Preparation method of human serum albumin and purification method thereof

Abstract

The present application provides a preparation method of human serum albumin, which comprises inoculating human serum albumin-producing Pichia pastoris into a culture medium for fermentation, and obtaining human serum albumin from the fermentation liquor; the culture medium is BSM culture medium with the concentration of all components reduced to at most 1 / 4 except for two components of glycerol and Pichia pastoris trace elements 1, preferably reduced to 1 / 4-1 / 2. The present application also provides a purification method of human serum albumin. The yield and the proportion of human serum albumin in total protein obtained by the preparation method of the present application are significantly higher than those of the prior art, the cost of the culture medium used in the preparation method is significantly reduced, and there is no risk of animal-derived virus contamination, thereby significantly reducing the cost of the preparation method. The recovery rate is significantly improved by using the purification method of the present application, and the purity of the final product is also significantly further improved.

Description

Technical Field

[0001] This invention relates to a method for preparing and purifying human serum albumin. Background Technology

[0002] Human serum albumin (HSA) is the most abundant protein in human plasma, accounting for 60% of total serum protein, approximately 35-40 g / L. Its main functions are maintaining plasma osmotic pressure and facilitating the transport of various substances. HSA is widely used in shock and burn treatment, postoperative recovery, drug delivery, and cell culture media. Global demand for HSA is approximately 600 tons per year, with my country accounting for about one-third of that demand. Currently, HSA is primarily extracted from human blood, and the corresponding purification process is low-temperature ethanol precipitation. However, its availability is severely limited by blood supply, posing a risk of bloodborne virus transmission and spread. Furthermore, the need for large amounts of organic reagents and low-temperature control during purification results in low efficiency.

[0003] The use of transgenic technology to construct engineered bacteria and produce exogenous proteins originated in the 1970s. Since then, a series of exogenous protein expression systems, including those based on *Escherichia coli*, *Saccharomyces cerevisiae*, *Pichia pastoris*, insects, animal and plant cells, and various plants and animals, have been established and developed rapidly. This has greatly promoted the industrial application of valuable protein drugs and more efficient and environmentally friendly industrial enzymes. Currently, transgenic technology has been used to achieve the exogenous expression and purification of human serum albumin in various expression systems, including *Escherichia coli*, *Saccharomyces cerevisiae*, *Pichia pastoris*, animal and plant cells, and various plants and animals.

[0004] Escherichia coli, a prokaryote, is widely used for the expression of exogenous proteins due to its clear genetic background, simple nutritional requirements, and safety. As early as 1981, Richard et al. successfully expressed HSA using E. coli, but only qualitatively detected the expression, with the yield unknown. D. Sleep et al. successfully expressed HSA using Saccharomyces cerevisiae in 1990, but the Saccharomyces cerevisiae expression system had low yields and caused significant immunogenicity due to its high glycosylation of HSA. Researchers at Wuhan University, including Yang He, applied transgenic technology to the traditional crop rice, achieving a remarkable 2.75g of high-purity HSA per kilogram of rice. However, the cultivation cycle of transgenic plants is long, highly susceptible to environmental factors, and prone to the spread of resistance genes. Scholars such as Yu-Kuo Liu achieved HSA expression of 75mg / L using an in vitro cell expression system of transgenic plants. However, overall, the yield of transgenic plant and animal cell expression systems is not optimistic, and the cultivation cost is high, so they are generally not the first choice for industrial production of HSA.

[0005] Pichia pastoris is a eukaryotic expression system discovered in 1969 by Koichi et al., which demonstrated its ability to grow using methanol as the sole carbon source. Its successful application for exogenous protein expression was first reported in 1987. Compared to traditional prokaryotic expression systems, Pichia pastoris offers significant advantages, including high expression levels, strong post-processing capabilities, and high stability of exogenous genes. Furthermore, compared to transgenic plants and animals and plant / animal cells, Pichia pastoris is characterized by simple genetic manipulation, large cell growth, low culture costs, and ease of industrialization. Currently, the Pichia pastoris expression system has achieved the expression of over 500 exogenous proteins. Merck researchers Sehoon Kim et al. achieved HSA expression of 10 g / L in the Pichia pastoris system by optimizing the initial induction cell volume (Kim S, Meehl M, d'Anjou M. Maximizationrecombinant hμmanserμmalbμmin production in a mutspichia pastoris strain [J]. Biotechnology progress, 2014,00(00):1-9.). Kobayashi et al. explored the fed-batch process and also achieved HSA expression of 11 g / L (Ohya T, Ohyama M, Kobayashi K. Optimization of hμmanserμmalbμmin production in methylotrophic yeast pichia pastoris by repeated fed-batch fermentation [J]. Biotechnology and bioengineering, 2005,90(7):876-887.). Based on the two studies with known high HSA expression levels, the methanol induction times were 450 hours and 250 hours, respectively, with corresponding target protein expression efficiencies of only 22 and 44 mg / (L*h). A patent published by Gao Jian et al. indicates that they also achieved HSA expression of approximately 10 g / L in Pichia pastoris, but the methanol induction time was at least 200 hours (Patent Application CN1854301A, Gao Jian, Jia Qian, Li Meiyan, Deng Jianhui. A vector and engineered bacteria for expressing human serum albumin [P]. 2005-04-29). Such a long induction time obviously prolongs the production cycle and increases time costs. Furthermore, the long-term storage of large quantities of methanol also poses a significant safety hazard.To overcome the drawbacks of low human serum albumin expression levels and long fermentation times, which hinder its industrialization, the inventors constructed and screened a Pichia pastoris engineered strain, GS115-pPIC9K-hsa-5-4, capable of efficiently producing human serum albumin, and applied for a related patent on December 25, 2017 (patent application number 201711421153.3). When using this engineered strain for on-board production of human serum albumin, the yield of the target protein reached 8.86 g / L after 96 hours of methanol induction, achieving a production efficiency of 92.29 mg / (L*h). Furthermore, sterile air was used throughout the fermentation process, eliminating the need for pure oxygen. The fermentation broth underwent simple separation and purification steps, including centrifugation, microfiltration, and a one-step affinity chromatography, achieving a recovery rate of 58.1% and a target protein purity of 96.47%. Although the production efficiency of human serum albumin at 92.29 mg / (L*h) is quite considerable, there is still room for improvement in the target protein yield of 8.86 g / L, the recovery rate of 58.1%, and the target protein purity of 96.47%. Furthermore, the BMGY medium used in the production process is relatively expensive (approximately 50 yuan / liter), and the tryptone contained in the BMGY medium poses a risk of animal-derived viral contamination.

[0006] Existing literature on reducing the salt concentration of BSM culture medium includes Zhao HL, Xue C, Wang Y, et al. Increasing the cell viability and heterologous protein expression of Pichia pastoris mutant deficient in PMR1 gene by culture condition optimization[J]. Appl Microbiol Biotechnol, 2008, 81(2):235-241. In this study, the HSA-IFN-α2b fusion protein was used as the target protein. After optimization of various conditions (including salt concentration of the culture medium, culture temperature, and extended production period), the expression level of this fusion protein reached 0.68 g / L, and only after adding peptone to the culture medium did it reach 1.26 g / L. Furthermore, the initial concentration of the target protein was already low, and optimization of the conditions made it relatively easy to increase the yield of the target protein accordingly.

[0007] Furthermore, in existing technologies, BMGY is typically used as the fermentation medium for exogenous expression of human serum albumin, and a three-step purification scheme of affinity chromatography-hydrophobic chromatography-ion exchange chromatography is employed. For example, see: Chen Yun; Huang Tengfei; Jin Jian. Preparation and preliminary crystallographic analysis of recombinant human serum albumin derived from Pichia pastoris system. Journal of Food and Biotechnology, 2014, 34(2), 151-157. This literature does not report the recovery rate, but based on the results of related master's and doctoral dissertations in this research group, the recovery rate is generally less than 50%. Summary of the Invention

[0008] To overcome the shortcomings of existing technologies using Pichia pastoris to express human serum albumin (PSA), such as unsatisfactory PSA yield, PSA percentage, downstream purification recovery rate, and final product purity, while maintaining production efficiency, the present invention provides a method for preparing and purifying PSA. The method described in this invention yields significantly higher PSA yields and a higher PSA percentage than existing technologies. Furthermore, the method significantly reduces the cost of the culture medium and eliminates the risk of animal-derived virus contamination, thus significantly lowering the overall cost. The purification method described in this invention also significantly improves the purification recovery rate and further enhances the purity of the final product, making it suitable for industrial production.

[0009] Because the expression level of exogenous proteins is related to many factors, including strain specificity, the properties of the exogenous protein itself, culture medium composition, culture environment, fermentation process, etc., there are multiple approaches to improving the expression level of exogenous proteins. The inventors unexpectedly discovered that using BSM medium with reduced salt concentration to ferment Pichia pastoris strains, which already exhibited high levels of human serum albumin expression, significantly increased the yield of human serum albumin by lowering the osmotic pressure of the medium. This discovery represents the first direct relationship between osmotic pressure and increased human serum albumin yield. In the art, methods using BSM with reduced salt concentration to culture yeast strains to increase the expression of exogenous proteins typically start from lower initial concentrations. However, through technical optimization, the inventors, starting with strains expressing a high initial concentration (8.86 g / L) of the target protein, ultimately achieved an optimized yield of 17.47 g / L for the target protein, which is extremely rare in the art.

[0010] In the process of protein purification, since the purification system is relatively fixed, those skilled in the art would not think of optimizing the pH of affinity chromatography. However, the inventors of this invention unexpectedly discovered that by changing the pH of the loading buffer for affinity chromatography and creatively applying the experimental scheme of affinity chromatography-hydrophobic chromatography-secondary affinity chromatography to the purification process of human serum albumin, the final product obtained has a purity of more than 99.9% and the recovery rate is greatly improved.

[0011] To solve the above-mentioned technical problems, the present invention provides a method for preparing human serum albumin, which includes inoculating Pichia pastoris, which produces human serum albumin, into a culture medium for fermentation, and obtaining human serum albumin from the fermentation broth;

[0012] The culture medium is a BSM medium in which the concentrations of all components except glycerol and Pichia pastoris trace element 1 are reduced to at most 1 / 4, preferably to 1 / 4 to 1 / 2.

[0013] Preferably, the reduced BSM medium (i.e., the 1 / 2 BSM medium in the prior art) comprises 4% glycerol, 1.335% phosphate, 0.0465% calcium sulfate dihydrate, 0.91% potassium sulfate, 0.745% magnesium sulfate heptahydrate, 0.206% potassium hydroxide and 0.4% Pichia pastoris trace element 1 (PTM1).

[0014] Preferably, the reduced BSM medium (i.e., the 1 / 4 BSM medium in the prior art) comprises 4% glycerol, 0.6675% phosphate, 0.02325% calcium sulfate dihydrate, 0.455% potassium sulfate, 0.3725% magnesium sulfate heptahydrate, 0.103% potassium hydroxide and 0.4% Pichia pastoris trace element 1.

[0015] Wherein, the percentages of the above-mentioned phosphate and the above-mentioned Pichia pastoris trace element 1 are the volume percentages of each component in the culture medium; the percentages of the above-mentioned remaining components are the mass volume percentages (g / mL) of each component in the culture medium.

[0016] In this invention, unless otherwise specified, the mass-volume percentage can be conventional in the art, such as grams per milliliter.

[0017] Preferably, the Pichia pastoris trace element 1 comprises 0.6% copper sulfate pentahydrate, 0.008% sodium iodide, 0.3% manganese sulfate monohydrate, 0.02% sodium molybdate dihydrate, 0.002% boric acid, 0.05% cobalt chloride, 2% zinc chloride, 6.5% ferrous sulfate heptahydrate, 0.02% biotin, and 0.5% sulfuric acid;

[0018] Wherein, the percentage of sulfuric acid is its volume percentage relative to the Pichia pastoris trace element 1; the percentages of the other components are the mass-volume percentages (g / mL) of each component relative to the Pichia pastoris trace element 1.

[0019] Preferably, the Pichia pastoris strain that produces human serum albumin is an engineered strain that integrates an expression vector containing the human serum albumin gene into Pichia pastoris, wherein the expression vector contains, in sequence, a promoter, an α-guide peptide, the human serum albumin gene, and a terminator.

[0020] The *Pichia pastoris* strain that produces human serum albumin may be the strain used in Example 5 of patent application number 201711421153.3 mentioned in the background, the entire contents of which are incorporated herein by reference.

[0021] More preferably, the promoter is the AOX1 promoter.

[0022] More preferably, the α-guide peptide is derived from Saccharomyces cerevisiae.

[0023] More preferably, the expression vector carries a gene against genipathic bacteria, and the genetically engineered bacteria are resistant to genipathic bacteria at concentrations of 0.25-5.0 mg / mL.

[0024] More preferably, the nucleotide sequence of the human serum albumin gene is shown in SEQ ID NO. 1 of the sequence listing.

[0025] More preferably, the backbone of the expression vector is the plasmid pPIC9K.

[0026] To address the aforementioned technical problems, this invention provides an application of a BSM culture medium, wherein the concentrations of all components except glycerol and Pichia pastoris trace element 1 are reduced to 1 / 4 or 1 / 2, in the bio-fermentation preparation of human serum albumin.

[0027] Preferably, the BSM medium reduced to 1 / 4 or 1 / 2 is the BSM medium reduced to 1 / 4 or 1 / 2 as described above.

[0028] Preferably, the bio-fermentation is carried out using the above-mentioned Pichia pastoris strain that produces human serum albumin.

[0029] To address the aforementioned technical problems, this invention provides a method for purifying human serum albumin, the purification method comprising the step of affinity chromatography of human serum albumin.

[0030] The loading buffer used in the affinity chromatography is pH ≥ 4.3, preferably pH ≥ 5;

[0031] The human serum albumin is obtained by fermenting Pichia pastoris, a yeast that produces human serum albumin.

[0032] In this invention, the fermentation can be conventional in the art and can be carried out in a fermentation tank, such as a 3L, 5L or 10L fermentation tank.

[0033] Preferably, in further research, the applicant discovered that the target protein and the impurity protein have very similar charge properties, and the separation effect of ion exchange in the prior art is poor. It was unexpectedly discovered that changing the last step of ion exchange to a second affinity chromatography has a better effect. Therefore, the purification method of the present invention also includes the steps of hydrophobic chromatography and a second affinity chromatography of human serum albumin.

[0034] 4.3 is the isoelectric point of human serum albumin. When the pH of the loading buffer used in affinity chromatography is less than 4.3, albumin may precipitate or become unstable. Therefore, excessively acidic conditions are not used.

[0035] The loading buffer used in the affinity chromatography described in this invention can be the loading buffer for affinity chromatography conventionally defined in the art, that is, the buffer used to dilute the sample to be loaded or the raw material solution to be purified during affinity chromatography.

[0036] Preferably, the human serum albumin is the human serum albumin obtained using the above-described preparation method.

[0037] Preferably, the loading buffer used in the affinity chromatography includes potassium phosphate buffer, and more preferably potassium phosphate buffer containing sodium chloride; the concentration of potassium phosphate is preferably 20-30 mmol / L, more preferably 25 mmol / L, and the concentration of sodium chloride is preferably ≤0.1 mol / L.

[0038] Preferably, during the affinity chromatography, the sample used before loading is mixed with the loading buffer used during the affinity chromatography, such that the sodium chloride concentration in the sample is ≤0.1 mol / L, preferably 0.09 mol / L; the sample used during the affinity chromatography is preferably a filtered sample; more preferably a sample filtered through a 0.45 μm microporous membrane.

[0039] Preferably, the loading buffer used in the affinity chromatography is used for column equilibration of the chromatography column used in the affinity chromatography.

[0040] Preferably, the column used in the affinity chromatography is filled with active blue F3GA (Cibacron blue F3GA, or simply blue gel), and the column used in the affinity chromatography is preferably Blue Sepharose 6FF.

[0041] Preferably, the elution solution used in the affinity chromatography is a potassium phosphate buffer containing sodium chloride, wherein the concentration of potassium phosphate is preferably 20-30 mmol / L, more preferably 25 mmol / L, and the concentration of sodium chloride is preferably 1.5-2.5 mol / L, more preferably 2 mol / L.

[0042] Preferably, the pH of the elution solution used in the affinity chromatography is 7.

[0043] Preferably, the hydrophobic chromatography uses the solution obtained after affinity chromatography as the sample for loading. Before loading, the solution obtained after affinity chromatography is preferably mixed with a potassium phosphate buffer containing sodium chloride, such that the sodium chloride concentration of the solution obtained after affinity chromatography is 1.5-2.5 mol / L, preferably 2 mol / L; wherein the concentration of potassium phosphate in the potassium phosphate buffer containing sodium chloride is preferably 20-30 mmol / L, more preferably 25 mmol / L.

[0044] Preferably, the solution obtained from the hydrophobic chromatography is used as the sample for the second affinity chromatography. Before loading the sample, it is preferable to mix the solution obtained from the hydrophobic chromatography with the elution solution used in the hydrophobic chromatography, so that the sodium chloride concentration of the solution obtained from the hydrophobic chromatography is ≤0.1 mol / L.

[0045] In this invention, the elution solution is the solution used to elute the target protein (for example, it may be referred to as chromatography solution B in the prior art), rather than the elution solution obtained after elution.

[0046] Preferably, the column used in the second affinity chromatography is filled with Reactive Blue F3GA, and the column used in the second affinity chromatography is preferably Blue Sepharose 6FF.

[0047] Preferably, the chromatography column used in the hydrophobic chromatography is a Phenyl Sepharose 6FF.

[0048] Preferably, the buffer solution used for column equilibration during hydrophobic chromatography or the elution solution used during the second affinity chromatography is a potassium phosphate buffer containing sodium chloride. The concentration of potassium phosphate is preferably 20-30 mmol / L, more preferably 25 mmol / L, and the concentration of sodium chloride is preferably 1.5-2.5 mol / L, more preferably 2 mol / L.

[0049] Preferably, the buffer solution used for column equilibration during the second affinity chromatography is the same as the loading buffer solution used during the affinity chromatography.

[0050] Preferably, the elution solution used in the hydrophobic chromatography is a potassium phosphate buffer solution, and the concentration of potassium phosphate is preferably 20-30 mmol / L, more preferably 25 mmol / L.

[0051] Preferably, the pH of the buffer solution used for column equilibration in the hydrophobic chromatography and the elution solution used in the hydrophobic chromatography or the second affinity chromatography is 7.

[0052] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0053] The reagents and raw materials used in this invention are all commercially available.

[0054] The positive and progressive effects of this invention are as follows:

[0055] (1) The yield of human serum albumin and the proportion of human serum albumin to total protein obtained by the preparation method described in this invention are significantly higher than those of the prior art.

[0056] In a preferred embodiment of the present invention, the relevant strain was cultured using 1 / 2 BSM medium for fermentation. After 192 hours of induction, a human serum albumin yield of 17.47 g / L was achieved (the yield in the background patent 201711421153.3 was 8.86 g / L). Compared with the human serum albumin-related literature and patents reported to date, the expression level and expression efficiency are significantly higher. Furthermore, the target protein accounted for as much as 76.86% of the total protein in the supernatant of the fermentation broth at the induction endpoint (the proportion of human serum albumin in the background patent 201711421153.3 was approximately 50.19%). This is more conducive to subsequent separation, purification, and processing.

[0057] (2) The culture medium used in the preparation method of the present invention has significantly reduced costs and has no risk of animal-derived viral contamination, thereby significantly reducing the cost of the preparation method.

[0058] The reduced-salt BSM medium (e.g., 1 / 2 BSM and 1 / 4 BSM) described in this invention is a fully synthetic medium, with all components and their contents precisely known. It exhibits excellent batch-to-batch stability and is inexpensive (approximately 3-4 RMB / L). Furthermore, this medium contains no animal-derived components, eliminating the risk of animal-derived viral contamination.

[0059] (3) The purification method described in this invention significantly improves the recovery rate and the purity of the final product is also significantly improved.

[0060] The purification method described in this invention employs an affinity chromatography-hydrophobic chromatography-secondary affinity chromatography separation and purification scheme, which can achieve a high purity (>99% in a preferred embodiment of this invention) of the target protein while simultaneously achieving a high recovery rate (up to 67.53% in a preferred embodiment of this invention), indicating that the downstream separation and purification scheme established in this invention has good industrialization prospects. Attached Figure Description

[0061] Figure 1 This is a schematic diagram of the electrophoresis of the supernatant protein in the fermentation broth under BMGY medium; M is the marker; 1-14 are electrophoresis diagrams of the supernatant of the fermentation broth at 0, 12, 24, 36, 48, 60, 72, 84, 96, 108, 120, 132, 144 and 156 hours after induction, respectively.

[0062] Figure 2 This is a schematic diagram of the electrophoresis of the supernatant protein in the fermentation broth under BSM medium; M is the marker; 1-9 are electrophoresis diagrams of the supernatant of the fermentation broth at 0, 12, 24, 36, 48, 60, 72, 84 and 96 hours after induction, respectively.

[0063] Figure 3 This is a schematic diagram of the electrophoresis of the supernatant protein in the fermentation broth under BSM medium; M is the marker; 1-9 are electrophoresis diagrams of the supernatant of the fermentation broth at 108, 120, 132, 144, 156, 168, 180, 192 and 204 hours after induction, respectively.

[0064] Figure 4 This is a schematic diagram comparing the wet weight of bacterial cells in BMGY and BSM media.

[0065] Figure 5 This is a schematic diagram comparing the yield of the target protein in BMGY and BSM media.

[0066] Figure 6 This is a schematic diagram of the electrophoresis of the supernatant protein in the fermentation broth under 1 / 2 BSM medium; M is the marker; 1-9 are electrophoresis diagrams of the supernatant of the fermentation broth at 0, 12, 24, 36, 48, 60, 72, 84, and 96 hours after induction, respectively.

[0067] Figure 7 The diagram shows the electrophoresis of the supernatant protein in the fermentation broth under 1 / 2 BSM medium; M is the marker; 1-9 are the electrophoresis diagrams of the supernatant of the fermentation broth at 108, 120, 132, 144, 156, 168, 180, 192 and 204 hours after induction, respectively.

[0068] Figure 8This is a schematic diagram of the electrophoresis of the supernatant protein in the fermentation broth under 1 / 4 BSM medium; M is the marker; 1-9 are electrophoresis diagrams of the supernatant of the fermentation broth at 0, 12, 24, 36, 48, 60, 72, 84, and 96 hours after induction, respectively.

[0069] Figure 9 The diagram shows the electrophoresis of the supernatant protein in the fermentation broth under 1 / 4 BSM medium; M is the marker; 1-9 are the electrophoresis results of the fermentation broth supernatant at 108, 120, 132, 144, 156, 168, 180, 192, and 204 hours after induction, respectively.

[0070] Figure 10 This is a schematic diagram comparing the wet weight of bacterial cells in BSM medium with three different salt concentrations.

[0071] Figure 11 This is a schematic diagram of the electrophoresis of supernatant proteins from fermentation broths in BSM medium with three different salt concentrations.

[0072] Figure 12 This is a schematic diagram comparing the osmotic pressure of fermentation broth under three different salt concentrations in BSM medium.

[0073] Figure 13 This is a schematic diagram comparing the proportion of target proteins in BMGY and 1 / 2 BSM media.

[0074] Figure 14 The effect of sample loading pH on Blue Sepharose 6FF affinity chromatography.

[0075] Figure 15 Elution curves for Blue Sepharose 6FF affinity chromatography.

[0076] Figure 16 For Blue Sepharose 6FF affinity chromatography protein electrophoresis analysis; M is the marker; 1-4 are 0, 333, 666 and 1000 mg / L HSA standards, respectively; 5 is the separation and purification raw material solution; 6 is the raw material solution dilution; 7 is the affinity chromatography loading solution; 8 is the flow-through solution; 9 is the elution solution; 10 is the regeneration solution.

[0077] Figure 17 The elution curves for Phenyl Sepharose 6FF(HS) hydrophobic chromatography are shown.

[0078] Figure 18 This is a Phenyl Sepharose 6FF(HS) hydrophobic chromatography protein electrophoresis analysis. M is the marker; 1-4 are 0, 333, 666 and 1000 mg / L HSA standards, respectively; 5 is the hydrophobic chromatography loading solution; 6 is the flow-through solution; 7 is the elution solution.

[0079] Figure 19 Elution curves for Blue Sepharose 6FF using secondary affinity chromatography.

[0080] Figure 20 For secondary affinity chromatography protein electrophoresis analysis using Blue Sepharose 6FF; M is the marker; 1-4 are 0, 333, 666 and 1000 mg / L HSA standards, respectively; 5 is the secondary affinity chromatography loading solution; 6 is the flow-through solution; 7 is the elution solution; 8 is the regeneration solution.

[0081] Figure 21 Comparison of protein electrophoresis in the elution buffers of the three-step chromatography.

[0082] Figure 22 This study aimed to determine the HPLC purity of the concentrated secondary affinity chromatography eluent. Detailed Implementation

[0083] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0084] Tris (tris(hydroxymethyl)aminomethane), acrylamide, ammonium persulfate (for preparing protein electrophoresis gels), TEMED (tetramethylethylenediamine), SDS (sodium dodecyl sulfate), DTT (dithiothreitol), biotin, YNB (yeast-based nitrogen), bromophenol blue, EDTA (ethylenediaminetetraacetic acid), glycine, modified BCA (diquinoline carboxylic acid) protein concentration assay kit, and 0.45 μm microporous membranes were purchased from Shanghai Sangon Biotech Co., Ltd.; Blue Sepharose 6FF affinity chromatography pre-packed columns and Phenly Sepharose 6FF (HS) hydrophobic chromatography pre-packed columns were purchased from Shanghai Borglon Biotechnology Co., Ltd.; tryptone and yeast extract were purchased from Oxiod Ltd. (UK); human serum albumin standard was purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.; other common reagents such as methanol, sodium chloride, and ammonia were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0085] Example 1: Comparative Experiment of Yields of Four Culture Media in a 5L Fermenter

[0086] The production of human serum albumin was compared using BMGY medium, BSM medium, 1 / 2 BSM medium, and 1 / 4 BSM medium at the 5L fermenter level. The composition of the medium is as follows:

[0087] BMGY (Glycerol-Buffered Complex Medium): 20 g / L tryptone, 10 g / L yeast extract, 40 g / L glycerol, 10 g / L ammonium sulfate, 3.4 g / L yeast basal nitrogen, 0.1 M potassium phosphate buffer (pH 6.0), 0.0004 g / L biotin. Price: Approximately 50 RMB / liter.

[0088] BSM (Basic Salt Medium): 40 g / L glycerol, 26.7 mL / L phosphate (85%), 0.93 g / L calcium sulfate dihydrate, 18.2 g / L potassium sulfate, 14.9 g / L magnesium sulfate heptahydrate, 4.13 g / L potassium hydroxide, 4.0 mL / L PTM1 (Pichia pastoris trace element 1, specific components: 6 g / L copper sulfate pentahydrate, 0.08 g / L sodium iodide, 3 g / L manganese sulfate monohydrate, 0.2 g / L sodium molybdate dihydrate, 0.02 g / L boric acid, 0.5 g / L cobalt chloride, 20 g / L zinc chloride, 65 g / L ferrous sulfate heptahydrate, 0.2 g / L biotin, 5 mL / L sulfuric acid). Price approximately 3-4 RMB / L (initial pH of the medium adjusted to 5.0).

[0089] 1 / 2 BSM medium: 40 g / L glycerol, 13.3 mL / L phosphate (85%), 0.46 g / L calcium sulfate dihydrate, 9.1 g / L potassium sulfate, 7.4 g / L magnesium sulfate heptahydrate, 2.06 g / L potassium hydroxide, 4.0 mL / L PTM1;

[0090] 1 / 4 BSM medium: 40 g / L glycerol, 6.6 mL / L phosphate (85%), 0.23 g / L calcium sulfate dihydrate, 4.5 g / L potassium sulfate, 3.7 g / L magnesium sulfate heptahydrate, 1.03 g / L potassium hydroxide, 4.0 mL / L PTM1.

[0091] The specific operation process is as follows:

[0092] (1) Take the engineered Pichia pastoris GS115-pPIC9K-hsa-5-4 glycerol strain (i.e. the strain used in Example 5 of the patent application 201711421153.3 mentioned in the background, the preparation method of which is cited in the entire contents of the patent) and plate it on YPD (yeast extract peptone glucose medium, the specific components of which are: 10g / L yeast extract, 20g / L tryptone, 20g / L glucose) plate for activation;

[0093] (2) Select single clones into 100 mL Erlenmeyer flasks containing 10 mL YPD liquid medium (the medium composition is: 10 g / L yeast extract, 20 g / L tryptone, 20 g / L glucose) and culture them at 28 °C and 230 rpm for about 24 hours as primary seeds;

[0094] (3) Take 5 mL of YPD culture obtained from (2) into a 500 mL Erlenmeyer flask containing 100 mL of YPD liquid culture medium and culture it at 28℃ and 230 rpm for about 12 hours as secondary seed. Measure the wet weight of the bacterial cells before inoculation and perform microscopic examination to ensure that there are no contaminants.

[0095] (4) Prepare BMGY or BSM medium, 1 / 2 BSM medium, 1 / 4 BSM medium, PTM1 (trace elements), and 50% glycerol feed solution according to the above formula (30g glycerol dissolved in water and brought to a final volume of 60mL); after calibrating the pH electrode and dissolved oxygen electrode, sterilize the BMGY or BSM medium, 1 / 2 BSM medium, and 1 / 4 BSM medium together with the fermenter; after sterilization, place the tank at room temperature and cool it to room temperature (BMGY has three components that cannot be autoclaved, so the volume of the medium in the tank is 700mL; BSM has no components that cannot be autoclaved, so the volume of the medium in the tank is 1L).

[0096] (5) The non-thermal sterilizable components (YNB, biotin, potassium phosphate buffer) in the culture medium are pumped into the fermenter through the feeding bottle and the fermenter feeding device; the fermentation system is stabilized for 1 hour at an initial temperature of 28°C, pH of 5.75 (BMGY) or 5.0 (BSM, 1 / 2BSM, 1 / 4BSM), initial rotation speed of 200 rpm, and 1 vvm (air volume / culture volume / min, aeration ratio). Then the dissolved oxygen is corrected to 100%. At the same time, the secondary seed liquid obtained in step (3) is pumped into the fermenter through the feeding bottle and the feeding device. The temperature is maintained at 28°C, pH of 5.75 (BMGY) or 5.0 (BSM, 1 / 2BSM, 1 / 4BSM), and the dissolved oxygen level (DO) and rotation speed (Agit) are coupled.

[0097] (6) After DO rebounds, replenish with glycerol-fed culture medium (50% glycerol [30g glycerol dissolved in water and brought to a final volume of 60mL] + 12mL PTM1 / L), maintain a rate of 15 mL / (L*h), replenish within 4 hours, for a total replenishment volume of 60mL, containing 30g glycerol, maintain a temperature of 28℃, pH of 5.75 (BMGY) or 5.0 (BSM, 1 / 2BSM, 1 / 4BSM), and couple DO to rotation speed;

[0098] (7) After DO rebounds, replenish the medium with methanol (100% methanol + 12 mL PTM1 / L), maintain the temperature at 28℃ and pH=5.75, turn off the coupling between DO and rotation speed and set the rotation speed to 900 rpm, reverse the correlation between the feed and DO and set the critical value to 20% (i.e., start the feed when DO is greater than 20% and stop the feed when DO is less than 20%).

[0099] Every 12 hours, samples are taken, examined under a microscope, wet weight is measured, and protein electrophoresis is performed to detect the concentration of human serum albumin in the fermentation broth (the target protein concentration can be measured by protein electrophoresis). The fermentation broth is removed from the tank when the target protein concentration shows a decreasing trend.

[0100] The results of protein electrophoresis in the fermentation broth supernatant of BMGY and BSM media are as follows: Figure 1 and Figure 2 , Figure 3 As shown, the wet weight of bacterial cells and the yield of human serum albumin under the two culture media induction are respectively as follows: Figure 4 and Figure 5 As shown.

[0101] The results showed that under both BMGY and BSM media, an initial 40 g / L glycerol sustained bacterial growth for approximately 20 hours, with a cell wet weight reaching around 120 g / L. During the glycerol feeding phase, a 4-hour period resulted in a cell wet weight of approximately 200 g / L. In the methanol induction phase, the growth rates in both BMGY and BSM media were nearly identical. Regarding target protein (human serum albumin) expression, although the accumulation rate was faster under BMGY media, the peak concentration of 9.1 g / L was reached at 132 hours after induction, followed by a decline. Under BSM induction, both cell density and target protein concentration achieved more sustained accumulation, with cell density reaching approximately 500 g / L at the end of induction, while the target protein concentration peaked at 11.46 g / L at 192 hours after induction.

[0102] Electrophoresis results of fermentation broth supernatant proteins in 1 / 2 BSM and 1 / 4 BSM media are as follows: Figure 6 , Figure 7 , Figure 8 and Figure 9 As shown, the wet weight of bacterial cells and the yield of human serum albumin induced by BSM culture in three different salt concentrations are as follows: Figure 10 and Figure 11As shown in the figure. The results indicate that, compared with BSM, the production strain exhibited significantly better growth on media with reduced salt concentrations (i.e., 1 / 2 BSM and 1 / 4 BSM). In 1 / 2 BSM medium, the cells quickly reached a peak wet weight of approximately 500 g / L and maintained this wet weight for up to 120 hours until the fermentation endpoint; while in 1 / 4 BSM medium, although the cells also grew rapidly, growth stagnated in the later stages of fermentation. Regarding target protein expression, 1 / 2 BSM medium achieved continuous and stable target protein accumulation, reaching a peak of 17.47 g / L at 192 hours after induction, significantly higher than BSM. In 1 / 4 BSM medium, the target protein accumulation rate was fastest in the early and middle stages of fermentation, although accumulation ceased in the later stages. However, the maximum target protein yield of 13.39 g / L was achieved at 156 hours after induction, also significantly higher than existing technologies.

[0103] It is evident that reducing the salt concentration in the BSM medium did not cause nutrient deficiency in the production strains; on the contrary, the reduction in salt concentration and osmotic pressure promoted cell growth and target protein expression.

[0104] This example further uses an osmometer to measure the osmotic pressure of the fermentation broth at various time points in different culture media. The results are as follows: Figure 12 As shown in the figure. The results indicate that the osmotic pressure of the original BSM medium is as high as 1400 mOsm / kg or more, far exceeding that of other commonly used media. Overall, the osmotic pressure decreases with the extension of culture time, because the cells absorb salt ions from the fermentation broth as nutrients into the cells, resulting in a continuous decrease in the salt ion concentration in the fermentation broth. In addition, the decrease in osmotic pressure of the culture medium coincides with the increase in the wet weight of the cells, so it is speculated that the trend of osmotic pressure change can well reflect the growth status of the production strain. For example, in the middle and late stages of fermentation in 1 / 4 BSM medium, the osmotic pressure of the fermentation broth remained below 100 mOsm / kg, suggesting that there were very few salt ions left as nutrients in the fermentation broth, and the cell growth had stagnated.

[0105] In addition, protein electrophoresis was performed on the supernatants of fermentation broths induced by BMGY and 1 / 2 BSM media for comparison, and the results were as follows: Figure 13 As shown, the proportion of extraneous proteins in 1 / 2 BSM culture was significantly lower than that in BMGY culture. Further analysis using a modified BCA protein concentration assay kit revealed that the total protein concentration in the fermentation broth supernatant after 192 hours of induction in 1 / 2 BSM culture was 22.73 g / L, indicating a target protein concentration of 76.86%, significantly higher than the 50.19% in BMGY culture.

[0106] The target protein concentration was measured in each culture medium as follows: Commercially available recombinant albumin (purity >99.9%) at known concentrations was used as standards and subjected to protein electrophoresis with diluted fermentation broth supernatant. The concentration of the standards and the gray values ​​of the scanned bands were fitted together. Data were considered valid when the linear correlation was greater than 99%. Based on the valid data, the concentration of the target protein could be calculated from the gray value of the target protein band in the fermentation broth supernatant. The total protein concentration was measured using a modified BCA protein concentration assay kit (purchased from Shanghai Sangon Biotech). The target protein concentration was divided by the total protein concentration to obtain the target protein percentage.

[0107] In summary, this invention uses a culture medium with reduced salt concentration (1 / 2 BSM as an example) to culture the production strain GS115-pPIC9K-hsa-5-4. From the initial seed culture to the fermentation endpoint induced by this culture medium, the total time taken was 252 hours (24 hours of primary seed culture + 12 hours of secondary seed culture + 24 hours of in-tank growth + 192 hours of in-tank induction). The culture medium cost was approximately 40 yuan (YPD secondary seed medium approximately 1.5 yuan + 1L 1 / 2 BSM approximately 3 yuan + 1.62L methanol approximately 26 yuan + 0.5L ammonia approximately 9 yuan; this cost is a reference price for purchasing analytical grade chemical reagents in a research laboratory. If large-scale procurement is carried out in subsequent industrial production, this cost can be further reduced). The total fermentation broth volume was approximately 2.5L, of which the supernatant was approximately 1.25L, and the total yield of the target protein was 21.84g. This production strain and fermentation process can achieve high target protein production with relatively low culture medium costs, and has good industrialization prospects.

[0108] Example 2: Isolation and purification of human serum albumin

[0109] In this example, the supernatant of the fermentation broth induced for 192 hours in 1 / 2 BSM medium was filtered through a 0.45 μm microporous membrane, and the filtrate was used as the separation and purification feed solution. When performing Blue Sepharose 6FF affinity chromatography, the pH of the loading buffer was selected as 5.

[0110] Affinity chromatography solutions A [25 mM KPB (potassium phosphate buffer) + 0.1 M NaCl] with different pH values ​​(5.0, 6.0, 7.0, 8.0, and 9.0) were prepared, mixed with fermentation supernatant at a 9:1 ratio, and then loaded onto the supernatant. Elution was performed using affinity chromatography solution B (25 mM KPB + 2.0 M NaCl) at pH 7.0. The flow-through and eluent were collected for protein gel electrophoresis. The results are shown below. Figure 14As shown in the figure, the electrophoresis results indicate that the amount of target protein flowing through the chromatography column tends to increase with increasing loading pH (pH value of the loading solution). This suggests that the binding ability of Blue Sepharose 6FF to the target protein decreases with increasing pH. When the pH is lower than the isoelectric point of human serum albumin (4.3), albumin may precipitate or become unstable. Therefore, it can be predicted that under acidic conditions above the isoelectric point of human serum albumin (4.3), Blue Sepharose 6FF will have a strong binding ability to the target protein. In a loading buffer system at pH 5, almost no target protein was observed in the flow-through electrophoresis results; in a loading buffer system at pH 6, the target protein was also very weak; while in other loading buffer systems at pH ≥ 7, the target protein was more abundant in the flow-through electrophoresis results.

[0111] (1) Blue Sepharose 6FF affinity chromatography

[0112] Take 0.4 mL of the purified feed solution and mix it thoroughly with 3.6 mL of affinity chromatography solution A (25 mM KPB [kalium (potassium) phosphate buffer] + 0.1 M NaCl, pH=5) to achieve a sample concentration of 0.09 M. Filter the mixture using a 0.45 μm microporous membrane; this filtrate is used as the affinity chromatography loading solution. After equilibrating the Blue Sepharose 6FF with affinity chromatography solution A, elute the column with the loading solution using the pump head and collect the flow-through (Ft). Elute with affinity chromatography solution B (25 mM KPB + 2.0 M NaCl, pH=7) and collect the eluent (Peak1). Regenerate the column with 0.1 M NaOH and collect the regeneration solution (Peak2). The elution curve is shown below. Figure 15 As shown; protein electrophoresis analysis was performed on the flow-through buffer, elution buffer, and regeneration buffer, and the results are as follows. Figure 16 As shown in the figure, the electrophoresis results indicate that the eluent contains the target protein with a certain purity, while the degradation fragments of the target protein are enriched in the strong alkaline regeneration solution. Furthermore, optimizing the culture medium from BMGY to 1 / 2 BSM and the pH for affinity chromatography loading from 7.0 to 5.0 significantly improved the elution recovery rate (the purity of the target protein was comparable to that in the background patent application 201711421153.3, and its recovery rate increased from 58% in the background patent application 201711421153.3 to 86%. See Table 1 for the specific recovery rate and purity data summarized in this experiment). Since the eluent remained pale yellow, it was presumed to contain pigment impurities that affinity chromatography could not remove. Therefore, hydrophobic chromatography was used in the next step of this example to remove the pigments.

[0113] (2) Phenyl Sepharose 6FF(HS) hydrophobic chromatography

[0114] Based on conductivity measurements, the actual NaCl content of the affinity chromatography eluent was estimated to be approximately 1.3 M. To ensure equilibrium during sample loading, the affinity chromatography eluent was mixed 1:1 with a buffer solution of 25 mM KPB + 2.7 M NaCl, pH=7, maintaining the final NaCl concentration at approximately 2 M. After column equilibration using Phenyl Sepharose 6FF hydrophobic chromatography solution A (25 mM KPB + 2.0 M NaCl, pH=7), the column was eluted with the mixture via a pump head, and the flow-through buffer (Ft) was collected. Elution was then performed using hydrophobic chromatography solution B (25 mM KPB, pH=7), and the eluent (Peak 1) was collected. Column regeneration was performed using 0.1 M NaOH. The elution curve is shown below. Figure 17 The elution curves show that the flow-through still contains a small amount of protein, while no protein residue remains in the column after elution with solution B. Protein electrophoresis analysis of the flow-through and elution solutions yielded the following results: Figure 18 The electrophoresis results show that the flow-through contained a very small amount of the target protein, but the vast majority remained in the elution buffer, which was transparent and colorless, indicating that the pigment was effectively removed (see Table 1 for the corresponding target protein purity and recovery rate data).

[0115] (3) Blue Sepharose 6FF secondary affinity chromatography

[0116] Because the hydrophobic eluent still contains trace amounts of target protein degradation fragments, a second affinity chromatography was performed using BlueSepharose 6FF in this example to further remove impurities. Since the hydrophobic eluent still contains a certain amount of NaCl, it was mixed with 25 mM KPB, pH=7 buffer at a ratio of 1:9 to achieve a final NaCl concentration between 0 and 0.1 M. After equilibrating BlueSepharose 6FF with affinity chromatography solution A (25 mM KPB + 0.1 M NaCl, pH=5), the column was eluted with the second affinity chromatography loading solution using the pump head, and the flow-through buffer (Ft) was collected. Elution was performed using affinity chromatography solution B (25 mM KPB + 2.0 M NaCl, pH=7), and the eluent (Peak1) was collected. Column regeneration was performed using 0.1 M NaOH, and the regeneration buffer (Peak2) was collected. The elution curve is shown below. Figure 19 The elution curves show that some proteins cannot be eluted by the high-salt solution B and can only be eluted by the strong alkali. Electrophoretic analysis of the flow-through, elution, and regeneration solutions yielded the following results: Figure 20The electrophoresis results show that the degradation fragments of the target protein were further enriched in the strong base elution buffer, while the high salt elution buffer only showed a single band of the target protein, indicating that the secondary affinity chromatography achieved a further purification effect (the corresponding target protein purity and recovery data are detailed in Table 1).

[0117] The purified samples obtained from the above three steps were analyzed by protein electrophoresis, and the results are as follows: Figure 21 .

[0118] The concentrated secondary affinity eluent was subjected to HPLC purity identification, and the results are as follows: Figure 22 The test results show that the secondary affinity eluent is a final product with high purity (>99%).

[0119] The separation and purification summary is shown in Table 1 below.

[0120] Table 1 Summary of Separation and Purification

[0121]

[0122] The final overall recovery rate of this separation and purification scheme reached 67.53%. Combined with the target protein concentration during fermentation in the tank, it is reasonable to conclude that if this scheme can be successfully scaled up, theoretically more than 10g of highly pure human serum albumin could be obtained per liter of fermentation broth. The excellent target protein expression performance of this production strain, combined with the efficient downstream fermentation process and purification scheme, demonstrates promising prospects for industrialization. SEQUENCE LISTING <110> Shanghai Pharmaceutical Industry Research Institute, China Pharmaceutical Industry Research Institute <120> A method for preparing and purifying human serum albumin <130> P180117303C <160> 1 <170> PatentIn version 3.5 <210> 1 <211> 1830 <212> DNA <213> Homo sapiens <400> 1 atgaagtggg taacctttat ttcccttctt tttctcttta gctcggctta ttccaggggt 60 gtgtttcgtc gagatgcaca caagagtgag gttgctcatc ggtttaaaga tttgggagaa 120 gaaaatttca aagccttggt gttgattgcc tttgctcagt atcttcagca gtgtccattt 180 gaagatcatg taaaattagt gaatgaagta actgaatttg caaaaacatg tgttgctgat 240 gagtcagctg aaaattgtga caaatcactt catacccttt ttggagacaa attatgcaca 300 gttgcaactc ttcgtgaaac ctatggtgaa atggctgact gctgtgcaaa acaagaacct 360 gagagtaatg aatgcttctt gcaacacaaa gatgacaacc caaacctccc ccgattggtg 420 agaccagagg ttgatgtgat gtgcactgct tttcatgaca atgaagagac atttttgaaa 480 aaatacctat atgaaattgc cagaagacat ccttactttt atgccccgga actccttttc 540 tttgctaaaa ggtataaagc tgcttttaca gaatgttgcc aagctgctga taaagctgcc 600 tgcctgttgc caaagctcga tgaacttcgg gatgaaggga aggcttcgtc tgccaaacag 660 agactcaagt gtgccagtct ccaaaaattt ggagaaagag ctttcaaagc atgggcagta 720 gctcgcctga gccagagatt tcccaaagct gagtttgcag aagtttccaa gttagtgaca 780 gatcttacca aagtccacac ggaatgctgc catggagatc tgcttgaatg tgctgatgac 840 agggcggacc ttgccaagta tatctgtgaa aatcaagatt cgatctccag taaatgaag 900 gaatgctgtg aaaaacctct gttggaaaaa tccactgca ttgccgaagt ggaaaatgat 960 gagatgcctg ctgacttgcc ttcattagct gctgattttg ttgaagtaa ggatgtttgc 1020 aaaaactatg ctgaggcaaa ggatgtcttc ctgggcatgt ttttgtatga atatgcaaga 1080 aggcatcctg attactctgt cgtgctgctg ctgagacttg ccaagacata tgaaaccact 1140 ctagagaagt gctgtgccgc tgcagatcct catgaatgct atgccaaagt gttcgatgaa 1200 tttaaacctc ttgtggaaga gcctcagaat ttaatcaaac aaaattgtga gctttttgag 1260 cagcttggag agtacaaatt ccagaatgcg ctattagttc gttacaccaa gaagtaccc 1320 caagtgtcaa ctccaactct tgtagaggtc tcaagaaacc taggaaaagt gggcagcaaa 1380 tgttgtaaac atcctggagc aaaaagaatg ccctgtgcag aagactatct atccgtggtc 1440 ctgaaccagt tatgtgtgtt gcatgaagaa acgccagtaa gtgacagagt caccaaatgc 1500 1560 tacgttccca aagagtttaa tgctgaaaca ttcaccttcc atgcagatat atgcacactt tctgagaagg agagacaaat caagaaacaa actgcacttg ttgagcttgt gaaacacaag cccaaggcaa caaaagagca actgaaagct gttatggatg atttcgcagc ttttgtagg aagtgctgca aggctgacga taaggagacc tgctttgccg aggggtaa aaaacttgtt gctgcaagtc aagctgcctt aggcttata

Claims

1. A method for preparing human serum albumin, characterized in that, include: With DO correction at 100%, Pichia pastoris, which produces human serum albumin, was inoculated into 1 / 2 BSM medium at pH 5.0 for fermentation, maintained at 28°C and pH 5.0, with DO and rotation speed coupled. After DO rebounds, glycerol-fed culture medium is added at a rate of 15 mL / (L*h) over 4 hours, maintaining a temperature of 28℃ and a pH of 5.

0. After DO rebounds, methanol-fed culture medium is added for induction. During this period, the coupling between DO and the fermentation speed is turned off, and the feed and DO are decoupled and the DO critical value is set to 20%. Human serum albumin can be obtained from the fermentation broth after 192 hours of induction. The 1 / 2 BSM medium is a BSM medium in which the concentrations of all components except glycerol and Pichia pastoris trace element 1 are reduced by 1 / 2. The 1 / 2 BSM medium comprises the following components: 4% glycerol, 1.335% phosphate, 0.0465% calcium sulfate dihydrate, 0.91% potassium sulfate, 0.745% magnesium sulfate heptahydrate, 0.206% potassium hydroxide, and 0.4% Pichia pastoris trace element 1; wherein the percentages of phosphate and Pichia pastoris trace element 1 are the volume percentages of each component in the medium; the percentages of the remaining components are the mass-volume percentages of each component in the medium. The glycerol-supplemented culture medium consists of 50% glycerol and 12 mL / L Pichia pastoris trace element 1; The methanol-fed culture medium consists of 100% methanol and 12 mL / L Pichia pastoris trace element 1; The *Pichia pastoris* strain producing human serum albumin is an engineered strain that integrates an expression vector containing the human serum albumin gene into *Pichia pastoris* GS-115. The expression vector sequentially contains a promoter, an α-guide peptide, the human serum albumin gene, and a terminator; the promoter is the AOX1 promoter; the α-guide peptide is derived from *Saccharomyces cerevisiae*; the nucleotide sequence of the human serum albumin gene is shown in SEQ ID NO: 1 in the sequence listing; and the backbone of the expression vector is plasmid pPIC9K. Obtaining human serum albumin from the fermentation broth includes further purification, which includes the steps of performing affinity chromatography, hydrophobic chromatography, and a second affinity chromatography on the fermentation supernatant.

2. The preparation method according to claim 1, characterized in that, The loading buffer used in the affinity chromatography is a potassium phosphate buffer containing sodium chloride at pH 5, wherein the concentration of potassium phosphate is 20-30 mmol / L and the concentration of sodium chloride is 0.1 mol / L. During the affinity chromatography, the sample and loading buffer are mixed at a ratio of 1:9 before loading. The sample is fermentation supernatant filtered through a 0.45 μm microporous membrane. The column is equilibrated with the loading buffer before loading. The chromatography column used in the affinity chromatography is a Blue Sepharose 6FF. The elution solution used in the affinity chromatography is a potassium phosphate buffer containing sodium chloride at pH 7, wherein the concentration of potassium phosphate is 20-30 mmol / L and the concentration of sodium chloride is 1.5-2.5 mol / L.

3. The preparation method according to claim 1, characterized in that, The components of the Pichia pastoris trace element 1 are as follows: 0.6% copper sulfate pentahydrate, 0.008% sodium iodide, 0.3% manganese sulfate monohydrate, 0.02% sodium molybdate dihydrate, 0.002% boric acid, 0.05% cobalt chloride, 2% zinc chloride, 6.5% ferrous sulfate heptahydrate, 0.02% biotin, and 0.5% sulfuric acid; Wherein, the percentage of sulfuric acid is its volume percentage relative to the Pichia pastoris trace element 1; the percentages of the other components are the mass-volume percentages of each component relative to the Pichia pastoris trace element 1.

4. The preparation method according to any one of claims 1-3, characterized in that, The expression vector carries a gene against genipathic mycotoxin, and the engineered bacteria exhibit resistance to genipathic mycotoxin at concentrations of 0.25–5.0 mg / mL.

5. The preparation method according to claim 2, characterized in that, The concentration of potassium phosphate in the loading buffer is 25 mmol / L; The elution solution used in the affinity chromatography contained potassium phosphate at a concentration of 25 mmol / L and sodium chloride at a concentration of 2 mol / L.

6. The preparation method according to claim 5, characterized in that, In hydrophobic chromatography, the affinity chromatography eluent is used as the sample for loading. Before loading, the affinity chromatography eluent is mixed with a potassium phosphate buffer containing sodium chloride, so that the concentration of sodium chloride in the mixed solution is 1.5-2.5 mol / L; wherein, the concentration of potassium phosphate in the potassium phosphate buffer containing sodium chloride is 20-30 mmol / L. And / or, during the second affinity chromatography, the hydrophobic chromatography eluent is used as the sample for loading. Before loading, the hydrophobic chromatography eluent is mixed with the eluent used in the hydrophobic chromatography so that the sodium chloride concentration in the mixed solution is 0.1 mol / L. And / or, the chromatographic column used in the second affinity chromatography is filled with Reactive Blue F3GA, and the chromatographic column used in the second affinity chromatography is Blue Sepharose 6FF; And / or, the chromatography column used in the hydrophobic chromatography is a Phenyl Sepharose 6FF; And / or, the buffer solution used for column equilibration during hydrophobic chromatography or the elution solution used during the second affinity chromatography is a potassium phosphate buffer containing sodium chloride, wherein the concentration of potassium phosphate is 20-30 mmol / L and the concentration of sodium chloride is 1.5-2.5 mol / L; And / or, the buffer solution used for column equilibration during the second affinity chromatography is the same as the loading buffer solution used during the affinity chromatography; And / or, the elution solution used in the hydrophobic chromatography is potassium phosphate buffer, wherein the concentration of potassium phosphate is 20~30 mmol / L; And / or, the buffer solution used for column equilibration during the hydrophobic chromatography, and the pH of the elution solution used during the hydrophobic chromatography or the second affinity chromatography, are 7.

7. The preparation method according to claim 6, characterized in that, In hydrophobic chromatography, the affinity chromatography eluent is used as the sample for loading. Before loading, the affinity chromatography eluent is mixed with a potassium phosphate buffer containing sodium chloride, so that the concentration of sodium chloride in the mixed solution is 2 mol / L; wherein, the concentration of potassium phosphate in the potassium phosphate buffer containing sodium chloride is 25 mmol / L. And / or, the buffer solution used for column equilibration during hydrophobic chromatography or the elution solution used during the second affinity chromatography is a potassium phosphate buffer containing sodium chloride, wherein the concentration of potassium phosphate is 25 mmol / L and the concentration of sodium chloride is 2 mol / L. And / or, the elution solution used in the hydrophobic chromatography is potassium phosphate buffer, wherein the concentration of potassium phosphate is 25 mmol / L.

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