WO2013023362A1 - 静注巨细胞病毒人免疫球蛋白及其制备方法 - Google Patents

静注巨细胞病毒人免疫球蛋白及其制备方法 Download PDF

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WO2013023362A1
WO2013023362A1 PCT/CN2011/078485 CN2011078485W WO2013023362A1 WO 2013023362 A1 WO2013023362 A1 WO 2013023362A1 CN 2011078485 W CN2011078485 W CN 2011078485W WO 2013023362 A1 WO2013023362 A1 WO 2013023362A1
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supernatant
concentration
ethanol
adjust
iii
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PCT/CN2011/078485
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English (en)
French (fr)
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张运佳
郭采平
丁玉江
宋清爽
张信
王锦才
吴恩应
张佩
陈玉琴
李慧
黄伟荣
戴美兰
张彦鹏
王春华
罗姗
黄倩云
刘永娣
吴开永
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深圳市卫武光明生物制品有限公司
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Priority to PCT/CN2011/078485 priority Critical patent/WO2013023362A1/zh
Priority to US13/821,991 priority patent/US20130172536A1/en
Publication of WO2013023362A1 publication Critical patent/WO2013023362A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/06—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from serum
    • C07K16/065—Purification, fragmentation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/36—Extraction; Separation; Purification by a combination of two or more processes of different types
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B65—CONVEYING; PACKING; STORING; HANDLING THIN OR FILAMENTARY MATERIAL
    • B65B—MACHINES, APPARATUS OR DEVICES FOR, OR METHODS OF, PACKAGING ARTICLES OR MATERIALS; UNPACKING
    • B65B1/00—Packaging fluent solid material, e.g. powders, granular or loose fibrous material, loose masses of small articles, in individual containers or receptacles, e.g. bags, sacks, boxes, cartons, cans, or jars
    • B65B1/04—Methods of, or means for, filling the material into the containers or receptacles
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/081—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from DNA viruses
    • C07K16/085—Herpetoviridae, e.g. pseudorabies virus, Epstein-Barr virus
    • C07K16/088—Varicella-zoster virus
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/081—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from DNA viruses
    • C07K16/085—Herpetoviridae, e.g. pseudorabies virus, Epstein-Barr virus
    • C07K16/089—Cytomegalovirus
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man

Definitions

  • Intravenous cytomegalovirus human immunoglobulin and preparation method thereof Intravenous cytomegalovirus human immunoglobulin and preparation method thereof
  • the invention relates to a human immunoglobulin and a preparation method thereof, in particular to a intravenous injection of a cytomegalovirus human immunoglobulin and a preparation method thereof.
  • Cytomegalovirus CMV (Cytomegalovirus) It is a DNA virus of the genus Herpesvirus, and the mortality rate of pregnant women, newborns, organ transplant patients, and immunosuppressed patients caused by cytomegalovirus can reach 50% to 80%.
  • the cytomegalovirus human immunoglobulin CMV-IgG is specifically used for the treatment of cytomegalovirus infections in pregnant women, neonates, immunosuppressed patients and organ transplants because of its ability to specifically neutralize cytomegalovirus.
  • the natural infection rate of cytomegalovirus in the general population can reach more than 80%.
  • the plasma containing high-valent cytomegalovirus IgG antibody is screened and collected from healthy people, and the cytomegalovirus human immunoglobulin drug is prepared by a specific separation and purification method. It has irreplaceable clinical application value for the treatment of severe infection caused by cytomegalovirus.
  • ethanol is a protein denaturant, which is easy to cause IgG structure change and deactivation in the separation process, resulting in low titer recovery rate and possibly new The formation of antigenic determinants;
  • the object of the present invention is to provide a cytomegalovirus human immunoglobulin and a preparation method thereof, and the technical problem to be solved is to improve the purity, yield and safety of the product.
  • the invention adopts the following technical scheme: a intravenous cytomegalovirus human immunoglobulin, the specific activity of the intravenous cytomegalovirus human immunoglobulin is not less than 2.5 PEI-U/mg, and the anti-CMV titer is not less than 100 PEI- U/ml, the purity is greater than 98.2%, and the protein content is 51-55 mg/ml.
  • a method for intravenously injecting cytomegalovirus human immunoglobulin comprising the steps of:
  • the plasma protein content was adjusted to 45-55 mg/ml with physiological saline, the pH was adjusted to 6.8-7.2 with glacial acetic acid, and ethanol or absolute ethanol having a volume fraction of 95% was added to adjust the ethanol concentration to 7.5-8.5%, and the reaction temperature was -2.5 ⁇ -2.0 °C, stir the reaction for 4 hours, the reaction is completed by centrifugation or pressure filtration separation to remove the FI precipitate, to obtain the supernatant, adjust the pH of the supernatant to 6.0-6.5 with glacial acetic acid, add 95% ethanol or absolute ethanol to adjust the ethanol concentration to 20 ⁇ 25%, the reaction temperature is -5.5 ⁇ -4.5°C, the stirring reaction is 4-6 hours, and the reaction is completed by centrifugation or pressure filtration to obtain FII+III precipitate;
  • FI+II+III or FII+III precipitates with a pH of 0.9 to 1.1 times the plasma amount Stirring for 8 to 16 hours at 2.8 to 5.2, a concentration of 20 to 80 mM sodium acetate buffer at 2 to 8 ° C, to fully dissolve the precipitate, and separating the supernatant by centrifugation or pressure filtration;
  • the supernatant is filtered with a pore size of 1.0 ⁇ m, and the filtration pressure is controlled to be no more than 0.25 MPa.
  • the pH of the filtrate is adjusted to 4.5-5.5 with 4 mol/L acetic acid or 0.5-1 mol/L sodium hydroxide, and the water for injection or octanoic acid is added to adjust the suspension.
  • Liquid octanoic acid concentration to 20 ⁇ 80mmol / L, in 20 ⁇ 30 Stir at °C for 1-2 hours, centrifuge or filter to separate the supernatant;
  • the supernatant is filtered through a 0.45 ⁇ m filter to control the filtration pressure to be no more than 0.25 MPa, and the filtrate is concentrated 15 to 20 times with a 30 KD ultrafiltration membrane, and the pH is adjusted to 8 to 10 times.
  • the phosphate buffer solution with a concentration of 6.0 to 7.1 and a concentration of 20 to 60 mmol/L is used as an equilibrium buffer column for 8 to 10 column volumes, and the sample protein is calculated according to 70 to 80% of the maximum load per ml of the filler. Amount, collect the penetrating solution after loading, and hang the column of the protein with a pH of 2mol/L NaCl. 6.0 to 7.1 of 20 to 60 mmol/L phosphate buffer;
  • control filtration pressure is not more than 0.25Mpa;
  • the filtrate is concentrated to a protein content of 80-100 through a 30KD ultrafiltration membrane.
  • Mg/ml using 8-10 times of water for ultrafiltration, after ultrafiltration, the control protein content is 80-150 mg/ml;
  • the method of the invention is sterilized and divided after the preparation step, filtered and sterilized by a 0.2 ⁇ m filter membrane, and the filtration pressure is controlled to be no more than 0.25 MPa, and the protein content is 51-55 mg/ml, and the titer is not less than 100 PEI-U/ml. Specifications are divided.
  • the method of the invention samples and determines the protein content of the product after the sub-assembly, the anti-CMV titer, the purity, the molecular size distribution, the octanoic acid residue, and the osmolality of the item quality index.
  • a filler is added, and the filler is selected from DEAE Sepharose Fast. Flow, TOYOPEARL DEAE 650M or Macro-Prep DEAE Media.
  • the invention Compared with the prior art whole process low temperature ethanol fractionation process, the invention has the following technical effects:
  • the mild octanoic acid precipitation and anion exchange chromatography processes are used to reduce the low temperature ethanol precipitation step, and the product yield can be effectively maintained while maintaining the purity of the product and improving the purity and yield.
  • the process titer recovery rate is 40-65%
  • the purification ratio is 5.30-8.14
  • the IgG recovery rate is greater than 4.9 g/L
  • the purity is greater than 98.2%
  • the IgG multimer is less than 0.1%.
  • the process of octanoic acid inactivation and nano membrane filtration and virus removal process can effectively inactivate and remove the virus, combined with anion exchange chromatography, and the virus reduction in the process is greater than 12 log 10 .
  • octanoic acid precipitation can effectively precipitate non-immunoglobulin impurities, while IgG, IgA and ceruloplasmin remain in the supernatant, and the antibody titer recovery rate in the precipitation process can reach over 90%.
  • Anion exchange chromatography can effectively remove multimers and acidic hybrid proteins. After purification, the final product does not contain impurities such as multimers and albumin.
  • the production cycle of the process of the invention is 5-7 days, and the production cycle of the prior art low temperature ethanol process is 28-30 days, which effectively improves the production efficiency, reduces the amount of ethanol used, and reduces energy consumption and labor intensity. Can effectively save production costs.
  • the preparation process of the cytomegalovirus human immunoglobulin used in the present invention not only improves product purity, yield and safety, but also saves energy and reduces production costs.
  • Fig. 1 is a flow chart showing the purification process of the intravenous cytomegalovirus human immunoglobulin of the present invention.
  • FIG. 2 is a flow chart of a prior art low temperature ethanol process for preparing ordinary human immunoglobulin.
  • Example 3 is a polyacrylamide gel electrophoresis pattern of the purified sample of the cytomegalovirus human immunoglobulin of Example 1.
  • Example 4 is a polyacrylamide gel electrophoresis pattern of the purified sample of the cytomegalovirus human immunoglobulin of Example 2.
  • Figure 5 is a polyacrylamide gel electrophoresis pattern of the purified sample of the cytomegalovirus human immunoglobulin of Example 3.
  • Figure 6 is a polyacrylamide gel electrophoresis pattern of the purified cytomegalovirus human immunoglobulin purified sample of Example 4.
  • Figure 7 is a polyacrylamide gel electrophoresis pattern of the purified cytomegalovirus human immunoglobulin purified sample of Example 5.
  • Figure 8 is a polyacrylamide gel electrophoresis pattern of a purified human immunoglobulin pH 4 purified sample of Comparative Example 1.
  • Embodiment 1 as shown in FIG. 1,
  • the supernatant is filtered through a 1.0 ⁇ m filter to control the filtration pressure to be no more than 0.25 MPa, and the pH is adjusted to 4.74 with 0.5 mol/L sodium hydroxide, and the octanoic acid concentration is adjusted to 22 mmol/L by adding water for injection, and stirred at 22 ° C.
  • the lipid enveloped virus was inactivated for 1 hour, and the supernatant was centrifuged.
  • the supernatant is filtered through a 0.45 ⁇ m filter to control the filtration pressure to be no more than 0.25 MPa, and the filtrate is concentrated 20 times with a 30 KD ultrafiltration membrane, and subjected to 8 times volume of pH 6.63, and a concentration of 25 mmol/L phosphate buffer ultrafiltration.
  • sample 2000ml protein content is 36.81 Mg/ml.
  • the filtrate was concentrated 10 times with 30KD ultrafiltration membrane, and ultrafiltration was performed with 8 times volume of water for injection.
  • the original solution was 600 ml
  • the protein content was 107.21 mg/ml, diluted with water for injection and added to maltose at 10 g/L.
  • the protein content was determined by Kjeldahl method, the titer was determined by enzyme-linked immunosorbent assay, and the purity and albumin residue were determined by non-reducing E-polyacrylamide gel electrophoresis SDS-PAGE.
  • the pH value was determined, and IgG monomer, dimer, multimer and lysate were determined by high performance liquid chromatography.
  • the residual amount of octanoic acid was determined by gas chromatography.
  • the osmolality was determined by osmolality. The test results are shown in Table 6. .
  • the SDS-PAGE electrophoresis pattern of the polyacrylamide gel electrophoresis sample of the cytomegalovirus human immunoglobulin purified sample of Example 1 was carried out, and the molecular weight of the IgG was 150KD-160KD, wherein 1, the loading buffer, 2, Anti-CMV high-priced plasma, 3, FI+II+III supernatant, 4, FI + II + III precipitation dissolved, 5, octanoic acid inactivated supernatant, 6, ethanol precipitation supernatant, 7, before DEAE chromatography, 8, penetration, 9, elution, 10, preparation of cytomegalovirus human immunoglobulin CMV-IgG.
  • the purity analysis of the map showed that the IgG purity of the caprylic acid inactivated supernatant was 89.72%, indicating that the caprylic acid precipitation and inactivation process can remove most of the heteroproteins such as albumin and fibrinogen.
  • the eluted electrophoresis band showed that anion exchange chromatography could effectively remove impurities such as multimers and residual albumin, and the penetration IgG purity was 98.76%.
  • the supernatant is filtered through a 1.0 ⁇ m filter to control the filtration pressure to be no more than 0.25 MPa, and the pH is adjusted to 5.17 with 1 mol/L sodium hydroxide, and the octanoic acid concentration is adjusted to 55 mmol/L by adding octanoic acid, and stirred at 25 ° C for 1.5 hours.
  • the lipid envelope virus was inactivated and the supernatant was centrifuged.
  • the supernatant is filtered through a 0.45 ⁇ m filter to control the filtration pressure to be no more than 0.25 MPa, and the filtrate is concentrated 15 times by a 30 KD ultrafiltration membrane, and subjected to 8 times volume of pH 6.93, and a concentration of 50 mmol/L phosphate buffer ultrafiltration. After ultrafiltration, the sample was collected in 2000 ml, and the protein content was 33.55 mg/ml.
  • the SDS-PAGE electrophoresis pattern of the polyacrylamide gel electrophoresis sample of the cytomegalovirus human immunoglobulin purified sample was as shown in FIG. 4, wherein 1, anti-CMV high-performance plasma, 2, FI+II+III The supernatant, 3, FI+II+III precipitate dissolved, 4, octanoic acid inactivated supernatant, 5, ethanol precipitation supernatant, 6, DEAE before chromatography, 7, penetration, 8, elution, 9, after preparation Cell virus human immunoglobulin CMV-IgG, 10. Loading buffer.
  • the electrophoresis pattern showed that the purification process parameters of the present example were consistent with the results of Example 1, and the IgG purity after preparation was 99.10%.
  • the supernatant is filtered through a 1.0 ⁇ m filter, the filtration pressure is controlled to be no more than 0.25 MPa, the pH is adjusted to 5.50 with 1 mol/L sodium hydroxide, octanoic acid is added, the octanoic acid concentration is adjusted to 78 mmol/L, and the mixture is stirred at 30 ° C.
  • the lipid enveloped virus was inactivated at the hour, and the supernatant was centrifuged.
  • the supernatant is filtered through a 0.45 ⁇ m filter to control the filtration pressure to be no more than 0.25 MPa, and the filtrate is concentrated 18 times by a 30 KD ultrafiltration membrane, and subjected to 10 times volume of pH 6.87, and a concentration of 40 mmol/L phosphate buffer ultrafiltration. After ultrafiltration, the sample was collected in 2000 ml, and the protein content was 34.54 mg/ml.
  • the filtrate was concentrated 10 times with 30KD ultrafiltration membrane, and ultrafiltration was performed with 8 times volume of water for injection, and 500 ml of the original solution was obtained, and the protein content was 126.57. Mg/ml, diluted with water for injection and added maltose in an amount of 10 g/L, adjusted to pH 3.85 with 1 mol/L hydrochloric acid, and sterilized by filtration through a 0.2 ⁇ m filter to control the filtration pressure to be no more than 0.25 MPa.
  • Example 2 Post-packing sampling The method described in Example 1 was used to determine protein content, potency, purity, pH, IgG monomer, dimer, multimer, lysate, albumin, octanoic acid residue and osmolality. Concentration, the test results are shown in Table 6.
  • the electropherogram showed that the purity of IgG in the foreign-injected human immunoglobulin reference product was 86.6%, the polymer contained 2.2%, the dimer contained 7.52%, and the albumin contained 2.51%.
  • the purification process parameters and examples in this example 1 consistent, the purity of IgG after preparation was 98.89%.
  • the supernatant is filtered through a 1.0 ⁇ m filter to control the filtration pressure to not more than 0.25 MPa.
  • the pH is adjusted to 5.21 with 1 mol/L sodium hydroxide, octanoic acid is added, the octanoic acid concentration is adjusted to 46 mmol/L, and the mixture is stirred at 27 ° C.
  • the lipid enveloped virus was inactivated at the hour, and the supernatant was centrifuged.
  • the supernatant is filtered through a 0.45 ⁇ m filter to control the filtration pressure to be no more than 0.25 MPa, and the filtrate is concentrated 20 times by a 30 KD ultrafiltration membrane, and the pH is 7.02 and the concentration is 60 mmol/L phosphate buffer ultrafiltration. After ultrafiltration, the sample was collected in 2000 ml, and the protein content was 37.17 mg/ml.
  • the SDS-PAGE electrophoresis pattern of the polyacrylamide gel electrophoresis sample of the cytomegalovirus human immunoglobulin purified sample of Example 4 was carried out, wherein 1, the loading buffer, 2, the anti-CMV high-valent plasma, 3, FI+II+III supernatant, 4, FI+II+III precipitation dissolved, 5, octanoic acid inactivated supernatant, 6, ethanol precipitation supernatant, 7, before DEAE chromatography, 8. Penetration, 9. Preparation of cytomegalovirus human immunoglobulin CMV-IgG, 10, elution, 11. Preparation of cytomegalovirus human immunoglobulin CMV-IgG, 12.
  • the main difference between this example and the examples 1-4 is that the plasma is separated by 8% ethanol precipitation reaction, and then the FII+III precipitate is prepared by 20% ethanol precipitation for subsequent purification.
  • the anion exchange chromatography step Use TOYOPEARL DEAE 650M (TOSOH, Japan) filler replaces DEAE Sepharose Fast Flow filler.
  • the supernatant is filtered through a 1.0 ⁇ m filter to control the filtration pressure to be no more than 0.25 MPa, and the pH is adjusted to 5.08 with 0.5 mol/L sodium hydroxide, and the octanoic acid concentration is adjusted to 38 mmol/L by adding water for injection, and stirring is carried out at 23 ° C.
  • the lipid enveloped virus was inactivated at the hour, and the supernatant was centrifuged.
  • the supernatant is filtered through a 0.45 ⁇ m filter to control the filtration pressure to be no more than 0.25 MPa, and the filtrate is concentrated 15 times by a 30 KD ultrafiltration membrane, and subjected to 10 times volume of pH 6.46, and a concentration of 25 mmol/L phosphate buffer ultrafiltration.
  • sample 170ml protein content is 38.09 Mg/ml.
  • the filtrate was concentrated 10 times with 30KD ultrafiltration membrane, and ultrafiltration was performed with 8 times volume of water for injection, and 50 ml of the original solution was obtained, and the protein content was 115.23. Mg/ml, diluted with water for injection and added maltose in an amount of 9 g/L, adjusted to pH 3.98 with 1 mol/L hydrochloric acid, and sterilized by filtration through a 0.2 ⁇ m filter to control the filtration pressure to be no more than 0.25 MPa.
  • Example 2 Post-packing sampling The method described in Example 1 was used to determine protein content, potency, purity, pH, IgG monomer, dimer, multimer, lysate, albumin, octanoic acid residue and osmolality. Concentration, the test results are shown in Table 6.
  • the electrophoresis pattern showed that
  • an ordinary human immunoglobulin is prepared by using the existing low-temperature ethanol method (the pharmacopoeia name is: intravenous immunoglobulin pH 4), as shown in FIG. 2, and the specific steps are as follows:
  • the FII precipitate was dissolved in 600 ml of water for injection, stirred at 2 to 8 ° C for 12 hours, and the supernatant was centrifuged. (7) The supernatant was concentrated to 100 ml with a 30 KD ultrafiltration membrane, and the alcohol was removed by ultrafiltration with 8 volumes of water for injection, and 150 ml was taken.
  • a comparative example 1 intravenous immunoglobulin pH4 purified sample polyacrylamide gel electrophoresis SDS-PAGE electrophoresis map wherein, 1, loading buffer, 2, ordinary human plasma, 3, diluted plasma 4, FI+II+III supernatant, 5, FI+II+III precipitation dissolved, 6, FI+III supernatant, 7, FI+III precipitation dissolved, 8, FII precipitation dissolved, 9, after preparation.
  • the electropherogram showed that the purity of the IgG after preparation was 96.7%.

Abstract

本发明公开了一种静注巨细胞病毒人免疫球蛋白及其制备方法。本发明的静注巨细胞病毒人免疫球蛋白比活性不小于2.5PEI-U/mg，抗-CMV效价不小于100PEI-U/ml，纯度大于98.2%，蛋白含量为51〜55mg/ml。本发明采用辛酸沉淀、阴离子交换层析代替传统低温乙醇法部分乙醇沉淀步骤，采用辛酸病毒灭活与纳米膜除病毒工艺。

Description

静注巨细胞病毒人免疫球蛋白及其制备方法 静注巨细胞病毒人免疫球蛋白及其制备方法
技术领域
本发明涉及一种人免疫球蛋白及其制备方法，特别是一种静注巨细胞病毒人免疫球蛋白及其制备方法。
背景技术
巨细胞病毒CMV (Cytomegalovirus) 是疱疹病毒科β属的DNA病毒，由巨细胞病毒引起的孕妇、新生儿、器官移植患者、免疫抑制患者感染死亡率可达50%～80%。巨细胞病毒人免疫球蛋白CMV-IgG因能特异中和巨细胞病毒，临床上主要用其治疗孕妇、新生儿、免疫抑制患者及器官移植手术中引发的巨细胞病毒重症感染。普通人群中的巨细胞病毒自然感染率可达80%以上，从健康人群中筛选并采集含高效价巨细胞病毒IgG抗体的血浆，采用特定的分离纯化方法制备巨细胞病毒人免疫球蛋白药物，对于治疗因巨细胞病毒引起的重症感染具有不可替代的临床应用价值。
目前，已上市的特异性人免疫球蛋白类药物大多采用低温乙醇法（Edwin J. Cohn, L.E. Strong, W.L. Hughes JR., D.J.Mulford, J.N. Ashworthm, M. Melin and H.L. Taylor, J. Am. Chem. Soc., 68(1946) 459-475.）进行分离提纯，该法由1946年美国哈佛大学Edwin.J.Cohn教授发明，通过调节工艺中的pH，蛋白浓度，温度，乙醇浓度和离子强度五个参数来实现不同蛋白的分离。长期实践表明，低温乙醇法存在以下几方面的缺陷：首先，乙醇是一种蛋白变性剂，分离过程中易引起IgG结构改变而变性失活，导致效价回收率较低，还可能导致新的抗原决定簇的形成；其次，相比柱层析技术，乙醇沉淀纯化效率较低，通常需要减少蛋白回收率来达到产品的纯度要求，因此工艺过程收率偏低，产品纯度不高；再次，因分离过程通常需在低温条件下进行，还存在硬件及运行成本高，劳动强度大等缺陷。
发明内容
本发明的目的是提供一种静注巨细胞病毒人免疫球蛋白及其制备方法，要解决的技术问题是提高产品的纯度、收率及安全性。
本发明采用以下技术方案：一种静注巨细胞病毒人免疫球蛋白，所述静注巨细胞病毒人免疫球蛋白比活性不小于2.5PEI-U/mg，抗-CMV效价不小于100PEI-U/ml，纯度大于98.2%，蛋白含量为51～55mg/ml。
一种静注巨细胞病毒人免疫球蛋白的方法，包括以下步骤：
（1）FI+II+III、FII+III沉淀制备
取经酶联免疫法测定的抗-CMV高效价的人血浆，2～30℃下融浆，合并混合；
①FI+II+III沉淀制备
用生理盐水调节血浆蛋白含量至45～55mg/ml，用冰乙酸调节pH至6.0～6.5，加入95%乙醇或无水乙醇调节乙醇浓度至20～25%，反应温度为-5.5～-4.5℃，搅拌反应为4～6小时，反应完毕离心或压滤分离获得FI+II+III沉淀；
②FII+III沉淀制备
用生理盐水调节血浆蛋白含量至45～55mg/ml，用冰乙酸调节pH至6.8～7.2，加入体积比分数为95%的乙醇或无水乙醇并调节乙醇浓度至7.5～8.5%，反应温度为-2.5～-2.0 ℃，搅拌反应4小时，反应完毕经离心或压滤分离去除FI沉淀，获得上清液，用冰乙酸调节上清液pH至6.0～6.5，加入95%乙醇或无水乙醇调节乙醇浓度至20～25%，反应温度为-5.5～-4.5℃，搅拌反应为4～6小时，反应完毕经离心或压滤分离获得FII+III沉淀；
（2）FI+II+III或FII+III沉淀溶解
FI+II+III或FII+III沉淀用0.9～1.1倍血浆量的pH 为4.8～5.2、浓度为20～80mM乙酸钠缓冲液在2～8 ℃下搅拌8～16小时，使沉淀充分溶解，经离心或压滤分离上清液；
（3）辛酸沉淀
用4mol/L乙酸或0.5～1mol/L氢氧化钠调节上清液pH至4.5～5.5，按10～100mmol/L浓度加入辛酸，在18～25 ℃下搅拌反应1～3小时，经离心或过滤分离上清液；
（4）辛酸病毒灭活
上清液用孔径为1.0μm滤膜过滤，控制过滤压力不大于0.25MPa，用4mol/L乙酸或0.5～1mol/L氢氧化钠调节滤液pH至4.5～5.5，补加注射用水或辛酸调节悬液辛酸浓度至20～80mmol/L，在20～30 ℃下搅拌1～2小时，离心或过滤分离上清液；
（5）乙醇沉淀
用0.5～1mol/L盐酸或氢氧化钠调节上清液pH至4.5～5.5，按12～16%浓度加入95%乙醇或无水乙醇进行沉淀反应，在-4.0～-2.5 ℃下搅拌反应2～8小时，离心或压滤分离上清液；
（6）超滤
上清液经0.45μm滤膜过滤，控制过滤压力不大于0.25MPa，滤液用30KD超滤膜15～20倍浓缩，经8～10倍体积pH 6.0～7.1的20～60mmol/L的磷酸盐缓冲液超滤透析，超滤后收样控制蛋白含量为25～40 mg/ml；
（7）阴离子交换层析
用pH 为6.0～7.1、浓度为20～60mmol/L的磷酸盐缓冲液作为平衡缓冲液平衡层析柱8～10柱体积，按每毫升填料不大于填料最大载量的70～80%计算上样蛋白量，上样后收集穿透液，挂柱杂蛋白经含2mol/LNaCl的pH 6.0～7.1 的20～60mmol/L的磷酸盐缓冲液洗脱；
（8）纳米膜除病毒过滤
用0.5～1mol/L的盐酸调节穿透液pH至4.2～5.0，经0.1μm滤膜预过滤后，用Novasip DV20纳米膜过滤除病毒，控制过滤压力不大于0.25Mpa；
（9）超滤
除病毒后滤液经30KD超滤膜浓缩蛋白含量至80～100 mg/ml，用8～10倍注射用水超滤，超滤后收样控制蛋白含量为80～150 mg/ml；
（10）配制
测定超滤后原液蛋白含量，用注射用水稀释调整制品蛋白含量至51～55 mg/ml，同时按9～11%的量加入麦芽糖，用0.5～1mol/L盐酸调节pH至3.8～4.2。
本发明的方法在所述配制步骤后除菌分装，经0.2μm滤膜过滤除菌，控制过滤压力不大于0.25MPa，按蛋白含量51～55mg/ml，效价不小于100PEI-U/ml规格分装。
本发明的方法抽样测定分装后制品蛋白含量，抗-CMV效价，纯度，分子大小分布，辛酸残留量，渗透压摩尔浓度的项目质量指标。
本发明的在阴离子交换层析步骤中加入填料，填料选自DEAE Sepharose Fast Flow、TOYOPEARL DEAE 650M或Macro-Prep DEAE Media。
本发明与现有技术的全过程低温乙醇分级分离工艺方法相比，具有的技术效果如下：
（1）采用了条件温和的辛酸沉淀及阴离子交换层析工艺，减少了低温乙醇沉淀的步骤，在提高产品收率的同时，能有效保持IgG的活性，提高纯度和收率。工艺过程效价回收率为40～65%，纯化倍数为5.30～8.14，IgG回收率大于4.9g/L，纯度大于98.2%，IgG多聚体小于0.1%。
（2）工艺过程中采用了辛酸病毒灭活和纳米膜过滤除病毒工艺，能有效灭活和去除病毒，结合阴离子交换层析，工艺过程病毒降低量大于12 log10。
（3）辛酸沉淀能有效沉淀非免疫球蛋白类杂质，而IgG、IgA、铜蓝蛋白保留在上清液中，沉淀过程抗体效价回收率可达90%以上。
（4）阴离子交换层析能有效去除多聚体及酸性杂蛋白，经纯化后最终产品不含多聚体、白蛋白等杂质。
（5）本发明工艺的生产周期为5～7天，现有技术的低温乙醇工艺生产周期为28～30天，有效提高了生产效率，同时减少了乙醇的使用量，降低能耗和劳动强度，能有效节约生产成本。
总之，本发明所用的巨细胞病毒人免疫球蛋白的制备工艺不仅提高了产品纯度、收率和安全性，还能节省能耗和降低生产成本。
附图说明
图1是本发明静注巨细胞病毒人免疫球蛋白纯化工艺流程图。
图2是现有技术低温乙醇法制备普通人免疫球蛋白工艺流程图。
图3是实施例1静注巨细胞病毒人免疫球蛋白纯化样品聚丙烯酰胺凝胶电泳电泳图谱。
图4是实施例2静注巨细胞病毒人免疫球蛋白纯化样品聚丙烯酰胺凝胶电泳电泳图谱。
图5是实施例3静注巨细胞病毒人免疫球蛋白纯化样品聚丙烯酰胺凝胶电泳电泳图谱。
图6是实施例4静注巨细胞病毒人免疫球蛋白纯化样品聚丙烯酰胺凝胶电泳电泳图谱。
图7是实施例5静注巨细胞病毒人免疫球蛋白纯化样品聚丙烯酰胺凝胶电泳电泳图谱。
图8是对比例1静注人免疫球蛋白pH4纯化样品聚丙烯酰胺凝胶电泳电泳图谱。
具体实施方式
下面结合实施例和附图对本发明作进一步详细说明。
实施例1，如图1所示，
（1）取经酶联免疫法测定抗-CMV高效价的人血浆20人份，25℃条件下融浆，混合后体积为11540ml。
（2）加入生理盐水2310ml调节血浆蛋白含量至49.53 mg/ml，加入冰乙酸调节pH至6.18，加入无水乙醇3926ml调节悬液乙醇浓度至22%，调节反应温度至-5.0℃，搅拌反应4小时，反应完毕离心分离获得FI+II+III沉淀。
（3）FI+II+III沉淀用pH为4.93、浓度为50mmol/L乙酸钠缓冲液11500ml进行溶解，4℃下搅拌12小时，离心分离上清液。
（4）用4mol/L乙酸调节上清pH至4.57，60mmol/L浓度加入辛酸，在21℃下搅拌反应3小时，离心分离上清液。
（5）上清液经1.0μm滤膜过滤，控制过滤压力不大于0.25MPa，用0.5mol/L氢氧化钠调节pH至4.74，补加注射用水调整辛酸浓度至22mmol/L，22℃下搅拌1小时灭活脂包膜病毒，离心分离上清液。
（6）用0.5mol/L 氢氧化钠调节上清pH至5.11，按14%浓度加入无水乙醇2070ml，在-3.8℃下反应7小时，离心分离上清液。
（7）上清液经0.45μm滤膜过滤，控制过滤压力不大于0.25MPa，滤液用30KD超滤膜20倍浓缩，经8倍体积pH为6.63、浓度为25mmol/L磷酸盐缓冲液超滤，超滤后收样2000ml，蛋白含量为36.81 mg/ml。
（8）用pH为6.63、浓度为25mmol/L磷酸盐缓冲液平衡DEAE Sepharose Fast Flow（GE Healthcare Bio-Sciences AB, 美国）柱10个柱体积，柱体积为200ml，将超滤后滤液过柱，收集穿透液7000ml，挂柱杂蛋白用含2mol/LNaCl的pH为6.63、浓度为25mmol/L的磷酸盐缓冲液洗脱。
（9）用1mol/L 盐酸调节穿透液pH至4.25，经0.1μm滤膜预过滤后，再用Novasip DV20纳米膜过滤除病毒，控制过滤压力不大于0.25MPa。
（10）除病毒后滤液用30KD超滤膜10倍浓缩，经8倍体积注射用水超滤，收获原液600ml，蛋白含量为107.21mg/ml，用注射用水稀释并按10g/L的量加入麦芽糖，用0.5mol/L盐酸调节pH至4.02，经0.2μm滤膜过滤除菌，控制过滤压力不大于0.25MPa，按蛋白含量51～55mg/ml，效价不小于100PEI-U/ml规格分装，分装后抽样用凯氏定氮法检测蛋白含量、用酶联免疫法法测定效价、非还原型E聚丙烯酰胺凝胶电泳SDS-PAGE法测定纯度及白蛋白残留、pH值测定法测定pH值，高效液相色谱法测定IgG单体、二聚体、多聚体、裂解体，气相色谱法测定辛酸残留量，渗透压摩尔浓度测定法测定渗透压摩尔浓度，检测结果见表6。
工艺过程蛋白及效价回收率见表1。
表1工艺过程蛋白及效价回收率
工艺步骤 蛋白含量（mg/ml） 抗-CMV效价（PEI-U/ml） 蛋白收率（g/L plasma） 效价回收率（%） 比活性（PEI-U/mg protein） 纯化倍数
血浆 59.45 20.79 59.45 100.00 0.35 1.00
FI+II+III 沉淀溶解 11.21 18.35 12.18 95.50 1.64 4.68
辛酸灭活上清 7.39 16.12 8.53 83.86 2.18 6.23
乙醇沉淀上清 4.90 12.24 6.78 75.13 2.50 7.14
层析前 32.73 82.58 6.38 68.54 2.52 7.21
穿透液 8.63 24.32 5.78 64.84 2.82 8.05
配制后 51.08 128.17 5.56 62.97 2.85 8.14
如图3所示，实施例1静注巨细胞病毒人免疫球蛋白纯化样品聚丙烯酰胺凝胶电泳SDS-PAGE电泳图谱，IgG分子量为150KD～160KD，其中，1、上样缓冲液，2、抗-CMV高效价血浆，3、FI+II+III上清， 4、FI+II+III沉淀溶解，5、辛酸灭活上清，6、乙醇沉淀上清，7、DEAE层析前， 8、穿透，9、洗脱，10、配制后巨细胞病毒人免疫球蛋白CMV-IgG。图谱纯度分析结果显示辛酸灭活上清的IgG纯度为89.72%，表明辛酸沉淀及灭活工艺能去除白蛋白、纤维蛋白原等大部分杂蛋白。洗脱的电泳条带显示阴离子交换层析能有效去除多聚体、残留白蛋白等杂质，穿透IgG纯度为98.76%。
实施例2
（1）取经酶联免疫法测定抗-CMV高效价的人血浆20人份，在30℃条件下融浆，混合后体积为11410ml。
（2）加入生理盐水2280ml调节血浆蛋白含量至50.30mg/ml，加入冰乙酸调节pH至6.48，加入无水乙醇4603ml调节悬液乙醇浓度至25%，调节反应温度至-4.5℃，搅拌反应6小时，反应完毕离心分离获得FI+II+III沉淀。
（3）FI+II+III沉淀用pH为5.16、浓度为50mmol/L乙酸钠缓冲液10500ml进行溶解，2.2℃搅拌12小时，经离心分离上清液。
（4）用4mol/L乙酸调节上清pH至5.07，按30mmol/L浓度加入辛酸，在25℃下搅拌2.5小时，离心分离上清液。
（5）上清液经1.0μm滤膜过滤，控制过滤压力不大于0.25MPa，用1mol/L氢氧化钠调节pH至5.17，补加辛酸调整辛酸浓度至55mmol/L，25℃下搅拌1.5小时灭活脂包膜病毒，离心分离上清液。
（6）用1mol/L氢氧化钠调节上清pH至5.48，按16%浓度加入无水乙醇2190ml，在-3.0℃下反应4小时，离心分离上清液。
（7）上清液经0.45μm滤膜过滤，控制过滤压力不大于0.25MPa，滤液经30KD超滤膜15倍浓缩，经8倍体积pH为6.93、浓度为50mmol/L磷酸盐缓冲液超滤，超滤后收样2000ml，蛋白含量为33.55mg/ml。
（8）用pH为6.93、浓度为50mmol/L磷酸盐缓冲液平衡DEAE Sepharose Fast Flow柱10个柱体积，柱体积为200ml，将超滤后滤液过柱，收集穿透液7000ml，挂柱杂蛋白用含2mol/L NaCl的pH为6.93、浓度为50mmol/L的磷酸盐缓冲液洗脱。
（9）用1mol/L盐酸调节穿透液pH至5.07，经0.1μm滤膜预过滤后，再Novasip DV20纳米膜过滤除病毒，控制过滤压力不大于0.25MPa。
（10）除病毒后滤液用30KD超滤膜10倍浓缩，经8倍体积注射用水超滤，收获原液750ml，蛋白含量为82.16mg/ml，用注射用水稀释并按10g/L的量加入麦芽糖，用1mol/L盐酸调节pH至4.12，经0.2μm滤膜过滤除菌，控制过滤压力不大于0.25MPa，分装后抽样用实施例1所述的方法进行测定蛋白含量、效价、纯度、pH值、IgG单体、二聚体、多聚体、裂解体、白蛋白、辛酸残留量和渗透压摩尔浓度，检测结果见表6。
工艺过程蛋白及效价回收率见表2。
表2工艺过程蛋白及效价回收率
工艺步骤 蛋白含量（mg/ml） 抗-CMV效价（PEI-U/ml） 蛋白收率（g/L plasma） 效价回收率（%） 比活性（PEI-U/mg protein） 纯化倍数
血浆 60.36 30.75 60.36 100.00 0.51 1.00
FI+II+III 沉淀溶解 12.75 29.93 12.63 96.39 2.35 4.60
辛酸灭活上清 8.73 26.86 8.42 84.21 3.08 6.03
乙醇沉淀上清 4.97 19.16 6.27 70.99 3.86 7.56
层析前 29.49 114.75 5.88 63.12 3.89 7.63
穿透 8.73 34.21 5.52 58.50 3.92 7.68
配制后 51.78 191.37 5.37 55.09 3.70 7.25
如图4所示，实施例2静注巨细胞病毒人免疫球蛋白纯化样品聚丙烯酰胺凝胶电泳SDS-PAGE电泳图谱，其中，1、抗-CMV高效价血浆，2、FI+II+III上清，3、FI+II+III沉淀溶解，4、辛酸灭活上清，5、乙醇沉淀上清，6、DEAE层析前，7、穿透，8、洗脱，9、配制后巨细胞病毒人免疫球蛋白CMV-IgG，10.上样缓冲液。电泳图谱显示本实施例纯化工艺参数效果与实施例1一致，配制后IgG纯度99.10%。
实施例3
（1）取经酶联免疫法测定抗-CMV高效价的人血浆20人份，15℃条件下融浆，混合后体积为11570ml。
（2）加入生理盐水2310ml调节血浆蛋白含量49.69 mg/ml，加入冰乙酸调节pH至6.32，加入无水乙醇4603ml调节悬液乙醇浓度至20%，调节反应温度至-4.8℃，搅拌反应6小时，反应完毕离心分离获得FI+II+III沉淀。
（3）FI+II+III沉淀用pH为5.02、浓度为80mmol/L乙酸钠缓冲液12500ml进行溶解，6.0℃下搅拌14小时，离心分离上清液。
（4）用1mol/L氢氧化钠调节上清pH至5.50，按40mmol/L浓度加入辛酸，在23℃下搅拌1小时，离心分离上清液。
（5）上清液经1.0μm滤膜过滤，控制过滤压力不大于0.25MPa，用1mol/L氢氧化钠调节pH至5.50，补加辛酸，调整辛酸浓度至78mmol/L，30℃下搅拌1小时灭活脂包膜病毒，离心分离上清液。
（6）用1mol/L盐酸调节上清pH至4.62，按12%浓度加入95%乙醇1765ml，在-2.5℃下反应3小时，离心分离上清液。
（7）上清液经0.45μm滤膜过滤，控制过滤压力不大于0.25MPa，滤液经30KD超滤膜18倍浓缩，经10倍体积pH为6.87、浓度为40mmol/L磷酸盐缓冲液超滤，超滤后收样2000ml，蛋白含量为34.54mg/ml。
（8）用pH为6.87、浓度为40mmol/L磷酸盐缓冲液平衡DEAE Sepharose Fast Flow，柱体积为200ml，将超滤后滤液过柱，收集穿透液6000ml，挂柱杂蛋白用含2mol/LNaCl的pH为6.87、浓度为40mmol/L的磷酸盐缓冲液洗脱。
（9）用1mol/L盐酸调节穿透液pH至4.62，经0.1μm滤膜预过滤后，再Novasip DV20纳米膜过滤除病毒，控制过滤压力不大于0.25MPa。
（10）除病毒后滤液用30KD超滤膜10倍浓缩，经8倍体积注射用水超滤，收获原液500ml，蛋白含量为126.57 mg/ml，用注射用水稀释并按10g/L的量加入麦芽糖，用1mol/L盐酸调节pH至3.85，经0.2μm滤膜过滤除菌，控制过滤压力不大于0.25MPa。分装后抽样用实施例1所述的方法进行测定蛋白含量、效价、纯度、pH值、IgG单体、二聚体、多聚体、裂解体、白蛋白、辛酸残留量和渗透压摩尔浓度，检测结果见表6。
工艺过程蛋白及效价回收率见表3。
表3工艺过程蛋白及效价回收率
工艺步骤 蛋白含量（mg/ml） 抗-HCMV效价（PEI-U/ml） 蛋白收率（g/L plasma） 效价回收率（%） 比活性（PEI-U/mg protein） 纯化倍数
血浆 59.61 27.83 59.61 100.00 0.47 1.00
FI+II+III 沉淀溶解 11.82 24.40 12.27 90.99 2.06 4.39
辛酸灭活上清 8.65 20.71 8.88 76.41 2.39 5.09
乙醇沉淀上清 4.93 13.68 6.31 61.26 2.77 5.90
层析前 35.97 100.56 5.92 58.40 2.80 5.95
穿透 9.77 28.62 5.67 53.33 2.93 6.23
配制后 52.23 155.47 5.46 50.70 2.98 6.33
如图5所示，实施例3静注巨细胞病毒人免疫球蛋白纯化样品聚丙烯酰胺凝胶电泳SDS-PAGE电泳图谱，其中，1、上样缓冲液，2.、抗-CMV高效价血浆，3、FI+II+III上清，4、FI+II+III沉淀溶解，5、辛酸灭活上清，6、乙醇沉淀上清，7、DEAE层析前，8、穿透，9、配制后巨细胞病毒人免疫球蛋白CMV-IgG，10、洗脱，11、配制后巨细胞病毒人免疫球蛋白CMV-IgG ，12、配制后巨细胞病毒人免疫球蛋白CMV-IgG，13、配制后巨细胞病毒人免疫球蛋白CMV-IgG，14、已上市静注人免疫球蛋白（国外），15，上样缓冲液。电泳图谱显示国外已上市静注人免疫球蛋白参考品IgG纯度为86.6%，多聚体含2.2%，二聚体含7.52%，白蛋白含2.51%，本实施例纯化工艺参数效果与实施例1一致，配制后IgG纯度为98.89%。
实施例4
（1）取经酶联免疫法测定抗-CMV高效价的人血浆20人份，4℃条件下融浆，混合后体积为11670ml。
（2）加入生理盐水2330ml调节血浆蛋白含量至49.27mg/ml，加入冰乙酸调节pH至6.02，加入95%乙醇4494ml调节悬液乙醇浓度至23%，调节反应温度至-5.5℃，搅拌反应6小时，反应完毕离心分离获得FI+II+III沉淀。
（3）FI+II+III沉淀用pH为4.81、浓度为30mmol/L乙酸钠缓冲液12000ml进行溶解，8.0℃下搅拌10小时，离心分离上清液。
（4）用1mol/L氢氧化钠调节上清pH至4.96，按10mmol/L浓度加入辛酸，在18℃下搅拌3小时，离心分离上清液。
（5）上清液经1.0μm滤膜过滤，控制过滤压力不大于0.25MPa，用1mol/L氢氧化钠调节pH至5.21，补加辛酸，调整辛酸浓度至46mmol/L，27℃下搅拌2小时灭活脂包膜病毒，离心分离上清液。
（6）用1mol/L盐酸调节pH至5.04，按15%浓度加入95%乙醇2396ml，在-3.5℃下反应5小时，离心分离上清液。
（7）上清液经0.45μm滤膜过滤，控制过滤压力不大于0.25MPa，滤液经30KD超滤膜20倍浓缩，经10倍体积pH为7.02、浓度为60mmol/L磷酸盐缓冲液超滤，超滤后收样2000ml，蛋白含量为37.17mg/ml。
（8）用pH为7.02、浓度为60mmol/L磷酸盐缓冲液平衡Macro-Prep DEAE Media（Bio-Rad, 美国）柱10个柱体积，柱体积为200ml，将超滤后滤液过柱，收集穿透液8000ml，挂柱杂蛋白用含2mol/LNaCl的pH为7.02、浓度为60mmol/L的磷酸盐缓冲液洗脱。
（9）用1mol/L 盐酸调节穿透液pH至4.47，经0.1μm滤膜预过滤后，再Novasip DV20纳米膜过滤除病毒，控制过滤压力不大于0.25MPa。
（10）除病毒后滤液用30KD超滤膜10倍浓缩，经8倍体积注射用水超滤，收获原液500ml，蛋白含量为134.27mg/ml，用注射用水稀释并按11g/L的量加入麦芽糖，用1mol/L盐酸调节pH至4.08，经0.2μm滤膜过滤除菌，控制过滤压力不大于0.25MPa。分装后抽样用实施例1所述的方法进行测定蛋白含量、效价、纯度、pH值、IgG单体、二聚体、多聚体、裂解体、白蛋白、辛酸残留量和渗透压摩尔浓度，检测结果见表6。
工艺过程蛋白及效价回收率见表4。
表4工艺过程蛋白及效价回收率
工艺步骤 蛋白含量（mg/ml） 抗-CMV效价（PEI-U/ml） 蛋白收率（g/L plasma） 效价回收率（%） 比活性（PEI-U/mg protein） 纯化倍数
血浆 59.11 21.60 59.11 100.00 0.37 1.00
FI+II+III 沉淀溶解 12.17 18.60 13.07 92.50 1.53 4.13
辛酸灭活上清 8.36 16.54 9.41 80.39 1.98 5.34
乙醇沉淀上清 4.87 11.47 6.86 68.24 2.36 6.37
层析前 36.29 87.70 6.37 66.98 2.42 6.53
穿透 8.09 20.51 6.02 65.10 2.54 6.85
配制后 52.81 140.20 5.74 61.19 2.65 7.18
如图6所示，实施例4静注巨细胞病毒人免疫球蛋白纯化样品聚丙烯酰胺凝胶电泳SDS-PAGE电泳图谱，其中，1、上样缓冲液，2、抗-CMV高效价血浆，3、 FI+II+III上清， 4、FI+II+III沉淀溶解，5、辛酸灭活上清，6、乙醇沉淀上清，7、DEAE层析前， 8、穿透，9、配制后巨细胞病毒人免疫球蛋白CMV-IgG，10、洗脱，11、配制后巨细胞病毒人免疫球蛋白CMV-IgG，12、配制后巨细胞病毒人免疫球蛋白CMV-IgG，13、已上市静注人免疫球蛋白（国外），14、已上市静注人免疫球蛋白（国内），15、上样缓冲液。电泳图谱对比了国内、国外的已上市静注人免疫球蛋白参考品的纯度，结果显示国内静注人免疫球蛋白IgG纯度为95.38%，多具体含0.8%，二聚体含2.37，白蛋白含0.52%。本实施例纯化工艺参数效果与实施例1一致，配制后IgG纯度为98.27%。
实施例5
本实施例与实施例1-4的主要区别在于，血浆先经过8%乙醇沉淀反应分离FI沉淀，再经20%乙醇沉淀反应制备FII+III沉淀用于后续纯化，此外，阴离子交换层析步骤用TOYOPEARL DEAE 650M（TOSOH, 日本）填料代替DEAE Sepharose Fast Flow填料。
（1）取经酶联免疫法测定抗-CMV高效价的人血浆2人份，20℃条件下融浆，混合后体积为1150ml。
（2）加入生理盐水230ml调节血浆蛋白含量50.72mg/ml，加入冰乙酸调节pH至7.00，加入95%乙醇130ml调节悬液乙醇浓度至8%，调节反应温度至-2.5℃，搅拌反应4小时，反应完毕经离心分离FI上清液，用冰乙酸调节上清pH至6.25，补加95%的乙醇247ml调节悬液乙醇浓度至20%，在-5.0℃下反应6小时，反应完毕经离心分离得到FII+III沉淀。
（3）FII+III沉淀用pH为4.98、浓度为40mmol/L乙酸钠缓冲液1150ml进行溶解，3.0℃下搅拌12小时，离心分离上清液。
（4）用4mol/L乙酸调节上清pH至4.78，按100mmol/L浓度加入辛酸，在22℃下搅拌2小时，离心分离上清液。
（5）上清液经1.0μm滤膜过滤，控制过滤压力不大于0.25MPa，用0.5mol/L氢氧化钠调节pH至5.08，加注射用水调整辛酸浓度至38mmol/L，23℃下搅拌1小时灭活脂包膜病毒，离心分离上清液。
（6）复测悬液pH至5.03，按14%浓度加入95%乙醇208ml，在-4.0℃下反应8小时，离心分离上清液。
（7）上清液经0.45μm滤膜过滤，控制过滤压力不大于0.25MPa，滤液经30KD超滤膜15倍浓缩，经10倍体积pH为6.46、浓度为25mmol/L磷酸盐缓冲液超滤，超滤后收样170ml，蛋白含量为38.09 mg/ml。
（8）用pH为6.46、浓度为25mmol/L磷酸盐缓冲液平衡TOYOPEARL DEAE 650M柱10个柱体积，柱体积为22ml，将超滤后滤液过柱，收集穿透液328ml，挂柱杂蛋白用含2mol/L NaCl的pH为6.46、浓度为25mmol/L的磷酸盐缓冲液洗脱。
（9）用1mol/L 盐酸调节穿透液pH至4.75，经0.1μm滤膜预过滤后，再Novasip DV20纳米膜过滤除病毒，控制过滤压力不大于0.25MPa。
（10）除病毒后滤液用30KD超滤膜10倍浓缩，经8倍体积注射用水超滤，收获原液50ml，蛋白含量为115.23 mg/ml，用注射用水稀释并按9g/L的量加入麦芽糖，用1mol/L盐酸调节pH至3.98，经0.2μm滤膜过滤除菌，控制过滤压力不大于0.25MPa。分装后抽样用实施例1所述的方法进行测定蛋白含量、效价、纯度、pH值、IgG单体、二聚体、多聚体、裂解体、白蛋白、辛酸残留量和渗透压摩尔浓度，检测结果见表6。
工艺过程蛋白及效价回收率见表5。
表5工艺过程蛋白及效价回收率
工艺步骤 蛋白含量（mg/ml） 抗-CMV效价（PEI-U/ml） 蛋白收率（g/L plasma） 效价回收率（%） 比活性（PEI-U/mg protein） 纯化倍数
血浆 60.86 32.42 60.86 100.00 0.53 1.00
FII+III 沉淀溶解 13.45 25.37 14.15 82.34 1.89 3.56
辛酸灭活上清 9.98 19.63 10.32 62.66 1.97 3.71
乙醇沉淀上清 5.62 13.41 6.79 50.00 2.39 4.50
层析前 36.00 95.21 5.63 45.97 2.64 4.99
穿透 15.79 43.07 5.24 44.13 2.73 5.15
配制后 51.52 144.75 4.92 42.71 2.81 5.30
如图7所示，实施例5静注巨细胞病毒人免疫球蛋白纯化样品聚丙烯酰胺凝胶电泳SDS-PAGE电泳图谱，其中，1、上样缓冲液，2、抗-CMV高效价血浆，3、FI上清，4、FII+III上清，5、FII+III沉淀溶解，6、乙醇沉淀上清，7、DEAE层析前，8、配制后巨细胞病毒人免疫球蛋白CMV-IgG，9、洗脱，10、已上市静注人免疫球蛋白（国内），11、已上市静注人免疫球蛋白（国外），12、已上市静注人免疫球蛋白（国外），13、已上市静注人免疫球蛋白（国外），14、已上市静注人免疫球蛋白（国外），15、上样缓冲液。电泳图谱显示本实施例纯化工艺参数效果与实施例1一致，配制后IgG纯度为99.82%。
对比例1，
本实施例为采用现有低温乙醇法工艺制备普通人免疫球蛋白（药典名称为：静注人免疫球蛋白pH4），如图2所示，具体步骤如下：
（1）取普通人血浆5人份，20℃条件下融浆，混合后体积为2850ml。
（2）加入生理盐水640ml调节血浆蛋白含量至47.36mg/ml，加入冰乙酸调节pH至6.28，加入无水乙醇930ml调节悬液乙醇浓度至20%，调节反应温度至-4.5℃，搅拌反应4小时，反应完离心分离FI+II+III沉淀。
（3）FI+II+III沉淀用pH5.07、浓度为20mmol/L磷酸盐缓冲液2300ml进行溶解，4℃下搅拌12小时，离心分离上清液。
（4）复测上清液pH至5.25，加入95%的乙醇456ml调节乙醇浓度至15%，-3.5℃下反应3小时，离心分离FI+III上清液。
（5）用1mol/L氢氧化钠调节FI+III上清液pH至7.05，补加95%的乙醇205ml调节乙醇浓度至20%，-7.0℃下反应6小时，经离心分离获得FII沉淀。
（6）FII沉淀用600ml注射用水溶解，2～8℃搅拌12小时，离心分离上清液。（7）上清液用30KD超滤膜浓缩至100ml，经8倍体积注射用水超滤脱醇，收样150ml。
（8）按蛋白含量大于50mg/ml、麦芽糖浓度10g/L进行配制，用1mol/L盐酸调节pH至3.97，经0.2μm滤膜过滤除菌，控制过滤压力不大于0.25MPa，
（9）除菌后配制样品放入病毒灭活间，23～25℃孵放21天，孵放完毕用Novasip DV50纳米膜过滤除病毒，控制过滤压力小于0.25MPa。分装后抽样用实施例1所述的方法进行测定蛋白含量、效价、纯度、pH值、IgG单体、二聚体、多聚体、裂解体、白蛋白、渗透压摩尔浓度。检测结果见表6。
表6巨细胞病毒人免疫球蛋白配制后样品质量指标
项目 实施例1 实施例2 实施例3 实施例4 实施例5 对比例1
蛋白含量（mg/ml） 51.08 51.78 52.23 52.81 51.52 51.58
效价（PEI-U/ml） 128.17 191.37 155.47 140.20 144.75 －
纯度（%） 98.76 99.10 98.89 98.27 99.82 96.7%
pH 4.02 4.12 3.85 4.08 3.98 3.97
IgG单体（%） 99.33 99.45 99.28 99.18 99.46 95.33%
二聚体（%） 0.62 0.52 0.67 0.74 0.47 2.52%
多聚体（%） 0.05 0.03 0.05 0.08 0.07 1.17%
裂解体（%） 0.00 0.00 0.00 0.00 0.00 0.46
白蛋白(%) 0.00 0.00 0.00 0.00 0.00 0.52
辛酸残留量（µmol/L） <50 <50 <50 <50 <50 －
渗透压摩尔浓度（mOsmol/kg） 326 337 316 342 307 331
如图8所示，对比例1静注人免疫球蛋白pH4纯化样品聚丙烯酰胺凝胶电泳SDS-PAGE电泳图谱，其中，1、上样缓冲液，2、普通人血浆，3、稀释后血浆，4、FI+II+III上清，5、FI+II+III沉淀溶解，6、FI+III上清，7、FI+III沉淀溶解，8、FII沉淀溶解，9、配制后。电泳图谱显示配制后IgG纯度为96.7%。

Claims (5)

  1. 一种静注巨细胞病毒人免疫球蛋白，其特征在于：所述静注巨细胞病毒人免 疫球蛋白比活性不 小于2.5PEI-U/mg，抗-CMV效价不小于100PEI-U/ml，纯度大于98.2%，蛋白含量为51～55mg/ml。
  2. 一种静注巨细胞病毒人免疫球蛋白的方法，包括以下步骤：
    （1）FI+II+III、FII+III沉淀制备
    取经酶联免疫法测定的抗-CMV高效价的人血浆，2～30℃下融浆，合并混合；
    ①FI+II+III沉淀制备
    用生理盐水调节血浆蛋白含量至45～55mg/ml，用冰乙酸调节pH至6.0～6.5，加入95%乙醇或无水乙醇调节乙醇浓度至20～25%，反应温度为-5.5～-4.5℃，搅拌反应为4～6小时，反应完毕离心或压滤分离获得FI+II+III沉淀；
    ②FII+III沉淀制备
    用生理盐水调节血浆蛋白含量至45～55mg/ml，用冰乙酸调节pH至6.8～7.2，加入体积比分数为95%的乙醇或无水乙醇并调节乙醇浓度至7.5～8.5%，反应温度为-2.5～-2.0 ℃，搅拌反应4小时，反应完毕经离心或压滤分离去除FI沉淀，获得上清液，用冰乙酸调节上清液pH至6.0～6.5，加入95%乙醇或无水乙醇调节乙醇浓度至20～25%，反应温度为-5.5～-4.5℃，搅拌反应为4～6小时，反应完毕经离心或压滤分离获得FII+III沉淀；
    （2）FI+II+III或FII+III沉淀溶解
    FI+II+III或FII+III沉淀用0.9～1.1倍血浆量的pH 为4.8～5.2、浓度为20～80mM乙酸钠缓冲液在2～8℃下搅拌8～16小时，使沉淀充分溶解，经离心或压滤分离上清液；
    （3）辛酸沉淀
    用4mol/L乙酸或0.5～1mol/L氢氧化钠调节上清液pH至4.5～5.5，按10～100mmol/L浓度加入辛酸，在18～25 ℃下搅拌反应1～3小时，经离心或过滤分离上清液；
    （4）辛酸病毒灭活
    上清液用孔径为1.0μm滤膜过滤，控制过滤压力不大于0.25MPa，用4mol/L乙酸或0.5～1mol/L氢氧化钠调节滤液pH至4.5～5.5，补加注射用水或辛酸调节悬液辛酸浓度至20～80mmol/L，在20～30℃下搅拌1～2小时，离心或过滤分离上清液；
    （5）乙醇沉淀
    用0.5～1mol/L盐酸或氢氧化钠调节上清液pH至4.5～5.5，按12～16%浓度加入95%乙醇或无水乙醇进行沉淀反应，在-4.0～-2.5 ℃下搅拌反应2～8小时，离心或压滤分离上清液；
    （6）超滤
    上清液经0.45μm滤膜过滤，控制过滤压力不大于0.25MPa，滤液用30KD超滤膜15～20倍浓缩，经8～10倍体积pH6.0～7.1的20～60mmol/L的磷酸盐缓冲液超滤透析，超滤后收样控制蛋白含量为25～40 mg/ml；
    （7）阴离子交换层析
    用pH为6.0～7.1、浓度为20～60mmol/L的磷酸盐缓冲液作为平衡缓冲液平衡层析柱8～10柱体积，按每毫升填料不大于填料最大载量的70～80%计算上样蛋白量，上样后收集穿透液，挂柱杂蛋白经含2mol/LNaCl的pH6.0～7.1 的20～60mmol/L的磷酸盐缓冲液洗脱；
    （8）纳米膜除病毒过滤
    用0.5～1mol/L的盐酸调节穿透液pH至4.2～5.0，经0.1μm滤膜预过滤后，用Novasip DV20纳米膜过滤除病毒，控制过滤压力不大于0.25Mpa；
    （9）超滤
    除病毒后滤液经30KD超滤膜浓缩蛋白含量至80～100mg/ml，用8～10倍注射用水超滤，超滤后收样控制蛋白含量为80～150mg/ml；
    （10）配制
    测定超滤后原液蛋白含量，用注射用水稀释调整制品蛋白含量至51～55mg/ml，同时按9～11%的量加入麦芽糖，用0.5～1mol/L盐酸调节pH至3.8～4.2。
  3. 根据权利要求2所述的静注巨细胞病毒人免疫球蛋白的方法，其特征在于：在所述配制步骤后除菌分装，经0.2μm滤膜过滤除菌，控制过滤压力不大于0.25MPa，按蛋白含量51～55mg/ml，效价不小于100PEI-U/ml规格分装。
  4. 根据权利要求3所述的静注巨细胞病毒人免疫球蛋白的方法，其特征在于：抽样测定分装后制品蛋白含量，抗-CMV效价，纯度，分子大小分布，辛酸残留量，渗透压摩尔浓度的项目质量指标。
  5. 根据权利要求2所述的静注巨细胞病毒人免疫球蛋白的方法，其特征在于：在阴离子交换层析步骤中加入填料，填料选自DEAE Sepharose Fast Flow、TOYOPEARL DEAE 650M或Macro-Prep DEAE Media。
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