Preparation method and product of human serum albumin

Abstract

The invention relates to a preparation method of human serum albumin. The preparation method comprises the following steps: a) providing a crude extract rich in human serum albumin; b) adding a protein stabilizer and ethanol into the crude extract to form a mixed solution containing ethanol; and c) heating the mixed solution obtained in the step b) at 60 + / -0.5 DEG C for more than 10 hours to synchronously realize precipitation purification of impure protein and inactivation of viruses. According to the method disclosed by the invention, a pasteurization step is integrated into an ethanol precipitation process environment, ethanol with a certain concentration is maintained or reintroduced to form an albumin-stabilizer-ethanol mixed system, and then the mixed system is directly subjected to pasteurization (heating at 60 DEG C for more than 10 hours); therefore, the virus inactivation is synchronously completed under the process condition of ethanol precipitation.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a method for preparing human serum albumin and a product thereof. Background Technology

[0002] Human serum albumin is an indispensable plasma product in clinical practice, and its safety is of paramount importance. Currently, the classic low-temperature ethanol method (Cohn method) is mainly used to separate albumin from human plasma through fractionation precipitation. This process mainly includes multiple low-temperature ethanol precipitation steps, ultimately yielding crude albumin (Fraction V). However, this process itself has limited ability to inactivate viruses.

[0003] To ensure virus safety, industry standards require a dedicated virus inactivation step in the purification process. Pasteurization (60°C, 10 hours) is a recognized effective method, but it is traditionally carried out in an aqueous solution system, i.e., after removing ethanol, a stabilizer is added to the albumin aqueous solution before heating. This step-by-step approach of "purification first, inactivation later" has the following shortcomings: 1. Discrete process: Purification and virus inactivation are two independent unit operations, with long processes and many equipment requirements.

[0004] 2. Limited virus inactivation spectrum: Aqueous pasteurization is sometimes not ideal for inactivating certain non-lipid-enveloped viruses.

[0005] 3. Potential risks: There is a theoretical risk of secondary contamination during the storage and transfer of intermediate products after purification and before virus inactivation. Summary of the Invention

[0006] To overcome the aforementioned technical deficiencies, the technical objective of this invention is to provide a method for preparing human serum albumin. This addresses the problems in existing technologies, such as the separation of purification and virus inactivation processes, lengthy processes, and the need to improve the virus inactivation spectrum.

[0007] To achieve the above-mentioned technical objectives, the technical solution adopted by the present invention is as follows: A method for preparing human serum albumin, the method comprising the following steps: a) Provide a crude extract rich in human serum albumin; b) Add a protein stabilizer and ethanol to the crude extract to form a mixture containing ethanol; c) The mixture obtained in step b) is heated at 60℃±0.5℃ for more than 10 hours to simultaneously precipitate and purify the impurities and inactivate the virus.

[0008] The crude extract rich in human serum albumin in step a) is preferably a complex solution of Fraction V precipitate obtained by low-temperature ethanol fractionation precipitation.

[0009] The protein stabilizer in step b) is preferably sodium caprylate and / or sodium acetyltryptophan. The stabilizer is added at a rate of 0.08 mmol to 0.24 mmol of sodium caprylate or an equimolar amount of sodium acetyltryptophan per 1 gram of protein.

[0010] The volume concentration of ethanol in the mixture in step b) is preferably 5% to 25%, more preferably 10% to 20%, and most preferably 15%.

[0011] The heating treatment in step c) is preferably performed at a temperature of 60°C ± 0.3°C for 10 to 11 hours. The pH of the mixture in step c) is maintained within the range of 6.5 to 7.5. Following step c), the mixture is further subjected to cooling and filtration to remove ethanol and impurities.

[0012] This invention provides a novel method for preparing human serum albumin based on the combined use of hot ethanol precipitation and pasteurization. The core of this method lies in the following: after obtaining the crude human serum albumin extract (such as Friction V), instead of immediately removing the ethanol, a protein stabilizer such as sodium caprylate is directly added to form an "ethanol-stabilizer-albumin" mixture. This system is then pasteurized at 60°C. This invention achieves deep synergistic effects by "pre-positioning" and "embedding" the pasteurization process within the ethanol precipitation environment. This not only simplifies the process but also significantly broadens the virus inactivation spectrum and enhances the killing ability against non-lipid-enveloped viruses (such as mouse parvovirus MVM) through the synergistic effect of heating and ethanol, thereby enabling the production of human serum albumin products with higher purity and stronger safety. Experimental verification shows that the human serum albumin product of the present invention has a protein purity of over 98% (the purity obtained by the traditional aqueous pasteurization process is about 96%), and the polymer content is ≤1.2%, with an LRV ≥5.2 for MVM virus inactivation (the traditional aqueous pasteurization process has a very poor effect on MVM inactivation, with an LRV ≤2.5).

[0013] Compared with the prior art, the present invention has the following significant advantages: 1. Process Integration and Synergistic Effect: The innovative combination of pasteurization and ethanol precipitation allows virus inactivation to be integrated into the purification process rather than a separate subsequent step. This combination produces a synergistic virus inactivation effect of heat and ethanol, significantly improving the inactivation capability against both lipid-enveloped and non-lipid-enveloped viruses, and broadening the inactivation spectrum.

[0014] 2. Simplified process and improved efficiency: It eliminates the step of removing ethanol before virus inactivation (such as ultrafiltration and dialysis) in traditional processes, shortens the production cycle, and reduces production costs and complexity.

[0015] 3. Enhanced safety: It avoids the potential risks of storing and transferring purified intermediate products in an ethanol-free environment. The entire critical step is conducted in an ethanol environment with antimicrobial activity, resulting in higher safety.

[0016] 4. Ensure product quality: Under the protection of stabilizers, albumin can still maintain the integrity of its structure and function in a hot ethanol environment, and the purity, yield and bioactivity of the final product meet the pharmacopoeia requirements. Detailed Implementation

[0017] The present invention will be further described below with reference to embodiments, but this is not intended to limit the invention in any way. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection of the present invention.

[0018] Example 1: A method (process) for preparing human serum albumin includes the following steps: 1. Raw material preparation: Collect plasma from healthy individuals, thaw and mix it, and then perform fractionation precipitation using a modified low-temperature ethanol method (such as the Cohn 6+9 method). Separate and remove cryoprecipitate, Fraction I, II+III and Fraction IV in sequence, and collect Fraction V precipitate.

[0019] 2. Reconstitution and Preparation of the Combined System: The Fraction V precipitate was reconstituted with an appropriate amount of water for injection under controlled conditions. Sodium caprylate stabilizer (or sodium acetyltryptophan) was added to the reconstituted solution to achieve a molar ratio of approximately 5:1 to albumin. Subsequently, ethanol was added to the solution and stirred thoroughly to adjust the ethanol concentration of the entire system to 15% (v / v). The pH was adjusted to 6.9 ± 0.1 using acid-base methods. This formed the combined treatment system.

[0020] 3. Core Step: Combined with hot ethanol pasteurization, the above mixture is transferred to a reactor with precise temperature and stirring control. The program is started, and the system temperature is uniformly raised to 60℃ ± 0.3℃ and maintained at this temperature for 10 hours. This step simultaneously achieves "precipitation purification in an ethanol environment (removal of heat-sensitive proteins)" and "efficient virus inactivation under heat."

[0021] 4. Post-processing: After heating, the material is rapidly cooled to room temperature. It is then subjected to a series of deep filtration and 0.22μm sterile filtration to remove denatured and precipitated proteins from the combined steps.

[0022] 5. Ultrafiltration and concentration: An ultrafiltration system is used to remove ethanol, excess stabilizers and small molecule impurities from the solution and to concentrate the albumin solution to the predetermined concentration.

[0023] 6. Final preparation and filling: Detect protein concentration, add stabilizer to the prescribed amount, and adjust to the target specification (e.g., 20% or 25%) with water for injection. After 0.22μm sterile filtration, perform aseptic filling.

[0024] Experiment Example 1: Result Verification Experiment To objectively and quantitatively evaluate the virus inactivation capability of the method of the present invention, we designed the following experiment in accordance with internationally accepted guidelines for virus validation research, comparing the method of the present invention (Example 1) with the traditional aqueous pasteurization method (as a comparative example).

[0025] Experimental group (this invention): Take the mixed solution from Example 1, in which Friction V precipitation has been redissolved and stabilizer (sodium caprylate, 0.16 mmol / g protein) and ethanol (final concentration 15% v / v) have been added.

[0026] Control group (traditional process): Take the same batch of Friction V precipitate reconstituted solution as the experimental group, add an equal amount of stabilizer, but completely remove ethanol by ultrafiltration to prepare an aqueous phase system.

[0027] The pH of both groups of samples was adjusted to 7.0.

[0028] Indicator viruses (pseudorabies virus PRV or mouse parvovirus MVM) were added to samples from both the experimental and control groups, with the initial viral titer controlled at approximately 1.0 × 10⁻⁶. 6 TCID 50 / mL The spiked sample is placed in a precision water bath, the program is started, the temperature is uniformly increased from room temperature to 60.0℃±0.3℃, and maintained at this temperature for 10 hours.

[0029] Samples were taken at the following time points during the heating process: 0 hours (start), 1 hour, 5 hours, and 10 hours (end).

[0030] Viral titration and calculation: Immediately after extraction, the samples were serially diluted and inoculated onto corresponding monolayers of cells. Cytopathic effects were observed, and the residual infectious virus titer (TCID) in the samples at each time point was calculated using the Reed-Muench method. 50 / mL).

[0031] The formula for calculating the log reduction value (LRV) of viral inactivation is: LRV =log 10 (V0 / Vt), Where V0 is the viral titer at 0 hours and Vt is the viral titer at time t hours.

[0032] The experimental results are shown in Tables 1 and 2:

[0033]

[0034] The results confirmed that, under the same heating temperature and time, the "combined" method of Example 1 of the present invention had significantly higher log reduction values ​​(LRV) for inactivation of indicator viruses (such as pseudorabies virus PRV and mouse parvovirus MVM) than the traditional aqueous phase method, especially for non-lipid-enveloped viruses (MVM).

[0035] Experiment Example 2: Screening Experiment for Ethanol Dosage The effects of different ethanol concentrations on virus inactivation and key product quality attributes (purity, polymers) were systematically evaluated to determine the optimal range of ethanol dosage.

[0036] Raw material: Friction V precipitate reconstituted solution from the same batch.

[0037] Stabilizer: Sodium octanoate, added at 0.16 mmol / g protein.

[0038] Pasteurization conditions: 60℃ ± 0.3℃, 10 hours.

[0039] Subsequent processing: consistent with Example 1.

[0040] Variable: Ethanol volume concentration, set at six gradients: 0% (water control), 5%, 10%, 15%, 20%, and 25%. Experimental results are shown in Table 3.

[0041] The results above show that an ethanol concentration of 10% to 20% can simultaneously achieve excellent virus inactivation (LRV ≥ 4.8 for non-lipid-enveloped viruses MVM) and optimal product quality (high purity, low polymer).

[0042] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for preparing human serum albumin, characterized in that, The method includes the following steps: a) Provide a crude extract rich in human serum albumin; b) Add a protein stabilizer and ethanol to the crude extract to form a mixture containing ethanol; c) The mixture obtained in step b) is heated at 60℃±0.5℃ for more than 10 hours to simultaneously precipitate and purify the impurities and inactivate the virus.

2. The method according to claim 1, characterized in that, The crude extract rich in human serum albumin in step a) is a complex solution of Friction V precipitate obtained by low-temperature ethanol fractionation precipitation.

3. The method according to claim 1, characterized in that, The protein stabilizer in step b) is sodium caprylate and / or sodium acetyltryptophan; the amount of stabilizer added is 0.08 mmol to 0.24 mmol of sodium caprylate or an equimolar amount of sodium acetyltryptophan per 1 gram of protein.

4. The method according to claim 1, characterized in that, The volume concentration of ethanol in the mixture in step b) is 5% to 25%.

5. The method according to claim 4, characterized in that, The volume concentration of the ethanol is 10% to 20%.

6. The method according to claim 1, characterized in that, The heating treatment in step c) is performed at a temperature of 60℃ ± 0.3℃ for 10 to 11 hours.

7. The method according to claim 1, characterized in that, In step c), the pH of the mixture is maintained in the range of 6.5 to 7.

5.

8. The method according to claim 1, characterized in that, Following step c), the process further includes cooling and filtering the treated mixture to remove ethanol and other proteins.

9. The human serum albumin product prepared by the method according to any one of claims 1-8.

10. The human serum albumin product according to claim 9, characterized in that, The protein purity of the product is above 98%, and the polymer content is ≤1.2%, with an LRV ≥5.2 for virus inactivation against MVM.

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