance the expression level of the target protein in CHO cells, increase cell-specific productivity, promote CHO cell growth, improve cell viability and yield, achieving a synergistic effect. Simultaneously, this technical solution can also reduce energy consumption and energy requirements in the biopharmaceutical manufacturing process, thereby lowering the production cost of biopharmaceuticals. Attached Figure Description

[0015] Figure 1 Western blot results for different treatment groups ( Figure 1In the diagram, A represents the Western blot bands from different treatment groups. Figure 1 In this context, B represents the relative expression level of HSA in different treatment groups.

[0016] Figure 2 The number of viable cells and cell viability in different treatment groups ( Figure 2 In this context, A represents the number of viable cells in different treatment groups. Figure 2 In this context, B represents the cell viability of different treatment groups. Detailed Implementation

[0017] This invention provides an application of palbociclib in the preparation of serum-free culture medium for CHO cells.

[0018] In this invention, the CHO cells include CHO-K1, CHO-S, or CHO-DG44 cells.

[0019] The present invention also provides a serum-free culture medium for CHO cells, comprising a basic serum-free culture medium and Palbociclib.

[0020] In this invention, the basic serum-free culture medium was purchased from Henan Punoyi Biological Products Research Institute Co., Ltd., and preferably includes DC1004a culture medium.

[0021] In this invention, the Palbociclib was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. The final concentration of the Palbociclib in this invention is preferably 2-50 μM, more preferably 20-45 μM, and even more preferably 40 μM.

[0022] In this invention, the serum-free culture medium for CHO cells also includes vitamin C.

[0023] In this invention, the preferred concentration ratio of Palbociclib and Vitamin C in the serum-free culture medium for CHO cells is (2-50) μM:15 μM, further preferably (25-45) μM:15 μM, and even more preferably 40 μM:15 μM.

[0024] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0025] Example 1

[0026] (I) Preparation of serum-free culture medium for CHO cells

[0027] Weigh 27.5g of serum-free culture medium powder DC1004a, add it to water for injection, dissolve thoroughly, adjust the pH to 7.2, and then filter sterilize using a 0.1μm pore size filter membrane to obtain the basic serum-free culture medium. Weigh 17.9mg of Palbociclib powder and add it to the above-obtained basic serum-free culture medium to obtain a serum-free culture medium containing 40μM Palbociclib. Weigh 2.6mg of vitamin C powder and add it to the above-obtained basic serum-free culture medium to obtain a serum-free culture medium containing 15μM vitamin C. Weigh 2.6mg of vitamin C powder and 17.9mg of Palbociclib powder and add them to the above-obtained basic serum-free culture medium to obtain a serum-free culture medium containing 40μM Palbociclib and 15μM vitamin C. Then, serum-free medium containing 40 μM Palbociclib and 15 μM Vitamin C was diluted with serum-free medium containing 15 μM Vitamin C to prepare serum-free medium of 2 μM Palbociclib and 15 μM Vitamin C, 5 μM Palbociclib and 15 μM Vitamin C, 10 μM Palbociclib and 15 μM Vitamin C, and 20 μM Palbociclib and 15 μM Vitamin C in sequence.

[0028] (II) Effects on the expression of recombinant protein HSA in CHO cells

[0029] To investigate the effects of palbociclib and / or vitamin C addition on cells, different treatment groups were set up.

[0030] Control group 1: Basic serum-free culture medium

[0031] Control group 2: Serum-free culture medium containing 15 μM vitamin C

[0032] Control group 3: Serum-free culture medium containing 40 μM Palbociclib

[0033] Experimental group 1: Serum-free culture medium containing 2 μM Palbociclib and 15 μM Vitamin C

[0034] Experimental group 2: Serum-free culture medium containing 5 μM Palbociclib and 15 μM Vitamin C

[0035] Experimental group 3: Serum-free culture medium containing 10 μM Palbociclib and 15 μM Vitamin C

[0036] Experimental group 4: Serum-free culture medium containing 20 μM Palbociclib and 15 μM Vitamin C

[0037] Experimental group 5: Serum-free culture medium containing 40 μM Palbociclib and 15 μM Vitamin C

[0038] 1. Cell resuscitation. Human serum albumin-containing CHO cells (purchased from Life Technologies, USA, catalog number A11557-01) were seeded in 24-well plates. After 24 hours, the cell density reached 95%. Cells were transfected with liposome 2000 and cultured at 37°C in a 5% CO2 incubator. After 48 hours, the medium was changed with medium containing 15 μg / mL BSD. After 10 days, all blank cells died. The concentration was then halved and the treatment was continued for 4 weeks to obtain a stable cell pool. Single clones were obtained using the limiting dilution method. Cells were diluted to 10 cells / mL in medium supplemented with 10% FBS. The cell dilution was evenly seeded in 96-well plates. Single clones were identified after 5 days. When the cell density reached 60%, the cells were scaled up for culture. The obtained HSA single-clone cell line was cryopreserved for later use.

[0039] Take out the frozen HSA monoclonal cell line, thaw it rapidly in a 37°C water bath, and use a pipette to transfer the cells into a 1.5 EP tube in a clean bench. Centrifuge at 1000 rpm for 5 min, discard the supernatant, add DMEM / F12 complete culture medium (Henan Punoyi Biological Products Research Institute Co., Ltd.), and pipette the cells evenly to form a single-cell suspension. Add the cell suspension to a cell culture flask, add 2 mL of complete culture medium, pipette evenly, and place in a 37°C, 5% CO2 cell culture incubator for culture.

[0040] 2. Cell passage. Remove the cell culture flask from the incubator, discard the old culture medium in a clean bench, and rinse with PBS. Add 1 mL of trypsin to digest the cells until they become rounded but do not detach, then aspirate the digestion solution. Add 6 mL of fresh complete culture medium to stop the digestion, and after thorough mixing, suspend the cells at 4000 cells / cm³. 2 Inoculate into another cell culture flask and add fresh complete culture medium to continue culturing.

[0041] 3. Cell suspension. Adherent cultured cells were collected and observed under a microscope. After washing the cells with PBS, they were digested with 1 mL of trypsin until the cells became rounded but did not detach. The digestion solution was removed, and 6 mL of fresh complete culture medium was added to terminate the digestion. The cell pellet was collected by centrifugation at 1000 rpm for 5 min and resuspended in the serum-free culture medium described in control groups 1-3 and experimental groups 1-5, respectively. The cells were then suspended at 4 × 10⁶ cells / mL. 5Cells were seeded at a density of 1 / mL in six-well cell culture plates and placed on a shaker at 120 rpm. The plates were then cultured in suspension at 37°C in a 5% CO2 incubator. Day 0 of suspension culture was the day of seeding. Cells were cultured for 7 days. On day 7, the plates were centrifuged at 1000 rpm, and the supernatant was collected for Western blot analysis. The relative expression level of HSA was obtained by grayscale analysis of the Western blot bands using ImageJ.

[0042] The results are as follows Figure 1 As shown, serum-free culture medium containing Palbociclib can enhance the expression of recombinant HSA in CHO cells. However, serum-free culture medium with both Palbociclib and vitamin C added has a more significant effect on enhancing the expression of recombinant HSA in CHO cells, indicating that Palbociclib and vitamin C have a synergistic effect. Among them, 40 μM is the optimal concentration of Palbociclib, and the relative expression level of HSA in experimental group 5 is the highest.

[0043] (III) Effects on CHO cell growth

[0044] The cells were cultured in suspension using the same serum-free medium as the control groups 1-3 and experimental groups 1-5, for 7 days. Samples were taken daily, and cells were counted using trypan blue staining. The number of viable cells and cell viability were recorded.

[0045] The results are as follows Figure 2 As shown, cells in serum-free culture medium containing palbociclib and vitamin C grew slowly, and the growth rate gradually decreased with increasing palbociclib concentration, while cell viability showed no significant difference. However, the increased protein expression may be due to the promotion of cell cycle arrest.

[0046] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Application of serum-free culture medium containing Palbociclib and vitamin C in increasing the expression level of recombinant human serum albumin in CHO cells.

2. The application according to claim 1, characterized in that, The CHO cells include CHO-K1, CHO-S, or CHO-DG44 cells.

3. The application according to claim 1, characterized in that, The concentration ratio of Palbociclib to vitamin C is (2-50) μM: 15 μM.

4. The application according to claim 1, characterized in that, The final concentration of Palbociclib is 2-50 μM.

5. The application according to claim 1, characterized in that, The final concentration of Palbociclib was 20-45 μM.

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