Process for the preparation of human immunoglobulin enriched in IgA and IgM from IgG chromatography waste

Abstract

The present application relates to a kind of from IgG chromatography waste material preparation rich in IgA and IgM human immunoglobulin, belong to the technical field of blood products.The method comprises the following steps: (1) Fractogel EMD TMAE anion exchange chromatography eluate is treated with ultrafiltration;(2) the filtrate obtained after ultrafiltration is subjected to affinity chromatography, and eluate is collected;(3) the eluate obtained in step (2) is subjected to ultrafiltration after ultrafiltration, and is subjected to pasteurization inactivation;(4) ultrafiltration and preparation are carried out according to specified concentration;(5) sterile filtration, dispensing, obtain finished product.The present application uses waste eluate as raw material, and rich in IgA and IgM human immunoglobulin can be obtained after ultrafiltration by one-step affinity chromatography.The prepared immunoglobulin contains IgM 19%-25%, IgA 15%-20%, IgG 55%-66%, and the purity can reach more than 95%.

Description

Technical Field

[0001] This invention relates to the technical field of blood products, and more specifically to a method for preparing human immunoglobulins rich in IgA and IgM from IgG chromatography waste. Background Technology

[0002] In the traditional preparation of intravenous immunoglobulin (IVIG), IgM and IgA are typically discarded as impurities. Studies have shown that these antibodies have good therapeutic effects on sepsis or septic shock. Considering the growing demand for human plasma-derived products, the scarcity of raw materials, and unmet clinical needs, industrial production requires further improvements in plasma utilization. Therefore, further purification of IgA and IgM-rich IgG products from IVIG production waste is highly valuable. Currently, three IgA and IgM-rich IgG products are being used in clinical research: Pentaglobin, Trimodulin, and Venimmun, all of which are intravenous formulations. Existing research indicates that Pentaglobin can improve vascular microcirculation and tissue perfusion in patients with severe sepsis and septic shock, and significantly reduce the mortality rate of neonatal sepsis. Trimodulin has significant potential in delaying the progression of early systemic inflammatory diseases and reducing mortality, and its efficacy in treating severe community-acquired pneumonia is currently under clinical investigation. Patients with recurrent miscarriage and steroid-resistant Crohn's disease can benefit from Venimmun treatment, making the development of IgG products rich in IgA and IgM of significant clinical importance. These IgM / IgA-rich formulations have reported an incidence of adverse events no higher than that of standard IVIGs, and the types of adverse reactions are similar to other IVIG formulations.

[0003] In existing chromatographic processes for producing IVIG, Fractogel® EMD TMAE anion exchange chromatography eluent contains approximately 60% IgG, IgA, and IgM. The remaining impurities include harmful residues (caprylic acid, ethanol, and aluminum), coagulation factor impurities (FXI and FXIa), and other plasma protein impurities (human fibronectin, haptoglobin, human serum albumin, α1 acid glycoprotein, ceruloplasmin, human antithrombin III, plasminogen activator, α1 antitrypsin, kallikrein activator, and C1 esterase inhibitors, etc.). If these impurities can be effectively removed to obtain high-purity IgG, IgA, and IgM, it could provide potential drugs for treating immunodeficiency and severe infections, aiding in the clinical treatment of hematological diseases. This also represents a new direction for the development of immunoglobulin products and is expected to fill a gap in the domestic market in this field. Summary of the Invention

[0004] To address the problem of ineffective utilization of IgG-containing chromatographic waste in existing IVIG production processes, this invention provides a method for preparing human immunoglobulins rich in IgA and IgM from IgG chromatographic waste, thus solving the aforementioned problem. This invention uses Fractogel® EMD TMAE anion exchange chromatography eluent from the IVIG production process as raw material. After ultrafiltration, a single affinity chromatography step yields human immunoglobulins rich in IgA and IgM. The prepared immunoglobulin fraction contains 19%–25% IgM, 15%–20% IgA, and 55%–66% IgG, with a purity exceeding 95%.

[0005] The technical solution of this invention is as follows: A method for preparing human immunoglobulins rich in IgA and IgM from IgG chromatography waste includes the following steps: (1) The Fractogel® EMD TMAE anion exchange chromatography eluent was subjected to ultrafiltration; (2) The filtrate obtained after ultrafiltration was subjected to affinity column chromatography to obtain the eluent; (3) After ultrafiltration, the eluent obtained in step (2) is then pasteurized and inactivated; (4) Perform ultrafiltration and preparation according to the specified concentration; (5) Sterilize and filter, then package to obtain the finished product.

[0006] Furthermore, in step (1), the specific steps are as follows: protein concentration ≤20mg / ml, molecular weight cutoff of ultrafiltration membrane ≤50kD, and transmembrane pressure controlled during ultrafiltration process ≤0.3MPa.

[0007] Further preferably, ultrafiltration is performed using a phosphate buffer solution containing 0.4-0.5M potassium sulfate, with the ultrafiltrate having a pH of 7.0-7.5.

[0008] Furthermore, in step (2), the specific steps are as follows: A. The chromatography packing material is PlasmidSelect Xtra gel; the column height is 10±2cm; B. Equilibrate the gel using phosphate buffer containing 0.4-0.5M potassium sulfate, processing 3-15 column volumes. The equilibration solution should be at pH 7.0-7.5. C. Load the ultrafiltration solution, 1-5 column volumes; D. Wash the chromatography column with phosphate buffer containing 0.5-0.65M potassium sulfate for 5-20 column volumes. The washing buffer should be a solution with pH 7.0-7.5. E. Elute with sodium phosphate solution containing 0-0.4 M potassium sulfate for 3-10 column volumes, and collect the elution peak based on the UV absorbance at a wavelength of 280 nm.

[0009] Further preferred, the flow rate is controlled to not exceed 300 cm / hour during the chromatography process.

[0010] More preferably, the eluent is a solution containing 15-30 mM sodium phosphate and pH=7.0-7.5.

[0011] Further, in step (3), the specific steps are as follows: Ultrafiltration dialysis is performed using a 50kD ultrafiltration membrane to remove the buffer components from the chromatography. Water for injection is used as the dialysate for constant-volume dialysis five times. Sorbitol is added to make the protein concentration 20-30g / L. The pH of the solution is adjusted to 4.8-5.2, and the sorbitol content is 35%±3%. Pasteurized virus is inactivated at 60℃ for 10 hours to obtain the pasteurized inactivated solution.

[0012] Further, in step (4), the specific steps are as follows: After pasteurization, the feed solution is first dialyzed with water for injection, and then ultrafiltered with glycine solution; it is prepared according to a 5% protein concentration, and the prepared product contains 0.15~0.35mol / L glycine and 20~80mg / L polysorbate 80(II). Glycine is added to the ultrafiltrate to adjust the pH to 4.5±0.5; then it is sterilized by 0.2μm pore size filtration and dispensed to obtain the finished product.

[0013] A further preferred formulation contains 0.25 mol / L glycine and 80 mg / L polysorbate 80 (II).

[0014] The beneficial effects of this invention are as follows: (1) The method provided by this invention can purify immunoglobulin preparations rich in IgA and IgM from IgG waste, further improving the comprehensive utilization rate of plasma. After low pH incubation, pasteurization virus inactivation, and three-step chromatography for virus inactivation / removal, it has higher virus safety.

[0015] (2) The method provided by this invention can control the residual impurities in the preparation at a lower level. The content of common harmful residues, coagulation factor impurities, and other plasma protein impurities in intravenous immunoglobulin preparations is low, and the production process is reliable, consistent, and stable. After multiple batches of production verification, the detected content of each residual impurity is as follows: Hazardous residues: Octanoic acid not detected, ethanol <0.025%, aluminum <20μg / L.

[0016] Coagulation factor impurities: FXI content <1.0 ng / ml, FXIa <0.6 mIU / ml, and maintained at extremely low levels throughout all stages of the process. Removal of activated coagulation factor impurities can significantly reduce adverse clinical reactions, decrease the risk of thrombosis, and ensure medication safety.

[0017] Other impurities: human fibronectin content <0.781 μg / ml, haptoglobin <16 ng / ml, α1 acid glycoprotein <5 ng / ml, ceruloplasmin <1 ng / ml, C1 esterase inhibitor <0.28 ng / ml, plasminogen <6.84 ng / ml, α1 antitrypsin <125 ng / ml, and kallikrein activator <10 IU / ml.

[0018] The preparation process of this invention has an excellent removal effect on the above impurities. From raw plasma to stock solution, the impurity removal efficiency is >99.9%, and the impurity content in the finished product is extremely low or below the lower limit of quantification, which improves the safety of medication.

[0019] (3) Compared with other existing preparation methods, the IgM ratio of the present invention is reasonable and the IVIG purity is high. It can not only exert the advantages of effective treatment, but also ensure the safety of medication, and will not pose a risk to diabetic patients. Detailed Implementation

[0020] To enable those skilled in the art to better understand the technical solutions of this invention, the technical solutions in the embodiments of this invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of this invention.

[0021] Example 1 A method for preparing human immunoglobulins rich in IgA and IgM from IgG chromatography waste includes the following steps: (1) Take Fractogel® EMD TMAE anion exchange chromatography eluent. The protein content in the raw material eluent is 15.13 g / L, the purity of IgG is 40.6 (%), the purity of IgA is 7.8 (%), and the purity of IgM is 11.6 (%). Use phosphate buffer containing 0.4M potassium sulfate to perform ultrafiltration to control the protein concentration to 15 g / L, and obtain the eluent.

[0022] (2) The eluent obtained in step (1) was subjected to affinity column chromatography, as follows: A. The chromatography packing material is PlasmidSelect Xtra, with a column height of 10cm and a column diameter of 1.6cm; B. Equilibrate the gel using phosphate buffer (pH=7.0) containing 0.4M potassium sulfate, and process 3 column volumes. C. Adjust the pH of the eluent obtained in step (1) to 7.0, and then load 1 column volume. D. Wash the column with phosphate buffer containing 0.5 M potassium sulfate (pH=7.0) to remove impurities, processing 5 column volumes. E. Elute three column volumes using an eluent containing 0.4 M potassium sulfate and 30 mM sodium phosphate (pH=7.0), and collect the elution peak based on the UV absorbance at a wavelength of 280 nm. During chromatography, the flow rate was controlled at 120 cm / hour to obtain the eluent.

[0023] (3) After ultrafiltration of the eluent obtained in step (2), add sorbitol to make the protein content in the product 20g / L, the prepared product contains 32% sorbitol, pH=4.8, and then heat to 60℃ for 10 hours for pasteurization and inactivation.

[0024] (4) Ultrafiltration and preparation were carried out at a protein concentration of 5%. The prepared product contained 0.35 mol / L glycine and 80 mg / L polysorbate (II). The pH was adjusted to 4.5.

[0025] (5) Sterilize and filter, and dispense to obtain human immunoglobulin preparations rich in IgA and IgM.

[0026] The prepared human immunoglobulin preparation was tested, and the results are shown in Table 1 below.

[0027] Table 1 - Results of Quality Characteristics Testing for Human Immunoglobulin Preparations

[0028] * indicates that the value is below the detection limit of the kit.

[0029] Example 2 A method for preparing human immunoglobulins rich in IgA and IgM from IgG chromatography waste includes the following steps: (1) Take Fractogel® EMD TMAE anion exchange chromatography eluent. The protein content in the raw material eluent is 14.60 g / L, the purity of IgG is 42.0 (%), the purity of IgA is 7.7 (%), and the purity of IgM is 12.7 (%). Use phosphate buffer containing 0.5M potassium sulfate to perform ultrafiltration to control the protein concentration to 20 g / L, and obtain the eluent.

[0030] (2) The eluent obtained in step (1) was subjected to affinity column chromatography, as follows: A. The chromatography packing material is PlasmidSelect Xtra, with a column height of 12cm and a column diameter of 1.6cm; B. Equilibrate the gel using phosphate buffer (pH=7.5) containing 0.5M potassium sulfate, and process 15 column volumes. C. Adjust the pH of the eluent obtained in step (1) to 7.5, and then load 5 column volumes; D. Wash the chromatography column with phosphate buffer (pH=7.5) containing 0.65M potassium sulfate to remove impurities, processing 20 column volumes; E. Elute 10 column volumes using elution buffer containing 15 mM sodium phosphate (pH=7.5), and collect the elution peak based on the UV absorbance at a wavelength of 280 nm. During chromatography, the flow rate was controlled at 300 cm / hour to obtain the eluent.

[0031] (3) After ultrafiltration of the eluent obtained in step (2), add sorbitol to make the protein content in the product 30g / L, the sorbitol content in the prepared product 37.5%, pH=5.2, and then heat to 60℃ for 10 hours for pasteurization and inactivation.

[0032] (4) Ultrafiltration and preparation were carried out at a protein concentration of 5%. The prepared product contained 0.15 mol / L glycine and 20 mg / L polysorbate 80 (II). The pH was adjusted to 5.0.

[0033] (5) Sterilize and filter, and dispense to obtain human immunoglobulin preparations rich in IgA and IgM.

[0034] The prepared human immunoglobulin preparation was tested, and the results are shown in Table 2 below.

[0035] Table 2 - Results of Quality Characteristics Testing for Human Immunoglobulin Preparations

[0036] * indicates that the value is below the detection limit of the kit.

[0037] Although the present invention has been described in detail by way of preferred embodiments, the present invention is not limited thereto. Various equivalent modifications or substitutions can be made to the embodiments of the present invention by those skilled in the art without departing from the spirit and essence of the invention, and such modifications or substitutions should all be within the scope of the present invention. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should also be covered within the protection scope of the present invention.

Claims

1. A method for preparing human immunoglobulins rich in IgA and IgM from IgG chromatography waste, characterized in that, Includes the following steps: (1) The Fractogel® EMD TMAE anion exchange chromatography eluent was subjected to ultrafiltration; (2) The filtrate obtained after ultrafiltration is subjected to affinity chromatography to obtain the eluent; (3) After ultrafiltration, the eluent obtained in step (2) is then pasteurized and inactivated; (4) Perform ultrafiltration and preparation according to the specified concentration; (5) Sterilize and filter, then package to obtain the finished product.

2. The method as described in claim 1, characterized in that, In step (1), the specific steps are as follows: protein concentration ≤20mg / ml, molecular weight cutoff of ultrafiltration membrane ≤50kD, and transmembrane pressure controlled during ultrafiltration process ≤0.3MPa.

3. The method as described in claim 2, characterized in that, Ultrafiltration was performed using a phosphate buffer solution containing 0.4–0.5 M potassium sulfate, with the ultrafiltrate having a pH of 7.0–7.

5.

4. The method as described in claim 1, characterized in that, The specific steps in step (2) are as follows: A. The chromatography packing material is PlasmidSelect Xtra gel; the column height is 10±2cm; B. Equilibrate the gel using phosphate buffer containing 0.4-0.5M potassium sulfate, processing 3-15 column volumes. The equilibration solution should be at pH 7.0-7.

5. C. Load the ultrafiltration solution, 1-5 column volumes; D. Wash the chromatography column with phosphate buffer containing 0.5-0.65M potassium sulfate for 5-20 column volumes. The washing buffer should be a solution with pH 7.0-7.

5. E. Elute with sodium phosphate solution containing 0-0.4 M potassium sulfate for 3-10 column volumes, and collect the elution peak based on the UV absorbance at a wavelength of 280 nm.

5. The method as described in claim 4, characterized in that, During chromatography, the flow rate should be controlled to not exceed 300 cm / hour.

6. The method as described in claim 5, characterized in that, The eluent is a solution containing 15-30 mM sodium phosphate and pH = 7.0-7.

5.

7. The method as described in claim 1, characterized in that, In step (3), the specific steps are as follows: use a 50kD ultrafiltration membrane for ultrafiltration dialysis to remove the buffer components in the chromatography, use water for injection as the dialysate for constant dialysis 5 times, add sorbitol to make the protein concentration 20~30g / L, adjust the pH of the solution to 4.8~5.2, and the sorbitol content to 35%±3%; perform pasteurization at 60℃ for 10 hours to obtain the pasteurized inactivated solution.

8. The method as described in claim 1, characterized in that, In step (4), the specific steps are as follows: After pasteurization, the feed solution is first dialyzed with water for injection, and then ultrafiltered with glycine solution; it is prepared according to a 5% protein concentration, and the prepared product contains 0.15~0.35mol / L glycine and 20~80mg / L polysorbate 80(II); glycine is added to the ultrafiltrate and the pH is adjusted to 4.5±0.5; then sterile filtration with a 0.2μm pore size is performed and the product is dispensed to obtain the finished product.

9. The method as described in claim 8, characterized in that, The prepared product contains 0.25 mol / L glycine and 80 mg / L polysorbate 80 (II).

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