Hypodermic human immune globulin preparation and preparation method thereof

Abstract

The invention provides a human immunoglobulin preparation for subcutaneous injection and a preparation method thereof. The preparation method comprises the following steps: S1) performing FI reaction, FI + II + III separation and purification on plasma to obtain a stock solution before concentration; s2) performing ultrafiltration concentration on the stock solution before concentration by utilizing unidirectional tangential flow ultrafiltration to obtain an ultrafiltration concentrated solution; and S3) mixing the ultrafiltration concentrated solution, the protective agent and the diluent to obtain the human immune globulin preparation for subcutaneous injection. The problem that a high-concentration human immunoglobulin preparation for subcutaneous injection prepared in the prior art is low in activity can be solved, and the method is suitable for the technical fields of biological pharmacy and blood products.

Description

Technical Field

[0001] The present invention relates to the technical field of biopharmaceuticals and blood products, and in particular to a subcutaneous human immunoglobulin preparation and a preparation method thereof. Background Art

[0002] Subcutaneous human immunoglobulin (SCIG) is a human immunoglobulin product derived from human plasma. Its main component is human immunoglobulin G (IgG). In addition, it also contains a small amount of IgA, IgM, etc. It is widely used as an effective drug for the treatment of primary and secondary immunodeficiency diseases and autoimmune diseases (such as Kawasaki disease and idiopathic thrombocytopenic purpura), and has an irreplaceable role for other drugs. This product contains broad-spectrum IgG antibodies against viruses, bacteria or other pathogens. In addition, the unique type of immunoglobulin and unique type antibodies can form a complex immune network, so it has the dual therapeutic effects of immune replacement and immune regulation. After subcutaneous infusion, it can quickly increase the IgG level in the recipient's blood and enhance the body's anti-infection ability and immune regulation function.

[0003] High-concentration subcutaneous human immunoglobulin (SCIG) preparations are upgraded products of the existing intravenous human immunoglobulin products on the market. They have the advantages of good tolerance, low incidence of adverse reactions, low economic cost, high patient satisfaction, stable physiological level of serum IgG concentration, self-administration, and easy self-management by patients. In recent years, with the continuous increase in clinical indications, the demand for high-concentration subcutaneous human immunoglobulin (SCIG) preparations has maintained a high growth momentum in plasma protein products, and its production and preparation has attracted attention from all parties. However, it is difficult to prepare high-concentration subcutaneous human immunoglobulin preparations with high biological activity using the preparation methods of subcutaneous human immunoglobulin preparations disclosed in the prior art. Summary of the invention

[0004] The main purpose of the present invention is to provide a subcutaneous human immunoglobulin preparation and a preparation method thereof, so as to solve the problem that the high-concentration subcutaneous human immunoglobulin preparation prepared in the prior art has low activity.

[0005] In order to achieve the above-mentioned purpose, according to the first aspect of the present invention, there is provided a method for preparing a subcutaneous human immunoglobulin preparation, the method comprising: S1) subjecting plasma to FI reaction, FI+II+III separation and purification to obtain a pre-concentration stock solution; S2) subjecting the pre-concentration stock solution to ultrafiltration and concentration using unidirectional tangential flow ultrafiltration to obtain an ultrafiltration concentrate; S3) mixing the ultrafiltration concentrate, a protective agent and a diluent to obtain a subcutaneous human immunoglobulin preparation.

[0006] Furthermore, the unidirectional tangential flow ultrafiltration includes a 50kDa three-stage unidirectional tangential flow ultrafiltration of PES material; preferably, the parameters of the unidirectional tangential flow ultrafiltration include: the pressure at the ultrafiltration inlet end is 3.0-4.0Bar, the ratio of the flow rate at the filtration end to the flow rate at the outlet end is 1:3~4.5, and the ultrafiltration time is ≤2h.

[0007] Furthermore, the protective agent includes glycine and L-proline; preferably, the subcutaneous human immunoglobulin preparation contains 1-3wt% glycine and 1-3wt% L-proline; preferably, the pH of the diluent is 3.5-4.5; preferably, the diluent is a 1-3wt% glycine solution with a pH of 3.5-4.5.

[0008] Furthermore, in the subcutaneous human immunoglobulin preparation, the protein concentration is 15wt% to 23wt%.

[0009] Furthermore, the FI reaction includes: adjusting the plasma to pH 6.90-7.20 with an acetate buffer solution of pH 4.0, adding 50% (v / v) ethanol until the ethanol concentration in the FI reaction system is 8% (v / v), and obtaining a FI reaction solution. During the FI reaction, the temperature of the FI reaction system is controlled to be -1 to -2°C.

[0010] Furthermore, the FI+II+III separation includes: adjusting the pH of the reaction system after the FI reaction to 6.75-6.85, adding 50% (v / v) ethanol until the ethanol concentration in the FI+II+III separation system is 20% (v / v), obtaining the FI+II+III separation system, and controlling the temperature of the FI+II+III separation system to -5.0-5.4°C; filtering the FI+II+III separation system to obtain a FI+II+III precipitate.

[0011] Further, the purification includes: dissolving the FI+II+III precipitate, precipitating impurities with octanoic acid, first dialysis, ion exchange chromatography, second dialysis, filtration and acidic pH incubation in sequence to obtain a pre-concentration stock solution; preferably, dissolving and precipitating impurities with octanoic acid includes: stirring and dissolving the FI+II+III precipitate in water at 2 to 8°C, adjusting the pH of the solution to 3.7 to 4.3; adding octanoic acid to a final concentration of 10 to 30 mM octanoic acid, and then adding 0.5 M The pH value is adjusted to 4.7-5.3 by using NaOH, and the filtrate is collected after filtering to obtain the system after the precipitation of octanoic acid; preferably, the first dialysis comprises: concentrating the system after the precipitation of octanoic acid by dialysis, adjusting the pH value and the conductivity, and obtaining the first dialysis product; the volume of the first dialysis product is 30%-70% of the volume of the system after the precipitation of octanoic acid, the pH value of the first dialysis product is 4.7-5.3, and the conductivity of the first dialysis product is 1.2ms / cm-1.8ms / cm; preferably, the ion exchange chromatography comprises: performing DEAE ion exchange chromatography and TMAE ion exchange chromatography on the first dialysis product to obtain the ion exchange chromatography product Preferably, the second dialysis comprises: ultrafiltration and concentration of the ion exchange chromatography product by an ultrafiltration membrane bag to obtain an ultrafiltration concentrate; the protein concentration in the ultrafiltration concentrate is 40-60 g / L, and the pH of the ultrafiltration concentrate is 3.8-4.4; more preferably, the dialysate used in the second dialysis is a 1-3 wt% glycine solution with a pH of 3.5-4.5; more preferably, a 2 wt% glycine solution with a pH of 3.95-4.05; preferably, the filtration comprises: filtering the ultrafiltration concentrate by a nanofiltration membrane to obtain a nanofiltration post-liquid; more preferably, the nanofiltration membrane comprises a hollow regenerated cellulose nanofiltration membrane, more preferably a Planova Bio-EX nanofiltration membrane; preferably, the acidic pH incubation comprises: adjusting the pH of the nanofiltration post-liquid to 3.8-4.4, and incubating at 24-26°C for 21 days to obtain a pre-concentration stock solution.

[0012] Further, the DEAE ion exchange chromatography comprises: loading the first dialysis product onto a DEAE ion exchange resin, collecting and mixing the flow-through liquid and the washing liquid, adjusting the pH to 5.0-6.0, and obtaining a DEAE flow-through washing liquid; the TMAE ion exchange chromatography comprises: loading the DEAE flow-through washing liquid onto a TMAE ion exchange resin, collecting and mixing the flow-through liquid and the washing liquid, and obtaining an ion exchange chromatography product; preferably, the balancing liquid and the washing liquid used in the DEAE ion exchange chromatography are 20-150 mmol / L sodium acetate solutions at pH 4.5-5.5, more preferably 50 mmol / L sodium acetate solutions at pH 4.95-5.05; preferably, the balancing liquid and the washing liquid used in the TMAE ion exchange chromatography are 20-50 mmol / L sodium acetate solutions at pH 5.0-6.0, more preferably 30 mmol / L sodium acetate solutions at pH 5.45-5.55.

[0013] In order to achieve the above object, according to the second aspect of the present invention, a subcutaneous human immunoglobulin preparation is provided. The subcutaneous human immunoglobulin preparation is prepared by the preparation method of the subcutaneous human immunoglobulin preparation.

[0014] Furthermore, the subcutaneous human immunoglobulin preparation contains 1-3 wt% of glycine and 1-3 wt% of L-proline, and the protein concentration of the subcutaneous human immunoglobulin preparation is 15 wt% to 23 wt%.

[0015] By applying the technical solution of the present invention, in the preparation method of the above-mentioned subcutaneous human immunoglobulin preparation, by adopting the unidirectional tangential flow ultrafiltration (SPTFF) technology, the shear force effect on the protein molecules during the high-concentration purification and concentration process is significantly reduced, and the precipitation and precipitation problems caused by the repeated circulation of IgG protein molecules in traditional ultrafiltration (TFF) are avoided, thereby improving the stability and quality of the product. In the unidirectional tangential flow ultrafiltration (SPTFF) process of the above-mentioned preparation method, the material does not circulate in the system, and the protein molecules only need to be sheared by the pump head once, which can significantly reduce the shear force effect on the material, so that a higher concentration can be achieved, and the activity retention is better, and the biological activity of the Fc segment is as high as 85% or more, which is much higher than the European Pharmacopoeia standard (60%). BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The drawings constituting a part of the present application are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0017] Figure 1 A schematic diagram of the process flow according to Example 1 of the present invention is shown.

[0018] Figure 2 The figure shows the electrophoresis detection result of detection example 1 according to the present invention. DETAILED DESCRIPTION

[0019] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

[0020] In December 2002, CSL's Vivaglobin was first approved in Germany, becoming the first immunoglobulin specifically for subcutaneous injection, and obtained FDA certification in January 2006. Since then, SCIGs of different concentrations and specifications have appeared one after another, such as CSL's Hizentra and Shire's Cuvitru. Information on SCIG drugs approved by the FDA in the prior art is shown in Table 1 (data source: working report of the American Academy of Allergy, Asthma, and Immunology).

[0021] Table 1

[0022]

[0023] However, as mentioned in the background technology, there are few disclosures in the prior art about the preparation methods of high-concentration subcutaneous human immunoglobulin, and only some preparation methods related to low-concentration subcutaneous human immunoglobulin are disclosed. It is difficult to prepare highly active subcutaneous human immunoglobulin using the methods in the prior art.

[0024] Therefore, in the present application, the inventors attempted to develop a novel method for preparing a subcutaneous human immunoglobulin preparation, and based on this, proposed a series of protection schemes of the present application.

[0025] In a first typical embodiment of the present application, a method for preparing a subcutaneous human immunoglobulin preparation is provided, characterized in that the preparation method comprises: S1) subjecting plasma to FI reaction, FI+II+III separation and purification to obtain a pre-concentration stock solution; S2) utilizing unidirectional tangential flow ultrafiltration (SPTFF) to ultrafilter and concentrate the pre-concentration stock solution to obtain an ultrafiltration concentrate; S3) mixing the ultrafiltration concentrate, a protective agent and a diluent to obtain a subcutaneous human immunoglobulin preparation.

[0026] In the prior art, for the preparation of human immunoglobulin products, especially high-concentration human immunoglobulin products, the main process difficulty lies in the instability of IgG protein molecules at ultra-high concentrations. In the prior art, patent CN112375141B discloses a method for preparing human immunoglobulin for subcutaneous injection, which discloses a specific ultrafiltration and concentration step to make the final product concentration reach more than 20%. However, in actual production, the inventors found that the high tangential flow rate of multiple ultrafiltration and concentration during the purification and concentration process will also bring higher shear force. In traditional ultrafiltration (TFF), the protein stock solution needs to be repeatedly circulated in the system. After repeated shearing, IgG protein molecules are very likely to be unstable, such as precipitation and precipitation, thereby affecting the integrity and conformation of the protein molecules, and ultimately affecting the quality and efficacy of immunoglobulin products, such as the Fc-terminal biological activity of IgG.

[0027] In the above preparation method of the present application, by adopting the SPTFF technology, not only the recovery rate of the protein is improved, but also the denaturation of the protein during the concentration process is reduced, thereby ensuring the biological activity and stability of the final preparation, and being suitable for large-scale production of high-quality subcutaneous human immunoglobulin preparations. The product prepared by this method has an Fc segment biological activity of more than 85%, which is much higher than the European Pharmacopoeia standard (60%).

[0028] In a preferred embodiment, the unidirectional tangential flow ultrafiltration includes a 50kDa three-stage unidirectional tangential flow ultrafiltration of PES material; preferably, the parameters of the unidirectional tangential flow ultrafiltration include: the pressure at the ultrafiltration inlet end is 3.0-4.0Bar, the ratio of the flow rate at the filtration end to the flow rate at the outlet end is 1:3~4.5; the ultrafiltration time is ≤2h, including but not limited to 0.1~2h.

[0029] The above-mentioned unidirectional tangential flow ultrafiltration parameter setting can more effectively remove low molecular weight impurities while keeping high molecular weight IgG intact, and is suitable for clinical treatments that require high purity IgG, such as the treatment of immunodeficiency diseases. The ultrafiltration time is controlled at ≤2h to prevent the loss of protein activity. The unidirectional tangential flow ultrafiltration using the above-mentioned parameters is used to concentrate the protein, reduce protein denaturation, and ensure the biological activity in the final product.

[0030] In a preferred embodiment, the protective agent includes glycine and L-proline; preferably, the subcutaneous human immunoglobulin preparation contains 1-3wt% glycine and 1-3wt% L-proline; preferably, the pH of the diluent is 3.5-4.5; preferably, the diluent is a 1-3wt% glycine solution with a pH of 3.5-4.5.

[0031] In the above preparation method, glycine and L-proline are used as protective agents to protect the human immunoglobulin in the preparation, and the concentration of glycine in the final product is 1-3wt%, and the concentration of L-proline is 1-3wt%. A preparation product with a clear and transparent appearance and a slight opalescence can be prepared, and the addition of L-proline can reduce the viscosity of the subcutaneous human immunoglobulin preparation. That is, the subcutaneous human immunoglobulin preparation prepared by the above preparation method does not have the common problems of heavy opalescence, high viscosity, and yellow appearance that are common in high-concentration human immunoglobulin products in the prior art. On the contrary, if glycine is used alone as a protective agent, the final product prepared is yellow, has a poor appearance, and has a high viscosity.

[0032] The plasma used as a raw material in the preparation method of the present application can be prepared by those skilled in the art using methods in the prior art, and the present application does not limit the specific source and preparation method of the plasma.

[0033] The preparation method used in this application is as follows:

[0034] 1. The collection and quality of plasma must comply with the provisions of "Human Plasma for Blood Product Production" in the 2020 edition (Part III) of the "Chinese Pharmacopoeia", and the plasma protein content should not be less than 50g / L.

[0035] 2. Plasma testing: Each plasma returned to the factory is retested one by one to test the appearance, protein content, alanine aminotransferase (ALT), HBsAg, syphilis spirochete, HIV-1 and HIV-2 antibodies, HCV antibodies, HBV DNA, HCVRNA, HIV RNA. Plasma that passes the retest and meets the quarantine period can be put into production. During the production process, mixed plasma is sampled and tested for HBsAg, HIV-1 and HIV-2 antibodies, HCV antibodies, hepatitis B virus surface antibodies, HBV DNA, HCVRNA, HIV RNA.

[0036] 3. After plasma is collected, it should be quickly frozen within 6 hours and stored at -20℃ or below. The storage period should not exceed 3 years from the date of plasma collection.

[0037] 4. The raw plasma used complies with the relevant regulations and requirements during the quarantine period.

[0038] In a preferred embodiment, the protein concentration of the subcutaneous human immunoglobulin preparation is 15 wt% to 23 wt%.

[0039] The above protein concentration is the concentration of total protein in the preparation, and the immunoglobulin content in the total protein can reach 99% or more. In the above preparation method, a subcutaneous human immunoglobulin preparation with a higher protein concentration can be prepared, which not only reduces the number of injections for patients and improves the convenience of treatment, but also reduces the volume of the preparation, making it easy to carry and use, and is suitable for patients with chronic diseases who need frequent injections for treatment.

[0040] In a preferred embodiment, the FI reaction includes: adjusting the plasma to pH 6.90-7.20 with a pH 4.0 acetate buffer, adding 50% (v / v) ethanol until the ethanol concentration in the FI reaction system is 8% (v / v), and obtaining a FI reaction solution. During the FI reaction, the temperature of the FI reaction system is controlled to be -1 to -2°C.

[0041] In the above preparation method, FI reaction refers to the preliminary precipitation reaction of plasma. Specifically, FI reaction is to change the solubility of protein in plasma by adjusting the pH value of plasma and adding ethanol, so that certain components in plasma, especially components other than IgG, such as fibrinogen, are precipitated, thereby performing preliminary purification. This process helps to improve the purity and yield of IgG in subsequent purification steps. Through this reaction, a large amount of non-IgG components can be effectively separated from plasma, providing purer raw materials for subsequent purification steps.

[0042] In a preferred embodiment, FI+II+III separation includes: adjusting the pH of the reaction system after the FI reaction to 6.75-6.85, the reagents used to adjust the pH include but are not limited to pH 4.0 acetate buffer, and then adding 50% (v / v) ethanol until the ethanol concentration in the FI+II+III separation system is 20% (v / v) to obtain the FI+II+III separation system, controlling the temperature of the FI+II+III separation system to -5.0 to -5.4°C; filtering the FI+II+III separation system to obtain a FI+II+III precipitate.

[0043] In the above preparation method, FI+II+III separation is a step for further purification and concentration of IgG, which is carried out after the FI reaction, and the purpose is to remove more non-IgG components in the plasma, improve the purity and concentration of IgG, and maintain its biological activity. The above specific reaction conditions can more effectively precipitate and separate IgG and reduce the co-precipitation of impurity proteins.

[0044] In a preferred embodiment, the purification comprises: dissolving the FI+II+III precipitate, precipitating impurities with octanoic acid, first dialysis, ion exchange chromatography, second dialysis, filtration and acidic pH incubation in sequence to obtain a pre-concentration stock solution; preferably, dissolving and precipitating impurities with octanoic acid comprises: stirring and dissolving the FI+II+III precipitate in water at 2 to 8°C, adjusting the pH of the solution to 3.7 to 4.3; adding octanoic acid to a final concentration of 10 to 30 mM octanoic acid, and then adding 0.5 M The pH value is adjusted to 4.7-5.3 by NaOH, and the filtrate is collected after filtration to obtain the system after the precipitation of octanoic acid; preferably, the first dialysis comprises: concentrating the system after the precipitation of octanoic acid by dialysis, adjusting the pH value and the conductivity, and obtaining the first dialysis product; the volume of the first dialysis product is 30%-70% of the volume of the system after the precipitation of octanoic acid, the pH value of the first dialysis product is 4.7-5.3, and the conductivity of the first dialysis product is 1.2ms / cm-1.8ms / cm; preferably, the ion exchange chromatography comprises: performing DEAE ion exchange chromatography and TMAE ion exchange chromatography on the dialysis product to obtain an ion exchange chromatography product ; Preferably, the second dialysis comprises: ultrafiltration and concentration of the ion exchange chromatography product using an ultrafiltration membrane bag to obtain an ultrafiltration concentrate; the protein concentration in the ultrafiltration concentrate is 40-60 g / L, and the pH of the ultrafiltration concentrate is 3.8-4.4; more preferably, the dialysate used in the second dialysis is a 1-3 wt% glycine solution with a pH of 3.5-4.5; more preferably, a 2 wt% glycine solution with a pH of 3.95-4.05; preferably, the filtration comprises: filtering the ultrafiltration concentrate using a nanofiltration membrane to obtain a nanofiltration post-liquid; more preferably, the nanofiltration membrane comprises a hollow regenerated cellulose nanofiltration membrane, more preferably a Planova Bio-EX nanofiltration membrane; preferably, the acidic pH incubation comprises: adjusting the pH of the nanofiltration post-liquid to 3.8-4.4, incubating at 24-26°C for 21 days, and obtaining a pre-concentration stock solution.

[0045] By using the above purification method and parameters, impurity proteins, bacteria, viruses, etc. in the product can be further removed, ensuring the safety and effectiveness of the final preparation. In the above purification method, caprylic acid is used as a precipitant for pretreatment, and then a high-purity, low-IgM, low-IgA IgG solution is obtained through chromatography technology. Finally, the residual viruses in the product are inactivated and removed by combining filtration technology and traditional low-pH incubation method, which not only ensures the quality of the product but also ensures the safety of the product. This process reduces the inactivation effect of ethanol on protein in the traditional process of human immunoglobulin products, as well as the consumption of protein by pasteurization, shortens the production cycle, improves the purity of the product, and at the same time improves the yield of the product.

[0046] In a preferred embodiment, DEAE ion exchange chromatography comprises: loading the first dialyzed product onto DEAE ion exchange resin, collecting and mixing the flow-through liquid and the washing liquid, adjusting the pH to 5.0-6.0, and obtaining a DEAE flow-through washing liquid; TMAE ion exchange chromatography comprises: loading the DEAE flow-through washing liquid onto TMAE ion exchange resin, collecting and mixing the flow-through liquid and the washing liquid, and obtaining an ion exchange chromatography product; preferably, the balancing liquid and the washing liquid used in the DEAE ion exchange chromatography are 20-150 mmol / L sodium acetate solutions at pH 4.5-5.5, more preferably 50 mmol / L sodium acetate solutions at pH 4.95-5.05; preferably, the balancing liquid and the washing liquid used in the TMAE ion exchange chromatography are 20-50 mmol / L sodium acetate solutions at pH 5.0-6.0, more preferably 30 mmol / L sodium acetate solutions at pH 5.45-5.55.

[0047] By fine-tuning the conditions of ion exchange chromatography, IgG can be separated and purified more effectively, and the loss of IgG can be reduced, making it suitable for clinical applications that require high yield and high purity IgG.

[0048] In a second typical embodiment of the present application, a subcutaneous human immunoglobulin preparation is provided. The subcutaneous human immunoglobulin preparation is prepared by the above-mentioned preparation method of subcutaneous human immunoglobulin preparation.

[0049] In a preferred embodiment, the human immunoglobulin preparation for subcutaneous injection contains 1-3 wt% of glycine and 1-3 wt% of L-proline, and the protein concentration of the human immunoglobulin preparation for subcutaneous injection is 15 wt% to 23 wt%.

[0050] By using the above-mentioned preparation method, a relatively high concentration subcutaneous human immunoglobulin preparation (i.e., SCIG preparation) can be prepared, and the prepared subcutaneous human immunoglobulin preparation has a clear and transparent appearance with a slight opalescence, and does not have the common problems of high-concentration human immunoglobulin products such as heavy opalescence, high viscosity, and yellowing appearance.

[0051] The beneficial effects of the present application will be further explained in detail below in conjunction with specific embodiments.

[0052] Example 1

[0053] Removal of plasma cryoprecipitate: fresh frozen human plasma is used as raw material, and the cryoprecipitate in the plasma is removed through processes such as thawing, mixing, and continuous centrifugation to obtain 10 kg of plasma centrifuge supernatant without cryoprecipitate;

[0054] FI reaction: adjust the plasma to pH 7.10 with pH 4.0 acetate buffer, add 50% ethanol to a reaction solution ethanol concentration of 8%, and control the reaction solution temperature at -1°C.

[0055] FI+II+III separation: adjust the pH value of the FI reaction solution to 6.80, add 50% ethanol until the ethanol concentration of the reaction solution is 20%. Control the temperature of the reaction solution at -5.0°C, add diatomaceous earth, stir and filter, and collect 1.1 kg of precipitate.

[0056] Precipitate dissolution and caprylic acid precipitation reaction: Dissolve FI+II+III precipitate 15 times in 4°C water for injection, stir and dissolve for 30 minutes, adjust the solution pH to 4.0, and continue stirring for 2 hours. Add caprylic acid (room temperature) directly to a final concentration of 20 mM, adjust the pH to 5.0 with 0.5 M NaOH, and stir slowly for 5 hours.

[0057] Ultrafiltration dialysis: Filter the caprylic acid precipitation reaction solution and concentrate it to 60% of the total volume. Adjust the sample pH to 5.0, the conductivity to 1.4 ms / cm, and collect 10 kg of the concentrate.

[0058] DEAE ion exchange chromatography: Load the equilibrated DEAE ion exchange chromatography column, wash the gel after injection, and collect 11.5 kg of flow-through and washing liquid. Adjust the pH of the flow-through washing liquid after column chromatography to 5.5, filter it, and use it in the next chromatography step.

[0059] TMAE ion exchange chromatography: The filtered solution was loaded onto the equilibrated TMAE ion exchange chromatography column. After the injection, the gel was washed and a total of 12.7 kg of the flow-through liquid and the washing liquid were collected.

[0060] Ultrafiltration dialysis: The flow-through washing liquid from the previous step of chromatography was concentrated to 1.1 kg using an ultrafiltration membrane package, and dialyzed 10 times with Buffer N. The final protein concentration reached 56 g / L. The ultrafiltration concentrate was collected and the pH of the ultrafiltration solution was adjusted to 4.2.

[0061] Nanomembrane filtration: After pre-filtration, the ultrafiltration dialysate was passed through the Planova Bio-EX nanofiltration membrane at a constant pressure to remove any possible residual viruses, and 1.13 kg of the post-nanofiltration liquid was collected.

[0062] Low pH incubation: After sterilization, the pH of the nanofiltration solution was adjusted to 3.8-4.4 according to the low pH incubation requirements, and the incubation temperature was controlled at 24-26°C for 21 days.

[0063] Ultrafiltration concentration: After mixing with low pH incubation liquid, use SPTFF ultrafiltration concentration method, use ultrafiltration membrane package for ultrafiltration concentration, control the ultrafiltration inlet pressure to 3.0-4.0Bar, the flow rate at the filtration end and the flow rate at the outlet end to be 1:3-4.5, and the ultrafiltration time is kept within 2h. When the protein concentration reaches above 195g / L, collect 280mL of ultrafiltration concentrate and adjust the pH to 6.0.

[0064] Liquid preparation: Add 28 mL of a mixed protective agent containing 20 wt% L-proline and 20 wt% glycine to the ultrafiltration concentrate, and the final concentrations of L-proline and glycine are both 2 wt%. After testing, the protein content of the original solution is 211 g / L, and 310 mL of filtrate is collected by filtering with a 0.22 μm filter head;

[0065] Packaging: Packaging according to the specification of 50.1mL / bottle, and a total of 6 bottles of high-concentration subcutaneous human immunoglobulin (SCIG) preparations are prepared.

[0066] The process flow diagram of Example 1 is as follows Figure 1 shown.

[0067] Test Example 1

[0068] The high-concentration subcutaneous human immunoglobulin (SCIG) preparation prepared in Example 1 was subjected to SDS-page electrophoresis to determine the distribution of the main protein species in the finished product. Detection operation steps:

[0069] (1) Wash and dry the glass plate;

[0070] (2) Fix the glass plate on the glue-filling bracket, and apply force evenly on both sides to prevent the glass plate from being damaged;

[0071] (3) Prepare 8% separation gel in proportion and quickly add it with a pipette until it reaches about 1 cm from the upper edge. Then add a small amount of water to seal the solution and let it stand for about 40 min.

[0072] (4) Pour out the water and absorb the remaining water with filter paper, prepare 5% concentrated glue, add concentrated glue continuously and steadily until it is 5 mm away from the edge, quickly insert the sample comb, and let it stand for 30-40 minutes;

[0073] (5) After adding buffer solution into the upper tank, pull out the sample comb to expel all bubbles;

[0074] (6) Sample loading: Take 4 times the volume of protein sample solution into an EP tube, then add 1 times the sample buffer (5X), and the sample volume is 10 μL; the marker is directly dissolved and loaded;

[0075] (7) Before adding the sample, the sample was heated in boiling water for 3 min to remove metastable polymerization;

[0076] (8) When adding samples, the height should be controlled to prevent diffusion or edge effects;

[0077] (9) Add buffer to the electrophoresis tank, turn on the power supply, and perform electrophoresis. Initially, the voltage is controlled at 80 V. After entering the separation gel, change it to 121 V. When the bromophenol blue is about 5 mm away from the edge of the gel, stop the electrophoresis.

[0078] (10) After electrophoresis, pry open the glass plate, mark the gel plate and place it in a large culture dish, add staining solution, stain for about 10 minutes, and then destain with destaining solution until the protein bands are clear.

[0079] The results of SDS-page electrophoresis are as follows Figure 2 As shown, the rightmost lane is the IgG standard, and the remaining lanes are all high-concentration subcutaneous human immunoglobulin preparations. The results show that the positions of the main protein bands of the high-concentration subcutaneous human immunoglobulin (SCIG) preparations prepared in Example 1 are basically consistent with those of the IgG standard, and the impure protein bands are almost invisible, indicating that the high-concentration subcutaneous human immunoglobulin (SCIG) preparations prepared in Example 1 have high purity and fewer impure protein types.

[0080] Test Example 2

[0081] The test results of all items of the finished products of high-concentration subcutaneous human immunoglobulin (SCIG) preparations produced in Example 1 and two other batches (hereinafter referred to as Batch A and Batch B) prepared by the same method as Example 1.

[0082] The finished products of high-concentration subcutaneous human immunoglobulin (SCIG) preparations produced in Example 1, batch A and batch B were tested for all items. The test items were set with reference to the quality standard of human immunoglobulin finished products in European Pharmacopoeia 8.0. The test results are shown in Table 2: All quality indicators of the above three batches of products were qualified, and they had high purity and high Fc segment biological activity. The biological activity of the Fc segment of the finished product was as high as 85%, which was much higher than the European Pharmacopoeia standard (60%), and it contained almost no impurities such as Ig A and Ig M. At the same time, the finished product had a clear and transparent appearance with a slight opalescence, and did not have common problems of high-concentration human immunoglobulin products such as heavy opalescence, high viscosity, and yellowing appearance.

[0083] Table 2

[0084]

[0085] Comparative Example 1

[0086] The only difference from the preparation method in Example 1 is that the feed solution incubated at low pH is concentrated by using the conventional TFF ultrafiltration concentration in the prior art instead of SPTFF ultrafiltration concentration.

[0087] The method of TFF ultrafiltration concentration is as follows:

[0088] Select a suitable ultrafiltration membrane package according to the molecular weight of the protein to be ultrafiltered. The molecular weight of the ultrafiltration membrane package is generally 1 / 3 to 1 / 6 of the molecular weight of the target protein. After the ultrafiltration system is cleaned and qualified, transfer the pre-treated feed liquid to the ultrafiltration tank, turn on the ultrafiltration system and the circulation pump, control the pressure at the inlet end of the ultrafiltration system to not exceed 3 bar, the pressure at the outlet end to not exceed 1 bar, and open the dialysis outlet. The solution enters the ultrafiltration system: the solution enters the ultrafiltration system through the feed pipe and starts the concentration process. During the concentration process, the target protein with a larger molecular weight than the ultrafiltration membrane package is retained, and the substance with a smaller molecular weight than the ultrafiltration membrane package is discharged through the dialysis outlet. During the concentration process, check regularly whether there is protein leakage in the dialysis outlet. After the ultrafiltration concentration is completed, use a suitable buffer to recover the residual protein in the ultrafiltration system and merge it into the concentrated solution.

[0089] The subsequent liquid preparation and packaging methods are the same as in Example 1.

[0090] Test Example 3

[0091] Three batches of samples were prepared using the method of Example 1, and two batches of samples were prepared using the method of Comparative Example 1. The Fc segment biological activity test was performed on the above samples, and the results are shown in Table 3.

[0092] Table 3 Results of biological activity detection of Fc segment by different ultrafiltration methods

[0093]

[0094] The results showed that the subcutaneous human immunoglobulin preparation prepared by the method of Example 1 (i.e., SPTFF ultrafiltration and concentration in Table 3) had a significantly higher biological activity of the Fc segment than the preparation prepared by the method of Comparative Example 1 (i.e., traditional TFF ultrafiltration and concentration in Table 3).

[0095] From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects: in the preparation method of the above subcutaneous human immunoglobulin preparation, by adopting the unidirectional tangential flow ultrafiltration (SPTFF) technology, the shear force effect on protein molecules during the high-concentration purification and concentration process is significantly reduced, the stability and quality of the product are improved, and a product with higher concentration can be prepared, and the activity retention is better. The biological activity of the Fc segment is as high as 85%, which is much higher than the European Pharmacopoeia standard (60%). And by using L-proline and glycine as protective agents, a product with a clear and transparent appearance and a slight opalescence can be prepared, which has a better appearance than the product using glycine alone as a protective group.

[0096] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing a subcutaneous human immunoglobulin preparation, characterized in that: The preparation method comprises: S1) performing FI reaction, FI+II+III separation and purification on the plasma to obtain a pre-concentration stock solution; S2) ultrafiltration and concentration of the pre-concentration stock solution by unidirectional tangential flow ultrafiltration to obtain an ultrafiltration concentrate; S3) mixing the ultrafiltration concentrate, the protective agent and the diluent to obtain the subcutaneous human immunoglobulin preparation.

2. The preparation method according to claim 1, characterized in that: The unidirectional tangential flow ultrafiltration includes a 50kDa three-stage unidirectional tangential flow ultrafiltration of PES material; Preferably, the parameters of the unidirectional tangential flow ultrafiltration include: the pressure at the ultrafiltration inlet end is 3.0-4.0 Bar, the ratio of the flow rate at the filtration end to the flow rate at the outlet end is 1:3-4.5, and the ultrafiltration time is ≤2h.

3. The preparation method according to claim 1, characterized in that: The protective agent includes glycine and L-proline; Preferably, the subcutaneous human immunoglobulin preparation contains 1 to 3 wt% of glycine and 1 to 3 wt% of L-proline; Preferably, the pH of the diluent is 3.5 to 4.5; Preferably, the diluent is a 1-3 wt% glycine solution with a pH of 3.5-4.

5.

4. The preparation method according to claim 1, characterized in that: In the subcutaneous human immunoglobulin preparation, the protein concentration is 15wt% to 23wt%.

5. The preparation method according to claim 1, characterized in that: The FI reaction includes: After adjusting the plasma to pH 6.90-7.20 with an acetate buffer of pH 4.0, 50% (v / v) ethanol is added until the ethanol concentration in the FI reaction system is 8% (v / v) to obtain a FI reaction solution. During the FI reaction, the temperature of the FI reaction system is controlled to be -1 to -2°C.

6. The preparation method according to any one of claims 1 to 5, characterized in that: The FI+II+III separation comprises: The pH of the reaction system after the FI reaction is adjusted to 6.75-6.85, 50% (v / v) ethanol is added until the ethanol concentration in the FI+II+III separation system is 20% (v / v), to obtain the FI+II+III separation system, and the temperature of the FI+II+III separation system is controlled to be -5.0-5.4°C; the FI+II+III separation system is filtered to obtain a FI+II+III precipitate.

7. The preparation method according to claim 6, characterized in that: The purification comprises: The FI+II+III precipitate is sequentially subjected to dissolution, octanoic acid precipitation of impurities, first dialysis, ion exchange chromatography, second dialysis, filtration and acidic pH incubation to obtain the pre-concentration stock solution; Preferably, said dissolving and said caprylic acid precipitating impurities comprises: The FI+II+III precipitate is stirred and dissolved in water at 2-8°C, and the pH of the solution is adjusted to 3.7-4.3; octanoic acid is added until the final concentration of octanoic acid is 10-30 mM, and then 0.5 M NaOH is added to adjust the pH to 4.7-5.3, and the filtrate is collected after filtering to obtain a system after octanoic acid precipitation; Preferably, the first dialysis comprises: The system after the octanoic acid precipitation is concentrated by dialysis, and the pH and conductivity are adjusted to obtain a first dialysis product; the volume of the first dialysis product is 30% to 70% of the volume of the system after the octanoic acid precipitation, the pH of the first dialysis product is 4.7 to 5.3, and the conductivity of the first dialysis product is 1.2 ms / cm to 1.8 ms / cm; Preferably, the ion exchange chromatography comprises: The first dialyzed product is subjected to DEAE ion exchange chromatography and TMAE ion exchange chromatography to obtain an ion exchange chromatography product; Preferably, the second dialysis comprises: The ion exchange chromatography product is ultrafiltered and concentrated using an ultrafiltration membrane package to obtain an ultrafiltration concentrate; the protein concentration in the ultrafiltration concentrate is 40 to 60 g / L, and the pH of the ultrafiltration concentrate is 3.8 to 4.4; More preferably, the dialysate used in the second dialysis is a 1-3 wt % glycine solution at pH 3.5-4.5; more preferably a 2 wt % glycine solution at pH 3.95-4.05; Preferably, the filtration comprises: filtering the ultrafiltration concentrate using a nanofiltration membrane to obtain a nanofiltration post-liquid; More preferably, the nanofiltration membrane comprises a hollow regenerated cellulose nanofiltration membrane, more preferably a Planova Bio-EX nanofiltration membrane; Preferably, the acidic pH incubation comprises: adjusting the pH of the post-nanofiltration solution to 3.8-4.4, incubating at 24-26° C. for 21 days, and obtaining the pre-concentration stock solution.

8. The preparation method according to claim 7, characterized in that: The DEAE ion exchange chromatography comprises: The first dialyzed product is loaded onto a DEAE ion exchange resin, and the flow-through liquid and the washing liquid are collected and mixed, and the pH is adjusted to 5.0 to 6.0 to obtain a DEAE flow-through washing liquid; The TMAE ion exchange chromatography comprises: Loading the DEAE flow-through washing liquid onto TMAE ion exchange resin, collecting and mixing the flow-through liquid and the washing liquid to obtain the ion exchange chromatography product; Preferably, the balancing solution and washing solution used in the DEAE ion exchange chromatography are 20-150 mmol / L sodium acetate solution at pH 4.5-5.5, more preferably 50 mmol / L sodium acetate solution at pH 4.95-5.05; Preferably, the balancing solution and washing solution used in the TMAE ion exchange chromatography are 20-50 mmol / L sodium acetate solution with a pH of 5.0-6.0, and more preferably 30 mmol / L sodium acetate solution with a pH of 5.45-5.

55.

9. A human immunoglobulin preparation for subcutaneous injection, characterized in that: The subcutaneous human immunoglobulin preparation is prepared by the method for preparing the subcutaneous human immunoglobulin preparation according to any one of claims 1 to 8.

10. The subcutaneous human immunoglobulin preparation according to claim 9, characterized in that: The subcutaneous human immunoglobulin preparation contains 1-3 wt% of glycine and 1-3 wt% of L-proline, and the protein concentration of the subcutaneous human immunoglobulin preparation is 15 wt%-23 wt%.

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