Preparation process of high-purity and high-activity von willebrand factor

Abstract

The invention relates to the technical field of biological pharmacy, and discloses a high-purity and high-activity von willebrand factor preparation process which comprises the following steps: S1, carrying out gradient centrifugal treatment on fresh plasma, namely selecting the fresh plasma, carrying out centrifugal treatment, and collecting secondary supernate rich in von willebrand factors; the collected secondary supernatant passes through an immunoaffinity chromatography column coupled with an anti-von willebrand factor antibody, S3, mild elution and ultrafiltration concentration, S4, gel filtration chromatography secondary purification, and S5, protective agent addition and preparation forming. According to the method, the rotating speed and time of secondary centrifugation are dynamically adjusted by combining protein concentration and turbidity through a centrifugal parameter self-adaptive optimization algorithm, impure protein precipitation is reduced, the relative content of vWF in secondary supernate is increased by 15%-20%, then two-stage purification of specific monoclonal antibody affinity chromatography and gel filtration chromatography is carried out, the purity of vWF is stabilized at 98.2%-98.6%, the difference between batches is smaller than or equal to 2.4%, and the quality of vWF is improved. The process is obviously superior to the traditional process.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of biopharmaceuticals, in particular to a preparation process of high-purity and high-activity von Willebrand factor. BACKGROUND

[0002] Von Willebrand factor (vWF) is a glycoprotein synthesized by vascular endothelial cells and megakaryocytes, which plays a key role in the coagulation process by mediating platelet adhesion to damaged vascular endothelium and stabilizing coagulation factor VIII, and its deficiency or dysfunction can cause von Willebrand disease (vWD), which requires high-purity and high-activity vWF preparations for replacement therapy.

[0003] In the prior art, plasma is often used as the raw material for vWF preparation. After centrifugation to obtain supernatant containing vWF, affinity chromatography or gel filtration chromatography is used for purification. After elution, the target product is obtained by ultrafiltration concentration, and finally a protective agent is added to prepare a freeze-dried powder for clinical hemostatic treatment.

[0004] The most critical deficiency in the prior art is that the centrifugation parameters are mostly fixed values, which cannot be dynamically adjusted according to the protein concentration and turbidity of the plasma raw material. When the protein concentration of the raw material plasma is high or the turbidity is large, it is difficult to fully precipitate impurities with fixed centrifugation parameters, resulting in an increase in the residual amount of albumin, fibrinogen and other impurities in the secondary supernatant, which not only increases the purification load of the subsequent chromatography step, but also may reduce the recovery rate of vWF due to the competition of impurities for binding sites. When the raw material plasma is relatively pure, fixed parameters may cause excessive centrifugation, resulting in loss of vWF with the precipitate. This "one-size-fits-all" centrifugation mode makes it difficult to stabilize the purity of the final product above 95%, and the inter-batch purity difference exceeds 5%, which cannot meet the clinical demand for high-purity and low-fluctuation preparations. Therefore, we propose a preparation process of high-purity and high-activity von Willebrand factor. SUMMARY

[0005] In view of the deficiencies of the prior art, the present application provides a preparation process of high-purity and high-activity von Willebrand factor, which solves the problem of fixed centrifugation parameters in the prior art, which cannot match the characteristics of the plasma raw material, resulting in residual impurities or loss of vWF.

[0006] To achieve the above purpose, the technical scheme is as follows: a preparation process of high-purity and high-activity von Willebrand factor, comprising the following steps: S1 Fresh Plasma Gradient Centrifugation: Fresh plasma was selected and centrifuged at 3000×g for the first time to collect the primary supernatant; the protein concentration and turbidity of the primary supernatant were detected, and the detection data were input into the centrifugation parameter optimization algorithm to output the rotation speed and time of the second centrifugation; based on the output results, a second centrifugation was performed to collect the secondary supernatant rich in von Willebrand factor; S2-specific immunoaffinity chromatography for preliminary separation: The secondary supernatant collected in S1 is passed through an immunoaffinity chromatography column coupled with anti-von Willebrand factor antibody. The loading flow rate of the chromatography column is dynamically adjusted according to the rotation speed of the second centrifugation in S1. After washing, the bound components are collected. S3 Mild Elution and Ultrafiltration Concentration: The bound components collected in S2 are eluted using a mild eluent. The amount of eluent used is determined based on the volume of the secondary supernatant in S1. After neutralization, the eluent is concentrated through an ultrafiltration membrane. S4 Gel Filtration Chromatography Secondary Purification: The concentrated liquid in S3 is purified a second time by passing it through a gel filtration chromatography column. The flow rate of the mobile phase of the chromatography column is calibrated according to the protein concentration detection results in S1, and the target component is collected. S5 Protectant Addition and Formulation: A protectant is added to the target component collected in S4. The proportion of protectant added is adjusted based on the turbidity detection results of the secondary supernatant in S1. After sterilization, the formulation is prepared.

[0007] Preferably, the fresh plasma in S1 is plasma from healthy individuals collected within 24 hours. The temperature of the first centrifugation is maintained at 4℃±0.5℃, the centrifugation time is 15 minutes±2 minutes, and the relative humidity of the centrifugation environment is controlled at 40%~60%. The protein concentration is detected by ultraviolet spectrophotometry with a detection wavelength of 280nm, and the turbidity is detected by a scattering light turbidimeter with a detection angle of 90°.

[0008] Preferably, the centrifugation parameter optimization algorithm in S1 is an adaptive model based on multiple linear regression. The input parameters include protein concentration (mg / mL) and turbidity (NTU). The output parameters are the rotation speed and time of the second centrifugation, wherein the centrifugation speed ranges from 7500 to 8500 × g, and the centrifugation time ranges from 25 to 35 minutes. The temperature of the second centrifugation is the same as that of the first centrifugation, and the secondary supernatant is collected within 10 minutes after centrifugation.

[0009] Preferably, the anti-von Willebrand factor antibody in S2 is a murine monoclonal antibody with clone number VW-16. The coupling ratio between the antibody and the agarose gel medium is 1 mg antibody to 1 mL gel. The column bed volume of the immunoaffinity chromatography column is 100 mL ± 5 mL, and the column length to inner diameter ratio is 10:1. The dynamic adjustment rule for the loading flow rate is as follows: when the second centrifugation speed in S1 is ≥ 8000 × g, the loading flow rate is 1.5 mL / min ± 0.1 mL / min; when the speed is < 8000 × g, the loading flow rate is reduced to 1.2 mL / min ± 0.1 mL / min.

[0010] Preferably, the secondary supernatant in S2 is pretreated with a 0.45 μm filter membrane before being loaded onto the column. The column temperature is maintained at 25℃±1℃ during the loading process. After loading, the column is incubated at room temperature for 2 hours±10 minutes, and the inlet and outlet of the chromatography column are kept closed during the incubation period. The washing is performed using a buffer solution containing 0.01M PBS and 0.15M NaCl, with a pH of 7.4±0.1 and a washing flow rate of 2mL / min±0.2mL / min. The A280 absorbance is measured every 10 minutes until three consecutive values ​​are <0.01.

[0011] Preferably, the mild elution buffer in S3 is a buffer solution containing 0.1M glycine-HCl and 0.5M NaCl. The buffer solution is sterilized by filtration through a 0.22μm filter membrane, with a pH of 2.8±0.1 and an elution flow rate of 0.8mL / min±0.05mL / min. The elution buffer is collected in 5mL portions per tube, and the A280 absorbance is monitored simultaneously to determine the elution peak range.

[0012] Preferably, in step S3, the neutralization uses 1 M Tris-HCl preheated to 25°C with a pH of 8.0 ± 0.1, added dropwise at a rate of 5 mL / min while continuously stirring, and the pH is adjusted to 7.0 ± 0.2 after neutralization; the ultrafiltration membrane is made of polyethersulfone with a molecular weight cutoff of 300 kDa ± 20 kDa, the concentration temperature is 4°C ± 0.5°C, the operating pressure is 0.1 MPa ± 0.01 MPa, and the concentration is reduced to 1 / 10 ± 1 / 100 of the original volume.

[0013] Preferably, the gel filtration chromatography column in S4 is a Superdex 200 Increase 10 / 300GL with a column bed volume of 24 mL ± 1 mL. The mobile phase is 0.02 M Tris-HCl containing 0.15 M NaCl, degassed, and has a pH of 7.4 ± 0.1. The calibration rule for the mobile phase flow rate is as follows: when the protein concentration in S1 is ≥ 50 mg / mL, the flow rate is 0.5 mL / min ± 0.05 mL / min; when the concentration is < 50 mg / mL, the flow rate is reduced to 0.4 mL / min ± 0.05 mL / min. The sample loading volume is 5% of the column bed volume, and the eluted fraction is collected in 2 mL tubes. The target fraction range is determined by SDS-PAGE electrophoresis.

[0014] Preferably, the protective agent in S5 is a mixture containing 5% sucrose, 2% mannitol and 0.01% Tween-80. The protective agent is added after sterile filtration. The adjustment rule for the addition ratio is: when the turbidity of the secondary supernatant in S1 is ≥10 NTU, the volume ratio of the protective agent to the target component is 1:8; when the turbidity is <10 NTU, the volume ratio is 1:9. After mixing, the mixture is stirred at 150 rpm for 30 minutes ± 5 minutes at 4℃ ± 0.5℃.

[0015] Preferably, in step S5, sterilization is performed using a 0.22μm polyethersulfone filter membrane, with the filtration pressure controlled at 0.05MPa-0.1MPa; the formulation is dispensed into 1mL vials, and freeze-drying is carried out by first freezing at -80℃±2℃ for 2 hours, then drying at a vacuum degree <10Pa and -50℃ for 24 hours, followed by heating to 25℃ and drying for another 24 hours.

[0016] This invention provides a process for preparing high-purity and highly active von Willebrand factor. It has the following beneficial effects: 1. This invention uses an adaptive optimization algorithm for centrifugation parameters to dynamically adjust the speed and time of the second centrifugation based on protein concentration and turbidity, reducing the precipitation of impurity proteins and increasing the relative content of vWF in the secondary supernatant by 15% to 20%. After two-stage purification by specific monoclonal antibody affinity chromatography and gel filtration chromatography, the purity of vWF is stabilized at 98.2% to 98.6%, with batch-to-batch variation ≤2.4%, which is significantly better than traditional processes.

[0017] 2. This invention uses a mild eluent (pH 2.8 ± 0.1) for immediate neutralization to avoid depolymerization induced by vWF polymeric acid. At the same time, the proportion of protective agent is dynamically adjusted according to the turbidity. When the turbidity is high, it is added at a ratio of 1:8. The structure is stabilized by hydrogen bonds, so that the activity retention rate is ≥91.8%, which is much higher than ≤80% of the traditional process.

[0018] 3. This invention uses a step-by-step parameter linkage design. For example, the loading flow rate in S2 is dynamically adjusted according to the centrifugation speed in S1. High speed corresponds to high flow rate to improve efficiency, while low speed reduces flow rate to ensure sufficient binding. The amount of elution buffer in S3 is directly related to the volume of secondary supernatant in S1, ensuring sufficient elution of the target protein. This results in a stable recovery rate of 79.5% to 82.3%, with batch-to-batch variation ≤2.4%. This avoids column overload and reagent waste while meeting the stability and efficiency requirements of large-scale production. Attached Figure Description

[0019] Figure 1 This is a flowchart illustrating the preparation process of this high-purity and highly active von Willebrand factor. Detailed Implementation

[0020] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0021] Please see the appendix Figure 1 This invention provides a process for preparing high-purity and highly active von Willebrand factor, comprising the following steps: S1. Fresh Plasma Gradient Centrifugation: Fresh plasma was selected and centrifuged at 3000×g for the first time, and the primary supernatant was collected. The protein concentration and turbidity of the primary supernatant were measured, and the data were input into the centrifugation parameter optimization algorithm to output the rotation speed and time for the second centrifugation. Based on the output results, a second centrifugation was performed to collect the secondary supernatant rich in von Willebrand factor. The adaptive optimization algorithm for centrifugation parameters based on linear regression is as follows: Plasma from healthy individuals collected within 24 hours was used as raw material. A first centrifugation was performed under conditions of 4°C ± 0.5°C and 40%–60% relative humidity. The centrifugation speed was set to 3000 × g, and the centrifugation time was 15 minutes ± 2 minutes. After centrifugation, the mixture was allowed to stand for 5 minutes, and the primary supernatant was collected. The protein concentration in the primary supernatant was determined using ultraviolet spectrophotometry at a wavelength of 280 nm and denoted as C, where C is in mg / mL, representing the total protein concentration in the primary supernatant, with a value ranging from 10 to 80 mg / mL. Simultaneously, turbidity was measured using a turbidimeter at a 90° detection angle and denoted as T, where T is in NTU, representing the turbidity caused by suspended particles in the primary supernatant, with a value ranging from 1 to 20 NTU. The detected C and T values ​​are input into an adaptive optimization model for centrifugation parameters based on multiple linear regression. This model calculates the rotational speed and time of the second centrifugation using the following formula, where the calculation formula for the second centrifugation rotational speed R (in xg) is: In the formula: 7500 is the base speed (xg); 20 represents the weight of the effect of protein concentration on rotation speed (xg / (mg / ml)); 50 represents the weight of the effect of turbidity on rotation speed (xg / NTU); The formula for calculating the second centrifugation time t (in minutes) is: In the formula: 25 is the base time (minutes); 0.2 represents the weight of the effect of protein concentration on time (minutes / (mg / mL)); 0.5 represents the weight of the effect of turbidity on time (minutes / NTU); The calculated R must be forcibly limited to the range of 7500-8500 xg. If the calculated value exceeds this range, the boundary value will be automatically taken. The calculated t must be forcibly limited to the range of 25-35 minutes. If the calculated value exceeds this range, the boundary value will be automatically taken. Then, a second centrifugation is performed based on the above R and t under the same temperature conditions as the first centrifugation. After centrifugation, the secondary supernatant rich in von Willebrand factor is collected within 10 minutes. At the same time, after each batch is processed, the vWF purity of the secondary supernatant must be detected by SDS-PAGE. If the purity is <90%, the coefficients 20, 50, 0.2, and 0.5 in the calculation formula for the next batch are dynamically corrected by ±5% to achieve continuous optimization of parameters.

[0022] S2. Preliminary separation by specific immunoaffinity chromatography: The secondary supernatant collected in S1 is passed through an immunoaffinity chromatography column coupled with anti-von Willebrand factor antibody. The loading flow rate of the chromatography column is dynamically adjusted according to the rotation speed of the second centrifugation in S1. After washing, the bound components are collected. The anti-von Willebrand factor antibody in S2 is a murine monoclonal antibody with clone number VW-16. The coupling ratio between the antibody and the agarose gel medium is 1 mg antibody to 1 mL gel. The column bed volume of the immunoaffinity chromatography column is 100 mL ± 5 mL, and the column length to inner diameter ratio is 10:1. The secondary supernatant in S2 was pretreated with a 0.45 μm filter membrane before being loaded onto the column. The column temperature was maintained at 25℃±1℃ during the loading process. After loading, the column was incubated at room temperature for 2 hours±10 minutes, and the inlet and outlet of the chromatography column were kept closed during the incubation period. The washing was performed using a buffer solution containing 0.01M PBS and 0.15M NaCl, with a pH of 7.4±0.1 and a washing flow rate of 2mL / min±0.2mL / min. The A280 absorbance was measured every 10 minutes until three consecutive values ​​were <0.01. In S2, the secondary supernatant is pretreated with a 0.45 μm filter membrane before being loaded onto the column. The column flow rate is dynamically adjusted based on R calculated in S1. When R ≥ 8000 × g, the column flow rate is set to 1.5 mL / min ± 0.1 mL / min. When R < 8000 × g, the column flow rate is reduced until the column inlet and outlet are closed. The logic behind R determining the flow rate is that at higher rotation speeds, there are fewer contaminating proteins in the secondary supernatant, so a higher flow rate can be used to improve efficiency. At lower rotation speeds, there are more contaminating proteins, so the flow rate needs to be reduced to ensure sufficient binding. S3. Mild elution and ultrafiltration concentration: The bound components collected in S2 are eluted using a mild elution buffer. The amount of elution buffer is determined based on the volume of the secondary supernatant in S1. After neutralization, the elution buffer is concentrated through an ultrafiltration membrane. The mild elution buffer in S3 is a buffer solution containing 0.1M glycine-HCl and 0.5M NaCl. The buffer solution is sterilized by filtration through a 0.22μm filter membrane, with a pH of 2.8±0.1. The elution flow rate is 0.8mL / min±0.05mL / min. The elution buffer is collected in 5mL portions per tube, and the A280 absorbance is monitored simultaneously to determine the elution peak range. In step S3, neutralization is achieved using 1 M Tris-HCl preheated to 25°C with a pH of 8.0 ± 0.1. Neutralization is performed by adding the HCl dropwise at a rate of 5 mL / min while continuously stirring. After neutralization, the pH is adjusted to 7.0 ± 0.2. The ultrafiltration membrane is made of polyethersulfone with a molecular weight cutoff of 300 kDa ± 20 kDa. The concentration temperature is 4°C ± 0.5°C, the operating pressure is 0.1 MPa ± 0.01 MPa, and the concentration is reduced to 1 / 10 ± 1 / 100 of the original volume. The amount of mild elution buffer used in S3 is determined based on the volume of the secondary supernatant in S1. The calculation formula is secondary supernatant volume (L) × 0.5 (L / L). The volume of the secondary supernatant is determined by R and t in S1. Generally, the higher R and the longer t, the smaller the volume of the secondary supernatant will be (approximately 5%–10%). The elution flow rate is set to 0.8 mL / min ± 0.05 mL / min. The elution buffer is collected in 5 mL portions per tube, and the A280 absorbance is monitored simultaneously to determine the elution peak range. The logic of the relationship between the amount of elution buffer and the volume of the secondary supernatant is to ensure that the elution buffer can fully cover the bound components in the chromatography column and avoid target protein residue due to insufficient amount. S4. Secondary purification by gel filtration chromatography: The concentrated liquid in S3 is purified again by passing it through a gel filtration chromatography column. The flow rate of the mobile phase of the chromatography column is calibrated according to the protein concentration detection results in S1, and the target component is collected. In S4, the gel filtration chromatography column is a Superdex 200 Increase 10 / 300GL with a column bed volume of 24 mL ± 1 mL. The mobile phase is 0.02 M Tris-HCl containing 0.15 M NaCl, degassed, and has a pH of 7.4 ± 0.1. The calibration rule for the mobile phase flow rate is as follows: based on the C obtained in S1, when C ≥ 50 mg / mL, the flow rate is set to 0.5 mL / min ± 0.05 mL / min; when C < 50 mg / mL, the flow rate is reduced to 0.4 mL / min ± 0.05 mL / min. The sample loading volume is 596 of the column bed volume. Elution fractions are collected at 2 mL per tube, and the target fraction range is determined by SDS-PAGE electrophoresis. The logic of the relationship between flow rate and C is that at higher protein concentrations, the flow rate needs to be appropriately increased to avoid fraction diffusion, while at lower concentrations, the flow rate should be reduced to ensure separation accuracy. S5. Addition of Protectant and Formulation: Add a protectant to the target component collected in S4. The proportion of protectant added is adjusted based on the turbidity detection results of the secondary supernatant in S1. When T≥10NTU, the volume ratio is set to 1:8, and when T<10NTU, the volume ratio is set to 1:9. After mixing, stir at 150rpm for 30 minutes ± 5 minutes at 4°C±0.5°C. The logic of the protectant ratio being related to T is that higher turbidity means that there may be more impurities that affect stability, and the proportion of protectant needs to be increased to maintain vWF activity. After sterilization, the formulation is prepared. The protective agent in S5 is a mixture of 5% sucrose, 2% mannitol and 0.01% Tween-80. The protective agent is added after sterile filtration. The adjustment rule for the addition ratio is: when the turbidity of the secondary supernatant in S1 is ≥10 NTU, the volume ratio of the protective agent to the target component is 1:8; when the turbidity is <10 NTU, the volume ratio is 1:9. After mixing, the mixture is stirred at 150 rpm for 30 minutes ± 5 minutes at 4℃ ± 0.5℃. The sterilization process in S5 uses a 0.22μm polyethersulfone filter membrane with a filtration pressure controlled at 0.05MPa-0.1MPa. The formulation is dispensed into 1mL vials, and freeze-drying is carried out by first freezing at -80℃±2℃ for 2 hours, then drying at a vacuum degree <10Pa and -50℃ for 24 hours, followed by heating to 25℃ and drying for 24 hours.

[0023] Example 1: The variable is protein concentration C (T = 5 NTU is fixed). S1 parameters: The protein concentration in the primary supernatant was C = 30 mg / mL, and the turbidity was T = 5 NTU. Second centrifugation speed (Within the range of 7500-8500×g); Second centrifugation time Minutes (within the range of 25-35 minutes); the volume of secondary supernatant is 80% of the initial plasma (assuming an initial plasma volume of 10 L and a secondary supernatant volume of 8 L). S2 parameters: because The loading flow rate was 1.5 mL / min; other parameters were fixed: the antibody was VW-16 monoclonal antibody, the column bed volume was 100 mL, the washing buffer was 0.01 M PBS + 0.15 M NaCl (pH 7.4), and the washing flow rate was 2 mL / min. S3 parameters: The volume of eluent used is 8L × 0.5 = 4L; the eluent is 0.1M glycine-HCl + 0.5M NaCl (pH 2.8), the elution flow rate is 0.8mL / min; the ultrafiltration membrane has a molecular weight cutoff of 300kDa, and the solution is concentrated to 0.8L; S4 parameters: because The mobile phase flow rate was 0.4 mL / min; the chromatography column was a Superdex 200 Increase 10 / 300 GL; and the mobile phase was 0.02 M Tris-HCl + 0.15 M NaCl (pH 7.4). S5 parameters: because The volume ratio of the protective agent to the target component is 1:9; the protective agent is 5% sucrose + 2% mannitol + 0.01% Tween-80. Stir for 30 minutes, sterilize, and freeze dry. Example 2: The variable is turbidity T (fixed C = 50 mg / mL) S1 parameters: The protein concentration of the primary supernatant was C = 50 mg / mL, and the turbidity was T = 15 NTU. The second centrifugation speed was R = 7500 + 20 × 50 + 50 × 15 = 7500 + 1000 + 750 = 9250 × g (out of range, take the boundary value of 8500 × g). The second centrifugation time was t = 25 + 0.2 × 50 + 0.5 × 15 = 25 + 10 + 7.5 = 42.5 minutes (out of range, take the boundary value of 35 minutes). The volume of the secondary supernatant was 75% of the initial plasma (10 L of initial plasma, 7.5 L of secondary supernatant). S2 parameters: Since R = 8500 ≥ 8000 × g, the loading flow rate is 1.5 mL / min; the other parameters are the same as in Example 1. S3 parameters: The eluent volume is 7.5L × 0.5 = 3.75L; other parameters are the same as in Example 1. S4 parameters: Since C=50≥50mg / mL, the mobile phase flow rate is 0.5mL / min; the other parameters are the same as in Example 1. S5 parameters: Since T=15≥10NTU, the volume ratio of the protective agent to the target component is 1:8; the other parameters are the same as in Example 1; Example 3: The variable is the second centrifugation speed R (achieved by adjusting C, with T=8 NTU fixed). S1 parameters: The protein concentration in the primary supernatant was C = 20 mg / mL, and the turbidity was T = 8 NTU. Second centrifugation speed ; Second centrifugation time minute; The volume of the secondary supernatant was 78% of the initial plasma (10L initial plasma, 7.8L secondary supernatant). S2 parameters: because The sample loading flow rate was 1.5 mL / min; other parameters were the same as in Example 1. S3-S5 parameters: Same as Example 1 (except that the amount of eluent used was adjusted to 3.9L due to the volume of the secondary supernatant = 7.8L × 0.5). Example 4: The variable is the proportion of the protective agent (achieved by adjusting T, with C fixed at 40 mg / mL). S1 parameters: Primary supernatant protein concentration C = 40 mg / mL, turbidity T = 10 NTU; The second centrifugation speed R = 7500 + 20 × 40 + 50 × 10 = 7500 + 800 + 500 = 8800g (take the boundary value of 8500g); The second centrifugation time t = 25 + 0.2 × 40 + 0.5 × 10 = 25 + 8 + 5 = 38 minutes (taking the boundary value of 35 minutes); The volume of the secondary supernatant was 76% of the initial plasma (10L initial plasma, 7.6L secondary supernatant). S2-S4 parameters: same as in Example 1 (S4 flow rate = 0.4 mL / min because C = 40 < 50 mg / mL); S5 parameters: Since T=10≥10NTU, the volume ratio of protective agent to target component is 1:8; other parameters are the same as in Example 1; Comparative Example 1: No centrifugation parameter optimization algorithm used (second centrifugation parameters fixed) In S1, the second centrifugation is not based on C and T calculations. The rotation speed is fixed at 8000×g and the time is fixed at 30 minutes. The remaining steps are the same as in Example 1 (C=30mg / mL, T=5NTU). Comparative Example 2: No specific monoclonal antibody used (polyclonal antibody used) In S2, an anti-VWF polyclonal antibody (not a VW-16 monoclonal antibody) is coupled to the immunoaffinity chromatography column, and the remaining steps and parameters are the same as in Example 1; Comparative Example 3: Elution buffer under non-mild conditions (pH deviates from range) The eluent in S3 was 0.1M glycine-HCl + 0.5M NaCl (pH 3.5, exceeding the range of 2.8 ± 0.1), and the remaining steps and parameters were the same as in Example 1; Comparative Example 4: Protectant ratio not adjusted based on T (fixed ratio 1:9) In S5, regardless of the T value, the volume ratio of the protective agent to the target component is fixed at 1:9, and the remaining steps and parameters are the same as in Example 2 (T=15NTU). Experimental process I. Experimental Materials and Instruments Materials: Fresh plasma from healthy individuals (within 24 hours of collection), anti-vWF monoclonal antibody (clone number VW-16), agarose gel medium, Superdex 200 Increase 10 / 300 GL chromatography column, sucrose, mannitol, Tween-80, and buffer reagents (analytical grade).

[0024] Instruments: Low-temperature centrifuge, ultraviolet spectrophotometer, turbidity meter, chromatography system (including pump and detector), ultrafiltration system, SDS-PAGE electrophoresis apparatus, freeze dryer.

[0025] II. Experimental Procedure Sample pretreatment: Take 10L of fresh plasma and process it according to the parameters of Examples 1-4 and Comparative Examples 1-4 in steps S1-S5. Repeat the experiment 3 times for each group.

[0026] Key indicator detection: Purity: SDS-PAGE electrophoresis was used, and the percentage of vWF bands was analyzed by gel imaging system.

[0027] Activity: The ristocetin cofactor activity (%) of vWF was detected by the coagulation factor activity assay, with the initial plasma activity as 100%.

[0028] Recovery rate: Calculate the ratio (%) of vWF mass in the final formulation to vWF mass in the initial plasma. Group Purity (%) Activity retention rate (%) Recovery rate (%) Batch difference (%) Example 1 98.6±0.2 93.5±1.1 82.3±2.0 2.1±0.3 Example 2 98.2±0.3 91.8±1.3 79.5±1.8 2.3±0.4 Example 3 98.5±0.2 92.7±1.0 81.7±1.9 2.2±0.3 Example 4 98.3±0.3 92.1±1.2 80.6±2.1 2.4±0.4 Comparative Example 1 92.5±0.5 85.3±1.5 75.2±2.3 4.8±0.6 Comparative Example 2 90.1±0.6 83.7±1.6 73.5±2.5 5.2±0.7 Comparative Example 3 97.8±0.3 72.4±2.1 78.6±2.2 3.5±0.5 Comparative Example 4 98.0±0.2 80.5±1.8 81.2±2.0 2.6±0.4 The standards involved in the experiment are as follows: Standard number Standard name Related content and application scenarios GB 18469 -2012 Quality Requirements of Whole Blood and Blood Components Fresh frozen plasma is used as raw material to clarify cryoprecipitate coagulation factors. The time for preparing fresh frozen plasma after blood collection, cryoprecipitate related indicators such as factor VIII and fibrinogen content and volume, etc. are regulated to control the quality of raw materials and finished products. WS 399 -2023 Standard for Blood Storage Fresh frozen plasma, cryoprecipitate coagulation factor storage temperature, shelf life, and cryoprecipitate thawing storage temperature and infusion time requirements are specified to standardize storage and use. Technical Operation Rules of Blood Station (2015 Edition) Technical Operation Rules of Blood Station The preparation method of cryoprecipitate, blood collection specification, plasma preparation time limit and quick freezing requirements are covered to standardize the operation process of blood stations. GB 14232 Plastic bags for human blood and blood components - Part 1: conventional blood bags The blood storage container is required to be free of damage, and the length of the transfer tube for preparing cryoprecipitate is retained to ensure the safety of storage and transportation. Chinese Pharmacopoeia 2020 Edition National Drug Standard The content and activity standards of main components such as coagulation factor I and fibrinogen in cryoprecipitate are specified for product quality control. EU GMP requirements Good Manufacturing Practice for Drugs The standards for sterility and pyrogen testing of cryoprecipitate are specified, and the limits for endotoxin and microorganisms are required to ensure product safety. ISO 3676 International Standard Standards are proposed for plasma thawing, centrifugation, concentration and drying, etc. to ensure the enrichment of coagulation factors. DIN 58988:2023 Reference method for blood enzymes - Factor VIII multimers in human citrate plasma or coagulation factor concentrate It is applicable to detect VWF multimers in human citrate plasma or coagulation factor concentrate. The sample processing, electrophoresis system configuration and detection performance requirements are specified, such as sample room temperature transportation, thawing conditions, different concentration of agarose gel application, and number of multimer band detection, etc. DIN 58924:2022 Reference method for blood enzymes - Determination of the collagen binding activity of VWF A reference method for determining the collagen binding activity of VWF is provided to standardize the detection process to accurately evaluate the collagen binding activity of VWF. DIN 58987:2023 Reference method for blood enzymes - Factor VIII antigen The reference method for factor VIII antigen is specified to provide a standard for accurate determination of VWF antigen content. Results Analysis I. Collaborative optimization effects of Examples 1-4 Examples 1-4 all employ the fully optimized process of this technical solution. Through the adaptive algorithm for centrifugation parameters and the coordinated design of each step, the purity, activity, and recovery rate of vWF are simultaneously improved, specifically as follows: Purity Stability: The vWF purity of all four examples remained stable at 98.2%–98.6%, with batch-to-batch variation ≤2.4%. This result is attributed to the precise control of the centrifugation parameter optimization algorithm in S1—dynamically adjusting the rotation speed and time of the second centrifugation by adjusting protein concentration C and turbidity T effectively reduced the precipitation of contaminating proteins (such as albumin, fibrinogen, etc.), increasing the relative content of vWF in the secondary supernatant by 15%–20% (compared to fixed centrifugation parameters). Simultaneously, the high affinity binding of the specific monoclonal antibody (VW-16) in S2 and the molecular sieving effect of gel filtration chromatography in S4 formed a "two-stage purification system," further removing contaminating proteins with similar molecular weights (such as coagulation factor VIII), ultimately achieving a purity exceeding 98%.

[0029] Highly efficient activity retention: The activity retention rates of all examples were ≥91.8%, significantly higher than those of traditional processes (typically ≤80%). The core reason is: The mild elution buffer (pH 2.8 ± 0.1) in S3, combined with the immediate neutralization step, avoids acid-induced depolymerization of vWF polymers (the activity of vWF depends on the integrity of the polymer structure). The proportion of the protectant in S5 is dynamically adjusted based on the turbidity T of S1. Under high turbidity conditions (such as T=15NTU in Example 2), the proportion of the protectant is increased (1:8) to stabilize the tertiary structure of vWF by utilizing the hydrogen bonding between sucrose and mannitol, thereby reducing the loss of activity during the freeze-drying process (only ≤5%).

[0030] Recovery rate and batch-to-batch consistency: The recovery rate of the example was stable at 79.5%–82.3%, with a batch-to-batch difference of ≤2.4%, demonstrating the scalability of the process. This is attributed to the parameter linkage design of S1–S5: for example, the loading flow rate of S2 is dynamically adjusted according to the centrifugation speed of S1 (high speed corresponds to high flow rate), which avoids column overload caused by excessive impurities at low speeds and improves the processing efficiency of high-purity raw materials; the eluent volume of S3 is directly related to the secondary supernatant volume of S1, ensuring that the target protein is fully eluted without wasting reagents, ultimately achieving a balance between recovery rate and efficiency.

[0031] II. Mechanism of Difference Between Comparative Examples and Implementation Examples Comparative Example 1 (without centrifugation parameter optimization algorithm): Fixing the second centrifugation parameters (8000×g, 30 minutes) resulted in significantly lower purity (92.5%) and activity (85.3%) compared to Example 1. This is because fluctuations in the protein concentration or turbidity of the raw plasma... The fixed parameters could not match the characteristics of the raw materials. The excessively low rotation speed resulted in insufficient removal of contaminating proteins (the content of contaminating proteins in the supernatant was 30% higher than in Example 1), directly increasing the purification load of subsequent chromatography. At the same time, the mismatch in centrifugation time caused some vWF to be lost with the precipitation, resulting in a 7% decrease in recovery rate (75.2% vs 82.3%). In addition, the batch-to-batch variation reached 4.8%, proving that the process stability decreased significantly without algorithm optimization.

[0032] Comparative Example 2 (using polyclonal antibodies): After replacing the VW-16 monoclonal antibody with a polyclonal antibody, the purity decreased to 90.1% and the activity decreased to 83.7%. Because polyclonal antibodies recognize multiple antigenic epitopes of vWF, they are prone to cross-reaction with other plasma proteins (such as fibronectin), leading to an increase in the amount of residual contaminating proteins (SDS-PAGE showed more than 3 contaminating bands); at the same time, non-specific binding competitively occupies the binding sites on the chromatography column, causing a 10% decrease in vWF binding rate, ultimately resulting in a decrease in recovery rate to 73.5% (8.8% lower than in Example 1).

[0033] Comparative Example 3 (elution buffer pH deviation): After the eluent pH was increased to 3.5 (beyond the 2.8±0.1 range), the activity retention rate plummeted to 72.4%, the lowest among all groups. The mechanism is that vWF's acid tolerance threshold is pH 2.5-3.0. When pH > 3.0, the conformational disruption of vWF by the eluent is weakened, but this leads to a decrease in the binding affinity between the antibody and vWF (dissociation constant K). d The increased concentration (by 2 times) prevents some highly active polymers (molecular weight > 2000 kDa) from being effectively eluted and leaves them in the column. At the same time, the amount of extraneous proteins eluted increases at higher pH, forming co-elution with vWF. Although the purity still reaches 97.8%, the loss of the active core components is severe.

[0034] Comparative Example 4 (fixed protectant ratio): Under high turbidity (T=15 NTU) conditions, the 1:9 protectant ratio was still used, and the activity retention rate decreased to 80.5% (11.3% lower than Example 2). High turbidity plasma contains more lipids and denatured proteins, which competitively bind to the hydrophilic groups on the surface of vWF molecules, accelerating their aggregation; while the fixed protectant ratio cannot fully encapsulate vWF molecules, leading to polymer breakage during freeze-drying (electron microscopy showed an average reduction of 40% in polymer length), ultimately resulting in a significant decrease in activity.

[0035] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A preparation process for a high-purity and highly active von Willebrand factor, characterized in that, Includes the following steps: S1. Gradient centrifugation of fresh plasma: Select fresh plasma, centrifuge it at 3000×g for the first time, and collect the primary supernatant; Protein concentration and turbidity of the primary supernatant are detected, and the detection data are input into the centrifugation parameter optimization algorithm to output the rotation speed and time of the second centrifugation. A second centrifugation was performed based on the output results to collect the secondary supernatant rich in von Willebrand factor; S2. Preliminary separation by specific immunoaffinity chromatography: The secondary supernatant collected in S1 is passed through an immunoaffinity chromatography column coupled with anti-von Willebrand factor antibody. The loading flow rate of the chromatography column is dynamically adjusted according to the rotation speed of the second centrifugation in S1. After washing, the bound components are collected. S3. Mild elution and ultrafiltration concentration: The bound components collected in S2 are eluted using a mild elution buffer. The amount of elution buffer is determined based on the volume of the secondary supernatant in S1. After neutralization, the elution buffer is concentrated through an ultrafiltration membrane. S4. Secondary purification by gel filtration chromatography: The concentrated liquid in S3 is purified again by passing it through a gel filtration chromatography column. The flow rate of the mobile phase of the chromatography column is calibrated according to the protein concentration detection results in S1, and the target component is collected. S5. Addition of Protectant and Formulation: A protectant is added to the target component collected in S4. The proportion of protectant added is adjusted based on the turbidity test results of the secondary supernatant in S1. After sterilization, the formulation is prepared.

2. The preparation process of a high-purity and highly active von Willebrand factor according to claim 1, characterized in that, The fresh plasma in S1 is plasma from healthy individuals collected within 24 hours. The temperature for the first centrifugation is maintained at 4℃±0.5℃, the centrifugation time is 15 minutes±2 minutes, and the relative humidity of the centrifugation environment is controlled at 40%~60%. Protein concentration is detected by ultraviolet spectrophotometry at a wavelength of 280nm, and turbidity is detected by a scattering light turbidimeter at a detection angle of 90°.

3. The preparation process of a high-purity and highly active von Willebrand factor according to claim 1, characterized in that, The centrifugation parameter optimization algorithm in S1 is an adaptive model based on multiple linear regression. The input parameters include protein concentration (mg / mL) and turbidity (NTU). The output parameters are the rotation speed and time of the second centrifugation, with the centrifugation speed ranging from 7500 to 8500 × g and the centrifugation time ranging from 25 to 35 minutes. The temperature of the second centrifugation is the same as that of the first centrifugation. The secondary supernatant is collected within 10 minutes after centrifugation.

4. The preparation process of a high-purity and highly active von Willebrand factor according to claim 1, characterized in that, The anti-von Willebrand factor antibody in S2 is a murine monoclonal antibody with clone number VW-16. The antibody-agarose gel medium coupling ratio is 1 mg antibody to 1 mL gel. The column bed volume of the immunoaffinity chromatography column is 100 mL ± 5 mL, and the column length to inner diameter ratio is 10:

1. The dynamic adjustment rule for the loading flow rate is as follows: when the second centrifugation speed in S1 is ≥8000×g, the loading flow rate is 1.5 mL / min ± 0.1 mL / min; when the speed is <8000×g, the loading flow rate is reduced to 1.2 mL / min ± 0.1 mL / min.

5. The preparation process of a high-purity and highly active von Willebrand factor according to claim 1, characterized in that, The secondary supernatant in S2 was pretreated with a 0.45 μm filter membrane before being loaded onto the column. The column temperature was maintained at 25℃±1℃ during the loading process. After loading, the column was incubated at room temperature for 2 hours±10 minutes, and the inlet and outlet of the chromatography column were kept closed during the incubation period. The washing was performed using a buffer solution containing 0.01M PBS and 0.15M NaCl, with a pH of 7.4±0.1 and a washing flow rate of 2mL / min±0.2mL / min. The A280 absorbance was measured every 10 minutes until three consecutive values ​​were <0.

01.

6. The preparation process of a high-purity and highly active von Willebrand factor according to claim 1, characterized in that, The mild elution buffer in S3 is a buffer solution containing 0.1M glycine-HCl and 0.5M NaCl. The buffer solution is sterilized by filtration through a 0.22μm filter membrane, with a pH of 2.8±0.

1. The elution flow rate is 0.8mL / min±0.05mL / min. The elution buffer is collected in 5mL portions per tube, and the A280 absorbance is monitored simultaneously to determine the elution peak range.

7. The preparation process of a high-purity and highly active von Willebrand factor according to claim 1, characterized in that, In step S3, neutralization is achieved using 1 M Tris-HCl preheated to 25°C with a pH of 8.0 ± 0.

1. Neutralization is performed by adding the HCl dropwise at a rate of 5 mL / min while continuously stirring. After neutralization, the pH is adjusted to 7.0 ± 0.

2. The ultrafiltration membrane is made of polyethersulfone with a molecular weight cutoff of 300 kDa ± 20 kDa. The concentration temperature is 4°C ± 0.5°C, the operating pressure is 0.1 MPa ± 0.01 MPa, and the concentration is reduced to 1 / 10 ± 1 / 100 of the original volume.

8. The preparation process of a high-purity and highly active von Willebrand factor according to claim 1, characterized in that, The gel filtration chromatography column in S4 is a Superdex 200 Increase 10 / 300GL with a column bed volume of 24 mL ± 1 mL. The mobile phase is 0.02 M Tris-HCl containing 0.15 M NaCl, degassed, and with a pH of 7.4 ± 0.

1. The calibration rule for the mobile phase flow rate is as follows: when the protein concentration in S1 is ≥ 50 mg / mL, the flow rate is 0.5 mL / min ± 0.05 mL / min; when the concentration is < 50 mg / mL, the flow rate is reduced to 0.4 mL / min ± 0.05 mL / min. The sample loading volume is 5% of the column bed volume, and the eluted fraction is collected in 2 mL tubes. The target fraction range is determined by SDS-PAGE electrophoresis.

9. The preparation process of a high-purity and highly active von Willebrand factor according to claim 1, characterized in that, The protective agent in S5 is a mixture of 5% sucrose, 2% mannitol and 0.01% Tween-80. The protective agent is added after sterile filtration. The adjustment rule for the addition ratio is: when the turbidity of the secondary supernatant in S1 is ≥10 NTU, the volume ratio of the protective agent to the target component is 1:8; when the turbidity is <10 NTU, the volume ratio is 1:

9. After mixing, the mixture is stirred at 150 rpm for 30 minutes ± 5 minutes at 4℃ ± 0.5℃.

10. The preparation process of a high-purity and highly active von Willebrand factor according to claim 1, characterized in that, The sterilization process in S5 uses a 0.22μm polyethersulfone filter membrane with a filtration pressure controlled at 0.05MPa-0.1MPa. The formulation is dispensed into 1mL vials, and freeze-drying is carried out by first freezing at -80℃±2℃ for 2 hours, then drying at a vacuum degree <10Pa and -50℃ for 24 hours, followed by heating to 25℃ and drying for 24 hours.

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