Serum-free medium of recombinant human erythropoietin, applications of serum-free medium and method for preparing recombinant human erythropoietin
A serum-free medium and erythropoietin technology, which is applied in artificial cell constructs, animal cells, vertebrate cells, etc., can solve the problems of complicated instruments and high investment costs, improve yield, improve buffer capacity, and prolong survival. effect of time
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Embodiment 1～7
[0042] The preparation method of the serum-free medium is as follows: take the CHO SFM medium as shown in Table 1, add sodium bicarbonate, 4-hydroxyethylpiperazineethanesulfonic acid, sodium n-butyrate, D-glucose, D- After adding a certain amount of water to dissolve galactose, D-mannose, and N-acetylglucosamine, add a certain amount of soybean protein hydrolyzate prepared by the above method and make the volume to 1L. Filter the prepared medium through a 0.22 μm filter membrane to obtain the osmolality of the medium is 320-360 mOsmol kg -1 , pH is 6.9-7.2.
[0043] Among them: the prototype medium adopts 302CHO SFM medium produced by GIBCO Company. The preparation method of soybean protein hydrolyzate is: take soybean protein powder 100g (protein ≥ 50% water content ≤ 7% fat ≤ 1% ash content ≤ 4% total fiber content ≤ 4%), add 85 ℃ hot water 800mL, keep constant temperature 30 Minutes, adjust the pH to 5.0-7.0, add 100ml of papain (2%), heat up to 90°C and keep for 30 minut...
experiment example 1
[0048] The recombinant human erythropoietin was cultured according to the formula serum-free medium of Examples 1-4, cultured at 36-37°C, and the supernatant was collected on the 7th day of serum-free culture, and detected by isoelectric focusing / immunoblotting. Its spectrum is as figure 1 Shown (from left to right are respectively embodiment 1~4). It can be seen from the test results that the upper part of the supernatant is invalid EPO, which has a low degree of glycosylation and low activity in vivo, and part needs to be removed during purification; the lower part is effective EPO, which has a high degree of glycosylation. High activity in vivo, part of which is retained in purification. Depend on figure 1 It can be seen that the effective EPO amount accounts for more than 50% of the total expressed EPO amount, and the purification yield is increased by at least 50%.
[0049] Adopt the serum-free culture medium of the embodiment of the present invention 1~4 to carry ou...
experiment example 2
[0054] According to the serum-free medium of the formula in Example 2, the culture of recombinant human erythropoietin was carried out. The number of culture bottles was 10 bottles (the specification was 3000ml, and the actual culture medium capacity was 600ml), cultivated at 36~37°C for 7 days, and harvested the cell culture medium. Afterwards, new serum-free medium was added for culture, and the cycle was repeated three times. The co-culture was 21 days, and harvested three times; the cell culture medium was harvested about 18L, the average expression of EPO was 19203IU / ml, and the total harvest was 3.45×10 8 IU. The relationship between expression level and culture time in the first cycle is as follows image 3 shown.
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