CN107033236B - Mixed mode chromatography method for separating human serum albumin from yeast fermentation liquor - Google Patents
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Abstract
The invention discloses a mixed mode chromatography method for separating human serum albumin from yeast fermentation liquor, belonging to a protein chromatography separation technology in the field of biochemical engineering. The method comprises the following specific steps: 1) pretreating fermentation liquor, taking yeast fermentation liquor containing human serum albumin, centrifuging to remove yeast cells, adding sodium caprylate, heating, removing foreign proteins and inactivating protease, centrifuging, and taking supernatant; 2) and (3) performing column chromatography, adopting a mixed-mode medium with tryptophan as a ligand, separating supernatant through fixed bed chromatography, loading the supernatant to a pH value of 4.0-4.5, eluting the supernatant to a pH value of 7.0-9.0, and collecting an elution peak. 3) Desalting and drying, desalting the collected liquid, and freeze-drying to obtain human serum albumin with the purity of more than 95%. The invention is characterized in that a new separation process is developed, and high-purity human serum albumin can be directly separated and obtained from yeast fermentation liquor. The key point of the method is that a mixed-mode medium taking tryptophan as a ligand is adopted, the yeast fermentation liquor does not need to adjust the ionic strength, is adsorbed under an acidic condition, is eluted in neutral and weak alkaline conditions, and has the characteristics of mild elution conditions, simple process, high separation efficiency and high yield.
Description
Technical Field
The invention belongs to the technical field of protein separation, and relates to a mixed mode chromatography method for separating human serum albumin from yeast fermentation liquor.
Background
Human albumin is the most abundant protein in human plasma, accounting for about 60% of plasma protein, and has the main physiological functions of maintaining plasma osmotic pressure, binding and transporting nutrients. The composition is clinically used for treating hemorrhagic shock, traumatic shock, acute hypovolemia, hypoalbuminemia and the like. Can also be used as an additive component, a medicament auxiliary agent, an excipient and the like for cell culture, and has wide application value.
The human serum albumin product used in clinic is mainly extracted and purified from human plasma, the preparation method comprises cold ethanol precipitation, ammonium sulfate precipitation, rivanol precipitation, caprylate precipitation, chromatography and the like, the raw materials are tense, the separation is complex, and the influence of viruses or other potential pathogenic factors cannot be eliminated. The yeast cell for expressing recombinant human serum albumin is constructed by utilizing the modern biotechnology, so that the virus infection can be avoided, the problem of blood source supply tension and the like can be solved, and the method has good application prospect. However, the requirement for the purity of medicinal human serum albumin is high, the components of yeast fermentation liquor are complex, and various impurities such as protease, heteroprotein, nucleic acid, fatty acid, pigment, polysaccharide, pyrogen and the like are contained, and the impurities can be removed only by adopting a multi-step chromatography method.
The pH value of the yeast fermentation liquor is about 6.0 generally, and the conductance is 15-25 mS/cm. The patent CN1127299 and US5521287 use the steps of heating, diluting, adjusting acid, cation exchange chromatography, hydrophobic interaction chromatography, metal chelating chromatography, anion exchange chromatography, calcium borate precipitation and the like to obtain the human serum albumin with higher purity. Patent US5962649 replaces cation exchange chromatography with cation exchange expanded bed adsorption, shortening the process. However, the two methods adopt cation exchange chromatography to capture human serum albumin, the separation selectivity is limited, and meanwhile, the feed liquid needs to be diluted, so that the concentration of a target substance is reduced, the adsorption capacity is reduced, the treatment capacity is increased, and the process time is prolonged. Patent CN1854155 discloses a Capto MMC medium capturing human serum albumin under acidic condition, eluting with high salt to remove impurities, and eluting under neutral and salt-added conditions to obtain human serum albumin, wherein the albumin yield obtained by the method is 65.7%. In patent CN 101768206A, the sample loading and elution process of patent CN1854155 is kept unchanged, and the intermediate leaching step is removed to obtain the human serum albumin with the purity of 92% and the yield of 71.3%. Patent CN101260145 also discloses a chromatographic purification process for separating recombinant human serum albumin and fusion protein thereof by Capto MMC, wherein the yield and purity are both above 80%. The Capto MMC-based patents have similar problems, the purity and yield are generally low, part of the processes need high-salt leaching to remove impurity adsorption, and the process is complex. Aiming at the characteristics of yeast fermentation liquor, the capture efficiency of human serum albumin is improved, and a novel separation method which has high selectivity, salt resistance, high yield and simple process is developed, so that the method is beneficial to simplifying the separation process of human serum albumin, improving the efficiency and saving the cost.
Mixed mode chromatography is a novel biological separation technology, and the ligand has multiple functional groups, can generate multiple interactions with target protein, mainly hydrophobic and electrostatic interactions. The ligand density of the mixed-mode medium is generally higher, the adsorption capacity is large, the mixed-mode medium has salt-tolerant adsorption characteristics, the elution condition is mild, and the mixed-mode medium is particularly suitable for large-scale separation and purification and has been applied to the separation and purification of proteins such as antibodies and the like. The MX-Trp-650M medium is a mixed-mode medium developed by TOSOH BIOSCIENCE company, and is prepared by taking polymethacrylate microspheres as a matrix, coupling the amino group of tryptophan onto the matrix through an amido bond to be used as a functional ligand, wherein the ligand structure is shown in figure 1, and the MX-Trp-650M medium is mainly used for refining and purifying antibodies, such as patent US0264685A 1. The application of MX-Trp-650M and similar media in separating human serum albumin from yeast fermentation liquor is not found in the reference of domestic and foreign patents and documents
The invention utilizes the advantages of mixed mode chromatography and the specificity of combination of human serum albumin and small molecules, selects mixed mode medium with tryptophan as main functional ligand, directly captures human serum albumin from yeast fermentation liquor, and develops a new method for separating human serum albumin by mixed mode chromatography.
Disclosure of Invention
The invention aims to provide a mixed mode chromatography method for separating human serum albumin from yeast fermentation liquor, which comprises the following steps:
1) taking yeast fermentation liquor containing human serum albumin, removing yeast cells through centrifugation, adjusting the pH value to 6.0, adding sodium caprylate to 5-20 mM, heating at 68 ℃ for 30min, centrifuging at 8000-10000 rpm for 10-20 min by using a centrifuge, and taking supernatant to obtain a crude human serum albumin solution;
2) adjusting the pH value of the human serum albumin crude product solution to 4.0-4.5, filtering with a 0.45-micrometer filter membrane, loading the filtered human serum albumin crude product solution into a chromatographic column filled with a mixed-mode medium taking tryptophan as a ligand, washing with a balance buffer solution, eluting with an elution buffer solution, and collecting the elution buffer solution corresponding to an elution peak to obtain a human serum albumin solution;
3) desalting the human serum albumin solution, and freeze-drying to obtain human serum albumin with the purity of more than 95%;
the yeast fermentation liquor containing the human serum albumin is fermentation liquor of pichia pastoris expression recombinant human serum albumin, or fermentation liquor of saccharomyces cerevisiae expression recombinant human serum albumin;
the mixed mode medium is MX-Trp-650M, or a chromatography medium taking amido bond coupling tryptophan amino as ligand;
the balance buffer solution is an acetic acid-sodium acetate buffer solution, and the pH value is 4.0-4.5;
the elution buffer solution is a sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution, a Tris-hydrochloric acid buffer solution or a glycine-sodium hydroxide buffer solution, the pH value is 7.0-9.0, and 0-0.6M NaCl is added;
the acid solution used for adjusting the pH value of the yeast fermentation liquid in the step 1) and the pH value of the human serum albumin crude product solution in the step 3) is acetic acid or citric acid solution, and the alkali solution used is sodium hydroxide solution.
Aiming at the characteristics of complex components and higher electrical conductivity of yeast fermentation liquor, the invention adopts mixed-mode medium to directly treat the pretreated yeast fermentation liquor, fully utilizes the advantages of large adsorption capacity, good selectivity, salt-tolerant property, mild elution condition and the like of mixed-mode chromatography, improves the separation efficiency, simplifies the separation steps, and obtains a novel method for directly separating human serum albumin from the yeast fermentation liquor. The invention has the advantages that: 1) the mixed-mode medium with tryptophan as a main functional ligand is adopted, the specific combination of the tryptophan and the human serum albumin is utilized, the adsorption selectivity of the human serum albumin is improved, the single-step chromatographic separation purity is up to more than 95%, and the pigment removal rate is up to more than 90%; 2) the mixed-mode medium has good salt-tolerant adsorption characteristic, the feed liquid is directly loaded without adjusting the ionic strength, the pretreatment step is simplified, and the separation efficiency is improved;
3) the adsorption capacity of the mixed-mode medium is large, the sample loading amount reaches more than 40mg/ml of medium, and the process handling capacity is large; 4) the elution condition is mild, effective elution is realized by utilizing electrostatic repulsion under the condition of neutral pH or alkalescence, and the recovery rate of human serum albumin is high and reaches more than 92 percent; 5) the process is simple and easy to amplify.
Drawings
FIG. 1 is a schematic representation of the ligand structure of a mixed mode medium.
FIG. 2 is the chromatogram separating chart of the mixed mode chromatography separating human albumin of the invention.
FIG. 3 is a high performance liquid chromatography analysis of the feed and eluted fractions of the mixed mode chromatography separation of the present invention.
Detailed Description
The invention provides a method for separating human serum albumin from yeast fermentation liquor. Taking yeast fermentation liquor containing human serum albumin, centrifuging and clarifying, adding sodium caprylate, heating, removing impurity protein and inactivating protease, centrifuging, and taking supernatant; separating the supernatant with mixed mode chromatographic column to obtain human serum albumin. The human serum albumin obtained by the method has the purity of more than 95 percent.
The method for separating the human serum albumin from the yeast fermentation liquor comprises the following steps:
1) taking yeast fermentation liquor containing human serum albumin, removing yeast cells through centrifugation, adjusting the pH value to 6.0, adding sodium caprylate to 5-20 mM, heating at 68 ℃ for 30min, centrifuging at 8000-10000 rpm for 10-20 min by using a centrifuge, and taking supernatant to obtain a crude human serum albumin solution;
2) adjusting the pH value of the human serum albumin crude product solution to 4.0-4.5, filtering with a 0.45-micrometer filter membrane, loading the filtered human serum albumin crude product solution into a chromatographic column filled with a mixed-mode medium taking tryptophan as a ligand, washing with a balance buffer solution, eluting with an elution buffer solution, and collecting the elution buffer solution corresponding to an elution peak to obtain a human serum albumin solution;
3) desalting the human serum albumin solution, and freeze-drying to obtain human serum albumin with the purity of more than 95%;
the yeast fermentation liquor containing the human serum albumin is fermentation liquor of pichia pastoris expression recombinant human serum albumin, or fermentation liquor of saccharomyces cerevisiae expression recombinant human serum albumin;
the mixed mode medium is MX-Trp-650M, or a chromatography medium taking amido bond coupling tryptophan amino as ligand;
the balance buffer solution is an acetic acid-sodium acetate buffer solution, and the pH value is 4.0-4.5;
the elution buffer solution is a sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution, a Tris-hydrochloric acid buffer solution or a glycine-sodium hydroxide buffer solution, the pH value is 7.0-9.0, and 0-0.6M NaCl is added;
the acid solution used for adjusting the pH value of the yeast fermentation liquid in the step 1) and the pH value of the human serum albumin crude product solution in the step 3) is acetic acid or citric acid solution, and the alkali solution used is sodium hydroxide solution.
Example 1
Taking pichia pastoris fermentation liquor containing recombinant human serum albumin, wherein the conductivity is about 24mS/cm, and the concentration of the human serum albumin is 10 mg/ml. Clarifying by centrifuging, adjusting pH to 6.0, adding sodium caprylate to 5mM, heating at 68 deg.C for 30min, precipitating impurity protein and inactivating protease, centrifuging at 8000rpm for 20min to obtain supernatant. Adjusting the pH value of the supernatant to 4.0, filtering with a 0.45 μm filter membrane, and taking 10ml as a sample. 2ml of MX-Trp-650M medium was filled in a chromatographic column (inner diameter 0.5cm), the equilibrium buffer solution was 20mM acetic acid-sodium acetate buffer solution (pH 4.0), the eluent was 20mM sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution (pH 7.0), the eluted fractions were collected, desalted and freeze-dried to give human serum albumin, which had an HPLC analytical purity of 96.6%, a yield of 96.0% and a pigment removal rate of 90.2%.
Example 2
Taking pichia pastoris fermentation liquor containing recombinant human serum albumin, wherein the conductivity is about 24mS/cm, and the concentration of the human serum albumin is 10 mg/ml. After the supernatant is clarified by centrifugation, the pH is adjusted to 6.0, sodium caprylate is added to 20mM, the mixture is heated at 68 ℃ for 30min, the mixed protein and the inactivated protease are precipitated, and the mixture is centrifuged at 10000rpm for 10min to obtain the supernatant. Adjusting the pH value of the supernatant to 4.5, filtering with a 0.45-micron filter membrane, and taking 10ml as a sample. The column (inner diameter 0.5cm) was packed with 2ml of MX-Trp-650M medium, the equilibrium buffer was 20mM acetic acid-sodium acetate buffer (pH 4.5), the eluent was 20mM sodium dihydrogen phosphate-disodium hydrogen phosphate buffer (pH 7.0) supplemented with 0.2M NaCl, and the fractions were collected, desalted, freeze-dried to give human serum albumin with an HPLC assay purity of 96.7%, yield of 97.8% and pigment removal rate of 91.4%.
Example 3
Taking pichia pastoris fermentation liquor containing recombinant human serum albumin, wherein the conductivity is about 24mS/cm, and the concentration of the human serum albumin is 10 mg/ml. After clarification by centrifugation, the pH was adjusted to 6.0, sodium caprylate was added to 15mM, heated at 68 ℃ for 30min, the impure protein and the inactivated protease were precipitated, and centrifuged at 9000rpm for 15min to obtain the supernatant. Adjusting the pH value of the supernatant to 4.2, filtering with a 0.45-micron filter membrane, and taking 10ml as a sample. The chromatography column (inner diameter 0.5cm) is filled with 2ml of MX-Trp-650M medium, the equilibrium buffer solution is 20mM acetic acid-sodium acetate buffer solution (pH 4.2), the eluent is 20mM sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution (pH 7.0) added with 0.4M NaCl, the elution components are collected, desalted and freeze-dried to obtain human albumin, the chromatographic separation spectrogram is shown in figure 2, the HPLC analysis purity is 98.5%, the yield is 97.4%, the pigment removal rate is 92.5%, and the liquid phase analysis spectrogram is shown in figure 3.
Example 4
Taking pichia pastoris fermentation liquor containing recombinant human serum albumin, wherein the conductivity is about 24mS/cm, and the concentration of the human serum albumin is 10 mg/ml. After clarification by centrifugation, the pH was adjusted to 6.0, sodium caprylate was added to 15mM, heated at 68 ℃ for 30min, the impure protein and the inactivated protease were precipitated, and centrifuged at 9000rpm for 15min to obtain the supernatant. Adjusting the pH value of the supernatant to 4.0, filtering with a 0.45 μm filter membrane, and taking 10ml as a sample. 2ml of MX-Trp-650M medium is filled in a chromatographic column (inner diameter is 0.5cm), an equilibrium buffer solution is 20mM acetic acid-sodium acetate buffer solution (pH 4.0), an eluent is 50mM Tris-hydrochloric acid buffer solution (pH 8.0), elution components are collected, desalted and freeze-dried to obtain the human serum albumin, the HPLC analysis purity is 96.8%, the yield is 96.3%, and the pigment removal rate is 91.1%.
Example 5
Taking pichia pastoris fermentation liquor containing recombinant human serum albumin, wherein the conductivity is about 24mS/cm, and the concentration of the human serum albumin is 10 mg/ml. Clarifying by centrifuging, adjusting pH to 6.0, adding sodium caprylate to 20mM, heating at 68 deg.C for 30min, precipitating impurity protein and inactivating protease, and centrifuging at 8000rpm for 20min to obtain supernatant. Adjusting the pH value of the supernatant to 4.5, filtering with a 0.45-micron filter membrane, and taking 10ml as a sample. 2ml of MX-Trp-650M medium was packed in a column (inner diameter 0.5cm), the equilibrium buffer was 20mM acetic acid-sodium acetate buffer (pH 4.5), the eluent was 50mM glycine-sodium hydroxide buffer (pH 9.0), and the fractions were collected, desalted and freeze-dried to give human serum albumin with an HPLC analytical purity of 95.2%, a yield of 95.3% and a pigment removal rate of 90.2%.
Example 6
And (3) taking the saccharomyces cerevisiae fermentation liquor containing the recombinant human serum albumin, wherein the conductivity is about 20mS/cm, and the concentration of the human serum albumin is 8 mg/ml. After clarification by centrifugation, the pH was adjusted to 6.0, sodium caprylate was added to 10mM, heated at 68 ℃ for 30min, the impure protein and the inactivated protease were precipitated, and centrifuged at 9000rpm for 15min to obtain the supernatant. Adjusting the pH value of the supernatant to 4.0, filtering with a 0.45 μm filter membrane, and taking 10ml as a sample. The column (inner diameter 0.5cm) was packed with 2ml of MX-Trp-650M medium, the equilibrium buffer was 20mM acetic acid-sodium acetate buffer (pH 4.0), the eluent was 20mM sodium dihydrogen phosphate-disodium hydrogen phosphate buffer (pH 7.0) supplemented with 0.6M NaCl, and the fractions were collected, desalted, freeze-dried to give human serum albumin with an HPLC assay purity of 96.8%, a yield of 97.5% and a pigment removal rate of 90.0%.
Example 7
Taking pichia pastoris fermentation liquor containing recombinant human serum albumin, wherein the conductivity is about 24mS/cm, and the concentration of the human serum albumin is 10 mg/ml. After clarification by centrifugation, the pH was adjusted to 6.0, sodium caprylate was added to 15mM, heated at 68 ℃ for 30min, the impure protein and the inactivated protease were precipitated, and centrifuged at 9000rpm for 15min to obtain the supernatant. Adjusting the pH value of the supernatant to 4.0, filtering with a 0.45 μm filter membrane, and taking 10ml as a sample. The chromatographic column (inner diameter 0.5cm) was filled with 2ml of an agarose medium using an amino group of an amide bond-coupled tryptophan as a ligand, an equilibrium buffer solution was 20mM acetic acid-sodium acetate buffer solution (pH 4.0), and an eluent was 20mM sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution (pH 7.0) to which 0.2M NaCl was added, and the eluate fractions were collected, desalted, and freeze-dried to obtain human serum albumin, the HPLC analysis purity was 96.7%, the yield was 97.0%, and the pigment removal rate was 92.2%.
Example 8
Taking pichia pastoris fermentation liquor containing recombinant human serum albumin, wherein the conductivity is about 24mS/cm, and the concentration of the human serum albumin is 10 mg/ml. After clarification by centrifugation, the pH was adjusted to 6.0, sodium caprylate was added to 15mM, heated at 68 ℃ for 30min, the impure protein and the inactivated protease were precipitated, and centrifuged at 9000rpm for 15min to obtain the supernatant. Adjusting the pH value of the supernatant to 4.0, filtering with a 0.45 μm filter membrane, and taking 10ml as a sample. The chromatographic column (inner diameter 0.5cm) was filled with 2ml of cellulose medium using the amino group of the amide bond-coupled tryptophan as a ligand, the equilibrium buffer was 20mM acetic acid-sodium acetate buffer (pH 4.0), the eluent was 20mM sodium dihydrogen phosphate-disodium hydrogen phosphate buffer (pH 7.0) to which 0.2M NaCl was added, and the eluted fractions were collected, desalted, and freeze-dried to obtain human serum albumin, the HPLC analysis purity was 97.2%, the yield was 96.5%, and the pigment removal rate was 91.1%.
Claims (4)
1. A mixed mode chromatography method for separating human serum albumin from yeast fermentation liquor is characterized by comprising the following steps:
1) taking yeast fermentation liquor containing human serum albumin, removing yeast cells by centrifugation, adjusting the pH to 6.0, adding sodium caprylate to 5-20 mM, 68%oC, heating for 30min, centrifuging for 10-20 min at 8000-10000 rpm by using a centrifuge, and taking supernatant to obtain a crude human serum albumin solution; the yeast fermentation liquor containing the human serum albumin is fermentation liquor of pichia pastoris expression recombinant human serum albumin, or fermentation liquor of saccharomyces cerevisiae expression recombinant human serum albumin;
2) adjusting the pH value of the human serum albumin crude product solution to 4.0-4.5, filtering with a 0.45-micrometer filter membrane, loading the filtered human serum albumin crude product solution into a chromatographic column filled with a mixed-mode medium taking tryptophan as a ligand, washing with a balance buffer solution, eluting with an elution buffer solution, and collecting the elution buffer solution corresponding to an elution peak to obtain a human serum albumin solution;
3) desalting the human serum albumin solution, and freeze-drying to obtain human serum albumin with the purity of more than 95%;
the mixed mode medium is a chromatography medium taking amido bond coupling tryptophan amido as ligand;
the pH value of the balance buffer solution is 4.0-4.5;
the pH value of the elution buffer solution is 7.0-9.0.
2. The mixed mode chromatography method for separating human serum albumin from yeast fermentation broth of claim 1, wherein said equilibration buffer is acetic acid-sodium acetate buffer.
3. The mixed mode chromatography method for separating human serum albumin from yeast fermentation broth of claim 1, wherein said elution buffer is monosodium phosphate-disodium phosphate buffer, Tris-hydrochloric acid buffer or glycine-sodium hydroxide buffer, and 0-0.6M NaCl is added.
4. The mixed-mode chromatography method for separating human serum albumin from yeast fermentation broth according to claim 1, wherein the acid solution used for adjusting the pH value of the yeast fermentation broth in step 1) and adjusting the pH value of the crude human serum albumin solution in step 3) is acetic acid or citric acid solution, and the alkali solution used is sodium hydroxide solution.
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