Varicella zoster intravenous injection human immune globulin and preparation process thereof
By combining low-temperature ethanol precipitation, octanoic acid precipitation, and polyethylene glycol 4000 precipitation with cation exchange and mixed-mode chromatography, and using histidine and sucrose stabilizers, the purity and stability issues of varicella-zoster immunoglobulin were resolved, achieving an efficient and safe preparation process that meets pharmacopoeia standards.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-23
- Publication Date
- 2026-04-03
AI Technical Summary
Existing technologies for preparing varicella-zoster immunoglobulin have limitations such as limited ability to remove impurities, difficulty in improving purity, poor stability, and difficulty in ensuring batch-to-batch consistency, which affect the safety and clinical efficacy of the product.
After low-temperature ethanol precipitation combined with octanoic acid precipitation, polyethylene glycol 4000 was added for co-precipitation, followed by cation exchange chromatography and mixed-mode chromatography. Histidine and sucrose were used as stabilizers, and the process conditions were optimized to maintain the integrity and stability of the IgG antibody.
It significantly improves product purity and stability, increases loading capacity by 40%, extends column life by 2.4 times, reduces production costs, maintains the integrity and biological activity of IgG subtypes, meets domestic and international pharmacopoeia standards, and provides safer and more effective treatment options.
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Figure CN121779547A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the preparation of immunoglobulins, and more particularly to a human immunoglobulin for varicella-zoster intravenous injection and its preparation process. Background Technology
[0002] Both chickenpox and shingles are caused by the varicella-zoster virus (VZV). Primary infection manifests as chickenpox, while secondary infection presents as shingles (HZ). In recent years, the incidence of shingles has increased significantly, and it is commonly seen in the elderly, immunocompromised individuals, and those using immunosuppressants.
[0003] Varicella-zoster immunoglobulin (VZIG) is an important passive immunizing agent for the prevention of varicella and shingles, and has significant clinical value for immunocompromised individuals exposed to VZV (such as organ transplant recipients, leukemia patients, pregnant women, and newborns). IgG1 and IgG3 are the main viral antibodies in the plasma of healthy blood donors, as well as in the plasma of recovered patients after primary and recurrent diseases. Studies of plasma samples collected from varicella patients during the 7-month period following the outbreak show a rapid decline in IgG1 and IgG3. Patients recovering from varicella show predominantly IgG3 antibodies, while those recovering from shingles show predominantly IgG1 antibodies. Therefore, it is important to maintain the integrity and effectiveness of the IgG subtypes in protective immunoglobulin products. After intravenous injection of varicella-zoster immunoglobulin, the level of IgG in the blood rapidly increases, neutralizing viral antigens, blocking immune-mediated immune responses, and enhancing the body's anti-infection ability and immune regulatory function.
[0004] Traditional immunoglobulin production processes often rely on low-temperature ethanol precipitation combined with caprylic acid precipitation and anion exchange chromatography. While this method is considered classic, it still has the following limitations: 1. Traditional processes have limited ability to remove impurities, especially IgA and PKA, and the purity of the product is usually maintained at 95%-98%, making it difficult to reach higher standards.
[0005] 2. Conventional stabilizer formulations are less effective at inhibiting polymerization and degradation, which are more likely to occur in high-purity IgG products, affecting potency and safety within the shelf life.
[0006] 3. Anion exchange chromatography relies on antibody collection in flow-through mode, which is extremely sensitive to fluctuations in loading conditions (such as pH and conductivity), resulting in low production tolerance and difficulty in ensuring batch-to-batch consistency.
[0007] 4. Strenuous purification conditions can easily lead to changes in the distribution of IgG subtypes and a decrease in the biological activity of the Fc fragment, affecting clinical efficacy.
[0008] Therefore, developing a VZIG purification process that can achieve higher purity, better safety, and better stability is of urgent industrial demand and significant clinical value. Summary of the Invention
[0009] The main objective of this invention is to provide an intravenous human immunoglobulin for varicella-zoster and its preparation process, aiming to solve at least one of the aforementioned technical problems.
[0010] To achieve the above objectives, the present invention provides a preparation process for intravenous human immunoglobulin for varicella-zoster virus, comprising the following steps: (1) Melt the raw plasma with qualified anti-varicella-zoster virus IgG antibody titer, and then centrifuge it to obtain plasma without cryoprecipitate; (2) The de-cryogenic plasma was subjected to stepwise precipitation using the low-temperature ethanol method to obtain fraction II+III precipitate; (3) Dissolve the precipitate of component II+III in water, first add calcium chloride, acetate buffer and octanoic acid in sequence to precipitate the impurities, then add polyethylene glycol 4000 to precipitate the impurities, and finally let it stand and filter to obtain the filtrate. (4) The filtrate is first subjected to ultrafiltration dialysis, and then purified by cation exchange chromatography column elution. The eluent is collected. (5) The eluent is first subjected to ultrafiltration dialysis, and then purified by mixed-mode chromatography column chromatography. The flow-through is collected. (6) The flow-through solution was subjected to ultrafiltration dialysis with a stabilizer buffer containing histidine and sucrose, and then diluted with water to obtain a dilute solution; (7) The diluted solution is first sterilized and filtered, then incubated for virus inactivation, and finally filtered to remove the virus, to obtain a protein solution containing varicella-zoster intravenous immunoglobulin.
[0011] Furthermore, in step (1), the qualified anti-varicella-zoster virus IgG antibody titer means ≥4 IU / mL.
[0012] Further, in step (3), the mass ratio of II+III precipitate to water is 1:11; the final concentration of calcium chloride is 0.15 mol / L; the pH of the acetate buffer is 4.0, and the amount added is to make the pH reach 4.4-4.6; the concentration of octanoic acid is 98.5 wt%, and the amount added is 40 mL / L; the process of treating the precipitate of impurities is to stir the reaction at 4-6℃ for 60-90 minutes.
[0013] Furthermore, in step (3), the final concentration of polyethylene glycol 4000 added is 8wt%; the impurity precipitation treatment process is to stir and react at 4-6℃ for 30 minutes; and the standing time is 3-5 hours.
[0014] Furthermore, in step (4), the ultrafiltration dialysis process uses a 30kDa PES membrane and 20mM sodium acetate buffer to dialyze until the protein concentration of the filtrate reaches 10wt%.
[0015] Further, in step (4), the medium of the cation exchange chromatography column is Capto S, the equilibration buffer is 20mM sodium acetate buffer, and the elution purification process is carried out by linear gradient elution with 0-150mM NaCl solution. The elution peak segment from which the UV 280nm absorption peak drops to more than 20% of the peak value is collected as the elution solution.
[0016] Further, in step (5), the ultrafiltration dialysis process uses a 30kDa PES membrane and 20mM phosphate buffer to dialyze until the protein concentration of the eluent reaches 5.5wt%; the medium of the mixed mode chromatography column is Capto MMC and the equilibration buffer is 20mM phosphate buffer.
[0017] Further, in step (6), the stabilizer buffer contains 10-50 mM histidine, 5-10 wt% sucrose, and has a pH of 4.0-4.5; the ultrafiltration dialysis treatment uses a 30 kDa PES membrane to dialyze until the protein concentration of the flow-through reaches 5.5 wt% or more.
[0018] Furthermore, it also includes the following steps: (8) After sterilizing and filtering the protein solution containing varicella-zoster intravenous immunoglobulin, it is aseptically dispensed and capped.
[0019] The present invention also provides a human immunoglobulin for varicella-zoster virus intravenous injection, which is prepared according to the preparation process described above.
[0020] In this invention, the raw material plasma is convalescent plasma from recovered patients with varicella-zoster virus, plasma from healthy individuals immunized with varicella-zoster virus vaccine, or convalescent plasma or immune plasma after virus inactivation.
[0021] The beneficial effects of this invention are reflected in: 1. This invention innovatively introduces a specific concentration of polyethylene glycol 4000 (PEG 4000) for co-precipitation after traditional octanoic acid precipitation. This combination produces a synergistic purification effect: octanoic acid mainly precipitates non-IgG proteins, while the subsequent PEG 4000 effectively precipitates residual impurities after octanoic acid treatment (such as lipoproteins, some polymers, and unstable components). This precipitation combination not only significantly reduces the turbidity of the filtrate from >18 NTU (without PEG) to <5 NTU, greatly improving the clarity of the filtrate, but more importantly, it increases the loading capacity of the subsequent cation exchange chromatography column by approximately 40% (from 45 g IgG / L resin to 64 g IgG / L resin), increases the filtration throughput by 3 times, extends the column life by 2.4 times, and significantly reduces production costs.
[0022] 2. This invention abandons traditional anion exchange chromatography and adopts a two-step chromatography process of cation exchange (CEX) + mixed-mode chromatography (MMC). CEX chromatography, under weakly acidic conditions (pH 4.5-4.8), efficiently adsorbs IgG through charge interactions, effectively removing impurities such as IgA and PKA. MMC chromatography, utilizing its multiple interactions (hydrophobic, ionic, and hydrogen bonding), under mild near-neutral conditions (pH 6.5-7.0), efficiently removes residual trace polymers and viruses, while minimizing activity loss. This combined chromatographic process is mild, effectively maintaining the integrity of IgG1 and IgG3 subtypes in IgG antibodies, and ensuring that the Fc fragment biological activity remains stable at over 90%.
[0023] 3. This invention uses histidine and sucrose as stabilizers. In this combination, histidine is not only an effective buffer but also works synergistically with sucrose to form a stable "protective shell" around the protein molecules, significantly inhibiting protein aggregation and degradation induced by low-pH incubation and long-term storage. Comparative experiments show that this formulation performs exceptionally well in accelerated stability tests, significantly outperforming commonly used citrate, glycine, or single-sugar stabilizers (see Experimental Example 3). 4. The final product of this invention has a stable purity of over 99.5%, a PKA content of less than 5 IU / ml, and a varicella-zoster antibody titer of over 25 IU / ml. Furthermore, accelerated stability testing (25±2℃, 6 months) shows that its molecular size distribution, titer, and Fc segment biological activity have not changed significantly. All indicators not only meet but are far superior to domestic and international pharmacopoeia standards, providing a safer and more effective treatment option for clinical use. Attached Figure Description
[0024] Figure 1 This is a flowchart illustrating the preparation process of the intravenous human immunoglobulin for varicella-zoster virus according to the present invention. Detailed Implementation
[0025] The preparation process of the varicella-zoster intravenous immunoglobulin of this invention is described in the following figure. Figure 1 To enable those skilled in the art to more clearly understand the technical solutions described in this invention, the following embodiments are provided for illustration. It should be noted that the following embodiments do not constitute a limitation on the scope of protection claimed by this invention.
[0026] Unless otherwise specified, the raw materials, reagents, or devices used in the following examples can be obtained from conventional commercial sources or by existing known methods; unless otherwise specified, the methods used in the embodiments of the present invention are methods mastered by those skilled in the art. The main buffer solution formulations are as follows: Acetic acid buffer (pH 4.0): Weigh 2.4 L of glacial acetic acid and 1.089 kg of sodium acetate, dissolve them in water for injection and bring the volume to 10 kg.
[0027] Sodium acetate buffer (20mM): Weigh 16.40g of anhydrous sodium acetate, dissolve it in water for injection and bring the volume to 10kg, then adjust the pH to 4.7 with glacial acetic acid.
[0028] Phosphate buffer (20mM): Weigh 31.20g of sodium dihydrogen phosphate and 71.63g of disodium hydrogen phosphate, dissolve them in water for injection and bring the volume to 10kg. Adjust the pH to 6.8 with 1M hydrochloric acid or sodium hydroxide.
[0029] Stabilizer buffer: Weigh 31.03g of histidine and 800g of sucrose, dissolve them in water for injection and bring the volume to 10kg. Adjust the pH to 4.2 with 1M hydrochloric acid or sodium hydroxide.
[0030] Example 1 Preparation of intravenous human immunoglobulin for varicella-zoster virus (1) The raw plasma with anti-VZV IgG antibody titer ≥4 IU / mL was melted at 0℃, and then centrifuged at 20000g. The outlet temperature was controlled at 0°C to obtain plasma without reflux precipitation. (2) Transfer the de-chilled plasma to a reaction vessel, stir for 30 min, centrifuge at 10000 g, and control the outlet temperature at -1 °C to obtain the supernatant protein solution of component I; add acetate buffer solution with pH 4.0 to the supernatant protein solution of component I, adjust the pH value to 6.7, then add ethanol (until the ethanol volume ratio reaches 22%), stir at -5 °C for 150 min, and finally let stand for 80 min. After pressure filtration, obtain the precipitate of component II + III.
[0031] (3) Dissolve the precipitate of component II + III in 11 times its mass of water for injection at 6°C to obtain a solution. Add calcium chloride to the solution (the amount added is to make the calcium ion concentration of the solution reach 0.15 mol / L). Then, while stirring, slowly add acetate buffer at pH 4.0 to adjust the pH value to 4.5. Then, slowly add 98.5 wt% octanoic acid (40 ml of octanoic acid per liter of solution). Stir at 5°C for 60 min. Then, while stirring, slowly add PEG 4000 until the final concentration reaches 8 wt%. Continue stirring at 5°C for 30 min. Finally, let it stand at 5°C for 3 hours. After pressure filtration (the pressure filtration pressure is controlled not higher than 0.2 MPa), obtain the filtrate.
[0032] (4) The filtrate was subjected to ultrafiltration dialysis. Ultrafiltration dialysis was performed using a 30kDa PES membrane and 5 volumes of 20mM sodium acetate buffer (pH 4.7) until the conductivity was about 1.5-1.8mS / cm. The filtrate was concentrated to a protein concentration of 10wt%. Then, the Capto S cation exchange chromatography column was equilibrated with 6 column volumes of 20mM sodium acetate buffer (pH 4.7) until the baseline was stable. The sample was loaded at a flow rate of 80cm / h and eluted with a linear gradient of 12 column volumes containing 0-150mM NaCl. The elution peaks from the peak value of UV280nm to the peak value and the peak value of more than 20% of the peak value were collected to obtain the eluent.
[0033] (5) The eluent was subjected to ultrafiltration dialysis. Ultrafiltration dialysis was performed using a 30kDa PES membrane and 5 volumes of 20mM phosphate buffer (pH 6.8) until the conductivity was about 1.8-2.2mS / cm, so that the eluent was concentrated to a protein concentration of 5.5wt%. Then, the Capto MMC mixed mode chromatography column was equilibrated with 6 column volumes of 20mM phosphate buffer (pH 6.8) until the baseline was stable. The sample was loaded at a flow rate of 80cm / h and the flow-through was collected.
[0034] (6) The flow-through solution was subjected to ultrafiltration dialysis. The ultrafiltration dialysis was performed using a 30kDa PES membrane and 6 times the volume of stabilizer buffer (containing 20mM histidine and 8wt% sucrose, pH 4.2) until the conductivity was about 0.7-1.1mS / cm. The flow-through solution was concentrated to a protein concentration of more than 5.5wt%. After sampling and testing the pH value and protein concentration, water for injection was added, and the pH was adjusted with 1M hydrochloric acid solution to obtain a dilute solution with pH 3.9 and protein content of 5.23wt%.
[0035] (7) The diluted solution was filtered through a 0.2μm sterile filter and then placed in an incubation room. It was incubated at 24℃ for 21 days. After incubation, it was filtered with a DV20 filter to remove the virus, and a protein solution containing varicella-zoster intravenous immunoglobulin was obtained.
[0036] (8) The protein solution containing varicella-zoster intravenous immunoglobulin was sterilized by filtering it through a 0.2μm filter cartridge and then sterilely dispensed and capped. The dispensing volume was 50ml per bottle to obtain the final product of varicella-zoster intravenous immunoglobulin.
[0037] Experimental Example 1 Impurity Removal Analysis Samples were taken from the intermediate products obtained after each of the corresponding treatments in Example 1, and protein recovery and impurity analysis were performed. The results are shown in Table 1.
[0038] Table 1: Study on Impurity Removal of Intermediate Products
[0039] The results showed that: ① Octanoic acid + PEG co-precipitation could significantly remove IgA; ② CEX chromatography reduced the polymer content from 1.5% to 0.8% and further removed PKA; ③ MMC chromatography reduced the polymer content to 0.5% with a yield loss of <1%.
[0040] Experiment Example 2 Study on the effect of PEG4000 on impurity removal Based on Example 1, step (3) "then slowly add PEG 4000 while stirring until the final concentration reaches 8wt%, and continue stirring at 5°C for 30 min" was omitted. All other processes and conditions were the same as in Example 1, serving as control group 1. Key quality indicators of Example 1 and control group 1 were tested, and the results are shown in Table 2.
[0041] Table 2: Comparison of Key Quality Indicators between Example 1 and Control Group 1
[0042] The results showed that increasing PEG precipitation significantly reduced the turbidity of the filtrate, increased the column loading, and extended the column life.
[0043] Experimental Example 3 Comparative Study of the Invention Process and Traditional Anion Exchange Process Based on Example 1, the following steps were modified: Step (4) "equilibrate the Capto S cation exchange chromatography column with 6 column volumes of 20 mM sodium acetate buffer (pH 4.7) until the baseline is stable, load the sample at a flow rate of 80 cm / h, elute with 12 column volumes of a linear gradient containing 0-150 mM NaCl, and collect the elution peak segment from the peak value of UV280 nm to the peak value and then decrease to more than 20% of the peak value," and Step (5) "equilibrate the Capto MMC mixed mode chromatography column with 6 column volumes of 20 mM phosphate buffer (pH 6.8) until the baseline is stable, load the sample at a flow rate of 80 cm / h, and collect the flow-through." were all replaced with "using DEAE Sepharose Fast..." Purification was performed using a Flow anion exchange chromatography column. The sample was equilibrated to a pH above 6.4 using 7 column volumes of 0.03M phosphate-sodium hydroxide buffer (pH 6.4). The flow-through was then collected and processed. All other processes and conditions were consistent with those in Example 1, serving as control group 2. Key quality indicators were tested for Example 1 and control group 2, and the results are shown in Table 3.
[0044] Table 3: Comparison of Key Quality Indicators between Example 1 and Control Group 2
[0045] Experiment Example 4 Study on the effect of different stabilizer formulations on product stability Based on Example 1, the stabilizer buffer was replaced with the following five different stabilizer buffers, while other processes and conditions remained the same as in Example 1.
[0046] Formula A (carbohydrate control): 8 wt% sucrose, pH 4.2 (no amino acids); Formula B (amino acid control): 20mM histidine, pH 4.2 (sucrose-free). Formula C (common formula comparison): 5wt% maltose + 100mM NaCl, pH 4.2; Formula D (common formula comparison): 20mM glycine + 8wt% sucrose, pH 4.2; Formula E (blank control): pH adjusted to 4.2 using only water for injection.
[0047] The protein liquid samples obtained in Example 1 and the above five samples were placed in an accelerated stability test chamber at 25±2°C. Key quality indicators were tested at 0 months, 3 months and 6 months, including molecular size distribution, anti-VZV antibody titer, FC segment biological activity and appearance clarity. The results are shown in Table 4.
[0048] Table 4: Comparison of Key Quality Indicators for Accelerated Stability Testing of Different Stabilizer Formulations
[0049] The results showed that, compared with single formulations (A, B) and other stabilizer formulations commonly used in biopharmaceuticals (C, D), the formulation of the present invention performed best in inhibiting polymerization, maintaining potency, and protecting Fc activity. After 6 months of accelerated testing, the monomer, potency, and Fc biological activity of the formulation of the present invention all decreased by less than 3%, and the solution remained clear, providing excellent long-term stability for VZIG.
[0050] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.
Claims
1. A preparation process for intravenous human immunoglobulin for varicella-zoster virus, characterized in that, Includes the following steps: (1) Melt the raw plasma with qualified anti-varicella-zoster virus IgG antibody titer, and then centrifuge it to obtain plasma without cryoprecipitate; (2) The de-cryogenic plasma was subjected to stepwise precipitation using the low-temperature ethanol method to obtain fraction II+III precipitate; (3) Dissolve the precipitate of component II+III in water, first add calcium chloride, acetate buffer and octanoic acid in sequence to precipitate the impurities, then add polyethylene glycol 4000 to precipitate the impurities, and finally let it stand and filter to obtain the filtrate. (4) The filtrate is first subjected to ultrafiltration dialysis, and then purified by cation exchange chromatography column elution. The eluent is collected. (5) The eluent is first subjected to ultrafiltration dialysis, and then purified by mixed-mode chromatography column chromatography. The flow-through is collected. (6) The flow-through solution was subjected to ultrafiltration dialysis with a stabilizer buffer containing histidine and sucrose, and then diluted with water to obtain a dilute solution; (7) The diluted solution is first sterilized and filtered, then incubated for virus inactivation, and finally filtered to remove the virus, to obtain a protein solution containing varicella-zoster intravenous immunoglobulin.
2. The preparation process of varicella-zoster intravenous immunoglobulin as described in claim 1, characterized in that, In step (1), the qualified anti-varicella-zoster virus IgG antibody titer means ≥4 IU / mL.
3. The preparation process of varicella-zoster intravenous immunoglobulin as described in claim 1 or 2, characterized in that, In step (3), the mass ratio of II+III precipitate to water is 1:11; the final concentration of calcium chloride added is 0.15 mol / L; the pH of the acetate buffer is 4.0, and the amount added is to make the pH reach 4.4-4.6; the concentration of octanoic acid is 98.5 wt%, and the amount added is 40 mL / L; the process of treating the precipitate of impurities is to stir the reaction at 4-6℃ for 60-90 minutes.
4. The preparation process of varicella-zoster intravenous immunoglobulin as described in claim 1 or 2, characterized in that, In step (3), the final concentration of polyethylene glycol 4000 added is 8wt%; the impurity precipitation treatment process is to stir and react at 4-6℃ for 30 minutes; and the standing time is 3-5 hours.
5. The preparation process of intravenous human immunoglobulin for varicella-zoster virus as described in claim 1 or 2, characterized in that, In step (4), the ultrafiltration dialysis process uses a 30kDa PES membrane and 20mM sodium acetate buffer to dialyze until the protein concentration of the filtrate reaches 10wt%.
6. The preparation process of varicella-zoster intravenous immunoglobulin as described in claim 1 or 2, characterized in that, In step (4), the medium of the cation exchange chromatography column is Capto S, the equilibration buffer is 20 mM sodium acetate buffer, and the elution purification process is carried out by linear gradient elution with 0-150 mM NaCl solution. The elution peak segment from which the UV 280 nm absorption peak drops to more than 20% of the peak value is collected as the elution solution.
7. The preparation process of varicella-zoster intravenous immunoglobulin as described in claim 1 or 2, characterized in that, In step (5), the ultrafiltration dialysis process uses a 30kDa PES membrane and 20mM phosphate buffer to dialyze until the protein concentration in the eluent reaches 5.5wt%; the medium of the mixed mode chromatography column is Capto MMC and the equilibration buffer is 20mM phosphate buffer.
8. The preparation process of varicella-zoster intravenous immunoglobulin as described in claim 1 or 2, characterized in that, In step (6), the stabilizer buffer contains 10-50 mM histidine, 5-10 wt% sucrose, and has a pH of 4.0-4.5; the ultrafiltration dialysis treatment uses a 30 kDa PES membrane to dialyze until the protein concentration in the flow-through reaches 5.5 wt% or higher.
9. The preparation process of varicella-zoster intravenous immunoglobulin as described in claim 1 or 2, characterized in that, It also includes the following steps: (8) After sterilizing and filtering the protein solution containing varicella-zoster intravenous immunoglobulin, it is aseptically dispensed and capped.
10. A varicella-zoster intravenous immunoglobulin, characterized in that, Prepared according to the preparation process described in any one of claims 1 to 9.