• 专利附录
  • 专利说明
  • 权利要求
  • 类似技术
  • 同族专利
  • 引用文献
  • 法律状态
  • 优先权
    • 专利摘要:
    Method for preparation of pure hepatitis B surface antigen HBsAg from human plasma, wherein the initial plasma after being treated to remove lipides and partially remove human plasma proteins, is submitted to digestion with pepsin in amount of 0.01-0.50 mg/mg of protein to selectively decompose the plasma proteins while the antigen remains intact, filtered in a phosphate buffered medium, up to a pH value of 7.1-7.3, through molecular filters passing molecules of up to 100,000 Daltons, the infectiosity being subsequently de-activated by means of formaldehyde, in proportion of 1:2000 by volume, for 96 hours.
    公开号:SU1409121A3
    申请号:SU823454639
    申请日:1982-06-09
    公开日:1988-07-07
    发明作者:Бжоско Витольд；Яниски Петр；Класковски Збигнев；Мадалиньски Казимеж；Донброва Анджей
    申请人:Акадэмия Мэдычна；
    IPC主号:
    专利说明:

    (21) 3454639 / 28-14
    (22) 09.06.82
    (31) P-231588
    (32) 10.06.81
    (33) PL
    (46) 07.07.88. Bul 25
    (71) Academi Medychna (PL)
    (72) Vitold Brzosko, Petr Janiski, Zbigniew Klaskowski, Kazimierz Madalinski and Andrzej Donbrova (PL)
    (53) 615.373 (088.8) (56) Eveline E. Reerink et al. Viral jHepatitis Jut. Symposium, edt. by Franklin Just. liess .. Sect V, 1981, 437-450.
    (54) (57) METHOD OF PREPARATION OF HEPATITIS B SURFACE ANTIGEN OF HUMAN BLOOD PLASMA comprising delipidi- zatsiyu and prior removal of plasma proteins by treating polyethylen glycol-f, otlichayuschiy- in that, in order to increase the purity of the final product, and after delipidization preliminarily removing plasma proteins, the starting product is treated with pepsin in an amount of 0.01-0.5 mg / mg protein, then filtered on a molecular filter that allows particles up to 100,000 daltons to pass into the phosphate-buffered medium at pH 7.1- 7.3, and deactivating forms aldehyde in the amount of 1: 2000 volume / volume for 96 hours.
    §
    WITH

    about
    to
     cm
    The invention relates to the production of hepatitis B surface antigen (HBsAg) from human blood plasma of the century and can be used to obtain HBS antibodies during treatment.
    The purpose of the invention in-. increase the purity of the final product.
    The method is carried out as follows.
    In the first stage, human blood plasma is delipidized. For this, to 1000 MP of plasma obtained from HBsAg donors, having a titer as determined by immuno-precipitation method (IEOP) of at least 1:10, 100 ml 0.1 mol, 0 is added.
    After the addition of manganese chloride, the plasma is mixed in an electromagnetic mixing device at an ice bath temperature for 1 hour. Then the material is centrifuged for 30 minutes at a speed of 6000 1 / min. After centrifugation, the precipitate that does not contain HBsAg is discarded. The liquid layer obtained by centrifugation was adjusted to pH 5.6, 0.1N HC1 was used, and polyethylene glycol 6000 (PEG 6000) was added in an amount of 75 g per 1000 ml of material. Then the material is stirred for 12 hours at 4 ° C in an electromagnetic re-masking device. The material is discharged from the mixer and centrifuged for 30 minutes at a speed of 6000 1 / min. The solder is free of antigens, discarded, and the precipitate obtained by centrifugation is collected for subsequent processing.
    This precipitate is dissolved in 200 ml of 0.9 M NaCl and the solution is diluted with deionized water to a volume that is p.1y equal to the volume of the material before centrifugation. After adding NaCl, the precipitate goes back into solution. This solution is again subjected to centrifugation for 30 minutes at a speed of 600 1 / min. After centrifugation, a layer of liquid containing HBs antigen is collected, the residue not containing it is discarded. By adding deionized water, the volume of the liquid layer is adjusted to 10,000 MP. A small amount of the residue remaining after dilution is removed by centrifugation for 30 minutes at a speed of 6000
    0 g
    with
    five
    0
    1 minute. In a subsequent stage of the antigen production process, a liquid mix is used resulting from this centrifugation.
    The material obtained during delipidization is digested with an enzyme. Dp this 1000 MP material is heated to a temperature of about 37 ° C is set by adding 1N. HCl pH 2.5. Sigma pepsin was added to the material thus obtained, crystallized several times in an amount of about 0.05 mg per 1 mg of protein solution. The protein content in the solution is determined by a spectrophotometric method. After 1 hour of digestion with pepsin, it is interrupted by adjusting the pH of the solution to 4.6 with a 1N effect. NaOH. After the digestion is complete, the solution is centrifuged for 30 minutes at a speed of 6000 1 / min. HBsAg is not detected in the precipitate formed after centrifuging, since it completely passes into the liquid layer.
    Digestion with pepsin causes the splitting of human plasma proteins into polypeptide units, the size of which does not exceed 30,000 daltons. However, the HBsAg protein is not digested.
    This is followed by a stage of purification of the material from plasma proteins, digested with pepsin, by means of molecular filtration on filters that pass particles of up to 100,000 daltons. In this, an Ami con Company (USA) closed-cycle filter system can be used, in which the material to be (i.e. filtered) is repeatedly filtered through a filtration zone, where the particles are separated from the solution by means of molecular filters. the size is less than the filtration limit. According to the proposed method, filtration is applied through filters of type HI "100, which allow passage of particles as large as 75,000 daltons. The full filtration cycle consists of 9-fold pumping of material in a volume of 80 liters at a pressure of 0, 63 atm. Material Before being fed to the filter system was diluted to volume with phosphate buffer based on the deionized water does not cause fever pH 7.1-7.3. Such water can be
    3
    obtained on equipment manufactured by the company EIGA Company (UK)
    In the course of filtration, the complete elution of plasma proteins, which have been previously digested with pepsin, occurs. The material obtained from the filtration system containing purified HBsAg is placed in 10,000 ml portions in a refrigerator for 96 hours, adding formaldehyde in an amount necessary to create its concentration in a 1: 2000 solution by volume. After the solution is removed from the refrigerator, formaldehyde is removed from the material by dedialization. Then, HBsAg is adsorbed on aluminum hydroxide added in an amount of 1 mg per I ml of solution. The finished product obtained in this way is placed in ampoules.
    Then it is analyzed for the presence of human plasma proteins in it by the method of double diffusion in agar, and no deposition lines are found at 30-crater dilution with human antiproteins by plasma. It also does not contain infectious particles of the hepatitis B virus.
    Example 1. From the HBsAg donor, plasma is collected using plasmaphoresis. HBsAg activity was determined by immuno-electrophoresis with a titer not lower than 1:16. Human blood plasma is subjected to the delipidization process by adding, per l of plasma, l l of I M and 150,000 international units of heparin in a volume of 30 ml. This material is incubated at room temperature in an electromagnetic device for 30 minutes. After this time, all the material is centrifuged in a Beckman S-6 centrifuge with an SA-10 motor at a speed of 6000 1 / min for 30 min. The resulting residue, which is HBsAg (-), is discarded, a layer of liquid is collected in a 2 l flask .
    The same amount of a 7.5% solution of polyethylene glycol with a molecular weight of 6000 is added to the product. The pH of the medium is adjusted to 5.5 with iH.HCl. The product is placed in a refrigerator and incubated overnight with constant stirring.
    The next day, centrifuged in a Behman centrifuge with a motor
    Jq 15 20
    25
    30 Q d5
    35
    50
    five
    121
    JA-100 at 600 1 / M1IH. The liquid layer, which is HBsAg, (-), is discarded, the residue is dissolved in 200 ml of 0.9% NaOH and diluted with deionized water to a volume of 2 liters. After 20–30 minutes, the copious sediment is removed by centrifuging for 30 minutes in a Beckman J-6 centrifuge with a JA-10 motor at a speed of 6000 1 / min.
    The resulting clear filtrate has HBsAg activity as determined by immunoelectroosmophoresis with a titer of 1: 8. The volume of the solution is increased to 10 liters by diluting deionized water (2 liters + 8 liters deionization water). After 30 minutes, a small amount of precipitate is observed, which is removed by centrifugation on a Beckman J-6 centrifuge with a JA-10 motor at a speed of 6000 1 / min for 30 minutes. A transparent layer of a liquid is obtained with a titer of about 1: 2 according to the test for immuno-electro-precipitation.
    90 g of NaCl are added to 10 liters of liquid to give a 0.9% NaCl solution.
    The product thus obtained was incubated at 37 ° C for 12 hours. After this time, pepsin was added to the liquid at 37 seconds and at pH 2.5 in an amount of 0.01 mg per 1 mg of proteins. The digestion of pepsin with the product takes 1 hour at 37 ° C. After 1 h, digestion is stopped by adding 0.1 n. NaOH to adjust pH to 3.5.
    The remaining precipitate is centrifuged for 20 minutes in a Beckman J-6 centrifuge with a JA-10 motor at 6000 1 / min, and then the precipitate is discarded.
    The liquid layer is subjected to molecular filtration in a DS 30 Amicon Company apparatus using an H 10 100 hollow fiber filter. The filtration is carried out in a phosphate buffer at pH 7.1.
    A 10-liter material is obtained as a result of molecular filtration, the titer is 1: 2 when determined by the method of immuno-electro-precipitation. At a 100-fold dilution, this material does not show the deposition lines according to the immunodiffusion technology using the blood plasma of animals with anti-IgG, anti-IgM, anti-IgA, human anti-proteins and human anti-protein plasma. Antigen, purified from pro51
    I f-iiHoa lIas 5 l, inactive with formaldehyde for 96 hours in the amount of i: 2000 by volume.
    Vaccination of laboratory animals such as guinea pigs or rabbits with a 100-fold dilution of the material does not give them a tunogenic response against the infection of the preparation with protein derived from human blood plasma. Only one deposition line is obtained with material containing HBsAg.
    The material obtained by the proposed method is highly immunogenic, as evidenced by vaccination of shim panza and 10 people.
    Chemical characteristics of the vaccine for viral hepatitis B:
    HBsAg, mm / cm 0,5 Total protein,
    mm / cm 0,5
    Polypeptides, mm / cm0,02
    Molecular Weight 30000
    NaCl,% 0.85
    g
    0
    five
    216
    1 and aluminum oxide, mg / cm 1
    Mertiolat, mm / cm3100
    The material does not contain HBsAg, HBsAg, or DNA polymerase, which has been proven using third-generation tests.
    Example 2. The starting material is processed according to the method of Example 1, using a greater amount of pepsin, i.e. 0.5 mg of pepsin per 1 mg of protein at. Next, the resulting product is filtered on a molecular filter, as in Example 1, using phosphate-buffered medium with a pH of 7.3 and deactivating with formaldehyde in an amount of 1: 2000 by volume for 96 hours. The required concentration of formaldehyde is obtained by adding 150 cm 4% formalin solution to 10 dm filtrate.
    The proposed method allows to increase the purity of HBsAg to 100% (by the known 15%).
    权利要求:
    Claims (1)
    [1]
    METHOD FOR PRODUCING HEPATITIS SURFACE ANTIGEN IN FROM HUMAN BLOOD PLASMA, including delipidization and preliminary removal of plasma proteins by treatment with polyethylene f glycol, characterized in that, in order to increase the purity of the final product, the plasma products are processed after delipidization and preliminary removal, in an amount of 0.01-0.5 mg / mg protein, then filtered on a molecular filter, allowing the passage of particles up to 100,000 daltons in a phosphate-buffered medium at a pH of 7.1-7.3, and the formaldehyde is deactivated house at 1: 2000 v / v for 96 hr.
    SU., 1409121 AZ
    140,912 I
    类似技术:
    公开号 | 公开日 | 专利标题
    Pope1938|Disaggregation of proteins by enzymes
    Ghosh et al.2000|Purification of lysozyme using ultrafiltration
    US3636191A|1972-01-18|Vaccine against viral hepatitis and process
    US3808189A|1974-04-30|Isolation of gamma globulin preparations enriched in iga and igm using polyethylene glycol
    US4386068A|1983-05-31|Antihemophilic factor concentrate and method for preparation
    US4017360A|1977-04-12|Method for purifying hepatitis B antigen
    SU1409121A3|1988-07-07|Method of producing surface antigen of hepatitis b from human blood plasma
    US4206014A|1980-06-03|Process for removing detergents from virus-antigen suspensions
    IL44008A|1977-08-31|Production of antisera
    JP4426720B2|2010-03-03|Method for removing endotoxin from vaccine
    US4349539A|1982-09-14|Separation of hepatitis B surface antigen
    US3434929A|1969-03-25|Process for purifying plasmin
    US4204989A|1980-05-27|Isolation of hepatitis B e antigen
    US3989818A|1976-11-02|Influenza virus vaccine
    CN109134622A|2019-01-04|The multistep continuous Integration purification process of foot-and-mouth disease virus antigen
    US4894444A|1990-01-16|Recovery and purification of immunogens from immunogen-alum complexes
    GB2038179A|1980-07-23|Meningo-encephalitis virus vaccines
    Aasletad et al.1972|Recovery of protective activity in rabies virus vaccines concentrated and purified by four different methods
    US3477911A|1969-11-11|Method of production of urokinase
    JPH05310799A|1993-11-22|Method for purifying mucin
    Neumüller1953|Chemical studies on the influenza hemagglutination inhibitor normal allantoic fluid
    JPH02223531A|1990-09-05|Vaccine of japanese encephalitis and production thereof
    RU2062617C1|1996-06-27|Method of antitoxic serum preparing
    US3477910A|1969-11-11|Method of production of urokinase
    JPH06116297A|1994-04-26|Separation of water-soluble protein in egg york
    同族专利:
    公开号 | 公开日
    HU186864B|1985-10-28|
    ES512971A0|1983-02-16|
    ES8303916A1|1983-02-16|
    FI822018A0|1982-06-07|
    DK260482A|1982-12-11|
    GB2099700B|1984-12-19|
    IT8221787D0|1982-06-09|
    FR2507478B1|1984-09-07|
    JPS584729A|1983-01-11|
    CH650682A5|1985-08-15|
    AU8471282A|1982-12-16|
    PL133476B1|1985-06-29|
    YU116882A|1985-03-20|
    RO84231B|1984-07-30|
    IT1151793B|1986-12-24|
    GB2099700A|1982-12-15|
    NO821927L|1982-12-13|
    RO84231A|1984-05-23|
    PT75031A|1982-07-01|
    FR2507478A1|1982-12-17|
    PL231588A1|1982-12-20|
    DD202506A5|1983-09-21|
    CA1203169A|1986-04-15|
    DE3221813A1|1983-02-17|
    PT75031B|1984-05-09|
    SE8203435L|1982-12-11|
    CS232726B2|1985-02-14|
    CS425482A2|1984-06-18|
    US4558011A|1985-12-10|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    US3636191A|1969-10-08|1972-01-18|Cancer Res Inst|Vaccine against viral hepatitis and process|
    DE2225548C3|1972-05-26|1980-05-29|Behringwerke Ag, 3550 Marburg|Intravenously tolerated vaccine against distemper and hepatite contagiosa canis|
    FR2303562B1|1975-03-14|1978-08-04|Community Blood Council Greate|
    US4017360A|1975-05-14|1977-04-12|Merck & Co., Inc.|Method for purifying hepatitis B antigen|
    SE420977B|1976-03-18|1981-11-16|Kabi Ab|PROCEDURE FOR CLEANING AND INSULATING HEPATITVIRUS FOR VACCINE PREPARATION|
    SU810811A1|1979-02-19|1981-03-07|Институт Полиомиелита И Вирусныхэнцефалитов Amh Cccp|Method of culturing hepatite a virus|US4683294A|1985-04-03|1987-07-28|Smith Kline Rit, S.A.|Process for the extraction and purification of proteins from culture media producing them|
    US4742158A|1986-04-25|1988-05-03|Merck & Co., Inc.|Purification of hepatitis pre-S antigens by polymerized serum albumin affinity binding|
    US5242812A|1989-02-07|1993-09-07|Bio-Technology General Corp.|Method for production and purification of hepatitis B vaccine|
    GB9822714D0|1998-10-16|1998-12-09|Smithkline Beecham Sa|Vaccines|
    法律状态:
    优先权:
    申请号 | 申请日 | 专利标题
    PL1981231588A|PL133476B1|1981-06-10|1981-06-10|Method of obtaining pure antigen hba from human serum|
    [返回顶部]