CN114907469A - Method for separating and purifying albumin in human serum - Google Patents
Method for separating and purifying albumin in human serum Download PDFInfo
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- CN114907469A CN114907469A CN202110180818.6A CN202110180818A CN114907469A CN 114907469 A CN114907469 A CN 114907469A CN 202110180818 A CN202110180818 A CN 202110180818A CN 114907469 A CN114907469 A CN 114907469A
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
- C07K14/765—Serum albumin, e.g. HSA
Abstract
The invention discloses a method for separating and purifying albumin in human serum. The method comprises the following steps: 1) pretreatment of the sample: unfreezing a human serum sample (prepared according to the requirements of national standard substances) at 4 ℃, and then filtering the human serum sample by using a water system disposable filter head with the diameter of 25mm and the pore diameter of 0.45 mu m to obtain a sample; 2) affinity chromatography: performing affinity chromatography on the sample to obtain a sample after the affinity chromatography; 3) anion exchange chromatography: and (3) carrying out anion exchange chromatography on the sample subjected to the affinity chromatography to obtain purified albumin. The purity of the albumin purified by the method can reach more than or equal to 99 percent.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for separating and purifying albumin in human serum.
Background
Common albumin purification methods can be classified into more conventional precipitation methods and chromatography methods developed by new techniques according to a large classification. In any method, the principle of separation and purification is to utilize the differences of physical and chemical properties. The precipitation method realizes the separation of target protein and impurities by a precipitation mode, and has the advantages of low cost, simple operation, high recovery rate, low equipment requirement and the like. However, the method has low selectivity to protein, and is suitable for primary purification and concentration of protein. Commonly used methods for precipitation of albumin are: salt precipitation, organic precipitation, thermal precipitation, isoelectric point precipitation, and the like. The chromatographic method is to separate and purify the target product through the difference of the physical and chemical properties and the biological properties of the interaction between the substance to be separated and the stationary phase. With the development of new material technology, the chromatographic technology has higher separation performance and wider application range, and has become a common method for separating and purifying protein. At present, the chromatographic methods mainly comprise molecular sieve gel chromatography, ion exchange chromatography, hydrophobic chromatography, affinity chromatography, reverse phase chromatography and the like.
The method for separating and purifying albumin in serum is reported in the literature. For example, in Japanese patent publication "Committee on Diabetes Mellitus industries of the Japan Society of Clinical Chemistry-derived reference measurement process and reference materials for purified albumin", purification by anion exchange method is proposed, the purity can only reach 95%, and the purification cost of the method is high, and the method is not suitable for mass production.
Disclosure of Invention
The invention aims to provide a method for separating and purifying albumin in human serum.
The method for separating and purifying the albumin in the human serum provided by the invention comprises the following steps:
1) pretreatment of the sample: thawing a human serum sample (prepared according to the requirements of national standard substances) at 4 ℃, and then filtering the human serum sample by using a water system disposable filter head with the diameter of 25mm and the pore diameter of 0.45 mu m to obtain a sample;
2) affinity chromatography: performing affinity chromatography on the sample to obtain a sample after the affinity chromatography;
3) anion exchange chromatography: and (3) carrying out anion exchange chromatography on the sample after affinity chromatography to obtain the purified albumin.
In step 1) of the method, the human serum sample is prepared according to the requirements of national standard substances.
The specific method comprises the following steps: serum from healthy persons was collected following ISO guidelines 34 and the guidelines for collection of serum raw materials for preparing standard substances in accordance with the national Standard substance technical Specifications. The serum was collected directly and mixed using 50ml polyethylene closed centrifuge tubes without any additives or preservatives. The collected serum tubes are numbered and the volume is recorded, and then the surface of the bottle body is disinfected by 75 percent alcohol or iodine and sealed, and then the bottle body is immediately placed into a refrigerator at the temperature of minus 80 ℃. All collected serum samples are clear and transparent, except for the abnormal characters such as extravasated blood, chyle and the like, and HIV antibody and hepatitis virus positive samples. Filtering and packaging to obtain human serum candidate standard substance.
In step 2) of the method, the chromatographic conditions of the affinity chromatography are as follows:
the instrument comprises: liquid chromatography of AKTApurifier UPC10 protein;
detection wavelength: 280 nm;
affinity chromatography column: blue Sepharose 6FF 50mL, specification 16mm x 30cm, packing height 26 cm;
flow rate: 2 mL/min;
loading buffer A: 20mM PB (phosphate buffer) contained 0.15M NaCl, pH 7.2;
elution buffer B: 20mM PB (phosphate buffer) contained 2M NaCl, pH 7.2;
the loading buffer A is a buffer used for diluting a sample to be loaded in affinity chromatography.
Preferably, in the affinity chromatography, the loading sample used before loading and the loading buffer A are mixed according to the volume ratio of 1: (2-50), specifically, the ratio of 1: 10 volume mix.
Preferably, 3-5 CV (column volume) of the loading buffer solution A is used for balancing the chromatographic column before column chromatography.
Preferably, the loading amount is 30 mL;
preferably, after the sample loading is finished, the sample loading buffer solution A is continuously used for leaching until the baseline is stable, then the elution buffer solution B is used for eluting, and the peak position is collected for sample receiving to obtain an eluted sample.
The amount of the elution buffer B is preferably 13 CV. The volume of the collected inoculum was 250 mL.
The step 2) further comprises the step of purifying the collected elution sample: desalting the collected eluate with MD44-3500 dialysis bag, and detecting Cl in dialysate with 0.1M silver nitrate - (ii) a The desalted eluate was concentrated (to about 30mL) using a 10kDa 50mL ultrafiltration tube for use.
In step 3) of the above method, the chromatographic conditions of the anion exchange chromatography are as follows:
the instrument comprises: liquid chromatography of AKTApurifier UPC10 protein;
detection wavelength: 280 nm;
anion exchange chromatography column: 20mL of Unigel-80DEAE, specification of 16mm multiplied by 30cm, and height of filler of 10 cm;
flow rate: 2 mL/min;
sampling: pump head sample loading step 2) affinity desalting concentrated sample;
loading buffer C20 mM PB (phosphate buffer), pH 7.2;
elution buffer D20 mM PB (phosphate buffer) containing 1M NaCl, pH 7.2;
carrying out sample loading on the sample subjected to affinity desalting and concentration in the step 2); after the sample loading is finished, eluting the sample with a sample loading buffer solution C until the baseline is stable, then carrying out gradient elution, and respectively carrying out sample receiving and collection on peak positions;
the mobile phase is as follows: the phase A is the elution buffer solution D, and the phase B is the loading buffer solution C;
the flow of the gradient elution is as follows:
(1)5CV 0-5% phase A, 5CV 5% phase A;
(2)5CV 5-10% phase A, 5CV 10% phase A;
(3)5CV 10-15% phase A, 5CV 15% phase A;
(4)5CV 15-30% phase A, 5CV 30% phase A.
Preferably, 3-5 CV (column volume) of the loading buffer solution C is used for balancing the anion exchange chromatography column before column chromatography.
Preferably, elution samples corresponding to 0-5% of elution buffer solution D, 5-10% of elution buffer solution D and 15-30% of elution buffer solution D in the gradient elution procedure are respectively collected; the collected samples were then mixed and desalted using MD44-3500 dialysis bags and concentrated using 10kDa 50mL ultrafiltration tubes.
The method further comprises the step of performing purity detection on the purified albumin.
The purity test here includes two methods:
the first method is to adopt a high-efficiency SEC gel filtration 23mL chromatographic column for purity detection, and inject 50 μ L of sample with 3-5 CV of equilibrium buffer (50mM phosphate buffer +0.15M NaCl, pH 7.4) at a flow rate of 0.6mL/min until the effluent conductance and pH are unchanged and are consistent with the equilibrium solution), and simultaneously compare with the sample of the albumin standard sample;
the purity is measured by a high-efficiency SEC column, and the chromatographic conditions are as follows:
the instrument comprises the following steps: AKTApurifier UPC10
Detection wavelength: 280nm
Column: high resolution gel filtration medium 23mL (column size 10mm X30 cm packing height 30cm)
Flow rate: 0.6mL/min
Buffer：50mM phosphate buffer+0.15MNaCl,pH7.4
Loading: 50 μ L.
The second method is to use a reverse phase chromatography column, which is washed with acetonitrile before loading and then equilibrated with 0.25% trifluoroacetic acid solution until the baseline is stable. Injection loading 10 μ L, elution protocol: 0-5min 100% of 0.25% of trifluoroacetic acid, 5-35min 100% -10% of 0.25% of trifluoroacetic acid, and 35-40min 10% -100% of 0.25% of trifluoroacetic acid. And simultaneously comparing with a albumin standard sample.
The purity of the reversed phase chromatographic column is measured, and the chromatographic conditions are as follows:
the instrument comprises the following steps: high performance liquid phase
Detection wavelength: 280nm
Column: agilent PLRP-S8 um
(250×4.6mm)
Flow rate: 1mL/min
Buffer A 1 : 0.25% trifluoroacetic acid
Buffer B 1 : acetonitrile
And (3) quantitative ring: 10 μ L
Elution process: 0-5min 100% Buffer A 1 ，0％Buffer B 1 ；
5-35min 100％-10％Buffer A 1 ，0％-90％Buffer B 1 ；
35-40min 10％-100％Buffer A 1 ，90％-0％Buffer B 1 。
Compared with the prior art, the invention has the following beneficial effects:
the albumin purification techniques are widely used from early precipitation methods to various separation techniques such as ion exchange chromatography, gel filtration chromatography, affinity chromatography, and membrane chromatography, which have been developed in recent years. Because the purity of albumin is required to be more than 95 percent in clinical experiments, three different purification methods are tried by experimental groups,
1. anion exchange chromatography (blue agarose gel filling) does not achieve the separation effect because the filling is not ideal, and the ES-502N 20C anion exchange column (silica gel filling) is selected as the post-improvement column, so that the separation effect is improved. The purity can reach 95 percent by HPLC, and the method is a Japanese purification method for reference, but the method is high in cost and cannot meet the requirement of large-scale purification.
SDS-PAGE electrophoresis and ammonia water extraction of albumin, which is convenient for gel preparation and low in cost, the obtained result has relatively ideal purity, the yield can reach over 90 percent, and the purity reaches electrophoretic purity but is lower than that of the previous method.
3. The anion exchange is combined with the molecular sieve for two-dimensional separation, and because the treated samples are relatively few, the treatment is improved into affinity chromatography combined with anion exchange, the cost of the column is saved by filling the column by a laboratory, the separation effect and the purity are better than those obtained by an ES-502N 20C anion exchange method, the purity can reach 99 percent through HPLC (high performance liquid chromatography), and the purification requirement of large sample amount is met.
Drawings
FIG. 1 shows a chromatogram of the HAS standard (1012595-6 MG).
FIG. 2 is a chromatogram of the albumin purified in example 1.
Detailed Description
The present invention will be further illustrated with reference to the following specific examples, but the present invention is not limited to the following examples. The method is a conventional method unless otherwise specified. The starting materials are commercially available from the open literature unless otherwise specified.
Example 1 purification scheme of Albumin
Pretreatment of the sample: human serum samples (prepared according to the requirements of national standard substances) are thawed at 4 ℃, and then filtered by a water system disposable filter head with the diameter of 25mm and the pore diameter of 0.45 mu m for standby.
The specific preparation method of the human serum sample comprises the following steps: serum from healthy persons was collected following ISO guidelines 34 and the collection guidelines for preparing standard substance serum raw materials in accordance with the national standard substance technical specifications. Serum was collected directly using 50ml polyethylene closed centrifuge tubes and mixed without any additives or preservatives. After the collected serum tubes are numbered and the volume is recorded, the surface of the bottle body is disinfected by 75 percent alcohol or iodine and sealed, and the bottle body is immediately placed into a refrigerator at the temperature of minus 80 ℃. All collected serum specimens are clear and transparent except abnormal characters such as hemolysis, chyle and the like and HIV antibody and hepatitis virus positive specimens. The serum was filtered and dispensed to obtain 600mL of a candidate standard substance (albumin concentration: 44 g/L).
The first step is affinity chromatography using a self-packed 50ml Blue Sepharose 6FF medium. The loaded 50mL column was applied to AKTApurifier UPC10 and the column was equilibrated with 3-5 CV Buffer a (20mM PB 0.15M NaCl pH 7.2) before column chromatography. And dissolving the sample solution subjected to membrane filtration in Buffer A for sample loading. After the sample loading is finished, the elution is continued by using Buffer A until the baseline is stable, then the elution is performed by using Buffer B (20mM PB 2M NaCl pH 7.2), and the sample collection and sample collection are performed on the position of the peak. The collected eluted sample was desalted using MD44-3500 dialysis bag and Cl-in the dialysate was detected with 0.1M silver nitrate. And concentrating the desalted eluent by using a 10KDa 50mL ultrafiltration tube for later use.
Operating parameters of the affinity chromatography purification process:
the instrument comprises the following steps: AKTApurifier UPC10
Detection wavelength: 280nm
Column: blue Sepharose 6FF 50mL (column size 16mm X30 cm packing height 26cm)
Flow rate: 2mL/min
Sampling: pump head sample 30mL of pretreated serum (sample and BufferA in a volume ratio of 1: 10 before sample loading.)
Buffer A：20mM PB 0.15M NaCl pH＝7.2
Buffer B：20mM PB2M NaCl pH＝7.2
13CV washing of impurities
Collecting: 250mL of sample is inoculated at the position of an elution peak
The eluate sample was collected, desalted using MD44-3500 dialysis bag and concentrated to about 30mL using 10kDa 50mL ultrafiltration tube.
The second step is anion exchange chromatography, and the chromatographic column uses self-packed Unigel-80DEAE 20mL medium. The column was equilibrated with 3-5 CV (column volume) Buffer C (20mM PB pH 7.2) before column chromatography. And (4) loading the eluent subjected to the first desalting and concentration step. And after the sample loading is finished, continuously leaching the sample with Buffer C until the baseline is stable. Then gradient elution is carried out, and sample collection is carried out on the peak positions respectively. The collected samples were desalted using MD44-3500 dialysis bags and concentrated using 10kDa 50mL ultrafiltration tubes.
Operating parameters of the anion exchange chromatography purification process:
the instrument comprises the following steps: AKTApurifier UPC10
Detection wavelength: 280nm
Column: unigel-80DEAE 20mL (column specification 16mm x 30cm packing height 10cm)
Flow rate: 2mL/min
Sampling: pump head sample loading, affinity desalting and concentration
Buffer C:20mM PB pH＝7.2
Buffer D:20mM PB 1M NaCl pH＝7.2
The mobile phase is as follows: the phase A is Buffer D, and the phase B is Buffer C;
gradient elution procedure:
(1)5CV 0-5％Buffer D 5CV 5％BufferD
(2)5CV 5-10％BufferD 5CV 10％BufferD
(3)5CV 10-15％Buffer D 5CV 15％BufferD
(4)5CV 15-30％Buffer D 5CV30％BufferD
(5)5CV 30-50％Buffer D 5CV 50％BufferD
(6)5CV 50-100％Buffer D 5CV 100％BufferD
respectively collecting elution samples corresponding to 0-5% of buffer D, 5-10% of buffer D and 15-30% of buffer D in the gradient elution program; the collected samples were then mixed, desalted using MD44-3500 dialysis bags and concentrated using 10KDa 50mL ultrafiltration tubes.
And the third step is to carry out purity detection on the sample.
The first method is to adopt a high-efficiency SEC gel filtration 23mL chromatographic column for purity detection, and inject 50 μ L of sample with 3-5 CV of equilibrium buffer (50mM phosphate buffer +0.15M NaCl, pH 7.4) at a flow rate of 0.6mL/min until the effluent conductance and pH are unchanged and are consistent with the equilibrium solution), and simultaneously compare with the sample of the albumin standard sample;
the purity is measured by a high-efficiency SEC column, and the chromatographic conditions are as follows:
the instrument comprises: AKTApurifier UPC10
Detection wavelength: 280nm
Column: high resolution gel filtration medium 23mL (column size 10mm X30 cm packing height 30cm)
Flow rate: 0.6mL/min
Buffer：50mM phosphate buffer+0.15MNaCl,pH7.4
Loading: 50 μ L.
The second method is to use a reverse phase chromatography column, which is washed with acetonitrile before loading and then equilibrated with 0.25% trifluoroacetic acid solution until the baseline is stable. Injection loading 10 μ L, elution protocol: 0-5min 100% of 0.25% of trifluoroacetic acid, 5-35min 100% -10% of 0.25% of trifluoroacetic acid, and 35-40min 10% -100% of 0.25% of trifluoroacetic acid. And simultaneously comparing with a albumin standard sample.
The purity of the reversed phase chromatographic column is measured, and the chromatographic conditions are as follows:
the instrument comprises the following steps: high performance liquid phase
Detection wavelength: 280nm
Column: agilent PLRP-S8 um
(250×4.6mm)
Flow rate: 1mL/min
Buffer A 1 : 0.25% trifluoroacetic acid
Buffer B 1 : acetonitrile
And (3) quantitative ring: 10 μ L
Elution procedure: 0-5min 100% Buffer A 1 ，0％Buffer B 1 ；
5-35min 100％-10％Buffer A 1 ，0％-90％Buffer B 1 ；
35-40min 10％-100％Buffer A 1 ，90％-0％Buffer B 1 ；
The purity of HAS standard (1012595-6MG) and albumin purified according to the above method was checked by HPLC. The results are shown in FIGS. 1 and 2.
As can be seen from the figure, FIG. 1 shows that the purity of the albumin standard substance is 99% or more, and FIG. 2 shows that the purity of the albumin purified by the method is 99% or more.
Claims (10)
1. A method for separating and purifying albumin in human serum comprises the following steps:
1) pretreatment of the sample: unfreezing a human serum sample at 4 ℃, and then filtering the human serum sample by using a water system disposable filter head with the diameter of 25mm and the pore diameter of 0.45 mu m to obtain a sample;
2) affinity chromatography: performing affinity chromatography on the sample to obtain a sample after the affinity chromatography;
3) anion exchange chromatography: and (3) carrying out anion exchange chromatography on the sample subjected to the affinity chromatography to obtain purified albumin.
2. The method of claim 1, wherein: in the step 2), the chromatographic conditions of the affinity chromatography are as follows:
the instrument comprises: liquid chromatography of AKTApurifier UPC10 protein;
detection wavelength: 280 nm;
affinity chromatography column: blue Sepharose 6FF 50mL, specification 16mm x 30cm, packing height 26 cm;
flow rate: 2 mL/min;
loading buffer solution A: 20mM PB, containing 0.15M NaCl, pH 7.2;
elution buffer B: 20mM PB, 2M NaCl, pH 7.2.
3. The method of claim 2, wherein: in the step 2), during the affinity chromatography, the volume ratio of the loading sample used before loading to the loading buffer A is 1: (2-50).
4. A method according to claim 2 or 3, characterized in that: in the step 2), 3-5 CV of the sample loading buffer solution A is used for balancing the chromatographic column before column chromatography is carried out on the chromatographic column.
5. The method according to any one of claims 2-4, wherein: the sample loading amount is 30 mL;
after the sample loading is finished, continuously leaching with a loading buffer solution A until the baseline is stable, then eluting with an elution buffer solution B, and collecting the sample at the peak position to obtain an elution sample;
the dosage of the elution buffer B is preferably 13 CV; the volume of the collected inoculum was 250 mL.
6. The method of claim 5, wherein: the step 2) further comprises the step of purifying the collected elution sample: desalting the collected eluate with MD44-3500 dialysis bag, and detecting Cl in dialysate with 0.1M silver nitrate - (ii) a And concentrating the desalted eluent by using a 10KDa 50mL ultrafiltration tube for later use.
7. The method of claim 1, wherein: in the step 3), the chromatographic conditions of the anion exchange chromatography are as follows:
the instrument comprises: liquid chromatography of AKTApurifier UPC10 protein;
detection wavelength: 280 nm;
anion exchange chromatography column: 20mL of Unigel-80DEAE, specification of 16mm multiplied by 30cm, and height of filler of 10 cm;
flow rate: 2 mL/min;
loading: pump head sample loading step 2) affinity desalting concentrated sample;
the loading buffer C is 20mM PB pH 7.2;
elution buffer D20 mM PB 1M NaCl pH 7.2;
carrying out sample loading on the sample subjected to affinity desalting and concentration in the step 2); after the sample loading is finished, eluting the sample with a sample loading buffer solution C until the baseline is stable, then carrying out gradient elution, and respectively carrying out sample receiving and collection on peak positions;
the mobile phase is as follows: the phase A is the elution buffer solution D, and the phase B is the loading buffer solution C;
the flow of the gradient elution is as follows:
(1)5CV 0-5% phase A, 5CV 5% phase A;
(2)5CV 5-10% phase A, 5CV 10% phase A;
(3)5CV 10-15% phase A, 5CV 15% phase A;
(4)5CV 15-30% phase A, 5CV 30% phase A.
8. The method of claim 7, wherein: and balancing the anion exchange chromatographic column by using the 3-5 CV loading buffer solution C before column chromatography.
9. The method of claim 7, wherein: respectively collecting elution samples corresponding to 0-5% of phase A, 5-10% of phase A and 15-30% of phase A in the gradient elution procedure; the collected samples were then mixed, desalted using MD44-3500 dialysis bags and concentrated using 10KDa 50mL ultrafiltration tubes.
10. The method according to any one of claims 1-9, wherein: the method also comprises the step of carrying out purity detection on the purified albumin.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1526732A (en) * | 1999-01-30 | 2004-09-08 | \ | Process for producing high pureness albumin solution |
| CN102234332A (en) * | 2010-04-26 | 2011-11-09 | 浙江海正药业股份有限公司 | Process for separating and purifying recombinant human serum albumin and fusion protein thereof |
| CN103694343A (en) * | 2013-12-26 | 2014-04-02 | 扬州艾迪生物科技有限公司 | Method for preparing human albumin from urine |
| CN112210002A (en) * | 2020-10-15 | 2021-01-12 | 湖南科众源创科技有限公司 | Purification method of recombinant human serum albumin |
-
2021
- 2021-02-08 CN CN202110180818.6A patent/CN114907469A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1526732A (en) * | 1999-01-30 | 2004-09-08 | \ | Process for producing high pureness albumin solution |
| CN102234332A (en) * | 2010-04-26 | 2011-11-09 | 浙江海正药业股份有限公司 | Process for separating and purifying recombinant human serum albumin and fusion protein thereof |
| CN103694343A (en) * | 2013-12-26 | 2014-04-02 | 扬州艾迪生物科技有限公司 | Method for preparing human albumin from urine |
| CN112210002A (en) * | 2020-10-15 | 2021-01-12 | 湖南科众源创科技有限公司 | Purification method of recombinant human serum albumin |
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