CN116731157A - Method for purifying recombinant human serum albumin from serum-free culture supernatant of human cells - Google Patents
Method for purifying recombinant human serum albumin from serum-free culture supernatant of human cells Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- 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
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Abstract
The invention provides a method for purifying recombinant human serum albumin from a serum-free culture supernatant of human cells, which comprises the following steps: a) Stabilizing recombinant human serum albumin in the serum-free culture supernatant of human cells with sodium octoate; b) Precipitating the hybrid protein by 9% ethanol; c) Treating the serum-free culture solution in a water bath at 68 ℃ for 30 minutes; d) Further precipitating the hybrid protein at the temperature of minus 8 ℃ with pH of 5.85 and 25 percent ethanol; e) Recombinant human serum albumin was precipitated at the isoelectric point of albumin, ph4.75, 40% ethanol, -8 ℃. The invention has the advantages of less material consumption, short process flow, low purification cost, high purity of the obtained albumin and capability of filling the huge market vacancy of human serum albumin.
Description
Technical Field
The invention relates to the field of biotechnology and protein purification, in particular to a method for purifying recombinant human serum albumin from serum-free culture solution of artificial cell lines expressing the recombinant human serum albumin.
Background
Human serum albumin (Human Serum Albumin, HSA) is an important molecule produced by liver cells and secreted into blood, and is the most abundant protein in plasma, and the content of human serum albumin in normal human blood is about 42 g/L, accounting for 60% of the total protein in plasma [1]. Human serum albumin is very stable in structure and has a half-life in vivo of up to 19 days. Human serum albumin has a variety of physiological functions, which are very important to the human body, and the functions include: maintaining the colloid osmotic pressure of plasma, combining and transporting important biological functional molecules, maintaining the permeability of capillary membranes, protecting nerve cells, stabilizing extracellular body fluid, promoting wound healing and resisting oxidation [2]. In particular, in the transport of biologically functional molecules, human serum albumin is capable of reversibly binding a variety of endogenous and exogenous substances, and thus is capable of acting as a universal carrier in the circulatory system for transporting proteins or other nutrients. Currently, human serum albumin is known as a carrier substance responsible for the transport of amino acids, hormones, fatty acids, inorganic salt ions, other biologically active molecules, certain drugs, etc. in the blood.
Human serum albumin has important clinical value as a medicine. Human serum albumin has been widely used in medicine and the pharmaceutical industry, including: as an injectable medicament for the treatment of various wounds, burns, blood loss during or after surgery, oedema, fluid replacement in critically ill patients, various hypoalbuminemia and many chronic wasting diseases, such as tumors, etc.; as stabilizers for active agents in pharmaceutical formulations, pharmaceutical carriers, vaccine adjuvants, excipients or placebo; as a cell culture medium additive in the pharmaceutical industry, etc. Human serum albumin as fusion peptide is fused with some protein medicines, and can prolong the retention time of the medicines in vivo, thereby prolonging the service life of the medicines [1,2]. Because of the important and wide functions, human serum albumin is a large clinical first-line drug, and has great strategic significance for clinical medical application.
Natural human serum albumin is currently obtained by extraction from plasma [3]. Cohn and coworkers have invented for the first time in the middle of the last century a method for separating active human serum albumin from plasma by cold ethanol precipitation. This method has become a widely used method for extracting human serum albumin based on the difference in solubility between human serum albumin and other plasma proteins [4]. The method has strong accessibility, low cost and easy scale up, and the method can ensure that the human serum albumin is free from virus and bacteria pollution because the ethanol has the effect of killing virus and bacteria, thereby having unique advantage in the aspect of safety [3]. In order to avoid protein denaturation caused by ethanol, various extraction methods combining cold ethanol and chromatography methods, including ion exchange chromatography, affinity chromatography, fluidized bed, molecular sieve and the like, have been developed so as to further improve the purity, activity and other aspects of human albumin [5-7].
Since natural human serum albumin is extracted from human blood plasma, it is difficult to satisfy a great market demand. Human serum albumin is in shortage worldwide, and more than half of human serum albumin for injection is imported in China. Recombinant human albumin production by recombinant technology is the final approach to solve the shortage of human albumin [2]]. Several protein expression systems including colibacillusEscherichia coli) Saccharomyces cerevisiaeSaccharomyces cerevisiae) Hansenula polymorpha (Hansenula polymorpha)Hansenula polymorpha) Pichia pastorisPichia pastoris) Transgenic plants (potato or rice) were developed to recombinantly express human serum albumin, respectively.
Recombinant human albumin was first expressed in bacterial E.coli by Genentech company in the United states as early as 1981 [8]. Since E.coli belongs to a prokaryote, and the cell lacks the protein folding, processing and secretion system of eukaryotic cells, the expression of recombinant human albumin in E.coli is not only low in yield, but also in incorrect conformation, and the recombinant product has no function, which strategy has been abandoned.
Pichia pastoris is the most excellent of yeast expression systems, and has certain advantages in protein folding, post-translational modification and secretion capacity because the expression system belongs to a eukaryotic expression system. The system has the characteristics of simple genetic operation, high-density culture, low cost of culture medium, easy scale up and the like, and is favored by most researchers [9]. Great amounts of manpower and material resources are successively invested by Delta biotechnology company, japanese Green Cross (Green Cross) and Merck (Merck company) in the United kingdom, recombinant albumin is successfully expressed in yeast, and the level of mass production is reached. The highest yields of recombinant human albumin in Pichia have been achieved to 10 g/l or even higher. But there are significant challenges encountered in extracting recombinant albumin from yeast fermentation broths. The process for extracting recombinant human serum albumin from yeast fermentation broth is complex, and is the speed limiting step of large-scale industrialized recombinant human serum albumin [3]. Because human serum albumin has wide binding capacity, host impurities, pigments and the like which are tightly combined with the albumin in a yeast fermentation system are difficult to remove, so that downstream purification is difficult, and the purification process is very complex, thereby having high cost [10-12]. The mass production of recombinant human serum albumin from yeast has become a reality through the optimization of culture medium composition [10], expression vector optimization and downstream purification process [2], and particularly the improvement of the purification process greatly improves the purity of the recombinant human serum albumin, so that the immunogenicity thereof is relatively weakened, and related research and development institutions have made great progress in the expression, purification and clinical test of the yeast heavy human serum albumin [2]. However, the extraction of recombinant human serum albumin from yeast requires multi-step chromatography, including ion exchange chromatography, hydrophobic chromatography and gel filtration chromatography, and combines the means of dilution of fermentation liquor, heating and incubation to promote the dissociation of polysaccharide impurities and pigments combined with albumin. There are also methods of removing the bound impurities by partial unfolding and crystallization [10].
Production of recombinant human serum albumin from plants, such as potato or rice, in a bioreactor is another strategy to address the shortage of recombinant human serum albumin from a cost and scale perspective [13,14], [15,16]. Research using rice as a reactor is mainly carried out in japan and the united states of america, and there is wuhan grass biotechnology limited in China. The Wuhan Heyuan Biotechnology Co-Ltd realizes the large-scale production of recombinant human serum albumin in rice through genetic engineering, and high-purity recombinant human serum albumin [15,17,18] is obtained through multi-step purification, thus the national clinical test permission is obtained. The recombinant human serum albumin extracted from rice can be finally obtained by the purification steps such as crushing, soaking, ion exchange, hydrophobic chromatography, gel filtration and the like.
In summary, the purification process of recombinant human serum albumin requires multi-step chromatography, both in yeast expression systems and plant expression systems. The chromatography medium belongs to expensive consumable materials, and increases the production cost of recombinant human serum albumin. In addition, multi-step chromatography reduces yields and increases time costs [3,19].
Therefore, it is necessary to provide a process for expressing and purifying human serum albumin with high efficiency and low cost, so as to reduce the production cost and time cost of human serum albumin.
In the previous work, human liver cells are used as a bioreactor to express recombinant human serum albumin (Chinese patent ZL 2021110440769.2), and particularly human liver cancer cells HepG2/C3A are used as the bioreactor to produce the recombinant human serum albumin. The liver cells in the recombinant expression system can be subjected to suspension expansion culture in a serum-free culture medium, and the recombinant human serum albumin produced by the liver cells is secreted into the serum-free culture medium. Since human serum albumin is also synthesized by hepatocytes and then secreted into the blood in vivo, our recombinant system closely resembles the synthetic and secretory pattern of human serum albumin in vivo.
The cold ethanol precipitation method is a popular purification method for industrially extracting natural human serum albumin from blood plasma [3]. Although the composition and many characteristics of the cell culture solution containing recombinant human serum albumin produced by the hepatocyte expression system are quite different from those of blood plasma, the recombinant expression system has a certain similarity with the production of human serum albumin in vivo, and it is inspired that we can try to purify the recombinant human serum albumin in the recombinant system by extracting the natural human serum albumin from the blood plasma.
Because of its high stability, human serum albumin is much more stable to heat and organic solvents than other plasma proteins, especially in the presence of stabilizers such as sodium octoate and acetyl tryptophan. The recombinant human serum albumin is purified from a starting material which is completely different from plasma, namely a culture supernatant of a human cell line by adopting sodium octoate to stabilize the recombinant human serum albumin and combining a thermal denaturation method and a cold ethanol precipitation method.
In view of this, the present invention has been made.
Disclosure of Invention
The invention provides a method for purifying recombinant human serum albumin from a serum-free culture supernatant of human cells, which comprises the following steps:
1. stabilization of recombinant human serum albumin in human cell serum-free culture supernatant with sodium octoate: firstly, centrifuging a cell culture solution of serum-free suspension culture at a temperature of 4 ℃ and at a speed of 8000rpm for 30 minutes, then adding sodium octoate into supernatant, and stirring to fully dissolve the sodium octoate;
2. ethanol precipitation of hybrid proteins: regulating the pH value of the cell culture supernatant by using 2M sodium acetate solution, then dropwise adding absolute ethyl alcohol while stirring at room temperature, and continuously stirring for 1 hour;
3. water bath treatment of serum-free culture solution: (a) placing a serum-free culture solution containing ethanol in a water bath, maintaining the temperature for 30 minutes after the temperature of the cell culture solution reaches a specified value, (b) placing the cell culture solution in a water bath at room temperature for cooling, standing for 1 hour, (c) taking out the cell culture solution, centrifuging at 8000rpm for 30 minutes at 4 ℃, removing the precipitate, and carrying out ethanol precipitation on the supernatant;
4. ethanol further precipitated the hybrid protein: measuring the volume of the supernatant fluid after centrifugation in the step 3 by using a measuring cylinder to be 975mL, filling the fluid into a beaker, adding acetic acid solution with the concentration of 2M while stirring to adjust the pH value of the fluid, (b) cooling the fluid to-8 ℃, slowly dripping absolute ethyl alcohol with the temperature of-20 ℃, maintaining the temperature of the fluid at-8 ℃ for 1 hour, continuously stirring the fluid for 1 hour, standing at-20 ℃ for overnight, (d) centrifuging at 4 ℃ for 30 minutes at 8000rpm, removing sediment, and performing ethanol sediment on the supernatant fluid in the next step;
5. ethanol precipitation of recombinant human serum albumin: (a) measuring the volume of the liquid after centrifugation in the step 4 by using a cylinder, (b) measuring the pH value of the liquid, adding an acetic acid solution with the concentration of 2M to adjust the pH value of the liquid, (c) cooling the liquid to-8 ℃, slowly dropwise adding absolute ethyl alcohol with the temperature of-20 ℃, maintaining the temperature of the liquid at-8 ℃, continuously stirring for 1 hour, standing at-20 ℃ overnight, (e) centrifuging at 4 ℃ for 30 minutes at 8000rpm, retaining precipitation, precipitating to be pure recombinant human albumin, and (f) dissolving the precipitated recombinant human albumin by using 30mL of sterile water.
The invention is further improved as follows: the addition amount of the sodium octoate in the step 1 is 632mg of sodium octoate per 950mL of supernatant;
the invention is further improved as follows: the pH value of the cell culture supernatant in the step 2 is regulated to 6.5, and the final concentration of the absolute ethyl alcohol is 9%;
the invention is further improved as follows: the water bath temperature in the step 3 is 68 ℃;
the invention is further improved as follows: the pH value of the feed liquid in the step 4 is adjusted to 5.85+/-0.05, and the final concentration of ethanol is 25%;
the invention is further improved as follows: and (5) regulating the pH value of the feed liquid in the step (5) to 4.75+/-0.05, and regulating the final concentration of ethanol to 40%.
The invention has the advantages that:
(1) The method for purifying the recombinant human serum albumin from the serum-free culture supernatant of the human cells only needs low-cost absolute ethyl alcohol without expensive chromatographic medium, only needs a centrifuge and general refrigeration equipment, and can purify the recombinant human serum albumin in the human cell culture solution to the purity of more than 96% through 3 times of centrifugation;
(2) The purity of the recombinant human serum albumin purified from the serum-free culture supernatant of the human cells is high and can reach more than 96 percent;
(3) The recombinant human serum albumin purified from the serum-free culture supernatant of the human cells provided by the invention is derived from the serum-free culture medium, so that potential virus and bacterial pollution of the recombinant human serum albumin is avoided, and the safety is high;
(4) The method for purifying recombinant human serum albumin from the serum-free culture supernatant of human cells provided by the invention is low in production cost and time cost, high in purity, and beneficial to filling the huge market gap of the human serum albumin at present.
Drawings
FIG. 1 shows the steps of purification of recombinant human serum albumin from human cell serum-free culture supernatants.
FIG. 2 shows the results of polyacrylamide gel electrophoresis of the pre-treatment samples, the treated supernatants, and the proteins in the pellet.
Detailed Description
The following detailed description of the invention is provided in connection with the accompanying drawings that illustrate the invention and are not intended to limit the scope of the invention.
The experimental methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are all commercially available.
EXAMPLE 1 stabilization of recombinant human serum Albumin in serum-free culture supernatant of human cells with sodium caprylate
(1) Cell culture medium of artificial cell line C3A810802 (see Chinese patent ZL 2021110440769.2) in serum-free suspension culture is centrifuged at 8000rpm at 4 ℃ for 30 minutes;
(2) 950mL of the supernatant was taken, 632mg of sodium octoate was added, and the mixture was stirred to dissolve it sufficiently.
Example 2, 9% ethanol precipitation of hybrid proteins
(1) Measuring the pH value of the cell culture solution supernatant after adding sodium octoate, and adding 4.5mL of acetic acid solution with the concentration of 2M to adjust the pH value to 6.5;
(2) Absolute ethanol was added dropwise at room temperature with stirring to give a final ethanol concentration of 9% (volume ratio) in the cell culture broth, and stirring was continued for 1 hour.
EXAMPLE 3 Artificial an engineering cell line C3A810802 serum-free Medium was treated in a 68℃water bath
(1) Placing the serum-free culture solution of the artificial cell line C3A810802 containing 9% ethanol in a water bath at 68 ℃, and maintaining the temperature of the cell culture solution at 68 ℃ for 30 minutes after the temperature of the cell culture solution reaches 68 ℃;
(2) Cooling the cell culture solution in a water bath at room temperature, and standing for 1 hour;
(3) Then, the cell culture broth was taken out and centrifuged at 8000rpm for 30 minutes at 4℃to remove the precipitate, and the supernatant was subjected to the next ethanol precipitation.
Example 4 further precipitation of the hybrid protein with ethanol
(1) Measuring 975mL of the supernatant fluid after centrifugation in example 3 by using a measuring cylinder, filling the material fluid into a beaker, and adding 2M acetic acid solution while stirring to adjust the pH value to 5.85+/-0.05;
(2) Cooling the feed liquid to-8deg.C;
(3) Slowly dripping absolute ethanol at-20deg.C to make final concentration of ethanol in the feed liquid 25%, maintaining the temperature of the feed liquid at-8deg.C, continuously stirring for 1 hr, and standing at-20deg.C overnight;
(4) Centrifuging at 8000rpm at 4deg.C for 30 min, removing precipitate, and collecting supernatant as ethanol precipitate.
EXAMPLE 5 ethanol precipitation of recombinant human serum albumin
(1) Measuring the volume of the centrifuged feed liquid in example 4 by using a measuring cylinder;
(2) Measuring the pH value of the feed liquid, and adding an acetic acid solution with the concentration of 2M to adjust the pH value of the feed liquid to 4.75+/-0.05;
(3) Cooling the feed liquid to-8deg.C;
(4) Slowly dripping absolute ethanol at-20deg.C to make the final concentration of ethanol in the feed liquid 40%, maintaining the temperature of the feed liquid at-8deg.C, continuously stirring for 1 hr, and standing at-20deg.C overnight;
(5) Centrifuging at 8000rpm for 30 min at 4deg.C, and keeping the precipitate to obtain pure recombinant human serum albumin;
(6) The precipitated recombinant human serum albumin was dissolved in 30mL of sterile water.
The purification of the recombinant human serum albumin is completed through the steps 1-5, and the purification step is shown in figure 1.
Example 6 analysis of proteins in pretreatment samples, treated supernatants and precipitations
The components before and after centrifugation in each of the above examples were sampled at 20. Mu.L, and protein and recombinant human serum Albumin in each component were detected by polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie brilliant blue staining, with commercially available Plasma-derived human serum Albumin (purity 96.6%) as a control. As shown in FIG. 2, after 9% ethanol precipitation, 68℃water bath treatment and 25% ethanol further precipitation of the hetero protein, the hetero protein in the supernatant of the cell culture broth was significantly reduced (lane: supernatant after centrifugation at pH 5.85% ethanol); after 40% ethanol and the recombinant human serum albumin is precipitated at the isoelectric point of albumin, the foreign protein in the precipitate of the cell culture solution is obviously reduced, the main band of the recombinant human serum albumin is more obvious (the recombinant human serum albumin is precipitated after the centrifugation of the ethanol with the pH of 4.7540% in a lane), and the content of the recombinant human serum albumin is equivalent to 2.5 micrograms of the pure human serum albumin. The results demonstrate that 9%, 25% and 40% three-step ethanol precipitation is effective in purifying high purity recombinant human serum albumin from human serum-free cultured cells.
Reference to the literature
1. Chuang, V.T. and M. Otagiri, Recombinant human serum albumin. Drugs Today (Barc), 2007. 43(8): p. 547-61.
2. Chen, Z., et al., Human serum albumin from recombinant DNA technology: challenges and strategies. Biochim Biophys Acta, 2013. 1830(12): p.5515-25.
3. Raoufinia, R., et al., Overview of Albumin and Its Purification Methods. Adv Pharm Bull, 2016. 6(4): p. 495-507.
4. Cohn, E.J., W.L. Hughes, Jr., and J.H. Weare, Preparation and properties of serum and plasma proteins; crystallization of serum albumins fromethanol water mixtures. J Am Chem Soc, 1947. 69(7): p. 1753-61.
5. Raoufinia, R., et al., Human albumin purification: a modified and concise method. J Immunoassay Immunochem, 2018. 39(6): p. 687-695.
6. Ge, C.T., et al., Rational design of specific ligands for human serum albumin separation and applications. J Sep Sci, 2020. 43(21): p.4028-4035.
7. Badgujar, S.B., et al., A cost-effective method for purification and characterization of human urinary albumin. J Chromatogr B Analyt TechnolBiomed Life Sci, 2019. 1114-1115: p. 31-44.
8. Lawn, R.M., et al., The sequence of human serum albumin cDNA and its expression in E. coli. Nucleic Acids Res, 1981. 9(22): p. 6103-114.
9. Zhu, W., et al., High level expression and purification of recombinant human serum albumin in Pichia pastoris. Protein Expr Purif, 2018.147: p. 61-68.
10. Zhu, W., et al., Medium optimization for high yield production of human serum albumin in Pichia pastoris and its efficientpurification. Protein Expr Purif, 2021. 181: p. 105831.
11. Qiu, R.D., et al., High Expression and Purification of Recombinant Human Serum Albumin from Pichia pastoris. Sheng WuHua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2000. 32(1): p. 59-62.
12. Harm, S., C. Schildbock, and J. Hartmann, Removal of stabilizers from human serum albumin by adsorbents and dialysis usedin blood purification. PLoS One, 2018. 13(1): p. e0191741.
13. Huang, L.F., et al., Production of human serum albumin by sugar starvation induced promoter and rice cell culture.Transgenic Res, 2005. 14(5): p. 569-81.
14. Zhang, Q., et al., Expression and purification of recombinant human serum albumin from selectively terminabletransgenic rice. J Zhejiang Univ Sci B, 2013. 14(10): p. 867-74.
15. He, Y., et al., Large-scale production of functional human serum albumin from transgenic rice seeds. Proc Natl Acad Sci US A, 2011. 108(47): p. 19078-83.
16. Sun, Q.Y., et al., Improved expression and purification of recombinant human serum albumin from transgenic tobaccosuspension culture. J Biotechnol, 2011. 155(2): p. 164-72.
17. Pang, J., J. Zhou, and D. Yang, Knock-in at GluA1 locus improves recombinant human serum albumin expression in rice grain.J Biotechnol, 2020. 321: p. 87-95.
18. Chen, Z., et al., Quantitation of the residual DNA from rice-derived recombinant human serum albumin. Anal Biochem,2014. 450: p. 4-10.
19. Raoufinia, R., et al., A methodological approach for purification and characterization of human serum albumin. JImmunoassay Immunochem, 2016. 37(6): p. 623-35.
Claims (8)
1. A method for purifying recombinant human serum albumin from a serum-free culture supernatant of human cells comprising the steps of:
(1) Stabilizing recombinant human serum albumin in the serum-free culture supernatant of human cells with sodium octoate;
(2) Precipitating the impurity protein by ethanol;
(3) Heating the serum-free culture solution in a water bath;
(4) Ethanol further precipitates the hybrid protein;
(5) Ethanol precipitation of recombinant human serum albumin.
2. The method of purifying recombinant human serum albumin from a serum-free culture supernatant of human cells according to claim 1, wherein: in the step (1), the cell culture medium of the serum-free suspension culture was centrifuged at 8000rpm at 4℃for 30 minutes, and then sodium octoate was added to the supernatant and stirred to be sufficiently dissolved.
3. The method of purifying recombinant human serum albumin from a serum-free culture supernatant of human cells according to claim 1, wherein: in the step (2), the pH of the cell culture supernatant was adjusted with a 2M sodium acetate solution, and then absolute ethanol was added dropwise with stirring at room temperature, followed by stirring for 1 hour.
4. The method of purifying recombinant human serum albumin from a serum-free culture supernatant of human cells according to claim 1, wherein: in step (3), (a) placing the serum-free cell culture solution containing ethanol in a water bath, after the temperature of the cell culture solution reaches a specified value, maintaining the temperature for further incubation for 30 minutes, (b) placing the cell culture solution in a water bath at room temperature for cooling, standing for 1 hour, (c) taking out the cell culture solution, centrifuging at 8000rpm for 30 minutes at 4 ℃, removing the precipitate, and taking the supernatant as the next ethanol precipitate.
5. The method of purifying recombinant human serum albumin from a serum-free culture supernatant of human cells according to claim 1, wherein: in the step (4), (a) the volume of the supernatant liquid after centrifugation in the step (3) is 975mL by using a measuring cylinder, the liquid is filled into a beaker, an acetic acid solution with the concentration of 2M is added while stirring to adjust the pH value, (b) the liquid is cooled to-8 ℃, absolute ethyl alcohol with the temperature of minus 20 ℃ is slowly dripped, the temperature of the liquid is maintained at minus 8 ℃ for continuously stirring for 1 hour, the liquid is placed at minus 20 ℃ overnight, and (d) centrifugation is carried out at 8000rpm for 30 minutes at 4 ℃, sediment is removed, and the supernatant liquid is subjected to the next ethanol sediment.
6. The method of purifying recombinant human serum albumin from a serum-free culture supernatant of human cells according to claim 1, wherein: in the step (5), (a) the volume of the feed liquid after centrifugation in the step (4) is measured by using a cylinder, (b) the pH value of the feed liquid is measured, an acetic acid solution with the concentration of 2M is added to adjust the pH value of the feed liquid, (c) the feed liquid is cooled to-8 ℃, absolute ethyl alcohol with the temperature of-20 ℃ is slowly added dropwise, the temperature of the feed liquid is maintained at-8 ℃ for 1 hour, the mixture is placed at-20 ℃ for overnight, (e) centrifugation is carried out at 4 ℃ for 30 minutes at 8000rpm, the precipitate is reserved, and the precipitated recombinant human albumin is dissolved by 30mL of sterile water.
7. The method of purifying recombinant human serum albumin from a serum-free culture supernatant of human cells according to claim 1, wherein:
in step (1): firstly, centrifuging a cell culture solution of serum-free suspension culture at a temperature of 4 ℃ and at a speed of 8000rpm for 30 minutes, then adding sodium octoate into supernatant, and stirring to fully dissolve the sodium octoate;
in step (2): regulating the pH value of the cell culture supernatant by using 2M sodium acetate solution, then dropwise adding absolute ethyl alcohol while stirring at room temperature, and continuously stirring for 1 hour;
in step (3): (a) placing a serum-free cell culture solution containing ethanol in a water bath, maintaining the temperature of the cell culture solution for 30 minutes after the temperature of the cell culture solution reaches a specified value, (b) placing the cell culture solution in a water bath at room temperature for cooling, standing for 1 hour, (c) taking out the cell culture solution, centrifuging at 8000rpm for 30 minutes at 4 ℃, removing the precipitate, and taking the supernatant as the next ethanol precipitate;
in step (4): measuring the volume of the supernatant fluid after centrifugation in the step (3) by using a measuring cylinder to be 975mL, filling the fluid into a beaker, adding an acetic acid solution with the concentration of 2M while stirring to adjust the pH value of the fluid, (b) cooling the fluid to-8 ℃, slowly dropwise adding absolute ethyl alcohol with the temperature of-20 ℃, maintaining the temperature of the fluid at-8 ℃ for continuous stirring for 1 hour, standing at-20 ℃ overnight, (d) centrifuging at 4 ℃ for 30 minutes at 8000rpm, removing sediment, and taking the supernatant fluid as the next ethanol sediment;
in step (5): (a) measuring the volume of the centrifuged feed liquid in the step (4) by using a cylinder, (b) measuring the pH value of the feed liquid, adding an acetic acid solution with the concentration of 2M to adjust the pH value of the feed liquid, (c) cooling the feed liquid to-8 ℃, slowly dropwise adding absolute ethyl alcohol with the temperature of-20 ℃, maintaining the temperature of the feed liquid at-8 ℃ for 1 hour, continuously stirring, standing at-20 ℃ overnight, (e) centrifuging at 4 ℃ for 30 minutes at 8000rpm, retaining precipitate, precipitating to be pure recombinant human serum albumin, and (f) dissolving the precipitated recombinant human serum albumin by using 30mL of sterile water.
8. The method of purifying recombinant human serum albumin from a serum-free culture supernatant of human cells according to any one of claims 1 to 7, wherein:
the sodium octoate in the step (1) is added in an amount of 632mg of sodium octoate per 950mL of supernatant;
the pH value of the cell culture supernatant in the step (2) is adjusted to 6.5, and the final concentration of the absolute ethyl alcohol is 9%;
the water bath temperature in the step (3) is 68 ℃;
the pH value of the feed liquid in the step (4) is adjusted to 5.85+/-0.05, and the final concentration of ethanol is 25%;
and (3) regulating the pH value of the feed liquid in the step (5) to 4.75+/-0.05, and regulating the final concentration of ethanol to 40%.
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