Industrial production method of highly glycosylated recombinant human erythrocyte growth stimulating protein

Abstract

The invention discloses an industrialized production method for high galactosylated modification recombination human erythrocyte growth stimulation protein utilizing serum-free medium used for steadily improving protein expression index of Darbepoetin Alpha. The method comprises the following steps: adopting serum-free medium to cultivate CHO cell strain in a suspending manner in a biological reactor; expressing the Darbepoetin Alpha in high efficiency in the CHO cell. The invention further discloses the serum-free medium adopted by the industrialized production method for high galactosylated modification recombination human erythrocyte growth stimulation protein. The serum-free medium component used for steadily improving protein expression index of Darbepoetin Alpha is utilized; meanwhile, the CHO cell can efficiently and steadily express Darbepoetin Alpha; expression is stable; cultivation method is brief, and is suitable for large scale cultivation.

Description

technical field

[0001] The invention belongs to the technical field of biomedicine, and relates to an industrial production method of highly glycosylated recombinant human erythrocyte growth-stimulating protein, in particular to a high-glucose high-glucose medium using a serum-free medium that can be used to stably increase the expression of Darbepoetinα The invention relates to an industrialized production method of kylated modified recombinant human erythrocyte growth stimulating protein. Background technique

[0002] Erythropoietin (EPO) is a glycoprotein mainly secreted by the kidney, with a molecular weight of about 34kD. It is the main hormone regulating erythroid hematopoiesis in the human body. Binding, stimulating the proliferation, differentiation and maturation of erythroid progenitor cells, including erythroid burst colony-forming units (BFU-E), erythroid colony-forming units (CFU-E) and proerythroid cells. Impaired renal function leads to abnormal secretion of ...

Examples

Embodiment 1

[0039] This embodiment provides a serum-free medium, which includes a serum-free basal medium and additives.

[0040] The serum-free basal medium ratio of the present embodiment is determined like this:

[0041] Resuscitate the seed cells and amplify the culture, prepare the cell suspension, and inoculate them in three serum-free mediums 1, 2, and 3 respectively. The weight ratios of medium 1, 2, and 3 are 9:1, 3:1, respectively. , 1:1 combination medium of Ex-cell302 medium and CP1027 medium. Cultivate at 36+0.2°C, conduct 6-day sugar batch experiment, start from the 4th day of culture, count the cells every day, collect the culture supernatant, centrifuge to remove the cells and freeze them, and use the ELISA method to detect the expression level of the cells. The proportion of the acidic part of the cell expression product was detected by the method of IEF. In the evaluation, the proportion of the acidic part is taken as the primary consideration index. Evaluation result...

Embodiment 2

[0046] This example provides an industrial production method of highly glycosylated recombinant human erythrocyte growth-stimulating protein, using the serum-free medium of Example 1, and adopting a 5L reactor of animal cells for high-density, suspension perfusion culture, including the following step:

[0047] 1. Cell inoculation stage control

[0048] Resuscitate a 20mL CHO seed cell into a shaker flask, subculture and expand every 3 days, until the cell is expanded to 500mL, and the cell is in the logarithmic growth phase (the density is about 3×106cells / mL), inoculate the 500ml seed cell To the serum-free medium in the 5L NBSCELLIGEN310 bioreactor, the stirring speed is 100-200 rpm. The cell density after inoculation was 5.1×10 5 cells / mL, the initial working volume of the reactor was 3L, and the serum-free medium inoculated with CHO cells was obtained.

[0049] 2. Cell growth stage control

[0050] Under the conditions of temperature 36+0.2°C, pH 6.90-7.20, and dissol...

Embodiment 3

[0057] This example provides an industrial production method of highly glycosylated recombinant human erythrocyte growth-stimulating protein, using the serum-free medium of Example 1, adopting high-density, suspension perfusion culture of animal cells in a 30L reactor, including the following steps :

[0058] 1. Cell inoculation stage control

[0059] Resuscitate a 20mL CHO seeded cell into the shake flask, subculture and expand every 3 days, until the cell is expanded to 5000mL, and the cell is in the logarithmic growth phase (the density is 3×10 6 cells / mL), the 5000ml seed cells were inoculated into the serum-free medium in the SartouriusStedium C-Plus30L bioreactor, and the stirring speed was 100-200 rpm. The cell density after inoculation was 6.6×10 5 cells / mL, the initial working volume of the reactor was 30L, and a serum-free medium inoculated with CHO cells was obtained.

[0060] 2. Cell growth stage control

[0061] Under the conditions of temperature 36+0.2°C, pH...

technical field

[0001] The invention belongs to the technical field of biomedicine, and relates to an industrial production method of highly glycosylated recombinant human erythrocyte growth-stimulating protein, in particular to a high-glucose high-glucose medium using a serum-free medium that can be used to stably increase the expression of Darbepoetinα The invention relates to an industrialized production method of kylated modified recombinant human erythrocyte growth stimulating protein. Background technique

[0002] Erythropoietin (EPO) is a glycoprotein mainly secreted by the kidney, with a molecular weight of about 34kD. It is the main hormone regulating erythroid hematopoiesis in the human body. Binding, stimulating the proliferation, differentiation and maturation of erythroid progenitor cells, including erythroid burst colony-forming units (BFU-E), erythroid colony-forming units (CFU-E) and proerythroid cells. Impaired renal function leads to abnormal secretion of ...

Examples

Embodiment 1

[0039] This embodiment provides a serum-free medium, which includes a serum-free basal medium and additives.

[0040] The serum-free basal medium ratio of the present embodiment is determined like this:

[0041] Resuscitate the seed cells and amplify the culture, prepare the cell suspension, and inoculate them in three serum-free mediums 1, 2, and 3 respectively. The weight ratios of medium 1, 2, and 3 are 9:1, 3:1, respectively. , 1:1 combination medium of Ex-cell302 medium and CP1027 medium. Cultivate at 36+0.2°C, conduct 6-day sugar batch experiment, start from the 4th day of culture, count the cells every day, collect the culture supernatant, centrifuge to remove the cells and freeze them, and use the ELISA method to detect the expression level of the cells. The proportion of the acidic part of the cell expression product was detected by the method of IEF. In the evaluation, the proportion of the acidic part is taken as the primary consideration index. Evaluation result...

Embodiment 2

[0046] This example provides an industrial production method of highly glycosylated recombinant human erythrocyte growth-stimulating protein, using the serum-free medium of Example 1, and adopting a 5L reactor of animal cells for high-density, suspension perfusion culture, including the following step:

[0047] 1. Cell inoculation stage control

[0048] Resuscitate a 20mL CHO seed cell into a shaker flask, subculture and expand every 3 days, until the cell is expanded to 500mL, and the cell is in the logarithmic growth phase (the density is about 3×106cells / mL), inoculate the 500ml seed cell To the serum-free medium in the 5L NBSCELLIGEN310 bioreactor, the stirring speed is 100-200 rpm. The cell density after inoculation was 5.1×10 5 cells / mL, the initial working volume of the reactor was 3L, and the serum-free medium inoculated with CHO cells was obtained.

[0049] 2. Cell growth stage control

[0050] Under the conditions of temperature 36+0.2°C, pH 6.90-7.20, and dissol...

Embodiment 3

[0057] This example provides an industrial production method of highly glycosylated recombinant human erythrocyte growth-stimulating protein, using the serum-free medium of Example 1, adopting high-density, suspension perfusion culture of animal cells in a 30L reactor, including the following steps :

[0058] 1. Cell inoculation stage control

[0059] Resuscitate a 20mL CHO seeded cell into the shake flask, subculture and expand every 3 days, until the cell is expanded to 5000mL, and the cell is in the logarithmic growth phase (the density is 3×10 6 cells / mL), the 5000ml seed cells were inoculated into the serum-free medium in the SartouriusStedium C-Plus30L bioreactor, and the stirring speed was 100-200 rpm. The cell density after inoculation was 6.6×10 5 cells / mL, the initial working volume of the reactor was 30L, and a serum-free medium inoculated with CHO cells was obtained.

[0060] 2. Cell growth stage control

[0061] Under the conditions of temperature 36+0.2°C, pH...

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