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WO1999066955A1 - Preparation ne contenant pas d'agent protecteur a double inhibition virale au moyen de la technologie utilisant l'immunoglobuline intraveineuse - Google Patents

Preparation ne contenant pas d'agent protecteur a double inhibition virale au moyen de la technologie utilisant l'immunoglobuline intraveineuse Download PDF

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Publication number
WO1999066955A1
WO1999066955A1 PCT/CN1999/000081 CN9900081W WO9966955A1 WO 1999066955 A1 WO1999066955 A1 WO 1999066955A1 CN 9900081 W CN9900081 W CN 9900081W WO 9966955 A1 WO9966955 A1 WO 9966955A1
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Prior art keywords
immunoglobulin
inactivation
virus
adjust
viruses
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French (fr)
Inventor
Aimin Chen
Shaowen Fan
Chao Zhou
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Chengdu Shuyang Pharmaceutical Factory
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Chengdu Shuyang Pharmaceutical Factory
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Priority to JP2000555641A priority Critical patent/JP2002518462A/ja
Priority to EP99926233A priority patent/EP1033136A4/en
Priority to KR1020007001911A priority patent/KR20010023277A/ko
Priority to US09/486,311 priority patent/US6338849B1/en
Priority to AU43576/99A priority patent/AU756017B2/en
Publication of WO1999066955A1 publication Critical patent/WO1999066955A1/zh
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2/00—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor
    • A61L2/0005—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor for pharmaceuticals, biologicals or living parts
    • A61L2/0011—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor for pharmaceuticals, biologicals or living parts using physical methods
    • A61L2/0023—Heat
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants

Definitions

  • the invention relates to a method for preparing biological products.
  • unprotected double virus inactivation is used to prepare human blood immunoglobulins for intravenous injection.
  • IVIG intravenous immunoglobulin
  • Placental blood IVIG was first introduced in China in 1985, and its manufacturing and testing procedures have been included in the “Chinese Biological Product Regulations”.
  • the Ministry of Health approved the trial production of human blood immunoglobulins for lyophilized low pH intravenous injection. Due to the large injection volume of intravenous immunoglobulins and the need for repeated injections, many cases of hepatitis C epidemics have been reported. There is increasing evidence that if there is no virus inactivation step during the production process Its security is difficult to guarantee.
  • virus inactivation has been prescribed by countries around the world as an indispensable step in IVIG production processes since the 1990s.
  • the earliest study of virus inactivation methods is the pasteurization method, that is, 60 to 10 hours heat inactivation method. Due to the rapid spread of AIDS, a method for destroying the lipid envelope of the virus with detergents has been developed for human immunodeficiency virus (HIV). There have been successively developed low pH incubation methods, dry heat methods, irradiation methods and filtration methods. Although these virus inactivation methods can inactivate HIV, human hepatitis B virus (HBV), human hepatitis C virus (HCV) and some common viral pathogenic factors, different methods can inactivate different viruses.
  • HBV human hepatitis B virus
  • HCV human hepatitis C virus
  • the purpose of the present invention is to overcome the shortcomings in the prior art mentioned above and provide a process for preparing an intravenous immunoglobulin by inactivating double viruses without protection, that is, using two different mechanisms of virus inactivation. In the absence of a protective agent, in order to achieve complete inactivation of the virus, the intravenous immunoglobulin can be more safely and effectively applied to the clinic.
  • the preparation process of the present invention is as follows:
  • the first step is pasteurization: Cohn's component II (F II) is used as a raw material to dissolve with 2 to 10 times ice-distilled water, adjust the pH to 3.5 to 5.0, and use 0.2 to 2.0 mmol / L.
  • Acetic acid solution molecular weight 10 ⁇ 100kDa (Dalton), ultrafiltration membrane, dealcoholization, desalting to sodium ion concentration of l-10mmol / L, adjusting immunoglobulin concentration to 0.5-2%, bottling, filling with C0 2
  • the internal pressure is 0.7-200kPa, sealed without any protective agent, 60 ° C 1 ° C, inactivated for 10 hours; the second step of low pH incubation treatment: pasteurized F ⁇ solution with 10 ⁇ 100kDa ultra-thin
  • the membrane is cut and refined.
  • the purity of the immunoglobulin is still less than 97%, the purity can be improved by gel adsorption purification, and then concentrated to an immunoglobulin concentration of 5-10%, adjusted to pH 4.1 ⁇ 0.3, sterilized and filtered, After standing at room temperature for 21 days, 5-10% glucose was added to adjust the osmotic pressure. After sterilization, semi-finished products were subjected to physical and chemical inspections and biological tests. After the semi-finished product passes the test, it is sterilized and filtered. It is divided into 25ml / bottles or 50ml / bottles, and a comprehensive quality inspection is performed according to the human blood immunoglobulin protocol for intravenous injection.
  • the intravenous immunoglobulin produced according to the above method was tested (tested by West China Medical University), and the indicator virus used was vesicular stomatitis virus ( VSV), polio virus (Polio-1), and Japanese encephalitis virus (Sindbis).
  • VSV vesicular stomatitis virus
  • polio virus Polio virus
  • Tindbis Japanese encephalitis virus
  • the methods of inactivating the virus include pasteurization, low pH inactivation, and dual virus inactivation (the present invention ), The results are shown in Table 1, Table 2, and Table 3.
  • Pasteurization Table 1 Inactivation effects of three viruses at 60 ° C
  • Control sample Process sample Control sample Process sample
  • VSV is very sensitive to the acid environment of pH 4.0 and is easily inactivated. From the 7th day after treatment, the virus is inactive. The Sindbis virus was slightly less sensitive to pH 4.0, and the virus was completely inactivated on the 21st day after treatment.
  • Double Virus Inactivation Table 3 Inactivation effect of dual virus inactivation method (the present invention) on three viruses
  • Sindbis basal virus titer 7.70 Log PFU / ml.
  • the minimum detection limit of the virus in this experiment is 0.50 Log TCID 50 /0.1ml
  • Sindbis base virus titer 8.35 Log PFU / ml
  • Test conclusion The samples submitted for testing were incubated at 22 ⁇ 24 ° C for 21 days at pH 4.0 ⁇ 0.2, inactivated VSV ⁇ 5.88—6.63 Log TCID 50 /0.1ml, HIV ⁇ 3.77— 4.17, Sindbis ⁇ 7.33—7.74 Log PFU / ml and three generations of negative transmission were negative.
  • the pasteurization method can inactivate four indicator viruses at 60 ° C for 10 hours, especially for VSV and Polio-1 viruses.
  • Low PH incubation method can effectively inactivate VSV, HIV and Sindbis virus. It has a strong inactivation effect on VSV, HIV and Sindbis, and can be completely inactivated in 7 days.
  • Double virus inactivation method After 10 hours of pasteurization, it can be stored at pH 4.0 for 21 days at room temperature, which can more safely inactivate all indicator viruses and ensure the safety of blood products.
  • the preparation of intravenous immunoglobulin according to the process of the present invention has the following advantages:
  • IVIG liquid formulations were selected instead of lyophilized formulations, which shortened the production cycle, reduced energy consumption, facilitated large-scale production, and its product quality was stable and reliable.
  • Example 1 Preparation of human blood immunoglobulin for intravenous injection with double inactivation without protective agent: the first step of pasteurization: take 16.5 kg of F II precipitate, dissolve by adding 165 liters of distilled water at 0 ° C, adjust pH 3.5, Use lmmol / L acetic acid 100kDa ultrafiltration membrane to remove alcohol, desalting to a sodium ion concentration of 1.2mmol / L, adjust the immunoglobulin concentration to 1.5%, bottle, fill with 03 2 so that the internal pressure is 100kPa, and seal under 60 ° C.
  • the second step of low pH incubation pasteurized F II solution, lOOkDa ultrafiltration membrane cutting and purification, if the immunoglobulin purity is still less than 97%, DEAE- Sephadex A-50 gel adsorption improves the purity to 98%, and then concentrates to an immunoglobulin concentration of 5%, adjusts the pH to 4.1, sterilizes and filters, and leaves it for 21 days, adds 5% glucose, and sends the semi-finished product for physical and chemical inspection after heat removal. Quality inspection and sterility test. After the semi-finished product passes the test, it is sterilized and filtered, and it is packed in 25ml / bottle or 50ml / bottle.
  • Example 2 Preparation of human blood immunoglobulin for intravenous injection by double inactivation without protective agent: the first step of pasteurization: take 15 kg of F II precipitate, add 150 liters of 0 ° C distilled water to dissolve, adjust pH 5.0, with 0.2mmol / L acetic acid 10kDa ultrafiltration membrane dealcoholation, desalting to sodium ion concentration of 10mmol / L, adjusting the immunoglobulin concentration of 2%, bottling, sealing the C0 2 so that the charge pressure of 0.7kPa conditions, 60 ⁇ 1 ° C, inactivated for 10 hours; the second step of low pH incubation: pasteurized F II solution, lOkDa ultrafiltration membrane cutting and purification, if the purity of immunoglobulin is still less than 97%, DEAE-Sephadex A-50 gel adsorption can be used to increase the purity to 98%, and then concentrated to an immunoglobulin concentration of 8%, and adjust the pH 4 .1 Sterilize
  • Example 3 Preparation of human blood immunoglobulin for intravenous injection by double inactivation without protective agent:
  • the first step of pasteurization take 16.5 kg of F II precipitate, dissolve by adding 165 liters of distilled water at 0 ° C, adjust pH 4.2, dealcoholation with 2.0mmol / L 50kDa ultrafiltration membrane acetate, desalted into a sodium ion concentration 5mmol / L, adjusting the immunoglobulin concentration of 0.5%, bottling, sealing the filling pressure of 200kPa conditions so that the C0 2, 60 Soil incubation at 1 ° C for 10 hours.
  • the second step is incubation at low pH: cut and refined by pasteurized F II solution, 50kDa ultrafiltration membrane.
  • DEAE- can be used.
  • Sephadex A-50 gel adsorption increased the purity to 98%, then concentrated to an immunoglobulin concentration of 10%, adjusted the pH to 4.1, sterilized and filtered, left it for 21 days, added 8% glucose, and performed the same operation as in Example 1.

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Description

无保护剂双重病毒灭活制备静注免疫球蛋白工艺 技术领域
本发明涉及生物制品的制备方法。 特别是无保护剂双重病毒灭活制 备静脉注射用人血免疫球蛋白。
技术背景
免疫球蛋白的生产开始于 1949年。 静脉注射用免疫球蛋白 （IVIG) 自六十年代问世至今，已陆续有 12个国家的 29个生产厂家的 30多种 IVIG 制品出现在国际市场。 我国 1985 年首次有人胎盘血 IVIG 问世， 其制造 与检定规程已载入 《中国生物制品规程》， 1992 年国家卫生部批准了冻干 低 pH静脉注射用人血免疫球蛋白的试生产。 由于静脉注射用免疫球蛋白 注射量大且常需反复多次注射， 而引起丙型肝炎流行的事例已有不少报 道， 越来越多的证据证明， 如果生产过程中没有病毒灭活步骤， 其安全 性是难以保证的。 故从九十年代起 "病毒灭活" 己作为 IVIG生产工艺中 必不可少的步骤被世界各国所规定。 病毒灭活方法研究最早的有巴氏灭 活法， 即 60Ό 10 小时加热灭活法。 由于艾滋病 （AIDS) 的快速传播， 针对人免疫缺陷病毒 （HIV) 又发展了用去污剂破坏病毒脂质性包膜的方 法。 相继发展的还有低 pH孵放法、 干热法、 照射法和过滤法。 以上这些 病毒灭活方法虽然可以对包括 HIV、 人乙型肝炎病毒 （HBV)、 人丙型肝 炎病毒 （HCV ) 和一些常见病毒致病因子进行灭活， 但不同的方法对不 同的病毒的灭活能力也有一定的差异， 尤其是保护剂存在时， 对病毒也 有一定保护作用， 制品中就可能残存微量病毒， 去污剂灭活法又存在有 机溶剂残留的问题， 不但影响制品的纯洁性， 患者在使用中还可能出现 一些不良反应。 目前国内外生产的 IVIG制品大都以蔗糖为保护及赋型剂 的冻干制剂， 在近年据文献报道 （参见 Murphy P. et al., J-Med- Virol,41(l):61-64,1993 ), 保护剂的存在尽管对保持蛋白质的天然活性起一 定的作用， 但也会对病毒产生一定的保护作用， 同时报道了数例因使用 以蔗糖作为保护剂， 所致急性肾功能衰竭的病例 (参见 Winward DB. et al., Pharmaotherapy,15(6):765-772,1995;Hifenhaus L. and Nowak Τ·, Vox Sang 67(Suppl) 1 :62-66, 1994;Horowitz B.et al., Blood, 86(11):4331-4336,1995)0 综上所述， 冻干 IVIG的临床副作用明显大于液体制品， 因为在冻干过程 中部分 IVIG分子发生聚合， 加之冻干制剂不利于大规模生产。
发明目的
本发明的目的正是为了克服上述现有技术中的不足之处而提供一种 无保护剂双重病毒灭活制备静注免疫球蛋白的工艺， 即采用两种不同机 理的病毒灭活方法， 在无保护剂条件下， 以达到对病毒的完全灭活作用， 使静注免疫球蛋白更安全有效地应用于临床。
发明内容
本发明的制备工艺如下： 第一步巴氏灭活： 以 Cohn's组分 II (F II ) 为原料用 2〜10倍的冰蒸馏水溶解， 调节 pH3.5〜5.0, 用 0.2〜2.0mmol/L 醋酸溶液， 分子量 10〜100kDa (道尔顿） 超滤膜超滤脱醇、 脱盐至钠离 子浓度为 l-10mmol/L， 调节免疫球蛋白浓度为 0.5-2%， 装瓶， 充 C02使 内压为 0.7-200kPa, 在无任何保护剂条件下封口， 60土 1 °C， 10小时灭活； 第二步低 pH孵放处理： 经巴氏灭活的 F Π溶液用 10〜100kDa超滤膜切 割精制， 如果免疫球蛋白纯度仍 < 97%时， 可通过凝胶吸附纯化来提高 纯度， 然后浓缩至免疫球蛋白浓度为 5-10%， 调 pH4.1 ±0.3， 除菌过滤， 室温放置 21 天， 加入 5-10%葡萄糖调节渗透压， 除菌后进行半成品理化 检査和生物学试验。 半成品检测合格后， 除菌过滤， 分装 25ml/瓶或 50ml/ 瓶， 按静脉注射用人血免疫球蛋白规程进行全面质量检査。
为有效地保证静注免疫球蛋白的质量及其安全性， 对按上述方法生 产的静注免疫球蛋白进行了检测 （由华西医科大学进行检测）， 采用的指 示病毒为水疱性口炎病毒 （VSV), 脊髓灰质炎病毒 （Polio- 1 )， 乙型脑 炎病毒 （Sindbis) 三种， 灭活病毒的方法采用了巴氏灭活、 低 pH灭活法 以及双重病毒灭活法 （本发明）， 其结果见表 1、 表 2、 表 3。
1. 巴氏灭活法 表 1 60°C加热对三种病毒的灭活效果
灭活 VSV Polio- T Sindbis 时间 Log TCID50/0.1ml Log TCID50/0.1ml Log PFU/ml
(小时）
对照样品 处理样品 对照样品 处理样品 对照样品 处理样品
0 6 6.5 4X106
1 6 4 6.5 2 4X106 104
4 6 3 6.3 一 4X106 < 104
5 5.8 2 6.3 一 4X106 < 103
7 5.8 1 6.3 一
10 5.8 ― 6 一 4X106 ― 从表 1中可见，巴氏灭活法对 Polio- - I病毒灭活效果相当显著，对 vs、 的灭活效果次之， 对 Sindbis的作用较差。
2. 低 pH灭活法
寸
X 表 2 pH4.0, 23°C放置对两种病毒的灭活效果
V
时间 VSV (LogTCID50/0.1ml) Sindbis (Log PFU/ml) o
(天）
对照样品 处理样品 对照样品 处理样品
0 6.5 4X 06
3 6.5 1 4X105 < 104
7 8 - 3.5X106 < 103
14 6 - 3.5X106 < 102
21 5.5 - 3.5X104 一 从表 2 中可见， VSV对 pH4.0的酸环境非常敏感容易被灭活， 从处 理后第 7天， 病毒已无活性。 而 Sindbis病毒对 pH4.0敏感性稍差， 在自 处理后的第 21天， 其病毒才被完全灭活。
3. 双重病毒灭活法 表 3 双重病毒灭活法 （本发明） 对三种病毒的灭活效果
灭活 VSV Polio- I Sindbis 时间 Log TCID50/0.1ml Log TCID50/0.1ml Log PFU/inl
(小时）
对照样品 处理样品 对照样品 处理样品 对照样品 处理样品
60 °C 0 6 6.5 4X106
5 6 2 6.5 ― 4X106 103 加热 10 6 ― 6.3 ― 4X106 一
PH4.0 7 5.5 ― 6 ― 3.5 X106 ―
23 °C 14天 5.5 ― 6 ― 3.5 X106 ― 放置 21天 5 ― 5.5 ― 3.5 X105 ― 从表 3 中可见， 巴氏灭活法 10小时， 三种指示病毒已被全部灭活， 继续进行低 pH灭活可使制品的安全性更佳。
将本发明所制备的产品送中国药品生物制品检定所和中国人民解放 军艾滋病检测确认实验室检测， 其结果见表 4、 表 5。
1. 巴氏灭活法
表 4 60°C加热对四种病毒的灭活效果
VSV Polio- I HIV Sindbis
Log TCID50/0.1ml Log TCID50/0.1ml Log TCID5C/0.1ml Log PFU/ml 时间
(小时） 对照 处理 对照 处理 对照 处理 对照 处理
样品 样品 样品 样品 样品 样品 样品 样品
0 7.00 5.00 6.00 3.13 6.00 5.77 ― 6.63
0.5 ^-0.50 0.50 -0.50 ≤Ξ0.50 一 ― ― ND
1 -0.50 0.50 ^-0.50 0.50 一 一 一 ND
4 -0.50 0.50 -0.50 0.50 ― ― ― ND
5 -0.50 0.50 -0.50 0.50 ― ― 一 ―
6 -0.50 0.50 sS-0.50 0.50 ― 一 ― ND
10 ίξ-0.50 0.50 -0.50 0.50 ^5.80 <2.00 一 一 本实验中病毒最低检测限为 0.50 Log TCID50/0.1mlc
Sindbis基础病毒滴度： 7.70Log PFU/ml。
ND未检测出病毒。 检测结论： 对送检样品， 经巴氏灭活法 （60°C加热 10 小时） 处理， 对指示病毒 VSV、 HIV, Sindbis及 Polio- 1灭活显著， 加热 30分钟后， 病毒滴度均降至试验检测限之下。病毒减少分别为 VSV: ^4.50— 4.63,Polio — I： 2.63—3.63， HIV: 3.77— 4.17(单位 Log TCID5。/0.1ml)Sindbis: ^ 6.32— 6.41 Log PFU/ml, 且对加热 6小时、 10小时样品盲传 3代， 均为 阴性。
2. 低 pH灭活法
表 5 低 pH室温孵放对三种病毒的灭活效果
Figure imgf000007_0001
本实验中病毒最低检测限为 0.50 Log TCID50/0.1ml
*: 巴氏灭活后样品
Sindbis基础病毒滴度： 8.35 Log PFU/ml
DN: 未检测出病毒
检测结论： 送检样品在 pH4.0±0.2经 22— 24°C孵放 21天， 灭活 VSV ^5.88—6.63 Log TCID50/0.1ml, HIV^3.77— 4.17, Sindbis ^7.33—7.74 Log PFU/ml且经盲传三代均为阴性。
综上所述实验结果得出结论：
1. 巴氏灭活法 60°C， 10 小时能灭活四种指示病毒， 尤其是对 VSV 和 Polio- 1病毒更有效。
2. 低 PH孵放法能有效灭活 VSV、 HIV和 Sindbis病毒。对 VSV、 HIV 和 Sindbis有很强的灭活作用， 7天即能彻底灭活。 3. 双重病毒灭活法， 巴氏灭活 10 小时后， 再经 pH4.0室温放置 21 天， 更能稳妥地灭活全部指示病毒， 确保血液制品的安全性。
按本发明工艺制作静注免疫球蛋白与现有技术相比具有的优点是：
1. 本工艺中不加任何保护剂， 不但能有效杀灭病毒， 保持 IVIC分子 的完整性和天然生物活性以及制品的纯洁性和安全性， 并有良好的稳定 性， 制品的病毒灭活效果及全面质量标准均通过国家卫生部的验证和检 定达到 《中国生物制品规程》 95年版标准。
2. 本工艺中选择了制备 IVIG液体剂型而不是冻干制剂， 縮短了生产 周期， 降低了能耗， 有利于大规模生产， 其产品质量稳定而可靠。
3. 在本制品中加入 5-10%葡萄糖调节溶液的等渗度， 避免了一些患 者在使用中出现的不良反应。
最佳实施例
实施例 1. 无保护剂双灭活制备静脉注射用人血免疫球蛋白工艺： 第 一步巴氏灭活： 取 F II沉淀 16.5千克， 加 0°C的蒸馏水 165升溶解， 调节 pH3.5 , 用 lmmol/L醋酸 lOOkDa超滤膜超滤脱醇、 脱盐至钠离子浓度为 1.2mmol/L, 调节免疫球蛋白浓度为 1.5%, 装瓶， 充 032使内压为 lOOkPa 条件下封口， 60士 1 °C, 10小时灭活； 第二步低 pH孵放处理： 经巴氏灭活 的 F II溶液， lOOkDa超滤膜切割精制， 如果免疫球蛋白纯度仍 <97%时， 可用 DEAE-Sephadex A-50凝胶吸附提高纯度至 98%， 然后浓缩至免疫 球蛋白浓度 5%， 调 pH4.1除菌过滤， 放置 21天， 加入 5%葡萄糖， 除菌 后送半成品做理化检查、 热原质检验及无菌试验。 半成品检测合格后， 除菌过滤， 分装 25ml/瓶或 50ml/瓶， 按静脉注射用人血免疫球蛋白规程 进行全面质量检查。
实施例 2. 无保护剂双灭活制备静脉注射用人血免疫球蛋白工艺： 第 一步巴氏灭活： 取 F II沉淀 15千克， 加 0°C的蒸馏水 150升溶解， 调节 PH5.0, 用 0.2mmol/L醋酸 10kDa超滤膜超滤脱醇、 脱盐至钠离子浓度为 10mmol/L, 调节免疫球蛋白浓度为 2%, 装瓶， 充 C02使内压为 0.7kPa条 件下封口， 60士 1 °C, 10小时灭活； 第二步低 pH孵放处理： 经巴氏灭活的 F II溶液， lOkDa超滤膜切割精制， 如果免疫球蛋白纯度仍 <97%时， 可 用 DEAE-Sephadex A-50凝胶吸附提高纯度至 98%， 然后浓缩至免疫球 蛋白浓度 8%， 调 pH4.1 除菌过滤， 放置 21天， 加入 10%葡萄糖， 以后 操作同实施例 1。
实施例 3. 无保护剂双灭活制备静脉注射用人血免疫球蛋白工艺： 第 一步巴氏灭活： 取 F II沉淀 16.5千克， 加 0°C的蒸馏水 165升溶解， 调节 pH4.2, 用 2.0mmol/L醋酸 50kDa超滤膜超滤脱醇、 脱盐至钠离子浓度为 5mmol/L, 调节免疫球蛋白浓度为 0.5%, 装瓶， 充 C02使内压为 200kPa 条件下封口， 60土 1 °C, 10小时灭活； 第二步低 pH孵放处理： 经巴氏灭活 的 F II溶液， 50kDa超滤膜切割精制， 如果免疫球蛋白纯度仍 <97%时， 可用 DEAE-Sephadex A-50凝胶吸附提高纯度至 98%， 然后浓缩至免疫 球蛋白浓度 10%， 调 pH4.1 除菌过滤， 放置 21天， 加入 8%葡萄糖， 以 后操作同实施例 1。

Claims

权利要求
1. 一种无保护剂双重病毒灭活制备静注免疫球蛋白工艺， 其特征在 于： 第一步巴氏灭活： 以 Cohn's组份 II为原料用 2〜10倍的冰蒸馏水溶 解,调节 pH3.5〜5.0, 用 0.2〜2.0mmol/L醋酸， 分子量 10〜100kDa超滤膜 超滤脱醇、 脱盐至钠离子浓度为 l-10mmol/L, 调节免疫球蛋白浓度为 0.5- 2%, 装瓶， 充（ 02使内压为 0.7-200kPa,在无任何条件下封口 ,60土 1 °C, 10 小时灭活； 第二步低 pH孵放处理： 经巴氏灭活的 F II溶液用 10〜100kDa 超滤膜切割精制， 然后浓缩至免疫球蛋白浓度为 5-10%， 调 pH4.1 ±0.3, 除菌过滤， 室温放置 21天， 加入 5-10%葡萄糖调节渗透压。
2. 如权利要求 1 所述的一种制备静注免疫球蛋白工艺， 其特征在于 所述第二步低 PH孵放处理中， 经巴氏灭活的 F II溶液用 10-lOOkDa超泸 膜切割精制后， 在免疫球蛋白纯度 <97%时， 可通过凝胶吸附纯化。
PCT/CN1999/000081 1998-06-25 1999-06-21 Preparation ne contenant pas d'agent protecteur a double inhibition virale au moyen de la technologie utilisant l'immunoglobuline intraveineuse Ceased WO1999066955A1 (fr)

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JP2000555641A JP2002518462A (ja) 1998-06-25 1999-06-21 保護剤を添加しない二重失活処理による静脈内注射用の免疫グロブリンの調製法。
EP99926233A EP1033136A4 (en) 1998-06-25 1999-06-21 METHOD FOR PRODUCING IMMUNOGLOBULIN SOLUTIONS FOR INTRAVENOUS INJECTION THAT DO NOT CONTAIN PROTECTIVE AGENTS BY DOUBLE STERILIZATION AGAINST VIRUSES
KR1020007001911A KR20010023277A (ko) 1998-06-25 1999-06-21 특정의 보호제를 첨가하지 않고 바이러스를 이중-멸균시킨정맥내 주사용 면역글로불린의 제조방법
US09/486,311 US6338849B1 (en) 1998-06-25 1999-06-21 Process of preparing immunoglobulin for intravenous injection by viruses double-sterilized without adding any protectant
AU43576/99A AU756017B2 (en) 1998-06-25 1999-06-21 The process of preparing immunoglobulin for intravenous injection by viruses double-sterilized without adding any protectant

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CNB98112108XA CN1137727C (zh) 1998-06-25 1998-06-25 无保护剂双重病毒灭活制备静注免疫球蛋白工艺

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ES2381828B1 (es) * 2012-03-20 2012-11-16 Grifols, S.A. PROCEDIMIENTO PARA OBTENER UNA COMPOSICION DE IgG MEDIANTE TRATAMIENTO TERMICO
CN108101981B (zh) * 2018-01-15 2019-06-04 四川远大蜀阳药业有限责任公司 一种静注免疫球蛋白的生产工艺

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AU4357699A (en) 2000-01-10
AU756017B2 (en) 2003-01-02
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CN1201694A (zh) 1998-12-16
CN1137727C (zh) 2004-02-11
JP2002518462A (ja) 2002-06-25

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