A method for preparing intravenous human immunoglobulin

Abstract

The present application belongs to the technical field of blood product preparation, and particularly relates to a preparation method of intravenous human immunoglobulin, comprising the following steps which are sequentially performed: Cohn FIII supernatant is pretreated to obtain a sample I to be loaded; the sample I to be loaded is subjected to first anion exchange chromatography to obtain a first chromatography product; the first chromatography product is pretreated to obtain a sample II to be loaded; the sample II to be loaded is subjected to second anion exchange chromatography to obtain a second chromatography product; the second chromatography product is pretreated to obtain a sample III to be loaded; the sample III to be loaded is subjected to heparin affinity chromatography to obtain a third chromatography product; and the third chromatography product is configured into human immunoglobulin finished product. The technical scheme can solve the technical problems of the existing preparation method of intravenous human immunoglobulin, such as introduction of new materials, poor effect of impurity protein precipitation, high content of anti-A and anti-B, and the like, and has an ideal application prospect.

Description

Technical Field

[0001] This invention belongs to the field of blood product preparation technology, specifically relating to a method for preparing intravenously injected human immunoglobulin. Background Technology

[0002] Intravenous immunoglobulin G (IVIG) is produced from healthy human plasma through separation, purification, removal of anti-complement activity and viruses, and inactivation. It is an effective drug for treating primary immunodeficiency diseases, secondary immunodeficiency diseases, and other conditions, and has become an important clinical treatment method. Its main component is immunoglobulin G (IgG), which is the main component of immunoglobulins in serum, accounting for up to 75% of the total immunoglobulin content. IgG, as the main antibody in the human body's anti-infective immunity, can be considered an important indicator of human immune function. Based on different heavy chain structures, it is divided into four subclasses: IgG1, IgG2, IgG3, and IgG4, each playing different roles in the occurrence and development of diseases.

[0003] Early IVIG products, due to incomplete IgG molecules, only retained the function of the F(ab)2 fragment, thus failing to perform the intended biological functions of IgG, such as treating bacterial infections. Research institutes in France, the United States, and Japan modified these molecules, using enzymatic hydrolysis to produce the first generation of IVIG products. These products contained a portion of complete IgG, effectively fulfilling their biological functions. Since the first-generation IVIG contained only 30-40% complete IgG, there was significant room for improvement. Therefore, Masuho et al. successfully prepared sulfonated immunoglobulins using sulfonation. These immunoglobulins maintained the integrity of the IgG molecule, had no anti-complement activity, and had a biological half-life comparable to normal IgG, thus officially introducing the second generation of IVIG products. However, research on IVIG did not stop there. Cutter, an American company, discovered that using Cohn fraction II as a starting material could eliminate the enzymatic digestion step, lower the pH of the immunoglobulin solution, and significantly increase the content and stability of IgG monomers. Further research revealed that IVIG, after treatment at pH 4.25, not only maintained its natural and complete biological functions but also inactivated lipid-enveloped viruses, thus ushering in the era of third-generation IVIG. However, third-generation IVIG also had its shortcomings in preparation, mainly manifested in low yield, a single virus inactivation method in the production process, and an inability to completely prevent the spread of non-lipid-enveloped viruses. Therefore, based on this, researchers at home and abroad combined virus inactivation methods with different mechanisms to form the fourth-generation IVIG production process. These include caprylate-chromatography and caprylate-PEG-chromatography, which mainly utilize the characteristic that γ-globulin is not easily precipitated, using Cohn fractions II+III as starting materials, and obtaining IVIG through several steps of chromatographic separation. This method can not only ensure the yield of IgG but also remove viruses.

[0004] Our research revealed that while the novel IVIG production method can optimize product production cycle and tolerability to some extent, it still has inherent drawbacks, such as the introduction of new materials, poor precipitation of contaminating proteins, and high levels of anti-A and anti-B antibodies. Therefore, further improvements to the IVIG preparation method are needed to ensure IgG yield and virus clearance rate, improve the precipitation of contaminating proteins, and reduce the content of antibodies against type A and type B blood cell surface antigens, without introducing new materials. Summary of the Invention

[0005] The present invention aims to provide a method for preparing intravenously injected human immunoglobulin, so as to solve the technical problems of existing methods for preparing intravenously injected human immunoglobulin, such as the introduction of new materials, poor precipitation of impurity proteins, and high content of anti-A and anti-B proteins.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A method for preparing intravenously administered human immunoglobulin includes the following steps performed sequentially:

[0008] S1: Obtain the supernatant from Cohn FIII;

[0009] S2: After ultrafiltration concentration, dialysis, dilution, conductivity adjustment, pH adjustment and filtration, the supernatant of Cohn FIII is used to obtain sample I to be loaded; sample I is subjected to the first anion exchange chromatography treatment to obtain the first chromatographic product;

[0010] S3: After the first chromatographic product is adjusted for conductivity and pH and filtered, sample II is obtained; sample II is subjected to a second anion exchange chromatography treatment to obtain the second chromatographic product.

[0011] S4: After the conductivity and pH of the second chromatographic product are adjusted and filtered, sample III to be loaded is obtained; sample III to be loaded is subjected to heparin affinity chromatography to obtain the third chromatographic product.

[0012] S5: The third chromatography product is processed through nanofiltration, pH adjustment, concentration, dialysis, and virus inactivation to prepare human immunoglobulin product.

[0013] Furthermore, in S2, the protein content of the sample to be loaded, I, is 10-50 g / L; the conductivity of the sample to be loaded, I, is 0.2-5.0 mS / cm, preferably 0.2-3.0 mS / cm, and more preferably 0.3-2.0 mS / cm; the pH of the sample to be loaded, I, is 5.0-7.0, preferably 5.2-6.8, and more preferably 5.4-6.2.

[0014] Furthermore, in S2, the chromatographic column used in the first ion exchange chromatography is a quaternary ammonium salt ion exchange packing material; the sample to be loaded, I, is loaded into the chromatographic column at a loading rate of 300-600 g / L and a flow rate of 5-15 ml / min; then the chromatographic column is washed with the first equilibration buffer until the baseline is reached, and the flow-through of the sample to be loaded, I, and the first equilibration buffer flowing out of the chromatographic column are collected to obtain the first chromatographic product;

[0015] The first equilibration solution is an acetate-sodium acetate buffer solution, and its pH and conductivity are consistent with those of the sample to be loaded, I.

[0016] Furthermore, in S3, the conductivity of the sample II to be loaded is 0.2-5.0 mS / cm, preferably 0.2-3.0 mS / cm, and more preferably 0.3-2.0 mS / cm; the pH of the sample II to be loaded is 5.0-7.0, preferably 5.2-6.8, and more preferably 5.4-6.2.

[0017] Furthermore, in S3, the chromatographic column used for the second ion exchange chromatography is a quaternary ammonium salt ion exchange packing material; the sample to be loaded, product II, is loaded into the chromatographic column at a loading rate of 600-1500 g / L and a flow rate of 3-10 ml / min; then the chromatographic column is washed with the second equilibration buffer until the baseline is reached, and the flow-through of product II and the second equilibration buffer flowing out of the chromatographic column are collected to obtain the second chromatographic product;

[0018] The second equilibration solution is an acetate-sodium acetate buffer solution, and its pH and conductivity are consistent with those of the sample to be loaded, product II.

[0019] Furthermore, in S3, the conductivity of the sample product III to be loaded is 0.2-3.0 mS / cm, preferably 0.5-1.5 mS / cm; the pH of the sample product III to be loaded is 5.0-7.0, preferably 5.2-6.8, and more preferably 5.4-6.2.

[0020] Further, in S3, the sample to be loaded, product III, is loaded into the chromatography column at a loading capacity of 800-1500 g / L and a flow rate of 5-15 ml / min. Then, the chromatography column is washed with the third equilibration buffer until the baseline is reached. The flow-through of product II and the third equilibration buffer flowing out of the chromatography column are collected to obtain the third chromatographic product.

[0021] The third equilibration solution is an acetate-sodium acetate buffer solution, and its pH and conductivity are consistent with those of the sample to be loaded, product III.

[0022] Further, the raw plasma is centrifuged, and the supernatant A is collected. The temperature of supernatant A is adjusted to -3.0 to -1.0℃, the protein concentration to 40-65 g / L, the pH to 6.80-7.30, the conductivity to 12-14 mS / cm, and the ethanol concentration to 7-10 vol.%. The reaction is carried out for 1-3 hours, and the supernatant B is collected by pressure filtration. The temperature of supernatant B is adjusted to -6.0 to -4.0℃, the protein concentration to 30-45 g / L, the pH to 5.70-6.30, and the conductivity to 5.0-8. At 0 mS / cm and an ethanol concentration of 18-22 vol.%, react for 1-3 h, then collect the precipitate by pressure filtration. Add water to the precipitate and dissolve for 2-4 h, with the water mass being 8-10 times the mass of the precipitate. Control the temperature at 0-5℃, add phosphate buffer to adjust the pH to 4.6-5.0, and react for 1-2 h. Then add phosphate buffer to adjust the pH to 5.0-5.4, and react for 1-2 h. Finally, adjust the ethanol concentration to 13-15 vol.%, react for 1-3 h, and collect the supernatant by pressure filtration to obtain the Cohn FIII supernatant.

[0023] Furthermore, in S5, the nanofiltration pore size is 20 nm; the low pH incubation parameters are pH 4.0-4.8 and incubation temperature 20-30℃.

[0024] This technical solution also provides a method for preparing intravenous immunoglobulin to obtain an intravenous immunoglobulin product.

[0025] The principle and beneficial effects of this technical solution are as follows:

[0026] This technical solution uses Cohn FIII supernatant as the starting material for IVIG production, and then obtains the immunoglobulin product through multi-step chromatography. Two-step ion exchange chromatography removes impurities from the material, and affinity chromatography further removes any coagulation factors that may be present. Using Cohn FIII supernatant as the starting material significantly reduces anti-A and anti-B thrombin impurities and IgA impurities, and avoids the use of traditional caprylate precipitation, thus reducing the introduction of impurities. This invention employs multi-step chromatography, resulting in a product with low levels of impurities (including IgA, IgM, albumin, fibrinogen, etc.), further ensuring clinical safety. The protein product prepared by this invention achieves an IgG yield of over 85%, effectively ensuring product purity and efficacy while improving yield, and fully utilizing plasma resources. This invention uses nanomembrane filtration and low-pH incubation to further ensure the safety of intravenous immunoglobulin virus inactivation. Attached Figure Description

[0027] Figure 1 This is the process flow diagram for this solution.

[0028] Figure 2 The image shows the electrophoretic image of the finished product from Example 1.

[0029] Figure 3 This is an electrophoresis image of the third chromatographic product of Comparative Example 4. Detailed Implementation

[0030] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art; the experimental methods used are all conventional methods; and the materials, reagents, etc. used are all commercially available.

[0031] Example 1

[0032] A process for producing intravenous immunoglobulin (IVIG) using Cohn FIII supernatant (hereinafter referred to as FIII supernatant) as the starting material for IVIG production includes the following steps (see the overall process flow diagram). Figure 1 ):

[0033] S1: Acquisition of Cohn FIII supernatant:

[0034] Raw plasma stored at -30℃ is placed at -5℃ overnight after being taken out of storage. Before use, the surface of the plasma bag is sterilized with 70%-75% ethanol solution, then the plasma bag is broken and thawed at a controlled temperature of 0-4℃. Raw plasma is the supernatant after centrifugation to remove cells from blood; it contains proteins, inorganic salts, and water, but does not contain blood cells. More specifically, raw plasma refers to human plasma as defined in the Chinese Pharmacopoeia: human plasma for blood product manufacturing refers to plasma from healthy individuals collected via apheresis for the production of plasma protein products. The Cohn FIII supernatant is obtained using existing techniques, specifically as follows:

[0035] After routine centrifugation of the raw plasma, collect the supernatant A. Adjust the temperature of supernatant A to -3.0 to -1.0℃, protein concentration to 40-65 g / L, pH to 6.80-7.30, conductivity to 12-14 mS / cm, and ethanol concentration to 7-10 vol.%, react for 1-3 hours, and then collect the supernatant B by routine pressure filtration. Adjust the temperature of supernatant B to -6.0 to -4.0℃, protein concentration to 30-45 g / L, pH to 5.70-6.30, conductivity to 5.0-8.0 mS / cm, and ethanol concentration to 18-22 vol.%, react for 1-3 hours, and then collect the precipitate by routine pressure filtration to obtain components II+III. Dissolve components II+III in water for 2-4 hours, with the water mass being 8-10 times the mass of components II+III. Then, control the temperature at 0-5℃, add phosphate buffer to adjust the pH to 4.6-5.0, and react for 1-2 hours; then add phosphate buffer to adjust the pH to 5.0-5.4, and react for 1-2 hours; adjust the ethanol concentration to 13-15 vol.%, react for 1-3 hours, and collect the supernatant C by pressure filtration, which is the Cohn FIII supernatant.

[0036] S2: First step of tomography:

[0037] (1) The FIII supernatant was concentrated by ultrafiltration using a 30-50kD membrane. The temperature was controlled at 2-8℃ during the ultrafiltration process, and the concentration was carried out at a ratio of FIII supernatant: ultrafiltration concentrated liquid = 100L: 10-15L. Dialysis was performed with an equal volume of dialysis buffer, 6-9 times the volume. After dialysis, the solution was concentrated to 5-8L and stored at 2-8℃. Ultrafiltration concentration and dialysis to remove impurities are standard operating procedures in existing technologies. The dialysis buffer consisted of ultrapure water.

[0038] (2) Dilute the protein concentration of the product obtained in step (1) with ultrapure water to 10-50 g / L, adjust the conductivity to 0.2-5.0 mS / cm (preferably 0.2-3.0 mS / cm, more preferably 0.3-2.0 mS / cm) with equilibration stock solution (0.5M acetate-sodium acetate buffer), and adjust the pH to 5.0-7.0 (preferably 5.2-6.8, more preferably 5.4-6.2) with 1M acetic acid solution. After the above adjustments, filter the product using a 0.22 ± 0.45 μm filter cartridge to obtain the sample I to be loaded, and detect its protein content.

[0039] (3) Pretreatment of the anion exchange chromatography column, preferably a column packed with Q packing (Quaternary Ammonium). The pretreatment process includes: treating 4-6 column volumes with 0.5M sodium hydroxide; treating 4-6 column volumes with a stock solution to balance the pH; and balancing 5-10 column volumes with a first equilibration solution to balance the pH and conductivity. The first stock solution is formulated as follows: 0.5M acetate-sodium acetate buffer, pH 5.4-6.2. The first equilibration solution is formulated as follows: an acetate-sodium acetate buffer prepared based on the stock solution, adjusting the pH and conductivity of the first equilibration solution to be consistent with the pH and conductivity of the sample I to be loaded.

[0040] (4) Load the protein solution (sample I to be loaded) described in step (2) into an anion exchange chromatography column. Calculate the loading capacity and control it at 300-600 g / L (preferably 300-500 g / L, where the loading capacity is the total protein content / column volume). Control the loading speed at 5-15 mL / min. Wash the chromatography column with the first equilibration solution described above until the baseline is reached, controlling the washing speed at 5-15 mL / min. Collect the protein flow-through (sample I to be loaded) and the first equilibration solution flowing out of the chromatography column used to wash the column to the baseline (collectively referred to as the first chromatographic product). The baseline refers to a straight line in the chromatogram representing the signal level when no target compound or impurity passes through the detector. Store the collected sample at 2-8°C.

[0041] S3: Second step of chromatography:

[0042] (1) Take the flow-through liquid after the first step of chromatography (the flow-through liquid of the sample to be loaded + the effluent washed to the baseline with equilibration solution, two parts, i.e. the first chromatography product), adjust the conductivity to 0.2-5.0 mS / cm (preferably 0.2-3.0 mS / cm, more preferably 0.3-2.0 mS / cm) with equilibration stock solution, adjust the pH to 5.0-7.0 (preferably 5.2-6.8, more preferably 5.4-6.2) with 1M acetic acid or 0.5M sodium hydroxide, filter with a 0.22μm filter to obtain sample II to be loaded, and detect its protein content.

[0043] (2) Pretreatment of the selected ion exchange chromatography column, preferably a column packed with Q packing (Quaternary Ammonium). The pretreatment steps mainly include: treating 4-6 column volumes with 0.5M sodium hydroxide; treating 4-6 column volumes with the second equilibration stock solution to equilibrate the pH; and equilibrating 5-20 column volumes with the second equilibration buffer to equilibrate the pH and conductivity. The equilibration stock solution is formulated as follows: 0.5M acetate-sodium acetate buffer, pH 5.4-6.2. The second equilibration buffer is formulated as follows: acetate-sodium acetate buffer prepared based on the equilibration stock solution, adjusting the pH and conductivity of the second equilibration buffer to be consistent with the pH and conductivity of the sample II to be loaded.

[0044] (3) Load the sample II to be loaded in step (1) into the chromatography column. Calculate the loading capacity and control it within the range of 600-2000 g / L (preferably 600-1500 g / L). Control the loading speed at 3-10 mL / min. Wash the chromatography column with the second equilibration solution mentioned above until the baseline is reached. Control the washing speed at 3-10 mL / min. Collect the flow-through of the protein solution (sample II to be loaded) and the second equilibration solution flowing out of the chromatography column for washing the chromatography column to the baseline (collectively referred to as the second chromatography product). Store the obtained product at 2-8℃.

[0045] S4: Third step of tomography:

[0046] (1) Take the protein product after the second step of chromatography (second chromatography product), adjust the conductivity to 0.2-3.0 mS / cm (preferably 0.5-1.5 mS / cm) with the equilibration mother liquor, adjust the pH to 5.0-7.0 (preferably 5.2-6.8, more preferably 5.4-6.2) with 1M acetic acid or 0.5M sodium hydroxide, filter the product with a 0.22μm filter cartridge to obtain sample III to be loaded, and detect its protein content.

[0047] (2) Pretreatment of the selected affinity chromatography column is preferred, with columns packed with heparin affinity packing material being the preferred option. For example, agarose or polymer packing material with heparin as the ligand can be selected. Heparin can be synthesized using conventional methods or extracted from animals; heparin affinity packing material products can also be obtained commercially. The pretreatment steps mainly include: treating 4-6 column volumes with 0.5M sodium hydroxide; treating 4-6 column volumes with the third equilibration stock solution to balance the pH; and equilibrating 5-10 column volumes with the third equilibration buffer to balance the pH and conductivity. The equilibration stock solution is 0.5M acetate-sodium acetate buffer solution with a pH of 5.4-6.2. The third equilibration buffer solution is prepared based on the equilibration stock solution, and its pH and conductivity are adjusted to be consistent with those of the sample III to be loaded.

[0048] (3) Perform affinity chromatography on the protein solution (sample III to be loaded) from step (1), calculate the loading capacity and control it within the range of 800-1500 g / L, and control the loading rate at 5-15 mL / min. Wash the chromatography column with the third equilibration buffer mentioned above until the baseline is reached, and control the washing rate at 5-15 mL / min. Collect the flow-through of the protein solution (sample III to be loaded) and the third equilibration buffer that flows out of the chromatography column and is used to wash the chromatography column to the baseline (collectively referred to as the third chromatography product). Store the obtained product at 2-8℃.

[0049] S5: Ultrafiltration Preparation

[0050] Take the protein product after the third-step chromatography (third-step chromatography product), and perform nanofiltration using a 20nm nanomembrane. Then adjust the pH of the product to 3.6-4.0. Concentrate by ultrafiltration using a 50kD membrane to a protein concentration of 30g / L-50g / L. Then add an equal volume of dialysis buffer (preferably water for injection) and dialyze 6-9 times (preferably 8 times). After dialysis, further concentrate by ultrafiltration to a protein concentration greater than 100g / L (preferably 130g / L-150g / L). Using 0.2-0.3M glycine as a protectant, prepare a high-concentration human immunoglobulin product with a protein concentration greater than 10% (preferably 10%). Dispense the product into molded bottles and incubate the prepared protein product at a low pH (pH 4.0-4.8, incubation temperature 20-30℃). The resulting product is the finished product.

[0051] Experimental Example 1: Study on the effects of first-step and second-step chromatography

[0052] Step 1: Tomography

[0053] (1) Concentrate 100L of FIII supernatant to 13L using ultrafiltration with a 50kD membrane, keeping the temperature around 4℃ during the ultrafiltration process. Then, use water as the dialysate for dialysis at an equal volume ratio of 8. After dialysis, concentrate to 6L and store at around 4℃.

[0054] To facilitate the experiment, the Cohn FIII supernatant was obtained using conventional methods, specifically prepared as follows: The raw plasma was centrifuged and supernatant A was collected. The temperature of supernatant A was adjusted to approximately -2.0℃, protein concentration to approximately 50 g / L, pH to approximately 7.00, conductivity to approximately 13 mS / cm, and ethanol concentration to approximately 8 vol.%, and the reaction was allowed to proceed for approximately 2 hours. Supernatant B was then collected by conventional pressure filtration. The temperature of supernatant B was adjusted to approximately -5.0℃, protein concentration to approximately 40 g / L, pH to approximately 6.00, conductivity to approximately 7.0 mS / cm, and ethanol concentration to approximately 20 vol.%, and the reaction was allowed to proceed for approximately 2 hours. The precipitate was then collected by conventional pressure filtration to obtain fractions II+III. Water was added to fractions II+III and dissolved for approximately 3 hours, with the water mass being 9 times the mass of fractions II+III. Then, the temperature was controlled at approximately 4°C, and phosphate buffer was added to adjust the pH to approximately 4.8, and the reaction was allowed to proceed for approximately 2 hours. Next, phosphate buffer was added again to adjust the pH to approximately 5.2, and the reaction was allowed to proceed for approximately 2 hours. Finally, the ethanol concentration was adjusted to approximately 14 vol.%, and the reaction was allowed to proceed for approximately 2 hours. The supernatant C was collected by pressure filtration, thus obtaining the Cohn FIII supernatant. Additionally, the precipitate of components II+III can also be used for the preparation of immunoglobulins (as a raw material for comparative experiments).

[0055] (2) Dilute the protein concentration of the product obtained in step (1) to 20 g / L, adjust the conductivity to about 1.5 mS / cm with the equilibration stock solution, and adjust the pH to about 5.8 with 1M acetic acid solution. After the above adjustments, filter the product using a 0.45 ± 0.22 μm filter cartridge to obtain the sample I to be loaded, and detect its protein content.

[0056] (3) Pretreatment of the anion exchange chromatography column is performed, specifically using anion exchange packing material packed with Q-ligands, more specifically Nanomicro 60Q packing material (other agarose-based Q-ligand packing materials can also be used). The ion exchange chromatography column has dimensions of 26 mm × 40 cm. The pretreatment process includes: treating 5 column volumes with 0.5 M sodium hydroxide; treating 5 column volumes with equilibration mother liquor to balance the pH; and equilibrating 5 column volumes with the first equilibration solution to balance the pH and conductivity.

[0057] The specific formulation of the equilibration stock solution is: 0.5M acetate-sodium acetate buffer, pH 5.8. The specific formulation of the first equilibration solution is: acetate-sodium acetate buffer prepared based on the equilibration stock solution, pH 5.8, conductivity 1.5mS / cm (to be consistent with the sample to be loaded I).

[0058] (4) Load the protein solution (sample I to be loaded) described in step (2) into an anion exchange chromatography column, and calculate the loading capacity to be controlled at approximately 400 g / L. Determine the chromatography flow rate to be 10 ml / min, and wash the chromatography column with the first equilibration solution mentioned above until the baseline is reached. Collect the flow-through of the protein solution (sample I to be loaded) and the first equilibration solution flowing out of the chromatography column used for washing the chromatography column to the baseline (collectively referred to as the first chromatography product). Store the collected product at approximately 4°C.

[0059] The collected sample (first chromatography product) was analyzed for contaminating proteins, and the content was compared with that in the FIII supernatant. The results showed that 60Q chromatography can significantly reduce small molecule contaminating proteins in the FIII supernatant, improve product purity, and enhance product safety and efficacy. The results are shown in Table 1.

[0060] Table 1: Content of various proteins before and after 60Q chromatography (all data in the table are converted to a protein concentration of 50 g / L).

[0061]

[0062] Second step: Chromatography

[0063] (1) Take the flow-through liquid after the first step of chromatography (the first chromatography product), adjust the conductivity to about 1.5 mS / cm with the equilibration mother liquor, adjust the pH to about 5.8 with 1M acetic acid or 0.5M sodium hydroxide, filter with a 0.22μm filter to obtain sample II to be loaded, and detect its protein content.

[0064] (2) Pretreatment of the selected ion exchange chromatography column was performed, with a preference for columns packed with 50Q packing material, such as Nanomicro 50Q (selected for specific experimental research) and Bojin 50Q. Nanomicro 50Q and Bojin 50Q packing materials have particularly large pore sizes, more than five times that of ordinary packing materials. Using large-pore packing materials results in better removal of impurities and proteins and a higher loading capacity; ordinary packing materials can only achieve a loading capacity of around 400 g / L, while large-pore packing materials can reach over 1000 g / L. The ion exchange chromatography column specifications are 16 mm × 40 cm. The pretreatment steps mainly include: treating 5 column volumes with 0.5 M sodium hydroxide; treating 5 column volumes with a equilibration mother solution to balance the pH; and equilibrating 10 column volumes with a second equilibration solution to balance the pH and conductivity.

[0065] The stock solution was prepared as follows: 0.5M acetate-sodium acetate buffer solution, pH 5.8. The second equilibration solution was prepared as follows: acetate-sodium acetate buffer solution prepared based on the stock solution, with the pH adjusted to 5.8 and the conductivity to 1.5 mS / cm (to be consistent with the sample to be loaded, product II).

[0066] (3) Load the sample II from step (1) into the chromatography column, and calculate the loading capacity to be controlled at approximately 1000 g / L. Control the flow rate for 8 min, and wash the chromatography column with the second equilibration buffer mentioned above until the baseline is reached. Collect the flow-through of the protein solution (sample II) and the second equilibration buffer that flows out of the chromatography column to wash the column to the baseline (collectively referred to as the second chromatography product). Store the obtained product at approximately 4°C.

[0067] The collected samples were tested for extraneous proteins and compared with the extraneous protein content after the first step of chromatography. The results showed that chromatography can further reduce extraneous proteins in the FIII supernatant, with particularly significant removal effect on large molecular extraneous proteins, which can improve product purity and quality. The results are shown in Table 2.

[0068] Table 2: Content of various proteins before and after 50Q chromatography (all data in the table are converted to a protein concentration of 50 g / L).

[0069]

[0070] Experimental Example 2: Study on the Effect of Affinity Chromatography

[0071] The first and second steps of ion exchange chromatography in this embodiment are performed in accordance with Example 1, with the following differences:

[0072] In the first step of chromatography, the conductivity of sample I to be loaded was adjusted to approximately 1.5 mS / cm. In the second step of chromatography, the conductivity of sample II to be loaded was also adjusted to approximately 1.5 mS / cm. In the second step of chromatography, the loading volume was controlled at approximately 800 g / L. In addition to the above differences, this experimental example also included a subsequent affinity chromatography step. Tests 1 and 2 tested two heparin affinity chromatography columns packed with different heparin affinity chromatography packing materials.

[0073] Test 1:

[0074] Third step: Tomography

[0075] (1) Take the protein product after the second step of chromatography (second chromatography product), adjust the conductivity to 1.5 mS / cm, adjust the pH to 5.8 with 1M acetic acid or 0.5M sodium hydroxide, filter the product with a 0.22μm filter to obtain sample III to be loaded, and detect its protein content.

[0076] (2) Pretreatment was performed on the selected affinity chromatography column. Unigel Heparin was selected as the packing material, and the column size was 16mm × 20cm. The pretreatment steps mainly included: treating 5 column volumes with 0.1M sodium hydroxide; treating 5 column volumes with equilibration mother liquor to balance the pH; and equilibrating 5 column volumes with a third equilibration solution to balance the pH and conductivity.

[0077] The specific formulation of the equilibration stock solution is: 0.5M acetate-sodium acetate buffer solution, pH 5.8. The specific formulation of the third equilibration solution is: acetate-sodium acetate buffer solution prepared based on the equilibration stock solution. The pH and conductivity of the third equilibration solution should be adjusted to be consistent with the pH and conductivity of the sample to be loaded, i.e.

[0078] (3) Perform affinity chromatography on the protein solution (sample III to be loaded) from step (1), calculate the loading capacity, and control it at approximately 800 g / L. Control the loading rate at approximately 10 mL / min, and wash the chromatography column with the aforementioned third equilibration buffer until baseline. Collect the flow-through of the protein solution (sample III to be loaded) and the third equilibration buffer that flows out of the chromatography column for washing the column to baseline (collectively referred to as the third chromatography product). Store the obtained product at approximately 4°C.

[0079] Ultrafiltration preparation

[0080] The protein product after the third step of chromatography was nanofiltered using a 20 nm nanometer-terminated membrane, and then the pH of the product was adjusted to approximately 3.8. Next, it was concentrated by ultrafiltration using a 50 kDa membrane to a protein concentration of approximately 40 g / L, and then dialyzed eight times with an equal volume of dialysis buffer (preferably water for injection). After dialysis, it was further concentrated by ultrafiltration to a protein concentration of approximately 140 g / L. A high-concentration human immunoglobulin product with a protein concentration of 10% was prepared using glycine as a preservative. The product was then tested for activation of factor XIa, anti-A, and anti-B titers, and contaminating protein bands were detected by electrophoresis. The electrophoresis results can be found in [link to electrophoresis results]. Figure 2From left to right: Well 1: Marker; Well 2: Pre-heparin affinity chromatography; Well 3: Heparin affinity eluent; Well 4: Heparin affinity flow-through buffer; Well 5: High-concentration human immunoglobulin product (IVIG). Pre-heparin affinity chromatography specifically refers to sample III to be loaded; heparin affinity flow-through buffer specifically refers to the product of the third chromatography step. The heparin affinity eluent is the product eluted from the column after heparin affinity chromatography, and is a step performed after obtaining the product of the third chromatography step. The eluent formulation is 1M sodium chloride + 0.02M acetate-sodium acetate, with a pH consistent with the pH used in affinity chromatography. The elution process is a step after equilibration treatment. In the heparin affinity eluent, bands distinctly different from those in the sample before heparin affinity chromatography appeared. This is because the protein molecules corresponding to these bands were enriched during chromatography and could be detected by electrophoresis in the eluent, further demonstrating the purification effect of heparin chromatography on protein products, which can remove some impurity molecules. The XIa factor content of the product obtained in this test 1 was below the detection limit, the anti-A and anti-B titers were 1:16 / 1:16, the molecular size distribution was greater than 99%, and the total protein yield after three-step chromatography was greater than 90% (yield = amount of IgG in the final product / amount of IgG in the raw plasma).

[0081] Test 2: The test method is basically the same as Test 1, except that the packing material of the affinity chromatography column is changed to Heparin FF. The XIa factor content of the product obtained in Test 1 is below the detection limit, the anti-A and anti-B titers are 1:16 / 1:16, and the yield and molecular size distribution are basically the same as in Test 1.

[0082] Therefore, it is evident that by employing heparin affinity chromatography, using heparin as the ligand for the packing material, and using various scaffolds, the goal of further improving immunoglobulin production efficiency and product quality can be achieved.

[0083] Comparative Example 1

[0084] Chinese patent CN116120435A (A method for preparing intravenous human immunoglobulin from plasma fraction FIII precipitate) uses a combination of octanoate precipitation, two-step anion exchange chromatography, and anti-A / anti-B affinity chromatography to separate and purify human immunoglobulin from Cohn FIII precipitate (not the Cohn FIII supernatant used in this method). Due to the different initial raw materials, this technique requires an octanoate precipitation step before chromatography, which results in residual octanoate and other substances in the finished product. Furthermore, the Cohn FIII precipitate contains a high content of anti-A and anti-B hemagglutinins, requiring specific anti-A / anti-B affinity chromatography for removal. Since anti-A / anti-B affinity packing material is 10 times more expensive than heparin affinity packing material, this method saves production costs by eliminating the need to purchase expensive packing material. The inventors have discovered for the first time that Cohn FIII supernatant contains fewer anti-A and anti-B hemagglutinin impurities. Using Cohn FIII supernatant as a raw material, without the need for specialized anti-A and anti-B affinity chromatography, the anti-A and anti-B titers can reach 1:16 / 1:16, meeting the standard requirements. The inventors analyzed that this is because Cohn FIII supernatant is obtained through component precipitation, thus reducing the content of anti-A and anti-B hemagglutinin impurities. The inventors also experimented by directly replacing the anti-A and anti-B affinity chromatography in this comparative example with the heparin affinity chromatography of this scheme (still using Cohn FIII precipitation as the starting material), which resulted in a product anti-A and anti-B titer much higher than 1:32 / 1:32. This indicates that using Cohn FIII supernatant as the starting material can ensure that anti-A and anti-B hemagglutinin impurities are minimized, reducing the difficulty and cost of subsequent affinity chromatography, where the removal of anti-A and anti-B hemagglutinin impurities does not need to be the primary objective.

[0085] In addition, this protocol employs heparin affinity chromatography to remove potentially present coagulation factors, particularly XIa, thus reducing the clinical risk of thrombosis. Factor XI is a serine protease precursor, which, upon activation, becomes factor XIa. Factor XIa can activate factor IX, thereby further advancing the coagulation process. Excessive factor XIa in globulin products may lead to adverse reactions or affect the safety and efficacy of the product. The affinity chromatography process in this protocol also additionally removes other contaminating proteins; electrophoresis results show a significant reduction in contaminating protein bands (see [link to relevant documentation]). Figure 2 ).

[0086] Comparative Example 2

[0087] Experimental studies were conducted on the Cohn FII+III (component II+III) precipitate obtained in Example 1. First, the Cohn FII+III precipitate was treated with octanoate precipitation: the Cohn FII+III precipitate was added to 10 times its mass of water for injection, dissolved at 4°C for 2 hours, the pH was adjusted to 4.2, and after reacting for 1 hour, sodium octanoate was added to a final concentration of 0.2 mol / L, the pH was adjusted again to 5.2, and the reaction continued for another hour. The filtrate was then separated by pressure filtration. The filtrate was subjected to two-step ion exchange chromatography according to the method in Example 1 to obtain the second chromatographic product. The second chromatographic product had a relatively high content of impurity proteins, far exceeding the level of impurity proteins in the second chromatographic product obtained in Example 1 (impurity proteins included IgA, IgM, fibrinogen, albumin, etc.). If the second chromatographic product obtained by the octanoate precipitation method is subjected to subsequent processing steps (such as affinity chromatography, nanofiltration, etc.), it will negatively impact the efficiency of subsequent processes, for example, affecting the nanofiltration loading capacity and leading to lower nanofiltration efficiency (the nanofiltration loading capacity will decrease, and the impurity protein content will increase). The inventors analyzed the causes of the above phenomena and adjusted the process flow and parameters. They found that the loading amount of sample I on the 60Q column was too high (approximately 400 g / L in Example 1), and the loading amount of sample II on the 50Q column was too high (approximately 1000 g / L in Example 1). This ultimately led to unsatisfactory results in the two-step ion exchange chromatography, thus affecting subsequent processes. Research revealed that if the octanoic acid process of this comparative example were used, the loading amount of the packing material for both anion exchange chromatography steps could only be adjusted to below 200 g / L to effectively reduce the impurity content in the products after the two-step ion exchange chromatography. Therefore, the process efficiency of this scheme is far superior to that of the comparative example. This scheme uses the supernatant chromatography process of component III, with the loading amount of the first-step anion exchange chromatography being greater than 300 g / L and the loading amount of the second-step anion exchange chromatography being greater than 600-800 g / L, resulting in a significant increase in chromatographic loading. In addition, using octanoic acid precipitation makes pressure filtration difficult, and octanoic acid itself has a high viscosity, making equipment cleaning difficult.

[0088] Comparative Example 3

[0089] This technical solution uses Cohn FIII supernatant as the starting material, thereby ensuring that anti-A and anti-B hemagglutinin impurities in the material are minimized. Therefore, it eliminates the need for octanoate precipitation and conventional anti-A and anti-B affinity chromatography, ensuring that the anti-A and anti-B titers in the final product meet requirements. The inventors have discovered a difference in the distribution of anti-A and anti-B hemagglutinin impurities between Cohn FIII supernatant and Cohn FII+III precipitate for the first time. Combined with the presence of a large amount of usable immunoglobulins in Cohn FIII supernatant, a new preparation process was developed based on this discovery. During the immunoglobulin preparation process using Cohn FIII supernatant, the inventors employ heparin affinity chromatography to remove XIa / XI from the Cohn FIII supernatant, further improving product quality.

[0090] If the octanoate precipitation method described in Comparative Example 2 is used to prepare immunoglobulin products, and the loading of the two-step ion chromatography is controlled to be less than 200 g / L, and subsequent affinity chromatography is not performed, the product from the second chromatography step is directly subjected to nanofiltration and other operations as described in Example 2 to obtain the final product. Even at the expense of process efficiency (lower loading in the chromatography process), the quality of the final product is not ideal. The anti-A and anti-B titers of the final product in this comparative example are 1:64 / 1:32. Due to the lack of additional anti-A and anti-B affinity chromatography, there is a relatively high amount of residual anti-A and anti-B hemagglutinin impurities. In addition, the final product of this comparative example was found to have a high impurity content, for example, the concentration of IgM ranges from 2.0 to 3.0 g / L, indicating a high impurity content. The process effect comparison (for the obtained immunoglobulin products) is detailed in Table 3. Therefore, using FIII supernatant results in lower impurity protein content, higher IgG purity, easier purification, higher chromatographic loading, and the use of FIII pressure filtration technology is more mature and the equipment is simpler. However, using octanoic acid precipitation is difficult due to pressure filtration, and octanoic acid itself has a high viscosity, making equipment cleaning difficult.

[0091] Table 3: Technical parameters of products obtained by different processes (statistical results for multiple preparation processes)

[0092] process FIII Supernatant Process (This Process) Octyl acid precipitation process (Comparative Example 3) equipment Filter press (mature technology) Centrifuge or filter press (filter pressing is difficult) IgG purity in supernatant 80-85％ 70-80％ IgA 1.5-2.0 (g / L) 3.5-4.4 (g / L) IgM 0.2-0.5 (g / L) 2.0-3.0 (g / L) Fibrin 0.02-0.1 (g / L) 0.02-0.05 (g / L) albumin Approximately 3.0 (g / L) 2.6-3.0 (g / L) Anti-A and anti-B 1:8 / 1:16 1:64 / 1:32

[0093] Comparative Example 4

[0094] This comparative example was conducted according to Test 1 of Example 2. The conductivity of sample III and the third equilibration solution was studied, and their conductivity was controlled to be 0.5 mS / cm, 1.5 mS / cm, 3.0 mS / cm, 5.0 mS / cm, 10.0 mS / cm, and 20.0 mS / cm, respectively. The third chromatographic product was obtained by affinity chromatography, and the electrophoresis results are shown below. Figure 3In the figure, channels 1-6 from left to right represent the third-stage chromatographic products obtained under conductivity conditions of 0.5 mS / cm, 1.5 mS / cm, 3.0 mS / cm, 5.0 mS / cm, 10.0 mS / cm, and 20.0 mS / cm, respectively. It can be seen that using conductivity between 0.5 mS / cm and 1.5 mS / cm results in fewer contaminating proteins in the third-stage chromatographic products, leading to a more ideal quality of the prepared immunoglobulins. At a conductivity of 3.0 mS / cm, a small amount of contaminating proteins begins to appear.

[0095] The above descriptions are merely embodiments of the present invention, and common knowledge such as specific technical solutions and / or characteristics are not described in detail here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solutions of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A method for preparing intravenously administered human immunoglobulin, characterized in that: The following steps are performed sequentially: S1: Obtain the supernatant from Cohn FIII; S2: After ultrafiltration concentration, dialysis, dilution, conductivity adjustment, pH adjustment and filtration, the supernatant of Cohn FIII is used to obtain sample I to be loaded; sample I is subjected to the first anion exchange chromatography treatment to obtain the first chromatographic product; S3: After the first chromatographic product is adjusted for conductivity and pH and filtered, sample II is obtained; sample II is subjected to a second anion exchange chromatography treatment to obtain the second chromatographic product. S4: After the conductivity and pH of the second chromatographic product are adjusted and filtered, sample III to be loaded is obtained; sample III to be loaded is subjected to heparin affinity chromatography to obtain the third chromatographic product. S5: The third chromatography product is processed through nanofiltration, pH adjustment, concentration, dialysis, and virus inactivation to prepare human immunoglobulin product.

2. The method for preparing intravenously injected human immunoglobulin according to claim 1, characterized in that: In S2, the protein content of the sample to be loaded, I, is 10-50 g / L; the conductivity of the sample to be loaded, I, is 0.2-5.0 mS / cm, preferably 0.2-3.0 mS / cm, and more preferably 0.3-2.0 mS / cm; the pH of the sample to be loaded, I, is 5.0-7.0, preferably 5.2-6.8, and more preferably 5.4-6.

2.

3. The method for preparing intravenously injected human immunoglobulin according to claim 2, characterized in that: In S2, the first ion exchange chromatography uses a quaternary ammonium salt ion exchange packing material; the sample to be loaded, I, is loaded into the chromatography column at a loading rate of 300-600 g / L and a flow rate of 5-15 ml / min. Then wash the chromatography column with the first equilibration buffer until the baseline, collect the flow-through of the sample I to be loaded and the first equilibration buffer flowing out of the chromatography column to obtain the first chromatography product; The first equilibration solution is an acetate-sodium acetate buffer solution, and its pH and conductivity are consistent with those of the sample to be loaded, I.

4. The method for preparing intravenously injected human immunoglobulin according to claim 1, characterized in that: In S3, the conductivity of the sample II to be loaded is 0.2-5.0 mS / cm, preferably 0.2-3.0 mS / cm, and more preferably 0.3-2.0 mS / cm; the pH of the sample II to be loaded is 5.0-7.0, preferably 5.2-6.8, and more preferably 5.4-6.

2.

5. The method for preparing intravenously injected human immunoglobulin according to claim 4, characterized in that: In S3, the chromatographic column used for the second ion exchange chromatography is a quaternary ammonium salt ion exchange packing material; the sample to be loaded, product II, is loaded into the chromatographic column at a loading rate of 600-1500 g / L and a flow rate of 3-10 ml / min. Then wash the chromatography column with the second equilibration buffer until the baseline, collect the flow-through of the sample II to be loaded and the second equilibration buffer flowing out of the chromatography column to obtain the second chromatography product; The second equilibration solution is an acetate-sodium acetate buffer solution, and its pH and conductivity are consistent with those of the sample to be loaded, product II.

6. The method for preparing intravenously injected human immunoglobulin according to claim 1, characterized in that: In S3, the conductivity of the sample to be loaded, product III, is 0.2-3.0 mS / cm, preferably 0.5-1.5 mS / cm; the pH of the sample to be loaded, product III, is 5.0-7.0, preferably 5.2-6.8, and more preferably 5.4-6.

2.

7. The method for preparing intravenously injected human immunoglobulin according to claim 6, characterized in that: In S3, sample III is loaded into the chromatography column at a loading rate of 800-1500 g / L and a flow rate of 5-15 ml / min. The column is then washed with the third equilibration buffer until the baseline is reached. The flow-through of sample II and the third equilibration buffer flowing out of the column are collected to obtain the third chromatographic product. The third equilibration solution is an acetate-sodium acetate buffer solution, and its pH and conductivity are consistent with those of the sample to be loaded, product III.

8. The method for preparing intravenously injected human immunoglobulin according to claim 1, characterized in that: In S1, the CohnFIII supernatant was prepared by the following method: After centrifuging the raw plasma, collect the supernatant A. Adjust the temperature of supernatant A to -3.0 to -1.0℃, protein concentration to 40-65 g / L, pH to 6.80-7.30, conductivity to 12-14 mS / cm, and ethanol concentration to 7-10 vol.%. React for 1-3 hours, then filter and collect supernatant B. Adjust the temperature of supernatant B to -6.0 to -4.0℃, protein concentration to 30-45 g / L, pH to 5.70-6.30, and conductivity to 5.0-8.0 mS / cm. With an S / cm concentration of 18-22 vol.%, react for 1-3 hours, then collect the precipitate by pressure filtration. Add water to the precipitate and dissolve for 2-4 hours, with the water mass being 8-10 times the mass of the precipitate. Control the temperature at 0-5℃, add phosphate buffer to adjust the pH to 4.6-5.0, and react for 1-2 hours. Then add phosphate buffer to adjust the pH to 5.0-5.4, and react for 1-2 hours. Finally, adjust the ethanol concentration to 13-15 vol.%, react for 1-3 hours, and collect the supernatant by pressure filtration to obtain the Cohn FIII supernatant.

9. The method for preparing intravenously injected human immunoglobulin according to claim 1, characterized in that: In S5, the nanofiltration pore size is 20 nm; the low pH incubation parameters are pH 4.0-4.8 and incubation temperature 20-30℃.

10. The intravenous immunoglobulin product obtained by the method for preparing intravenous immunoglobulin according to any one of claims 1-9.

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