Method for preparing a recombinant serum albumin with preserved sulfhydryl groups and conformation

Abstract

This invention provides a method for preparing recombinant serum albumin with thiol preservation and conformational fidelity, relating to the field of recombinant protein preparation. Its key feature is the use of a dual protection system—low temperature and temperature control throughout the process, and specificity—to directionally protect the free thiol group at the Cys34 position and maintain the protein's native conformation. Combined with a two-stage purification process of first-stage ultrafiltration and second-stage chromatography, impurities and aggregates are efficiently removed. The final product is obtained through conformational locking and aseptic filtration. The advantages of this invention are: the recombinant serum albumin exhibits a Cys34 free thiol group retention rate ≥90%, purity ≥98%, and aggregate content ≤1%, demonstrating high activity, high stability, and high formulation compatibility. The process is animal-free, industrially scalable, and has high patent barriers, making it particularly suitable for high-end cosmetic raw materials.

Description

Technical Field

[0001] This invention relates to the field of recombinant protein preparation technology, and in particular to a method for preparing recombinant serum albumin with thiol preservation and conformational fidelity. Background Technology

[0002] Recombinant human serum albumin is a non-animal-derived albumin prepared using recombinant DNA technology. It possesses excellent biocompatibility, antioxidant properties, and moisturizing and repairing capabilities, making it a core functional ingredient in high-end cosmetics and biopharmaceuticals. Currently, industrial production of recombinant human serum albumin primarily utilizes microbial expression systems, followed by fermentation and crude purification before refining.

[0003] Existing preparation processes suffer from the following technical defects: The core active site is easily oxidized and inactivated. The free thiol group at position 34 of recombinant human serum albumin is the core functional site for its antioxidant activity. This site is chemically reactive, and temperature fluctuations, pH changes, and oxidative environments during conventional purification processes can rapidly oxidize it to a disulfide bond, leading to a loss of the protein's antioxidant activity and rendering it ineffective in cosmetics. The spatial conformation is easily disrupted. Traditional purification methods employ high temperature, high shear, and wide pH fluctuations, which easily disrupt the natural secondary and tertiary conformations of recombinant human serum albumin, causing protein misfolding and the formation of large amounts of aggregates, resulting in turbidity, layering, and discoloration in cosmetic formulations. Core indicators cannot be synergistically optimized. Existing technologies can only optimize thiol protection or purity control individually, making it difficult to simultaneously achieve thiol preservation, conformational fidelity, and oligomer control, resulting in poor product stability and low formulation compatibility. The process barriers are low, and homogenization is severe. Industry technology focuses on protein expression and crude purification, with insufficient research on refined processes for subsequent purification, activity locking, and precise structural maintenance, failing to meet the high purity, high activity, and high stability requirements of high-end cosmetic raw materials.

[0004] Therefore, there is an urgent need for a method to prepare highly active recombinant serum albumin that can simultaneously achieve thiol group preservation, conformational fidelity, and oligomer control, and is suitable for industrial production under mild conditions, in order to solve the above-mentioned technical problems. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing recombinant serum albumin with thiol preservation and conformational fidelity. Through a dual protection system of low temperature and mild control throughout the process and specific thiol groups, the free thiol group at Cys34 is systematically protected, the native conformation of the protein is maintained, and aggregate formation is effectively controlled, thereby obtaining highly active and highly stable recombinant serum albumin.

[0006] To achieve the above-mentioned objectives, the technical solution adopted by this invention is as follows:

[0007] A method for preparing recombinant serum albumin with thiol preservation and conformational fidelity includes the following steps:

[0008] S1. Pre-dispersion and in-situ protection: The crude recombinant serum albumin was added to a cryoprotectant and dispersed at low speed under conditions of 4℃~8℃ and pH 6.5~7.2 to form a homogeneous protein solution.

[0009] The low-temperature environment of 4℃~8℃ inhibits the oxidation reaction rate and prevents the oxidation of free thiol groups at Cys34 from the source; the neutral to weakly alkaline environment of pH 6.5~7.2 closely resembles the physiological state of proteins and avoids denaturation; low-speed stirring reduces shear force and prevents mechanical damage to protein conformation; the protective solution immediately coats the free thiol groups at Cys34, providing immediate protection and achieving in-situ protection of free thiol groups;

[0010] S2, First-stage ultrafiltration purification: The protein solution obtained in step S1 is purified by ultrafiltration membrane separation at a temperature of 8℃~15℃. Cross-flow filtration is used to remove small molecule impurities, salt ions and degradation fragments, so as to keep the native conformation of the protein intact.

[0011] The ultrafiltration membrane has a molecular weight cutoff that matches recombinant human serum albumin, enabling selective separation of the target protein; cross-flow filtration reduces membrane fouling and protein shear damage, protecting conformation; and a temperature range of 8℃~15℃ balances purification efficiency and protein stability, reducing energy consumption for industrial applications.

[0012] S3. Two-stage chromatographic purification: The product obtained in step S2 is precisely purified by ion exchange chromatography, with the elution pH controlled at 6.8~7.5 and the temperature at 10℃~18℃. The elution peak of the target protein is collected, effectively removing aggregates and impurities, increasing the protein purity to ≥98%, and reducing the aggregate content to ≤1%.

[0013] Ion exchange chromatography separates proteins based on their charge differences, precisely removing aggregates and other proteins; pH 6.8~7.5 ensures the charge stability of recombinant human serum albumin; temperature 10℃~18℃ provides mild conditions to prevent conformational changes.

[0014] S4. Conformation locking and stabilization: Add conformation stabilizer and osmotic pressure regulator to the purified protein solution collected in step S3, mix at low speed for 15-30 min at 10℃~20℃ to lock the protein spatial structure, and then concentrate under low pressure to obtain the protein solution of the target concentration.

[0015] Conformation stabilizers are used to anchor the secondary and tertiary structures of proteins and prevent folding errors; osmotic pressure regulators are adapted to cosmetic formulation systems to improve product stability; low-speed, short-time mixing avoids shear forces from damaging the protein structure; low-pressure concentration ensures gentle concentration without the loss of activity caused by high temperature and high pressure.

[0016] S5. Aseptic filtration and finished product: The protein solution obtained in step S4 is filtered through a 0.22μm aseptic filter membrane to remove bacteria, thereby obtaining a recombinant serum albumin product with thiol preservation, conformational fidelity, high activity, and high stability.

[0017] The 0.22μm filter membrane can completely remove bacteria and fungi, ensuring the product is sterile; the entire process involves no strong chemical reagents or high-temperature treatment, thus fully preserving the activity of protein thiol groups and their natural conformation.

[0018] Preferably, the cryogenic protective liquid in step S1 contains a thiol protective agent, which includes at least one of reduced glutathione, cysteine, dithiothreitol, and ethylenediaminetetraacetic acid.

[0019] Preferably, the conformational stabilizer in step S4 includes at least one of trehalose, sucrose, sorbitol, mannitol, and glycine.

[0020] The beneficial effects of this invention are as follows:

[0021] 1. High thiol retention: The retention rate of free thiol groups at Cys34 is ≥90%, significantly enhancing the antioxidant activity of the protein;

[0022] 2. High conformational fidelity: The protein's natural secondary structure is intact, with an aggregate content of ≤1%, resulting in higher activity and greater stability;

[0023] 3. Significantly enhanced stability: Improved heat resistance, shear resistance, and acid and alkali resistance, resulting in better formula compatibility;

[0024] 4. High purity and high safety: Protein purity ≥98%, low endotoxin content, no animal-derived risk, and meets cosmetic safety standards;

[0025] 5. Exclusive process with high barriers to entry: The combination of mild control, dual protection, and two-stage purification technology has not been disclosed in existing patents, there are no similar solutions, and the authorization rate is extremely high;

[0026] 6. Suitable for industrial-scale production: The process conditions are mild, easy to scale up, and the operation is stable, making it suitable for large-scale production. Attached Figure Description

[0027] Figure 1 This is a flowchart of a method for preparing recombinant serum albumin with thiol preservation and conformational fidelity according to the present invention. Detailed Implementation

[0028] To make the content of this invention easier to understand, the technical solutions of the embodiments of this invention will be clearly and completely described below with reference to the accompanying drawings. Identical components are indicated by the same reference numerals.

[0029] like Figure 1As shown, a method for preparing recombinant serum albumin with thiol preservation and conformational fidelity includes the following steps:

[0030] S1. Pre-dispersion and in-situ protection: The crude recombinant serum albumin was added to a cryoprotectant and dispersed at low speed under conditions of 4℃~8℃ and pH 6.5~7.2 to form a homogeneous protein solution.

[0031] S2, First-stage ultrafiltration purification: The protein solution obtained in step S1 is purified by ultrafiltration membrane separation at a temperature of 8℃~15℃. Cross-flow filtration is used to remove small molecule impurities, salt ions and degradation fragments, so as to keep the native conformation of the protein intact.

[0032] S3. Two-stage chromatographic purification: The product obtained in step S2 is precisely purified by ion exchange chromatography, with the elution pH controlled at 6.8~7.5 and the temperature at 10℃~18℃. The elution peak of the target protein is collected, effectively removing aggregates and impurities, increasing the protein purity to ≥98%, and reducing the aggregate content to ≤1%.

[0033] S4. Conformation locking and stabilization: Add conformation stabilizer and osmotic pressure regulator to the purified protein solution collected in step S3, mix at low speed for 15-30 min at 10℃~20℃ to lock the protein spatial structure, and then concentrate under low pressure to obtain the protein solution of the target concentration.

[0034] S5. Aseptic filtration and finished product: The protein solution obtained in step S4 is filtered through a 0.22μm aseptic filter membrane to remove bacteria, thereby obtaining a recombinant serum albumin product with thiol group preservation, conformational fidelity, high activity, and high stability.

[0035] Example 1:

[0036] This embodiment provides a method for preparing highly active recombinant human serum albumin with thiol group preservation and conformational fidelity. The specific steps are as follows:

[0037] S1. Pre-dispersion and in-situ protection:

[0038] Take 10L of high-density fermentation broth of recombinant human serum albumin expressed by Pichia pastoris, centrifuge and collect the supernatant as crude product.

[0039] Under aseptic conditions, the crude product was slowly added to 20 L of cryoprotectant solution pre-cooled to 4 °C. The cryoprotectant solution was formulated as follows: 20 mM sodium phosphate buffer containing 0.5 mM reduced glutathione, 0.1 mM ethylenediaminetetraacetic acid solution, and 50 mM sodium chloride.

[0040] The entire addition process was carried out in a jacketed temperature-controlled stirred tank, maintaining the tank temperature at 4±1℃, stirring at 80 rpm, and dispersing and mixing for 30 minutes to obtain a uniform protein solution.

[0041] S2, First-stage ultrafiltration purification:

[0042] The protein solution obtained in step S1 was concentrated and washed using a tangential flow ultrafiltration system. Operating conditions: inlet pressure ≤30 psi, reflux pressure 5 psi, and temperature controlled at 10±2℃.

[0043] First, wash the protein solution with 5 times its volume of pre-cooled ultrafiltration wash buffer to remove small molecule impurities, GSH, and salt ions. Then concentrate the protein solution to 5L. The ultrafiltration wash buffer formula is: 20mM sodium phosphate and 50mM sodium chloride.

[0044] When the conductivity of the effluent is consistent with that of the washing buffer, the ultrafiltration is terminated, and the ultrafiltration retentate is obtained.

[0045] S3, Two-stage analytical refining:

[0046] Anion exchange chromatography was used. The ultrafiltration retentate obtained in step 2 was loaded at a flow rate of 5 mL / min. After loading, the sample was washed with equilibration buffer until the UV baseline was stable. Then, isocratic elution was performed with elution buffer, and the elution peak was collected. The temperature during the elution process was controlled at 15±1℃.

[0047] S4. Conformation locking and stable modulation:

[0048] Slowly add the sterile filtered stabilizer stock solution to the chromatographic eluent collected in step S3. Mix at a low stirring speed of 60 rpm for 20 minutes at 15±2℃. Subsequently, concentrate under low pressure using a 30 kDa ultrafiltration membrane to adjust the protein concentration to 10 mg / mL.

[0049] S5. Aseptic filtration and finished product:

[0050] In a biosafety cabinet, the protein solution obtained in step 4 is filtered and sterilized by passing it through a series of 0.45 μm pre-filters and 0.22 μm final filters. The aseptically collected protein is then placed in a final container to obtain the recombinant human serum albumin product of this invention, which is stored at 2-8°C protected from light.

[0051] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing recombinant serum albumin with thiol preservation and conformational fidelity, characterized in that, Includes the following steps: S1. Pre-dispersion and in-situ protection: The crude recombinant serum albumin was added to the cryoprotectant and dispersed by low-speed stirring to form a homogeneous protein solution. S2, First-stage ultrafiltration purification: The protein solution obtained in step S1 is purified by ultrafiltration membrane separation at a temperature of 8℃~15℃; S3. Two-stage chromatographic purification: The product obtained in step S2 is precisely purified by ion exchange chromatography, and the elution peak of the target protein is collected. S4. Conformation locking and stabilization: Add conformation stabilizer and osmotic pressure regulator to the purified protein solution collected in step S3, mix at 10℃~20℃ to lock the protein spatial structure, and then concentrate under low pressure to obtain the protein solution of the target concentration. S5. Aseptic filtration and finished product: The protein solution obtained in step S4 is filtered through an aseptic filter membrane to remove bacteria, and the recombinant serum albumin product is obtained.

2. The method for preparing recombinant serum albumin with thiol preservation and conformational fidelity according to claim 1, characterized in that: The cryogenic protective liquid in step S1 contains a thiol protective agent, which includes at least one of reduced glutathione, cysteine, dithiothreitol, and ethylenediaminetetraacetic acid.

3. The method for preparing recombinant serum albumin with thiol preservation and conformational fidelity according to claim 1, characterized in that: The low-speed stirring and dispersion conditions in step S1 are: temperature 4℃~8℃, pH 6.5~7.

2.

4. The method for preparing recombinant serum albumin with thiol preservation and conformational fidelity according to claim 1, characterized in that: The operating conditions for step S3 are: temperature 10℃~18℃, pH 6.8~7.

5.

5. The method for preparing recombinant serum albumin with thiol preservation and conformational fidelity according to claim 1, characterized in that: The conformational stabilizer mentioned in step S4 includes at least one of trehalose, sucrose, sorbitol, mannitol, and glycine.

6. The method for preparing recombinant serum albumin with thiol preservation and conformational fidelity according to claim 1, characterized in that: The mixing in step S4 is a low-speed stirring mixture at 10-60 rpm for 15-30 minutes.

7. The method for preparing recombinant serum albumin with thiol preservation and conformational fidelity according to claim 1, characterized in that: In step S5, the pore size of the sterile filter membrane is 0.22 μm.

8. The highly active recombinant serum albumin prepared by the preparation method according to any one of claims 1-7, characterized in that: The recombinant serum albumin has a Cys34 free sulfhydryl group retention rate of ≥90%, an aggregate content of ≤1%, and a protein purity of ≥98%.

9. The use of the recombinant serum albumin according to claim 8 in the preparation of cosmetics, cell culture media or pharmaceutical excipients.

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