A method for preparing a human coagulation factor IX stock solution
The preparation process of human coagulation factor IX stock solution was simplified by using A-50 gel adsorption combined with S/D inactivating agent, which solved the problems of complex steps and high residue in the existing technology and achieved efficient and safe production.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- SHENZHEN WEIGUANG BIOLOGICAL PROD
- Filing Date
- 2026-02-10
- Publication Date
- 2026-05-19
AI Technical Summary
The preparation process of human coagulation factor IX stock solution in the existing technology is complicated and has a high residual amount of S/D inactivating agent, which leads to a decrease in product competitiveness and an increase in production costs.
The method of using A-50 gel adsorption combined with S/D inactivator reduces S/D inactivator residue through multiple washing and inactivation steps, simplifies subsequent purification steps, and allows direct affinity chromatography, thereby reducing production costs and improving efficiency.
It significantly reduced the residual amounts of tributyl phosphate and polysorbate 80, increased the specific activity of human coagulation factor IX, simplified the production process, reduced costs, and improved product safety.
Smart Images
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Abstract
Description
Technical Field
[0001] This invention relates to the field of biopharmaceutical technology, specifically to a method for preparing human coagulation factor IX stock solution. Background Technology
[0002] Hemophilia B, also known as Christmas disease, is an X-linked recessive inherited bleeding disorder caused by mutations in the factor IX gene, leading to reduced or absent factor IX activity. The clinical manifestations of hemophilia B are primarily related to bleeding tendency due to factor IX deficiency, and its severity is closely related to the level of factor IX activity in the individual.
[0003] Currently, clinical treatment for hemophilia B mainly includes replacement therapy, preventive therapy, inhibitor management, and gene therapy. Replacement therapy, as the primary and effective treatment, has evolved from early prothrombin complex concentrate injections to blood-derived coagulation factors, reducing the incidence of thrombosis. Domestically produced blood-derived human coagulation factor IX has demonstrated good efficacy and safety in multicenter phase III clinical trials, with no viral infection or inhibitor production observed.
[0004] In my country, the separation and purification process is currently widely used to obtain human coagulation factor IX from human plasma. In the preparation process, virus inactivation is required to ensure the safety of the product. This process often uses organic solvent / detergent mixtures, i.e., S / D inactivating agents (including tributyl phosphate and polysorbate 80), for inactivation. Controlling the residual amount of S / D inactivating agents is urgent.
[0005] Existing methods for removing the S / D inactivating agent during the preparation of human coagulation factor IX stock solution include: one method involves removing most of the tributyl phosphate and polysorbate 80 residues from the initial pure human coagulation factor IX suspension via ultrafiltration or direct chromatography after S / D inactivation, followed by ultrafiltration to prepare the stock solution. For example, Chinese invention patent application CN110257358A, "A Production Method of High-Purity Human Coagulation Factor IX Preparation," obtains the stock solution via affinity chromatography after S / D inactivation. However, this method only involves one affinity chromatography purification step after S / D inactivation, resulting in a high residual level of tributyl phosphate and polysorbate 80 in the finished product, significantly reducing its market competitiveness.
[0006] Another method involves purifying the stock solution after inactivation with an S / D inactivating agent through a complex multi-step chromatography and ultrafiltration process. For example, Chinese invention patent application CN111378029A, entitled "A method for preparing high-stability, high-purity human coagulation factor IX", involves obtaining the stock solution after inactivation with an SD agent through anion exchange chromatography, heparin affinity chromatography, hydrophobic chromatography, and ultrafiltration. This method is cumbersome and has a low recovery rate. Patent application CN115975997A, entitled "A secondary ultrafiltration dialysate and purification method for human coagulation factor IX", involves obtaining the stock solution after inactivation with an SD agent through secondary anion exchange chromatography, ultrafiltration dialysis, re-affinity chromatography, and ultrafiltration. This method also involves many steps and is cumbersome, resulting in a low recovery rate. Summary of the Invention
[0007] To address the shortcomings of existing technologies, the present invention aims to provide a method for preparing human coagulation factor IX stock solution, thereby solving the problems of complex production processes and high residual S / D inactivating agent levels in the preparation of human coagulation factor IX stock solution in existing technologies.
[0008] The technical problem to be solved by the present invention is achieved through the following technical solution:
[0009] A method for preparing human coagulation factor IX stock solution, comprising the following steps:
[0010] S1. Combined plasmas were centrifuged and filtered to obtain the supernatant, and after adjusting the pH, A-50 gel adsorption was performed.
[0011] S2. After washing with A-50 washing solution, retain the washing solution, add the protectant and S / D inactivator, and stir to inactivate; S3. Wash again with A-50 washing solution, then add A-50 elution solution to elute, and combine the eluents;
[0012] S4. After chromatographic washing and elution, ultrafiltration was used to prepare human coagulation factor IX stock solution.
[0013] Further, step S2 specifically includes: draining the adsorbed plasma suspension obtained in S1, adding A-50 washing buffer at 5-15% w / v of the plasma volume, stirring and reacting, draining the washing buffer, repeating the operation 2-4 times, and retaining the last washing buffer; adding an inactivating protective agent to the retained washing buffer, followed by adding an S / D inactivating agent, and continuing to stir and inactivate for 6-9 hours. The inactivating protective agent and the S / D inactivating agent are added at a weight of 1 / 10-1 / 9 of the washing buffer weight.
[0014] Further, step S3 specifically includes: after the inactivation time is completed, drain the washing solution, add A-50 washing solution at 5-15% w / v of the plasma volume, stir to react, drain the washing solution, and repeat the operation 2-4 times; add A-50 elution solution, stir to react, drain the elution solution, repeat the elution, and combine the elution suspensions.
[0015] After washing 3-5 times in step S2, most of the impurities in the plasma that cannot be adsorbed by the A-50 gel have been removed, significantly reducing the total protein content in the suspension and preventing poor inactivation of the S / D inactivating agent due to excessive protein content. The S / D inactivating agent is added directly to the retained washing buffer for inactivation. During the washing and elution process in step S3, most of the S / D inactivating agent is removed by the washing buffer, reducing the need for separate inactivation and ultrafiltration steps, and simultaneously reducing the impact of the S / D inactivating agent on the lifespan of the expensive affinity chromatography packing material. In the subsequent S4, affinity chromatography can be performed directly after a simple dilution operation.
[0016] Furthermore, after each addition of A-50 washing solution, the reaction time should be 5-15 minutes.
[0017] Furthermore, the A-50 washing solution contains 5-20 mmol / L sodium citrate and 100-140 mmol / L sodium chloride, has a pH of 6.8-7.2, a conductivity of 12.00-19.00 mS / cm, and a temperature of 0.0-6.0℃.
[0018] Furthermore, the inactivating protectant contains 10-30 mmol / L sodium citrate and 40-60 mmol / L sucrose.
[0019] Furthermore, the S / D inactivating agent contains 3.0–3.5% w / v tributyl phosphate, 10–12% w / v polysorbate 80, and 10–30 mmol / L sodium citrate.
[0020] Furthermore, the inactivation time by stirring is 6-9 hours.
[0021] Furthermore, the amount of A-50 eluent added is 1-5% w / v of the plasma volume.
[0022] Furthermore, the A-50 eluent contains 10-20 mmol / L sodium citrate and 2 mol / L sodium chloride, with a pH of 6.9 ± 0.2 and a temperature of 0.0-6.0 °C.
[0023] Furthermore, in step S4, the affinity chromatography packing material is IX Select or HiScreen IX Select;
[0024] The affinity chromatography equilibration buffer contains 10-50 mmol / L of tris(hydroxymethyl)aminomethane and 100-200 mmol / L of sodium chloride, with a pH of 6.9 ± 0.2; preferably, it contains 20 mmol / L of tris(hydroxymethyl)aminomethane and 150 mmol / L of sodium chloride, with a pH of 6.9.
[0025] The affinity chromatography eluent contains 10-50 mmol / L of tris(hydroxymethyl)aminomethane, 2.0 mol / L of magnesium chloride, and a pH of 6.9 ± 0.2; preferably, it contains 20 mmol / L of tris(hydroxymethyl)aminomethane, 2 mol / L of magnesium chloride, and a pH of 6.9.
[0026] The ultrafiltration dialysis buffer contains 15-25 mmol / L sodium citrate, 200-300 mmol / L sodium chloride, 3-10 g / L arginine hydrochloride and 3-10 g / L lysine hydrochloride, with a pH of 7.0±0.1 and a temperature of 0-6℃; preferably, it contains 18 mmol / L sodium citrate, 250 mmol / L sodium chloride, 5 g / L arginine hydrochloride and 5 g / L lysine hydrochloride, with a pH of 7.0 and a temperature of 4℃.
[0027] The present invention has the following beneficial effects:
[0028] The preparation method of human coagulation factor IX stock solution of the present invention significantly reduces production steps, lowers production costs, and improves production efficiency. Using the preparation method of the present invention, tributyl phosphate is not detected in the stock solution, and the residual amount of polysorbate 80 is no higher than 5 μg / ml, greatly improving the safety of the product. At the same time, the specific activity of human coagulation factor IX is no less than 600 IU / mg, significantly higher than that of traditional methods. Detailed Implementation
[0029] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.
[0030] The present invention will be further illustrated by the following embodiments.
[0031] Example 1
[0032] This embodiment provides a method for preparing human coagulation factor IX stock solution, specifically including the following steps:
[0033] S1. Combine the plasmas, centrifuge and filter to obtain the supernatant, and add hydrochloric acid or sodium hydroxide solution to the supernatant to adjust the pH of the supernatant to 7.0;
[0034] Weigh 2g of A-50 dry gel per kilogram of plasma, add A-50 swelling solution to swell for 24 hours, drain the swelling solution, add A-50 equilibration solution, stir evenly, drain the equilibration solution, repeat the equilibration operation until the pH of the drained A-50 equilibration solution is 7.0, add supernatant and adsorb for 30 minutes to obtain the adsorbed plasma suspension.
[0035] S2. After draining the adsorbed plasma suspension, add the prepared A-50 washing solution at 10% w / v of the plasma volume, stir and react for 10 min, drain the washing solution, repeat the operation 3 times, and retain the washing solution from the 3rd time.
[0036] Add the inactivating protective agent to the remaining washing solution at 1 / 9 of the washing solution weight, then add the S / D inactivating agent at 1 / 10 of the washing solution weight, and continue stirring for 6 hours to inactivate.
[0037] S3. After the inactivation time is over, drain the washing solution and add the prepared A-50 washing solution again at 10% of the plasma volume. Stir and react for 10 minutes, drain the washing solution, and repeat the operation 3 times.
[0038] Add A-50 elution buffer at 3% w / v of plasma volume, stir and react for 10 min, drain the elution buffer, repeat elution 3 times, and combine the 3 elution suspensions;
[0039] Dilute S4 and A-50 elution suspension by 1:1 and directly load onto a well-equilibrated affinity chromatography column. Wash with 6 column volumes of affinity chromatography washing buffer. After washing, elute with 10 column volumes of affinity chromatography elution buffer to obtain human coagulation factor IX suspension. The human coagulation factor IX suspension is then prepared by ultrafiltration to obtain the stock solution.
[0040] The A-50 washing solution contains 10 mmol / L sodium citrate and 130 mmol / L sodium chloride, has a pH of 7.0, a conductivity of 15.00 mS / cm, and a temperature of 4.0℃.
[0041] The inactivating protectant contains 20 mmol / L sodium citrate and 50 mmol / L sucrose; the S / D inactivating agent contains 3.3% w / v tributyl phosphate, 11% w / v polysorbate 80 and 20 mmol / L sodium citrate;
[0042] The eluent contained 15 mmol / L sodium citrate and 2 mol / L sodium chloride, with a pH of 6.9 and a temperature of 4.0℃.
[0043] The affinity chromatography equilibration buffer contains 20 mmol / L tris(hydroxymethyl)aminomethane and 150 mmol / L sodium chloride, with a pH of 6.9;
[0044] The affinity chromatography eluent contains 20 mmol / L tris(hydroxymethyl)aminomethane, 2 mol / L magnesium chloride, and pH 6.9;
[0045] The ultrafiltration dialysis solution contained 18 mmol / L sodium citrate, 250 mmol / L sodium chloride, 5 g / L arginine hydrochloride and 5 g / L lysine hydrochloride, with a pH of 7.0 and a temperature of 4℃.
[0046] Example 2
[0047] This embodiment provides a method for preparing human coagulation factor IX stock solution, specifically including the following steps:
[0048] S1. Combine the plasmas, centrifuge and filter to obtain the supernatant, and add hydrochloric acid or sodium hydroxide solution to the supernatant to adjust the pH of the supernatant to 6.5;
[0049] Weigh 3g of A-50 dry gel per kilogram of plasma, add A-50 swelling solution to swell for 27 hours, drain the swelling solution, add A-50 equilibration solution, stir evenly, drain the equilibration solution, repeat the equilibration operation until the pH of the drained A-50 equilibration solution is 6.5, add supernatant and adsorb for 60 minutes to obtain the adsorbed plasma suspension.
[0050] S2. After draining the adsorbed plasma suspension, add the prepared A-50 washing solution at 5% w / v of the plasma volume, stir and react for 5 min, drain the washing solution, repeat the operation twice, and retain the second washing solution.
[0051] Add the inactivating protective agent to the remaining washing solution at 1 / 9 of the washing solution weight, then add the S / D inactivating agent at 1 / 10 of the washing solution weight, and continue stirring for 8 hours to inactivate.
[0052] S3. After the inactivation time is over, drain the washing solution and add the prepared A-50 washing solution again at 5% w / v of the plasma volume. Stir and react for 5 minutes, drain the washing solution, and repeat the operation 3 times.
[0053] Add A-50 elution buffer at 5% w / v of plasma volume, stir and react for 15 min, drain the elution buffer, repeat elution 4 times, and combine the 4 elution suspensions;
[0054] S4 and A-50 elution suspensions were diluted 2-fold and directly loaded onto a pre-equilibrated affinity chromatography column. The column was then washed with 3 column volumes of affinity chromatography washing buffer. After washing, the column was eluted with 6 column volumes of affinity chromatography elution buffer to obtain human coagulation factor IX suspension. The human coagulation factor IX suspension was then prepared by ultrafiltration to obtain the stock solution.
[0055] The A-50 washing solution contains 20 mmol / L sodium citrate and 140 mmol / L sodium chloride, has a pH of 7.2, a conductivity of 12.00 mS / cm, and a temperature of 1.0℃.
[0056] The inactivating protectant contains 10 mmol / L sodium citrate and 60 mmol / L sucrose; the S / D inactivating agent contains 3.0% w / v tributyl phosphate, 10% w / v polysorbate 80 and 30 mmol / L sodium citrate;
[0057] The eluent contained 10 mmol / L sodium citrate and 2 mol / L sodium chloride, with a pH of 6.7 and a temperature of 6.0℃.
[0058] The affinity chromatography equilibration buffer contains 10 mmol / L of tris(hydroxymethyl)aminomethane and 200 mmol / L of sodium chloride, with a pH of 6.9.
[0059] The affinity chromatography eluent contains 50 mmol / L tris(hydroxymethyl)aminomethane, 2 mol / L magnesium chloride, and pH 7.1.
[0060] The ultrafiltration dialysis solution contained 25 mmol / L sodium citrate, 300 mmol / L sodium chloride, 3 g / L arginine hydrochloride and 10 g / L lysine hydrochloride, with a pH of 7.1 and a temperature of 6℃.
[0061] Example 3
[0062] This embodiment provides a method for preparing human coagulation factor IX stock solution, specifically including the following steps:
[0063] S1. Combine the plasmas, centrifuge and filter to obtain the supernatant, and add hydrochloric acid or sodium hydroxide solution to the supernatant to adjust the pH of the supernatant to 7.5;
[0064] Weigh 1g of A-50 dry gel per kilogram of plasma, add A-50 swelling solution to swell for 30 hours, drain the swelling solution, add A-50 equilibration solution, stir evenly, drain the equilibration solution, repeat the equilibration operation until the pH of the drained A-50 equilibration solution is 7.5, add supernatant and adsorb for 90 minutes to obtain the adsorbed plasma suspension.
[0065] S2. After draining the adsorbed plasma suspension, add the prepared A-50 washing solution at 15% w / v of the plasma volume, stir and react for 8 min, drain the washing solution, repeat the operation 4 times, and retain the washing solution from the 4th time.
[0066] Add the inactivating protective agent to the remaining washing solution at 1 / 10 of the washing solution weight, then add the S / D inactivating agent at 1 / 9 of the washing solution weight, and continue stirring for 9 hours to inactivate.
[0067] S3. After the inactivation time is over, drain the washing solution and add the prepared A-50 washing solution again at 15% w / v of the plasma volume. Stir and react for 8 minutes, drain the washing solution, and repeat the operation 3 times.
[0068] Add A-50 elution buffer at 1% w / v of plasma volume, stir for 5 min, drain the elution buffer, repeat elution twice, and combine the two elution suspensions;
[0069] Dilute S4 and A-50 elution suspension by 1:1 and directly load onto a well-equilibrated affinity chromatography column. Wash with 10 column volumes of affinity chromatography washing buffer. After washing, elute with 3 column volumes of affinity chromatography elution buffer to obtain human coagulation factor IX suspension. The human coagulation factor IX suspension can be prepared by ultrafiltration to obtain the stock solution.
[0070] The A-50 washing solution contains 5 mmol / L sodium citrate and 100 mmol / L sodium chloride, has a pH of 6.8, a conductivity of 19.00 mS / cm, and a temperature of 6.0℃.
[0071] The inactivating protectant contains 30 mmol / L sodium citrate and 40 mmol / L sucrose; the S / D inactivating agent contains 3.5% w / v tributyl phosphate, 12% w / v polysorbate 80 and 10 mmol / L sodium citrate;
[0072] The eluent contained 20 mmol / L sodium citrate and 2 mol / L sodium chloride, with a pH of 7.1 and a temperature of 1.0℃.
[0073] The affinity chromatography equilibration buffer contains 50 mmol / L tris(hydroxymethyl)aminomethane and 100 mmol / L sodium chloride, with a pH of 6.9;
[0074] The affinity chromatography eluent contains 10 mmol / L tris(hydroxymethyl)aminomethane, 2 mol / L magnesium chloride, and pH 6.7.
[0075] The ultrafiltration dialysate contained 15 mmol / L sodium citrate, 200 mmol / L sodium chloride, 10 g / L arginine hydrochloride and 3 g / L lysine hydrochloride, with a pH of 6.9 and a temperature of 1℃.
[0076] Comparative Example 1
[0077] This comparative example illustrates a method for preparing human coagulation factor IX stock solution, specifically including the following steps:
[0078] (1) Combine the plasmas, centrifuge and filter to obtain the supernatant, add hydrochloric acid or sodium hydroxide solution to the supernatant to adjust the pH of the supernatant to 7.0;
[0079] Weigh 2g of A-50 dry gel per kilogram of plasma, add A-50 swelling solution to swell for 24 hours, drain the swelling solution, add A-50 equilibration solution, stir evenly, drain the equilibration solution, repeat the equilibration operation until the pH of the drained A-50 equilibration solution is 7.0, add supernatant and adsorb for 30 minutes to obtain the adsorbed plasma suspension.
[0080] (2) After the plasma suspension is drained, add the prepared A-50 washing solution at 10% w / v of the plasma volume, stir and react for 10 min, drain the washing solution, and repeat the operation 3 times.
[0081] (3) Add A-50 elution buffer at 3% w / v of plasma volume, stir and react for 10 min, drain the elution buffer, repeat the elution 3 times, and combine the 3 elution suspensions;
[0082] (4) The eluent is pre-concentrated by 1 / 4, and then dialyzed with chromatographic equilibration buffer 4 to 6 times to obtain the pre-inactivation suspension;
[0083] (5) Add the inactivating protective agent to the pre-inactivation suspension at 1 / 9, then add the S / D inactivating agent at 1 / 10, and continue stirring for 6 hours to inactivate;
[0084] (6) After equilibrating the chromatography column with affinity chromatography equilibration buffer, perform chromatography purification; wash with affinity chromatography wash solution for 6 column volumes, and elute with affinity chromatography elution solution for 10 column volumes to obtain human coagulation factor IX suspension; human coagulation factor IX suspension is prepared by ultrafiltration to obtain the original solution.
[0085] Comparative Example 2
[0086] This comparative example illustrates a method for preparing human coagulation factor IX stock solution, specifically including the following steps:
[0087] (1) Combine the plasmas, centrifuge and filter to obtain the supernatant, add hydrochloric acid or sodium hydroxide solution to the supernatant to adjust the pH of the supernatant to 7.0;
[0088] Weigh 2g of A-50 dry gel per kilogram of plasma, add A-50 swelling solution to swell for 24 hours, drain the swelling solution, add A-50 equilibration solution, stir evenly, drain the equilibration solution, repeat the equilibration operation until the pH of the drained A-50 equilibration solution is 7.0, add supernatant and adsorb for 30 minutes to obtain the adsorbed plasma suspension.
[0089] (2) After the plasma suspension is drained, add the prepared A-50 washing solution at 10% w / v of the plasma volume, stir and react for 10 min, drain the washing solution, and repeat the operation 3 times.
[0090] (3) Add A-50 elution buffer at 3% w / v of plasma volume, stir and react for 10 min, drain the elution buffer, repeat the elution 3 times, and combine the 3 elution suspensions;
[0091] (4) The eluent is pre-concentrated by 1 / 4, and then dialyzed with chromatographic equilibration buffer 4 to 6 times to obtain the pre-inactivation suspension;
[0092] (5) Add the inactivating protective agent to the pre-inactivation suspension at 1 / 9, then add the S / D inactivating agent at 1 / 10, and continue stirring for 6 hours to inactivate;
[0093] (6) Dilute the SD inactivation solution to a level close to that of the DEAE equilibration buffer before loading the sample;
[0094] (7) After loading the sample, wash the column with DEAE equilibration buffer to level the baseline, and then wash the column with washing buffer.
[0095] (8) Elute with elution buffer and collect the DEAE chromatography eluent;
[0096] (9) Dilute the elution buffer from step (8) with heparin equilibration buffer and adjust the pH to 7.2 with 1M hydrochloric acid before loading the heparin column;
[0097] (10) After loading the sample, wash the chromatography column with heparin washing buffer, then elute with elution buffer and collect the heparin chromatography eluent.
[0098] (11) Add 1 times the conductivity adjustment solution to the heparin chromatography eluent to adjust the conductivity, and then load it onto a hydrophobic chromatography column;
[0099] (12) After the sample loading is completed, the chromatography column is rinsed with hydrophobic chromatography equilibration buffer and the hydrophobic chromatography flow-through and rinsing buffer are collected. The hydrophobic chromatography collection is ultrafiltered and dialyzed through a 10kD ultrafiltration membrane to obtain the ultrafiltration dialysis collection. The ultrafiltration dialysis collection is then filtered through a deep filter to obtain the original solution.
[0100] The total process time, specific activity of human coagulation factor IX, and residual amounts of tributyl phosphate and polysorbate 80 were measured for Examples 1 and Comparative Examples 1 and 2, respectively. The total process time was obtained by comparing the time taken for each step before and after the process. Potency: A single-stage method was used. Protein content: The Lowry method (Chinese Pharmacopoeia 2025 Edition, Part III, General Chapter 0731, Method II, Lowry Method) was used to determine protein content. Specific activity = potency / protein content. The residual amount of tributyl phosphate was determined by gas chromatography (Chinese Pharmacopoeia 2025 Edition, Part III, General Chapter 3205, Determination of Tributyl Phosphate Residue), and the residual amount of polysorbate 80 was determined by colorimetry (Chinese Pharmacopoeia 2025 Edition, Part III, General Chapter 3203, Determination of Polysorbate 80 Residue). The results are shown in Tables 1, 2, and 3, respectively.
[0101] Table 1. Overall potency recovery rate and original solution detection results for each step in the example.
[0102]
[0103] *The detection limit is 1 μg / ml.
[0104] Table 2. Overall potency recovery rate and original solution detection results for each step in Comparative Example 1.
[0105]
[0106] Table 3. Overall potency recovery rate and original solution detection results for each step in Comparative Example 2.
[0107]
[0108] The results of Example 1 (see Table 1) and Comparative Example 1 (see Table 2) of this invention show that: by inactivating the virus during the A-50 gel adsorption and washing process, this invention can not only fully inactivate the virus, but also significantly reduce the residual amounts of tributyl phosphate and polysorbate 80, and obtain a human coagulation factor IX product with higher specific activity. The results of Example 1 (see Table 1) and Comparative Example 2 (see Table 3) of this invention show that by inactivating the virus during the A-50 gel adsorption and washing process, this invention not only significantly reduces process steps, working time, and production costs, but also achieves a total recovery rate of no less than 74%, far exceeding the approximately 25% recovery rate of Comparative Example 2.
[0109] The above embodiments merely illustrate the implementation of the present invention, and their descriptions are relatively specific and detailed. However, they should not be construed as limiting the scope of the present invention. Any technical solutions obtained by adopting equivalent substitutions or equivalent transformations should fall within the protection scope of the present invention.
Claims
1. A method for preparing human coagulation factor IX stock solution, characterized in that, Includes the following steps: S1. Combined plasmas were centrifuged and filtered to obtain the supernatant, and after adjusting the pH, A-50 gel adsorption was performed. S2. After washing with A-50 washing solution, retain the washing solution, add inactivating protectant and S / D inactivating agent, and stir to inactivate; S3. Add A-50 washing solution again to wash, then add A-50 elution solution to elute, and combine the eluents; S4. After chromatographic washing and elution, ultrafiltration was used to prepare human coagulation factor IX stock solution; Step S2 specifically includes: draining the adsorbed plasma suspension obtained in S1, adding A-50 washing solution at 5-15% w / v of the plasma volume, stirring and reacting, draining the washing solution, repeating the operation 2-4 times, and retaining the last washing solution; adding an inactivating protectant to the retained washing solution, then adding S / D inactivating agent, and continuing to stir and inactivate for 6-9 hours. The inactivating protective agent comprises 10-30 mmol / L sodium citrate and 40-60 mmol / L sucrose; The A-50 washing solution contains 5-20 mmol / L sodium citrate and 100-140 mmol / L sodium chloride, has a pH of 6.8-7.2, a conductivity of 12.00-19.00 mS / cm, and a temperature of 1.0-6.0℃. The S / D inactivating agent comprises 3.0-3.5% w / v tributyl phosphate, 10-12% w / v polysorbate 80 and sodium citrate 10-30 mmol / L; The A-50 eluent contains 10-20 mmol / L sodium citrate and 2 mol / L sodium chloride, with a pH of 6.9±0.2 and a temperature of 1.0-6.0℃; Step S4 specifically includes: diluting the A-50 elution suspension 1-2 times, directly loading it onto a pre-equilibrated affinity chromatography column, and washing with 3-10 column volumes of affinity chromatography washing buffer; after washing, eluting with 3-10 column volumes of affinity chromatography elution buffer to obtain human coagulation factor IX suspension; the human coagulation factor IX suspension is then prepared by ultrafiltration to obtain the stock solution; The ultrafiltration dialysis solution contains 15-25 mmol / L sodium citrate, 200-300 mmol / L sodium chloride, 3-10 g / L arginine hydrochloride and 3-10 g / L lysine hydrochloride, with a pH of 7.0±0.1 and a temperature of 1.0-6℃. The affinity chromatography equilibration buffer contains 10–50 mmol / L of tris(hydroxymethyl)aminomethane and 100–200 mmol / L of sodium chloride, with a pH of 6.
9. The affinity chromatography eluent contains 10–50 mmol / L of tris(hydroxymethyl)aminomethane, 2.0 mol / L of magnesium chloride, and a pH of 6.9 ± 0.
2.
2. The method for preparing human coagulation factor IX stock solution according to claim 1, characterized in that, Step S3 specifically includes: after the inactivation time is completed, drain the washing solution, add A-50 washing solution at 5-15% w / v of the plasma volume, stir to react, drain the washing solution, and repeat the operation 2-4 times; add A-50 elution solution, stir to react, drain the elution solution, repeat the elution, and combine the elution suspensions.
3. The method for preparing human coagulation factor IX stock solution according to claim 1, characterized in that, Each time A-50 washing solution is added, the reaction time is 5-15 minutes.
4. The method for preparing human coagulation factor IX stock solution according to claim 1, characterized in that, The amount of A-50 elution buffer added is 1-5% w / v of the plasma volume.