A method for purifying human serum albumin from supernatant of low temperature ethanol precipitated fraction II+III by affinity chromatography

Abstract

The application belongs to the field of biological medicine, and provides a method for purifying human serum albumin from supernatant of low-temperature ethanol precipitation component II+III by affinity chromatography. The supernatant of component II+III is clarified after pH adjustment, concentrated by ultrafiltration, and buffer replacement, and then is loaded into a fatty acid affinity chromatography column; the column is sequentially washed by an equilibrium buffer, a high-salt washing buffer to remove transferrin and other impurities, and then is specifically eluted by an elution buffer containing sodium octanoate; the target peak is collected, subjected to ultrafiltration and buffer replacement, pasteurization and sterilization, and then is divided into finished products. The application innovatively proposes a combination strategy of "weak acid conformation recognition loading-high salt charge shielding washing", and realizes efficient purification by single-step affinity chromatography. The obtained HSA has a purity of ≥99% and a recovery rate of >90%, and the production cycle is shortened by about 60% compared with the traditional low-temperature ethanol method. The process has strong stability, is easy to linearly enlarge, significantly reduces the production cost and operation risk, and is suitable for industrial large-scale production.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and more specifically to a method for affinity chromatography purification of human serum albumin from the supernatant of low-temperature ethanol precipitation fraction II+III. Background Technology

[0002] Human serum albumin (HSA) is the most abundant protein in blood plasma, accounting for approximately 50%-60% of total plasma protein. With a molecular weight of about 66.5 kDa, it plays various physiological roles, including maintaining plasma colloid osmotic pressure, transporting nutrients and drugs, and acting as an antioxidant. In the medical field, HSA is widely used in shock resuscitation, burn treatment, correction of hypoalbuminemia, treatment of ascites in cirrhosis, and as a drug carrier. Global annual demand exceeds 500 tons, making it an important plasma-derived biological product.

[0003] Currently, the main industrial method for producing human serum albumin is the Cohn low-temperature ethanol precipitation method and its improved processes (such as the Kistler-Nitschmann method). This method utilizes the differences in plasma protein solubility under different ethanol concentrations, temperatures, and pH conditions to obtain various protein components through multi-step precipitation separation. Among them, the supernatant of component II+III is the liquid phase produced during the separation process of the Cohn 6 method or the Kistler-Nitschmann method under conditions of ethanol concentration of approximately 18%-25% and pH 5.8-7.2, and it is the main enriched phase of albumin.

[0004] However, the traditional low-temperature ethanol method has some technical drawbacks, such as a lengthy and complex process, typically requiring more than five ethanol precipitation steps, resulting in a production cycle of several days. It also places high demands on low-temperature reaction equipment and energy consumption, making process control difficult. Furthermore, the product purity is limited; the albumin purity obtained by traditional processes is generally around 96%, which is insufficient to meet the requirements of some high-end medical applications for ultra-high purity albumin (≥99%). The yield is also low, as multiple precipitation steps lead to albumin adsorption and denaturation losses at the solid-liquid interface, resulting in a total yield of only 60%-75%. Finally, impurity control is difficult; impurities such as transferrin (Tf, molecular weight approximately 76 kDa, isoelectric point approximately 5.9) and ceruloplasmin (Cp) have similar physicochemical properties to albumin, making them difficult to remove efficiently using traditional methods. The residual amount of transferrin is typically higher than 0.1%, affecting product safety.

[0005] To overcome the aforementioned problems, the industry has attempted to introduce chromatography techniques for albumin purification. For example, techniques such as ion exchange chromatography, hydrophobic chromatography, and gel filtration chromatography have been reported for albumin purification. However, these conventional chromatography methods suffer from limited resolution, low throughput, and high media costs, and typically require multiple steps in series, making the process still relatively complex.

[0006] Affinity chromatography has attracted much attention in the field of biopharmaceutical purification due to its high selectivity and high resolution. Existing literature reports methods for purifying albumin using dye-ligand chromatography (such as Blue Sepharose conjugated with Cibacon Blue F3G-A), metal chelate chromatography, or biomimetic affinity ligands. However, these methods have limited selectivity for albumin in their affinity media, often accompanied by non-specific adsorption of other plasma proteins, and there is a potential risk of dye ligand shedding, limiting their application in the production of injectable albumin.

[0007] The paper, titled "Study on the Purification of Human Serum Albumin by Electrostatic Coupling Affinity Chromatography," published in 2023, describes DASA-Sepharose as a novel affinity medium using cross-linked agarose as a matrix and octyl succinic anhydride affinity ligands coupled to a 3,5-diaminobenzoic acid spacer arm. Albumin is adsorbed electrostatically via the carboxyl groups on the spacer arm, and then the octyl succinic anhydride affinity ligand specifically binds to the fatty acid binding sites of albumin, achieving electrostatic coupling affinity adsorption. The paper found in adsorption equilibrium experiments that NaCl concentrations of 0.025-0.06 mol / L had almost no effect on the static adsorption capacity Qm of albumin, while Qm decreased significantly at NaCl concentrations of 0.1 mol / L and above. Based on this finding, the paper added 0.035 mol / L NaCl to the equilibration buffer for elution to avoid high salt concentrations disrupting the electrostatic adsorption of albumin, leading to a decrease in Qm and yield; the final product purity was only 98.20%.

[0008] Therefore, developing a method for efficient purification of high-purity human serum albumin directly from the supernatant of components II+III based on DASA Sepharose affinity media is of significant industrial value for simplifying the process, improving product quality, and reducing production costs. Summary of the Invention

[0009] The first aspect of the present invention provides a method for affinity chromatography purification of human serum albumin from the supernatant of low-temperature ethanol precipitation fraction II+III, the method comprising the following steps:

[0010] S1. Raw material pretreatment: Collect the supernatant of components II+III from the low-temperature ethanol separation process of human plasma, adjust the pH value to 5.0-7.0, centrifuge or filter to remove insoluble precipitates, and obtain a clear liquid;

[0011] S2, Ultrafiltration Concentration and Buffer Replacement: The clarified solution obtained in S1 is concentrated by ultrafiltration and replaced with a balanced buffer system to obtain the loading solution;

[0012] S3. Chromatographic purification: The sample solution obtained in S2 is fed into the chromatography column, the chromatography column is washed with washing buffer, and then eluted with elution buffer. The elution peak is collected, which is the purified human serum albumin chromatographic product.

[0013] S4. Formulation and Virus Inactivation: The purified human serum albumin obtained in S3 is replaced with a formulation buffer, and then pasteurized, sterilized, filtered, and dispensed to obtain the human serum albumin product.

[0014] Specifically, the low-temperature ethanol component II+III supernatant mentioned in S1 is the liquid phase produced during the Cohn separation process under conditions of ethanol concentration of 19%-25%.

[0015] Specifically, the ultrafiltration described in S2 is performed using an ultrafiltration membrane with a molecular weight cutoff of 10kDa-50kDa.

[0016] Specifically, the equilibration buffer described in S2 comprises 10-50 mM phosphate buffer or acetate-sodium acetate buffer and 0-0.2 M sodium chloride, pH 5.0-7.0.

[0017] More specifically, the concentration of the equilibration buffer is 10-20 mM.

[0018] More specifically, the concentration of the equilibration buffer is 20-50 mM.

[0019] More specifically, the concentration of sodium chloride in the equilibration buffer is 0-0.15M.

[0020] More specifically, the concentration of sodium chloride in the equilibration buffer is 0.15-0.2M.

[0021] More specifically, the pH of the equilibration buffer is 5.0-6.0.

[0022] More specifically, the pH of the equilibration buffer is 6.0-6.5.

[0023] More specifically, the pH of the equilibration buffer is 6.5-7.0.

[0024] Specifically, the sample loading solution described in S2 has a pH of 5.0-7.0 and an ionic strength of 0.05-0.2M.

[0025] Specifically, the chromatography described in S3 is affinity chromatography, and the chromatography column is packed with DASA Sepharose affinity gel.

[0026] In this invention, the term "DASA Sepharose" refers to an electrostatic coupling affinity chromatography medium, which is obtained by covalently coupling n-octyl succinic anhydride affinity ligands with cross-linked agarose (Sepharose) as the solid matrix and 3,5-diaminobenzoic acid as the spacer arm.

[0027] More specifically, the DASA Sepharose preparation method is the same as in "Study on the Purification of Human Serum Albumin by Electrostatic Coupling Affinity Chromatography", specifically as follows:

[0028] (1) Microsphere activation: Measure 10 mL of Sepharose 6 FF microspheres, wash them thoroughly with deionized water, completely replace the ethanol preservation solution, and dry them; add 8 mL of 1 mol / L NaOH, 20 mL of epichlorohydrin, and 8 mL of dimethyl sulfoxide in sequence, shake gently to fully suspend the medium, seal and place in a shaker for shaking (conditions: 40℃, 150 r / min, 2.5 h).

[0029] (2) Spacer arm connection: The activated medium is thoroughly washed with pre-cooled deionized water. Add 2 mL of 1.4 mol / L sodium thiosulfate and 0.2 mL of phenolphthalein reagent to the washing solution until the washing solution does not turn red after vigorous shaking, indicating that the washing is complete; dry it again and add an equal volume of ethanol solution of 3,5-diaminobenzoic acid, and shake again (conditions: 25℃, 150 r / min, 14 h).

[0030] (3) Ligand linkage: Remove the medium connecting the spacer arms, dry it, and wash it sequentially with deionized water, 0.5 mol / L NaCl, 0.1 mol / L sodium acetate (pH=4.70), and 50 mmol / L PB (pH=7.40) and dry it again. Place the medium in an aqueous solution of dioxane at a ratio of 14 mL:7 mL (v / v=2:1); dissolve 2 g of octyl succinic anhydride in 20 mL of dioxane, and slowly add the ligand to the medium suspension while maintaining the pH at 7.60~8.50; shake again (25℃, 130 r / min, 2 h). The coupling ligand medium was washed sequentially with deionized water, 0.5 mol / L NaCl, 0.1 mol / L sodium acetate (pH=4.70), and 50 mmol / L PB (pH=7.40) and then dried. Finally, the medium was stored in 20% ethanol-water solution. The 3,5-diaminobenzoic acid mesoarm octyl succinic anhydride ligand affinity medium (abbreviated as DASA-Sepharose) was thus prepared.

[0031] Specifically, the volume ratio of the loading solution to the DASA Sepharose affinity medium in S3 is 0.5-2:1.

[0032] Specifically, the washing buffer described in S3 comprises 10-50 mM phosphate buffer and 0.15-1.0 M sodium chloride, pH 6.0-8.0.

[0033] More specifically, the washing buffer solution has a concentration of 10-20 mM.

[0034] More specifically, the washing buffer solution has a concentration of 20-50 mM.

[0035] More specifically, the concentration of sodium chloride in the washing buffer is 0.15-0.5M.

[0036] More specifically, the concentration of sodium chloride in the washing buffer is 0.5-0.75M.

[0037] More specifically, the sodium chloride concentration in the washing buffer is 0.75-1.0M.

[0038] Specifically, the elution buffer described in S3 comprises 10-50 mM buffer, 4-30 g / L sodium octanoate and 0.1-0.5 M sodium chloride, pH 6.0-8.5.

[0039] More specifically, the concentration of sodium octanoate in the elution buffer is 4-10 g / L.

[0040] More specifically, the concentration of sodium octanoate in the elution buffer is 10-30 g / L.

[0041] More specifically, the buffer solution includes phosphate buffer or Tris-HCl buffer.

[0042] A second aspect of the present invention provides a human serum albumin, which is prepared by any of the methods described above.

[0043] Beneficial effects:

[0044] 1. Innovative Process Route: This invention abandons the traditional five-step precipitation process for purifying albumin using low-temperature ethanol precipitation. Instead, it directly utilizes the high selectivity of DASA Sepharose media to capture albumin from the supernatant of fractions II+III in a single step. Compared to existing technologies, this shortens the production cycle by approximately 60%, reduces the consumption of ethanol and buffer solutions, and, due to the reduced number of steps, achieves higher separation efficiency, resulting in a yield that is 20-30% higher than the traditional low-temperature ethanol precipitation process.

[0045] 2. Solving the Impurity Problem: To address the unique impurities such as transferrin and immunoglobulins in the supernatant of components II+III, this invention innovatively introduces a combined strategy of "weakly acidic loading + high-salt washing." Experiments have shown that this strategy effectively blocks the non-specific binding of transferrin to the medium, ensuring that the purity of the final product remains stable at over 99%, superior to the 96% purity of traditional processes.

[0046] 3. Media performance optimization: It was found that DASA Sepharose media has excellent stability under weakly acidic conditions at pH 5-7, and the conformation of albumin is more conducive to the binding of specific ligands under these conditions, thereby improving the dynamic loading capacity of the media and reducing production costs. Attached Figure Description

[0047] Figure 1 This is a schematic diagram of the affinity chromatography purification process.

[0048] Figure 2 High-performance liquid chromatography analysis of affinity chromatography eluted components. Detailed Implementation

[0049] The present invention can be better understood through the following examples.

[0050] Example 1

[0051] 1. Pretreatment: Human plasma was treated with ethanol at low temperature until the concentration reached approximately 8% and the pH reached 7.2. Fibrinogen (fraction I) was precipitated and removed, yielding supernatant I. Ethanol was then added to supernatant I until the concentration reached 19%, the pH was adjusted to 5.8, and the temperature was controlled at -5°C. This precipitated immunoglobulins and other proteins into a solid phase, fractions II and III. The resulting liquid phase after separation was the supernatant of fractions II and III.

[0052] Take 50 mL of Cohn fraction II+III supernatant, concentrate by ultrafiltration (10 kDa ultrafiltration membrane) using equilibration buffer (20 mM PBS, pH 6.5, 0.15 M NaCl) and replace with buffer to obtain pretreated sample.

[0053] 2. Affinity chromatography:

[0054] Pack a DASA Sepharose affinity column (10 mL column volume) and equilibrate it with 30 mL of equilibration buffer (20 mM PBS, pH 6.5, 0.15 M NaCl) at a flow rate of 2 mL / min.

[0055] 20 mL of the pretreated sample was fed at a flow rate of 1 mL / min.

[0056] Rinse with 40 mL of equilibration buffer (20 mM PBS, pH 6.5, 0.15 M NaCl);

[0057] Wash 40 mL with washing buffer (10 mM PBS, pH 6.5, 0.50 M NaCl) until the effluent A280 < 0.05;

[0058] A gradient elution was performed using elution buffer (50 mM PBS, pH 7.0, 0.5 M NaCl, sodium octanoate concentration 10 g / L) at a flow rate of 2 mL / min. The elution peaks were collected, and the affinity chromatography purification process was as follows. Figure 1 As shown, the high-performance liquid chromatography analysis of the eluted components is as follows: Figure 2 As shown.

[0059] 3. Post-processing: Ultrafiltration concentration (10kDa ultrafiltration membrane) was performed using 20 mM pH 7.0 phosphate buffer (4.37 g disodium hydrogen phosphate dodecahydrate; 1.22 g sodium dihydrogen phosphate dihydrate; 1 L water added, pH adjusted to 7.0) to achieve a protein concentration of 200 g / L. The sodium caprylate concentration was then adjusted to 32 mM to obtain the albumin product.

[0060] Test results: The final product was a colorless to pale yellow clear liquid. The albumin purity was 99.6% (SEC-HPLC, Superdex 75 10 / 300 GL column, flow rate 0.6 mL / min; mobile phase: 200 mM PBS, pH 7.0, with 1% isopropanol added; detection wavelength: 280 nm, column temperature: 25-30 ℃, injection volume: 20 µL). The transferrin residue was 0.015 g / L, ceruloplasmin was not detected, and the albumin recovery rate was 92.3%.

[0061] Example 2

[0062] 1. Pretreatment: Same as in Example 1.

[0063] 2. Affinity chromatography:

[0064] Pack a DASA Sepharose affinity column (10 mL column volume) and equilibrate it with 30 mL of equilibration buffer (20 mM PBS, pH 6.5, 0.15 M NaCl) at a flow rate of 2 mL / min.

[0065] Feed 10 mL of the pretreated sample at a flow rate of 1 mL / min.

[0066] Washing: First wash 30 mL with equilibration buffer, then wash 30 mL with washing buffer (50 mM PBS, pH 8.0, 1.0 M NaCl) until the outflow A280 < 0.05;

[0067] Elution: Gradient elution was performed using elution buffer (50 mM Tris-HCl buffer, pH 8.5, 0.5 M NaCl, sodium octanoate concentration 30 g / L) at a flow rate of 2 mL / min, and the elution peak was collected.

[0068] 3. Post-processing: Ultrafiltration concentration and buffer replacement were performed using 20 mM pH 7.0 phosphate buffer.

[0069] 4. Test Results: The final product was clear in appearance. SEC-HPLC analysis showed that the albumin purity was 99.0%. The residual transferrin content was 0.020 g / L. The albumin recovery rate was 90.1%. This example demonstrates that even under high pH (8.5) and high concentrations of sodium caprylate (30 g / L), the method of this invention can still effectively elute the target product and maintain high purity.

[0070] Example 3

[0071] 1. Pretreatment: Same as in Example 1.

[0072] 2. Affinity chromatography:

[0073] Pack a DASA Sepharose affinity column (10 mL column volume), equilibrate with 30 mL of equilibration buffer at a flow rate of 2 mL / min;

[0074] Feed 10 mL of the pretreated sample at a flow rate of 1 mL / min.

[0075] Washing: Wash 30 mL with washing buffer (10 mM PBS, pH 6.0, 0.75 M NaCl) until the effluent A280 < 0.05;

[0076] Elution: Gradient elution was performed using elution buffer (10 mM PBS, pH 7.0, 0.1 M NaCl, sodium octanoate concentration 8 g / L) for 30 mL at a flow rate of 2 mL / min, and the elution peaks were collected.

[0077] Post-processing: Ultrafiltration concentration and buffer replacement were performed using 20 mM pH 7.0 phosphate buffer.

[0078] Test results: The final product was colorless and clear. SEC-HPLC analysis showed an albumin purity of 99.3%. The residual transferrin content was 0.016 g / L. The albumin recovery rate was 91.5%. This example validated the process robustness under low buffer concentration (10 mM) and low salt (0.1 M NaCl) elution conditions.

[0079] Comparative Example 1

[0080] Except for using a equilibration buffer for washing, the other operations are the same as in Example 1.

[0081] Results: The purity of the obtained product was only 94.2%, and obvious transferrin bands were visible in the electrophoresis pattern. This indicates that without high-salt washing, the co-adsorbed contaminating proteins cannot be effectively removed when directly processing the supernatant of fractions II+III.

[0082] Comparative Example 2

[0083] Except for adjusting the pH of the equilibration buffer and the feed sample to 7.5, the rest of the operation is the same as in Example 1.

[0084] Results: The dynamic loading of the medium decreased by approximately 15%, and the product purity decreased to 96.0%. This indicates that DASA Sepharose media exhibits stronger specific recognition of albumin and better resistance to interference in a weakly acidic environment of pH 6.5.

[0085] Comparative Example 3

[0086] Except for adjusting the sodium octanoate concentration in the elution buffer to 3 g / L, the rest of the operation is the same as in Example 1.

[0087] Results: The elution yield of the medium decreased to 55%, and the product purity decreased to 95.0%. This indicates that the sodium octanoate concentration has a significant impact on the elution yield; when the sodium octanoate concentration is below 3 g / L, the elution yield of affinity chromatography is below 55%.

[0088] Comparative Example 4

[0089] The difference from Example 1 is that Cytiva's Blue Sepharose dye affinity chromatography medium was used. ™ 6 Fast Flow, Cytiva 17-0948-01).

[0090] Results: The purity of albumin purified by blue gel chromatography was 89.6%, and the yield was 83.4%. This indicates that the purification effect of DASA chromatography is significantly better than that of blue gel dye affinity chromatography, and the dynamic adsorption capacity of DASA medium is more than twice that of blue gel medium, which is more conducive to large-scale industrial applications.

[0091] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for affinity chromatography purification of human serum albumin from the supernatant of low-temperature ethanol precipitation fraction II+III, characterized in that, The method includes the following steps: S1. Raw material pretreatment: Collect the supernatant of components II+III from the low-temperature ethanol precipitation process of human plasma, adjust the pH value to 5.0-7.0, centrifuge or filter to remove insoluble precipitates, and obtain a clear liquid; S2, Ultrafiltration Concentration and Buffer Replacement: The clarified solution obtained in S1 is concentrated by ultrafiltration and replaced with a balanced buffer system to obtain the loading solution; S3. Chromatographic purification: The sample solution obtained in S2 is fed into a chromatography column packed with DASA Sepharose affinity medium. The chromatography column is washed with washing buffer and then eluted with elution buffer. The elution peak is collected, which is the purified human serum albumin chromatographic product. S4. Formulation and Virus Inactivation: The purified human serum albumin obtained in S3 is replaced with a formulation buffer, and then pasteurized, sterilized, filtered, and dispensed to obtain the human serum albumin product.

2. The method according to claim 1, characterized in that, The supernatant of component II+III mentioned in S1 is the liquid phase produced during the Cohn separation process under conditions of 19%-25% ethanol concentration.

3. The method according to claim 1, characterized in that, The ultrafiltration described in S2 uses an ultrafiltration membrane with a molecular weight cutoff of 10kDa-50kDa.

4. The method according to claim 1, characterized in that, The equilibration buffer described in S2 includes 10-50 mM phosphate buffer or acetate-sodium acetate buffer and 0-0.2 M sodium chloride, pH 5.0-7.

0.

5. The method according to claim 1, characterized in that, The sample loading solution described in S2 has a pH of 5.0-7.0 and an ionic strength of 0.05-0.2M.

6. The method according to claim 1, characterized in that, The volume ratio of the loading solution to the DASA Sepharose affinity medium in S3 is 0.5-2:

1.

7. The method according to claim 1, characterized in that, The washing buffer described in S3 comprises 10-50 mM phosphate buffer and 0.15-1.0 M sodium chloride, pH 6.0-8.

0.

8. The method according to claim 1, characterized in that, The elution buffer described in S3 comprises 10-50 mM buffer, 4-30 g / L sodium octanoate and 0.1-0.5 M sodium chloride, pH 6.0-8.

5.

9. The method according to claim 8, characterized in that, The buffer solution includes phosphate buffer or Tris-HCl buffer.

10. A human serum albumin, characterized in that, The human serum albumin is prepared by the method according to any one of claims 1-9.

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