Classifications

• • 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

Definitions

• the present invention relates to a method of preparing a blood product, and more particularly to a method of preparing a human immunoglobulin.
• immunoglobulins There are at least five immunoglobulins: IgG, IgM, IgA, IgE, and IgD, of which IgG is the most important immunoglobulin, and its lack leads to low body defense.
• immunoglobulin preparations intramuscular immunoglobulin, specific immunoglobulin, and intravenous immunoglobulin.
• immunoglobulin preparations are mainly separated from blood plasma.
• U.S. Patent Application Serial No. 6,307,028 discloses a purification method using a two-step anion exchange chromatography with octanoic acid precipitation to prepare an immunoglobulin preparation having a high purity, IgG subclass distribution identical to that of a normal blood plasma.
• IgM content in Chinese blood paddles accounts for about 10% of total protein content
• IgM content in foreign blood plasmas accounts for 1%-3% of total protein content.
• the immunoglobulin with high purity is prepared, and the column required for adsorbing a large amount of IgM by the column is large in volume and high in cost, and it is difficult to realize large-scale industrial production.
• the present invention provides a novel method for preparing human immunoglobulin.
• the present invention provides a method for preparing human immunoglobulin, which comprises the steps of: (1) dissolving: dissolving Cohn component I + ⁇ + ⁇ or Cohn component II + ⁇ in water for injection; (2) octanoic acid precipitation: Precipitate impurities with octanoic acid or octanoate, and filter to obtain a filtrate;
• the first step of anion exchange chromatography the filtrate obtained in the step (2) is adjusted to pH 5.2, and purified by anion exchange chromatography to obtain a flow through solution;
• Precipitating IgM The conductivity of the flow through solution obtained in step (3) is adjusted to 500-1000 us/cm with water, and then the pH is adjusted to 6.0-7.3, allowed to stand for l-2 h, and filtered to obtain a filtrate;
• step (1) is: taking Cohn component I + II + ⁇ or Cohn component II + III, adding water for injection, stirring at 2-8 ° C until it is dissolved, and adjusting the pH to 3.8-4.9.
• amount of water for injection is: The volume ratio of water for injection to Cohn component I + ⁇ + ⁇ or Cohn component ⁇ + ⁇ is 10: 1-15: 1.
• the step (2) is: adding octanoic acid or octoate at a concentration of 10 mM to 20 mM, and adjusting the pH to 5.0-5.3.
• the filler of the anion exchange chromatography in the step (3) is Capto Q, Gigacap Q or Unosphere Q, and the volume of the exchange chromatography column is 1/105 ⁇ 1/50 of the sample volume.
• the pH of the step (4) is preferably 6.3 to 6.74, more preferably 6.51 to 6.7, still more preferably 6.51 or 6.7.
• the filler of the anion exchange chromatography in the step (5) is Macracap Q, and the volume of the exchange chromatography column is 1/50 to 1/35 of the sample volume.
• the invention also provides a human immunoglobulin prepared by the aforementioned method.
• the present invention finally provides a pharmaceutical composition prepared by using the aforementioned human immunoglobulin as an active ingredient together with a pharmaceutically acceptable adjuvant or auxiliary ingredient.
• the preparation provided by the invention skillfully increases the step of precipitating IgM between the two-step anion chromatography, and the second step of the anion exchange chromatography has a significantly smaller volume, lowers the production cost, and has a good market application prospect.
• FIG. 1 Schematic diagram of the preparation method of the human immunoglobulin of the present invention
• Cohn component ⁇ + ⁇ + ⁇ or component ⁇ + ⁇ Prepared according to the low temperature ethanol precipitation method described in Medical Biotechnology (People's Health Publishing House), Second Edition, page 1194.
• Hydrochloric acid, octanoic acid, Capto Q filler, Gigacap Q filler, Unosphere Q filler, Macrocap Q, diatomaceous earth and Beckman lMMAGE IgA test kits are all commercially available.
• Cohn component I + II +III Take 4kg Cohn component ⁇ + ⁇ + ⁇ precipitate dissolved in 40L 5 V WFI (water for injection), stir for 1h, adjust pH to 4.20 with 25M hydrochloric acid, 25 ° C Stir in a water bath for 2 h ;
• (2) octanoic acid precipitation directly add octanoic acid to a final concentration of 15 mM, adjust the pH to 5.20 with 0.5M NaOH, stir in a water bath at 25 °C for 2 h, and let stand at 8 ° C for 2 h;
• the first step of anion exchange chromatography the octanoic acid precipitation reaction suspension is filtered with a common deep filter plate, the filtrate is adjusted to pH 5.20 with 0.5 M hydrochloric acid or 0.5 M NaOH, and the first step anion exchange chromatography is carried out with Capto Q filler. , chromatography pH 5.20, line flow rate 200 cm / h, exchange column volume is 1 / 75 of the sample volume;
• the second step of anion exchange chromatography filtration with diatomaceous earth, adjust the pH of the filtrate to 5.6, and carry out the second step of anion exchange chromatography.
• the filler used is Macrocap Q, the line flow rate is 75 cm/h, and the exchange column volume is 1/40 of the sample volume;
• Product IgA content testing was performed on a Beckman IMMAGE blood protein analyzer
• the immunoturbidimetric assay is performed using the Beckman lMMAGE IgA test kit.
• specific assay methods refer to the test kit instructions.
• the indicators of human immunoglobulin prepared by the process of the present invention all meet the requirements of the pharmacopoeia.
• the process of the invention can improve the purity of the product, reduce the content of the polymer and IgA, can further improve the safety under the condition of large-dose infusion, and the method for preparing the human immunoglobulin of the invention The yield is high and is increased by more than 10% compared with the existing low-temperature ethanol method.
• Steps of the method of the invention (4) Screening of conductivity and pH in precipitated IgM for screening:
• the IgM removal rate and the yield of IgG are balanced, and the method (4) of the method of the present invention has a conductivity adjustment of 500 to 1000 us/cm and a pH adjustment of 6.0 to 7.0.
• the following comparative experiments are provided:
• Cohn component I + II + ⁇ dissolution Take 4kg Cohn component ⁇ + ⁇ + ⁇ precipitate dissolved in 40L 5 V WFI (water for injection), stir for 1h, adjust pH to 4.20 with 25M hydrochloric acid, 25 ° C Stir in a water bath for 2 h ;
• octanoic acid precipitation directly add octanoic acid to a final concentration of 15 mM, adjust the pH to 5.20 with 0.5M NaOH, heat and stir for 2 hours in a 25 ° C water bath, and let stand at 8 ° C for 2 h;
• the first step of anion exchange chromatography The octanic acid precipitation reaction suspension is filtered with a common deep filter plate, the filtrate is adjusted to pH 5.20 with 0.5 M hydrochloric acid or 0.5 M NaOH, and the first step anion exchange chromatography is carried out with Capto Q filler. Chromatography pH 5.20, line flow rate 200 cm / h ;
• the second step of anion exchange chromatography collect the chromatographic flow through the solution, add diatomaceous earth to filter, adjust the pH of the filtrate to 5.6, and carry out the second step of anion exchange chromatography.
• the filler used is Macracap Q, line flow rate. 75cm/h, the volume of the exchange column is 1/19 of the sample volume;
• the detection method is the same as in the first embodiment.
• the volume of the exchange chromatography column of the preparation method of the present invention is 1/40 of the sample volume
• the volume of the exchange chromatography column of the comparison method is 1/19 of the sample volume.
• the method of the present invention significantly reduces the column volume required for the second step of anion exchange chromatography as compared to the comparative method.
• the method provided by the present invention solves the problem of high production cost of human immunoglobulin caused by excessive IgM content in Chinese blood plasma, greatly reduces production cost, and enables large-scale industrial production of human immunoglobulin. , has a good market application prospects.

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• Chemical & Material Sciences (AREA)
• Organic Chemistry (AREA)
• Health & Medical Sciences (AREA)
• General Health & Medical Sciences (AREA)
• Biochemistry (AREA)
• Biophysics (AREA)
• Life Sciences & Earth Sciences (AREA)
• Genetics & Genomics (AREA)
• Medicinal Chemistry (AREA)
• Molecular Biology (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Immunology (AREA)
• Analytical Chemistry (AREA)
• Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
• Peptides Or Proteins (AREA)

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• 2012
  • 2012-02-22 CN CN2012100410986A patent/CN102532307B/zh active Active
• 2013
  • 2013-02-22 IN IN7228DEN2014 patent/IN2014DN07228A/en unknown
  • 2013-02-22 BR BR112014020734-8A patent/BR112014020734B1/pt active IP Right Grant
  • 2013-02-22 WO PCT/CN2013/071754 patent/WO2013123889A1/zh active Application Filing
  • 2013-02-22 RU RU2014135665A patent/RU2614119C9/ru active
• 2014
  • 2014-08-22 PH PH12014501909A patent/PH12014501909B1/en unknown
  • 2014-09-18 CO CO14207099A patent/CO7141451A2/es unknown

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