Nutritional supplement as well as preparation method and application thereof

Abstract

The invention discloses a nutritional supplement as well as a preparation method and application thereof, and relates to the technical field of biological medicines. The invention provides a nutritional supplement capable of maintaining and even improving the cell viability at the later stage of cell culture. The nutritional supplement comprises the following components in parts by weight: 0.05-0.6 part of choline chloride, 0.1-0.6 part of lithium chloride and 0.1-0.2 part of taurine. By compounding the taurine, the lithium chloride and the choline chloride, the invention provides the nutritional supplement capable of maintaining and even improving the cell viability in the later period of cell culture.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a nutritional supplement and a preparation method and application thereof. Background Art

[0002] The therapeutic principle of biological drugs is mainly to stimulate the body's immune system to produce immune substances to exert its efficacy, produce humoral immune cell immunity or cell-mediated immunity in the human body, so as to achieve the therapeutic effect. The raw materials of biological drugs are mainly natural biological materials, so the safety is relatively high and the side effects are relatively small. Biological drugs are of great significance to human life and health, and are considered to be one of the most promising industries in the 21st century. Cell culture has a wide range of applications in biopharmaceuticals. It is a method of producing drugs by using the ability of cells to grow and divide. Through cell culture technology, we can produce various drugs on a large scale, such as protein drugs, antibody drugs and vaccines. The application of cell culture technology in biopharmaceuticals can not only improve the production efficiency of drugs, but also reduce dependence on animal experiments, thereby reducing the cost of drug research and development.

[0003] At present, the process of producing biopharmaceuticals using cell culture is still subject to many constraints, such as growth restriction, low yield, low pressure resistance and relatively high cost. In particular, the rapid decline in cell viability in the later stage of cell culture has a huge impact on cell target products, such as protein expression and virus titer. The current mainstream method is to use the culture medium itself to provide nutrients to cells, maintain environmental stability, promote cell adhesion and diffusion, control cell proliferation and differentiation, and provide necessary support for the normal growth and metabolism of cells. However, the culture medium has more than eighty to one hundred components, and the components are complex. When the concentration of each component reaches a certain amount, it not only cannot maintain the normal growth of cells, but even has a toxic effect on cells. The low-concentration culture medium can only support the growth and nutritional needs of cells in the early stage, and cannot effectively support the viability requirements of cell culture in the later stage.

[0004] Therefore, a nutritional supplement that can maintain or even improve cell viability in the later stages of cell culture has broad application prospects. Summary of the invention

[0005] Based on this, the purpose of the present invention is to overcome the shortcomings of the above-mentioned prior art and provide a nutritional supplement that can maintain or even improve cell viability in the later stage of cell culture, as well as a preparation method and application thereof.

[0006] To achieve the above object, the technical solution adopted by the present invention is: a nutritional supplement, comprising the following components in parts by weight: 0.05-0.6 parts of choline chloride, 0.1-0.6 parts of lithium chloride, and 0.1-0.2 parts of taurine.

[0007] The present invention provides a nutritional supplement that can maintain or even improve cell viability in the later stage of cell culture by combining taurine, lithium chloride and choline chloride. Among them, taurine can promote cell proliferation, reduce cell apoptosis, and enhance cell function. It can also be used as a nutritional factor and participate in cell metabolism, etc., and these effects jointly improve the effect of cell culture and the survival rate of cells. Lithium chloride can promote cell proliferation, inhibit cell migration, inhibit cell apoptosis, and has an immunomodulatory effect. Choline chloride is not only an important component of cell membrane, but also participates in methylation reaction in cells, has physiological and biochemical effects, and can improve the enzyme activity of microorganisms and improve the survival rate of cells. The three components of taurine, lithium chloride and choline chloride are used in combination under a specific ratio, which can produce a synergistic effect in terms of anti-oxidation and improving the activity of intracellular enzymes, thereby further improving the survival rate of cells.

[0008] Preferably, the weight ratio of choline chloride, lithium chloride and taurine is choline chloride: lithium chloride: taurine = (2-4): (2-5): 1.5. Further preferably, the weight ratio of choline chloride, lithium chloride and taurine is choline chloride: lithium chloride: taurine = 3: 5: 1.5.

[0009] The inventors found in actual experiments that the weight ratio of choline chloride, lithium chloride and taurine affects the cell viability in the later stage of cell culture. When the above specific weight ratio is used, the cell viability is higher.

[0010] In addition, the present invention provides a method for preparing the nutritional supplement, which is as follows: choline chloride, lithium chloride and taurine are uniformly mixed to obtain the nutritional supplement.

[0011] Furthermore, the present invention provides the use of the nutritional supplement in the later stage of cell culture.

[0012] Preferably, the late stage of cell culture is when the cells have entered the cell growth plateau phase and are about to enter the cell death phase.

[0013] Preferably, the cells in the cell culture are one of CHO cell line and HEK293 cell line.

[0014] Preferably, the CHO cell line includes one of CHO-K1 cells, CHO-DXB11 cells, CHO-DG44 cells, and CHO-S cells, and the HEK293 cell line includes Expi293F.

[0015] Preferably, the nutritional supplement accounts for 1% to 10% of the culture volume in the later stage of cell culture.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: the present invention provides a nutritional supplement that can maintain or even improve cell viability in the later stage of cell culture by combining three components: taurine, lithium chloride and choline chloride. Among them, taurine can promote cell proliferation, reduce cell apoptosis, and enhance cell function. At the same time, it can be used as a nutritional factor and participate in cell metabolism, etc. These effects jointly improve the effect of cell culture and the survival rate of cells. Lithium chloride can promote cell proliferation, inhibit cell migration, inhibit cell apoptosis, and has an immunomodulatory effect. Choline chloride is not only an important component of the cell membrane, but also participates in the methylation reaction in the cell, has physiological and biochemical effects, and can improve the enzyme activity of microorganisms and improve the survival rate of cells. DETAILED DESCRIPTION

[0017] To better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments, the purpose of which is to understand the content of the present invention in detail, rather than to limit the present invention. All other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention. The experimental reagents and instruments involved in the implementation of the present invention are all commonly used ordinary reagents and instruments unless otherwise specified.

[0018] Examples and Comparative Examples

[0019] The nutritional supplement of the present invention has the components shown in Tables 1-2. The preparation method of the nutritional supplement described in Table 1 is as follows: choline chloride, lithium chloride and taurine are uniformly mixed to obtain the nutritional supplement. The preparation method of the nutritional supplement described in Table 2 is the same as that in Table 1, except that if there are no relevant components, no relevant components are added, and if there are other components, they can be mixed and added.

[0020] Table 1

[0021]

[0022] Table 2

[0023]

[0024]

[0025] Performance Test 1 - CHO-K1 ATCC Cell Test.

[0026] Test method:

[0027] Step 1: Cell recovery and passaging: Take out a CHO-K1 ATCC cell from liquid nitrogen for recovery. Thaw the cell in a water bath at 37.0℃ and transfer it to OPM-CHO CD07 Medium (hereinafter referred to as "CD07"). The recovery culture volume is 20ml. The viable cell density (VCD) after recovery is 0.50±(0.20)×10 6 cells / ml. The cells were subcultured every three days. At the N-1 generation (seed culture of the previous generation), the screening pressure in the subculture medium needed to be removed, i.e., the subculture medium was CD07 (with 0.5% ACA added). The parameters of the seed cell culture shaker were as follows: temperature was 37.0±0.5℃, CO2 concentration was 8.0±1.0%, relative humidity was 80±10%, and the shaker speed was 120±10rpm (amplitude 50mm).

[0028] Step 2: Preparation of nutritional supplements with different ingredients; Measure 90% of the preparation volume of deionized water at room temperature (18°C-30°C) and start stirring. Slowly add the different nutritional supplements obtained in Table 1-Table 2 into the container and continue stirring. Use a calibrated container and dilute to a preparation volume of 100 ml with deionized water. Stir for 10 minutes. Measure the pH value of 7.0 and check and record the osmotic pressure of 300mOsmo / kg, and filter using a sterilizing filter membrane.

[0029] Step 3: Effects of different nutritional supplements on CHO-K1 ATCC cells; Take a certain volume of seed cells and add 0.50 (± 0.10) × 10 6 The initial density of cells / ml was inoculated into shake flasks A and 1-14 respectively, the basal medium was CD07 (supplemented with 0.5% ACA), the feed medium was OPM-CHO CDF36 (hereinafter referred to as "CDF36") and OPM-CHO CDFS36 (hereinafter referred to as "CDFS36"), and the culture volume was 60 ml.

[0030] 0.2 ml samples were taken from the shake flask every day and counted using a Beckman cell counter and a Roche Glucose concentration was measured by BioAnalyzer.

[0031] The shake flask A was supplemented with 5% CDF36 and 0.5% CDFS36 on day 3, 10% CDF36 and 1% CDFS36 on day 5, 10% CDFS36 and 1% CDFS36 on day 7, 10% CDF36 and 1% CDFS36 on day 9, and 5% CDF36 and 1% CDFS36 on day 11. Glucose was fed from less than 2 g / l to 6 g / l.

[0032] Shake flasks 1-14 were supplemented with 5% CDF36 and 0.5% CDFS36 on day 3, 10% CDF36 and 1% CDFS36 on day 5, 10% CDF36 and 1% CDFS36 on day 7, 10% CDF36 and 1% CDFS36 and 2% nutritional supplements on day 9, and 5% CDF36 and 1% CDFS36 and 2% nutritional supplements on day 11. Glucose was fed according to the principle of supplementing from less than 2 g / l to 6 g / l. The culture was terminated until the viability was less than 65% or on day 14 of culture, and the cell culture fluid was centrifuged to take the supernatant to detect cell output.

[0033] Among them, the nutritional supplements inoculated in shake flasks 1-14 are nutritional supplements No. 1-14 respectively, that is, the nutritional supplements inoculated in shake flask 1 throughout the entire process are nutritional supplement No. 1, and so on.

[0034] Cell density of shake flask A and shake flasks 1-14 (unit: ×10 6 The data of cell viability (unit: %) of shake flask A and shake flasks 1-14 are shown in Table 3, and the data of cell viability (unit: %) of shake flask A and shake flasks 1-14 are shown in Table 4, which are tested by Beckman cell counter, the same below. The protein yield at harvest is shown in Table 5, and the detection method is as follows: the culture supernatant is first purified by Protein-A affinity chromatography column (HiTrapProtein AHPcolumns, GE), and the operation is carried out according to the standard Protein-A instructions; the purified monoclonal antibody is quantified by high-performance affinity gel chromatography (1525Binary HPLC pump, 717plus Autosampler, 2487Dualλabsorbance detector, Waters, TSK-GELG3000SWXL, Tosoh, Japan).

[0035] The chromatographic operating conditions are as follows: mobile phase (2.4 g / L NaH2PO4, 11.7 g / L NaCl, pH = 7.4); detection wavelength (204 nm); flow rate (0.5 ml / min); time (30 min); injection volume (20 ul). The peak area was obtained by integration, and the concentration of each sample was calculated by comparing it with the peak area of ​​the standard.

[0036] Table 3

[0037]

[0038]

[0039] Table 4

[0040]

[0041]

[0042]

[0043] Table 5

[0044]

[0045] According to the cell density data and cell viability data tables of each shake flask, the specific ratio of nutritional supplements 1-5 (choline chloride / lithium chloride / taurine) performed best in maintaining the density and viability of cells in the later stage of culture. However, shake flask 1 without nutritional supplements was only cultured for thirteen days, and the viability at the end of culture was only 61.1%.

[0046] Nutritional supplement 9 (aluminum chloride / nicotinic acid) had an adverse effect on cell growth and viability maintenance. After being added on the 9th day of culture, the cell density and viability dropped rapidly, and the viability was only 50.3% on the 11th day of culture. Although nutritional supplement 6 (choline chloride / lithium chloride / taurine / betaine / nicotinic acid) had a certain effect on maintaining cell viability compared with shake flask 1 without nutritional supplements, it was still different from the shake flask with nutritional supplement 1. Combined with the data of shake flask F, it is possible that the addition of betaine and nicotinic acid affected the effects of other ingredients.

[0047] Performance Test 2 - CHO-DG44 cell test.

[0048] Step 1: Cell recovery and passaging

[0049] A CHO-DG44 cell was taken out from liquid nitrogen for recovery. The cells were thawed in a water bath at 37.0°C and transferred to OPM-CHO CD07 Medium (hereinafter referred to as "CD07"). The recovery culture volume was 20 ml. The viable cell density (VCD) after recovery was 0.50 (±0.20) × 10 6 cells / ml. The cells were subcultured every three days. At the N-1 generation (seed culture of the previous generation), the screening pressure in the subculture medium needed to be removed, i.e., the subculture medium was CD07 (with 0.5% ACA added). The parameters of the seed cell culture shaker were as follows: temperature was 37.0±0.5℃, CO2 concentration was 8.0±1.0%, relative humidity was 80±10%, and the shaker speed was 120±10rpm (amplitude 50mm).

[0050] Step 2: Cell culture

[0051] Take a certain volume of seed cells and add 0.50 (± 0.10) × 10 6The initial density of cells / ml was inoculated into shake flasks B and 1-14 respectively, the basal medium was CD07 (supplemented with 0.5% ACA), the feed medium was OPM-CHO CDF18 (hereinafter referred to as "CDF18") and OPM-CHO CDF26 (hereinafter referred to as "CDF26"), and the culture volume was 60 ml.

[0052] 0.2 ml samples were taken from the shake flask every day and counted using a Beckman cell counter and a Roche Glucose concentration was measured by BioAnalyzer. Shake flask A was supplemented with 5% CDF18 and 0.5% CDF26 on day 3, 10% CDF18 and 1% CDF26 on day 5, 10% CDF18 and 1% CDF26 on day 7, 10% CDF18 and 1% CDF26 on day 9, and 5% CDF18 and 1% CDF26 on day 11. Glucose was fed from less than 2 g / l to 6 g / l. Shake flasks 1-14 were supplemented with 5% CDF18 and 0.5% CDF26 on day 3, 10% CDF18 and 1% CDF26 on day 5, 10% CDF18 and 1% CDF26 on day 7, 10% CDF18 and 1% CDF26, 2% nutritional supplements on day 9, and 5% CDF18 and 1% CDF26, 2% nutritional supplements on day 11. Glucose was fed according to the principle of supplementing from less than 2 g / l to 6 g / l.

[0053] The culture was terminated on the 14th day, and the cell culture medium was centrifuged to obtain the supernatant to detect cell output.

[0054] Among them, the nutritional supplements inoculated in shake flasks 1-14 are nutritional supplements 1-14, that is, the nutritional supplements inoculated in shake flask 1 throughout the whole process are nutritional supplements 1, and so on. The cell density of shake flasks B and shake flasks 1-14 (unit: ×10 6The data of 100 cells / ml) are shown in Table 6, and the data of cell viability (unit: %) of shake flask B and shake flasks 1-14 are shown in Table 7. Protein yield detection method at harvest: The culture supernatant was first purified by Protein-A affinity chromatography column (HiTrap ProteinAHPcolumns, GE), and the operation was carried out according to the standard Protein-A instructions; the purified monoclonal antibody was quantified by high-performance affinity gel chromatography (1525Binary HPLC pump, 717plus Autosampler, 2487Dualλabsorbancedetector, Waters, TSK-GELG3000SWXL, Tosoh, Japan). The chromatographic operating conditions are as follows: mobile phase (2.4g / LNaH2PO4, 11.7g / L NaCl, pH=7.4); detection wavelength (204nm); flow rate (0.5ml / min); time (30min); injection volume (20ul). The peak area was obtained by integration, and the concentration of each sample was calculated by comparing with the peak area of ​​the standard. The test data is shown in Table 8.

[0055] Table 6

[0056]

[0057]

[0058]

[0059] Table 7

[0060]

[0061]

[0062] Table 8

[0063]

[0064] From the table above, we can see that the cells in shake flask A reached the highest density of 24.5×10 on the 8th day of culture. 6 cells / ml, the cell density and viability began to decline rapidly, and the cell viability was only 57% at the end of the culture. On the other hand, the cell density of shake flask B with nutritional supplements reached the highest density of 25×10 cells / ml on the 8th day of culture. 6 cells / ml, and then maintained a relatively good cell density and viability. At the end of the culture, the cell density was 15.0×10 6 cells / ml, and the cell viability was 79.2%, which was significantly superior to that of shake flask A without adding nutritional supplements.

[0065] From the perspective of cell protein expression, the protein expression level at the end of culture in shake flask A was 2 g / L, and the protein expression level at the end of culture in shake flask B was 4 g / L. Shake flask B with nutritional supplements added had a significant advantage in increasing protein expression compared to shake flask A without nutritional supplements added.

[0066] Performance test 3 - Expi293F cell test.

[0067] Step 1: Cell recovery and passaging

[0068] Take out a piece of Expi293F cells from liquid nitrogen for recovery. Thaw the cells in a water bath at 37.0℃ and transfer them to OPM-CHO CD05 Medium (hereinafter referred to as "CD05") by a robotic arm. The recovery culture volume is 20ml. The viable cell density (VCD) after recovery is 0.30 (±0.20)×10 6 cells / ml. Subculture every two to three days. Seed cell culture shaker parameter settings: temperature 37.0±0.5℃, CO2 concentration 8.0±1.0%, relative humidity 80±10%, shaker speed 100±10rpm (amplitude 50mm).

[0069] Step 2: Cell culture

[0070] The cells were expanded until the viable cell density reached about 2-3×10 ^6 cells / mL, cell viability ≥95%. Take a certain volume of seed cells and add 0.30 (±0.20) × 10 6 The initial density of cells / ml was inoculated into shake flasks C and 1-14, respectively, the basic culture medium was CD05, and the culture volume was 60 ml.

[0071] When the cell density reaches 5×10 6 When the transfection rate is 1:1, the plasmid DNA is diluted with OPM-293CD05 medium, and the PEI is diluted with OPM-293CD05 medium. The diluted PEI is mixed with the released plasmid DNA in a volume ratio of 1:1 and incubated at room temperature for 15-20 minutes. The mixture is slowly added to shake flasks C and 1-14. 16-22 hours after transfection, 5% OPM-293ProFeed v / v is added to shake flask C, and 5% OPM-293ProFeed v / v and 2% nutrient supplements are added to shake flask D. The flow addition of glucose in the two shake flasks is based on the principle of supplementing from less than 2g / l to 6g / l.

[0072] When the cell survival rate was lower than 50% or on the 14th day of culture, the cells were harvested, and the cell culture fluid was centrifuged to obtain the supernatant to detect the cell protein expression.

[0073] Among them, the nutritional supplements inoculated in shake flasks 1-14 are nutritional supplements No. 1-14 respectively, that is, the nutritional supplements inoculated in shake flask 1 throughout the entire process are nutritional supplement No. 1, and so on.

[0074] Cell density of shake flask C and shake flasks 1-14 (unit: ×10 6 The data of cell viability (unit: %) of shake flask C and shake flasks 1-14 are shown in Table 9, and the data of cell viability (unit: %) of shake flask C and shake flasks 1-14 are shown in Table 10. Protein yield detection method at harvest: The culture supernatant was first purified by Protein-A affinity chromatography column (HiTrap Protein AHPcolumns, GE), and the operation was carried out according to the standard Protein-A instructions; the purified monoclonal antibody was quantified by high-performance affinity gel chromatography

[0075] (1525Binary HPLC pump, 717plus Autosampler, 2487Dualλabsorbancedetector, Waters, TSK-GELG3000SWXL, Tosoh, Japan). Chromatographic operating conditions are as follows: mobile phase (2.4g / LNaH2PO4, 11.7g / L NaCl, pH=7.4); detection wavelength (204nm); flow rate (0.5ml / min); time (30min); injection volume (20ul). The peak area was obtained by integration, and the concentration of each sample was calculated by comparing with the peak area of ​​the standard. The test data are shown in Table 11.

[0076] Table 9

[0077]

[0078]

[0079] Table 10

[0080]

[0081]

[0082]

[0083] Table 11

[0084]

[0085] From the above cases, it can be seen that the specific types and dosages of nutritional supplements (choline chloride / lithium chloride / taurine) of the present invention have obvious effects on maintaining cell viability in different cell lines (CHO-DG44 / CHO-K1 / Expi293F) at the late stage of cell culture.

[0086] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. A nutritional supplement, characterized in that: The invention comprises the following components in parts by weight: 0.05-0.6 parts of choline chloride, 0.1-0.6 parts of lithium chloride and 0.1-0.2 parts of taurine.

2. The nutritional supplement according to claim 1, characterized in that The weight ratio of the choline chloride, lithium chloride and taurine is choline chloride:lithium chloride:taurine=(2-4):(2-5):1.

5.

3. The nutritional supplement according to claim 1, characterized in that The weight ratio of the choline chloride, lithium chloride and taurine is choline chloride: lithium chloride: taurine = 3:5:1.

5.

4. A method for preparing the nutritional supplement according to any one of claims 1 to 3, characterized in that: The preparation method is as follows: choline chloride, lithium chloride and taurine are evenly mixed to obtain the nutritional supplement.

5. Use of the nutritional supplement according to any one of claims 1 to 3 in the later stage of cell culture.

6. The use according to claim 5, characterized in that The cells in the cell culture are one of CHO cell line and HEK293 cell line.

7. The use according to claim 6, characterized in that The CHO cell line includes one of CHO-K1 cells, CHO-DXB11 cells, CHO-DG44 cells, and CHO-S cells, and the HEK293 cell line includes Expi293F cells.

8. The use according to claim 5, characterized in that The nutritional supplement accounts for 1% to 10% of the culture volume in the later stage of cell culture.

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