阅读说明：本技术 一种重组人血清白蛋白纯化过程去除色素的方法 (Method for removing pigment in purification process of recombinant human serum albumin ) 是由 齐智 常贝贝 艾云鹏 高磊 柏凯 于 2021-09-10 设计创作，主要内容包括：本发明提供了一种重组人血清蛋白纯化过程去除色素的方法,所述采用脱色树脂结合硼酸填料作为脱色剂,可快速有效的脱除重组人血白蛋白生产中产生的杂质色素,并且不影响蛋白收率和活性,具有广阔的应用前景。(The invention provides a method for removing pigment in the purification process of recombinant human serum albumin, which adopts decolorizing resin and boric acid filler as decolorizing agent, can rapidly and effectively remove impurity pigment generated in the production of recombinant human serum albumin, does not influence the yield and activity of protein, and has wide application prospect.)

1. The filler composition for removing the pigment in the purification process of the recombinant human serum albumin comprises anion decolorizing resin and boric acid filler, wherein the combination ratio of the anion decolorizing resin to the boric acid filler is 1-8: 1.

2. A method for removing pigments in a recombinant human serum albumin purification process, comprising the steps of:

(1) performing fermentation culture on a seed solution obtained by performing activation culture on the recombinant yeast strain containing the HSA gene;

(2) after fermentation is finished, taking fermentation liquor containing rHSA, and removing yeast cells through centrifugation to obtain rHSA concentrated fermentation supernatant;

(3) passing the rHSA concentrated fermentation supernatant obtained in step (2) through a decolorizing column, wherein the filler in the decolorizing column is the filler composition of claim 1.

3. The method of claim 2, wherein the volume ratio of the filler composition to the rHSA concentrated fermentation supernatant is from 1:10 to 1: 20.

4. The method of claim 3, wherein the filler composition has a combined anionic decolorizing resin and boric acid filler ratio of 4: 1.

5. The method of claim 3, wherein the volume ratio of the filler composition to rHSA concentrated fermentation supernatant is 1: 20.

6. The method of claim 3, wherein the step (2) comprises the steps of: taking fermentation liquor containing rHSA, removing yeast cells by a laminated centrifuge, wherein the rotating speed is as follows: 12000rpm, feed liquid temperature: 15 ℃, solid content: 20-30%, and performing microfiltration on the centrifugal supernatant with the pump frequency of 25-35 HZ; concentrating the permeate at 30KD under inlet pressure of not more than 0.2MPa, adding anhydrous ethanol and sodium caprylate to final concentration of 15mM in the concentrated solution at volume ratio of 40%, and heating at 68 deg.C for 30min to obtain 30KD concentrated solution.

7. The method of claim 3, wherein the step (3) comprises the steps of:

soaking the anion decolorizing resin in purified water for 24 hr, and swelling and dissolving; eluting the boric acid filler by a gradient of NaCl concentration from low to high, wherein the NaCl concentration gradient is set between 0.1M and 1M, and balancing by 20mMPB and PH7.0 sodium phosphate buffer solution after elution; mixing anion decolorizing resin and boric acid filler at a ratio of 4:1, loading into a filler column, passing the obtained concentrated fermentation supernatant through a decolorizing column, wherein the volume ratio of the anion decolorizing resin and boric acid filler composition to the sample solution is 1: 20.

Technical Field

The invention belongs to the technical field of bioengineering, and particularly relates to a method for quickly and effectively removing pigments in a purification process of gene recombinant albumin.

Background

Human Serum Albumin (HSA) is the most abundant protein in human plasma, accounts for 50% -60% of total plasma protein, has a concentration of 38-48 g/L, is the most main protein in human plasma, maintains the nutrition and osmotic pressure of an organism, and has important physiological functions in the organism. The liver synthesizes approximately 12g of albumin per day. HSA belongs to a single-chain non-glycosylated protein, which comprises 585 amino acids, has a molecular weight of 66.5kD, and an isoelectric point of 4.7-4.9. Human serum albumin, known as the "gold life-saving drug" in the blood, is used clinically mainly for the following indications: (1) shock caused by blood loss trauma and burn; (2) maintenance therapy for patients with advanced cancer; (3) elevated cranial pressure caused by cerebral edema and injury; (4) edema or ascites due to cancer, cirrhosis and nephropathy; (5) prevention and treatment of hypoproteinemia; (6) hyperbilirubinemia of the newborn; (7) cardiopulmonary bypass, burn or hemodialysis adjuvant therapy, adult respiratory distress syndrome, is a special medicine for clinical first aid.

In the field of biological medicine, human serum albumin can be used as a medicine for direct intravenous injection, can also be used as an excellent medicine carrier to prolong the in vivo half-life period of the medicine and realize long-acting effect of the medicine, and can also be used as a stabilizer of biological preparations such as vaccines and the like or used as a culture medium of cells such as CHO and the like. However, human serum albumin is derived from blood products, and blood resources are relatively scarce in China, so the price of human serum albumin is relatively expensive. And the method for extracting the human serum albumin by artificially culturing the human serum albumin by using the gene technology to replace the traditional human blood extraction method effectively widens the acquisition sources of the human serum albumin.

Professor e.j.cohn, harvard university, usa and his working group invented a process called the low temperature ethanol method, which for the first time purified human serum albumin from human blood. At present, the gene recombinant human serum albumin (rHSA) is mainly produced by using methanol yeast, and two impurities are generated in the production process: one is impurities that do not interact directly with albumin and these impurities can be removed using conventional precipitation, chromatographic separation, and the like. The other is small molecules such as pigments and the like which are wrapped inside or have strong interaction with the albumin. Once the pigment impurities are input into the human body, the life may be threatened. Therefore, pigment removal is a difficult problem which needs to be solved for reaching the pharmaceutical grade purity and is the most critical link.

Disclosure of Invention

In order to solve the above technical problems, the present application provides a simple method for removing pigments present in the purification process of rHSA, which efficiently performs decolorization by combining macroporous anion decolorizing resin with boric acid filler.

In a first aspect of the present invention, there is provided a filler composition for pigment removal in a recombinant human serum albumin purification process, the filler composition comprising an anionic decolorizing resin and a boric acid filler, wherein the combined ratio of the anionic decolorizing resin and the boric acid filler is 1-8:1, preferably, the combined ratio is 4: 1.

in a second aspect of the present invention, there is provided a method for removing pigments in a recombinant human serum albumin purification process, the method comprising the steps of:

(1) performing fermentation culture on a seed solution obtained by performing activation culture on the recombinant yeast strain containing the HSA gene;

(2) after fermentation is finished, taking fermentation liquor containing rHSA, and removing yeast cells through centrifugation to obtain rHSA concentrated fermentation supernatant;

(3) passing the rHSA concentrated fermentation supernatant obtained in step (2) through a decolorizing column, wherein the filler in the decolorizing column is the filler composition of claim 1.

In one embodiment, the volume ratio of the filler composition to the rHSA concentrated fermentation supernatant is 1:10 to 1:20, preferably, the volume ratio is 1: 20.

in one embodiment, the step (2) comprises the steps of: taking fermentation liquor containing rHSA, removing yeast cells by a laminated centrifuge, wherein the rotating speed is as follows: 12000rpm, feed liquid temperature: 15 ℃, solid content: 20-30%, and performing microfiltration on the centrifugal supernatant with the pump frequency of 25-35 HZ; concentrating the permeate at 30KD under inlet pressure of not more than 0.2MPa, adding 40 vol% ethanol and sodium caprylate to 15mM, and heating at 68 deg.C for 30min to obtain 30KD concentrated solution.

In one embodiment, the step (3) comprises the steps of: soaking the anion decolorizing resin in purified water for 24 hr, and swelling and dissolving; eluting the boric acid filler by a gradient of NaCl concentration from low to high, wherein the NaCl concentration gradient is set between 0.1M and 1M, and balancing by 20mMPB and PH7.0 sodium phosphate buffer solution after elution; mixing anion decolorizing resin and boric acid filler at a ratio of 4:1, loading into a filler column, passing the obtained concentrated fermentation supernatant through a decolorizing column, wherein the volume ratio of the anion decolorizing resin and boric acid filler composition to the sample solution is 1: 20.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention. In the drawings:

FIG. 1 is an SDS-PAGE pattern of a decolorized sample according to example of the present invention.

Detailed Description

The preferred embodiments of the present invention will be described in conjunction with the accompanying drawings, and it will be understood that they are described herein for the purpose of illustration and explanation and not limitation.

EXAMPLE 1 screening of purification conditions

The purification reagent A was selected from anionic decolorizing resin (model: POSITISIL decolorizing # 2) available from Beijing Enlaik scientific development Co., Ltd.

The purifying reagent B was obtained from Shanghai pharmaceutical company Shanghai Hu test AR grade (product number 10004818) by selecting a boric acid filler (hereinafter referred to as pb filler). The boric acid filler is a novel deglycosylated filler, and is mainly used for removing glycosylated proteins in the protein purification process.

Example 2 depigmentation procedure

1. Inoculating a seed solution obtained by activating and culturing a recombinant pichia pastoris strain containing an HSA gene into a primary seed tank for culturing; the jar contains YPD medium (YPD medium comprises glucose, yeast extract powder, and tryptone, and can be mixed at a certain ratio, or purchased from commercial product), the capacity of the first-stage seeding jar can be 30L, the culture is generally carried out for 12 hr, and when dissolved oxygen begins to rise, the jar is transferred into the second-stage seeding jar;

2. the secondary seed tank contains YPD culture medium, the capacity of the secondary seed tank can be 300L, the culture is carried out for about 8 hours, and the secondary seed tank is transferred into a fermentation tank when dissolved oxygen begins to rise;

3. adding an organic synthetic culture medium F121.6kg, phosphoric acid 18690mL, potassium hydroxide 2.891kg and glycerol 28kg into a fermentation tank, adding vitamin C and synthetic p1 supplementary solution before transferring into the fermentation liquid, wherein the formula of the synthetic p1 supplementary solution is as follows: 2g of copper sulfate, 0.02g of sodium iodide, 1g of manganese sulfate monohydrate, 0.1g of sodium molybdate dihydrate, 0.005g of boric acid, 5g of zinc chloride, 25g of ferrous sulfate and 5ml of sulfuric acid, and the volume of water is fixed to 1L; the organic synthetic medium F1 comprises the following components: peptone ═ 2: 1. When the wet weight ratio of the fermentation broth (the ratio of the fermentation broth to the cells) is greater than 26, methanol induction is performed. When the wet weight ratio (the ratio of the fermentation liquid to the thallus) of the fermentation liquid is more than or equal to 50 and the OD is more than or equal to 2.0, the fermentation is stopped. The total fermentation time is about 192 h;

4. after fermentation is finished, taking fermentation liquor containing rHSA, removing yeast cells through a laminated centrifuge, wherein the rotating speed is as follows: 12000rpm, feed liquid temperature: 15 ℃, solid content: 20 to 30 percent. Microfiltering the centrifugal clear liquid, and pumping at a frequency of 25-35 HZ; the inlet pressure does not exceed 0.2 MPa. Concentrating the permeate with 30KD, adding alcohol (40% by volume), adding sodium caprylate to final concentration of 15mM, and heating at 68 deg.C for 30min to obtain 30KD concentrate;

5. and (3) carrying out three-time parallel detection on the impurity content of the obtained concentrated solution by using a spectrophotometer, wherein the specific detection results are as shown in the following table 1:

TABLE 1

Remarking: the measurement results of different models of photometers have errors

When the ratio of A350/A280 is less than or equal to 0.05, the content of the pigment and the impurities in the solution can be ignored, the content of the pigment and the impurities is more when the ratio is larger, and the content of the pigment and the impurities is more when the ratios of A450/A280 and A500/A280 are larger. Therefore, as can be seen from the above table, the fermentation concentrate contains many impurities and pigments.

6. Decolouring column

Soaking anion decolorizing resin in purified water for 24 hr to swell and dissolve fully;

eluting the boric acid filler in a gradient of NaCl concentration from low to high, wherein the NaCl concentration gradient is set between 0.1M and 1M, and balancing the eluted boric acid filler with 20mMPB and pH7.0 sodium phosphate buffer solution;

thirdly, fully and uniformly mixing the anion decolorizing resin and the boric acid filler according to the proportion of 1:1,2:1,4:1 and 8:1, and filling the mixture into a filler column (arranging two fillers to be respectively and independently filled into the column as a comparison);

selecting the optimal proportion in the step (c), enabling the concentrated fermentation supernatant obtained in the step (4) to pass through a decolorizing column, wherein the volume ratio of the composition of the anion decolorizing resin and the boric acid filler to the sample solution is as follows: 1:10-1:20, and verifying the decoloring effect and yield.

Example 3 decolorization results

1. The decolorization of the fermentation broth by using the anionic decolorizing resin alone is specifically shown in the following table 2: (flow rate 50ml/min)

TABLE 2

2. The decolorization of the fermentation broth with boric acid alone is shown in table 3 below: (flow rate 50ml/min)

TABLE 3

As shown in the above results, when the anionic decolorizing resin or boric acid was used alone as a filler, the decolorizing effect on the fermentation broth was not significant.

3. The 60min decolorization condition of the fermentation liquor by using a plurality of anion decolorizing resins and boric acid combined fillers is specifically shown in the following table 4:

TABLE 4

The results show that the method has the advantages of high yield,the anion decolorizing resin and boric acid composite filler can obviously reduce the impurities and color of the fermentation liquor And (4) element. Mixing anion resin and boric acid filler at a mass ratio of 4:1, pretreating the fermentation broth, wherein A350/A280 can be When the temperature is reduced to 0.05, A450/A280 and A500/A280 are both 0 or lower than the detection line, and each index meets the requirement.

4. The decolorization conditions of the loading solutions and the decolorizing resin composite boric acid filler (anion decolorizing resin: boric acid filler 4:1) with different volume ratios in the elution time of 60min are specifically shown in the following table 5:

TABLE 5

The result shows that the decolorization effect is best when the volume ratio of the sample loading liquid to the decolorized resin composite boric acid filler is 1: 20.

5. The SDS-PAGE results of the decolorized samples are shown in FIG. 1, and from the results, the decolorized resin/boric acid filler does not substantially adsorb the target protein.

6. Protein yield

Firstly, mixing the sample solution and the decolorizing resin composite boric acid filler according to the proportion of 1:20, and then performing column filling after uniform mixing

② washing and eluting the chromatographic column with water for injection for 2 BV

③ sampling 200ml of purified concentrated solution, sampling and detecting PH, liquid phase and protein yield

The detection results are as follows:

as a result, it can be seen that when the decolorized resin/boric acid filler ratio is 4:1, the protein yield is relatively high.

It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.