A method for recovering albumin from waste components by low-temperature ethanol precipitation

Abstract

The present application relates to the technical field of blood product production, and particularly relates to a method for recovering albumin from waste components in low-temperature ethanol precipitation method. Based on in-depth research on the distribution rule of albumin in the process flow of low-temperature ethanol precipitation method, two efficient and scalable albumin recovery paths using component II supernatant as raw material are established: one is anion exchange chromatography process, and the other is cation exchange-hydrophobic interaction combined process. Both schemes achieve selective enrichment and purification of albumin in a low-concentration and high-heteroprotein background. The technical scheme can effectively solve the problems existing in the prior art: in the traditional albumin preparation process, the waste part still contains considerable amount of albumin, which is not effectively recovered, causing waste of plasma resources. The present application has important practical value for improving the resource utilization efficiency of the blood product industry and enhancing the clinical albumin supply capacity.

Description

Technical Field

[0001] This invention relates to the field of blood product manufacturing technology, specifically to a method for recovering albumin from waste components in a low-temperature ethanol precipitation process. Background Technology

[0002] Human serum albumin is an important blood product extracted from the plasma of healthy individuals. It has the highest content of plasma proteins, accounting for approximately 50-60% of total protein. Primarily synthesized by the liver, it is a single-chain polypeptide formed by 585 amino acids linked by 17 disulfide bonds, with a molecular weight of approximately 69 kDa. Since its first clinical application in the 1940s, human serum albumin has become an indispensable therapeutic protein preparation in clinical practice due to its unique physicochemical properties and multiple key physiological functions.

[0003] Human serum albumin plays multiple roles in maintaining homeostasis. First, due to its large molecular size and negative charge, it is difficult to cross the vascular endothelial barrier, thus it is a major component in maintaining plasma colloid osmotic pressure, accounting for approximately 70-80% of this pressure. This makes it crucial for regulating intravascular and extravascular water distribution and maintaining effective circulating blood volume. Second, albumin possesses highly reversible small molecule binding capacity. Its molecular structure contains numerous hydrophobic regions and multiple specific binding sites, enabling it to form complexes with various endogenous substances (such as bilirubin and fatty acids) and exogenous drugs (such as antibiotics and antitumor drugs), thereby participating in the transport and metabolic regulation of substances within the body. Furthermore, albumin also possesses antioxidant and free radical scavenging capabilities, inhibiting the production of reactive oxygen species by polymorphonuclear leukocytes, reducing oxidative stress damage, and providing protection in various pathological states such as inflammation, infection, trauma, and liver and kidney dysfunction. Recent studies have also found that albumin may participate in coagulation balance by inhibiting platelet aggregation and regulating coagulation factor activity, exhibiting certain anticoagulant properties.

[0004] Given its irreplaceable physiological and clinical value, human serum albumin is widely used in the treatment of various critical illnesses, including hypoalbuminemia, shock, burns, ascites due to cirrhosis, and acute respiratory distress syndrome. However, due to the scarcity of raw material (healthy human plasma) and ethical constraints, there has been a long-standing global shortage of albumin. Against this backdrop, maximizing the efficient recovery of albumin from limited plasma resources has become crucial for increasing production capacity and ensuring clinical supply.

[0005] Currently, the mainstream industrial albumin preparation process is still based on the classic Cohn low-temperature ethanol precipitation method or its modified version. This method achieves the fractional precipitation of different protein components in plasma by gradually adjusting parameters such as ethanol concentration, pH value, ionic strength, and temperature. In this multi-step separation process, albumin is mainly concentrated in fraction V and precipitated, while IgG is enriched in fraction II precipitation. The supernatant of fraction II is the part discarded during the IgG acquisition process. However, a large amount of analytical data shows that these "discarded" fractions still contain a considerable proportion of albumin, especially in the supernatant of fraction II. Although the albumin concentration is lower than that in the main collection section, the total amount is not negligible. Because traditional processes lack effective means to recover these low-concentration albumins, some usable albumin is directly discharged with the waste liquid, which not only wastes valuable plasma resources but also reduces the overall yield and exacerbates the supply and demand imbalance.

[0006] More significantly, with the continuous growth in clinical demand and the increasing demands for the safety and purity of blood products, the bottlenecks in improving albumin yield using existing processes are becoming increasingly apparent. On the one hand, to ensure product purity and viral safety, process design often tends to sacrifice some yield; on the other hand, traditional precipitation methods themselves suffer from limited resolution, narrow operating windows, and large batch-to-batch variations, further limiting the effective capture of low-abundance albumin. Therefore, without significantly increasing cost and complexity, how to systematically identify and reduce albumin loss throughout the entire preparation process, especially addressing the "hidden loss" of albumin in non-target fractions such as Component II, has become a core technical challenge that the blood products industry urgently needs to overcome. Summary of the Invention

[0007] The present invention aims to provide a method for recovering albumin from waste components in a low-temperature ethanol precipitation process, in order to solve the following technical problem: In the existing albumin preparation process, waste fractions such as the supernatant of component II still contain a considerable amount of albumin, which leads to waste of plasma resources and low overall albumin yield due to ineffective recovery.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: A method for recovering albumin from waste components obtained by low-temperature ethanol precipitation includes the following steps performed sequentially: S1: Adjust the pH of the supernatant of component II to 7.0-7.5 and the conductivity to 1.0-4.0 mS / cm to obtain the anion exchange chromatography product. S2: Load the anion exchange chromatography product to be anion exchanged onto the anion exchange chromatography column, then wash the anion exchange chromatography column, then elute, collect the eluent, and obtain the preliminarily purified albumin product. S3: The preliminarily purified albumin product is prepared, pasteurized, filtered, and packaged to obtain the finished product; Alternatively, it may include the following steps performed in sequence: SS1: After the supernatant of component II is concentrated by ultrafiltration and dialysis, the protein content is adjusted to 10-20 g / L, the pH value is 5.1-5.5, and the conductivity is 1.0-3.5 mS / cm to obtain the product to be cation exchange chromatography. SS2: Load the sample to be cation exchange chromatography onto the cation exchange chromatography column, collect the corresponding flow-through, and obtain the preliminarily purified albumin product; SS3: Adjust the pH of the preliminarily purified albumin product to 7.0-7.6 and the conductivity to 15-45 mS / cm to obtain the product to be subjected to hydrophobic chromatography; SS4: Load the sample to be hydrophobically chromatographically analyzed onto the hydrophobic chromatography column, collect the corresponding flow-through, and obtain the albumin product that has been purified again. SS5: The purified albumin product is prepared, pasteurized, filtered, and dispensed to obtain the finished product.

[0009] Furthermore, in S2, the packing material of the anion exchange chromatography column is a weak anion exchange packing material.

[0010] Preferably, the packing material of the anion exchange chromatography column is a weak anion exchange packing material with a functional group of diethylaminoethyl.

[0011] Preferably, the packing material of the anion exchange chromatography column is a weak anion exchange packing material based on a polymer matrix.

[0012] Preferably, the packing material of the anion exchange chromatography column is a weak anion exchange packing material based on a polymer matrix and having a functional group of diethylaminoethyl.

[0013] Preferably, the packing material for the anion exchange chromatography column is UniGel 80 DEAE.

[0014] Weak anion exchange resins are chromatographic media with weakly basic functional groups, used to adsorb and separate negatively charged biomolecules (such as proteins and nucleic acids), for example: DEAE (diethylaminoethyl), ANX (aminoethyl), etc.

[0015] Furthermore, in S2, the buffer solution used to wash the anion exchange chromatography column is a solution containing 0.02-0.04M disodium hydrogen phosphate, with a pH of 7.0-7.5 and a conductivity of 1.0-4.0 mS / cm.

[0016] Furthermore, in S2, the buffer solution used for eluting the anion exchange chromatography column is a solution containing 0.1-0.3M disodium hydrogen phosphate, with a pH of 7.0-7.4 and a conductivity of 15-30 mS / cm.

[0017] Furthermore, in S2, the loading rate of the anion exchange chromatography product is 0.5-1.5 cm / min, and the loading volume is 70-90 L / L of column packing material.

[0018] Furthermore, in S2, the amount of buffer used to wash the anion exchange chromatography column is 3-5 column volumes.

[0019] Furthermore, in S3, the preliminarily purified albumin product is formulated into a crude product with a protein concentration >100g / L, containing 0.120-0.200mmol / g of protein in sodium caprylate, 50-150mmol / L in sodium chloride, and a pH value of 6.2-7.6. The pasteurization parameters were set as follows: 60℃±0.5℃, inactivation for 10 hours; after pasteurization, the inactivated product was obtained. The inactivated product was filtered through 0.45μm and 0.22μm filter cartridges, sterilized, and then packaged to obtain the finished product.

[0020] Furthermore, in SS2, the packing material of the cation exchange chromatography column is a strong cation exchange packing material.

[0021] Preferably, the packing material of the cation exchange chromatography column is a strong cation exchange packing material with a functional group of sulfopropyl group.

[0022] Preferably, the packing material of the cation exchange chromatography column is a strong cation exchange packing material based on a polymer matrix.

[0023] Preferably, the packing material of the cation exchange chromatography column is a strong cation exchange packing material with sulfopropyl functional groups based on a polymer matrix.

[0024] Preferably, the packing material for the cation exchange chromatography column is UniGel 80 SP.

[0025] Strong cation exchange resins are chromatography media with strongly acidic functional groups, used to adsorb and separate positively charged biomolecules, such as SP (sulfopropyl) and S (sulfomethyl).

[0026] Furthermore, the sample to be cation exchanged is pumped into the cation exchange chromatography column at a flow rate of 0.1-1.5 cm / min, and the sample loading is controlled at 130-190 g protein / L column packing.

[0027] Furthermore, in SS4, the hydrophobic chromatography column is packed with moderately hydrophobic chromatography packing material.

[0028] Preferably, the packing material for the hydrophobic chromatography column is butyl-modified hydrophobic chromatography packing material. The hydrophobicity of butyl is between that of phenyl (strongly hydrophobic) and methyl (weakly hydrophobic).

[0029] Preferably, the packing material for the hydrophobic chromatography column is Diamond Butyl.

[0030] Moderate hydrophobic interaction chromatography resins refer to chromatography media with medium-length alkyl chains bonded together. They utilize the hydrophobic interactions between the hydrophobic regions on the surface of biomolecules and the packing material for separation. Examples include Butyl and Hexyl.

[0031] Furthermore, the hydrophobic chromatographic sample to be chromatographically analyzed is pumped into the chromatography column at a flow rate of 0.1-1.5 cm / min, and the sample loading is controlled at 450-650 g protein / L column packing material.

[0032] Furthermore, in SS5, the purified albumin product is formulated into a crude product with a protein concentration >100g / L, containing 0.120-0.200mmol / g of protein in sodium caprylate, 50-150mmol / L in sodium chloride, and a pH value of 6.2-7.6. The pasteurization parameters were set as follows: 60℃±0.5℃, inactivation for 10 hours; after pasteurization, the inactivated product was obtained. The inactivated product was filtered through 0.45μm and 0.22μm filter cartridges, sterilized, and then packaged to obtain the finished product.

[0033] Furthermore, the supernatant of component II was obtained by the following method: After the raw plasma is melted, the supernatant is collected by centrifugation to obtain supernatant A; Adjust the parameters of supernatant A to: temperature -3 to 1℃, protein concentration 60–70 g / L, pH 5.8–7.8, conductivity 11–13 mS / cm, and final ethanol concentration 9–11 vol.%; after reacting at a constant temperature for 1.5–2.5 h, collect supernatant B by pressure filtration. Adjust the parameters of supernatant B as follows: temperature -7–-5℃, protein concentration 25-35 g / L, pH 4.7-6.7, conductivity 5.0-7.0 mS / cm, and final ethanol concentration 16-20 vol.%; after reacting at a constant temperature for 1.5-2.5 h, collect the precipitate by pressure filtration. This precipitate is component II+III precipitate. Add 7-9 times the mass of water to the precipitate of components II+III, stir and dissolve at -2 to -2℃ for 2.5-3.5 hours; adjust the pH to 4.0-5.0 and react for 1.5-2.5 hours; adjust the pH to 4.5-5.5 again and continue the reaction for 1.5-2.5 hours; then adjust the ethanol concentration to 14-16 vol.% and react for 2.5-3.5 hours; then filter and collect the supernatant C. Adjust the temperature of supernatant C to -2 to 0℃ and the pH value to 6.8 to 8.0. Adjust the ethanol concentration to 20 to 30 vol.% and react for 2.0 to 4.0 h. Then filter and collect supernatant D. Supernatant D is the supernatant of component II.

[0034] The technical principle of this technical solution is as follows: This invention is based on in-depth research into the distribution patterns of albumin in the process fractions of the low-temperature ethanol precipitation method. Taking into account the fact that the supernatant of component II, a traditional waste fraction, still contains a considerable amount of albumin, two efficient and scalable recovery pathways are constructed: one is anion exchange chromatography, and the other is a cation exchange-hydrophobic interaction combined mode. Both schemes fully utilize the differences in charge characteristics and hydrophobic behavior of albumin under specific physicochemical conditions to achieve selective enrichment and purification of albumin from a low-concentration, high-contamination protein background.

[0035] In the anion exchange pathway, by precisely controlling the pH (7.0–7.5) and low conductivity (1.0–4.0 mS / cm) of the supernatant of component II, albumin is placed in a moderately negatively charged state, while most other proteins are preferentially adsorbed onto the UniGel 80 DEAE medium due to their higher isoelectric point or conformational differences. Subsequently, gradient elution is used to precisely elute bound albumin with a phosphate buffer system, achieving effective capture of latent albumin in the waste stream.

[0036] In this dual-flow-through pathway, the present invention creatively utilizes the property that albumin has a near-neutral or even slightly positive net charge under weakly acidic conditions (pH 5.1–5.5), allowing it to flow directly through a UniGel 80SP cation exchange column without binding, while a large number of positively charged impurities are effectively retained. The resulting flow-through solution is then subjected to Diamond Butyl hydrophobic chromatography under optimized low-salt conditions, where albumin again flows through in a flow-through manner, further removing hydrophobic impurities. This dual-flow-through strategy avoids the elution step, significantly simplifies the operation, improves the recovery rate, and effectively ensures the integrity of the product structure.

[0037] Compared with the prior art, the present invention has the following significant advantages: Significantly improved total albumin recovery: Residual albumin was successfully recovered from the previously discarded component II supernatant, greatly alleviating the pressure of plasma resource shortage.

[0038] High process compatibility and easy industrialization: The chromatography media used (UniGel 80 DEAE / SP, Diamond Butyl) are all high-rigidity, high-pressure resistant packing materials, supporting high flow rates and large-scale scale-up.

[0039] Dual assurance of product purity and safety: Through a multi-mode orthogonal purification mechanism (charge + hydrophobic), impurities, DNA, and potential viral risk factors are effectively removed; subsequent pasteurization and dual-stage sterilization filtration further ensure that the final product complies with the Chinese Pharmacopoeia and international standards; Reduce production costs and environmental burden: Reduce the ineffective discharge of high-value plasma proteins while avoiding the introduction of complex chemical reagents or additional precipitation steps.

[0040] In summary, this invention not only solves the long-standing technical bottleneck of albumin loss in the low-temperature ethanol method, but also provides an economical, robust, and scalable solution for the high-value utilization of waste components, which has significant practical implications for improving the resource utilization efficiency and clinical supply capacity of the blood products industry. Attached Figure Description

[0041] Figure 1 The image shows the UV absorption spectrum of UniGel 80SP cation exchange chromatography in Example 2.

[0042] Figure 2 The image shows the UV absorption spectrum of Diamond Butyl hydrophobic chromatography in Example 2.

[0043] Figure 3 The UV absorption spectrum of UniGel 80 DEAE anion exchange chromatography in Example 3 is shown. Detailed Implementation

[0044] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto. Unless otherwise specified, the technical means used in the following embodiments and experimental examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used can all be obtained commercially.

[0045] Example 1: Obtaining the supernatant of component II The specific process for obtaining the supernatant of component II is as follows: (1) Pretreatment of raw plasma After the raw plasma is discharged from the warehouse, the surface of the plasma bag is disinfected (for example, by disinfecting the surface of the plasma bag with 75 vol.% ethanol solution). After the bag is broken, the plasma is thawed at 0°C (optional range: -2 to 2°C).

[0046] The thawed plasma was combined and centrifuged at 10,000 RCF (optional range: 8,000-12,000 RCF). The supernatant after centrifugation was collected and denoted as supernatant A.

[0047] Raw plasma is the supernatant after centrifuging blood to remove cells. It contains proteins, inorganic salts, and water, but no blood cells. More specifically, raw plasma refers to human plasma as defined in the Chinese Pharmacopoeia: human plasma used in the production of blood products is plasma from healthy individuals collected using apheresis to produce plasma protein products.

[0048] (2) First step: Low-temperature ethanol precipitation (supernatant A → supernatant B) Adjust the parameters of supernatant A as follows: temperature -1.0℃ (selectable range: -3 to 1℃), protein concentration 65 g / L (selectable range: 60–70 g / L), pH 6.80 (selectable range: 5.8–7.8), conductivity 12 mS / cm (selectable range: 11–13 mS / cm), and final pure ethanol concentration 10 vol.% (selectable range: 9–11 vol.%). Under the above conditions, react at a constant temperature for 2 h (selectable range: 1.5–2.5 h), then collect supernatant B by pressure filtration.

[0049] (3) Second step: Low-temperature ethanol precipitation (supernatant B → component II + III precipitation) Adjust the parameters of supernatant B as follows: temperature -6.0℃ (selectable range: -7–-5℃), protein concentration 30 g / L (selectable range: 25-35 g / L), pH 5.70 (selectable range: 4.7-6.7), conductivity 6.0 mS / cm (selectable range: 5.0-7.0 mS / cm), and final ethanol concentration 18 vol.% (selectable range: 16-20 vol.%). Under the above conditions, react at a constant temperature for 2 h (selectable range: 1.5-2.5 h), then collect the precipitate by pressure filtration. This precipitate is the fraction II+III precipitate.

[0050] (4) Precipitation and purification of component II+III (precipitate of component II+III → supernatant C → supernatant D) Add 8 times (optional range: 7-9 times) the mass of the precipitate (II+III) of water for injection to the precipitate. Stir and dissolve at 0℃ (optional range: -2-2℃) for 3 hours (optional range: 2.5-3.5 hours) until completely dissolved. Maintain the temperature at 0℃ (optional range: -2-2℃), add phosphate buffer to adjust the pH to 4.6 (optional range: 4.0-5.0), and react for 2 hours (optional range: 1.5-2.5 hours). Then, add more phosphate buffer to adjust the pH to 5.0 (optional range: 4.5-5.5), and continue reacting for 2 hours (optional range: 1.5-2.5 hours). Next, adjust the ethanol concentration to 15 vol.% (optional range: 14-16 vol.%), react for 3 hours (optional range: 2.5-3.5 hours), and collect the supernatant C by pressure filtration. The supernatant C obtained in this step is the supernatant of component III. Further processing of the component III supernatant is then performed.

[0051] In existing technologies, supernatant C is typically used to prepare component II precipitate. The subsequent process after obtaining supernatant C is as follows: adjust the parameters of supernatant C to: temperature -1.0℃ (optional range: -2-0℃), pH 7.4 (optional range: 6.8-8.0), and add ethanol to achieve a final concentration of 26 vol.% (optional range: 20-30 vol.%). Under these conditions, after a constant temperature reaction for 3 hours (optional range: 2.0-4.0 hours), filter by pressure to collect the precipitate and supernatant (supernatant D). This precipitate is component II precipitate (existing technologies use it for the separation and enrichment of IgG), and supernatant D is the starting material component II supernatant in this scheme.

[0052] This process utilizes the core mechanism of low-temperature ethanol precipitation: ethanol reduces protein solubility, and pH and temperature regulate protein charge properties. Through a two-step gradient of ethanol concentration and pH / temperature regulation, plasma impurities are gradually removed, ultimately yielding the supernatant of component II.

[0053] Example 2: Albumin was separated from the supernatant of component II (cation exchange chromatography). (1) Ultrafiltration The supernatant of Component II obtained in Example 1 was first concentrated by ultrafiltration (concentration mode) using a 10KD membrane to concentrate the protein concentration to 40-120 g / L. Then, 8 times the volume of the concentrated solution (2-8°C) of low-temperature injection water was slowly added, and the ultrafiltration system was started, operating in dialysis mode. After dialysis, the conductivity dropped to below 1 mS / cm, and the residual ethanol content dropped to below 0.5 vol.%. The purpose of dialysis is to effectively remove residual ethanol, small molecule salts, broken protein fragments, and other impurities from the supernatant of Component II, preventing them from contaminating the subsequent chromatography column packing or affecting the adsorption / flow-through effect of the target protein. The entire process was conducted at low temperature (2-8°C), which effectively prevented albumin denaturation and inactivation, ensuring the activity and structural stability of the target protein. After concentration and dialysis, the obtained protein solution was used for further processing; this protein solution is referred to below as the ultrafiltration product.

[0054] (2) Product preparation The protein content of the ultrafiltration product was adjusted to 10 g / L-20 g / L, the pH value to 5.1-5.5, and the conductivity to 1.0-3.5 mS / cm. The protein content of the solution could be adjusted to these levels through conventional dilution and concentration. pH adjustment was performed using conventional acid-base addition methods, preferably with disodium hydrogen phosphate solution and citric acid solution. For conductivity adjustment, based on the initial conductivity of the ultrafiltration product, conventional methods were selected to decrease conductivity by dilution with water for injection or to increase conductivity by adding electrolytes such as sodium chloride solution. After adjusting the protein concentration, pH value, and conductivity to ensure the pH value and conductivity of the material were within the above ranges, the mixture was filtered using a 0.22 μm filter cartridge to obtain the product to be chromatographically analyzed (the product to be used for cation exchange chromatography). The preferred parameters for the product to be used in subsequent experimental studies were: protein content 13 g / L, pH value 5.4, and conductivity adjusted to 2.0 mS / cm.

[0055] (3) Cation exchange chromatography This step utilizes the cation exchange properties of the UniGel 80SP packing material and the charge difference of albumin at pH 5.1-5.5 to achieve efficient separation of albumin from other proteins through a "flow-through mode." Albumin carries a negative charge within this pH range and does not bind to the cationic packing material, flowing through with the feed solution; other proteins (such as globulins) carry a positive charge and bind to the packing material, being retained. The flow-through solution is then collected to obtain the preliminarily purified albumin product. The specific operation process is as follows: (3.1) Pretreatment of chromatography column and packing material Chromatography column preparation: Select a chromatography column that matches the production scale (diameter 10-20mm, column height 20-50cm; subsequent experimental studies use: diameter 16mm, column height 20cm). Before installation, flush the column cavity with purified water and check the sealing of the pipeline connections to ensure no leakage and no dead zones.

[0056] Pretreatment of packing material: Take the UniGel 80SP cation exchange packing material from Nanomicro and rinse it thoroughly with an appropriate amount of first equilibration solution to remove residual protective agents (such as ethanol) during the storage of the packing material. At the same time, it can make the packing material particles evenly dispersed to avoid local voids or agglomeration after the column is packed.

[0057] UniGel 80SP uses highly cross-linked polyacrylate microspheres as a matrix with an average particle size of approximately 80 μm. Its functional group is sulfopropyl (SP), a strong cation exchange group that maintains a stable charge over a wide pH range.

[0058] The first equilibration solution is formulated as follows: 0.01-0.03M disodium hydrogen phosphate, dissolved in water for injection; the pH of the first equilibration solution is 5.2-5.5 (adjusted with citric acid), and the conductivity is 1.0-3.5 mS / cm. The first equilibration solution is sterilely filtered through a 0.22μm filter cartridge before use, and the entire process can be carried out at room temperature. The preferred first equilibration solution is: 0.015M disodium hydrogen phosphate, pH 5.4, and conductivity 2.0 ± 0.2 mS / cm.

[0059] Column packing operation: The conventional wet packing method is adopted. The general process is as follows: the rinsed packing suspension is slowly injected into the chromatography column. After the packing naturally settles to a stable height on the column bed, the column bed is backwashed with the first equilibration solution for 1-2 column volumes (CV) to compact the packing and ensure the uniformity of the column bed.

[0060] (3.2) Chromatographic system equilibrium System equilibration operation: Pump the prepared first equilibration solution into the chromatography system and continue equilibration for 3-5 CVs until the pH and conductivity of the liquid at the column outlet are basically consistent with the first equilibration solution at the inlet.

[0061] (3.3) Sample loading and flow-through collection Sample loading procedure: The sample to be chromatographically prepared in the previous steps (the sample to be cation exchange chromatography, protein content: 10-20 g / L, pH value: 5.1-5.5, conductivity: 1.0-3.5 mS / cm) is pumped into the chromatography column at a flow rate of 0.5-1.5 cm / min (preferably 1 cm / min). The sample loading rate is controlled at 130-190 g protein / L column packing material (preferably 170 g protein / L) to avoid excessive sample loading, which may cause impurities to penetrate and contaminate the target product.

[0062] Flow-through monitoring and collection: Flow-through collection begins immediately after sample loading, with real-time monitoring of the UV absorbance at 280 nm at the column outlet. Collection continues until the post-equilibration step (after sample loading, the first equilibration solution is pumped back into the chromatography system for post-equilibration). Chromatographic observation shows that the UV signal has not yet returned to baseline during the post-equilibration stage, indicating that the target protein is still present in the effluent. Therefore, all flow-through samples from this period are combined as the preliminary purified albumin product. During the experiment, detailed records must be kept of the sample loading volume, total flow-through volume, and protein concentration. Figure 1 Representative chromatographic patterns based on UniGel 80 SP packing material are shown, reflecting the changes in UV absorption at each stage of the operation; the intervals marked by the two downward arrows in the figure are the collected preliminary purified albumin fractions.

[0063] The baseline refers to the stable UV signal value (usually close to 0 or a low background value) before the start of chromatography or at the initial stage of sample loading, when no protein flows out. Flow-through refers to the liquid portion that is not retained by the packing material and passes directly through the column.

[0064] (4) Hydrophobic Chromatography Hydrophobic chromatography was performed on the flow-through solution (preliminarily purified albumin product) obtained in the previous step.

[0065] (4.1) Sample preparation The collected, preliminarily purified albumin product was prepared by adjusting the pH to 7.0-7.6 and the conductivity to 15-45 mS / cm using 2M sodium chloride. After filtration through a 0.22 μm filter cartridge, the product to be chromatographically purified (the hydrophobic chromatographic product) was obtained. The preferred parameters for the hydrophobic chromatographic product are: pH 7.6 and conductivity 30 mS / cm.

[0066] (4.2) Pretreatment of chromatography column and packing material Chromatography column preparation: Select a chromatography column that matches the production scale (diameter 10-20mm, column height 20-50cm; subsequent experimental studies use: diameter 16mm, column height 20cm). Before installation, flush the column cavity with purified water and check the sealing of the pipeline connections to ensure no leakage and no dead zones.

[0067] Pretreatment of packing material: Take Borglon Diamond Butyl packing material and rinse it thoroughly with an appropriate amount of second equilibration solution to remove residual protective agents (such as ethanol) during the packing material storage process. At the same time, it can make the packing material particles evenly dispersed to avoid local voids or agglomeration after the column is packed.

[0068] Among them, Borglon Diamond Butyl is a hydrophobic interaction chromatography (HIC) packing material developed by Bestchrom. Its framework is high-rigidity agarose and its functional group is butyl.

[0069] The second equilibration solution is formulated as follows: 0.02-0.04M tris(hydroxymethyl)aminomethane and 0.10-0.60 mol / L sodium chloride, dissolved in water for injection; the pH is adjusted to 7.0-7.6 with hydrochloric acid, and the conductivity is 15 mS / cm-45 mS / cm. The preferred second equilibration solution is: 0.025M tris(hydroxymethyl)aminomethane and 0.40 mol / L sodium chloride; pH 7.6; conductivity 30 mS / cm.

[0070] The column packing method is the same as "(3) cation exchange chromatography", except that the first equilibration solution is replaced with the second equilibration solution.

[0071] (4.3) Chromatographic system equilibrium System equilibration operation: Pump the prepared second equilibration solution into the chromatography system and continue equilibration for 3-5 CVs until the pH and conductivity of the liquid at the column outlet are basically consistent with those of the second equilibration solution at the inlet.

[0072] (4.4) Sample loading and flow-through collection Sample loading procedure: The sample to be chromatographically analyzed (hydrophobic sample; pH: 7.0-7.6, conductivity: 15-45 mS / cm) prepared in the previous steps is pumped into the chromatography column at a flow rate of 0.5-1.5 cm / min (preferably 1 cm / min). The sample loading rate is controlled at 450-650 g protein / L column packing material (preferably 550 g protein / L column packing material) to avoid excessive sample loading, which may cause impurities to penetrate and contaminate the target product.

[0073] Flow-through monitoring and collection: Flow-through collection begins immediately after sample loading, with real-time monitoring of the UV absorbance at 280 nm at the column outlet. Collection continues until the post-equilibration step (after sample loading, a second equilibration solution is pumped into the chromatography system for post-equilibration), yielding the purified albumin product. Detailed records of sample loading volume, total flow-through volume, and protein concentration must be kept throughout the experiment. Figure 1 Representative chromatographic patterns based on Diamond Butyl packing material are shown, reflecting changes in UV absorption at each stage of the process. The intervals marked by the two downward arrows in the figure represent the collected pre-purified albumin fraction.

[0074] (5) Preparation of albumin products The albumin product, purified again after hydrophobic chromatography, was pre-filtered using a 0.45μm + 0.20μm sterile filter cartridge, and then subjected to ultrafiltration dialysis using an ultrafiltration system (10kDa ultrafiltration membrane pack). Dialysis was performed first with 5 volumes of physiological saline, followed by 5 volumes of injectable water. After dialysis, the product was concentrated to a protein concentration of over 100g / L.

[0075] The albumin product obtained through the above steps is then used to prepare a crude product containing 0.120-0.200 mmol / g sodium caprylate and 50-150 mmol / L sodium chloride, with a pH of 6.2-7.6. The ultrafiltered albumin product is then further prepared by adding sodium caprylate at a ratio of 0.17 mmol / g protein and sodium chloride at a ratio of 0.1 mol / L (semi-finished product volume), adjusting the pH to 7.0.

[0076] (6) Inactivation of Parvovirus The crude product was pasteurized to obtain the inactivated product. The parameters were set as follows: 60℃±0.5℃, inactivation time 10 hours.

[0077] (7) Filtration and dispensing The inactivated product was filtered through 0.45μm and 0.22μm filter cartridges, sterilized, and then packaged to obtain the finished product.

[0078] Example 3: Albumin was separated from the supernatant of component II (anion exchange chromatography). (1) Product preparation The supernatant of component II was taken and directly prepared without ultrafiltration to obtain the chromatographic product, which is simpler than the method in Example 2. The pH of the supernatant of component II obtained in Example 1 was adjusted to 7.0-7.5, and the conductivity was adjusted to 1.0-4.0 mS / cm. After adjusting the pH and conductivity to ensure they were within the above ranges, the mixture was filtered using a 0.22 μm filter cartridge to obtain the chromatographic product (the anion exchange chromatographic product). The preferred parameters for the anion exchange chromatographic product used in subsequent experimental studies were: pH 7.3 and conductivity adjusted to 2.0 mS / cm. Since the anion exchange chromatographic product was prepared directly from the unfiltered supernatant of component II, the protein concentration was relatively low, estimated to be around 0.15-0.30 g / L.

[0079] (2) Anion exchange chromatography The pretreatment of the chromatography column and packing material, as well as the equilibration of the chromatography system, were carried out using conventional methods, as described in Example 2. A third equilibration solution was used, and the packing material employed was Nanomicro UniGel 80 DEAE. UniGel 80 DEAE is a weak anion exchange packing material with a particle size of approximately 80 μm, a rigid polymer matrix, and a functional group of diethylaminoethyl-DEAE.

[0080] The third equilibration solution is formulated as follows: 0.02-0.04M disodium hydrogen phosphate, dissolved in water for injection; acetic acid is used to adjust the pH to 7.0-7.5 and the conductivity to 1.0-4.0 mS / cm. The third equilibration solution is sterilely filtered through a 0.22μm filter cartridge before use, and can be prepared at room temperature throughout the process. The preferred formulation of the third equilibration solution is: 0.02M disodium hydrogen phosphate, pH 7.3, and conductivity adjusted to 2.0 mS / cm.

[0081] The next steps involve loading the product sample and collecting the target protein. The specific operating procedure is as follows: Pump the third equilibration solution into the chromatography column to equilibrate the column. Continue equilibration for 3-5 CV cycles until the pH and conductivity of the eluent at the column outlet are basically the same as those of the equilibration solution at the inlet.

[0082] The chromatographic sample (anion exchange chromatography sample; pH: 7.0-7.5, conductivity: 1.0-4.0 mS / cm) prepared in the aforementioned steps is pumped into the chromatography column at a flow rate of 0.5-1.5 cm / min (preferably 1 cm / min), with the loading volume controlled at 70-90 L of chromatographic sample / L of column packing material (preferably 80 L of chromatographic sample / L of column packing material). Under pH 7.0-7.4 conditions, albumin has an isoelectric point of approximately 4.7 and carries a strong negative charge, allowing it to electrostatically adsorb onto the positively charged groups on the surface of the UniGel 80DEAE packing material. However, some other proteins (such as globulins) have different charge properties or charge amounts, resulting in weaker or no binding ability.

[0083] After sample loading, immediately switch to pumping in the third equilibration buffer and flush the column for 3-5 CVs at a flow rate of 0.5-1.5 cm / min (preferably 1 cm / min). Monitor the UV absorbance throughout the process until the reading returns to baseline. The eluent at this stage mainly contains unbound proteins and free impurities and should be discarded entirely. The purpose of washing is to remove residual sample solution and weakly bound proteins from the column bed voids to prevent contamination of the subsequent target protein elution peak.

[0084] Switch to the elution buffer and maintain a flow rate of 0.5-1.5 cm / min (preferably 1 cm / min) to begin elution.

[0085] The entire process involves continuous monitoring with a UV detector to obtain chromatographic spectra. Representative UniGel 80DEAE chromatographic spectra from this protocol are detailed below. Figure 3 The changes in UV absorbance at different operational stages are illustrated. The fraction corresponding to the albumin peak is collected as a pre-purified albumin product for subsequent processes.

[0086] The eluent is formulated as follows: 0.1-0.3M disodium hydrogen phosphate, dissolved in water for injection; the entire process can be carried out at room temperature. Acetic acid is used to adjust the pH to 7.0-7.4 and the conductivity to 15-30 mS / cm. The eluent is filtered through a 0.22μm filter and used for later use; it can be prepared at room temperature throughout the process. A preferred eluent formulation is: 0.2M disodium hydrogen phosphate, with acetic acid adjusting the pH to 7.3 and the conductivity to 20 mS / cm.

[0087] (3) Preparation of albumin products Similar to step (5) of Example 2, the crude product was prepared using the preliminarily purified albumin product obtained in step (2).

[0088] (4) Inactivation of Parvovirus Same as step (6) in Example 2.

[0089] (5) Filtration and dispensing Same as step (7) in Example 2.

[0090] Comparative Example 1: A50 Anion Exchange Medium Chromatography Following the method described in Example 2, the supernatant of component II obtained in Example 1 was subjected to ultrafiltration to obtain the ultrafiltration product. The pH value of the ultrafiltration product was adjusted to approximately 6.00, the conductivity to approximately 5.00 ms / cm, and the protein concentration to approximately 10 g / L to obtain the product to be chromatographically analyzed.

[0091] This protocol uses DEAE Sephadex A-50 (diethylaminoethyl-dextran A-50; matrix is ​​cross-linked dextran Sephadex, functional group is diethylaminoethyl DEAE) packing material for anion exchange chromatography. The pretreatment of the chromatography column and packing material, and the equilibration of the chromatography system are performed according to Examples 2 and 3, which are conventional methods in the prior art. The only difference is that the column packing material is replaced with DEAE Sephadex A-50 packing material, and its corresponding equilibration buffer is used. The equilibration buffer is a buffer solution containing 0.01 mol / L sodium citrate and 0.08 mol / L sodium chloride, with a pH of approximately 6.0.

[0092] The chromatography process was performed according to the optimal method described in Example 3. After loading the sample as described in Example 3, the column was washed for 2-3 CVs with the aforementioned equilibration buffer (0.01 mol / L sodium citrate + 0.08 mol / L sodium chloride, pH 6.0). Then, the column was washed for 2-3 CVs with another equilibration buffer (0.01 mol / L sodium citrate + 0.15 mol / L sodium chloride, pH 6.0). After washing, elution was performed with a buffer solution containing 0.01 mol / L sodium citrate and 2 mol / L sodium chloride, with a pH of approximately 7.0. The eluent was collected as described in Example 3 to obtain the preliminarily purified albumin product. Then, the product was prepared, pasteurized, filtered, and dispensed according to the method described in Example 3 to obtain the final product. The aforementioned equilibration buffer and washing method are optimal for DEAE Sephadex A-50 packing material.

[0093] When using A50 anion exchange chromatography to process the supernatant of fraction II, ultrafiltration is necessary. The supernatant of fraction II has a relatively low protein content (estimated at approximately 0.15-0.30 g / L), a large volume, and a high ethanol concentration. Directly performing A50 anion exchange chromatography on the supernatant of fraction II, after only adjusting the pH and conductivity, results in very poor albumin purification and enrichment. Therefore, Comparative Example 1 employed a process such as ultrafiltration dialysis of the supernatant of fraction II to remove impurities such as ethanol and concentrate the protein. Even with the aforementioned ultrafiltration operations, the quality and efficiency of the albumin product obtained using the method in Comparative Example 1 were still inferior to those in Examples 2 or 3. In particular, Example 3, which also used anion exchange chromatography, achieved a higher albumin concentration and lower polymer content without ultrafiltration of the supernatant of fraction II. This demonstrates that the 80DEAE column chromatography in Example 3 achieved unexpected technical effects compared to existing technologies, and is suitable for situations where the protein content of the supernatant of Component II is low and the ethanol concentration is high, thus achieving a dual improvement in product quality and process efficiency.

[0094] Example 4: Indicator Detection Examples 1-3 and Comparative Example 1 all used the described optimal method to prepare albumin products, followed by albumin purity, polymer content, and yield testing. Albumin content was detected using a Beckman Coulter specific protein analysis system, and total protein content was detected using an Yilanbei fully automated biochemical analyzer. Albumin purity was calculated as the ratio of albumin content to total protein content in the sample. Albumin yield was calculated by detecting the amount of albumin in the finished product and converting it to the amount of albumin (g) obtained per ton of component II supernatant. The polymer content of albumin products refers to the percentage of dimers, polymers, and aggregates other than albumin monomers in the total protein, as analyzed by size exclusion chromatography or electrophoresis. It is a key indicator reflecting albumin purity, structural integrity, and product stability. Higher polymer content results in stronger immunogenicity and a greater likelihood of adverse reactions; therefore, the polymer content of the product should be minimized.

[0095] Table 1: Test results of finished products from the examples and comparative examples

[0096] The experimental data above show that Comparative Example 1 used anion exchange chromatography (DEAE Sephadex A-50 packing material) to separate and purify albumin from the supernatant of Component II, but it had obvious defects: the albumin purity was not ideal, the polymer content was high, and the yield was low, with only about 300g of albumin obtained from one ton of Component II supernatant.

[0097] This technical solution has been systematically optimized based on Comparative Example 1, resulting in two process routes: Example 2 and Example 3. The specific advantages are as follows: Example 3 used UniGel 80 DEAE packing material to replace DEAE Sephadex A-50 packing material for anion exchange chromatography, achieving three breakthroughs: First, significantly improved product quality. The polymer content in Example 3 was 1.3%, only 50% of that in Comparative Example 1 (2.6%), greatly reducing the polymer content in the finished product and effectively improving albumin purity. Second, significantly increased yield. After using UniGel 80 DEAE packing material, approximately 550g of albumin could be obtained from one ton of fraction II supernatant, an increase of over 80% compared to 300g in Comparative Example 1. This yield improvement far exceeded expectations, greatly improving process efficiency and raw material utilization. Third, significantly simplified process flow. Using the process in Example 3, only the pH and conductivity of the fraction II supernatant need to be adjusted for direct sample loading, eliminating pretreatment operations such as ultrafiltration, further improving overall process efficiency.

[0098] Regarding Example 3, those skilled in the art would generally believe that replacing the chromatography packing material would only achieve limited improvements in quality or yield. However, Example 3, using UniGel 80 DEAE packing material, significantly improved product quality (reducing polymer content by 50%) while substantially increasing yield by over 80%, and also simplified the process flow. This synergistic effect of simultaneous optimization of quality, yield, and efficiency far exceeds the reasonable expectations of those skilled in the art, especially in terms of the quantitative technical effects of polymer content and yield.

[0099] Example 2 employs a combined process of "cation exchange chromatography + hydrophobic chromatography," further optimizing product quality based on Example 3. The advantages are as follows: First, product quality reaches optimal levels. The polymer content in Example 2 is as low as 0.6%, only 46% of Example 3 (1.3%) and 23% of Comparative Example 1 (2.6%). The polymer content is controlled below 50% of that in Example 3, significantly improving the quality of the finished product. Second, the yield is still significantly better than existing technologies. Although the yield of Example 2 is slightly lower than that of Example 3, it is still more than 50% higher than that of Comparative Example 1, maintaining a good yield level while pursuing high quality. Third, it can flexibly adapt to different production needs. Example 2 provides an optimal solution for application scenarios with higher requirements for finished product quality, allowing for a trade-off between quality and yield based on actual needs.

[0100] Regarding Example 2, according to conventional knowledge in the art, adding a chromatography step (cation exchange + hydrophobic chromatography) usually leads to increased loss of the target protein and a significant decrease in yield due to the increased number of operating units. However, in Example 2, even with the addition of a purification step, the polymer content dropped to an extremely low level of 0.6%, while the yield was still more than 50% higher than that of Comparative Example 1. This effect of increasing yield despite adding steps effectively reduces protein loss during purification and achieves unexpected technical results.

[0101] Both Embodiments 2 and 3 provided in this technical solution are significantly superior to the prior art (Comparative Example 1). The two process routes each have their own focus, and both have achieved unexpected technical effects: Example 3: A good balance is achieved between quality and yield. The polymer content is reduced by 50% while the yield is increased by more than 80%. The process is simplified and the synergistic optimization of the three factors far exceeds expectations, making it suitable for conventional large-scale production.

[0102] Example 2: With an emphasis on ultimate quality, the polymer content is as low as 0.6%. Even with the addition of purification steps, the yield is still more than 50% higher than that of existing technologies, demonstrating the synergistic effect between chromatography processes. This is suitable for high-end applications with strict requirements for product quality.

[0103] The aforementioned technical effects are beyond what a person skilled in the art could reasonably expect based on existing technology, fully demonstrating the ingenuity and technological advancement of this technical solution. Users can flexibly choose the most suitable process route according to their actual production needs.

[0104] The above descriptions are merely embodiments of the present invention, and common knowledge such as specific technical solutions and / or characteristics are not described in detail here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solutions of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A method for recovering albumin from waste components obtained by low-temperature ethanol precipitation, characterized in that: The following steps are performed sequentially: S1: Adjust the pH of the supernatant of component II to 7.0-7.5 and the conductivity to 1.0-4.0 mS / cm to obtain the anion exchange chromatography product. S2: Load the anion exchange chromatography product to be anion exchanged onto the anion exchange chromatography column, then wash the anion exchange chromatography column, then elute, collect the eluent, and obtain the preliminarily purified albumin product. S3: The preliminarily purified albumin product is prepared, pasteurized, filtered, and packaged to obtain the finished product; Alternatively, it may include the following steps performed in sequence: SS1: After the supernatant of component II is concentrated by ultrafiltration and dialysis, the protein content is adjusted to 10-20 g / L, the pH value is 5.1-5.5, and the conductivity is 1.0-3.5 mS / cm to obtain the product to be cation exchange chromatography. SS2: Load the sample to be cation exchange chromatography onto the cation exchange chromatography column, collect the corresponding flow-through, and obtain the preliminarily purified albumin product; SS3: Adjust the pH of the preliminarily purified albumin product to 7.0-7.6 and the conductivity to 15-45 mS / cm to obtain the product to be subjected to hydrophobic chromatography; SS4: Load the sample to be hydrophobically chromatographically analyzed onto the hydrophobic chromatography column, collect the corresponding flow-through, and obtain the albumin product that has been purified again. SS5: The purified albumin product is prepared, pasteurized, filtered, and dispensed to obtain the finished product.

2. The method for recovering albumin from waste components by low-temperature ethanol precipitation according to claim 1, characterized in that: In S2, the packing material of the anion exchange chromatography column is a weak anion exchange packing material; The buffer solution used for washing the anion exchange chromatography column is a solution containing 0.02-0.04M disodium hydrogen phosphate, with a pH of 7.0-7.5 and a conductivity of 1.0-4.0 mS / cm.

3. The method for recovering albumin from waste components by low-temperature ethanol precipitation according to claim 2, characterized in that: In S2, the buffer solution used for eluting the anion exchange chromatography column is a solution containing 0.1-0.3M disodium hydrogen phosphate, with a pH of 7.0-7.4 and a conductivity of 15-30 mS / cm.

4. The method for recovering albumin from waste components by low-temperature ethanol precipitation according to claim 3, characterized in that: In S2, the loading rate of the anion exchange chromatography product is 0.5-1.5 cm / min, and the loading volume is 70-90 L / L of column packing material.

5. The method for recovering albumin from waste components by low-temperature ethanol precipitation according to claim 4, characterized in that: In S2, the amount of buffer used to wash the anion exchange chromatography column is 3-5 column volumes.

6. The method for recovering albumin from waste components of low-temperature ethanol precipitation according to claim 5, characterized in that: In S3, the preliminarily purified albumin product is formulated into a crude product with a protein concentration >100g / L, containing 0.120-0.200mmol / g of protein in sodium caprylate, 50-150mmol / L in sodium chloride, and a pH value of 6.2-7.

6. The pasteurization parameters were set as follows: 60℃±0.5℃, inactivation for 10 hours; after pasteurization, the inactivated product was obtained. The inactivated product was filtered through 0.45μm and 0.22μm filter cartridges, sterilized, and then packaged to obtain the finished product.

7. The method for recovering albumin from waste components by low-temperature ethanol precipitation according to claim 1, characterized in that: In SS2, the packing material of the cation exchange chromatography column is a strong cation exchange packing material; The sample to be cation exchanged is pumped into the cation exchange chromatography column at a flow rate of 0.1-1.5 cm / min, and the loading is controlled at 130-190 g protein / L column packing.

8. The method for recovering albumin from waste components by low-temperature ethanol precipitation according to claim 7, characterized in that: In SS4, the hydrophobic chromatography column is packed with moderately hydrophobic chromatography packing material; Pump the hydrophobic chromatographic sample into the chromatography column at a flow rate of 0.1-1.5 cm / min, and control the sample loading at 450-650 g protein / L column packing material.

9. The method for recovering albumin from waste components by low-temperature ethanol precipitation according to claim 8, characterized in that: In SS5, the purified albumin product is formulated into a crude product with a protein concentration >100g / L, containing 0.120-0.200mmol / g protein of sodium caprylate, 50-150mmol / L of sodium chloride, and a pH of 6.2-7.

6. The pasteurization parameters were set as follows: 60℃±0.5℃, inactivation for 10 hours; after pasteurization, the inactivated product was obtained. The inactivated product was filtered through 0.45μm and 0.22μm filter cartridges, sterilized, and then packaged to obtain the finished product.

10. A method for recovering albumin from waste components of low-temperature ethanol precipitation according to any one of claims 1-9, characterized in that: The supernatant of component II was obtained by the following method: After the raw plasma is melted, the supernatant is collected by centrifugation to obtain supernatant A; Adjust the parameters of supernatant A to: temperature -3 to 1℃, protein concentration 60–70 g / L, pH 5.8–7.8, conductivity 11–13 mS / cm, and final ethanol concentration 9–11 vol.%; after reacting at a constant temperature for 1.5–2.5 h, collect supernatant B by pressure filtration. Adjust the parameters of supernatant B as follows: temperature -7–-5℃, protein concentration 25-35 g / L, pH 4.7-6.7, conductivity 5.0-7.0 mS / cm, and final ethanol concentration 16-20 vol.%; after reacting at a constant temperature for 1.5-2.5 h, collect the precipitate by pressure filtration. This precipitate is component II+III precipitate. Add 7-9 times the mass of water to the precipitate of components II+III, stir and dissolve at -2 to -2℃ for 2.5-3.5 hours; adjust the pH to 4.0-5.0 and react for 1.5-2.5 hours; adjust the pH to 4.5-5.5 again and continue the reaction for 1.5-2.5 hours; then adjust the ethanol concentration to 14-16 vol.% and react for 2.5-3.5 hours; then filter and collect the supernatant C. Adjust the temperature of supernatant C to -2 to 0℃ and the pH value to 6.8 to 8.

0. Adjust the ethanol concentration to 20 to 30 vol.% and react for 2.0 to 4.0 h. Then filter and collect supernatant D. Supernatant D is the supernatant of component II.

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