A method for extracting and separating IgM and coagulation factor VII from plasma

Abstract

The present invention relates to the technical field of blood product production, and specifically relates to a method for extracting and separating IgM and coagulation factor VII from plasma. The method comprises the following steps: after the cold glue plasma is adjusted in pH value and conductivity, it is loaded onto a first anion exchange chromatography column, and then sequentially eluted with a first eluent and a second eluent to obtain a first ion chromatography product; after the first ion chromatography product is ultrafiltered, dialyzed, and adjusted in pH value and conductivity, it is loaded onto a gel filtration chromatography column, and the flow-through solution corresponding to the first ultraviolet peak and the flow-through solution corresponding to the third ultraviolet peak are respectively collected to obtain a first gel filtration chromatography product containing IgM and a second gel filtration chromatography product containing coagulation factor VII. This technical solution can solve the technical problem that the existing preparation process cannot simultaneously purify and enrich IgM and human coagulation factor VII, and at the same time ensure the product quality, improve the utilization rate of plasma raw materials, and has ideal practical application value.

Description

Technical Field

[0001] The present invention relates to the technical field of blood product production, and particularly relates to a method for extracting and separating IgM and coagulation factor VII from plasma. Background Art

[0002] IgM exhibits extremely strong antigen neutralization and immune regulation effects in antibody-mediated humoral immune responses, etc. It is the first antibody produced by the body after anti-infection and immune stimulation. IgM is also the earliest antibody to appear in the primary humoral immune response and is the "vanguard" of collective infection. IgM accounts for 5% - 10% of the total serum immunoglobulin. Monomeric IgM is expressed on the cell surface in a membrane-bound form, constituting the B-cell antigen receptor. Secretory IgM is a pentamer and is the immunoglobulin with the largest molecular weight, mainly present in the blood and having a strong antigen-binding ability. IgM is the earliest synthesized and secreted antibody during individual development, and fetuses in the late stage of embryonic development can produce IgM. Therefore, an increase in cord blood IgM indicates intrauterine infection in the fetus. The existing immunoglobulin production technologies internationally mainly adopt methods such as the cold ethanol method and the combination of the cold ethanol method and chromatography technology, and all are for separating IgG, removing IgM, IgA, etc. as impurity proteins.

[0003] Coagulation factors are protein components involved in the blood coagulation process. There are more than a dozen coagulation factors involved in blood coagulation in plasma. Among them, human coagulation factor VII is the starting factor that triggers the coagulation cascade reaction in the extrinsic coagulation pathway and is a vitamin K-dependent zymogen with serine protease hydrolysis activity synthesized by hepatocytes. Human coagulation factor VII is mainly used clinically to treat coagulation factor VII deficiency, that is, hemophilia. Currently, the separation process of human coagulation factor VII mainly uses plasma as a raw material to first produce human prothrombin complex (PCC) and then purify it. This process is complex and has a low recovery rate.

[0004] Considering the differences in the performance of IgM and coagulation factors, to ensure the quality of IgM and coagulation factor products, in the existing technology, the separation and enrichment of the above two components are almost carried out separately, and there is no relevant report on an industrialized production process technology for simultaneously purifying the two substances. Summary of the Invention

[0005] The present invention aims to provide a method for extracting and separating IgM and coagulation factor VII from plasma to solve the technical problem that the existing preparation process cannot directly purify and enrich IgM and human coagulation factor VII from plasma simultaneously.

[0006] To achieve the above object, the present invention adopts the following technical scheme:

[0007] A method for extracting and separating IgM and coagulation factor VII from plasma, comprising the following steps carried out in sequence:

[0008] S1: After the cold glue-free plasma is adjusted in pH value and conductivity, it is loaded onto the first anion exchange chromatography column and then successively eluted with the first eluent and the second eluent to obtain the first ion chromatography product.

[0009] S2: After the first ion chromatography product is ultrafiltered, dialyzed, and adjusted in pH value and conductivity, it is loaded onto a gel filtration chromatography column. The flow-through solution corresponding to the first UV peak and the flow-through solution corresponding to the third UV peak are respectively collected to obtain the first gel filtration chromatography product containing IgM and the second gel filtration chromatography product containing coagulation factor VII.

[0010] The principle and beneficial effects of adopting the above technical solution are as follows:

[0011] The chromatography process separation method can be used for primary separation, medium separation, and fine separation. The chromatography separation has high precision, simple equipment, and convenient operation. According to different principles, chromatography methods are widely used in the separation and purification of proteins. Chromatography is an effective separation means and an important protein purification tool. With the continuous development of polymer chemistry, the gel chromatography process is more and more widely used in various aspects. The chromatography medium can be reused, has good separation effect and high repeatability, and the most prominent is the high recovery rate of samples. This technical solution uses the full chromatography method to separate and purify coagulation factor VII and IgM, realizes the simultaneous separation of two substances from plasma, and the obtained coagulation factor VII product and IgM product have ideal quality and meet the application requirements. This technical solution greatly improves the utilization rate of the hard-won plasma raw materials, simplifies the operation steps, and has great practical application value.

[0012] Regarding the differences in the performance of IgM and coagulation factors, to ensure the quality of IgM and coagulation factor products, in the prior art, the separation and enrichment of the above two components are almost carried out separately. During the continuous exploration process, the inventor found that when using chromatography to adsorb factors II, VII, IX, and X from plasma, immunoglobulin IgM can be adsorbed simultaneously, and it does not affect other protein components such as IgG and albumin, which are also immunoglobulins, in the flow-through solution. Factors II, VII, IX, and X can be used to prepare prothrombin complex (i.e., PCC). The inventor unexpectedly found in a large number of screening studies that by adopting the chromatography method of this technical solution, factors II, IX, and X can also be separated from factors VII and immunoglobulin IgM through the first anion exchange chromatography, and factors II, IX, and X are retained on the chromatography column. Factors II, IX, and X can be used to prepare high-concentration factor IX products, and factors VII and immunoglobulin IgM are further separated to prepare high-purity factor VII and IgM products respectively.

[0013] In the initial stage of the research, the inventor explored various methods for separating Factor VII from Factor II, Factor IX, Factor X, etc. Under the process sequence and conditions of adopting this technical solution, not only was the effective separation of Factor VII from Factor II, Factor IX, and Factor X achieved, but also Factor VII and IgM could be co-separated simultaneously, obtaining an unexpected technical effect. Next, the two can be further separated by the difference in molecular weight, and the operation process is simple.

[0014] Furthermore, it also includes S3-1: After the first gel filtration chromatography product is adjusted in pH and conductivity, it is loaded onto the second anion exchange chromatography column, and the flow-through liquid is collected to obtain the second ion chromatography product. And before loading, the second anion exchange chromatography column is equilibrated with the third equilibration solution; the third equilibration solution is 0.015 - 0.075 mol / L sodium acetate-acetic acid buffer solution with a pH value of 4.5 - 5.5.

[0015] Furthermore, in S3-1, the packing material of the second anion exchange chromatography column is an anion exchange chromatography packing material; the pH value of the first gel filtration chromatography product is adjusted to 4.5 - 5.5, and the conductivity is adjusted to 1.5 - 7.5 mS / cm, and then it is loaded.

[0016] Furthermore, in S3-1, the second ion chromatography product is ultrafiltered, dialyzed, and adjusted in concentration, pH, and conductivity, and sorbitol and glycine are added to obtain a crude IgM product; the crude IgM product is inactivated by pasteurization, and then ultrafiltered, dialyzed, and adjusted in concentration and pH, and maltose is added and filtered and sub-packed to obtain a finished IgM product.

[0017] Furthermore, it also includes S3-2: The second gel filtration chromatography product is ultrafiltered, dialyzed, and adjusted in concentration and pH, and glycine, arginine, and lysine hydrochloride are added to obtain a crude Factor VII product; the crude Factor VII product is sub-packed and then freeze-dried and heat-inactivated to obtain a finished Factor VII product.

[0018] Furthermore, in S1, the packing material of the first anion exchange chromatography column is an anion exchange packing material; before loading, the first anion exchange chromatography column is equilibrated with the first equilibration solution; the first equilibration solution is 10 - 20 mM sodium citrate with a pH value of 6.3 - 7.6 and a conductivity of 9 - 13 mS / cm.

[0019] Furthermore, in S1, the first eluent is 10 - 20 mM sodium citrate with a pH value of 6.3 - 7.6 and a conductivity of 15 - 30 mS / cm; the second eluent is 0.5 - 1 M sodium glutamate solution with a pH value of 6.8 - 7.7 and a conductivity of 19 - 30 mS / cm.

[0020] Further, in S1, the cold glue-removed plasma is adjusted to a pH value of 6.3 - 7.6 and a conductivity of 9 - 13 mS / cm, and then loaded onto the column.

[0021] Further, in S2, the packing material of the gel filtration chromatography column is Sephadex; before loading, the gel filtration chromatography column is equilibrated with a second equilibration solution; the second equilibration solution is a mixed solution containing 10 - 20 mM sodium citrate and 0.13 - 0.17 M sodium chloride, with a pH value of 7.2 - 8.0 and a conductivity of 14 - 20 mS / cm.

[0022] Further, in S2, the pH value of the first ion chromatography product is adjusted to 7.2 - 8.2, and the conductivity is adjusted to 14 - 20 mS / cm.

[0023] In summary, the beneficial effects of the present technical solution are as follows:

[0024] (1) The entire chromatography process has no low-temperature ethanol precipitation step, does not require low temperature, saves energy and reduces consumption; does not require ethanol, reduces safety risks, and improves the safety of the workshop;

[0025] (2) The entire chromatography process has simple operation, requires a single piece of equipment, the chromatography medium can be reused, has good separation effect, high repeatability, and most prominently, has high recovery rates of factor VII and IgM.

[0026] (3) The present technology combines means such as anion exchange and gel filtration in the separation and purification of factor VII, simplifies the separation of different proteins in plasma, and at the same time can greatly improve the utilization rate of the hard-won plasma raw materials, especially separating IgM and factor VII from plasma simultaneously.

[0027] (4) The specific activity of factor VII can reach 25 IU / mg protein, far higher than the 2 IU / mg protein specified in the European Pharmacopoeia.

[0028] (5) The purity of IgM protein can reach over 90%, and the total recovery rate of IgM is ideal. Description of the Drawings

[0029] Figure 1 It is a process flow chart for extracting and separating IgM and coagulation factor VII from plasma in Example 1.

[0030] Figure 2 It is a typical elution profile of the first anion exchange chromatography in Example 1.

[0031] Figure 3 It is a typical elution profile of the gel filtration chromatography in Example 1. Detailed Embodiments

[0032] The following further elaborates on the present invention in conjunction with embodiments, but the implementation manners of the present invention are not limited thereto. Unless otherwise specified, the technical means used in the following embodiments and experimental examples are conventional means well-known to those skilled in the art, and the materials, reagents, etc. used can all be obtained through commercial channels.

[0033] Example 1:

[0034] The overall process flow chart for extracting and separating IgM and coagulation factor VII from plasma is shown in Figure 1 , and the detailed process is as follows:

[0035] S1: Co-enrichment of IgM and coagulation factor VII

[0036] (1) Raw plasma treatment: After the raw plasma is taken out of the warehouse, the surface of the plasma bag is disinfected with a 70% - 75% ethanol solution, and then the plasma bag is broken. The temperature is controlled at 0 - 4°C for melting. After melting, the plasma is centrifuged (10000 rpm - 15000 rpm) using a centrifuge, and the supernatant is collected, which is the cold precipitate-free plasma. The plasma (raw plasma) used in this step is the human plasma referred to in the "Chinese Pharmacopoeia": The human plasma used for the production of blood products is the healthy human plasma collected by plasmapheresis for the production of plasma protein products. It is the supernatant after centrifuging blood to remove cells, containing proteins, inorganic salts, water, etc., and no blood cells.

[0037] (2) First anion exchange chromatography: By adjusting the pH value of the cold precipitate-free plasma to 6.3 - 7.6 and the conductivity to 9 - 13 mS / cm, after terminal filtration with a 0.22 μm filter element, the first anion exchange chromatography experiment is carried out. In the first anion exchange chromatography, the formula of the equilibration solution (the first equilibration solution) is 10 - 20 mM sodium citrate equilibration buffer, the pH value is adjusted to 6.3 - 7.6 using hydrochloric acid, and the conductivity is adjusted to 9 - 13 mS / cm using sodium chloride; the formula of the first eluent (eluent 1) is 10 - 20 mM sodium citrate equilibration buffer, the pH value is adjusted to 6.3 - 7.6 using hydrochloric acid, and the conductivity is adjusted to 15 - 30 mS / cm using sodium chloride. The formula of the second eluent (eluent 2) is 0.5 - 1 M sodium glutamate solution, the pH value is adjusted to 6.8 - 7.7 using hydrochloric acid, and the conductivity is adjusted to 19 - 30 mS / cm using sodium chloride, or it is 10 mM sodium citrate solution, the pH value is adjusted to 6.8 - 7.7 using hydrochloric acid, and the conductivity is adjusted to 19 - 30 mS / cm for elution. Through the first anion exchange chromatography, a protein solution containing coagulation factor VII and IgM is obtained for further purification.

[0038] The packing material that can be used for the first anion exchange chromatography is an anion exchange chromatography packing material (either a strong anion exchange chromatography packing material or a weak anion exchange chromatography packing material can be selected). Before use, equilibrate the chromatography column with the equilibration solution described above, and then load the cold plasma with adjusted pH and conductivity onto the chromatography column. The loading volume is 10 - 25 mL of cold plasma per mL of gel. After loading, elute the chromatography column with the first eluent (eluent 1). After completion, elute the chromatography column with the second eluent (eluent 2), and collect the flow-through of the second eluent (the first ion chromatography product), which contains factor VII and IgM. A typical elution profile is shown in Figure 2 .

[0039] (3) Ultrafiltration and dialysis: The first ion chromatography product is ultrafiltered using a 10KD ultrafiltration membrane package and dialyzed with 6 - 8 times the dialysis solution. Both ultrafiltration and dialysis are conventional methods in the art. The dialysis solution formula is 10 - 20 mM sodium citrate + 0.13 - 0.17 M sodium chloride, adjusted to pH 7.2 - 8.2 with hydrochloric acid, and adjusted to a conductivity of 14 - 20 mS / cm with sodium chloride to obtain the product after ultrafiltration and dialysis.

[0040] S2: Chromatographic separation of IgM and coagulation factor VII

[0041] (4) Gel filtration chromatography: The product after ultrafiltration and dialysis is processed using gel filtration chromatography. The column packing material is Sephadex - G200, and the column height is 30 - 60 cm. After equilibrating the chromatography column with the equilibration solution (the second equilibration solution), then load and perform chromatography. Among them, the equilibration solution is 10 - 20 mM sodium citrate + 0.13 - 0.17 M sodium chloride, adjusted to pH 7.2 - 8.0 with hydrochloric acid, and adjusted to a conductivity of 14 - 20 mS / cm with sodium chloride. Pre - equilibrate the gel packing material with 3 - 5 column volumes, and the loading volume is 3 - 8% of the gel packing material volume. When the first UV peak starts to rise to 150 mAU, start collecting the flow - through and stop collecting when the first UV peak ends. The collected product is the IgM - containing product (the first gel filtration chromatography product), as shown in Figure 3 . At the same time, collect the flow - through corresponding to the third UV peak, which is the product containing factor VII (the second gel filtration chromatography product), as shown in Figure 3 . The first gel filtration chromatography product and the second gel filtration chromatography product are respectively used for subsequent processing.

[0042] S3 - 1: Preparation of IgM product

[0043] (5) Second anion exchange chromatography of IgM: Take the product of the first gel filtration chromatography, further adjust the sample loading pH value to 4.5 - 5.5 and the conductivity to 1.5 - 7.5 mS / cm, and then perform the second anion exchange chromatography packing. Use the equilibration solution to equilibrate the anion exchange chromatography packing for 3 - 5 CVs and then load the sample, collect the flow-through fraction to obtain the second ion chromatography product. Among them, the formula of the equilibration solution (the third equilibration solution) is 0.015 - 0.075 mol / L sodium acetate - acetic acid buffer solution, with pH 4.5 - 5.5 and conductivity 1.5 - 7.5 mS / cm. The packing that can be used for the second anion exchange chromatography is anion exchange chromatography packing (preferably strong anion exchange chromatography packing).

[0044] (6) Ultrafiltration preparation: The second ion chromatography product is ultrafiltered using a 50KD ultrafiltration membrane package, dialyzed and concentrated with 6 - 10 times of injection water until the protein content reaches 120 - 140 g / L. Finally, prepare a crude IgM product with a protein concentration of 50 g / L, a sorbitol content of 30 - 50%, and a glycine content of 5 - 20%, and adjust the pH to 5.6 - 7.6 and the conductivity to 5 - 12 mS / cm.

[0045] (7) Pasteur inactivation: Treat the crude IgM product according to the conventional Pasteur inactivation method, with Pasteur inactivation at 60°C for 10 hours.

[0046] (8) Ultrafiltration preparation: Adjust the pH of the inactivated product to 4.30 with 1M acetic acid, filter it through a 0.22μm filter element at the end, ultrafilter and concentrate it using a 50KD ultrafiltration membrane package, and then dialyze and concentrate it with 10 - 20 times the volume of injection water until the protein content reaches 90 - 100 g / L. Then, prepare a product with a protein concentration of 50 g / L, a maltose content of 10%, adjust the pH to 4.0 - 4.3 with 1M acetic acid, and filter and dispense it through a 0.22μm filter element at the end.

[0047] S3-2: Preparation of coagulation factor VII product

[0048] (9) Ultrafiltration preparation of factor VII: Collect the product of the third ultraviolet peak of gel filtration (the second gel filtration chromatography product), ultrafilter and concentrate it using a 10KD ultrafiltration membrane package, and then dialyze it 3 - 5 times with the dialysis solution. Prepare according to a factor VII titer of 10 IU / ml, with a glycine addition of 1.5 - 3.5 g / L, arginine of 2.5 - 4.5 g / L, and hydrochloric acid lysine of 0.5 - 2.0 g / L, and adjust the pH of the product to 6.80 - 7.20.

[0049] (10) Dispensing of factor VII: Dispense at 200 IU / vial.

[0050] (11) Freeze-drying and dry heat inactivation of Factor VII: The filled Factor VII products are freeze-dried, stoppered, and capped, and then the samples are further placed in a heating cabinet for dry heat inactivation at 99.5°C ± 0.5°C for 30 - 35 minutes.

[0051] S4: Quality inspection

[0052] (12) Product quality inspection: The experimental equipment uses GE Healthcare Aavant150, GE Healthcare Pure150. The detection of IgM and Factor VII content uses the Beckman specific protein analysis system, the detection of total protein content uses the Yilanbei automatic biochemical analyzer, the Stago automatic coagulation analyzer, and the detection of protein content uses an ultraviolet spectrophotometer.

[0053] The calculation method of IgM purity is obtained by the ratio of the IgM content of the sample to the total protein content, and the IgM recovery rate is calculated according to the ratio of the IgM content in different experimental steps to the IgM sample before chromatography to obtain the IgM recovery rate of different steps.

[0054] The specific activity of Factor VII is obtained by the ratio of the Factor VII titer to the total protein content, and the Factor VII titer recovery rate is calculated according to the ratio of the Factor VII titer in different experimental steps to the Factor VII titer of the sample before chromatography to obtain the Factor VII recovery rate of different steps.

[0055] Experimental Example 1

[0056] To verify the effect of this process flow, the following experiments were carried out with reference to Example 1 (the following experiments use the following process conditions if not otherwise specified):

[0057] S1: Co-enrichment of IgM and Factor VII

[0058] (1) Raw plasma treatment: After the raw plasma is taken out of the warehouse, the surface of the plasma bag is disinfected with 75% ethanol solution, and then the plasma bag is broken, and the temperature is controlled at 2°C for melting. After melting, continuous flow centrifugation of the plasma is carried out at 0 - 4°C (12000 rpm, 10 min), and the supernatant is collected, which is the cold precipitate-free plasma.

[0059] (2) First anion exchange chromatography: By adjusting the pH value of the defrosted plasma to 7.5 and the conductivity to 10 mS / cm, and performing terminal filtration with a 0.22 μm filter element, the first anion exchange chromatography experiment was carried out. In the first anion exchange chromatography, the formulation of the equilibration buffer was 15 mM sodium citrate equilibration buffer, adjusted to pH 7.0 with hydrochloric acid and with a conductivity of 11 mS / cm; the formulation of the first eluent (eluent 1) was 15 mM sodium citrate equilibration buffer, adjusted to pH 7.0 with hydrochloric acid and with a conductivity of 22 mS / cm. The formulation of the second eluent (eluent 2) was 0.9 M sodium glutamate solution, with a pH of 7.2 and a conductivity of 28 mS / cm. Through the first anion exchange chromatography, a protein solution containing factor VII and IgM was obtained for further purification.

[0060] To demonstrate the effect, in subsequent experimental studies, TMAE was specifically used as the packing material (Fractgel EMD TMAE (M)), and the specifications of the chromatography column used for the experimental study were 26 mm × 25 cm. Before use, the chromatography column was equilibrated with the equilibration buffer described above for 5 column volumes, and then the defrosted plasma with adjusted pH and conductivity was loaded onto the chromatography column, with a loading volume of 20 mL of defrosted plasma / mL of gel. After the loading was completed, the chromatography column was eluted with 3 column volumes of the first eluent (eluent 1). After completion, the chromatography column was eluted with the second eluent (eluent 2) for 3 column volumes, and the flow-through of the second eluent (the first ion chromatography product) was collected, which contained factor VII and IgM.

[0061] The IgM yield of the first ion chromatography product was detected, as well as the yield of factor VII.

[0062] (3) Ultrafiltration and dialysis: The first ion chromatography product was ultrafiltered using a 10 KD ultrafiltration membrane package and dialyzed with 7 times the dialysis solution. The formulation of the dialysis solution was 15 mM sodium citrate + 0.15 M sodium chloride, adjusted to pH 7.8 with hydrochloric acid and adjusted to a conductivity of 17 mS / cm with sodium chloride to obtain the ultrafiltered and dialyzed product.

[0063] S2: Chromatographic separation of IgM and factor VII

[0064] (4) Gel filtration chromatography: The product after ultrafiltration and dialysis was processed by gel filtration chromatography. The column packing was sephadex-G200, and the column height was 45 cm. After equilibrating the chromatography column with the equilibration buffer, sample loading and chromatography were then carried out. Among them, the equilibration buffer was 15 mM sodium citrate + 0.14 M sodium chloride, and its pH was adjusted to 7.8 with hydrochloric acid, and the conductivity was adjusted to 17 mS / cm with sodium chloride. The gel packing was pre-equilibrated with 4 column volumes, the sample loading volume was 5% of the gel packing volume, and the flow-through was collected starting from when the first UV peak began to rise to 150 mAU until the end of the first UV peak. The collected product was the IgM-containing product (the first gel filtration chromatography product). At the same time, the flow-through corresponding to the third UV peak was collected, which was the product containing Factor VII (the second gel filtration chromatography product). The first gel filtration chromatography product and the second gel filtration chromatography product were respectively used for subsequent processing.

[0065] The IgM purity and IgM recovery of the first gel filtration chromatography product were detected; the specific activity of Factor VII and the Factor VII recovery of the second gel filtration chromatography product were detected.

[0066] S3-1: Preparation of IgM product

[0067] (5) Second anion exchange chromatography of IgM: Take the first gel filtration chromatography product, further adjust the sample loading pH value to 5.0 and the conductivity to 1.5 mS / cm, and then perform the second anion exchange chromatography packing. After equilibrating the anion exchange chromatography packing with 4 CV of the equilibration buffer, sample loading was carried out, and the flow-through was collected to obtain the second ion chromatography product. Among them, the equilibration buffer formula was 0.05 mM sodium acetate-acetic acid buffer, pH 5.0, and the conductivity was 5.5 mS / cm. In subsequent experimental studies, Fractogel EMD TMAE was specifically used as the packing, and the specification of the chromatography column used in the experimental study was 26 mm × 25 cm.

[0068] The IgM recovery and purity of the second ion chromatography product were detected.

[0069] According to the above operation process, the experimental conditions were studied:

[0070] Experiments 1-6 studied the effects of the conductivity and pH value of the cold gel-free plasma on the IgM recovery during the first anion exchange chromatography. The operation process and the selection of various parameters are referred to above, but the selection of the conductivity and pH value of the cold gel-free plasma is shown in Table 1 in detail. The calculation method of the IgM recovery of the first ion chromatography product is: the content of IgM in the first ion chromatography product / the content of IgM in the adjusted cold gel-free plasma for sample loading × 100%.

[0071] Table 1: Experimental conditions and results of IgM in the first ion exchange chromatography

[0072]

[0073] As can be seen from the above experimental data, adjusting the conductivity of the cold-ethanol-depleted plasma to 10 mS / cm and the pH to 7.3 - 7.5 can greatly improve the IgM recovery rate of the eluate. When the conductivity of the cold-ethanol-depleted plasma is adjusted to 12 mS / cm and the pH to 6.8 - 7.65, although the IgM recovery rate decreases to a certain extent, it can still remain above 64%. This IgM recovery rate is a technically acceptable parameter in the actual production process, indicating that this solution can achieve the enrichment of IgM and its separation from other components.

[0074] Experiments 7 - 10 studied the effects of the conductivity and pH value of the cold-ethanol-depleted plasma, as well as the formula, pH, and conductivity of the eluate, on the recovery rate of coagulation factor VII during the first anion exchange chromatography process. The operation process and the selection of each parameter are as described above. However, the selection of the conductivity and pH value of the cold-ethanol-depleted plasma, as well as the formula, pH, and conductivity of the eluate, are shown in Table 2 in detail. The calculation method for the recovery rate of coagulation factor VII in the first ion chromatography product is: the titer of coagulation factor VII in the first ion chromatography product / the titer of coagulation factor VII in the adjusted cold-ethanol-depleted plasma used for loading × 100%.

[0075] Table 2: Experimental conditions of the eluate for the first ion exchange chromatography and results of factor VII

[0076]

[0077] The above experimental results show that for the first anion exchange chromatography of Factor VII, if the pH value of the cold-precipitated plasma is adjusted to 7.2 - 7.5 and the conductivity is adjusted to 10 - 12 (Experiment 8 and Experiment 11), and sodium glutamate is used as the second eluent (Eluent 2, pH 7.2 - 7.5, conductivity 28 - 29 mS / cm), the recovery rate of Factor VII in the eluent can reach about 97%. However, in Experiment 11, due to the relatively low conductivity of the cold-precipitated plasma, there was also a certain residue of Factor II in the first ion chromatography product, and Factors VII, II, IX, and X could not be fully separated. If the pH value of the cold-precipitated plasma is reduced to 6.8 and at the same time the pH of Eluent 2 is reduced to 6.8 (Experiment 7), the recovery rate of Factor VII in the eluent will drop significantly to 87%, and Factor X cannot be separated from Factor VII. If the pH value of the cold-precipitated plasma is adjusted too high and at the same time the conductivity of Eluent 2 is reduced, the recovery rate of Factor VII will decrease and it cannot be fully separated from other coagulation factors (Experiment 10). If the formulation of Eluent 2 is adjusted to sodium citrate (Experiment 9), it will also cause a very significant reduction in the recovery rate of Factor VII in the eluent. Moreover, when using sodium citrate as Eluent 2, even if a series of adjustments are made to the pH of the cold-precipitated plasma, the pH of the eluent, etc., it is impossible to ensure the recovery rate of Factor VII and the separation of Factor VII from other coagulation factors (Experiment 12).

[0078] Therefore, in the first ion exchange chromatography, in order to fully recover Factor VII and separate it from Factors II, IX, and X, it is the best choice to adjust the pH value of the cold-precipitated plasma to 7.2 and the conductivity to 12. Under this parameter setting, the deficiencies of the prior art can be overcome, and the full separation of Factor VII from other coagulation factors can be achieved. Under the above conditions, although the recovery rate of IgM in the first ion exchange chromatography will be negatively affected to a certain extent, it can still reach 64%, which is acceptable in the actual production process.

[0079] Referring to the operation methods (S1 and S2) in the previous experimental examples, the purity detection and yield detection of IgM, the specific activity detection of coagulation factor VII, and the yield detection of coagulation factor VII were respectively carried out on the first gel filtration chromatography product and the second gel filtration chromatography product after gel filtration. The calculation method of the IgM yield of the first gel filtration chromatography product is: the content of IgM in the first gel filtration chromatography product / the content of IgM in the product after ultrafiltration and dialysis × 100%. The calculation method of the yield of coagulation factor VII for the second gel filtration chromatography product is: the titer of coagulation factor VII in the second gel filtration chromatography product / the titer of coagulation factor VII in the product after ultrafiltration and dialysis × 100%. The calculation method of the specific activity of gel filtration factor VII is: the titer of coagulation factor VII in the second gel filtration chromatography product / the total protein content in the second gel filtration chromatography product. The purity of gel filtration IgM is: the content of IgM in the first gel filtration chromatography product / the total protein content in the first gel filtration chromatography product × 100%. Under the process conditions, the purity of gel filtration IgM can reach 88%, the yield of gel filtration IgM can reach 88%, the specific activity of gel filtration factor VII (IU / mg protein) can reach 25 IU / mg protein, and the yield of gel filtration factor VII can reach 95%.

[0080] Experiments 13 - 18 studied the influence of the parameter conditions of the second anion exchange chromatography (the adjusted pH and conductivity of the first gel filtration chromatography product, and the selection of the chromatography column packing material). The relevant parameter settings and experimental results are shown in Table 3, and other parameters are referred to above. The calculation method of the IgM yield of the second ion chromatography product is: the content of IgM in the second ion chromatography product / the content of IgM in the adjusted first gel filtration chromatography product for sample loading × 100%. The purity of IgM in the second ion chromatography product is: the content of IgM in the second ion chromatography product / the total protein content in the second ion chromatography product × 100%.

[0081] Table 3: Experimental Conditions and Results of the Second Ion Exchange Chromatography of IgM

[0082]

[0083] From the above experimental data, it can be seen that both Fractogel EMD TMAE and Eshmuno Q have good chromatography effects as packing materials. The key to affecting the chromatography effect lies in the pH value and conductivity of the first gel filtration chromatography product. When the pH value of the first gel filtration chromatography product is 5.0 and the conductivity is 1.5 mS / cm, both the IgM yield and purity are at the most ideal levels.

[0084] Comparative Example 1

[0085] The applicant previously studied a method for simultaneously separating and purifying coagulation factors IX, X, and VII from human plasma. See the prior patent (CN109651502B). In the patent technical solution, in order to isolate factor VII, it is necessary to first extract the prothrombin complex (containing IX, X, VII, etc.) from plasma. The prothrombin complex contains relatively few other proteins, mainly coagulation factors, and there are fewer other influencing factors for the separation of coagulation factors. Under the above composition, factor VII can be separated from other coagulation factors through an anion exchange resin column, Toyopearl DEAE 650M. From this technical solution, it is very difficult to directly use an anion exchange resin column to separate various coagulation factors in plasma because the content of coagulation factors in raw plasma is relatively low, mainly globulins, albumins, etc., and the plasma components are complex. For example, the inventor once tried to directly separate the coagulation factors in plasma using the method in this patent. For the specific operation method, see the description in this patent "2. Separation of coagulation factors VII and IX from the prothrombin complex by anion exchange chromatography", and change the treatment object from the prothrombin complex to cold ethanol-precipitated plasma. Although the inventor tried various ways to adjust the pH value and conductivity, the recovery rate of factor VII was very unsatisfactory, only about 20-30%, and a large amount of other coagulation factors were mixed in factor VII, making it impossible to separate the coagulation factors from each other. In addition, using this patent method, it is impossible to purify IgM from plasma.

[0086] Compared with this patent technology, in this technical solution, plasma can be directly used for column treatment. By controlling the pH and conductivity of the cold ethanol-precipitated plasma appropriately, as well as the type of eluent, etc., factor VII can be directly separated from other coagulation factors, and at the same time, IgM in plasma can be separated. Subsequently, through gel filtration, IgM and factor VII with large differences in molecular weight can be separated. This technical solution has achieved unexpected technical effects in substance separation compared with this patent solution, and has simplified the treatment process, achieving unexpected technical effects.

[0087] Comparative Example 2

[0088] The applicant has previously studied a method for purifying human coagulation factor VII from human plasma. See the prior patent (CN111500563A). The technical solution of this patent separates human coagulation factor VII from plasma through two ion exchange chromatographies (UniGel-80Q gel chromatography and TMAE ion exchange packing chromatography in sequence). The present technical solution uses one anion exchange chromatography to achieve the separation of coagulation factor VII from other coagulation factors. Then, through gel filtration chromatography, the separation and purification of coagulation factor VII can be finally achieved, and gel filtration chromatography is simpler than anion exchange chromatography. In addition, through the present technical solution, the co-separation of IgM and coagulation factor VII in anion exchange chromatography can also be achieved, which is a process method not found in the prior art.

[0089] The above are only embodiments of the present invention, and common general technical solutions and / or characteristics and the like that are well known in the art are not described in detail herein. It should be noted that for those skilled in the art, without departing from the technical solution of the present invention, several modifications and improvements can be made, and these should also be regarded as the protection scope of the present invention, and these will not affect the implementation effect of the present invention and the practicality of the patent. The protection scope claimed in this application shall be subject to the content of its claims, and the specific implementation manners and the like recorded in the specification can be used to interpret the content of the claims.

Claims

1. A method for extracting and separating IgM and coagulation factor VII from plasma, characterized in that: it includes the following steps carried out in sequence: S1: Adjust the pH value of the cold-removed plasma to 6.3 - 7.6 and the conductivity to 9 - 13 mS / cm, load it onto the first anion exchange chromatography column that has been equilibrated with the first equilibration solution, and then carry out elution treatment with the first eluent and the second eluent in sequence to obtain the first ion chromatography product; The packing material of the first anion exchange chromatography column is TMAE; The first equilibration solution is 10 - 20 mM sodium citrate with a pH value of 6.3 - 7.6 and a conductivity of 9 - 13 mS / cm; The first eluent is 10 - 20 mM sodium citrate with a pH value of 6.3 - 7.6 and a conductivity of 15 - 30 mS / cm; The second eluent is 0.5 - 1 M sodium glutamate solution with a pH value of 6.8 - 7.7 and a conductivity of 19 - 30 mS / cm; S2: After the first ion chromatography product is ultrafiltered and dialyzed, adjust the pH value to 7.2 - 8.2 and the conductivity to 14 - 20 mS / cm, load it onto the gel filtration chromatography column that has been equilibrated with the second equilibration solution, collect the flow-through liquid corresponding to the first UV peak and the flow-through liquid corresponding to the third UV peak respectively, and obtain the first gel filtration chromatography product containing IgM and the second gel filtration chromatography product containing coagulation factor VII respectively; The packing material of the gel filtration chromatography column is Sephadex; The second equilibration solution is a mixed solution containing 10 - 20 mM sodium citrate and 0.13 - 0.17 M sodium chloride, with a pH value of 7.2 - 8.0 and a conductivity of 14 - 20 mS / cm; S3: Adjust the pH value of the first gel filtration chromatography product to 4.5 - 5.5 and the conductivity to 1.5 - 7.5 mS / cm, then load it onto the second anion exchange chromatography column that has been equilibrated with the third equilibration solution, and collect the flow-through liquid to obtain the second ion chromatography product; the second ion chromatography product is ultrafiltered, dialyzed, and adjusted in concentration, pH, and conductivity, and sorbitol and glycine are added to obtain the crude IgM product; the crude IgM product is subjected to pasteurization inactivation, and then ultrafiltered, dialyzed, and adjusted in concentration and pH, and maltose is added and filtered and sub-packed to obtain the finished IgM product; the packing material of the second anion exchange chromatography column is TMAE; the third equilibration solution is 0.015 - 0.075 mol / L sodium acetate - acetic acid buffer solution with a pH value of 4.5 - 5.5; The second gel filtration chromatography product is ultrafiltered, dialyzed, and adjusted in concentration and pH, and glycine, arginine, and lysine hydrochloride are added to obtain the crude coagulation factor VII product; The crude coagulation factor VII product is sub-packed and subjected to freeze-drying and dry heat inactivation treatment to obtain the finished coagulation factor VII product.

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