• 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:
    The invention relates to medicine and relates to a method for the preparation of the main proteins of placental hemolyzed blood. The purpose of the invention is to increase the productivity of the process. The method is carried out as follows. 150 kg of human placenta are crushed in frozen form, dispersed in 150 l of water and 22 l of ethanol at T = ° C to a final concentration of 8%, then this suspension is stirred, filtered, hydrochloric acid of normal concentration is added to the resulting liquid to bring the pH to 5.1 with stirring T = 0 ° C. The clarified blood is subjected to ultrafiltration and after concentration to 60 L, it is diafiltered.
    公开号:SU1590031A3
    申请号:SU843764663
    申请日:1984-07-05
    公开日:1990-08-30
    发明作者:Тайо Жан-Луи；Анник Гаттель Поль；Андре Тарди Мишель
    申请人:Энститю Мерье (Фирма)；
    IPC主号:
    专利说明:

    The invention relates to medicine and relates to a process for the preparation of the main proteins of placental hemolyzed blood, namely albumin and hemoglobin.
    The aim of the invention is to increase the productivity of the process.
    PRI measure 1. 1. The stage of clarification of hemolyzed placental blood. 150 kg of the whole placenta are crushed frozen and dispersed in 150 liters of water, to which 22 liters of ethanol are added at 0 ° C to a final concentration of 8%. The suspension thus obtained is vigorously stirred, then filtered on a 400 l press equipped with a metal mesh with a porosity of 50 µm to separate
    blood juice. from placental tissues. The resulting liquid takes up a volume of 270 liters. Either acetic acid or hydrochloric acid of normal concentration is added to it (the choice of acid does not matter) to bring the RP to-5.1 with stirring and temperature.
    After 2 h, the suspension is introduced at a rate of 300 l / h into a centrifuge, the precipitate is removed in the amount of 7-10 kg (on different days in different ways). The resulting blood is completely transparent and takes up about 260 l.
    2. Stage of concentration and diafiltration of clarified blood.
    An ultrafilter with a membrane having an absorption threshold, equal to
    with
    00

    s
    fO
    10,000 dal. For example, two spiral filter elements of 5 m i each from Millipore are well suited for this function. After concentration to about 60 L, the pH is adjusted to 5.5 s. With a normal solution of sodium hydroxide, a is diafiltered at a constant volume with about 200 liters of softened water. The removal rate of the ultrafiltrate is approximately 50 liters / hr at an inlet pressure of 4 bar. The whole operation lasts about 8 hours at. The final pH is close to 5.25 and does not change. After regeneration, the ultrafilter can be used without clogging another 200 times. After settling in. for 15 h at 4c, concentrated blood co
    On one column, the exercise was carried out, but more than 50 cycles without losing the effect of I TIVNOSTI.
    4. The final separation of gamma globulins and hemoglobin,
    The concentration of sodium chloride in the previous filtrate (135 L) is adjusted. equal to 8 g / l, pH 7, O, and the filtrate is cooled to. Then, 45 l of oOH-11 ° C ethanol is gradually added to -20 ° C with stirring and at the end of the addition the final temperature is set to, After 15 nights, the resulting suspension is centrifuged at 300 l / h. The gamma globulin pellet weighs an average of 900 grams (data from several batches) and then it should be subjected to other purification operations that are known to produce immunoglobulins suitable for use in medicine.
    The sediment layer is completely transparent. It contains almost free of immunoglobulin hemoglobin. It is diluted with 300 liters of water at 0 ° C in order to reduce the concentration below 10%. This solution
    25
    keeps a light euglobulin pellet which is removed by centrifugation. The resulting precipitate has a weight of 100 - 300 g, depending on the party. Concentrated clarified blood in a volume of 120 liters is filtered on a membrane with a porosity of 0.2 mmk and maintained under sterile conditions until the next stage.
    3. The chromatographic step is concentrated by ultrafiltration. The column with a diameter of 16 cm and walkie-talkies according to the described system, it can be filled with a height of 1 m. 9 kg of Sphero can be diafiltered at the end to adjust the ionic strength and composition of the solution in accordance with its future use. The amount of hemoglodex-EE thus obtained, equilibrated in phosphate buffer, 0.01 11, -PH 5.25 under sterile conditions. Filtered and confined 120 liters of blood, which were obtained at the previous stage,
    injected into the column at a rate of 40 L / H.
    The column is then washed with 40 p of PO buffer, 0.01 M, pH 5.25. The filtrate in a volume of 135 l containing hemoglobin and immunoglobulins is completely devoid of albumin. It is maintained at a temperature of 2 ° C to the next stage. The albumin recorded on the column along with other proteins is then eluted by introducing 60 liters of NaCl solution with a concentration of 20 g / l. A volume of 50 liters is sufficient to quantitatively extract albumin, i.e. about 1200 g (average 8 g / kg placenta). This albumin solution should then be subjected to and; light purification steps, after which it can be used in medical
    practice. After washing with a solution with a concentration of 0.1 N and alcohol, the column is ready for a new cycle.
    fO
    i 20
    5900314
    On one column, a bout was carried out, but more than 50 cycles without losing the effect of tivality.
    4. The final separation of gamma globulins and hemoglobin,
    The concentration of sodium chloride in the previous filtrate (135 L) is adjusted. equal to 8 g / l, pH 7, O, and the filtrate is cooled to. Then, 45 l of oOH-11 ° C ethanol is gradually added to -20 ° C with stirring and at the end of the addition the final temperature is set to, After 15 nights, the resulting suspension is centrifuged at 300 l / h. The gamma globulin pellet weighs an average of 900 grams (data from several batches) and then it should be subjected to other purification operations that are known to produce immunoglobulins suitable for use in medicine.
    The sediment layer is completely transparent. It contains almost free of immunoglobulin hemoglobin. It is diluted with 300 liters of water at 0 ° C in order to reduce the concentration below 10%. This solution
    25
    d is concentrated by ultrafiltration according to the described system; it can
    five
    0
    five
    0
    five
    finally be diafiltered to control the ionic strength and composition of the solution in accordance with its future use. The amount of hemoglobin thus obtained is equal to 3700 g, i.e. its yield is close to 25 g / kg of the placenta.
    Example 2, Example 2 is repeated as in Example 1, except that in step 1 of the clarification, the alcohol concentration is 15%,
    Example 3: The procedure was carried out as in Example 1, but with the exception that at the end of stage 2, the concentration and diafiltration of the clarified
    In the blood, the RP value was adjusted to 6.8. At the end of the ten chromatography cycles, it is ascertained that the column is slightly tinted, which corresponds to a small amount of protein. However, the column is not clogged yet, which indicates significant progress compared with the prior art,
    EXAMPLE 4 The conditions of example 1 are repeated, but with the exception that at the end of the stage of concentration and diafiltration of clarified blood we have 1590031
    The pH is adjusted to 4.8. The chromatographic separation, which proceeds unhindered, but the yield of albumin is reduced, is carried out.
    Example 5. It is carried out analogously to example 1, except that the chromatography is carried out on a column with DEAE -: - safarose. The column is equilibrated and washed with the same buffer. The filtrate, containing hemoglobin and gamma globulins, is completely purified from al | bumin and is recovered by elution by means of an NaCl solution at a concentration of 20 g / l.
    Example 6 was carried out analogously to example 1, replacing the column for chromatographic separation with DEAE trisacryl. The results obtained are the same as in example 1. The properties of the obtained albumin and hemoglobin are the same as in example 1.
    The advantage of the proposed method over known, is in the possibility of industrial processing of significant amounts of placental hemolyzed blood by separating the main blood proteins on a chromatographic column with its almost continuous use without replacement.
    The proposed method can be processed by chromatography many tons of placental blood per day, not replace
    the chromatographic column, at the same time, the
    As for a known column, it is necessary to change it at the end of ten passages, while in the proposed one they remain unchanged. In all other methods, when hemolyzed blood is chromatographed, the columns are systematically clogged.
    on spherodex-SS, or on DEAE-Sepha rose, or DEAE-trisacryl at pH 4 6.8, and the remaining gamma globulin is removed from hemoglobin fraction 40 by alcohol-sedimentation, and albumin is eluted from the column with buffer containing 20 g / l sodium chloride.
    6
    As for methods that do not use chromatography, for example, selective precipitation with ethanol or another solvent, for example ammonium sulfate, capric acid, rivanol, they lead to the denaturation of hemoglobin, which cannot be used, while gemoglobin obtained by the proposed method undenatured.
    As for albumin, obtained by the proposed method, it contains 100% monomers, the content of the aggregates is almost undetectable. Albumin is stable when heated at a temperature of 50 hours.
    权利要求:
    Claims (1)
    [1]
    Invention Formula
    The method of obtaining albumin and hemoglobin from placental blood by treating hemolyzed placental blood with ethanol, filtration and chromatographic separation on an anion exchanger, characterized in that, in order to reverse the process performance,
    the ethanol treatment is carried out at a concentration of 8–15% after filtration, the filtrate is acidified to pH 5.1 and the clarified blood is subjected to ultrafiltration and diafiltration, and chroman spherodex-SS, or on DEAE-sepharose, or DEAE-trisacryle at pH 4 , 8-6,8, and the remaining gamma globulin is removed from the hemoglobin fraction 40 by alcohol-sedation, and albumin is eluted from the column with buffer containing 20 g / l of sodium chloride.
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    同族专利:
    公开号 | 公开日
    FR2548670A1|1985-01-11|
    EP0132178B1|1987-11-19|
    EP0132178A1|1985-01-23|
    CA1234047A|1988-03-15|
    ES8504837A1|1985-04-16|
    FR2548670B1|1985-10-25|
    DE3467504D1|1987-12-23|
    AT30844T|1987-12-15|
    JPH0533239B2|1993-05-19|
    ES534008A0|1985-04-16|
    JPS60149526A|1985-08-07|
    US4764279A|1988-08-16|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    DE1907014A1|1969-02-12|1970-09-03|Haller Dr Wolfgang|Process for separating blood components, in particular immunologically active globulins, from other components|
    US3657116A|1971-05-05|1972-04-18|Wolfgang Haller|Process for the separation of blood components|
    LU73094A1|1975-07-29|1977-03-24|
    FR2321932B1|1975-08-28|1980-04-11|Rhone Poulenc Ind|
    US4100149A|1975-08-28|1978-07-11|Rhone-Poulenc Industries|Method of separating proteins by ion exchange|
    SE405549B|1975-10-09|1978-12-18|Pharmacia Fine Chemicals Ab|PROCEDURE FOR ISOLATING ALBUMIN FROM PLASMA PRODUCTS BY CHROMATOGRAPHIC FRACTIONING|
    DE2718326A1|1977-04-25|1978-10-26|Behringwerke Ag|NEW PROTEIN AND METHOD FOR THE PRODUCTION THEREOF|US4639513A|1984-02-02|1987-01-27|Cuno Inc.|Intravenously injectable immunoglobulin Gand method for producing same|
    FR2596988B1|1986-04-14|1990-06-08|Merieux Inst|PROCESS FOR THE PREPARATION OF LIPIDS RICH IN LONG CHAIN POLYUNSATURATED FATTY ACIDS|
    US4810391A|1987-11-06|1989-03-07|Bio-Rad Laboratories, Inc.|Separation of hemoglobin A2 from hemoglobin mixture|
    US4980058A|1987-11-06|1990-12-25|Bio-Rad Laboratories, Inc.|Separation of hemoglobin A2 from hemoglobin mixture|
    US5277818A|1988-10-31|1994-01-11|The Green Cross Corporation|Albumin preparation and process for preparing the same|
    US5250662A|1989-10-05|1993-10-05|Alpha Therapeutic Corporation|Albumin purification|
    CA2022165C|1989-10-05|1999-11-23|Chong E. Chang|Albumin purification|
    US5650388A|1989-11-22|1997-07-22|Enzon, Inc.|Fractionated polyalkylene oxide-conjugated hemoglobin solutions|
    US5250663A|1990-04-19|1993-10-05|Miles Inc.|Preparing essentially monomeric normal human serum albumin|
    US5264555A|1992-07-14|1993-11-23|Enzon, Inc.|Process for hemoglobin extraction and purification|
    US5840851A|1993-07-23|1998-11-24|Plomer; J. Jeffrey|Purification of hemoglobin|
    CA2106612C|1993-09-21|2001-02-06|Diana Pliura|Displacement chromatography process|
    US5545328A|1993-09-21|1996-08-13|Hemosol Inc.|Purification of hemoglobin by displacement chromatography|
    US5631219A|1994-03-08|1997-05-20|Somatogen, Inc.|Method of stimulating hematopoiesis with hemoglobin|
    US6242417B1|1994-03-08|2001-06-05|Somatogen, Inc.|Stabilized compositions containing hemoglobin|
    US5741894A|1995-09-22|1998-04-21|Baxter International, Inc.|Preparation of pharmaceutical grade hemoglobins by heat treatment in partially oxygenated form|
    CA2333747C|1998-06-01|2008-02-19|Genentech, Inc.|Separation of protein monomers by use of ion-exchange chromatography|
    法律状态:
    优先权:
    申请号 | 申请日 | 专利标题
    FR8311323A|FR2548670B1|1983-07-07|1983-07-07|PROCESS FOR THE PREPARATION OF THE MAIN BLOOD HEMOLYSIS PROTEINS IN UNDENATURED FORM|
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