A two-step mixed-mode chromatography method for separating recombinant human albumin

Abstract

The present invention discloses a two-step mixed mode chromatography method for separating recombinant human albumin, which belongs to protein chromatography separation technology. The specific steps are as follows: 1) fermentation broth pretreatment, taking yeast fermentation broth containing recombinant human albumin, adding sodium octanoate, heating to remove foreign proteins and inactivate proteases, centrifugation, and taking the supernatant as feed liquid; 2) the first step of mixed mode chromatography, using (2-(1H-indole-3-yl) acetyl) cysteine ​​as a ligand medium, loading feed liquid, eluting and collecting eluent; 3) the second step of mixed mode chromatography, using 4-amino-N-(2-pyridyl) benzenesulfonamide as a ligand medium, the loading conditions are consistent with the first step elution conditions, collecting the flow-through liquid, and obtaining recombinant human albumin. The present invention develops a two-step purification process, using two mixed mode media, efficiently separating recombinant human albumin from yeast fermentation broth, with mild separation conditions, high separation efficiency, and high impurity removal rate.

Description

Technical Field

[0001] The invention belongs to the technical field of protein separation and relates to a two-step mixed mode chromatography method for separating recombinant human serum albumin. Background Art

[0002] Human albumin is the most abundant protein in human plasma. It consists of 585 amino acid residues and has three homologous regions. Its main functions are to maintain plasma osmotic pressure and transport exogenous substances and metabolites. Human albumin is widely used to treat traumatic shock, burns, acute liver failure, hypoproteinemia, etc., and has wide application value. In recent years, the global demand for human albumin has exceeded 600 tons / year. At present, human albumin products for injection are mainly purified from human plasma, which is limited by the insufficient plasma source and the potential risk of pathogen infection. Using yeast cells to express recombinant human albumin can avoid the risk of pathogen infection and solve problems such as insufficient plasma source.

[0003] Patent US5521287 discloses a method of combining cation exchange chromatography, hydrophobic interaction chromatography, and metal chelate affinity chromatography, which can separate recombinant human albumin with high purity from yeast fermentation broth. Patent CN102190722A discloses a method for purifying recombinant human albumin, which uses anion exchange expanded bed adsorption to capture recombinant human albumin from yeast fermentation broth, and then undergoes heating, hydrophobic interaction chromatography, cation exchange chromatography, ultrafiltration, chelation, precipitation and other steps to obtain recombinant human albumin with a purity greater than 99%. However, this method has many steps and is complicated, with a yield of 32.2%. Patent CN1191545 discloses a method for purifying recombinant human albumin from yeast fermentation broth by ion exchange chromatography. Due to the poor salt tolerance of the ion exchange medium, the fermentation broth needs to be diluted, resulting in a low separation effect. Patent US5962649 uses cation exchange expanded bed adsorption to treat yeast fermentation broth. The feed liquid needs to be diluted and the pH is adjusted to acidic conditions, resulting in partial degradation of recombinant human albumin. The above method has the problems of complex process, fermentation broth needs pretreatment, low yield, etc. Developing a new separation method with high selectivity, simple process and high yield will help improve process efficiency and save preparation costs.

[0004] Mixed-mode chromatography is a new and efficient bio-separation technology. The ligand has multiple functional groups, high separation selectivity, large adsorption capacity, and mild elution conditions. Patent CN110681362A discloses a mixed-mode medium with carboxyl and indole as functional groups, which exhibits advantages such as large adsorption capacity, high selectivity, and good salt tolerance for human albumin. The article (Journal of Separation Science, 43(21):4028-4035, 2020 and Journal of Chemical Engineering of Colleges and Universities, 35(3):492-499, 2021) uses a mixed-mode medium with (2-(1H-indol-3-yl)acetyl)cysteine ​​as a ligand to separate recombinant human albumin from fermentation broth. The purity and yield can reach 90%, but it still contains trace impurities such as aggregates, pigments, and host cell proteins that need to be removed. Summary of the invention

[0005] In view of the above problems, the present invention designs a two-step mixed-mode chromatography method for separating recombinant human albumin. The first step is an adsorption-elution mode, using a mixed-mode medium with (2-(1H-indol-3-yl)acetyl)cysteine ​​as a ligand to directly capture recombinant human albumin from yeast fermentation broth; the second step is a flow-through mode, using a mixed-mode medium with 4-amino-N-(2-pyridyl)benzenesulfonamide as a ligand to adsorb impurities and allow recombinant human albumin to flow through, thereby obtaining high-purity recombinant human albumin.

[0006] The purity of human serum albumin obtained by the method of the present invention is greater than 95%, and the host cell protein removal rate reaches 99%. It has the characteristics of mild separation conditions, high separation efficiency and high impurity removal rate.

[0007] The present invention provides a two-step mixed mode chromatography method for separating recombinant human serum albumin, which specifically comprises the following steps:

[0008] 1) taking yeast fermentation broth containing recombinant human albumin and pretreating it to obtain a crude solution containing recombinant human albumin;

[0009] 2) adjusting the pH of the crude recombinant human albumin solution to 4.0-5.0, filtering and loading the solution into a chromatography column filled with a mixed mode medium with (2-(1H-indol-3-yl)acetyl)cysteine ​​as a ligand, washing with an equilibrium buffer, eluting with an elution buffer, and collecting the eluate corresponding to the elution peak;

[0010] 3) The eluate obtained in step 2) is loaded into a chromatography column filled with a mixed mode medium with 4-amino-N-(2-pyridyl)benzenesulfonamide as a ligand, washed with equilibrium buffer, the recombinant human albumin flows through, the impurities are adsorbed, and the flow-through liquid is collected to obtain a recombinant human albumin solution.

[0011] As a preferred embodiment of the present invention, in the step 1), the pretreatment method is: taking yeast fermentation broth containing recombinant human albumin, the recombinant human albumin concentration is 2-10 mg / mL, adjusting the pH to 6.0, adding sodium octanoate to a concentration of 10-20 mM sodium octanoate, heating at 68°C for 30 min, rapidly cooling to room temperature, centrifuging and taking the supernatant to obtain a crude solution containing recombinant human albumin.

[0012] As a preferred embodiment of the present invention, the acid solution used to adjust the pH value of the yeast fermentation liquid is 0.1M acetic acid or citric acid solution, and the alkaline solution used is 0.1M sodium hydroxide solution.

[0013] As a preferred embodiment of the present invention, in the step 1), the yeast fermentation broth containing recombinant human albumin is Pichia pastoris fermentation broth or Saccharomyces cerevisiae fermentation broth.

[0014] As a preferred embodiment of the present invention, the ligand density of the mixed mode medium used in step 2) is 100-200 μmol / L.

[0015] As a preferred embodiment of the present invention, the equilibrium buffer in step 2) is acetic acid-sodium acetate buffer with a pH value of 4.0-5.0.

[0016] As a preferred embodiment of the present invention, the filtration in step 2) uses a filter membrane with a pore size of 0.45 μm.

[0017] As a preferred embodiment of the present invention, the elution buffer in step 2) is a phosphate buffer added with 0.2-0.6M NaCl or a Tris-hydrochloric acid buffer added with 0.2-0.6M NaCl, and the pH value is 8.0-9.0.

[0018] As a preferred embodiment of the present invention, the ligand density of the mixed mode medium used in step 3) is 30-100 μmol / L.

[0019] As a preferred embodiment of the present invention, the equilibration buffer in step 3) is a phosphate buffer added with 0.2-0.6M NaCl or a Tris-hydrochloric acid buffer added with 0.2-0.6M NaCl, and the pH value is 8.0-9.0.

[0020] The present invention makes full use of the advantages of mixed-mode chromatography such as large adsorption capacity, good selectivity, mild elution conditions, etc., to form a two-step mixed-mode chromatography, improve separation efficiency, simplify separation steps, and achieve efficient separation of recombinant human albumin from yeast fermentation broth.

[0021] The advantages of the present invention are:

[0022] 1) Optimize the combination of adsorption elution mode and flow-through mode to form a new two-step mixed mode chromatography separation process, which is simple and efficient;

[0023] 2) The adsorption-elution mode uses a mixed mode medium with (2-(1H-indol-3-yl)acetyl)cysteine ​​as a ligand to achieve the initial separation of recombinant human albumin and remove most impurities;

[0024] 3) The flow-through mode uses a mixed mode medium with 4-amino-N-(2-pyridyl)benzenesulfonamide as the ligand to achieve efficient adsorption of residual impurities, with a host cell protein removal rate of up to 99%;

[0025] 4) The purity of recombinant human albumin obtained by two-step chromatography reaches more than 95% and the yield is more than 80%;

[0026] 5) The two-step separation process is easy to connect. The elution conditions of the first mixed mode chromatography are used as the loading conditions of the second mixed mode chromatography. No feed pretreatment or buffer exchange is required, and the appropriate pH and salt concentration are the key. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 is a schematic diagram of the structure of (2-(1H-indol-3-yl)acetyl)cysteine ​​ligand;

[0028] Figure 2 4-amino-N-(2-pyridyl)benzenesulfonamide ligand structure schematic diagram;

[0029] Figure 3 These are HPLC analysis diagrams of different samples, which are respectively a crude recombinant human albumin solution, a flow-through component of the first mixed mode chromatography, an elution component of the first mixed mode chromatography, and a flow-through component of the second mixed mode chromatography. DETAILED DESCRIPTION

[0030] The present invention provides a two-step mixed mode chromatography method for separating recombinant human albumin. Yeast fermentation broth containing recombinant human albumin is taken, sodium octanoate is added, heated to remove impurities and inactivated proteases, rapidly cooled to room temperature, centrifuged, and the supernatant is taken; the supernatant is separated by adsorption elution mode through a chromatography column equipped with a mixed mode medium containing (2-(1H-indol-3-yl)acetyl)cysteine ​​as a ligand, the eluate is collected, and the eluate is separated by flow-through mode through a chromatography column equipped with a mixed mode medium containing 4-amino-N-(2-pyridyl)benzenesulfonamide as a ligand to obtain recombinant human albumin. The purity of human albumin obtained by the method of the present invention is greater than 95%, and the host cell protein removal rate reaches 99%.

[0031] The two-step mixed mode chromatography method for separating recombinant human albumin comprises the following steps:

[0032] 1) taking yeast fermentation broth containing recombinant human albumin, adjusting the pH to 6.0, adding sodium octanoate to a sodium octanoate concentration of 10-20 mM, heating at 68° C. for 30 min, rapidly cooling to room temperature, centrifuging and taking the supernatant to obtain a crude recombinant human albumin solution;

[0033] 2) The pH of the crude recombinant human albumin solution was adjusted to 4.0-5.0, filtered with a 0.45 μm filter membrane, and then loaded into a chromatography column filled with a mixed mode medium with (2-(1H-indol-3-yl)acetyl)cysteine ​​as a ligand. The ligand structure is as follows: Figure 1 As shown, the equilibration buffer is used for washing, the elution buffer is used for elution, and the eluate corresponding to the elution peak is collected;

[0034] 3) After filtering the elution buffer with a 0.45 μm filter membrane, the sample is loaded into a chromatography column filled with a mixed mode medium with 4-amino-N-(2-pyridyl)benzenesulfonamide as a ligand. The ligand structure is as follows: Figure 2 As shown, the flow-through liquid corresponding to the flow-through peak is collected to obtain a recombinant human albumin solution; the final protein purity is greater than 95%, and the host cell protein removal rate reaches 99%.

[0035] Example 1

[0036] Take the Pichia fermentation broth containing recombinant human albumin, the conductivity is about 20mS / cm, and the concentration of recombinant human albumin is 2.0mg / mL. Adjust the pH to 6.0, add sodium octanoate to 10mM, heat at 68℃ for 30min, precipitate impurities and inactivate proteases, quickly cool to room temperature, centrifuge and take the supernatant to obtain a crude solution. Adjust the pH value of the crude solution to 4.0, filter with a 0.45μm filter membrane, and take 20mL as the injection sample. Fill the chromatography column (inner diameter 0.5cm) with 1mL of a chromatography medium with (2-(1H-indol-3-yl)acetyl)cysteine ​​as a ligand, the ligand density is 100μmol / L, the equilibrium buffer is 20mM acetic acid-sodium acetate buffer (pH 4.0), and the eluent is 20mM sodium dihydrogen phosphate-disodium hydrogen phosphate buffer (pH 8.0) with 0.2M NaCl added. Collect the eluted components and take 12mL as the sample for subsequent separation. A chromatography column (inner diameter 0.5 cm) was filled with 1 mL of a chromatography medium with 4-amino-N-(2-pyridyl)benzenesulfonamide as a ligand, the ligand density was 30 μmol / L, the equilibrium buffer was 20 mM sodium dihydrogen phosphate-disodium hydrogen phosphate buffer (pH 8.0) added with 0.2 M NaCl, the sample was loaded, and the flow-through was collected to obtain recombinant human serum albumin, the purity of which was 95.1% and the yield was 81.2% according to HPLC analysis, and the host cell protein removal rate was 99.8% according to ELISA analysis.

[0037] Example 2

[0038] Take the Pichia fermentation broth containing recombinant human albumin, the conductivity is about 20mS / cm, and the concentration of recombinant human albumin is 2.4mg / mL. Adjust the pH to 6.0, add sodium octanoate to 10mM, heat at 68℃ for 30min to precipitate impurities and inactivate proteases, quickly cool to room temperature, centrifuge and take the supernatant to obtain a crude solution. Adjust the pH value of the crude solution to 4.0, filter with a 0.45μm filter membrane, and take 16mL as the injection sample. A chromatography column (inner diameter 0.5 cm) was filled with 1 mL of a chromatography medium with (2-(1H-indol-3-yl)acetyl)cysteine ​​as a ligand, the ligand density was 100 μmol / L, the equilibrium buffer was 20 mM acetic acid-sodium acetate buffer (pH 4.0), the eluent was 20 mM sodium dihydrogen phosphate-disodium hydrogen phosphate buffer (pH 8.0) added with 0.4 M NaCl, the eluted fractions were collected, and 12 mL was taken as a sample for subsequent separation. A chromatography column (inner diameter 0.5 cm) was filled with 1 mL of a chromatography medium with 4-amino-N-(2-pyridyl)benzenesulfonamide as a ligand, the ligand density was 65 μmol / L, and the equilibrium buffer was 20 mM sodium dihydrogen phosphate-disodium hydrogen phosphate buffer (pH 8.0) added with 0.4 M NaCl. The flow-through was collected to obtain recombinant human albumin, with a purity of 95.3% and a yield of 80.1% according to HPLC analysis. The host cell protein removal rate according to ELISA analysis was 99.2%.

[0039] Example 3

[0040] Take the Pichia fermentation broth containing recombinant human albumin, the conductivity is about 20mS / cm, and the concentration of recombinant human albumin is 5.1mg / mL. Adjust the pH to 6.0, add sodium octanoate to 10mM, heat at 68℃ for 30min, precipitate impurities and inactivate proteases, quickly cool to room temperature, centrifuge and take the supernatant to obtain a crude solution. Adjust the pH value of the crude solution to 4.5, filter with a 0.45μm filter membrane, and take 8.0mL as the injection sample. A chromatography column (inner diameter 0.5 cm) was filled with 1 mL of a chromatography medium with (2-(1H-indol-3-yl)acetyl)cysteine ​​as a ligand, the ligand density was 100 μmol / L, the equilibrium buffer was 20 mM acetic acid-sodium acetate buffer (pH 4.5), the eluent was 20 mM sodium dihydrogen phosphate-disodium hydrogen phosphate buffer (pH 8.0) added with 0.6 M NaCl, the eluted fractions were collected, and 8.0 mL was taken as a sample for subsequent separation. A chromatography column (inner diameter 0.5 cm) was filled with 1 mL of a chromatography medium with 4-amino-N-(2-pyridyl)benzenesulfonamide as a ligand, the ligand density was 100 μmol / L, the equilibrium buffer was 20 mM sodium dihydrogen phosphate-disodium hydrogen phosphate buffer (pH 8.0) added with 0.6 M NaCl, the flow-through was collected to obtain recombinant human albumin, the purity of HPLC analysis was 94.3%, the yield was 80.2%, and the host cell protein removal rate of ELISA analysis was 99.5%.

[0041] Example 4

[0042] Take the fermentation broth of Saccharomyces cerevisiae containing recombinant human albumin, the conductivity is about 20mS / cm, and the concentration of recombinant human albumin is 10mg / mL. Adjust the pH to 6.0, add sodium octanoate to 10mM, heat at 68℃ for 30min, precipitate impurities and inactivate proteases, quickly cool to room temperature, centrifuge and take the supernatant to obtain a crude solution. Adjust the pH value of the crude solution to 4.5, filter with a 0.45μm filter membrane, and take 5mL as the injection sample. Fill the chromatography column (inner diameter 0.5cm) with 1mL of a chromatography medium with (2-(1H-indol-3-yl)acetyl)cysteine ​​as a ligand, the ligand density is 150μmol / L, the equilibrium buffer is 20mM acetic acid-sodium acetate buffer (pH 4.5), and the eluent is 20mM Tris-hydrochloric acid buffer (pH 9.0) with 0.2M NaCl added. Collect the eluted components and take 12mL as the sample for subsequent separation. A chromatography column (inner diameter 0.5 cm) was filled with 1 mL of a chromatography medium with 4-amino-N-(2-pyridyl)benzenesulfonamide as a ligand, the ligand density was 30 μmol / L, and the equilibrium buffer was 20 mM Tris-hydrochloric acid buffer (pH 9.0) with 0.2 M NaCl added. The flow-through was collected to obtain recombinant human albumin, with a purity of 95.1% and a yield of 81.3% according to HPLC analysis. The host cell protein removal rate was 99.5% according to ELISA analysis.

[0043] Example 5

[0044] Take the Pichia fermentation broth containing recombinant human albumin, the conductivity is about 20mS / cm, and the concentration of recombinant human albumin is 2.4mg / mL. Adjust the pH to 6.0, add sodium octanoate to 10mM, heat at 68℃ for 30min to precipitate impurities and inactivate proteases, quickly cool to room temperature, centrifuge and take the supernatant to obtain a crude solution. Adjust the pH value of the crude solution to 4.5, filter with a 0.45μm filter membrane, and take 20mL as the injection sample. A chromatography column (inner diameter 0.5 cm) was filled with 1 mL of a chromatography medium with (2-(1H-indol-3-yl)acetyl)cysteine ​​as a ligand, the ligand density was 150 μmol / L, the equilibrium buffer was 20 mM acetic acid-sodium acetate buffer (pH 4.5), the eluent was 20 mM sodium dihydrogen phosphate-disodium hydrogen phosphate buffer (pH 8.0) added with 0.2 M NaCl, the eluted fractions were collected, and 10 mL was taken as a sample for subsequent separation. The chromatography column (inner diameter 0.5 cm) was filled with 1 mL of a chromatography medium with 4-amino-N-(2-pyridyl)benzenesulfonamide as a ligand, the ligand density was 65 μmol / L, and the equilibrium buffer was 20 mM sodium dihydrogen phosphate-disodium hydrogen phosphate buffer (pH 8.0) with 0.2 M NaCl added. The flow-through was collected to obtain recombinant human albumin, with a purity of 95.1% and a yield of 81.3% according to HPLC analysis. The host cell protein removal rate according to ELISA analysis was 99.5%. The HPLC analysis of different samples is shown in the attached figure. Figure 3 Part (a) is the yeast fermentation broth sample pretreated in step 1, which can be found to contain more impurities; Part (b) is the flow-through obtained after the chromatographic separation in step 2, which can be found that most of the impurities are removed by the flow-through; Part (c) is the eluate obtained after the chromatographic separation in step 2, which can be found to obtain relatively pure recombinant human albumin; Part (d) is the flow-through obtained after the chromatographic separation in step 3, which can be found to be further purified and the impurity content is further reduced.

[0045] Example 6

[0046] Take the Pichia fermentation broth containing recombinant human albumin, the conductivity is about 20mS / cm, and the concentration of recombinant human albumin is 5.1mg / mL. Adjust the pH to 6.0, add sodium octanoate to 10mM, heat at 68℃ for 30min to precipitate impurities and inactivate proteases, quickly cool to room temperature, centrifuge and take the supernatant to obtain a crude solution. Adjust the pH value of the crude solution to 4.5, filter with a 0.45μm filter membrane, and take 10mL as the injection sample. A chromatography column (inner diameter 0.5 cm) was filled with 1 mL of a chromatography medium with (2-(1H-indol-3-yl)acetyl)cysteine ​​as a ligand, the ligand density was 150 μmol / L, the equilibrium buffer was 20 mM acetic acid-sodium acetate buffer (pH 4.5), the eluent was 20 mM sodium dihydrogen phosphate-disodium hydrogen phosphate buffer (pH 9.0) added with 0.4 M NaCl, the eluted fractions were collected, and 8.0 mL was taken as a sample for subsequent separation. A chromatography column (inner diameter 0.5 cm) was filled with 1 mL of a chromatography medium with 4-amino-N-(2-pyridyl)benzenesulfonamide as a ligand, the ligand density was 100 μmol / L, and the equilibrium buffer was 20 mM sodium dihydrogen phosphate-disodium hydrogen phosphate buffer (pH 9.0) added with 0.4 M NaCl. The flow-through was collected to obtain recombinant human albumin, with a purity of 95.0% and a yield of 80.3% according to HPLC analysis. The host cell protein removal rate was 99.1% according to ELISA analysis.

[0047] Example 7

[0048] Take the Pichia fermentation broth containing recombinant human albumin, the conductivity is about 20mS / cm, and the concentration of recombinant human albumin is 10mg / mL. Adjust the pH to 6.0, add sodium octanoate to 10mM, heat at 68℃ for 30min, precipitate impurities and inactivate proteases, quickly cool to room temperature, centrifuge and take the supernatant to obtain a crude solution. Adjust the pH value of the crude solution to 4.5, filter with a 0.45μm filter membrane, and take 6.0mL as the injection sample. Fill the chromatography column (inner diameter 0.5cm) with 1mL of a chromatography medium with (2-(1H-indol-3-yl)acetyl)cysteine ​​as a ligand, the ligand density is 200μmol / L, the equilibrium buffer is 20mM acetic acid-sodium acetate buffer (pH4.5), and the eluent is 20mM sodium dihydrogen phosphate-disodium hydrogen phosphate buffer (pH 8.0) with 0.6M NaCl added. Collect the eluted components and take 12mL as the sample for subsequent separation. A chromatography column (inner diameter 0.5 cm) was filled with 1 mL of a chromatography medium with 4-amino-N-(2-pyridyl)benzenesulfonamide as a ligand, the ligand density was 30 μmol / L, and the equilibrium buffer was 20 mM sodium dihydrogen phosphate-disodium hydrogen phosphate buffer (pH 8.0) added with 0.6 M NaCl. The flow-through was collected to obtain recombinant human albumin, with a purity of 95.0% and a yield of 81.0% according to HPLC analysis. The host cell protein removal rate was 99.4% according to ELISA analysis.

[0049] Example 8

[0050] Take the Pichia fermentation broth containing recombinant human albumin, the conductivity is about 20mS / cm, and the concentration of recombinant human albumin is 5.0mg / mL. Adjust the pH to 6.0, add sodium octanoate to 10mM, heat at 68℃ for 30min to precipitate impurities and inactivate proteases, quickly cool to room temperature, centrifuge and take the supernatant to obtain a crude solution. Adjust the pH value of the crude solution to 5.0, filter with a 0.45μm filter membrane, and take 12mL as the injection sample. A chromatography column (inner diameter 0.5 cm) was filled with 1 mL of a chromatography medium with (2-(1H-indol-3-yl)acetyl)cysteine ​​as a ligand, the ligand density was 200 μmol / L, the equilibrium buffer was 20 mM acetic acid-sodium acetate buffer (pH 5.0), the eluent was 20 mM sodium dihydrogen phosphate-disodium hydrogen phosphate buffer (pH 8.0) added with 0.2 M NaCl, the eluted fractions were collected, and 10 mL was taken as a sample for subsequent separation. A chromatography column (inner diameter 0.5 cm) was filled with 1 mL of a chromatography medium with 4-amino-N-(2-pyridyl)benzenesulfonamide as a ligand, the ligand density was 65 μmol / L, and the equilibrium buffer was 20 mM sodium dihydrogen phosphate-disodium hydrogen phosphate buffer (pH 8.0) added with 0.2 M NaCl. The flow-through was collected to obtain recombinant human albumin, with a purity of 95.1% and a yield of 81.2% according to HPLC analysis. The host cell protein removal rate was 99.5% according to ELISA analysis.

[0051] Example 9

[0052] Take the fermentation broth of Saccharomyces cerevisiae containing recombinant human albumin, the conductivity is about 20mS / cm, and the concentration of recombinant human albumin is 5.0mg / mL. Adjust the pH to 6.0, add sodium octanoate to 10mM, heat at 68℃ for 30min, precipitate impurities and inactivate proteases, quickly cool to room temperature, centrifuge and take the supernatant to obtain a crude solution. Adjust the pH value of the crude solution to 5.0, filter with a 0.45μm filter membrane, and take 12mL as the injection sample. Fill the chromatography column (inner diameter 0.5cm) with 1mL of a chromatography medium with (2-(1H-indol-3-yl)acetyl)cysteine ​​as a ligand, the ligand density is 200μmol / L, the equilibrium buffer is 20mM acetic acid-sodium acetate buffer (pH 5.0), and the eluent is 20mM Tris-hydrochloric acid buffer (pH 8.0) with 0.6M NaCl added. Collect the eluted components and take 8.0mL as the sample for subsequent separation. A chromatography column (inner diameter 0.5 cm) was filled with 1 mL of a chromatography medium with 4-amino-N-(2-pyridyl)benzenesulfonamide as a ligand, the ligand density was 100 μmol / L, and the equilibrium buffer was 20 mM Tris-hydrochloric acid buffer (pH 8.0) added with 0.6 M NaCl. The flow-through was collected to obtain recombinant human albumin, with a purity of 95.1% and a yield of 81.1% according to HPLC analysis. The host cell protein removal rate was 99.7% according to ELISA analysis.

[0053] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the present invention. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the attached claims.

Claims

1. A two-step mixed mode chromatography method for separating recombinant human albumin, characterized in that The steps include: 1) Taking yeast fermentation broth containing recombinant human albumin, pre-treating to obtain a crude solution containing recombinant human albumin; the pre-treatment method is: taking yeast fermentation broth containing recombinant human albumin, adjusting the pH to 6.0, adding sodium octanoate to a concentration of 10 mM, heating at 68°C for 30 min, rapidly cooling to room temperature, centrifuging to obtain the supernatant, and obtaining a crude solution containing recombinant human albumin; 2) Adjust the pH of the crude recombinant human albumin solution to 4.0-5.0, filter and load it into a chromatography column filled with a mixed-mode medium with (2-(1H-indol-3-yl)acetyl)cysteine ​​as a ligand, wash with an equilibrium buffer, elute with an elution buffer, and collect the eluate corresponding to the elution peak; the ligand density of the mixed-mode medium used in step 2) is 100-200 μmol / L; the elution buffer in step 2) is a phosphate buffer with 0.2-0.6 M NaCl or a Tris-hydrochloric acid buffer with 0.2-0.6 M NaCl, with a pH of 8.0-9.0; 3) The eluate obtained in step 2) is loaded onto a chromatography column filled with a mixed-mode medium with 4-amino-N-(2-pyridyl)benzenesulfonamide as a ligand, washed with an equilibrium buffer, the recombinant human albumin flows through, the impurities are adsorbed, and the flow-through is collected to obtain a recombinant human albumin solution; the ligand density of the mixed-mode medium used in step 3) is 30-100 μmol / L; the equilibrium buffer in step 3) is a phosphate buffer with 0.2-0.6 M NaCl added or a Tris-hydrochloric acid buffer with 0.2-0.6 M NaCl added, and the pH value is 8.0-9.

0.

2. A two-step mixed mode chromatography method for separating recombinant human albumin according to claim 1, characterized in that: In the step 1), the yeast fermentation broth containing recombinant human albumin is Pichia pastoris fermentation broth or Saccharomyces cerevisiae fermentation broth.

3. A two-step mixed mode chromatography method for separating recombinant human albumin according to claim 1, characterized in that The acid solution used to adjust the pH value of the yeast fermentation liquid is acetic acid or citric acid solution, and the alkali solution used is sodium hydroxide solution.

4. A two-step mixed mode chromatography method for separating recombinant human albumin according to claim 1, characterized in that The equilibrium buffer in step 2) is acetic acid-sodium acetate buffer with a pH value of 4.0-5.

0.

5. A two-step mixed mode chromatography method for separating recombinant human albumin according to claim 1, characterized in that The filtration in step 2) uses a filter membrane with a pore size of 0.45 μm.

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