CN112759643B - Degreasing method for serum albumin - Google Patents
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Abstract
The invention discloses a degreasing method of serum albumin. The invention provides application of tangential flow filtration technology in degreasing serum albumin. The invention also provides a degreasing method of serum albumin, which comprises the following steps: separating serum albumin from the conjugate by tangential flow filtration under specific solution conditions; the specific solution conditions are solution conditions that enable separation of albumin from the conjugate. The degreasing effect of the albumin can reach the level that the fatty acid content in the albumin is not higher than 5 mug/g albumin, which is superior to the degreasing level of the albumin which can be reached by other technical schemes at present; the recovery rate of the serum albumin is not lower than 97%, and is superior to the activated carbon adsorption degreasing technology; the tangential flow filtration membrane can be repeatedly used for a plurality of times, the process is simple, and feed liquid with different volumes can be processed by linearly amplifying the membrane area of the tangential flow device, thereby being beneficial to industrialized application and popularization.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a degreasing method of serum albumin.
Background
Albumin is the most abundant protein in plasma and is widely present in the lymphatic system and muscle tissue. The physiological functions of albumin are mainly to maintain blood osmotic pressure balance, bind and transport endogenous or exogenous small molecule substances, resist oxidation, exhibit lipase catalytic activity and the like.
Albumin is an important biological product, and human serum albumin is used in the fields of plasma expansion, drug delivery, stem cell culture and the like, and bovine serum albumin is used as a stabilizer to be used as a stabilizing component of biological reagents.
Albumin is the most important nonspecific transport protein in the blood system, and can bind anions and cations; not only can it bind to transport endogenous metabolites (e.g., fatty acids, hormones, and enzymes), but also exogenous drugs, particularly lipid-soluble drugs, can be transported.
Defatted albumin refers to albumin from which all or part of lipid conjugates carried by albumin are removed, and has higher exogenous molecule binding capacity due to removal of originally bound fatty acid molecules, and is commonly used for preparing drug binding and release systems. Defatted albumin is also used in the field of stem cell culture.
Current techniques for degreasing albumin are activated carbon treatment (application number 201480030956.4), ion exchange treatment (Scheider W, fuller j.an effective method for defatting albumin using resin columns [ J ]. Biochimica et Biophysica Acta,1970,221 (2): 376-378.Cameron R,Davies J,Adcock W,et al.The Removal of Model Viruses,Poliovirus type 1and Canine Parvovirus,During the Purification of Human Albumin using Ion-exchange Chromatographic Procedures [ J ]. Biologicals,1997,25 (4): 391-401 ]), and heat treatment (Tanaka K, shigueoka E M, sawatani E, et al, purification of human albumin by the combination of the method of Cohn with liquid chromatography [ J ]. Brazilian Journal of Medical and Biological Research,1998,31 (11): 1383-1388.). The activated carbon treatment method needs to add activated carbon with high specific area into albumin solution, and part of albumin is adsorbed on the activated carbon due to non-specific adsorption property of the activated carbon, which is non-selective to small molecules such as protein, so that the recovery rate of albumin is low. The ion exchange treatment method needs to develop a process of separating albumin and lipid molecules through chromatography, the process development process is complex, the process comprises the steps of balancing, loading and adsorbing, eluting, regenerating and the like, the process is complex, and the process method needs to be supported by a whole set of chromatography purification system and has large fixing investment. The disclosed method of heat treatment requires the removal of fatty acid precipitate formed after heating by centrifugation, which has a limited treatment volume and requires a long process time.
Disclosure of Invention
The present invention aims at providing one kind of technology of eliminating serum albumin lipid conjugate to prepare defatted albumin. In order to achieve the aim, the invention adopts a scheme taking tangential flow ultrafiltration technology as a main body, and can realize the effect of separating albumin from lipid conjugates thereof under specific conditions.
In a first aspect, the invention claims the use of tangential flow filtration technology for degreasing serum albumin.
In a second aspect, the invention claims a method of degreasing serum albumin.
The degreasing method of serum albumin claimed by the invention can comprise the following steps: serum albumin is separated from the conjugate by tangential flow filtration under specific solution conditions.
The specific solution conditions are solution conditions that enable separation of serum albumin from the conjugate.
Further, the specific solution conditions include:
(1) Solution pH;
the pH of the solution is 3.0-4.2 or 9.5-10.5;
(2) The content of surfactant and/or organic solvent in the solution;
the volume percentage of the surfactant in the solution is 0-1.2 percent (without 0 point); the percentage of the organic solvent in the solution is 0-35% (without 0 point).
Wherein the surfactant includes, but is not limited to, polysorbate 80 or/and triton X100. The organic solvent includes but is not limited to ethanol and/or tributyl phosphate.
In a specific embodiment of the invention, the volume percentage of polysorbate 80 in the solution is 0.2-1.2%; the volume percentage of the triton X100 in the solution is 0-0.8% (without 0 point).
In a specific embodiment of the invention, the volume percentage of ethanol in the solution is 10-35%; the volume percentage of tributyl phosphate in the solution is 0-0.7% (without 0 point).
Still further, the specific solution conditions further include:
(3) The solution contains buffer components;
further, the buffer component is glycine. When the pH of the solution is 3.0-4.2, hydrochloric acid is adopted to adjust the pH (at the moment, glycine and hydrochloric acid form a buffer pair); when the pH of the solution is 9.5-10.5, sodium hydroxide is used to adjust the pH (in this case, glycine forms a buffer pair with sodium hydroxide).
In a specific embodiment of the invention, the glycine content of the solution is 3.8g/L.
In the present invention, the tangential flow filtration technology employs a filter membrane having a retention pore size of no more than 30 kilodaltons, preferably 10 kilodaltons.
In the present invention, the tangential flow filtration technique employs a replacement solution that is an aqueous solution that is free of serum albumin and meets the specific solution conditions.
In the present invention, the tangential flow filtration technique employs a displacement solution in a volume that is 3-10 times (e.g., 10 times) the volume of the serum albumin solution (containing the surfactant and/or the organic solvent) that was on-stream when the tangential flow filtration was performed.
In the present invention, the conjugates include, but are not limited to, fatty acids, hormones, or enzymes, etc. In the embodiment of the invention, the examination index selects fatty acid. The principle of fatty acid binding is similar to that of small molecules such as hormone, and the removal effect of fatty acid can be extended to binding substances such as hormone.
In the present invention, all steps of the method may be performed at room temperature or at a low temperature (e.g., 2 to 8 ℃ C., particularly, 4 ℃ C.).
In a specific embodiment of the invention, the method specifically comprises the following steps:
(A) Preparing serum albumin solution;
in a first embodiment of the present invention, the serum albumin solution has a pH of 3.0, the solute is human serum albumin, glycine, polysorbate 80, ethanol, and the solvent is water. The serum albumin solution contains 15% of human serum albumin, 3.8g/L glycine, 0.2% of polysorbate 80 and 10% of ethanol by volume.
In a second embodiment of the present invention, the serum albumin solution has a pH of 4.2, the solute is human serum albumin, glycine, ethanol, and the solvent is water. In the serum albumin solution, 15% volume percent of human serum albumin, 3.8g/L glycine, and 35% volume percent of ethanol are contained.
In a third embodiment of the present invention, the serum albumin solution has a pH of 9.5, the solute is bovine serum albumin, glycine, triton X100, and the solvent is water. The serum albumin solution contains 25g/L bovine serum albumin, 3.8g/L glycine and 0.8 percent by volume of triton X100.
In a fourth embodiment of the present invention, the serum albumin solution has a pH of 10.5, the solute is bovine serum albumin, glycine, polysorbate 80, tributyl phosphate, and the solvent is water. In the serum albumin solution, 25g/L bovine serum albumin, 3.8g/L glycine, 1.2% by volume of polysorbate 80, and 0.7% by volume of tributyl phosphate were contained.
(B) Preparing a displacement solution for tangential flow filtration;
the substitution solution for tangential flow filtration differs from the serum albumin solution of step (a) only in that it does not contain serum albumin (such as human serum albumin or bovine serum albumin).
The volume of the displacement solution for tangential flow filtration is 10 times the volume of the serum albumin solution of step (a).
(C) Tangential flow filtration is performed;
washing a tangential flow filtration membrane with the tangential flow filtration replacement solution in the step (B), filtering the serum albumin solution in the step (A) by using the washed tangential flow filtration membrane, concentrating the solution to 4/5 of the original volume, adding the tangential flow filtration replacement solution in the step (B) to the original volume, and repeatedly concentrating and adding the replacement solution until the tangential flow filtration replacement solution in the step (B) is exhausted.
In the present invention, the serum albumin may be human serum albumin or bovine serum albumin.
In the present invention, the tangential flow filtration is performed with a tangential flow inlet pressure of no more than 0.3bar.
In a third aspect, the invention claims the use of the method according to the second aspect of the preceding claims for the preparation of defatted albumin.
Compared with the prior art, the invention has the following advantages and effects:
the degreasing effect of the albumin can reach the level that the fatty acid content in the albumin is not higher than 5 mug/g albumin, which is superior to the degreasing level of the albumin which can be reached by other technical schemes at present; the recovery rate of serum albumin is not lower than 97%, the method is superior to the activated carbon adsorption degreasing technology, the process is simple, different volumes of feed liquid can be treated by linearly amplifying the membrane area of the tangential flow device, the tangential flow filter membrane can be repeatedly used for many times, and the method is beneficial to industrialized application and popularization.
Drawings
FIG. 1 is a tangential flow filtration device employed in the present invention.
Detailed Description
The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.
The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
The tangential flow filtration device used in the present invention is shown in FIG. 1 (model VIVAFLOW50 10000MWCO PES, manufactured by Sidoris Corp., germany).
Example 1
1. Preparation of serum Albumin solution
15ml of human serum albumin (self-produced by Hualan bioengineering Co., ltd., product registration number S10950007) is measured, 60ml of water for injection is firstly added for dilution, then 0.38g of glycine (product of Hubei eight-peak pharmaceutical Co., ltd.) is added for stirring and dissolution, the pH value of the solution is regulated to 3.0 by using 0.1mol/L of hydrochloric acid solution, 0.2ml of polysorbate 80 (product of Hunan Erkang pharmaceutical Co., ltd.) and 10ml of ethanol are added, and after stirring and mixing uniformly, water is used for constant volume to 100ml.
2. Preparing a replacement solution
3.8g glycine is weighed and added into 700ml water to be stirred and dissolved, the pH value of the solution is regulated to 3.0 by using 0.1mol/L hydrochloric acid solution, 2ml polysorbate 80 and 100ml ethanol are added, and after stirring and mixing evenly, the volume is fixed to 1000ml by water.
3. 100ml of the displacement solution prepared in step 2 was taken to rinse the tangential flow filtration membrane (product of Sidoris Corp., MWCO 10 kD).
4. Filtering the serum albumin solution prepared in the step 1 by using a tangential flow filtration membrane after rinsing, concentrating the solution to 80ml, adding 20ml of the replacement solution prepared in the step 2, repeatedly concentrating and adding the replacement solution until the replacement solution prepared in the step 2 is exhausted, controlling the flow rate of a peristaltic pump until the tangential flow inlet pressure is not more than 0.3bar, and performing at room temperature.
5. The filtered solution was collected. Sampling was performed to detect protein content and fatty acid content, respectively.
6. The protein content is detected by adopting a BCA method, and the BCA kit is a product of Biyun Tian biotechnology limited company; the fatty acid content detection adopts a methanol-chloroform extraction method and a NEFA method fatty acid content detection kit (product of Beijing Lidermann sublimation technology Co., ltd.). The results are summarized in Table 1.
The methanol-chloroform extraction method in the fatty acid content detection is operated as follows: 1ml of albumin solution is taken, 2ml of methanol and 1ml of chloroform (both methanol and chloroform are Kemeou reagent) are added, shaking is carried out vigorously for 10 minutes, then centrifugation is carried out for 10 minutes by using 10000g of centrifugal force, the centrifuged lower solution is taken, dried, and then 0.1ml of isopropanol (Kemeou reagent) is added for dissolving and preparing for detection.
Example 2
1. Preparation of serum Albumin solution
15ml of human serum albumin (Wallace bioengineering Co., ltd., product registration number S10950007) is measured, 30ml of water is added for dilution, 0.38g of glycine is added for stirring and dissolution, 0.1mol/L of hydrochloric acid solution is used for regulating the pH value of the solution to 4.2, meanwhile, the solution is cooled to 4 ℃, 35ml of ethanol is added, after stirring and mixing uniformly, water is used for constant volume to 100ml, and the solution is cooled to 4 ℃.
2. Preparing a replacement solution
3.8g glycine is weighed and added into 700ml water to be stirred and dissolved, 0.1mol/L hydrochloric acid solution is used for adjusting the pH value of the solution to 4.7, 350ml ethanol is added, after stirring and mixing evenly, water is used for fixing the volume to 1000ml, and the solution is cooled to 4 ℃.
3. 100ml of the displacement solution prepared in step 2 was taken and washed and passed tangentially through a filter (product of Sidoris Corp., MWCO 10 kD).
4. Filtering the serum albumin solution prepared in the step 1 by using a tangential flow filtration membrane after rinsing, concentrating the solution to 80ml, adding 20ml of the replacement solution prepared in the step 2, repeatedly concentrating and adding the replacement solution until the replacement solution prepared in the step 2 is exhausted, controlling the flow rate of a peristaltic pump during the period to ensure that the tangential flow inlet pressure is not more than 0.3bar, and the temperature of the solution in the ultrafiltration process is between 2 and 8 ℃.
5. The filtered solution was collected and sampled for detection of protein content and fatty acid content, respectively.
6. Protein content detection adopts BCA method, and BCA kit is purchased from Biyun Tian biotechnology Co., ltd; the fatty acid content was measured by using a methanol-chloroform extraction method (same as in example 1) and a NEFA method fatty acid content measuring kit (Beijing Lidaman sublimation technology Co., ltd.). The results are summarized in Table 1.
Example 3
1. Preparation of serum Albumin solution
2.5g of bovine serum albumin (product of Biyun biotechnology Co., ltd.) is weighed, dissolved in 90ml of water, added with 0.38g of glycine, stirred and dissolved, the pH value of the solution is adjusted to 9.5 by using 0.1mol/L of sodium hydroxide solution, added with 0.8ml of triton X100 (product of sigma-aldrich), stirred and mixed uniformly, and then the volume is fixed to 100ml by using water.
2. Preparing a replacement solution
3.8g glycine is weighed and added into 900ml water to be stirred and dissolved, 0.1mol/L sodium hydroxide (Hunan' er Kang Chanpin) solution is used for adjusting the pH value of the solution to 9.5, 8ml of triton X100 is added, and after stirring and mixing uniformly, water is used for constant volume to 1000ml.
3. 100ml of the displacement solution prepared in step 2 was taken and washed and passed tangentially through a filter (product of Sidoris Corp., molecular cut-off pore size 10 kilodaltons).
4. Filtering the serum albumin solution prepared in the step 1 by using a tangential flow filtration membrane after rinsing, concentrating the solution to 80ml, adding 20ml of the replacement solution prepared in the step 2, and repeating the operations of concentrating and adding the replacement solution until the replacement solution prepared in the step 2 is exhausted.
5. The filtered solution was collected. Sampling was performed to detect protein content and fatty acid content, respectively.
6. Protein content detection adopts BCA method, and BCA kit is purchased from Biyun Tian biotechnology Co., ltd; the fatty acid content was measured by using a methanol-chloroform extraction method (same as in example 1) and a NEFA method fatty acid content measuring kit (Beijing Lidaman sublimation technology Co., ltd.). The results are summarized in Table 1.
Example 4
1. Preparation of serum Albumin solution
Weighing 2.5g of bovine serum albumin (product of Biyun biotechnology Co., ltd.) and dissolving in 90ml of water, adding 0.38g of glycine, stirring and dissolving, and adjusting the pH value of the solution to 10.5 by using 0.1mol/L sodium hydroxide solution; 1.2ml of polysorbate 80 and 0.7ml of tributyl phosphate (sigma-aldrich product) were mixed, added to the protein solution together, stirred and mixed, and then the volume was fixed to 100ml with water.
2. Preparing a replacement solution
3.8g glycine is weighed and added into 700ml water for stirring and dissolution, 0.1mol/L sodium hydroxide solution is used for adjusting the pH value of the solution to 10.5, 12ml polysorbate 80 and 7ml tributyl phosphate are added into the water solution after stirring and mixing evenly, and water is used for fixing the volume to 1000ml.
3. 100ml of the displacement solution prepared in step 2 was taken and washed and passed tangentially through a filter (product of Sidoris Corp., molecular cut-off pore size 10 kilodaltons).
4. Filtering the serum albumin solution prepared in the step 1 by using a tangential flow filtration membrane after rinsing, concentrating the solution to 80ml, adding 20ml of the replacement solution prepared in the step 2, and repeating the operations of concentrating and adding the replacement solution until the replacement solution prepared in the step 2 is exhausted.
5. The filtered solution was collected. Sampling was performed to detect protein content and fatty acid content, respectively.
6. Protein content detection adopts BCA method, and BCA kit is purchased from Biyun Tian biotechnology Co., ltd; the fatty acid content was measured by using a methanol-chloroform extraction method (same as in example 1) and a NEFA method fatty acid content measuring kit (same as in example 1). The results are summarized in Table 1.
Table 1 serum Albumin degreasing Effect in examples
The present invention is described in detail above. It will be apparent to those skilled in the art that the present invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with respect to specific embodiments, it will be appreciated that the invention may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.
Claims (6)
1. A method for degreasing serum albumin, comprising the steps of: separating serum albumin from the conjugate by tangential flow filtration under specific solution conditions;
the specific solution conditions are solution conditions that enable separation of serum albumin and the conjugate;
the specific solution conditions include:
(1) Solution pH;
the pH of the solution is 3.0-4.2 or 9.5-10.5;
(2) The content of surfactant and/or organic solvent in the solution;
the volume percentage of the surfactant in the solution is 0-1.2%, and no 0 point is contained; the volume percentage of the organic solvent in the solution is 0-35%, and no 0 point is contained;
the tangential flow filtration technology adopts a filter membrane with a interception aperture of 10 kilodaltons;
the surfactant is polysorbate 80 or/and triton X100; the organic solvent is ethanol or/and tributyl phosphate.
2. The method according to claim 1, characterized in that: the tangential flow filtration technique employs a replacement solution that is an aqueous solution that is free of serum albumin and meets the specific solution conditions.
3. The method according to claim 1, characterized in that: the volume of the replacement solution adopted by the tangential flow filtration technology is 3-10 times of the volume of the serum albumin solution on the machine when the tangential flow filtration is carried out.
4. A method according to any one of claims 1-3, characterized in that: the conjugate comprises a fatty acid, a hormone or an enzyme.
5. A method according to any one of claims 1-3, characterized in that: the serum albumin is human serum albumin or bovine serum albumin.
6. Use of the method according to any one of claims 1-5 for the preparation of defatted albumin.
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