Method for extracting human alpha1-antitrypsin from plasma by full chromatography

Abstract

The invention relates to a method for extracting human alpha1-antitrypsin from plasma by a full chromatography method. According to the method, specifically, human plasma or a flow-through liquid obtained after Pg, Fg, IgG and Alb are sequentially extracted from the human plasma is used as a raw material, chromatography preparation is conducted through a two-step chromatography method of CM FF cation exchange chromatography and DEAE FF ion exchange chromatography, according to the method, the yield of human alpha1-antitrypsin is larger than 400 g on the basis of 1000 L of plasma, and the relative purity of a product is larger than 99%.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically, it relates to a method for extracting α1-antitrypsin from plasma using a full chromatography method. Background Technology

[0002] α1-Antirtypsin (α1-AT, AAT) is a serine protease inhibitor mainly synthesized by hepatocytes. In electrophoresis, its migration site is located in the α1 protein band, hence it is also known as an α1-proteinase inhibitor (α1-AT, A1PI). α1-AT deficiency leads to inflammatory tissue damage in the lungs, causing chronic obstructive pulmonary disease (COPD). Current emerging research interests have shifted to its role in cancer-related inflammation and the tumor microenvironment, as well as investigating the mechanism of action of α1-AT in treating sepsis through animal model experiments.

[0003] α1-AT has a molecular weight of 52-55 kDa and an isoelectric point of approximately 4.9-5.1. The concentration of α1-AT in normal human plasma is (2.90 + / - 0.45) g / L, and its half-life in vivo is 6 days. Since α1-AT used for treatment is primarily derived from the precipitation of human plasma fraction IV, the antigen recovery rate of α1-AT in the raw material is almost half that of α1-AT in plasma. Currently, the antigen recovery rate of publicly available human α1-AT production methods is around 20%. To meet the needs of clinical patients, the recovery rate of plasma-derived α1-AT should be maximized, while simultaneously improving its purity.

[0004] Based on this, the present invention is proposed. Summary of the Invention

[0005] This invention first relates to a method for extracting human α1-antitrypsin (α1-AT) from plasma using a full chromatography method. The method includes the following steps:

[0006] (1) The starting material is human plasma or flow-through after extracting proteins Pg, Fg, IgG and Alb from human plasma by chromatography. The starting material is concentrated by ultrafiltration and the replacement buffer is B1 (4.8-5.2 mmol / L Tris, pH 7.5-8.3).

[0007] (2) Adjust the sample obtained in step (1) with adjustment buffer B2 (0.93-1.50 mmol / L NaOAc, pH 5.0-5.4) to make the final NaOAc content in the sample 20-32 mmol / L, and adjust the pH of the sample to 5.0-5.4 with acid or base. Let the adjusted A2 sample stand for 60-120 min, preferably 90 min.

[0008] (3) The sample was rinsed with buffer B3 (20-32 mmol / L NaOAc, pH 5.0-5.4), and then the sample was collected after clarification and filtration as the loading sample for CM FF chromatography.

[0009] (4) CMFF cation exchange chromatography: After the packed CMFF cation exchange chromatography column is equilibrated with buffer B3, the sample is loaded and purified by chromatography. The flow-through peak sample A4 is collected, and the pH and conductivity of sample A4 are detected and recorded. After the flow-through peak sample A4 is collected to the baseline, the chromatography column is eluted with buffer B4 (20-32 mmol / L NaOAc, 0.8-1.1 mol / L NaCl, pH 5.0-5.6), and the elution peak sample A5 is collected.

[0010] (6) DEAE FF ion exchange chromatography: The DEAE FF chromatography column was equilibrated with buffer B3, sample A4 was loaded, and the flow-through sample was collected and recorded as A6; the chromatography column was further washed with buffer B5 (20-32 mmol / L NaOAc, 0.07-0.11 mol / L NaCl, pH 5.0-5.4), and the sample was collected and recorded as A7;

[0011] (7) Sample A7 ultrafiltration concentration: Use buffer B6 for ultrafiltration concentration to the protein content of 25-33 g / L; the concentrated sample is recorded as A8, which is α1-antitrypsin;

[0012] And optional

[0013] (8) Pasteurization of α1-antitrypsin (sample A8): Add potassium acetate and sucrose to the sample to make their final contents 1.2-1.6 mol / L and 1.7-2.1 mol / L, respectively, and stir in a 30°C water bath to dissolve the solids. After the sample is completely dissolved, incubate in a 60°C water bath for at least 10 hours.

[0014] (9) Ultrafiltration concentration and preparation for dispensing: The inactivated sample was diluted with buffer B6, and after a 4-fold dilution, the pH was 6.7–7.7; further ultrafiltration concentration was performed using buffer B7 (14–21 mmol / L sodium phosphate, 47–55 mmol / L NaCl, pH 6.7–7.7). The concentration was carried out until the α1-antitrypsin (α1-AT) content was 25–33 g / L.

[0015] (10) Freeze-dry and dry heat inactivate, add mannitol, glycine, histidine and other protective agents for freeze-drying, and further dry heat inactivate at 100±2℃ for 60 to 90 minutes.

[0016] Furthermore,

[0017] The pH of the sample obtained after concentration in step (1) should be in the range of 7.5 to 8.3, and the conductivity should be less than 1 mS / cm.

[0018] The conductivity of the sample after adjustment in step (2) is no greater than 5.0 mS / cm.

[0019] Furthermore,

[0020] The amount of α1-AT prepared by the method, when the starting material is the flow-through liquid after extracting proteins Pg, Fg, IgG and Alb from human plasma by chromatography, the average recovery rate of the α1-AT antigen content in the sample is >90%.

[0021] Correspondingly, when using component IV prepared by the low-temperature ethanol method as the starting material in the traditional method, the average recovery rate of the antigen content preparation step for α1-AT is only about 50%.

[0022] When the starting material is the flow-through liquid after extracting proteins Pg, Fg, IgG and Alb from human plasma by chromatography, the proportion of α1-AT in the starting material is 15% to 18%; after the treatment of steps (1)-(4), the relative purity of α1-AT in the flow-through liquid A4 is >50%.

[0023] In the product of step (7), the average relative purity of α1-AT is >99%;

[0024] The method yields more than 400g of α1-AT per 1000L of plasma.

[0025] In each step of the method, the specific activity of the target protein α1-AT is as follows: approximately 0.02 in human plasma, approximately 0.04 in starting material, approximately 0.2-0.3 after adjustment (acid precipitation) of starting material, >0.7 in sample A5, >1.2 in sample A7, >1.1 after pasteurization, and >1 after lyophilization and / or dry heat inactivation.

[0026] The present invention also relates to the use of the method in the preparation of a medicine or pharmaceutical composition comprising, a therapeutically effective amount of human α1-antitrypsin (α1-AT), and necessary pharmaceutical excipients.

[0027] The beneficial effects of this invention are as follows:

[0028] The α1-AT production process proposed in this paper uses the flow-through liquid after extracting albumin (Alb), globulin (IgG), human plasminogen (Pg), and human fibrinogen (Fg) as the starting material. This process does not affect the recovery rate of the main proteins and improves the yield of α1-AT. The two-step virus inactivation improves the safety of the product and is suitable for large-scale production of α1-AT. It also improves the comprehensive utilization rate of plasma and increases the variety of blood products.

[0029] I. Improved recovery rate

[0030] This invention uses human plasma as raw material. The flow-through after sequentially extracting Pg, Fg, IgG, and Alb is used as the starting material for extracting α1-AT. A comparison of the α1-AT yields using plasma component IV precipitate prepared by the low-temperature ethanol method as the starting material shows that:

[0031] (1) When the component IV was prepared by the low temperature ethanol method, the recovery rate of α1-AT (comparative original plasma) was only 52.4%, while the recovery rate of the present invention can reach 90%.

[0032] (2) The effects of sequential extraction of Pg and Fg from human plasma on the recovery rates of the major proteins IgG and Alb were further analyzed. The results showed that the recovery rates of IgG and Alb antigens in the flow-through were approximately 96% and 99%, respectively. Therefore, We used the full chromatography method. Purification of α1-AT does not affect the yield of IgG and Alb, while increasing the variety of blood products. .

[0033] II. Optimization of Chromatography Methods

[0034] This invention employs a two-step ion exchange chromatography method to purify α1-AT, and explores different affinity packing materials for α1-AT purification.

[0035] (1) The main impurities in the starting materials are IgG, IgA, IgM, TRF, Alb, α2M, HPT, and AAG. The target protein α1-AT is an acidic glycoprotein with an isoelectric point of 4.7–5.0. Its potency is largely lost below pH 4.5. Select CM FF chromatography The pH is 5.0–5.6. α1-AT carries a negative charge during chromatography, therefore α1-AT exists in the CM FF flow-through solution. .

[0036] (2) The isoelectric points of impurity proteins AAG, Alb, α2M and HPT are 2.7-3.5, 4.7-4.9, 5.5 and 6.0, respectively. AAG, Alb and most of the α2M proteins flow through α1-AT, while a small portion of α2M and HPT bind weakly to CM FF.

[0037] (3) The loading conditions of CM FF chromatography were optimized, the standing time of acid precipitation was adjusted, and the physicochemical properties of impurity proteins were combined to remove most of the impurity proteins. The relative purity of the target protein in the flow-through solution increased from 21.872% of the starting material to 54.971%.

[0038] (4) The collected CM FF flow-through was subjected to DEAE FF chromatography. According to the isoelectric point of α1-AT, α1-AT was enriched in the eluent. Since the content of impurity proteins in the eluent was extremely small, we tested the antigen content of the protein after ultrafiltration concentration of this sample. The results showed that the relative purity of α1-AT in the concentrated eluent increased to 99.572%, of which AAG was the main impurity protein. AAG was more firmly bound to the DEAE FF chromatography column than α1-AT. Therefore, we reduced the salt ion elution concentration. A small part of α1-AT and AAG were still bound to the chromatography column during the elution process, which resulted in a sacrifice of some α1-AT yield while increasing the purity of α1-AT.

[0039] (5) The average recovery rates of the three batches of CM FF chromatography and DEAE FF chromatography were 76.445% and 74.816%, respectively. The CV of the recovery rates of the three batches were all less than 6%, indicating that the chromatography process had good repeatability.

[0040] III. Optimization of Inactivation, Freeze-drying, and Dry Heat Methods

[0041] During the preparation of α1-AT, pasteurization, lyophilization, and dry heat can cause α1-AT to lose its activity and fail to perform its function. Therefore, it is necessary to add a suitable protective agent to preserve the activity of α1-AT. This process involves screening different formulations and studying their proportions.

[0042] (1) Based on the potency test results of α1-AT, the pasteurization inactivation additives were determined to be sucrose and potassium acetate, with final contents of 1.2–1.6 mol / L and 1.7–2.1 mol / L, respectively.

[0043] (2) The freeze-drying formula is mannitol, glycine, or histidine.

[0044] The specific activity of α1-AT in each step from human plasma to finished product was analyzed. Both steps of chromatography increased the specific activity of α1-AT. Pasteurization and lyophilization / dry heat treatment both led to a slight decrease in specific activity, but the specific activity of the finished product was 54 times higher than that of plasma. Moreover, the specific activity CV of the intermediate and finished products prepared in the three batches was less than 10%. Therefore, the pasteurization and lyophilization / dry heat treatment formulations in this study can effectively protect the activity of α1-AT.

[0045] This invention employs a two-step virus removal process: inactivating porcine pseudorabies virus (PRV) with pasteurized virus and inactivating porcine parvovirus (PPV) with dry fever virus. Virus inactivation was verified separately for each method. The results showed that both pasteurized virus inactivation and dry fever virus inactivation could reduce the virus titer by ≥4.0 log. Therefore, these two virus inactivation methods are effective in this process, improving the safety of the product.

[0046] In summary, in the prior art, α1-AT can be extracted from 1000L of plasma by using plasma fraction IV precipitation as raw material, yielding 180g to 200g of α1-AT. However, under the process of the present invention, 400g to 500g of α1-AT can be extracted from 1000L of plasma, which improves the yield and is more suitable for large-scale production of α1-AT.

[0047] Accelerated stability studies were conducted on the α1-AT product obtained by the present invention. The potency recovery rate was greater than 90%, which initially reflects that the α1-AT product prepared by the process of the present invention has good stability and that the freeze-drying heat protectant can effectively protect the activity of α1-AT.

[0048] In summary, the α1-AT preparation process of this invention can obtain high-quality, high-yield, and low-risk α1-AT. The process has good stability and repeatability, making it suitable for large-scale production. It also increases the variety of blood products and improves the overall utilization rate of blood products. Attached Figure Description

[0049] Figure 1 The process flow diagram of this application.

[0050] Figure 2 The specific activity trend of AAT in the process flow of this invention.

[0051] Figure 3 PRV activity kinetic curve during pasteurization.

[0052] Figure 4 PPV activity kinetic curve during dry heat inactivation process. Detailed Implementation

[0053] Instruments and Materials

[0054] Sodium acetate, sodium chloride, Tris, disodium hydrogen phosphate, and sodium dihydrogen phosphate were all purchased from Sinopharm Chemical Reagent Co., Ltd.

[0055] α1-AT standard (lot number: D00117675) was purchased from CalBiochem GmbH, Germany;

[0056] α1-AT quality control material (batch number: PI2011-02) was purchased from Athens Research & Tech, USA.

[0057] Porcine pancrease lastase (PPE) was purchased from CalBiochem, Germany.

[0058] The elastase substrate [Suc-(Ala)3-pNa,BAPNA] was purchased from Sigma-Aldrich, USA.

[0059] Immunoturbidimetric assay reagents: α1-AT (AAT), albumin (Alb), immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM), transferrin (TRF), α2-macroglobulin (α2M), haptoglobulin (HPT), α1-acid glycoprotein (AAG), ceruloplasmin (CER), fibrinogen (Fg), and human plasminogen (Pg), purchased from Siemens (China) Ltd., Germany.

[0060] The chromatography media (CM sepharose fast flow, CM FF; DEAE sepharose fast flow, DEAEFF) and chromatography columns were purchased from Cytiva.

[0061] Replacement buffer B1: 4.8–5.2 mmol / L Tris, pH 7.5–8.3

[0062] Adjust buffer B2: 0.93–1.50 mmol / L NaOAc, pH 5.0–5.4

[0063] Buffer B3: 20–32 mmol / L NaOAc, pH 5.0–5.4

[0064] Buffer B4: 20–32 mmol / L NaOAc, 0.8–1.1 mol / L NaCl, pH 5.0–5.6

[0065] Buffer B5: 20–32 mmol / L NaOAc, 0.07–0.11 mol / L NaCl, pH 5.0–5.4

[0066] Buffer B6 (38–45 mmol / L sodium phosphate, pH 6.9–7.5)

[0067] Buffer B7 (14–21 mmol / L sodium phosphate, 47–55 mmol / L NaCl, pH 6.7–7.7)

[0068] Example 1: Preparation process of α1-AT

[0069] Human plasma, after undergoing three-step chromatography to capture Pg, Fg, IgG, and ALB, yields a flow-through (FT) solution, which serves as the starting material for α1-AT. The FT starting material is then concentrated by ultrafiltration and subsequently subjected to cation exchange chromatography, anion exchange chromatography, ultrafiltration followed by pasteurization, sterilization and aliquoting after ultrafiltration, lyophilization, and dry heat inactivation. The process flow diagram is shown below. Figure 1 As shown.

[0070] During the preparation process, we performed online pH and conductivity measurements on samples from key steps. The process has strict requirements on the conductivity and pH of the samples. By analyzing the results, we can identify deviations in parameters during the purification process and make timely adjustments. The pH and conductivity of the samples in the α1-AT preparation process need to be controlled within the specified ranges.

[0071] 1. Sample preparation process including sample configuration, pretreatment, and chromatographic loading.

[0072] 1.1 Initial conditioning of samples for CMFF cation exchange chromatography

[0073] The starting material for preparing α1-AT is human plasma or flow-through after extracting proteins Pg, Fg, IgG, and Alb from human plasma by chromatography. The starting material is concentrated by ultrafiltration. The replacement buffer is B1 (4.8–5.2 mmol / L Tris, pH 7.5–8.3). The pH of the dialysis endpoint sample should be in the range of 7.5–8.3, and the conductivity should be less than 1 mS / cm.

[0074] 1.2 Secondary adjustment of dialysis-collected samples

[0075] The sample collected by dialysis in step 1 is designated A1. Sample A1 is adjusted with adjusting buffer B2 (0.93–1.50 mmol / L NaOAc, pH 5.0–5.4) to achieve a final NaOAc concentration of 20–32 mmol / L. The pH of the sample is then adjusted to 5.0–5.4 using acid or alkali. The conductivity of the adjusted sample A2 is tested, ensuring it does not exceed 5.0 mS / cm before the next clarification filtration step. The adjusted sample A2 is allowed to stand for 60–120 minutes.

[0076] At pH levels below 5.0, the potency recovery rate of α1-AT was less than 90%. Table 1 shows that adjusting the pH to 5.2 removed impurities (significantly improving potency recovery) without affecting the protein recovery rate of α1-AT. Table 2 shows that after adjusting the pH to approximately 5.2 and allowing the sample to stand for 30 min, 60 min, and 90 min, the impurity removal rate tended to increase with the extension of the standing time. Based on these results, we ultimately chose to allow the sample to stand for 90 min before proceeding with the next step of clarification and filtration. Before adjustment, α1-AT accounted for 15% of the total protein in the sample, and this percentage increased after adjustment.

[0077] Table 1. Effects of different pH values ​​on α1-AT titer

[0078]

[0079] Table 2 Comparison of the effects of adjusting samples to pH 5.10–5.40

[0080]

[0081] 1.3 Clarification and filtration of sample A2

[0082] Before use, the clarifying filter should be rinsed with buffer B3 (20–32 mmol / L NaOAc, pH 5.0–5.4). Sample A2 collected after clarification filtration is numbered A3 and used as the loading sample for CM FF chromatography.

[0083] 2. Preparation of α1-AT using total chromatography

[0084] 2.1 CMFF Cation Exchange Chromatography

[0085] After the packed chromatography column is equilibrated with buffer B3 (20-32 mmol / L NaOAc, pH 5.0-5.4), sample A3 is loaded for chromatography purification, the flow-through peak sample A4 is collected, and the pH and conductivity of sample A4 are detected and recorded.

[0086] After the flow-through peak was collected to baseline, the column was eluted with buffer B4 (20–32 mmol / L NaOAc, 0.8–1.1 mol / L NaCl, pH 5.0–5.6), and the elution peak sample A5 was collected.

[0087] 2.2 DEAE FF Ion Exchange Chromatography

[0088] DEAE FF chromatography columns are equilibrated with buffer B3 (20–32 mmol / L NaOAc, pH 5.0–5.4). The amount of equilibration buffer B3 used should be controlled within 4 column volumes (CV). The pH of the equilibration flow-through solution is collected and checked to ensure that the pH is < 6.0.

[0089] After equilibrating the chromatography column, load sample A4 and collect flow-through sample A6 to the baseline. After collecting sample A6, take a small sample for testing.

[0090] The chromatography column was further washed with buffer B5 (20–32 mmol / L NaOAc, 0.07–0.11 mol / L NaCl, pH 5.0–5.4), and the collected samples were numbered A7.

[0091] 2.3 Ultrafiltration Concentration of Sample A7

[0092] This step uses buffer B6 (38–45 mmol / L sodium phosphate, pH 6.9–7.5). The sample after ultrafiltration concentration is designated as A8, and the protein content should be controlled between 25 and 33 g / L.

[0093] 2.4 Pasteurization of Sample A8

[0094] Potassium acetate and sucrose were added to the sample to achieve final concentrations of 1.2–1.6 mol / L and 1.7–2.1 mol / L, respectively. The sample was then stirred in a water bath (set to 30°C) to dissolve the solids. The sample that was completely dissolved and incubated in a 60°C water bath for at least 10 hours was designated as A9.

[0095] 2.5. Ultrafiltration concentration and preparation / dispensing

[0096] After incubating in a water bath for 10 hours, sample A9 was taken out and diluted with buffer B6. The pH of sample A10 after 4-fold dilution was measured to be between 6.7 and 7.7.

[0097] Sample A10 was concentrated by ultrafiltration using buffer B7 (14–21 mmol / L sodium phosphate, 47–55 mmol / L NaCl, pH 6.7–7.7). After dialysis, A10 was concentrated to a target protein α1-AT concentration of 25–33 g / L, and the sample was designated A11.

[0098] 1.3.8 Freeze-drying and dry heat inactivation

[0099] Sample A11 was freeze-dried with mannitol, glycine, histidine and other protective agents, and then further subjected to dry heat inactivation at 100±2℃ for 60 to 90 minutes.

[0100] Example 2: Sample testing and process repeatability verification at each step

[0101] 1. Comparative Analysis of Raw Materials for the Preparation of α1-AT

[0102] We simultaneously detected the antigen content of α1-AT in the low-temperature ethanol-based FIV precipitate and the starting material FT, and detected the corresponding main protein antigen content in the starting materials Pg, Fg, IgG, and ALB. We calculated the step recoveries of α1-AT, Pg, Fg, IgG, and ALB respectively, and evaluated the feasibility of sequentially extracting Pg, Fg, IgG, ALB, and α1-AT from human plasma.

[0103] The results show:

[0104] (1) The average recovery rate of α-AT from the starting material after three batches of low-temperature ethanol extraction of FIV precipitate was 52.429%.

[0105] (2) The average recovery rate of α1-AT in the starting material after extracting Pg, Fg, IgG and Alb by the chromatography method of Example 1 was 90.392%.

[0106] (3) The target proteins Pg and Fg were extracted sequentially by chromatography and used as the starting materials for albumin and globulin. The recovery rates of IgG and Alb antigens were both >95%.

[0107] Table 3. α1-AT recovery rates of different starting materials

[0108]

[0109]

[0110] 2. Chromatographic steps for impurity removal analysis

[0111] The contents of α1-AT and other impurities in the starting raw materials FT, CM FF ion exchange FT, and DEAE FF ion exchange flow-through solution (Elution, Elu) were determined respectively. The relative purity of α1-AT in the samples of each step (the ratio of α1-AT content to the total protein content of the sample) was calculated, and the CV value of the relative purity of α1-AT in the three batches of preparation process was calculated.

[0112] Content detection results show:

[0113] (1) α1-AT accounts for 15% to 25% of the starting materials, and the standard deviation (CV) of α1-AT antigen content in the three batches of raw materials is 9.862%.

[0114] (2) The average percentage of α1-AT in the CM FF flow-through solution was 54.971%, and the standard deviation (CV) of α1-AT antigen content in the three batches of CM FF flow-through solutions was 3.331%.

[0115] (3) The average relative purity of α1-AT after concentration of DEAE FF eluent was 99.572%, and the standard deviation (CV) of α1-AT antigen content in the three batches of concentrated eluent was 0.215%.

[0116] Table 4. Relative purity of α1-AT collected by two-step chromatography

[0117]

[0118] 3. Chromatographic Steps: Antigen and Protein Recovery Analysis. Antigen content is also protein content, referring to the content of a specific protein. The detection method is immunoturbidimetry. Total antigen content refers to the sum of the antigen content of all measurable proteins in the sample detected by immunoturbidimetry. Total protein content is determined by the BCA method to detect the total protein content of the sample.

[0119] The recovery rates of the α1-AT antigen preparation steps in three batches were detected and calculated, and the CV values ​​of the recovery rates for the three batches were calculated. The yield of α1-AT per ton of plasma was evaluated based on the overall recovery rate of the three batches of α1-AT antigen.

[0120] The results showed that the average antigen recovery rates of the two-step ion exchange steps were 76.445% and 74.816%, respectively, and the CV values ​​of the antigen recovery rates of the three batches were less than 6%. The three batches of α1-AT product from 1000L of plasma yielded 400.4g, 461.8g, and 496.4g, respectively.

[0121] 4. Specific activity analysis

[0122] The potency and total protein content of α1-AT in three batches prepared by the key process were detected. The specific activity of α1-AT was calculated, and the ratio of α1-AT potency to total protein content is the specific activity. The coefficient of variation (CV) of the specific activity for the three batches was also calculated. The impact of each step on the activity of α1-AT was evaluated based on the trend of the specific activity of α1-AT in the three batches.

[0123] The results showed that the mean specific activity of α1-AT in the three batches of key steps were as follows: human plasma 0.02, starting material 0.043, acid precipitation 0.235, CM FF 0.736, DEAE FF 1.235, pasteurization 1.131, lyophilization 1.108, and dry heat 1.075. The specific activity CV of the three batches of key steps was less than 10%. The specific activity trend plot showed that the two-step ion exchange increased the specific activity of α1-AT, while the specific activity of pasteurization and lyophilization / dry heat decreased slightly. The specific activity of α1-AT in the finished product was 54 times higher than that in human plasma. Figure 2 ).

[0124] 5. Verification of virus inactivation effect

[0125] Porcine pseudorabies virus (PRV) or porcine parvovirus (PPV) was added to the samples before inactivation for inactivation. The change in virus titer before and after inactivation was detected to verify the effectiveness of pasteurization inactivation of lipid-enveloped virus PRV and dry heat inactivation of non-lipid-enveloped virus PPV.

[0126] The results are as follows Figure 3 , Figure 4 ,

[0127] Pasteurization can effectively inactivate PRV, with a viral titer reduction of ≥4.91 Lg TCID50 / 0.1 ml;

[0128] Dry heat inactivation can effectively inactivate PPV, reducing the viral titer to ≥4.17 LgTCID50 / 0.1 ml.

[0129] 6. Stability analysis of finished product valence

[0130] Accelerated stability studies were conducted on samples after dry heat inactivation. The samples were placed in a 25°C stability chamber and samples were taken in January, March and June to detect the potency of α1-AT. The stability of the α1-AT product prepared by the α1-AT preparation process was evaluated by comparing the potency at 0 months with that at 0 months.

[0131] The test results showed that the potency recovery rates of the three batches of finished products were 95.009%, 91.198%, and 98.863% respectively after being stored for 6 months.

[0132] Table 5. Stability test results

[0133]

[0134] Finally, it should be noted that the above embodiments are only used to help those skilled in the art understand the essence of the present invention, and are not intended to limit the scope of protection of the present invention.

Claims

1. A method for extracting human α1-antitrypsin (α1-AT) from plasma using a full chromatography method. The method comprises the following steps: (1) The starting material is human plasma or flow-through after extracting proteins Pg, Fg, IgG and Alb from human plasma by chromatography. The starting material is concentrated by ultrafiltration and the replacement buffer is B1 (4.8-5.2 mmol / L Tris, pH 7.5-8.3). (2) Adjust the sample obtained in step (1) with adjustment buffer B2 (0.93-1.50 mmol / L NaOAc, pH 5.0-5.4) to make the final NaOAc content in the sample 20-32 mmol / L, and adjust the pH of the sample to 5.0-5.4 with acid or base. Let the adjusted A2 sample stand for 60-120 min, preferably 90 min. (3) The sample was rinsed with buffer B3 (20-32 mmol / L NaOAc, pH 5.0-5.4), and then the sample was collected after clarification and filtration as the loading sample for CM FF chromatography. (4) CMFF cation exchange chromatography: After the packed CMFF cation exchange chromatography column is equilibrated with buffer B3, the sample is loaded and purified by chromatography. The flow-through peak sample A4 is collected, and the pH and conductivity of sample A4 are detected and recorded. After the flow-through peak sample A4 is collected to the baseline, the chromatography column is eluted with buffer B4 (20-32 mmol / L NaOAc, 0.8-1.1 mol / L NaCl, pH 5.0-5.6), and the elution peak sample A5 is collected. (6) DEAE FF ion exchange chromatography: The DEAE FF chromatography column was equilibrated with buffer B3, sample A4 was loaded, and the flow-through sample was collected and recorded as A6; the chromatography column was further washed with buffer B5 (20-32 mmol / L NaOAc, 0.07-0.11 mol / L NaCl, pH 5.0-5.4) and the sample was collected and recorded as A7; (7) Sample A7 ultrafiltration concentration: Use buffer B6 for ultrafiltration concentration to concentrate to a protein content of 25-33 g / L; the concentrated sample is recorded as A8, which is α1-antitrypsin.

2. The method according to claim 1, characterized in that, The method further includes the following steps: (8) Pasteurization of α1-antitrypsin (sample A8): Add potassium acetate and sucrose to the sample to make their final contents 1.2-1.6 mol / L and 1.7-2.1 mol / L, respectively, and stir in a 30°C water bath to dissolve the solids. After the sample is completely dissolved, incubate in a 60°C water bath for at least 10 hours; (9) Ultrafiltration concentration and preparation for dispensing: The inactivated sample was diluted with buffer B6, and after a 4-fold dilution, the pH was 6.7–7.7; further ultrafiltration concentration was performed using buffer B7 (14–21 mmol / L sodium phosphate, 47–55 mmol / L NaCl, pH 6.7–7.7). The concentration was increased until the α1-antitrypsin (α1-AT) content was 25–33 g / L. (10) Freeze-dry and dry heat inactivate, add mannitol, glycine, histidine and other protective agents for freeze-drying, and further dry heat inactivate at 100±2℃ for 60 to 90 minutes.

3. The method according to claim 1 or 2, characterized in that, The pH of the sample obtained after concentration in step (1) should be in the range of 7.5 to 8.3, and the conductivity should be less than 1 mS / cm. The conductivity of the sample after adjustment in step (2) is no greater than 5.0 mS / cm.

4. The method according to any one of claims 1-3, characterized in that, When the starting material mentioned in step (1) is the flow-through liquid after extracting proteins Pg, Fg, IgG and Alb from human plasma by chromatography, the proportion (relative purity) of α1-AT in the starting material is 15% to 25%; after the treatment of steps (1)-(4), the relative purity of α1-AT in the flow-through liquid A4 is >50%. In the product of step (7), the average relative purity of α1-AT is >99%.

5. The method according to any one of claims 1-4, characterized in that, The amount of α1-AT prepared by the method, when the starting material is the flow-through liquid after extracting proteins Pg, Fg, IgG and Alb from human plasma by chromatography, the average recovery rate of α1-AT in the sample is >90%. The method yields more than 400g of α1-AT per 1000L of plasma. In each step of the method, the specific activity of the target protein α1-AT is as follows: approximately 0.02 in human plasma, approximately 0.04 in starting material, approximately 0.2-0.3 after adjustment (acid precipitation) of starting material, >0.7 in sample A5, >1.2 in sample A7, >1.1 after pasteurization, and >1 after lyophilization and / or dry heat inactivation.

6. Use of the method of any one of claims 1-5 in the preparation of a medicament or pharmaceutical composition, wherein the medicament or pharmaceutical composition comprises, a therapeutically effective amount of human α1-antitrypsin (α1-AT), and necessary pharmaceutical excipients.

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