Process for the preparation of immunoglobulins

Abstract

The application provides a preparation method of immunoglobulin, which comprises the following steps: (1) dissolving plasma components I+II+III / II+III precipitate, filtering, and collecting supernatant; (2) concentrating the supernatant, then purifying and performing nanofiltration treatment, and collecting filtrate; and (3) incubating at a temperature of 35 DEG C to 40 DEG C and a low pH for 20 h or less, to obtain immunoglobulin liquid. The method can effectively inactivate viruses, improve the stability of proteins, prevent agglomeration and precipitation, and ensure the safety and effectiveness of the product. Moreover, the method does not need to rely on high-temperature virus inactivation, avoids the loss of protein activity caused by high temperature, shortens the incubation time at a low pH, is safe, and is suitable for large-scale production and application.

Description

Technical Field

[0001] This application relates to the field of biomedicine. Specifically, this application relates to a method for preparing immunoglobulins. Background Technology

[0002] Immunoglobulins (Ig) are essential components of the human immune system. They are a class of animal proteins with antibody activity, playing a crucial role in recognizing bacteria and viruses and activating complement in the human body. Human plasma immunoglobulins can be classified into five classes: IgG, IgA, IgM, IgD, and IgE, with IgG being the most prevalent. The most significant characteristic of IgG is its ability to specifically recognize antigens and bind to them to form complexes. These complexes are then phagocytosed by phagocytes, rendering the antigens non-toxic and non-pathogenic.

[0003] Human immunoglobulins can be prepared by separating, purifying, inactivating, and removing viruses from the plasma or serum of healthy individuals. The safety of blood products has always been a major concern. Despite rigorous donor screening and plasma testing, viral contamination during the "window period" is unavoidable. Therefore, virus inactivation measures must be employed in the blood product manufacturing process. Although ethanol used in the low-temperature ethanol method is generally recognized as effective at inactivating viruses, additional virus inactivation measures are necessary to ensure greater product safety. Furthermore, the chosen virus inactivation method must ensure that product quality and biological activity are not affected, and that the impact on product yield is minimal.

[0004] Currently, the virus inactivation / removal processes in IgG production generally employ low pH incubation, S / D method, nanofiltration, and pasteurization. However, existing technologies still have many limitations: excessively long low pH incubation time, cumbersome virus inactivation steps, and high virus inactivation temperature all have adverse effects on production efficiency and product quality.

[0005] For example, patent CN201810036376.6 provides a production process for intravenous immunoglobulin, which uses a traditional incubation method, involving 21 days of low-pH incubation at 25°C followed by 21 days of low-pH treatment at 25°C. Although this method can obtain a product with a molecular size distribution of 99.2% for IgG monomers and dimers and has a certain virus removal effect, the long incubation time significantly reduces production efficiency and is difficult to meet the needs of modern industrial production.

[0006] Therefore, developing a virus inactivation process that is efficient, safe, and has minimal impact on product quality has become a pressing technical challenge in the current IgG production field. Summary of the Invention

[0007] This application aims to at least partially address the technical problems existing in the prior art. To this end, this application proposes a method for preparing immunoglobulins. This method effectively inactivates viruses while improving protein stability, preventing aggregation and precipitation, and ensuring the safety and efficacy of the product. Furthermore, this method does not rely on high-temperature virus inactivation, avoiding protein activity loss caused by high temperatures, shortening the low-pH incubation time, and offering high safety, making it suitable for large-scale production applications.

[0008] This application discloses a method for preparing immunoglobulins. According to embodiments of this application, the method includes:

[0009] (1) Dissolve the plasma component I+II+III / II+III precipitate, filter, and collect the supernatant;

[0010] (2) The supernatant is concentrated and then purified and nanofiltration is performed to collect the filtrate;

[0011] (3) Incubate at a low pH of 35℃~40℃ for less than 20 hours to obtain immunoglobulin solution.

[0012] According to the method described in this application, a two-step virus inactivation process is employed, consisting of first performing nanofiltration followed by low-pH incubation (temperature controlled at 35–40°C, incubation time less than 20 hours), achieving highly efficient virus inactivation of immunoglobulins. This method not only simplifies the virus inactivation steps but also significantly shortens the incubation time, while maintaining protein stability, avoiding aggregation and precipitation. Furthermore, it eliminates the need for a high-temperature inactivation process, thereby effectively killing viruses and improving the efficiency of the preparation process and the safety of the product.

[0013] According to embodiments of this application, the above-described method for preparing immunoglobulins may further include the following additional technical features:

[0014] According to an embodiment of this application, the incubation time is 10h to 20h.

[0015] According to an embodiment of this application, the pH is lowered to 3-5, more specifically to 4.0, and the mixture is incubated at 37°C at low pH for 10-15 hours to obtain the immunoglobulin solution.

[0016] According to embodiments of this application, the method does not include the step of virus inactivation treatment at a temperature above 60°C.

[0017] According to an embodiment of this application, the purification process includes a chromatography process; the chromatography process includes a first chromatography process, a second chromatography process, and a third chromatography process.

[0018] According to an embodiment of this application, the first chromatography process includes: passing the supernatant through an anion exchange chromatography column A, and collecting the anion exchange chromatography column A, which includes a Fractogel EMDDEAE, CaptoQ, or UNOsphereQ anion exchange chromatography column;

[0019] The second chromatography process includes passing the flow-through solution obtained from the first chromatography process through an anion exchange chromatography column B, wherein the anion exchange chromatography column B includes a Fractogel EMDTMAE, CaptoQXP, or NuviaHPQ anion exchange chromatography column.

[0020] The third chromatography process includes passing the flow-through solution obtained from the second chromatography process through a chromatography column C, wherein the chromatography column C includes an EshmunoPAnti-A / Anti-B mixed column, an EshmunoPAnti-A / Anti-B tandem column, an EshmunoPAnti-A column, or an EshmunoPAnti-B column.

[0021] According to an embodiment of this application, the equilibration solution for the first chromatography process includes a first sodium acetate buffer solution, the concentration of which is 5 mM to 20 mM and the pH value is 4.50 to 5.50. The first sodium acetate buffer solution is used for equilibration and sample loading at a linear velocity of 60 cm / h to 200 cm / h.

[0022] According to an embodiment of this application, the equilibration solution for the second chromatography process includes a second sodium acetate buffer solution, the concentration of which is 5 mM to 30 mM and the pH value is 5.50 to 6.50. The second sodium acetate buffer solution is used for equilibration and sample loading at a linear velocity of 10 cm / h to 100 cm / h.

[0023] According to an embodiment of this application, the equilibration solution for the third chromatography treatment includes at least one of acetate, citrate, phosphate, and chloride; the total concentration of at least one of acetate, citrate, phosphate, and chloride is 5 mM to 200 mM, and the pH value is 4.50 to 7.30; at least one of acetate, citrate, phosphate, and chloride is equilibrated and loaded at a linear velocity of 50 cm / h to 200 cm / h.

[0024] According to an embodiment of this application, the method for obtaining the plasma component I+II+III / II+III precipitate includes: precipitating the plasma with ethanol.

[0025] According to an embodiment of this application, the plasma component I+II+III / II+III precipitate is dissolved in water for injection, mixed with caprylic acid or caprylate, and the pH is adjusted to 4.0-5.0. The mixture is then filtered under pressure, and the supernatant is collected.

[0026] According to the embodiments of this application, the final concentration of caprylic acid or caprylate is 15-35 mmol / L. The pH value is adjusted to 4.5-5.2, then a filter aid is added and stirred, filtered by pressure, and the supernatant is collected.

[0027] According to an embodiment of this application, in step (3), the filtrate is incubated together with a protective agent.

[0028] According to embodiments of this application, the protective agent is selected from any one or more of polysorbate, maltose, glycine, sodium caprylate, and sorbitol.

[0029] According to embodiments of this application, the protective agent is selected from polysorbate.

[0030] According to embodiments of this application, the polysorbate is selected from any one or more of polysorbate 20, polysorbate 40, polysorbate 60, and polysorbate 80.

[0031] In a specific embodiment of this application, the polysorbate is polysorbate 80.

[0032] According to an embodiment of this application, the protective agent is selected from polysorbate, and the amount of the protective agent added based on 1 kg of the filtrate is 5 mg to 35 mg, preferably 15 mg to 25 mg;

[0033] Alternatively, the protective agent is selected from maltose, and the amount of the protective agent added is 80g to 120g based on 1L of the filtrate;

[0034] Alternatively, the protective agent is selected from glycine, and the amount of the protective agent added is 10g to 40g based on 1L of the filtrate;

[0035] Alternatively, the protective agent may be selected from sodium octanoate, and the final concentration of the protective agent may be 0.1 mM to 0.2 mM.

[0036] According to an embodiment of this application, before step (3), the filtrate is ultrafiltered and concentrated to a protein concentration of 15-25 g / L.

[0037] According to an embodiment of this application, the method further includes step (4): concentrating the reaction solution obtained from incubation by ultrafiltration, adding 1 w / w% to 5 w / w% glycine for preparation, adjusting the pH value to 4.2 to 5.4, and obtaining the immunoglobulin solution.

[0038] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0039] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0040] Figure 1 A flowchart illustrating a method for preparing an immunoglobulin according to an embodiment of this application is shown. Detailed Implementation

[0041] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0042] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0043] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0044] In this document, the terms “comprising” or “including” are open-ended expressions, meaning that they include the contents specified in this invention, but do not exclude other aspects.

[0045] In this document, the terms “optionally,” “optionally,” or “optionally” generally refer to an event or condition that may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0046] This application discloses a method for preparing immunoglobulins. The immunoglobulins prepared by this method can be used for intravenous injection or subcutaneous injection. In one embodiment provided in this application, the immunoglobulin is used for intravenous injection.

[0047] According to an embodiment of this application, see Figure 1 The method includes: S100 dissolving plasma component precipitate and collecting supernatant, S200 purification treatment, S300 nanofiltration treatment, and S400 low pH incubation. Each step will be described in detail below.

[0048] S100 dissolves plasma component precipitates and collects the supernatant.

[0049] In this step, the plasma component I+II+III precipitate or plasma component II+III precipitate is dissolved, precipitated with caprylic acid, filtered, and the supernatant is collected.

[0050] According to embodiments of this application, the method for obtaining plasma component I+II+III precipitation or plasma component II+III precipitation includes: precipitating plasma with ethanol. Ethanol is used to treat the plasma, causing protein components (such as albumin, immunoglobulins, etc.) to precipitate. In this step, the Cohn 6 method is used to precipitate the proteins in the plasma stepwise according to their different solubilities, obtaining component I+II+III or II+III. The precipitate contains multiple proteins, among which immunoglobulins (such as IgG) are the main target components.

[0051] According to embodiments of this application, the plasma component I+II+III precipitate or plasma component II+III precipitate is dissolved in water for injection, mixed with caprylic acid or caprylate, and the pH is adjusted to 4.0–5.0. The mixture is then filtered under pressure, and the supernatant is collected. Caprylic acid or caprylate has a hydrophobic effect, which can promote the precipitation of acidic proteins in the plasma. IgG molecules with a higher isoelectric point have sufficient charge to neutralize the hydrophobic effect of the caprylate ion, thus remaining in the supernatant. Separation can be achieved by pressure filtration. In some embodiments, the caprylate is selected from sodium caprylate, and the amount of water for injection used is 8–12 times (e.g., 8, 9, 10, 11, or 12 times) of ethanol for precipitation.

[0052] According to embodiments of this application, the final concentration of caprylic acid or caprylate is 15–35 mmol / L, for example, 15 mmol / L, 15 mmol / L, 16 mmol / L, 18 mmol / L, 20 mmol / L, 22 mmol / L, 24 mmol / L, 25 mmol / L, 26 mmol / L, 28 mmol / L, 30 mmol / L, 32 mmol / L, 34 mmol / L, or 35 mmol / L. This allows for sufficient precipitation of acidic proteins by caprylic acid or caprylate.

[0053] According to embodiments of this application, the pH value is adjusted to 4.5–5.2, then a filter aid is added and stirred, followed by pressure filtration and collection of the supernatant. This facilitates more complete precipitation of acidic proteins, which is beneficial for immunoglobulin extraction. In some embodiments, the filter aid is diatomaceous earth.

[0054] S200 purification treatment

[0055] In this step, the supernatant is purified to further separate immunoglobulins, improve product purity and yield, reduce impurities, and enhance product safety.

[0056] According to embodiments of this application, the purification process includes a first chromatographic process, a second chromatographic process, and a third chromatographic process; the first chromatographic process includes: passing the supernatant through an anion exchange chromatography column A, wherein the anion exchange chromatography column A includes a Fractogel EMDDEAE, CaptoQ, or UNOsphereQ anion exchange chromatography column; the second chromatographic process includes: passing the flow-through solution obtained from the first chromatographic process through an anion exchange chromatography column B, wherein the anion exchange chromatography column B includes a Fractogel EMDTMAE, CaptoQXP, or NuviaHPQ anion exchange chromatography column; the third chromatographic process includes: passing the flow-through solution obtained from the second chromatographic process through a chromatography column C, wherein the chromatography column C includes an EshmunoPAnti-A / Anti-B mixed column, an EshmunoPAnti-A column, or an EshmunoPAnti-B column. The supernatant after sodium octanoate precipitation was purified using the three different chromatography columns mentioned above to further remove impurities, improve the purity and yield of immunoglobulins in the product, and ensure that the product components and contents meet the requirements of the Chinese Pharmacopoeia.

[0057] According to an embodiment of this application, the equilibration solution for the first chromatography treatment includes a first sodium acetate buffer solution, the concentration of which is 5 mM to 20 mM (e.g., 5 mM, 6 mM, 8 mM, 10 mM, 12 mM, 14 mM, 15 mM, 16 mM, 18 mM, 20 mM), and the pH value is 4.50 to 5.50 (e.g., 4.50, 4.60, 4.70, 4.80, 4.90, 5.00, 5.10, 5.20, 5.30). The first sodium acetate buffer solution (5.40, 5.50) is equilibrated and loaded at a linear velocity of 60 cm / h to 200 cm / h (e.g., 60 cm / h, 70 cm / h, 80 cm / h, 90 cm / h, 100 cm / h, 110 cm / h, 120 cm / h, 130 cm / h, 140 cm / h, 150 cm / h, 160 cm / h, 170 cm / h, 180 cm / h, 190 cm / h, 200 cm / h). Using the above chromatographic conditions, the target immunoglobulin can be effectively separated from the mixed protein while removing impurities. This not only improves the purity and yield of the final product but also ensures that the content and quality of each component in the product meet the stringent standards of the Chinese Pharmacopoeia, resulting in high product safety and efficacy.

[0058] According to an embodiment of this application, the equilibration solution for the second chromatography treatment includes a second sodium acetate buffer solution, the concentration of which is 5 mM to 30 mM (e.g., 5 mM, 6 mM, 8 mM, 10 mM, 12 mM, 14 mM, 15 mM, 16 mM, 18 mM, 20 mM, 22 mM, 24 mM, 25 mM, 26 mM, 28 mM, 30 mM), and the pH value is 5.50 to 6.50 (e.g., 5.50, 5.60, 5.70, 5.80, 5.9). The second sodium acetate buffer (0, 6.00, 6.10, 6.20, 6.30, 6.40, 6.50) is used for equilibration and loading at a linear velocity of 10 cm / h to 100 cm / h (e.g., 10 cm / h, 20 cm / h, 40 cm / h, 50 cm / h, 60 cm / h, 70 cm / h, 80 cm / h, 90 cm / h, 100 cm / h, 110 cm / h, 120 cm / h, 130 cm / h, 140 cm / h, 150 cm / h). Using these chromatographic conditions, the target immunoglobulin can be effectively separated from the mixed protein while removing impurities. This not only improves the purity and yield of the final product but also ensures that the content and quality of each component in the product meet the stringent standards of the Chinese Pharmacopoeia, resulting in high product safety and efficacy.

[0059] According to an embodiment of this application, the equilibration solution for the third chromatography treatment includes at least one selected from acetate, citrate, phosphate, and chloride; the total concentration of at least one of acetate, citrate, phosphate, and chloride is 5 mM to 200 mM (e.g., 5 mM, 6 mM, 8 mM, 10 mM, 12 mM, 14 mM, 15 mM, 16 mM, 18 mM, 20 mM, 40 mM, 60 mM, 80 mM, 100 mM, 120 mM, 140 mM, 160 mM, 180 mM, 200 mM), and the pH value is 4.50 to 7.30 (e.g., 4.50). The chromatographic conditions are as follows: 5.00, 5.50, 6.00, 6.50, 7.00, 7.30), at least one of the acetate, citrate, phosphate, and chloride salts is equilibrated and loaded at a linear velocity of 50 cm / h to 200 cm / h (e.g., 50 cm / h, 60 cm / h, 70 cm / h, 80 cm / h, 90 cm / h, 100 cm / h, 110 cm / h, 120 cm / h, 130 cm / h, 140 cm / h, 150 cm / h, 160 cm / h, 170 cm / h, 180 cm / h, 190 cm / h, 200 cm / h). Using these chromatographic conditions, the target immunoglobulin can be effectively separated from the mixed protein while removing impurities. This not only improves the purity and yield of the final product but also ensures that the content and quality of each component in the product meet the stringent standards of the Chinese Pharmacopoeia, resulting in high product safety and efficacy.

[0060] S300 nanofiltration process.

[0061] In this step, the purified liquid is subjected to nanofiltration, and the filtrate is collected. This process helps to remove impurities and viruses.

[0062] S400 low pH incubation

[0063] In this step, the filtrate obtained from nanofiltration is incubated at a low pH of 35°C–40°C for at least 10 hours to obtain an immunoglobulin solution. Incubation under the above conditions after nanofiltration not only effectively inactivates the virus and improves product safety, but the inventors also unexpectedly discovered that these conditions can improve protein stability, preventing precipitation or aggregation. This eliminates the need for high-temperature virus inactivation (such as pasteurization), reducing inactivation steps and time, and avoiding the impact of high-temperature sterilization on protein structure. Furthermore, before low-pH incubation, the filtrate obtained from nanofiltration is subjected to ultrafiltration for concentration.

[0064] According to embodiments of this application, the incubation time is 10h to 20h, for example, 10h, 11h, 12h, 13h, 14h, 15h, 16h, 17h, 18h, 19h, and 20h. Therefore, within the above-mentioned incubation time, not only can the virus be efficiently inactivated, improving product safety, but protein stability can also be improved, enhancing the overall quality of the product. This eliminates the need for high-temperature virus inactivation, maintains protein activity, and shortens preparation time.

[0065] According to embodiments of this application, the low pH is 3 to 5, for example 3, 3.2, 3.5, 3.8, 4.0, 4.2, 4.5, 4.8, 5.0, preferably 4.0.

[0066] According to an embodiment of this application, the immunoglobulin solution is obtained by incubating at a low pH of 37°C for 10-15 hours. Preferably, the pH of the filtrate is adjusted to 4.0 and incubated at 37°C for 10 hours. Therefore, utilizing the above-mentioned low pH incubation conditions not only efficiently inactivates viruses and improves product safety, but also enhances protein stability and overall product quality. This eliminates the need for high-temperature virus inactivation, helps maintain protein activity, and shortens preparation time.

[0067] According to embodiments of this application, the method does not include a step of virus inactivation treatment at temperatures above 60°C. The method of this application can effectively inactivate viruses without relying on high-temperature inactivation, avoiding protein activity loss caused by high temperatures, and simultaneously improving protein stability, preventing aggregation and precipitation.

[0068] According to an embodiment of this application, in step S400, the filtrate is incubated together with a protective agent. Because proteins are inherently unstable and easily affected by external conditions such as temperature, vibration, shear force, buffer systems, and ionic strength, especially in this application where the filtrate needs to be incubated for a long time (less than 20 hours, e.g., 10-20 hours) at a temperature of 35°C to 40°C and a low pH. To avoid the adverse effects of relatively high temperature and low pH on the protein, a protective agent is added during incubation. Furthermore, the inventors selected the type of protective agent and found that using any one or more of polysorbate, maltose, glycine, sorbitol, and sodium caprylate can effectively maintain protein stability. After incubation, the solution remains clear without the formation of flocculent matter. Moreover, adding the above-mentioned protective agents also facilitates virus inactivation, resulting in a lower residual virus content in the final product. Among these, polysorbate shows the best overall effect. In some embodiments, the polysorbate is selected from any one or more of polysorbate 20, polysorbate 40, polysorbate 60, and polysorbate 80, with polysorbate 80 being preferred.

[0069] According to embodiments of this application, the protective agent is selected from polysorbate, and the amount of protective agent added based on 1 kg of the filtrate is 5 mg to 35 mg, for example, 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, or 35 mg. This further improves the stability of immunoglobulins.

[0070] According to embodiments of this application, the protective agent is selected from maltose, and based on 1L of the filtrate, the amount of the protective agent added is 80g to 120g, for example, 80g, 90g, 100g, 110g, or 120g. This further improves the stability of the immunoglobulins.

[0071] According to embodiments of this application, the protective agent is selected from glycine, and the amount of protective agent added based on 1L of the filtrate is 10g to 40g, for example, 10g, 20g, 30g, or 40g. This further improves the stability of the immunoglobulin.

[0072] According to embodiments of this application, the protective agent is selected from sodium caprylate, and the final concentration of the protective agent is 0.1 mM to 0.2 mM, for example, 0.1 mM, 0.12 mM, 0.14 mM, 0.15 mM, 0.16 mM, 0.18 mM, or 0.20 mM. This further improves the stability of immunoglobulins.

[0073] According to an embodiment of this application, before step S400, the filtrate is concentrated by ultrafiltration to a protein concentration of 15-25 g / L.

[0074] According to an embodiment of this application, the method further includes step S500: concentrating the reaction solution obtained from incubation by ultrafiltration, adding 1 w / w% to 5 w / w% glycine (e.g., 1 w / w%, 1.5 w / w%, 2 w / w%, 2.5 w / w%, 3 w / w%, 3.5 w / w%, 4 w / w%, 4.5 w / w%, 5 w / w%) to adjust the pH to 4.2 to 5.4, thereby obtaining the immunoglobulin solution. Glycine acts as a protein protectant, improving protein stability.

[0075] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the invention, both endpoints of each numerical range and any value in between may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art.

[0076] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0077] Example 1

[0078] 1. Precipitate I+II+III by reacting healthy human plasma with ethanol at -5℃, dissolve it with 10 times the amount of WFI, adjust the pH to 4.5 with acetic acid, and stir for 1 hour.

[0079] 2. Sodium octanoate precipitation

[0080] Add sodium octanoate to a final concentration of 20 mmol / L, adjust the pH to 4.8, add diatomaceous earth at a concentration of 300 g / kg of precipitate, stir, filter under pressure, and collect the supernatant.

[0081] 3. One-step chromatography

[0082] After ultrafiltration to concentrate the supernatant, anion exchange chromatography was performed using Capto-Q anion exchange gel, with the column equilibrated with ≥5 column volumes of 20 mM sodium acetate.

[0083] The chromatography conditions were: pH 5.2, 20 mM sodium acetate buffer, equilibration at a linear velocity of 150 cm / h, sample loading, and collection of flow-through and washing buffer.

[0084] 4. Two-step chromatography

[0085] The Niuva HP-Q anion exchange gel column was used, and the column was equilibrated with >5 column volumes of Buffer D (50 mM sodium acetate).

[0086] The chromatography conditions were: pH 5.9, 30 mM sodium acetate buffer, equilibration at a linear velocity of 50 cm / h, sample loading, and collection of flow-through and washing buffer.

[0087] 5. Three-step chromatography

[0088] After ultrafiltration and concentration of the washing buffer, the sample was loaded onto an Eshmuno P Anti-A / B chromatography column and equilibrated with >5 column volumes of 160 mM sodium acetate.

[0089] The chromatography conditions were: pH 5.5, 20 mM sodium acetate buffer, equilibration at a linear velocity of 125 cm / h, sample loading, and collection of flow-through wash buffer.

[0090] 6. Nanofiltration

[0091] The above-mentioned products are concentrated and then filtered through a nanofiltration membrane to remove the virus. The nanofiltration membrane has a pore size of 20 nm.

[0092] 7. Ultrafiltration concentration and formulation

[0093] The nanofiltration solution was dialyzed by ultrafiltration to adjust the protein concentration to 20 g / L. After sterilization, it was incubated at 37°C at a low pH (pH 4.0) for 10 hours.

[0094] After incubation, the solution was concentrated by ultrafiltration to a protein concentration of 105 g / L, 2% (w / w) glycine was added, the pH was adjusted to 4.5, filtered through a 0.2 μm sterile filter cartridge, and then dispensed to obtain intravenous immunoglobulin with a protein concentration of 10%.

[0095] Example 2

[0096] The difference from Example 1 is that the incubation temperature in step 7 is 36°C and the time is 12 hours.

[0097] Example 3

[0098] The difference from Example 1 is that the incubation temperature in step 7 is 35°C and the time is 15 hours.

[0099] Example 4

[0100] The difference from Example 1 is that the incubation temperature in step 7 is 38°C and the time is 10 hours.

[0101] Comparative Example 1

[0102] The difference from Example 1 is that: after step (5), the flow-through wash solution after three-step chromatography was ultrafiltered and dialyzed to adjust the protein concentration to 20 g / L. After sterilization, it was incubated at 37°C at a low pH (pH 4.0) for 10 days. The incubated solution was nanofiltered and concentrated to a protein concentration of 105 g / L. 2% (w / w) glycine was added to prepare the solution, which was then sterilized, dispensed, and the pH was adjusted to 4.5 to obtain intravenous immunoglobulin with a protein concentration of 10%.

[0103] Comparative Example 2

[0104] The difference from Example 1 is that: after sodium caprylate precipitation, the supernatant obtained by pressure filtration was subjected to ultrafiltration dialysis to adjust the protein concentration to 20 g / L. After sterilization, it was incubated at 37°C at a low pH (pH 4.0) for 10 hours. The incubated solution was then subjected to steps 3-5.

[0105] The chromatographic flow-through washing buffer obtained in step 5 was concentrated by ultrafiltration to a protein concentration of 105 g / L, then subjected to nanofiltration, and prepared with 2% (w / w) glycine. After sterilization, it was dispensed and the pH was adjusted to 4.5 to obtain intravenous immunoglobulin with a protein concentration of 10%.

[0106] Comparative Example 3

[0107] The difference from Example 1 is that: after sodium caprylate precipitation, the supernatant obtained by pressure filtration was subjected to ultrafiltration dialysis to adjust the protein concentration to 20 g / L. After sterilization, it was incubated at a low pH (pH 4.0) at 37°C for 9 hours. The incubated solution was then subjected to steps 3-5.

[0108] The chromatographic flow-through washing buffer from step 5 was concentrated by ultrafiltration to a protein concentration of 105 g / L, then subjected to nanofiltration and Pasteurella virus inactivation (inactivation temperature 60℃, inactivation time 10 h). 2% (w / w) glycine was added to prepare the solution, which was then sterilized and dispensed. The pH was adjusted to 4.5 to obtain intravenous immunoglobulin with a protein concentration of 10%.

[0109] Comparative Example 4

[0110] The difference from Example 1 is that in step 7, the incubation time is 25°C.

[0111] Comparative Example 5

[0112] The difference from Example 1 is that in step 7, the incubation temperature is 25°C and the incubation time is 21 days.

[0113] Comparative Example 6

[0114] The difference from Example 1 is that in step 7, the incubation temperature is 32°C and the time is 20 hours.

[0115] Comparative Example 7

[0116] The difference from Example 1 is that in step 7, the incubation temperature is 40°C and the time is 8 hours.

[0117] Example 5

[0118] 1. The intravenous immunoglobulins prepared in Examples 1-4 and Comparative Examples 1-7 were subjected to 4V / V% viral titers of vesicular stomatitis virus (VSV), Sindbis virus, and pseudorabies virus (PRV). The residual viral titers were calculated using the 96-well cytopathic effect assay and the Karber method. The initial VSV titer was 6.25 μg TCID. 50 / 0.1mL, initial Sindbis titer 6.00 Lg TCID 50 / 0.1mL, initial PRV titer 5.25 Lg TCID 50 / 0.1mL.

[0119] 2. Observe the state of the system after the low pH incubation of Examples 1-4 and Comparative Examples 1-7 respectively.

[0120] 3. The molecular size distribution (MBD) of the intravenous immunoglobulins prepared in Examples 1-4 and Comparative Examples 1-7 was determined by measuring the monomer content and dimer content (%). The detection methods were based on the General Technical Requirements and Guidelines 3122 of the Chinese Pharmacopoeia (Volume IV). Higher monomer content indicates greater protein stability, while higher dimer and polymer content indicates less stable protein.

[0121] In Comparative Example 1, the cells were first incubated at low pH at room temperature and then nanofiltration was performed; in Comparative Example 2, the cells were incubated at low pH before chromatography; in Comparative Example 3, the cells were incubated at low pH before chromatography and a pasteurization step was added; in Comparative Examples 4–7, the incubation conditions were changed.

[0122] The results are shown in Table 1. It can be seen that, compared with Comparative Examples 1-7, the preparation methods of Examples 1-4 can effectively inactivate the virus, and the resulting intravenous immunoglobulins have high stability and are not prone to protein denaturation and precipitation.

[0123] As can be seen from the comparison of Example 1, Comparative Example 1 and Comparative Example 2, low pH incubation significantly affects the virus inactivation effect and protein stability. When the supernatant obtained in step 2 is subjected to three-step chromatography, nanofiltration and low pH incubation in sequence, the virus inactivation effect and protein stability at the inactivation endpoint are both better.

[0124] As can be seen from the comparison of Example 1, Comparative Example 2 and Comparative Example 3, after incubating the supernatant obtained in step 2 at low pH and performing chromatography, even if pasteurization virus inactivation treatment is performed subsequently, although the virus can be inactivated to a certain extent, the effect is still not as good as that of Example 1. Although pasteurization virus inactivation treatment in Comparative Example 3 can inactivate the virus to a certain extent, the high temperature will affect the protein activity, resulting in poor stability and precipitation after long-term storage. The treatment method of Example 1 can effectively avoid these problems, and the obtained protein stability and virus inactivation effect are both better.

[0125] As can be seen from the comparison of Examples 1 and Comparative Examples 4-7, Comparative Example 4, with its low-pH incubation at low temperature for a short period, could not effectively inactivate the virus. It required up to 21 days (Comparative Example 5) to achieve an inactivation effect similar to that of Example 1, significantly reducing preparation efficiency. Furthermore, the prolonged low-pH incubation in Comparative Example 5 was also detrimental to protein stability. The low-pH incubation temperature in Comparative Example 6 was too low; even with extended incubation time, its virus inactivation effect was poor, and the protein remained unstable at the endpoint of low-pH incubation. In Comparative Example 7, the incubation time was shortened and the incubation temperature was increased, resulting in better virus inactivation, but poor stability.

[0126] Table 1. Low pH inactivation status and stability of intravenous immunoglobulin virus.

[0127]

[0128]

[0129] Note: In Table 1, residual VSV virus titer, Sindbis virus titer, and PRV virus titer ≤0.50LgTCID50 / 0.1mL represent undetectable (below the limit of detection); "—" indicates not detected; the above test data are the average values ​​of test data from three batches of samples, and the same applies to the following tables.

[0130] Example 6

[0131] In this embodiment, intravenous immunoglobulin was prepared according to the method of Example 1, except that in step 7, after sterilization, a protective agent was added and incubated at 37°C at a low pH (pH 4.0) for 10 hours. The added protective agents were added according to the table below to prepare the product.

[0132] 1. The prepared intravenous immunoglobulin was tested for viral titers of vesicular stomatitis virus (VSV), Sindbis virus, and pseudorabies virus (PRV) at 4V / V%, following the specific methods described in Example 5. The initial VSV titer was 6.25 μg TCID. 50 / 0.1mL, initial Sindbis titer 6.00 Lg TCID 50 / 0.1mL, initial PRV titer 5.25 Lg TCID 50 / 0.1mL.

[0133] 2. Observe the state of the system after the low pH incubation is completed.

[0134] 3. The molecular size distribution of the prepared intravenous immunoglobulin monomer content and molecular size distribution of the dimer content (%) were detected respectively. The detection method refers to the general technical requirements and guidelines 3122 of the Chinese Pharmacopoeia (Volume IV).

[0135] The results are shown in Table 2. Adding polysorbate 80, maltose, glycine, and sodium caprylate to the low pH incubation system effectively protected the protein, maintained its stability, and helped promote virus inactivation. Among these, polysorbate 80 showed the best virus inactivation effect.

[0136] Table 2. Low pH inactivation status and stability under different protective agents.

[0137]

[0138] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A method for preparing immunoglobulin, characterized in that, include: (1) Dissolve the plasma component I+II+III / II+III precipitate, filter, and collect the supernatant; (2) The supernatant is concentrated and then purified and nanofiltration is performed to collect the filtrate; (3) Incubate at a low pH of 35℃~40℃ for less than 20 hours to obtain immunoglobulin solution.

2. The method according to claim 1, characterized in that, The incubation time is 10h to 20h.

3. The method according to claim 1, characterized in that, The low pH is 3-5, and more specifically 4.0; Optionally, the immunoglobulin solution is obtained by incubation at 37°C and low pH for 10-15 hours.

4. The method according to claim 1, characterized in that, The method does not include the step of virus inactivation treatment at temperatures above 60°C.

5. The method according to claim 1, characterized in that, The purification process includes chromatography. Optionally, the chromatography process includes a first chromatography process, a second chromatography process, and a third chromatography process; Optionally, the first chromatographic treatment includes: passing the supernatant through an anion exchange chromatography column A, and collecting the anion exchange chromatography column A, which includes a Fractogel EMDDEAE, CaptoQ, or UNOsphereQ anion exchange chromatography column; The second chromatography process includes passing the flow-through solution obtained from the first chromatography process through an anion exchange chromatography column B, wherein the anion exchange chromatography column B includes a Fractogel EMDTMAE, CaptoQXP, or NuviaHPQ anion exchange chromatography column. The third chromatography process includes passing the flow-through solution obtained from the second chromatography process through a chromatography column C, wherein the chromatography column C includes an EshmunoPAnti-A / Anti-B mixed column, an EshmunoPAnti-A / Anti-B tandem column, an EshmunoPAnti-A column, or an EshmunoPAnti-B column.

6. The method according to claim 5, characterized in that, The equilibration solution for the first chromatography process includes a first sodium acetate buffer solution with a concentration of 5 mM to 20 mM and a pH of 4.50 to 5.

50. The first sodium acetate buffer solution is used for equilibration and sample loading at a linear velocity of 60 cm / h to 200 cm / h. Optionally, the equilibration solution for the second chromatography process includes a second sodium acetate buffer, the concentration of which is 5 mM to 30 mM and the pH value is 5.50 to 6.

50. The second sodium acetate buffer is used for equilibration and sample loading at a linear velocity of 10 cm / h to 100 cm / h. Optionally, the equilibration solution for the third chromatography treatment includes at least one of acetate, citrate, phosphate, and chloride; the total concentration of at least one of acetate, citrate, phosphate, and chloride is 5 mM to 200 mM, and the pH value is 4.50 to 7.30; at least one of acetate, citrate, phosphate, and chloride is equilibrated and loaded at a linear velocity of 50 cm / h to 200 cm / h.

7. The method according to claim 1, characterized in that, The method for obtaining the plasma component I+II+III / II+III precipitate includes: precipitating the plasma with ethanol; Further, the plasma component I+II+III / II+III precipitate was dissolved in water for injection, mixed with caprylic acid or caprylate, and the pH was adjusted to 4.0-5.

0. The mixture was then filtered and the supernatant was collected. Optionally, the final concentration of the octanoic acid or octanoate is 15–35 mmol / L, the pH is adjusted to 4.5–5.2, then a filter aid is added and stirred, the mixture is filtered under pressure, and the supernatant is collected.

8. The method according to any one of claims 1 to 7, characterized in that, In step (3), the filtrate and the protective agent are incubated together; Optionally, the protective agent is selected from any one or more of polysorbate, maltose, glycine, sodium caprylate, and sorbitol; Optionally, the protective agent is selected from polysorbate; Optionally, the polysorbate is selected from any one or more of polysorbate 20, polysorbate 40, polysorbate 60, and polysorbate 80; Optionally, the polysorbate is polysorbate 80.

9. The method according to claim 8, wherein the protective agent is selected from polysorbate, and the amount of the protective agent added based on 1 kg of the filtrate is 5 mg to 35 mg, preferably 15 mg to 25 mg; Alternatively, the protective agent is selected from maltose, and the amount of the protective agent added is 80g to 120g based on 1L of the filtrate; Alternatively, the protective agent is selected from glycine, and the amount of the protective agent added is 10g to 40g based on 1L of the filtrate; Alternatively, the protective agent may be selected from sodium octanoate, and the final concentration of the protective agent may be 0.1 mM to 0.2 mM.

10. The method according to any one of claims 1 to 9, characterized in that, The process also includes step (4): concentrating the reaction solution obtained from the incubation by ultrafiltration, adding 1 w / w% to 5 w / w% glycine for preparation, adjusting the pH value to 4.2 to 5.4, and obtaining the immunoglobulin solution.

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