Separating and purifying method for recombinant human serum albumin
A technology for the separation and purification of human serum albumin, which is applied in the direction of serum albumin, albumin peptide, and peptide preparation methods, can solve the problems of complex human blood sources and blood source pollution, and achieve low purification costs and short time-consuming , the effect of easy operation
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Embodiment 1
[0043] Embodiment 1, utilize hot ethanol precipitation method to carry out crude purification to rHSA in transgenic pig plasma, comprise the following steps:
[0044] A) Anticoagulant treatment of plasma and centrifugation to remove blood cells: Take a few milliliters of fresh transgenic pig blood, mix it with 80g / L sodium citrate solution at a volume ratio of 19:1, centrifuge at 2000×g for 30min, and obtain the supernatant, namely transgenic Pig plasma was frozen at -20°C for later use.
[0045] B) Cryoprecipitation: After slowly thawing the frozen plasma in step A at 4°C, centrifuge at 5000×g for 15 minutes to remove a little yellow-white precipitate to obtain a plasma supernatant.
[0046] C) Hot ethanol precipitation method: take 100mL of the plasma supernatant in step B and place it in the reactor, first add 100mL of an aqueous solution containing 0.6g sodium octanoate and 0.8g sodium chloride, then add 10mL of ethanol, mix well and adjust the pH to 6.5. Under the condit...
Embodiment 2
[0047] Example 2, the first step of refining and purifying the above-mentioned rHSA-containing crude extract by using anion exchange chromatography includes the following steps:
[0048] The rHSA crude extract in Example 1 was desalted and concentrated, and then loaded on an anion exchange chromatography. The chromatographic column used is a DEAE weak anion exchange chromatographic column, and its chromatographic conditions: mobile phase A is 0.02mol / L Tris-HCl (pH 8.8), mobile phase B is 0.02mol / L Tris-HCl (pH 8.8), 0.3 mol / L NaCl (pH 8.8); flow rate 1mL / min; detection wavelength 280nm; column temperature 25°C (room temperature); linear gradient elution program: 0 to the end of the 5th minute, 100% A; from the 6th minute to the end of the 50th minute , 100% A ~ 0% A; from the 51st minute to the end of the 60th minute, 0% A. (Preparative chromatogram see attached figure 1 a). Collect the target fraction eluate. The purity of rHSA characterized by RP-HPLC was 85.0%.
Embodiment 3
[0049] Embodiment 3, the second step of refining and purifying the product after anion exchange chromatography by reverse phase chromatography comprises the following steps:
[0050] The eluate in Example 2 was desalted and concentrated and then divided into two equal parts of the DEAE sample; one of them was subjected to secondary purification by reverse phase chromatography; the chromatographic column used was a C4 reverse phase chromatographic column, and its chromatographic conditions were : mobile phase A is 2% (volume ratio) acetonitrile-0.1% trifluoroacetic acid solution, mobile phase B is 98% acetonitrile-0.1% trifluoroacetic acid solution; flow rate 0.7mL / min; detection wavelength 280nm; column temperature 35 ℃; The linear gradient elution program is: 0 to the end of the 10th minute, 100% A; from the 11th minute to the end of 45 minutes, 100% A to 5% A; from the 46th minute to the end of the 50th minute, 5% A; from the 51st minute to the 53rd minute At the end, 5% A ~...
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