WO2019137435A1 - 一种静注免疫球蛋白的生产工艺 - Google Patents

一种静注免疫球蛋白的生产工艺 Download PDF

Info

Publication number
WO2019137435A1
WO2019137435A1 PCT/CN2019/071165 CN2019071165W WO2019137435A1 WO 2019137435 A1 WO2019137435 A1 WO 2019137435A1 CN 2019071165 W CN2019071165 W CN 2019071165W WO 2019137435 A1 WO2019137435 A1 WO 2019137435A1
Authority
WO
WIPO (PCT)
Prior art keywords
chromatography
column
intravenous immunoglobulin
eshmuno
producing
Prior art date
Application number
PCT/CN2019/071165
Other languages
English (en)
French (fr)
Inventor
郑炎
王伟
张飞官
柏婷
郑仙娇
Original Assignee
四川远大蜀阳药业有限责任公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 四川远大蜀阳药业有限责任公司 filed Critical 四川远大蜀阳药业有限责任公司
Priority to RU2020125016A priority Critical patent/RU2742655C1/ru
Priority to EP19738798.8A priority patent/EP3741775A4/en
Publication of WO2019137435A1 publication Critical patent/WO2019137435A1/zh

Links

Images

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
    • 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
    • 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
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/18—Ion-exchange chromatography
    • 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
    • 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
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/34—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against blood group antigens
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production

Definitions

  • the invention relates to the field of biopharmaceuticals, in particular to a production process of intravenous immunoglobulin.
  • Immunoglobulin is an important component of the human immune system. It is a kind of animal protein with antibody activity, which plays an important role in recognizing bacteria, viruses and activating complement in human body.
  • the immunoglobulins in human plasma can be classified into five types of IgG, IgA, IgM, IgD, and IgE, among which mainly IgG.
  • the most prominent feature of IgG is its ability to specifically recognize and bind to the antigen to form a complex. After the complex is phagocytosed by phagocytic cells, the antigen loses toxicity and pathogenicity. Since Cohn developed the ethanol precipitation method, human plasma enriched with immunoglobulin components has been widely studied and used for the treatment of various infections or congenital defects.
  • immunoglobulins obtained at home and abroad are mainly purified from plasma or component II, and few are prepared from components I+II+III or II+III, because component I+II+III Or the composition of II+III is complicated, and it is difficult to separate and purify, and it is difficult to analyze.
  • a method for obtaining an immunoglobulin, especially an IgG concentrate, in addition to selectively precipitating the protein with ethanol may also include various other methods, such as polyethylene glycol precipitation, proteolytic enzyme method, etc., and the usual production method is low temperature ethanol centrifugation. (Pressing) method, product yield is low, purity is low, and stability is not good.
  • Chinese patent CN 103394084 B provides an immunoglobulin G concentrate which removes anti-A, anti-B antibody and multi-reactive immunoglobulin IgG, preferably removes anti-A and anti-B antibodies, and obtains IgG concentrate Containing anti-A antibody does not exceed 23 ng / mg, especially between 19 and 23 ng / mg; anti-B antibody does not exceed 20 ng / mg, especially between 12 and 20 ng / mg, but its product yield And purity is investigated.
  • the existing research results show that in order to obtain IgG products with higher yield and purity and to remove anti-A and anti-B antibodies, there is a high requirement for the preparation process, and the existing processes fail to achieve good results at the same time.
  • the existing preparation process still has the following defects that cannot be solved: 1) When the flow-through solution after two chromatography is filtered through the nano membrane, the anti-A and anti-B indexes of the final product reach the requirements of the Chinese Pharmacopoeia (2010).
  • the ACA content is relatively high, and the ACA index is difficult to meet the standard after incubation; 2) After octanoic acid precipitation treatment and then two times of chromatography to ultrafiltration preparation, the osmolality is likely to occur naturally, which makes the sample unable to detect the molar penetration.
  • the pressure or osmotic pressure does not meet the standard, and the yield is low, the washing peak is long and the column is difficult to clean.
  • the object of the present invention is to solve the above technical problems, and to provide a production process of intravenous immunoglobulin, which not only can obtain a product with high yield and purity, but more importantly, anti-A of the obtained product.
  • the anti-B index and the ACA index are greatly reduced, which is in line with the requirements of the Chinese Pharmacopoeia (2010).
  • the technical scheme adopted by the present invention is: a production process of intravenous immunoglobulin, comprising the following steps:
  • the concentration of octanoic acid is preferably 10-30 mM; more preferably, the octanoic acid concentration is 22-25 mM;
  • Chromatography B flow through the wash mixture through anion chromatography column B, which includes Fractogel EMD TMAE or CaptoQ XP or Nuvia HR Q anion chromatography column, preferably Fractogel EMD TMAE anion chromatography column;
  • the chromatographic conditions are: pH 5.50 ⁇ 6.50, 5-20 mM sodium acetate buffer, conductance 0.5ms/cm ⁇ 2.5ms/cm, balance and load at a linear velocity of 60-150cm/h; preferably pH 5.70 ⁇ 6.30,8 ⁇ 16mM sodium acetate buffer, conductance 0.8ms/cm ⁇ 1.8ms/cm, balance and load at line speed 70 ⁇ 100cm/h; more preferably pH 6.0 ⁇ 6.2, 12-14mM sodium acetate buffer, conductance 1.0- 1.2ms/cm, balance and load at a line speed of 90-100cm/h;
  • Chromatography C The flow-through washing mixture is concentrated by ultrafiltration with a buffer to a protein content of 20 to 60 g/L, preferably 30 to 50 g/L, more preferably 35 to 45 g/L; the buffer is: pH 4.50 to 7.30 acetate or citrate or phosphate + NaCl, preferably pH 5.00 to 6.00, 5 to 20 mM acetate or citrate or phosphate + 80 to 150 mM NaCl, more preferably pH 5.30 ⁇ 5.50 acetate or citrate or phosphate + 100 ⁇ 120 mM NaCl; then ultrafiltration dialysis pH to the same as the buffer, the upper column C for affinity chromatography, the column C includes Eshmuno Affinity chromatography was carried out on a P Anti-A/Anti-B mixed column or an Eshmuno P Anti-A column or an Eshmuno P Anti-B column; the conditions of the affinity chromatography were: at a linear velocity of 60 ⁇ under buffer conditions Balanced and loaded at
  • the above chromatographic operation comprises performing a chromatography operation in the order of chromatography A + chromatography B + chromatography C, or performing a chromatography operation in the order of chromatography A + chromatography C, or performing chromatography in the order of chromatography B + chromatography C. operating;
  • octanoic acid precipitation and chromatographic conditions are key factors, and since the octanoic acid concentration, the protein content, the pH value during the chromatography, the conductivity, and the buffer index all have an important influence on the chromatographic results, Due to the different chromatographic conditions, there is a large gap in the index of purified immunoglobulin IgG.
  • the present invention finally shows that a good anti-A, anti-B index and ACA index can be obtained by using the above chromatographic conditions through a large number of comparative experiments.
  • the anti-A, anti-B index will not achieve such an excellent removal effect, and in addition, the yield of the product is difficult to increase to the level of the present invention.
  • the buffer conditions used in the present invention have a significant effect on the yield of the product and the osmotic pressure index. With the buffer conditions selected by the present invention, the osmotic pressure of the sample can well meet the standard, and under other buffer conditions, The osmotic pressure of the sample did not reach the standard and it was easy to produce natural crystallization.
  • the chromatography A is subjected to chromatographic treatment using an anion chromatography column, and mainly three kinds of anion chromatography columns of Fractogel EMD DEAE or Capto Q or UNO sphere Q can be used, and both can be obtained well.
  • Anti-A, anti-B index and ACA index but a more preferred way is to use Fractogel EMD DEAE anion chromatography column, which can obtain higher yield immunity by the chromatographic conditions described in the present invention.
  • Globulin; and the yield of immunoglobulin after treatment with CaptoQ and UNO sphere Q anion chromatography column is slightly lower.
  • the anion chromatography column used in chromatography B is mainly Fractogel EMD TMAE or CaptoQ XP or Nuvia HR Q anion chromatography column.
  • the three anion chromatography conditions can obtain better anti-A, anti-B index and ACA index, but A more preferred way is to use a Fractogel EMD TMAE anion chromatography column which, in combination with the chromatographic conditions described in the present invention, can obtain higher yields of immunoglobulin; and CaptoQ XP and Nuvia HR Q anions The yield of immunoglobulin after column treatment is slightly lower.
  • the three chromatographic conditions may be in the following order: 1) three-step chromatography in the order of chromatography A, chromatography B, and chromatography C; 2) chromatography A, layer The sequence of C was subjected to two-step chromatography; 3) Two-step chromatography was carried out in the order of chromatography B and chromatography C.
  • the above chromatographic methods can all have a good effect of removing anti-A and anti-B.
  • the anion chromatography column A is an anion chromatography gel with a height of 18 to 23 cm and a gel loading of 300 to 700 g precipitated on an XK50/30 column.
  • 400 mL gel preferably 350-650 g precipitate / 400 mL gel, more preferably 450-550 g precipitate / 400 mL gel.
  • the anion chromatography column B is an X-ray 50/30 column with an anion chromatography gel of 18-23 cm height, and the gel loading is 300-700 g precipitate/ 400 mL gel, preferably 350-650 g precipitate / 400 mL gel, more preferably 450-550 g precipitate / 400 mL gel.
  • the gel loading also has a certain influence on the effect of chromatography.
  • the present invention finally determines that a good chromatographic effect can be obtained under the above gel loading by a large number of experiments and process conditions.
  • the Eshmuno P Anti-A/Anti-B mixed column or the Eshmuno P Anti-A column or the Eshmuno P Anti-B column in the chromatography C is on the XK 16/20 column.
  • the Eshmuno P Anti-A and/or Eshmuno P Anti-B affinity gel is loaded with a 5-25 cm high column, preferably a 6-20 cm high column; the Eshmuno P Anti-A/Anti-B hybrid column is Eshmuno P Anti-A and Eshmuno P Anti-B affinity gels were mixed at a ratio of 1 to 2:2 to 3 and packed.
  • a product with high immunoglobulin yield can be obtained, and the anti-A or anti-B index in the product can well meet the Chinese Pharmacopoeia (2010) standard. .
  • the technical scheme further adopted by the present invention is that in the chromatography B, the flow-through washing mixture is firstly adjusted to pH and then allowed to stand at 2-8 ° C for more than 1 h, and then the supernatant is filtered, and the supernatant is passed through the anion layer.
  • the component I+II+III precipitate or the component II+III precipitate is obtained first, and the operation is to prepare the component I+II+III precipitate by reacting healthy human plasma with low temperature ethanol. Or component II+III precipitates.
  • the component I+II+III is precipitated or the component II+III is precipitated and then dissolved, and the operation is to precipitate the component I+II+III or the component II+III to precipitate 10-25 times.
  • WFI is dissolved step by step, the temperature of the solution is controlled to be 2-25 ° C, the pH is adjusted to 4.00-5.00, and the mixture is stirred for more than 1 h.
  • the pH of the solution is further adjusted to 4.80-5.80 with NaOH, and then octanoic acid is added dropwise to a final concentration of 10-30 mM. After the octanoic acid is added, the pH is adjusted to 4.80 with NaOH. 5.80, stirring was continued for 1 h or more, followed by filtration to obtain a clear liquid after octanic acid precipitation.
  • the flow-through washing mixture is ultrafiltered or diluted to a protein content of 5-100 g/L, and then 1-5% (w/w) glycine or proline is added to adjust the pH. Prepare after 3.8-4.4.
  • virus inactivation includes the following operations:
  • the filtered solution is combined, sterilized, and then incubated at 25 ° C, low pH incubation, and then the solution is mixed, prepared, and sterilized to obtain intravenous immunoglobulin;
  • the pH is first hatched at 25 ° C, and then filtered through a 20 nm or 15 nm nano membrane; the filtrate is mixed and prepared, and the intravenous immunoglobulin is obtained after sterilizing.
  • the total IgG yield can reach above 90.9%, the purity is above 99.9%, the total protein is about 56.69g, the IgA, IgM, albumin in the finished product are completely removed, the appearance and thermal stability are qualified, and the penetration
  • the molar concentration is 285mOsmol/kg, which is in line with the Chinese Pharmacopoeia (2010) standard.
  • FIG. 1 is a flow chart showing the production process of a intravenous immunoglobulin according to the present invention.
  • WFI water for injection
  • a production process of intravenous immunoglobulin is carried out according to the production process flow of the first route in FIG. 1 , and specifically includes the following steps:
  • the healthy human plasma is prepared by low-temperature ethanol reaction to obtain the component I+II+III precipitate, and then the component I+II+III precipitate is dissolved stepwise by 10 times WFI, and the temperature of the solution is controlled to 2 ° C to acetic acid. Adjust the pH to 4.00 and stir for 1 h;
  • (2) octanoic acid precipitation adjust the pH of the solution to 4.80 with NaOH, add octanoic acid to the solution in 40 minutes to a final concentration of 10 mM, and after the octanoic acid is added, adjust the pH to 4.80 with NaOH, stir for 1 hour, and filter the plate. Obtaining a clear liquid after octanoic acid precipitation;
  • a total of about 56.67 g of total protein was obtained with a product yield of 7.06 g/L plasma.
  • the albumin, IgA and IgM in the flow-through washing mixture obtained by Fractogel EMD DEAE chromatography have good removal effect.
  • Eshmuno P Anti-A/Anti-B chromatography the anti-A and anti-B removal effects are good, single-step IgG The yield was ⁇ 95%.
  • DEAE, TMAE, Anti-A/Anti-B mixed chromatography and ultrafiltration the total yield of IgG was 88.3%, the purity was 99.8%, and the Alb, IgA and IgM were cleaned and infiltrated.
  • the molar concentration was 281 mOsmol/kg, which was in line with the standard.
  • the intravenous immunoglobulin product is prepared according to the production process of the second route in Fig. 1, and the operation steps are as follows:
  • the healthy human plasma was prepared by low-temperature ethanol reaction to obtain the component II+III precipitate, and then the component II+III precipitate was dissolved stepwise by 25 times WFI, the temperature of the solution was controlled to 25 ° C, and the pH was adjusted to 5.00 with hydrochloric acid. Stir for 3h;
  • (2) octanoic acid precipitation adjust the pH of the solution to 5.80 with NaOH, add octanoic acid to the solution in 60 minutes to a final concentration of 30 mM, and after the octanoic acid is added, adjust the pH to 5.80 with NaOH, stir for 3 hours, and filter the plate. Obtaining a clear liquid after octanoic acid precipitation;
  • a Fractogel EMD DEAE anion chromatography column was prepared by using a 23 cm high Fractogel EMD DEAE anion gel on a XK50/30 column with a gel loading of 700 g precipitate/400 mL gel; step (2) The obtained clear liquid was concentrated by filtration to a protein content of 20 g/L, and then passed through a Fractogel EMD DEAE anion chromatography column.
  • the chromatographic conditions were: pH 5.50, 20 mM sodium acetate buffer, conductance 2.0 ms/cm, at a line speed of 150 cm/h. Balance and load;
  • a Fractogel EMD TMAE anion chromatography column was prepared by using a 23 cm high Fractogel EMD TMAE anion gel on a XK50/30 column with a gel loading of 700 g precipitate/400 mL gel; step (3) The flow through washing mixture was passed through a Fractogel EMD TMAE anion chromatography column; the chromatographic conditions were: pH 6.50, 20 mM sodium acetate buffer, conductance 2.5 ms/cm, equilibrated at a line speed of 150 cm/h, and loaded;
  • the IgG concentrate product was prepared by using the component I+II+III as a raw material, and the operation was as follows:
  • step (1) the precipitate of component I+II+III is dissolved in steps by 18 times of WFI, the temperature of the solution is controlled to be 15 ° C, the pH is adjusted to 4.35, and stirred for 2 hours;
  • step (2) the pH of the solution is adjusted to 5.20, and octanoic acid is added dropwise to the solution to a final concentration of 22 mM, and stirred for 2 hours, followed by filtration;
  • step (3) the Fractogel EMD DEAE gel loading is 350 g precipitate/400 mL gel, the column height is 20 cm, the clear liquid is filtered and concentrated to a protein content of 5 g/L, and the Fractogel EMD DEAE chromatography condition is pH 4.70. 8 mM sodium acetate buffer, conductance 0.8 ms/cm, balance and load at a line speed of 70 cm/h;
  • step (4) the Fractogel EMD TMAE gel loading is 350 g precipitate / 400 mL gel, the column height is 20 cm, and the Fractogel EMD TMAE chromatography conditions are: pH 5.7, 8 mM sodium acetate buffer, conductance 0.8 ms/cm, Balance and load at a line speed of 70 cm/h;
  • step (5) Eshmuno P Anti-A and Eshmuno P Anti-B affinity gel were mixed in a ratio of 1:1, the height was 6 cm, and the flow through washing mixture was buffered with sodium citrate + 80 mM NaCl at pH 5.0. The solution was concentrated by ultrafiltration to a protein content of 30 g/L, then adjusted to pH 5.0, and equilibrated at a line speed of 70 cm/h.
  • step (6) the flow-through washing mixture was ultrafiltered to a protein content of 80 g/L, 3% glycine was added, and the pH was adjusted to 4.1 with hydrochloric acid.
  • a total of about 57.21 g of total protein was obtained with a product yield of 7.12 g/L plasma.
  • the albumin, IgA and IgM in the flow-through washing mixture obtained by Fractogel EMD DEAE chromatography have good removal effect.
  • Eshmuno P Anti-A/Anti-B chromatography the anti-A and anti-B removal effects are good, single-step IgG The yield was ⁇ 95%.
  • DEAE, TMAE, Anti-A/Anti-B mixed chromatography and ultrafiltration the total IgG yield was 89.4%, the purity was 99.8%, and the Alb, IgA and IgM were cleaned and infiltrated.
  • the molar concentration is 285 mOsmol/kg, which meets the standard.
  • the IgG concentrate was prepared by using the precipitate of component II+III as the raw material, and the operation was as follows:
  • step (1) the component II+III precipitate is dissolved stepwise by 12 times WFI, the temperature of the solution is controlled to be 6 ° C, the pH is adjusted to 4.80, and stirred for 2.5 h;
  • step (2) the pH of the solution is adjusted to 5.60, and octanoic acid is added dropwise to the solution to a final concentration of 25 mM;
  • step (3) the Fractogel EMD DEAE gel loading is 650 g precipitate/400 mL gel, the column height is 22 cm, the clear liquid is filtered and concentrated to a protein content of 15 g/L, and the Fractogel EMD DEAE chromatography condition is pH 5.30. 16 mM sodium acetate buffer, conductance 1.8 ms/cm, balance and load at a line speed of 120 cm/h;
  • step (4) the Fractogel EMD TMAE gel loading is 650 g precipitate / 400 mL gel, the column height is 22 cm, and the Fractogel EMD TMAE chromatography conditions are: pH 6.30, 16 mM sodium acetate buffer, conductance 1.8 ms/cm, Balance and load at a line speed of 100 cm/h;
  • Eshmuno P Anti-A and Eshmuno P Anti-B affinity gel are mixed in a ratio of 2:3, height is 20 cm, flow through the washing mixture with pH 6.00, 20 mM sodium citrate + 150 mM
  • the NaCl buffer was concentrated by ultrafiltration to a protein content of 50 g/L, then adjusted back to pH 6.00, and equilibrated at a line speed of 130 cm/h.
  • step (6) the flow-through washing mixture was diluted to a protein content of 20 g/L, 4% proline was added, and the pH was adjusted to 3.9 with hydrochloric acid.
  • a total of about 57.27 g of total protein was obtained with a product yield of 7.12 g/L plasma.
  • the albumin, IgA and IgM in the flow-through washing mixture obtained by Fractogel EMD DEAE chromatography have good removal effect.
  • Eshmuno P Anti-A/Anti-B chromatography the anti-A and anti-B removal effects are good, single-step IgG
  • the yield was ⁇ 96%.
  • DEAE, TMAE, Anti-A/Anti-B mixed chromatography and ultrafiltration the total IgG yield was 89.32%, the purity was 99.8%, and the Alb, IgA and IgM were cleaned and infiltrated.
  • the molar concentration was 287 mOsmol/kg, which was in accordance with the standard.
  • the finished product of intravenous immunoglobulin IgG concentrate was prepared according to the method of Example 1 using human plasma component II+III as a raw material, and the operation was different in:
  • step (5) After completing the Fractogel EMD DEAE chromatography of step (3), the Eshmuno P Anti-A/Anti-B chromatography operation of step (5) is directly carried out.
  • the molar concentration is 268 mOsmol/kg, which is in line with the standard.
  • the finished product of the intravenous immunoglobulin IgG concentrate was prepared according to the method of Example 2 using the human plasma component I+II+III as a raw material, and the operation was different in that:
  • a total of about 54.35 g of total protein was obtained with a product yield of 6.85 g/L plasma.
  • the albumin, IgA and IgM in the flow-through washing mixture obtained by Fractogel EMD TMAE were well removed, and the anti-A and anti-B removal effects were good after Eshmuno P Anti-A/Anti-B chromatography.
  • the yield was ⁇ 94%.
  • the total IgG yield was 89.1%, the purity was 99.4%, and the Alb, IgA and IgM were cleaned and infiltrated.
  • the molar concentration is 263 mOsmol/kg, which is in line with the standard.
  • the finished product of intravenous immunoglobulin IgG concentrate was prepared according to the method of Example 3 using human plasma component II+III as a raw material, and the operation was different in:
  • step (1) component II+III is obtained and precipitated
  • step (6) after adding 3% glycine and adjusting the pH to 4.1, the preparation solution is firstly incubated at 25 ° C for 21 days at a low pH (pH 4.1), then filtered through a 20 nm nano membrane, and finally mixed. After preparation, sterilizing and dispensing, a intravenous immunoglobulin IgG concentrate is obtained.
  • a total of about 57.73 g of total protein was obtained with a product yield of 7.11 g/L plasma.
  • the albumin, IgA and IgM in the flow-through washing mixture obtained by Fractogel EMD DEAE chromatography have good removal effect.
  • Eshmuno P Anti-A/Anti-B chromatography the anti-A and anti-B removal effects are good, single-step IgG The yield was ⁇ 97%.
  • DEAE, TMAE, Anti-A/Anti-B mixed chromatography and ultrafiltration the total IgG yield was 91.3%, the purity was 99.9%, and the Alb, IgA and IgM were cleaned and infiltrated.
  • the molar concentration was 287 mOsmol/kg, which was in accordance with the standard.
  • the IgG concentrate product was prepared by the method of Example 4 using human plasma component I+II+III as a raw material, and the operation was as follows:
  • step (1) the component I+II+III is obtained and precipitated
  • step (3) the Fractogel EMD DEAE gel loading is 450g precipitate/400mL gel, the column height is 20cm, the clear liquid is filtered and concentrated to a protein content of 8g/L, and the Fractogel EMD DEAE chromatography condition is: pH 4.80. , 12 mM sodium acetate buffer, conductance 1.0 ms / cm, balance and load at a line speed of 90 cm / h;
  • step (4) the Fractogel EMD TMAE gel loading is 450 g precipitate / 400 mL gel, the column height is 20 cm, and the Fractogel EMD TMAE chromatography conditions are: pH 6.00, 12 mM sodium acetate buffer, conductance 1.0 ms/cm, Balance and load at a line speed of 90 cm/h;
  • step (5) the flow-through washing mixture was concentrated by ultrafiltration with a pH of 5.30, 10 mM sodium citrate + 100 mM NaCl buffer to a protein content of 35 g/L, and then adjusted to a pH of 5.30, and equilibrated at a linear velocity of 90 cm/h. Load it.
  • a total of about 56.69 g of total protein was obtained with a product yield of 7.09 g/L plasma.
  • the albumin, IgA and IgM in the flow-through washing mixture obtained by Fractogel EMD DEAE chromatography have good removal effect.
  • Eshmuno P Anti-A/Anti-B chromatography the anti-A and anti-B removal effects are good, single-step IgG The yield was ⁇ 97%.
  • DEAE, TMAE, Anti-A/Anti-B mixed chromatography and ultrafiltration the total IgG yield was 90.9%, the purity was 99.9%, and the Alb, IgA and IgM were cleaned and infiltrated.
  • the molar concentration is 285 mOsmol/kg, which meets the standard.
  • the finished product of intravenous immunoglobulin IgG concentrate was prepared according to the method of Example 4 using human plasma component II+III as a raw material, and the operation was different in that:
  • step (3) the Fractogel EMD DEAE gel loading is 550 g precipitate/400 mL gel, the column height is 20 cm, the clear liquid is concentrated by filtration to a protein content of 10 g/L, and the Fractogel EMD DEAE chromatography condition is pH 5.00. 14 mM sodium acetate buffer, conductance 1.2 ms/cm, equilibrate and load at a line speed of 100 cm/h;
  • step (4) the Fractogel EMD TMAE gel loading is 450 g precipitate / 400 mL gel, the column height is 20 cm, and the Fractogel EMD TMAE chromatography conditions are: pH 6.00, 12 mM sodium acetate buffer, conductance 1.0 ms/cm, Balance and load at a line speed of 90 cm/h;
  • step (5) the flow-through washing mixture was concentrated by ultrafiltration with a pH of 5.50, 15 mM sodium citrate + 120 mM NaCl buffer to a protein content of 45 g/L, and then adjusted back to pH 5.50, and equilibrated at a linear velocity of 110 cm/h. Load it.
  • a total of about 55.51 g of total protein was obtained with a product yield of 6.96 g/L plasma.
  • the albumin, IgA and IgM in the flow-through washing mixture obtained by Fractogel EMD DEAE chromatography have good removal effect.
  • Eshmuno P Anti-A/Anti-B chromatography the anti-A and anti-B removal effects are good, single-step IgG The yield is ⁇ 95%.
  • the total IgG yield is 90.1%, the purity is 99.8%, and the Alb, IgA and IgM are cleaned and infiltrated.
  • the molar concentration was 271 mOsmol/kg, which was in line with the standard.
  • the finished product of intravenous immunoglobulin IgG concentrate was prepared according to the method of Example 5 using human plasma component II+III as a raw material, and the operation was as follows:
  • step (6) after adding glycine and adjusting the pH, the preparation solution is firstly incubated at 25 ° C for 20 days at a low pH (pH 4.4), then filtered through a 15 nm nano membrane, and finally mixed, prepared, and sterilized. Aliquoting, a intravenous immunoglobulin IgG concentrate was obtained.
  • a total of about 55.13 g of total protein was obtained with a product yield of 6.91 g/L plasma.
  • the albumin, IgA and IgM in the flow-through washing mixture obtained by Fractogel EMD DEAE chromatography have good removal effect.
  • Eshmuno P Anti-A/Anti-B chromatography the anti-A and anti-B removal effects are good, single-step IgG The yield was ⁇ 93%.
  • DEAE, TMAE, Anti-A/Anti-B mixed chromatography and ultrafiltration the total IgG yield was 87.5%, the purity was 99.2%, and the Alb, IgA and IgM were cleaned and infiltrated.
  • the molar concentration was 267 mOsmol/kg, which was in line with the standard.
  • the finished product of intravenous immunoglobulin IgG concentrate was prepared according to the method of Example 6 using human plasma component I+II+III as a raw material, and the operation was as follows:
  • step (6) after adding glycine and adjusting, the preparation solution is firstly incubated at 25 ° C for low pH (pH 4.4) for 21 days, then filtered through a 20 nm nano membrane, and finally mixed, prepared, and sterilized. Packed to obtain a intravenous immunoglobulin IgG concentrate.
  • a total of about 54.15 g of total protein was obtained with a product yield of 6.84 g/L plasma.
  • the albumin, IgA and IgM in the flow-through washing mixture obtained by Fractogel EMD TMAE were well removed, and the anti-A and anti-B removal effects were good after Eshmuno P Anti-A/Anti-B chromatography.
  • the yield was ⁇ 94%.
  • the total yield of IgG was 88.7%, the purity was 99.6%, and the Alb, IgA and IgM were cleaned and infiltrated.
  • the molar concentration was 271 mOsmol/kg, which was in line with the standard.
  • the finished product of intravenous immunoglobulin IgG concentrate was prepared according to the method of Example 1 using human plasma component II+III as a raw material, and the operation was different in:
  • step (5) affinity chromatography was performed using a single column of Eshmuno P Anti-A, and the column height was 6 cm.
  • the finished product of the intravenous immunoglobulin IgG concentrate was prepared according to the method of Example 2 using the human plasma component I+II+III as a raw material, and the operation was different in that:
  • step (5) affinity chromatography was performed using a single column of Eshmuno P Anti-B, and the column height was 25 cm.
  • the finished product of intravenous immunoglobulin IgG concentrate was prepared according to the method of Example 5 using human plasma component II+III as a raw material, and the operation was also different in:
  • step (5) affinity chromatography was carried out using a single column of Eshmuno P Anti-A, and the column height was 6 cm.
  • the finished product of the intravenous immunoglobulin IgG concentrate was prepared according to the method of Example 6 using the human plasma component I+II+III as a raw material, and the operation was also different in:
  • step (2) After the completion of the step (2), the steps (4) and (5) are directly carried out, wherein in the step (5), affinity chromatography is carried out using a single column of Eshmuno P Anti-B, and the column height is 25 cm.
  • the IgG concentrate product was prepared according to the starting materials of Example 1 and specific procedures, and the operation was as follows:
  • the IgG concentrate product was prepared according to the raw materials of Example 2 and specific procedures, and the operation was as follows:
  • the finished IgG concentrate was prepared on the pilot scale of the component I+II+III precipitate.
  • the yield statistics are shown in Table 2 below:
  • a finished intravenous immunoglobulin IgG concentrate was prepared according to the materials and methods of Example 1.
  • the protein content was increased to 30 g/L, and the gel loading was increased to 800 g of precipitation/400 mL of gel.
  • the removal of IgM and IgA in the flow-through washing mixture obtained by Fractogel EMD DEAE chromatography was incomplete.
  • the IgM and IgA residues in the product are at least 2 to 5%.
  • a finished intravenous immunoglobulin IgG concentrate was prepared according to the materials and methods of Example 2.
  • the chromatographic conditions were changed to: pH 6.50, 25 mM sodium acetate buffer, conductance 3.0 ms/cm, equilibrated at a line speed of 170 cm/h, and loaded; flow-through washing after Fractogel EMD DEAE chromatography
  • the removal of IgM and IgA in the mixed solution is incomplete, and the residual of IgM and IgA in the product is at least 3 to 6%.
  • a finished intravenous immunoglobulin IgG concentrate was prepared according to the materials and methods of Example 5.
  • the buffer in the step (5) is concentrated by ultrafiltration using a sodium acetate + 60 mM NaCl buffer having a pH of 4.20 to a protein content of 80 g/L, and the total IgG yield is 68.3%, and the Alb, IgA, and IgM are all removed.
  • -A 1:32
  • Anti-B 1:16
  • the osmolality does not meet the Chinese Pharmacopoeia (2010) standard, and natural crystallization is visible to the naked eye.
  • a finished intravenous immunoglobulin IgG concentrate was prepared according to the materials and methods of Example 6.
  • the buffer in step (5) was diluted with sodium phosphate + 180 mM NaCl buffer having a pH of 4.10 to a protein content of 10 g/L, and the total IgG yield was 57.1%.
  • a finished intravenous immunoglobulin IgG concentrate was prepared according to the materials and methods of Example 1.
  • a finished intravenous immunoglobulin IgG concentrate was prepared according to the materials and methods of Example 7.
  • a finished intravenous immunoglobulin IgG concentrate was prepared according to the materials and methods of Example 8.
  • octanoic acid was added dropwise to the lysate to a final concentration of 5 mM in 40 minutes, and the subsequent operation was carried out by pressure filtration, resulting in the Fractogel EMD DEAE gel splicing.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Immunology (AREA)
  • Analytical Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Microbiology (AREA)
  • Mycology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Peptides Or Proteins (AREA)

Abstract

本发明提供了一种静注免疫球蛋白的生产工艺，其是从健康人血浆中分离得到组分Ⅰ+Ⅱ+Ⅲ/Ⅱ+Ⅲ；然后经辛酸沉淀过滤得到澄清液；并将澄清液依次通过以下层析：阴离子层析A、阴离子层析B、亲合层析C；层析后的流涤液进行超滤浓缩或稀释后配制，然后经纳米膜过滤，低pH值孵放，孵放后将溶液混合、配制、除菌得静注免疫球蛋白。本发明提供的生产工艺能够获得收率和纯度均较高的IgG产品，更为关键的是所得产品的抗A、抗B指标和ACA指标均大大降低，很好符合了《中国药典》的要求，而且样品的渗透压摩尔浓度也很好符合要求。

Description

一种静注免疫球蛋白的生产工艺 技术领域
本发明涉及生物制药领域，具体涉及一种静注免疫球蛋白的生产工艺。
背景技术
免疫球蛋白(Ig)是人体免疫系统的重要组成部分，是一类具有抗体活性的动物蛋白，其在人体中发挥着识别细菌、病毒以及活化补体的重要作用。人血浆中的免疫球蛋白可分为IgG、IgA、IgM、IgD、IgE五类，其中主要是IgG。IgG最显著的特点是能够特异性地识别抗原，并与之结合形成复合物，复合物被吞噬细胞吞噬后，使抗原失去毒性和致病性。自从Cohn研制出了乙醇沉淀法，富集免疫球蛋白成分的人血浆用于各种感染或者先天性缺陷疾病的治疗手段已被广泛研究和使用。
目前国内外获取免疫球蛋白主要是从血浆或组分Ⅱ中纯化制备，而很少有从组分Ⅰ+Ⅱ+Ⅲ或Ⅱ+Ⅲ中提取制备的，这是因为组分Ⅰ+Ⅱ+Ⅲ或Ⅱ+Ⅲ的成分复杂，分离提纯难度大，不易分析。
获得免疫球蛋白尤其是IgG浓缩物的方法，除了用乙醇使蛋白选择性沉淀，也可以包含其它各种方法，例如聚乙二醇沉淀法，蛋白水解酶法等，通常生产方法为低温乙醇离心(压滤)法，产品收率低，纯度低，稳定性不好。
为了提高IgG产品的收率和纯度，国内外学者已进行了大量的研究工作，其中主要以采用两步压滤和二步层析以及一步层析的方法为主，基本可以获得较高的产品收率和纯度。中国专利CN 103554253 B公开了一种静注人免疫球蛋白的制备方法，其通过两步压滤分离出Ⅱ+Ⅲ沉淀，低温乙醇压滤去除组分Ⅲ沉淀，然后通过两步层析得到的产品生物活性、收率和纯度较高，产品得率每升血浆不低于6.5g，产品纯度达到99％以上但是该专利并未对抗A和抗B进行检测，由于没有采用专用的去除抗A、抗B凝胶，因此其所得IgG产品中抗A和抗B抗体的含量较高，容易超出《中国药典》规定。中国专利CN 103394084 B提供了一种除去抗A、抗B抗体以及多反应性免疫球蛋白IgG的免疫球蛋白G浓缩物，较好地去除了抗A和抗B抗体，所获得的IgG浓缩物中含抗A抗体不超过23ng/mg，特别是在19和23ng/mg之间；含抗B抗体不超过20ng/mg，特别是在12和20ng/mg之间，但是其未对产品收率和纯度进行考察。现有研究结果表明，要想获得较高收率和纯度的IgG产品以及很好去除抗A、抗B抗体，对制备工艺有着较高的要求，现有工艺未能同时达到很好效果。
另外，现有的制备工艺还存在以下缺陷无法解决：1)当两次层析后的流穿液经纳米膜 过滤，最终制品抗A、抗B指标达到《中国药典》(2010)要求的同时，ACA含量较高，经孵放后ACA指标难以符合标准；2)经辛酸沉淀处理再经两次层析后至超滤配制，渗透压摩尔浓度容易出现自然结晶，致使样品无法测出摩尔渗透压或者渗透压不符合标准，且容易使收率较低，洗涤峰拖尾较长且不易清洗层析柱。因此，需要提供能够解决上述问题的IgG生产方法，不仅能获得较高收率和纯度的产品，而且最终制品中抗A、抗B指标和ACA指标均能符合《中国药典》(2010)规定。
发明内容
本发明的目的就是为了解决上述技术问题，而提供一种静注免疫球蛋白的生产工艺，该生产工艺不仅能够获得收率和纯度均较高的产品，更为关键的是所得产品的抗A、抗B指标和ACA指标均大大降低，很好符合了《中国药典》(2010)的要求。
为了实现上述目的，本发明采用的技术方案为：一种静注免疫球蛋白的生产工艺，包括以下步骤：
(1)从血浆中分离得到组分Ⅰ+Ⅱ+Ⅲ或者分离得到组分Ⅱ+Ⅲ；
(2)辛酸沉淀：采用辛酸对组分Ⅰ+Ⅱ+Ⅲ或者组分Ⅱ+Ⅲ进行沉淀处理，然后过滤得到澄清液；
此步骤中，辛酸的浓度优选为10-30mM；更为优选的，辛酸浓度为22-25mM；
(3)层析A：将澄清液过滤浓缩至蛋白含量为2-20g/L，优选为5-15g/L，更优选为8-10g/L；然后过阴离子层析柱A，所述阴离子层析柱A包括Fractogel EMD DEAE或CaptoQ或UNO sphere Q阴离子层析柱，优选过Fractogel EMD DEAE阴离子层析柱，层析条件为：pH4.50～5.50，5～20mM醋酸钠缓冲液，电导0.5ms/cm～2.0ms/cm，以线速度60～150cm/h平衡、上样；优选为pH 4.70～5.30，8～16mM醋酸钠缓冲液，电导0.8ms/cm～1.8ms/cm，以线速度70～120cm/h平衡、上样；更优选为pH 4.8～5.0，12-14mM醋酸钠缓冲液，电导1.0-1.2ms/cm，以线速度90-100cm/h平衡、上样；
层析B：将流穿洗涤混合液过阴离子层析柱B，所述阴离子层析柱B包括Fractogel EMD TMAE或CaptoQ XP或Nuvia HR Q阴离子层析柱，优选过Fractogel EMD TMAE阴离子层析柱；层析条件为：pH 5.50～6.50，5～20mM醋酸钠缓冲液，电导0.5ms/cm～2.5ms/cm，以线速度60～150cm/h平衡、上样；优选为pH 5.70～6.30，8～16mM醋酸钠缓冲液，电导0.8ms/cm～1.8ms/cm，以线速度70～100cm/h平衡、上样；更优选为pH 6.0～6.2，12-14mM醋酸钠缓冲液，电导1.0-1.2ms/cm，以线速度90-100cm/h平衡、上样；
层析C：将流穿洗涤混合液用缓冲液超滤浓缩至蛋白含量为20～60g/L，优选为30～50g/L， 更优选为35～45g/L；所述缓冲液为：pH 4.50～7.30的醋酸盐或柠檬酸盐或磷酸盐+NaCl，优选为pH 5.00～6.00、浓度为5～20mM的醋酸盐或柠檬酸盐或磷酸盐+80～150mM NaCl，更优选为pH 5.30～5.50的醋酸盐或柠檬酸盐或磷酸盐+100～120mM NaCl；然后超滤透析pH至与缓冲液一致，上层析柱C进行亲合层析，所述层析柱C包括Eshmuno P Anti-A/Anti-B混合柱或Eshmuno P Anti-A柱或Eshmuno P Anti-B柱进行亲合层析；所述亲合层析的条件为：在缓冲液条件下以线速度60～150cm/h平衡、上样；优选线速度70～130cm/h平衡、上样；更优选线速度90～110/h平衡、上样；
上述层析操作包括按层析A+层析B+层析C的顺序进行层析操作，或者按层析A+层析C的顺序进行层析操作，或者按层析B+层析C的顺序进行层析操作；
(4)超滤浓缩或稀释、病毒灭活、配制分装，得到静注免疫球蛋白，优选用甘氨酸或脯氨酸进行配制。
采用本发明的生产工艺，其中辛酸沉淀和层析条件是关键因素，由于辛酸浓度、蛋白含量、层析过程中的pH值、电导率以及缓冲液指标均会对层析结果产生重要影响，而由于层析条件的不同，纯化所得免疫球蛋白IgG的指标是有较大差距的。本发明通过大量对比实验，最终表明采用上述层析条件能够获得很好的抗A、抗B指标以及ACA指标。特别是在本发明的优选方案下，制备得到的样品中Anti-A＝1:4，Anti-B＝1:4，ACA＝2％，且渗透压摩尔浓度可达285mOsmol/kg，PKA＜10IU/mL，均很好达到《中国药典》(2010)的要求。而不在本发明的层析条件之内，抗A、抗B指标将无法获得如此优良的去除效果，另外，产品的收率也难以提高到本发明的水平。特别是本发明采用的缓冲液条件，对产品的收率以及渗透压指标均有显著影响，采用本发明选用的缓冲液条件，样品的渗透压能够很好符合标准，而其他缓冲液条件下，样品的渗透压达不到标准，且容易产生自然结晶。
在本发明的步骤(3)中，层析A是采用阴离子层析柱进行层析处理，主要可采用Fractogel EMD DEAE或Capto Q或UNO sphere Q三种阴离子层析柱，均可获得较好的抗A、抗B指标和ACA指标，但是较为优选的方式是采用Fractogel EMD DEAE阴离子层析柱，该阴离子凝胶层析柱配合本发明所述的层析条件，能够获得更高收率的免疫球蛋白；而CaptoQ和UNO sphere Q阴离子层析柱处理后的免疫球蛋白收率稍低。
层析B中采用的阴离子层析柱主要是Fractogel EMD TMAE或CaptoQ XP或Nuvia HR Q阴离子层析柱，三种阴离子层析条件均可获得较好的抗A、抗B指标和ACA指标，但是较为优选的方式是采用Fractogel EMD TMAE阴离子层析柱，该阴离子凝胶层析柱配合本发明所述的层析条件，能够获得更高收率的免疫球蛋白；而CaptoQ XP和Nuvia HR Q阴离子层 析柱处理后的免疫球蛋白收率稍低。
在本发明的步骤(3)中，三种层析条件可以按照以下顺序：1)按层析A、层析B、层析C的顺序进行三步层析；2)按层析A、层析C的顺序进行两步层析；3)按层析B、层析C的顺序进行两步层析。上述层析方式均能起到很好的去除抗A、抗B效果。
作为本发明的一种实施方式，在层析A中阴离子层析柱A为选取XK50/30层析柱装18～23cm高相应的阴离子层析凝胶，凝胶载量为300～700g沉淀/400mL凝胶，优选为350～650g沉淀/400mL凝胶，更优选为450～550g沉淀/400mL凝胶。
作为本发明的一种实施方式，在层析B中阴离子层析柱B为选取XK50/30层析柱装18～23cm高相应的阴离子层析凝胶，凝胶载量为300～700g沉淀/400mL凝胶，优选为350～650g沉淀/400mL凝胶，更优选为450～550g沉淀/400mL凝胶。
凝胶载量对层析的效果也有一定影响，本发明通过大量实验和工艺条件的摸索，最终确定在上述凝胶载量下可获得良好的层析效果。
作为本发明的一种实施方式，在层析C中Eshmuno P Anti-A/Anti-B混合柱或Eshmuno P Anti-A柱或Eshmuno P Anti-B柱均是在XK 16/20层析柱上以Eshmuno P Anti-A和/或Eshmuno P Anti-B亲合凝胶装5～25cm高柱，优选为装6-20cm高柱；所述Eshmuno P Anti-A/Anti-B混合柱是以Eshmuno P Anti-A和Eshmuno P Anti-B亲合凝胶按1～2:2～3比例混合后装柱。
在层析C步骤中，无论是采用混合柱还是单柱，均能获得免疫球蛋白收率高的产品，且产品中抗A或抗B指标能够很好符合《中国药典》(2010)的标准。
本发明进一步采用的技术方案是，层析B中是先将流穿洗涤混合液调pH后于2-8℃下静置1h以上，然后过滤得到上清液，再将上清液过阴离子层析柱B。
进一步的是，步骤(1)中是先获得组分Ⅰ+Ⅱ+Ⅲ沉淀或者组分Ⅱ+Ⅲ沉淀，其操作为将健康人血浆经低温乙醇反应后制备得到组分Ⅰ+Ⅱ+Ⅲ沉淀或者组分Ⅱ+Ⅲ沉淀。
进一步的是，获得组分Ⅰ+Ⅱ+Ⅲ沉淀或者组分Ⅱ+Ⅲ沉淀后再进行溶解，其操作为将组分Ⅰ+Ⅱ+Ⅲ沉淀或者组分Ⅱ+Ⅲ沉淀用10-25倍的WFI分步溶解，控制溶解液温度为2-25℃，调节pH至4.00-5.00，搅拌1h以上。
进一步的是，在用WFI分步溶解过后，进一步将溶解液用NaOH调pH至4.80-5.80，然后滴加辛酸至终浓度为10-30mM，辛酸滴加完毕后用NaOH调回pH至4.80-5.80，继续搅拌1h以上后过滤，得到辛酸沉淀后的澄清液。
进一步的是，步骤(4)中是将流穿洗涤混合液超滤浓缩或稀释至蛋白含量为5-100g/L，然后加1-5％(w/w)甘氨酸或脯氨酸，调节pH至3.8-4.4后进行配制。
进一步的是，所述病毒灭活包括以下操作：
先经20nm或15nm纳米膜过滤，过滤后的溶液经合并、除菌后于25℃下进行低pH值孵放，再将所得溶液混合、配制，除菌后分装得到静注免疫球蛋白；
或者先于25℃下进行低pH值孵放，然后经20nm或15nm纳米膜过滤；再将过滤液混合、配制，除菌后分装得到静注免疫球蛋白。
本发明的有益效果如下：
按照本发明的辛酸处理，再经层析A、层析B、层析C三步层析处理或者层析A、层析C两步层析处理或者层析B、层析C两步层析处理后，至超滤配制，总IgG收率可达90.9％以上，纯度为99.9％以上，总蛋白约56.69g，成品中IgA、IgM、白蛋白都完全去除，外观与热稳定性合格，渗透压摩尔浓度为285mOsmol/kg，符合《中国药典》(2010)标准。经层析C处理后Anti-A＝1:4，Anti-B＝1:4，ACA＝2％，PKA＜10IU/mL，均达到《中国药典》(2010)要求。
附图说明
图1为本发明一种静注免疫球蛋白的生产工艺流程图。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合实施例对本发明进行具体描述，有必要指出的是，以下实施例仅仅用于对本发明进行解释和说明，并不用于限定本发明。本领域技术人员根据上述发明内容所做出的一些非本质的改进和调整，仍属于本发明的保护范围。
如本领域技术人员所知，“WFI”为注射用水“water for injection”的英文缩写。
在下述实施例中，静注免疫球蛋白产品质量检测指标按表1中如下标准：
表1
Figure PCTCN2019071165-appb-000001
Figure PCTCN2019071165-appb-000002
实施例1
一种静注免疫球蛋白的生产工艺，是按照如图1中路线一的生产工艺流程进行，具体包括如下操作步骤：
(1)将健康人血浆经低温乙醇反应制备得到组分Ⅰ+Ⅱ+Ⅲ沉淀，然后用10倍的WFI分步溶解组分Ⅰ+Ⅱ+Ⅲ沉淀，控制溶解液温度为2℃，以醋酸调节pH至4.00，搅拌1h；
(2)辛酸沉淀：用NaOH调节溶解液pH至4.80，在40min内向溶解液中滴加辛酸至终浓度为10mM，辛酸滴加完毕后用NaOH调回pH至4.80，搅拌1h后板框过滤，得到辛酸沉淀后的澄清液；
(3)层析A：选取XK50/30层析柱装18cm高Fractogel EMD DEAE阴离子凝胶制成Fractogel EMD DEAE阴离子层析柱，凝胶载量为300g沉淀/400mL凝胶；将步骤(2)所得澄清液进行过滤浓缩至蛋白含量为2g/L，然后过Fractogel EMD DEAE阴离子层析柱，层析条件为：pH 4.50，5mM醋酸钠缓冲液，电导0.5ms/cm，以线速度60cm/h平衡、上样；
(4)层析B：选取XK50/30层析柱装18cm高Fractogel EMD TMAE阴离子凝胶制成Fractogel EMD TMAE阴离子层析柱，凝胶载量为300g沉淀/400mL凝胶；将步骤(3)的流穿洗涤混合液过Fractogel EMD TMAE阴离子层析柱；层析条件为：pH 5.50，5mM醋酸钠缓冲液，电导0.5ms/cm，以线速度60cm/h平衡、上样；
(5)层析C：将Eshmuno P Anti-A和Eshmuno P Anti-B亲合凝胶按1:2的比例混合后装6cm高于XK 16/20层析柱上，制备成Eshmuno P Anti-A/Anti-B混合柱；将步骤(4)的流穿洗涤混合液用pH为4.50的醋酸钠+60mM NaCl缓冲液超滤浓缩至蛋白含量为20g/L，然后超滤透析pH至4.50，上Eshmuno P Anti-A/Anti-B混合柱进行亲合层析，以线速度60cm/h平衡、上样；
(6)配制：将流穿洗涤混合液超滤透析至蛋白含量为5g/L，加1％(w/w)甘氨酸，用盐酸调pH至3.8，然后通过20nm纳米膜过滤，过滤量为80.63g/24h，将纳滤后的溶液经合并、除菌后于25℃下进行低pH值(pH 3.8)孵放21天，孵放后溶液经混合、配制、除菌后分装，得到静注免疫球蛋白IgG浓缩液。
共得到约56.67g总蛋白，产品收率为7.06g/L血浆。经Fractogel EMD DEAE层析后所得流穿洗涤混合液中白蛋白、IgA和IgM去除效果良好，经Eshmuno P Anti-A/Anti-B层析后 产品抗A、抗B去除效果好，单步IgG收率≥95％，经DEAE、TMAE、Anti-A/Anti-B混合层析后至超滤配制，IgG总收率为88.3％，纯度为99.8％，Alb、IgA、IgM均去除干净，渗透压摩尔浓度为281mOsmol/kg，符合标准。IgG单体及二聚体分子大小分布为99.2％，Anti-A＝1:16，Anti-B＝1:8，ACA＝10％，PKA＜10IU/mL，均达到《中国药典》(2010)要求。
实施例2
按照图1中路线二的生产工艺流程制备静注免疫球蛋白产品，其操作步骤如下：
(1)将健康人血浆经低温乙醇反应制备得到组分Ⅱ+Ⅲ沉淀，然后用25倍的WFI分步溶解组分Ⅱ+Ⅲ沉淀，控制溶解液温度为25℃，以盐酸调节pH至5.00，搅拌3h；
(2)辛酸沉淀：用NaOH调节溶解液pH至5.80，在60min内向溶解液中滴加辛酸至终浓度为30mM，辛酸滴加完毕后用NaOH调回pH至5.80，搅拌3h后板框过滤，得到辛酸沉淀后的澄清液；
(3)层析A：选取XK50/30层析柱装23cm高Fractogel EMD DEAE阴离子凝胶制成Fractogel EMD DEAE阴离子层析柱，凝胶载量为700g沉淀/400mL凝胶；将步骤(2)所得澄清液进行过滤浓缩至蛋白含量为20g/L，然后过Fractogel EMD DEAE阴离子层析柱，层析条件为：pH 5.50，20mM醋酸钠缓冲液，电导2.0ms/cm，以线速度150cm/h平衡、上样；
(4)层析B：选取XK50/30层析柱装23cm高Fractogel EMD TMAE阴离子凝胶制成Fractogel EMD TMAE阴离子层析柱，凝胶载量为700g沉淀/400mL凝胶；将步骤(3)的流穿洗涤混合液过Fractogel EMD TMAE阴离子层析柱；层析条件为：pH 6.50，20mM醋酸钠缓冲液，电导2.5ms/cm，以线速度150cm/h平衡、上样；
(5)层析C：将Eshmuno P Anti-A和Eshmuno P Anti-B亲合凝胶按1:3的比例混合后装25cm高于XK 16/20层析柱上，制备成Eshmuno P Anti-A/Anti-B混合柱；将步骤(4)的流穿洗涤混合液用pH为7.30的磷酸钠+180mM NaCl缓冲液超滤浓缩至蛋白含量为60g/L，然后超滤透析至pH 7.30，上Eshmuno P Anti-A/Anti-B混合柱进行亲合层析，以线速度150cm/h平衡、上样；
(6)配制：将流穿洗涤混合液超滤浓缩至蛋白含量为100g/L，加5％(w/w)脯氨酸，用磷酸调pH至4.4，然后于25℃下进行低pH值(pH 4.4)孵放19天，再经15nm纳米膜过滤，过滤量为140g/24h，将纳滤后的溶液混合、配制、除菌后分装，得到静注免疫球蛋白IgG浓缩液。
共得到约55.56g总蛋白，产品收率为6.89g/L血浆。经Fractogel EMD DEAE层析后所得流穿洗涤混合液中白蛋白、IgA和IgM去除效果良好，经Eshmuno P Anti-A/Anti-B层析后 产品抗A、抗B去除效果好，单步IgG收率≥95％，经DEAE、TMAE、Anti-A/Anti-B混合层析后至超滤配制，IgG收率为87.8％，纯度为99.7％，Alb、IgA、IgM均去除干净，渗透压摩尔浓度为277mOsmol/kg，符合标准。IgG单体及二聚体分子大小分布为99.1％，Anti-A＝1:8，Anti-B＝1:16，ACA＝12％，PKA＜10IU/mL，均达到《中国药典》(2010)要求。
实施例3
按照图1中路线一的生产工艺流程和实施例1的具体操作步骤以组分Ⅰ+Ⅱ+Ⅲ沉淀为原料制备IgG浓缩液产品，操作不同之处如下：
步骤(1)中用18倍的WFI分步溶解组分Ⅰ+Ⅱ+Ⅲ沉淀，控制溶解液温度为15℃，调节pH至4.35，搅拌2h；
步骤(2)中调节溶解液pH至5.20，向溶解液中滴加辛酸至终浓度为22mM，搅拌2h后过滤；
步骤(3)中Fractogel EMD DEAE凝胶载量为350g沉淀/400mL凝胶，装柱高为20cm，澄清液过滤浓缩至蛋白含量为5g/L，Fractogel EMD DEAE层析的条件为：pH 4.70，8mM醋酸钠缓冲液，电导0.8ms/cm，以线速度70cm/h平衡、上样；
步骤(4)中Fractogel EMD TMAE凝胶载量为350g沉淀/400mL凝胶，装柱高为20cm，Fractogel EMD TMAE层析的条件为：pH 5.7，8mM醋酸钠缓冲液，电导0.8ms/cm，以线速度70cm/h平衡、上样；
步骤(5)中Eshmuno P Anti-A和Eshmuno P Anti-B亲合凝胶按1:1的比例混装，高度为6cm，流穿洗涤混合液用pH为5.0的柠檬酸钠+80mM NaCl缓冲液超滤浓缩至蛋白含量为30g/L，然后调pH至5.0，以线速度70cm/h平衡、上样；
步骤(6)中将流穿洗涤混合液超滤浓缩至蛋白含量为80g/L，加3％甘氨酸，用盐酸调pH至4.1。
共得到约57.21g总蛋白，产品收率为7.12g/L血浆。经Fractogel EMD DEAE层析后所得流穿洗涤混合液中白蛋白、IgA和IgM去除效果良好，经Eshmuno P Anti-A/Anti-B层析后产品抗A、抗B去除效果好，单步IgG收率≥95％，经DEAE、TMAE、Anti-A/Anti-B混合层析后至超滤配制，IgG总收率为89.4％，纯度为99.8％，Alb、IgA、IgM均去除干净，渗透压摩尔浓度为285mOsmol/kg，符合标准。IgG单体及二聚体分子大小分布为98.9％，Anti-A＝1:8，Anti-B＝1:8，ACA＝9％，PKA＜10IU/mL，均达到《中国药典》(2010)要求。
实施例4
按照图1中路线二的生产工艺流程和实施例2的具体操作步骤以组分Ⅱ+Ⅲ沉淀为原料制 备IgG浓缩液，操作不同之处如下：
步骤(1)中用12倍的WFI分步溶解组分Ⅱ+Ⅲ沉淀，控制溶解液温度为6℃，调节pH至4.80，搅拌2.5h；
步骤(2)中调节溶解液pH至5.60，向溶解液中滴加辛酸至终浓度为25mM；
步骤(3)中Fractogel EMD DEAE凝胶载量为650g沉淀/400mL凝胶，装柱高为22cm，澄清液过滤浓缩至蛋白含量为15g/L，Fractogel EMD DEAE层析的条件为：pH 5.30，16mM醋酸钠缓冲液，电导1.8ms/cm，以线速度120cm/h平衡、上样；
步骤(4)中Fractogel EMD TMAE凝胶载量为650g沉淀/400mL凝胶，装柱高为22cm，Fractogel EMD TMAE层析的条件为：pH 6.30，16mM醋酸钠缓冲液，电导1.8ms/cm，以线速度100cm/h平衡、上样；
步骤(5)中Eshmuno P Anti-A和Eshmuno P Anti-B亲合凝胶按2:3的比例混装，高度为20cm，流穿洗涤混合液用pH为6.00，20mM的柠檬酸钠+150mM NaCl缓冲液超滤浓缩至蛋白含量为50g/L，然后调回pH至6.00，以线速度130cm/h平衡、上样；
步骤(6)中将流穿洗涤混合液稀释至蛋白含量为20g/L，加4％脯氨酸，用盐酸调pH至3.9。
共得到约57.27g总蛋白，产品收率为7.12g/L血浆。经Fractogel EMD DEAE层析后所得流穿洗涤混合液中白蛋白、IgA和IgM去除效果良好，经Eshmuno P Anti-A/Anti-B层析后产品抗A、抗B去除效果好，单步IgG收率≥96％，经DEAE、TMAE、Anti-A/Anti-B混合层析后至超滤配制，IgG总收率为89.32％，纯度为99.8％，Alb、IgA、IgM均去除干净，渗透压摩尔浓度为287mOsmol/kg，符合标准。IgG单体及二聚体分子大小分布为99.5％，Anti-A＝1:4，Anti-B＝1:8，ACA＝7％，PKA＜10IU/mL，均达到《中国药典》(2010)要求。
实施例5
按照实施例1的方法以人体血浆组分Ⅱ+Ⅲ沉淀为原料制备静注免疫球蛋白IgG浓缩液成品，操作不同之处在于：
在完成步骤(3)的Fractogel EMD DEAE层析之后，直接进行步骤(5)的Eshmuno P Anti-A/Anti-B层析操作。
共得到约55.46g总蛋白，产品收率为6.94g/L血浆。经Fractogel EMD DEAE层析后所得流穿洗涤混合液中白蛋白、IgA和IgM去除效果良好，经Eshmuno P Anti-A/Anti-B层析后产品抗A、抗B去除效果好，单步IgG收率≥93％，经DEAE、TMAE、Anti-A/Anti-B混合层析后至超滤配制，IgG总收率为86.3％，纯度为99.1％，Alb、IgA、IgM均去除干净，渗 透压摩尔浓度为268mOsmol/kg，符合标准。IgG单体及二聚体分子大小分布为98.5％，Anti-A＝1:16，Anti-B＝1:8，ACA＝12％，PKA＜10IU/mL，均达到《中国药典》(2010)要求。
实施例6
按照实施例2的方法以人体血浆组分Ⅰ+Ⅱ+Ⅲ沉淀为原料制备静注免疫球蛋白IgG浓缩液成品，操作不同之处在于：
在完成步骤(2)的辛酸沉淀之后，直接进行步骤(4)和步骤(5)的层析操作。
共得到约54.35g总蛋白，产品收率为6.85g/L血浆。经Fractogel EMD TMAE层析后所得流穿洗涤混合液中白蛋白、IgA和IgM去除效果良好，经Eshmuno P Anti-A/Anti-B层析后产品抗A、抗B去除效果好，单步IgG收率≥94％，经DEAE、TMAE、Anti-A/Anti-B混合层析后至超滤配制，IgG总收率为89.1％，纯度为99.4％，Alb、IgA、IgM均去除干净，渗透压摩尔浓度为263mOsmol/kg，符合标准。IgG单体及二聚体分子大小分布为99.2％，Anti-A＝1:16，Anti-B＝1:8，ACA＝14％，PKA＜10IU/mL，均达到《中国药典》(2010)要求。
实施例7
按照实施例3的方法以人体血浆组分Ⅱ+Ⅲ沉淀为原料制备静注免疫球蛋白IgG浓缩液成品，操作不同之处在于：
步骤(1)中是获得组分Ⅱ+Ⅲ沉淀并溶解；
步骤(6)中在加入3％甘氨酸并调pH至4.1后，先将配制液于25℃下进行低pH值(pH4.1)孵放21天，然后过20nm纳米膜过滤，最后经混合、配制、除菌后分装，得到静注免疫球蛋白IgG浓缩液。
共得到约57.73g总蛋白，产品收率为7.11g/L血浆。经Fractogel EMD DEAE层析后所得流穿洗涤混合液中白蛋白、IgA和IgM去除效果良好，经Eshmuno P Anti-A/Anti-B层析后产品抗A、抗B去除效果好，单步IgG收率≥97％，经DEAE、TMAE、Anti-A/Anti-B混合层析后至超滤配制，IgG总收率为91.3％，纯度为99.9％，Alb、IgA、IgM均去除干净，渗透压摩尔浓度为287mOsmol/kg，符合标准。IgG单体及二聚体分子大小分布为99.8％，Anti-A＝1:4，Anti-B＝1:4，ACA＝3％，PKA＜10IU/mL，均达到《中国药典》(2010)要求。
实施例8
按照实施例4的方法以人体血浆组分Ⅰ+Ⅱ+Ⅲ沉淀为原料制备IgG浓缩液产品，操作不同之处如下：
步骤(1)中是获得组分Ⅰ+Ⅱ+Ⅲ沉淀并溶解；
步骤(3)中Fractogel EMD DEAE凝胶载量为450g沉淀/400mL凝胶，装柱高为20cm， 澄清液过滤浓缩至蛋白含量为8g/L，Fractogel EMD DEAE层析的条件为：pH4.80，12mM醋酸钠缓冲液，电导1.0ms/cm，以线速度90cm/h平衡、上样；
步骤(4)中Fractogel EMD TMAE凝胶载量为450g沉淀/400mL凝胶，装柱高为20cm，Fractogel EMD TMAE层析的条件为：pH 6.00，12mM醋酸钠缓冲液，电导1.0ms/cm，以线速度90cm/h平衡、上样；
步骤(5)中流穿洗涤混合液用pH为5.30，10mM的柠檬酸钠+100mM NaCl缓冲液超滤浓缩至蛋白含量为35g/L，然后调回pH至5.30，以线速度90cm/h平衡、上样。
共得到约56.69g总蛋白，产品收率为7.09g/L血浆。经Fractogel EMD DEAE层析后所得流穿洗涤混合液中白蛋白、IgA和IgM去除效果良好，经Eshmuno P Anti-A/Anti-B层析后产品抗A、抗B去除效果好，单步IgG收率≥97％，经DEAE、TMAE、Anti-A/Anti-B混合层析后至超滤配制，IgG总收率为90.9％，纯度为99.9％，Alb、IgA、IgM均去除干净，渗透压摩尔浓度为285mOsmol/kg，符合标准。IgG单体及二聚体分子大小分布为99.9％，Anti-A＝1:4，Anti-B＝1:4，ACA＝2％，PKA＜10IU/mL，均达到《中国药典》(2010)要求。
实施例9
按照实施例4的方法以人体血浆组分Ⅱ+Ⅲ沉淀为原料制备静注免疫球蛋白IgG浓缩液成品，操作不同之处在于：
步骤(3)中Fractogel EMD DEAE凝胶载量为550g沉淀/400mL凝胶，装柱高为20cm，澄清液过滤浓缩至蛋白含量为10g/L，Fractogel EMD DEAE层析的条件为：pH 5.00，14mM醋酸钠缓冲液，电导1.2ms/cm，以线速度100cm/h平衡、上样；
步骤(4)中Fractogel EMD TMAE凝胶载量为450g沉淀/400mL凝胶，装柱高为20cm，Fractogel EMD TMAE层析的条件为：pH 6.00，12mM醋酸钠缓冲液，电导1.0ms/cm，以线速度90cm/h平衡、上样；
步骤(5)中流穿洗涤混合液用pH为5.50，15mM的柠檬酸钠+120mM NaCl缓冲液超滤浓缩至蛋白含量为45g/L，然后调回pH至5.50，以线速度110cm/h平衡、上样。
共得到约55.51g总蛋白，产品收率为6.96g/L血浆。经Fractogel EMD DEAE层析后所得流穿洗涤混合液中白蛋白、IgA和IgM去除效果良好，经Eshmuno P Anti-A/Anti-B层析后产品抗A、抗B去除效果好，单步IgG收率≥95％，经DEAE、TMAE、Anti-A/Anti-B混合层析后至超滤配制，IgG总收率为90.1％，纯度为99.8％，Alb、IgA、IgM均去除干净，渗透压摩尔浓度为271mOsmol/kg，符合标准。IgG单体及二聚体分子大小分布为99.8％，Anti-A＝1:4，Anti-B＝1:4，ACA＝5％，PKA＜10IU/mL，均达到《中国药典》(2010)要求。
实施例10
按照实施例5的方法以人体血浆组分Ⅱ+Ⅲ沉淀为原料制备静注免疫球蛋白IgG浓缩液成品，操作不同之处还有：
步骤(6)中在加入甘氨酸并调pH后，先将配制液于25℃下进行低pH值(pH 4.4)孵放20天，然后过15nm纳米膜过滤，最后经混合、配制、除菌后分装，得到静注免疫球蛋白IgG浓缩液。
共得到约55.13g总蛋白，产品收率为6.91g/L血浆。经Fractogel EMD DEAE层析后所得流穿洗涤混合液中白蛋白、IgA和IgM去除效果良好，经Eshmuno P Anti-A/Anti-B层析后产品抗A、抗B去除效果好，单步IgG收率≥93％，经DEAE、TMAE、Anti-A/Anti-B混合层析后至超滤配制，IgG总收率为87.5％，纯度为99.2％，Alb、IgA、IgM均去除干净，渗透压摩尔浓度为267mOsmol/kg，符合标准。IgG单体及二聚体分子大小分布为98.3％，Anti-A＝1:16，Anti-B＝1:8，ACA＝12％，PKA＜10IU/mL，均达到《中国药典》(2010)要求。
共得到约53.85g总蛋白，产品收率为6.81g/L血浆。经Fractogel EMD TMAE层析后所得流穿洗涤混合液中白蛋白、IgA和IgM去除效果良好，经Eshmuno P Anti-A/Anti-B层析后产品抗A、抗B去除效果好，单步IgG收率≥95％，经DEAE、TMAE、Anti-A/Anti-B混合层析后至超滤配制，IgG总收率为89.6％，纯度为99.7％，Alb、IgA、IgM均去除干净，渗透压摩尔浓度为268mOsmol/kg，符合标准。IgG单体及二聚体分子大小分布为99.4％，Anti-A＝1:8，Anti-B＝1:16，ACA＝11％，PKA＜10IU/mL，均达到《中国药典》(2010)要求。
实施例11
按照实施例6的方法以人体血浆组分Ⅰ+Ⅱ+Ⅲ沉淀为原料制备静注免疫球蛋白IgG浓缩液成品，操作不同之处还有：
步骤(6)中在加入甘氨酸并调后，先将配制液于25℃下进行低pH值(pH 4.4)孵放21天，然后过20nm纳米膜过滤，最后经混合、配制、除菌后分装，得到静注免疫球蛋白IgG浓缩液。
共得到约54.15g总蛋白，产品收率为6.84g/L血浆。经Fractogel EMD TMAE层析后所得流穿洗涤混合液中白蛋白、IgA和IgM去除效果良好，经Eshmuno P Anti-A/Anti-B层析后产品抗A、抗B去除效果好，单步IgG收率≥94％，经DEAE、TMAE、Anti-A/Anti-B混合层析后至超滤配制，IgG总收率为88.7％，纯度为99.6％，Alb、IgA、IgM均去除干净，渗透压摩尔浓度为271mOsmol/kg，符合标准。IgG单体及二聚体分子大小分布为99.5％，Anti-A＝1:8，Anti-B＝1:8，ACA＝14％，PKA＜10IU/mL，均达到《中国药典》(2010)要求。
实施例12
按照实施例1的方法以人体血浆组分Ⅱ+Ⅲ沉淀为原料制备静注免疫球蛋白IgG浓缩液成品，操作不同之处在于：
步骤(5)中是采用Eshmuno P Anti-A单柱进行亲合层析，装柱高6cm。
共得到约56.32g总蛋白，产品收率为6.98g/L血浆。经Fractogel EMD DEAE层析后所得流穿洗涤混合液中白蛋白、IgA和IgM去除效果良好，经Eshmuno P Anti-A柱层析后产品抗A去除效果好，单步IgG收率≥95％，经DEAE、TMAE、Anti-A层析后至超滤配制，IgG总收率为88.4％，纯度为99.7％，Alb、IgA、IgM均去除干净，渗透压摩尔浓度为277mOsmol/kg，符合标准。IgG单体及二聚体分子大小分布为99.3％，Anti-A＝1:8，Anti-B＝1:32，ACA＝15％，PKA＜10IU/mL，均达到《中国药典》(2010)要求。
实施例13
按照实施例2的方法以人体血浆组分Ⅰ+Ⅱ+Ⅲ沉淀为原料制备静注免疫球蛋白IgG浓缩液成品，操作不同之处在于：
步骤(5)中是采用Eshmuno P Anti-B单柱进行亲合层析，装柱高25cm。
共得到约56.11g总蛋白，产品收率为6.92g/L血浆。经Fractogel EMD DEAE层析后所得流穿洗涤混合液中白蛋白、IgA和IgM去除效果良好，经Eshmuno P Anti-B柱层析后产品抗B去除效果好，单步IgG收率≥95％，经DEAE、TMAE、Anti-B层析后至超滤配制，IgG总收率为88.9％，纯度为99.6％，Alb、IgA、IgM均去除干净，渗透压摩尔浓度为272mOsmol/kg，符合标准。IgG单体及二聚体分子大小分布为99.1％，Anti-A＝1:32，Anti-B＝1:8，ACA＝11％，PKA＜10IU/mL，均达到《中国药典》(2010)要求。
实施例14
按照实施例5的方法以人体血浆组分Ⅱ+Ⅲ沉淀为原料制备静注免疫球蛋白IgG浓缩液成品，操作不同之处还在于：
完成步骤(3)后直接进行步骤(5)时，是采用Eshmuno P Anti-A单柱进行亲合层析，装柱高6cm。
共得到约55.67g总蛋白，产品收率为6.88g/L血浆。经Fractogel EMD DEAE层析后所得流穿洗涤混合液中白蛋白、IgA和IgM去除效果良好，经Eshmuno P Anti-A柱层析后产品抗A去除效果好，单步IgG收率≥95％，经DEAE、TMAE、Anti-A层析后至超滤配制，IgG总收率为89.2％，纯度为99.6％，Alb、IgA、IgM均去除干净，渗透压摩尔浓度为278mOsmol/kg，符合标准。IgG单体及二聚体分子大小分布为99.1％，Anti-A＝1:4，Anti-B＝1:32， ACA＝14％，PKA＜10IU/mL，均达到《中国药典》(2010)要求。
实施例15
按照实施例6的方法以人体血浆组分Ⅰ+Ⅱ+Ⅲ沉淀为原料制备静注免疫球蛋白IgG浓缩液成品，操作不同之处还在于：
完成步骤(2)后直接进行步骤(4)和步骤(5)操作，其中在步骤(5)时，是采用Eshmuno P Anti-B单柱进行亲合层析，装柱高25cm。
共得到约56.31g总蛋白，产品收率为6.98g/L血浆。经Fractogel EMD DEAE层析后所得流穿洗涤混合液中白蛋白、IgA和IgM去除效果良好，经Eshmuno P Anti-B柱层析后产品抗B去除效果好，单步IgG收率≥95％，经DEAE、TMAE、Anti-B层析后至超滤配制，IgG总收率为89.2％，纯度为99.7％，Alb、IgA、IgM均去除干净，渗透压摩尔浓度为266mOsmol/kg，符合标准。IgG单体及二聚体分子大小分布为99.2％，Anti-A＝1:32，Anti-B＝1:4，ACA＝9％，PKA＜10IU/mL，均达到《中国药典》(2010)要求。
实施例16
按照实施例1的原料和具体操作步骤制备IgG浓缩液产品，操作不同之处如下：
(3)层析A中，选取Capto Q凝胶制成阴离子层析柱，层析条件与实施例1一致；
(4)层析B中，选取CaptoQ XP凝胶制成阴离子层析柱，层析条件与实施例1一致；
最终共得到约51.16g总蛋白，产品收率为6.06g/L血浆。经Capto Q层析后所得流穿洗涤混合液中白蛋白、IgA和IgM去除效果良好，经Eshmuno P Anti-A/Anti-B层析后产品抗A、抗B去除效果好，单步IgG收率≥90％，经Capto Q、CaptoQ XP、Anti-A/Anti-B混合层析后至超滤配制，IgG总收率为78.5％，纯度为96.7％，Alb、IgA、IgM均去除干净。IgG单体及二聚体分子大小分布为98.1％，Anti-A＝1:16，Anti-B＝1:16，ACA＝14％，PKA＜10IU/mL，均达到《中国药典》(2010)要求。
实施例17
按照实施例2的原料和具体操作步骤制备IgG浓缩液产品，操作不同之处如下：
(3)层析A中，选取UNO sphere Q凝胶制成阴离子层析柱，层析条件与实施例2一致；
(4)层析B中，选取Nuvia HR Q凝胶制成阴离子层析柱，层析条件与实施例2一致；
最终共得到约50.83g总蛋白，产品收率为5.98g/L血浆。经UNO sphere Q层析后所得流穿洗涤混合液中白蛋白、IgA和IgM去除效果良好，经Eshmuno P Anti-A/Anti-B层析后产品抗A、抗B去除效果好，单步IgG收率≥90％，经UNO sphere Q、Nuvia HR Q、Anti-A/Anti-B混合层析后至超滤配制，IgG总收率为77.6％，纯度为95.9％，Alb、IgA、IgM均去除干净。 IgG单体及二聚体分子大小分布为98.2％，Anti-A＝1:16，Anti-B＝1:16，ACA＝13％，PKA＜10IU/mL，均达到《中国药典》(2010)要求。
实施例18
按照图1的生产工艺流程和实施例1的具体操作步骤以组分Ⅰ+Ⅱ+Ⅲ沉淀为原料中试规模制备成品IgG浓缩液，收率统计如下表2：
表2
Figure PCTCN2019071165-appb-000003
对比例1
按照实施例1的原料和方法制备静注免疫球蛋白IgG浓缩液成品。其中步骤(3)中将蛋白含量提升至30g/L，凝胶载量提升至800g沉淀/400mL凝胶时，经Fractogel EMD DEAE层析后所得流穿洗涤混合液中IgM、IgA去除不完全，产品中IgM、IgA残留至少2～5％。
对比例2
按照实施例2的原料和方法制备静注免疫球蛋白IgG浓缩液成品。其中步骤(3)中将层析条件变为：pH 6.50，25mM醋酸钠缓冲液，电导3.0ms/cm，以线速度170cm/h平衡、上样；经Fractogel EMD DEAE层析后所得流穿洗涤混合液中IgM、IgA去除不完全，产品中IgM、IgA残留至少3～6％。
对比例3
按照实施例5的原料和方法制备静注免疫球蛋白IgG浓缩液成品。其中步骤(5)中的缓冲液采用pH为4.20的醋酸钠+60mM NaCl缓冲液超滤浓缩至蛋白含量为80g/L，总IgG收率为68.3％，Alb、IgA、IgM均去除干净，Anti-A＝1:32，Anti-B＝1:16，渗透压摩尔浓度不符合《中国药典》(2010)标准，出现肉眼可见自然结晶现象。
对比例4
按照实施例6的原料和方法制备静注免疫球蛋白IgG浓缩液成品。其中步骤(5)中缓冲液采用pH为4.10的磷酸钠+180mM NaCl缓冲液稀释至蛋白含量为10g/L，总IgG收率为57.1％，Alb、IgA、IgM均去除干净，Anti-A＝1:32，Anti-B＝1:16，渗透压摩尔浓度不符合《中国药典》(2010)标准，出现肉眼可见自然结晶现象。
对比例5
按照实施例1的原料和方法制备静注免疫球蛋白IgG浓缩液成品。其中不采用步骤(5)，而是步骤(4)做完直接进行步骤(6)的配制成品操作，总IgG收率为87.5％，Alb、IgA、IgM均去除干净，Anti-A＝1:64，Anti-B＝1:128，不符合《中国药典》(2010)标准。
对比例6
按照实施例7的原料和方法制备静注免疫球蛋白IgG浓缩液成品。其中将步骤(5)中缓冲液换为pH 4.00，10mM的PBS+80mM NaCl进行层析，总IgG收率为87.5％，Alb、IgA、IgM均去除干净，Anti-A＝1:4，Anti-B＝1:4，渗透压摩尔浓度不符合《中国药典》(2010)标准，出现肉眼可见自然结晶现象。
对比例7
按照实施例8的原料和方法制备静注免疫球蛋白IgG浓缩液成品。其中将步骤(2)中在40min内向溶解液中滴加辛酸至终浓度为5mM，压滤后进行后续操作，结果导致Fractogel EMD DEAE凝胶板结。

Claims (20)

  1. 一种静注免疫球蛋白的生产工艺，其特征在于，包括以下步骤：
    (1)从血浆中分离得到组分Ⅰ+Ⅱ+Ⅲ或者分离得到组分Ⅱ+Ⅲ；
    (2)辛酸沉淀：采用辛酸对组分Ⅰ+Ⅱ+Ⅲ或者组分Ⅱ+Ⅲ进行沉淀处理，然后过滤得到澄清液；
    (3)层析A：将澄清液过滤浓缩至蛋白含量为2～20g/L；然后过阴离子层析柱A，所述阴离子层析柱A包括Fractogel EMD DEAE或CaptoQ或UNO sphere Q阴离子层析柱，层析条件为：pH 4.50～5.50，5～20mM醋酸钠缓冲液，电导0.5ms/cm～2.0ms/cm，以线速度60～150cm/h平衡、上样；
    层析B：将流穿洗涤混合液过阴离子层析柱B，所述阴离子层析柱B包括Fractogel EMD TMAE或CaptoQ XP或Nuvia HR Q阴离子层析柱，层析条件为：pH 5.50～6.50，5～20mM醋酸钠缓冲液，电导0.5ms/cm～2.5ms/cm，以线速度60～150cm/h平衡、上样；
    层析C：将流穿洗涤混合液用缓冲液超滤浓缩至蛋白含量为20～60g/L，所述缓冲液为：pH 4.50～7.30的醋酸盐或柠檬酸盐或磷酸盐+NaCl，然后超滤透析pH至与缓冲液一致，上层析柱C进行亲合层析，所述层析柱C包括Eshmuno P Anti-A/Anti-B混合柱或Eshmuno P Anti-A柱或Eshmuno P Anti-B柱；所述亲合层析的条件为：在缓冲液条件下以线速度60～150cm/h平衡、上样；
    上述层析操作包括按层析A+层析B+层析C进行层析操作，或者按层析A+层析C进行层析操作，或者按层析B+层析C进行层析操作；
    (4)超滤浓缩或稀释、病毒灭活、配制分装，得到静注免疫球蛋白。
  2. 根据权利要求1所述的静注免疫球蛋白的生产工艺，其特征在于，层析A中阴离子层析柱A为选取层析柱装18～23cm高阴离子凝胶，凝胶载量为300～700g沉淀/400mL凝胶。
  3. 根据权利要求1所述的静注免疫球蛋白的生产工艺，其特征在于，层析B中阴离子层析柱B为选取层析柱装18～23cm高阴离子层析凝胶，凝胶载量为300～700g沉淀/400mL凝胶。
  4. 根据权利要求1所述的静注免疫球蛋白的生产工艺，其特征在于，层析C中Eshmuno P Anti-A/Anti-B混合柱或Eshmuno P Anti-A柱或Eshmuno P Anti-B柱均是在层析柱上，以Eshmuno P Anti-A和/或Eshmuno P Anti-B亲合凝胶装5～25cm高柱；所述Eshmuno P Anti-A/Anti-B混合柱是以Eshmuno P Anti-A和Eshmuno P Anti-B亲合凝胶按1～2:2～3比例混合后装柱。
  5. 根据权利要求1-4任一项所述的静注免疫球蛋白的生产工艺，其特征在于，层析B中 是先将流穿洗涤混合液用缓冲液调节pH后于2～8℃下静置1h以上，然后过滤得到上清液，再将上清液过阴离子层析柱B。
  6. 根据权利要求1-4任一项所述的静注免疫球蛋白的生产工艺，其特征在于，步骤(1)中是先获得组分Ⅰ+Ⅱ+Ⅲ沉淀或者组分Ⅱ+Ⅲ沉淀，其操作为将健康人血浆经低温乙醇反应后制备得到组分Ⅰ+Ⅱ+Ⅲ沉淀或者组分Ⅱ+Ⅲ沉淀。
  7. 根据权利要求6所述的静注免疫球蛋白的生产工艺，其特征在于，获得组分Ⅰ+Ⅱ+Ⅲ沉淀或者组分Ⅱ+Ⅲ沉淀后再进行溶解，其操作为将组分Ⅰ+Ⅱ+Ⅲ沉淀或者组分Ⅱ+Ⅲ沉淀用5～25倍的WFI分步溶解，控制溶解液温度为2～25℃，调节pH至4.00～5.00，搅拌1h以上。
  8. 根据权利要求7所述的静注免疫球蛋白的生产工艺，其特征在于，在用WFI分步溶解过后，进一步将溶解液用NaOH调pH至4.80～5.80，然后滴加辛酸至终浓度为10～30mM，辛酸滴加完毕后用NaOH调回pH至4.80～5.80，继续搅拌至少1h后过滤，得到辛酸沉淀后的澄清液。
  9. 根据权利要求1所述的静注免疫球蛋白的生产工艺，其特征在于，步骤(4)中是将流穿洗涤混合液超滤浓缩或稀释至蛋白含量为5～100g/L，然后加1～5％(w/w)甘氨酸或脯氨酸，调节pH至3.8～4.4后进行配制。
  10. 根据权利要求1所述的静注免疫球蛋白的生产工艺，其特征在于，步骤(4)中所述病毒灭活包括以下操作：
    先经20nm或15nm纳米膜过滤，过滤后的溶液经合并、除菌后于25℃下进行低pH值孵放，再将所得溶液混合、配制，除菌后分装得到静注免疫球蛋白；
    或者先于25℃下进行低pH值孵放，然后经20nm或15nm纳米膜过滤，再将过滤液混合、配制，除菌后分装得到静注免疫球蛋白。
  11. 根据权利要求1所述的静注免疫球蛋白的生产工艺，其特征在于，在步骤(2)中，所述辛酸的浓度为10～30mM。
  12. 根据权利要求11所述的静注免疫球蛋白的生产工艺，其特征在于，在步骤(2)中，所述辛酸的浓度为22～25mM。
  13. 根据权利要求1所述的静注免疫球蛋白的生产工艺，其特征在于，在步骤(3)所述的层析A中，所述蛋白含量为5～15g/L，所述阴离子层析柱A为Fractogel EMD DEAE阴离子层析柱，所述层析条件为：pH 4.70～5.30，8～16mM醋酸钠缓冲液，电导0.8ms/cm～1.8ms/cm，以线速度70～120cm/h平衡、上样；在步骤(3)所述的层析B中，所述阴离子层析柱B为Fractogel EMD TMAE阴离子层析柱，所述层析条件为：pH 5.70～6.30，8～16mM醋 酸钠缓冲液，电导0.8ms/cm～1.8ms/cm，以线速度70～100cm/h平衡、上样；在步骤(3)所述的层析C中，所述蛋白含量为30～50g/L，所述缓冲液为pH 5.00～6.00、浓度为5～20mM的醋酸盐或柠檬酸盐或磷酸盐+80～150mM NaCl，所述亲合层析的条件为：在缓冲液条件下以线速度70～130cm/h平衡、上样。
  14. 根据权利要求1所述的静注免疫球蛋白的生产工艺，其特征在于，在步骤(4)中，采用甘氨酸或脯氨酸进行配制。
  15. 根据权利要求2所述的静注免疫球蛋白的生产工艺，其特征在于，在步骤(3)所述的层析A中，所述凝胶载量为450～550g沉淀/400mL凝胶。
  16. 根据权利要求15所述的静注免疫球蛋白的生产工艺，其特征在于，所述阴离子层析柱A的层析柱型号为XK50/30。
  17. 根据权利要求3所述的静注免疫球蛋白的生产工艺，其特征在于，在步骤(3)所述的层析B中，所述凝胶载量为450～550g沉淀/400mL凝胶。
  18. 根据权利要求17所述的静注免疫球蛋白的生产工艺，其特征在于，所述阴离子层析柱B的层析柱型号为XK50/30。
  19. 根据权利要求4所述的静注免疫球蛋白的生产工艺，其特征在于，所述层析柱C中的Eshmuno P Anti-A和/或Eshmuno P Anti-B亲和凝胶装6～20cm高柱。
  20. 根据权利要求19所述的静注免疫球蛋白的生产工艺，其特征在于，所述层析柱C的层析柱型号为XK 16/20。
PCT/CN2019/071165 2018-01-15 2019-01-10 一种静注免疫球蛋白的生产工艺 WO2019137435A1 (zh)

Priority Applications (2)

Application Number Priority Date Filing Date Title
RU2020125016A RU2742655C1 (ru) 2018-01-15 2019-01-10 Способ производства иммуноглобулина для внутривенного введения
EP19738798.8A EP3741775A4 (en) 2018-01-15 2019-01-10 PROCESS FOR MANUFACTURING IMMUNGLOBULIN FOR INTRAVENOUS INJECTION

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201810036376.6 2018-01-15
CN201810036376.6A CN108101981B (zh) 2018-01-15 2018-01-15 一种静注免疫球蛋白的生产工艺

Publications (1)

Publication Number Publication Date
WO2019137435A1 true WO2019137435A1 (zh) 2019-07-18

Family

ID=62218708

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2019/071165 WO2019137435A1 (zh) 2018-01-15 2019-01-10 一种静注免疫球蛋白的生产工艺

Country Status (4)

Country Link
EP (1) EP3741775A4 (zh)
CN (1) CN108101981B (zh)
RU (1) RU2742655C1 (zh)
WO (1) WO2019137435A1 (zh)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3741775A4 (en) * 2018-01-15 2021-10-27 Sichuan Yuanda Shuyang Pharmaceutical Co., Ltd. PROCESS FOR MANUFACTURING IMMUNGLOBULIN FOR INTRAVENOUS INJECTION
WO2022008658A1 (en) * 2020-07-10 2022-01-13 Grifols Worldwide Operations Limited Method for obtaining a composition comprising human plasma-derived immunoglobulin m

Families Citing this family (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109575129B (zh) * 2018-12-29 2022-04-26 贵州泰邦生物制品有限公司 一种静注人免疫球蛋白的制备工艺
CN110128532A (zh) * 2019-05-09 2019-08-16 上海莱士血液制品股份有限公司 一种利用人血浆组份ii+iii生产制备ivig的方法
CN111234010A (zh) * 2020-01-20 2020-06-05 华兰生物工程重庆有限公司 降低静注人免疫球蛋白中aca的处理方法
CN111499736B (zh) * 2020-04-28 2021-04-30 国药集团武汉血液制品有限公司 一种静注covid-19人免疫球蛋白的制备方法
CN111848784B (zh) * 2020-06-29 2022-03-11 博雅生物制药集团股份有限公司 一种高浓度静注人免疫球蛋白的分离纯化方法
CN112111004B (zh) * 2020-08-27 2023-07-14 成都生物制品研究所有限责任公司 一种兔免疫球蛋白的生产工艺
CN111944043B (zh) * 2020-09-01 2023-05-09 华兰生物工程重庆有限公司 一种从血浆废弃物中提取IgM的方法
CN112375142B (zh) * 2020-11-18 2022-03-18 深圳市卫光生物制品股份有限公司 静注新型冠状病毒人免疫球蛋白的制备方法
CN112500477B (zh) * 2020-12-05 2023-06-06 贵州泰邦生物制品有限公司 一种快速从血浆提取人免疫球蛋白的方法
BE1029863B1 (nl) * 2022-05-10 2023-05-12 Prothya Biosolutions Belgium WERKWIJZEN VOOR HET PRODUCEREN VAN IMMUNOGLOBULINE G (IgG) PREPARATEN EN/OF OPLOSSINGEN

Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1201694A (zh) * 1998-06-25 1998-12-16 成都蜀阳制药厂 无保护剂双重病毒灭活制备静注免疫球蛋白工艺
WO2011038894A1 (en) * 2009-10-01 2011-04-07 F. Hoffmann-La Roche Ag Protein a chromatography
CN102532307A (zh) * 2012-02-22 2012-07-04 成都蓉生药业有限责任公司 一种制备人免疫球蛋白的方法
CN103394084A (zh) 2005-12-26 2013-11-20 分馏和生物工艺法国实验室公司 除去抗A、抗B抗体以及多反应性免疫球蛋白IgG的免疫球蛋白G(IgG)浓缩物
CN103554253A (zh) 2013-11-15 2014-02-05 同路生物制药股份有限公司 一种静注人免疫球蛋白的制备方法
CN103687874A (zh) * 2011-07-11 2014-03-26 法国分裂与生物技术实验室 多价免疫球蛋白的浓缩物的制造方法
CN105254754A (zh) * 2015-11-28 2016-01-20 上海洲跃生物科技有限公司 一种静注人免疫球蛋白（ph4）的制备方法
CN105358228A (zh) * 2013-07-05 2016-02-24 法国血液分割暨生化制品实验室 亲和层析基质
CN106103473A (zh) * 2014-03-11 2016-11-09 法国血液分割暨生化制品实验室 制备人血浆蛋白的方法
CN108101981A (zh) * 2018-01-15 2018-06-01 四川远大蜀阳药业股份有限公司 一种静注免疫球蛋白的生产工艺

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2824568B1 (fr) * 2001-05-11 2004-04-09 Lab Francais Du Fractionnement Procede de preparation de concentres d'immunoglobulines humaines a usage therapeutique
CN102584934B (zh) * 2012-03-19 2013-11-06 江西博雅生物制药股份有限公司 一种静注人免疫球蛋白的制备工艺
RU2768003C2 (ru) * 2013-03-08 2022-03-22 Джензим Корпорейшн Интегрированное непрерывное производство терапевтических белковых лекарственных веществ
US20170066839A1 (en) * 2015-09-08 2017-03-09 Merck Patent Gmbh Novel affinity chromatography media for removal of anti-a and/or anti-b antibodies

Patent Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1201694A (zh) * 1998-06-25 1998-12-16 成都蜀阳制药厂 无保护剂双重病毒灭活制备静注免疫球蛋白工艺
CN103394084A (zh) 2005-12-26 2013-11-20 分馏和生物工艺法国实验室公司 除去抗A、抗B抗体以及多反应性免疫球蛋白IgG的免疫球蛋白G(IgG)浓缩物
WO2011038894A1 (en) * 2009-10-01 2011-04-07 F. Hoffmann-La Roche Ag Protein a chromatography
CN103687874A (zh) * 2011-07-11 2014-03-26 法国分裂与生物技术实验室 多价免疫球蛋白的浓缩物的制造方法
CN102532307A (zh) * 2012-02-22 2012-07-04 成都蓉生药业有限责任公司 一种制备人免疫球蛋白的方法
CN105358228A (zh) * 2013-07-05 2016-02-24 法国血液分割暨生化制品实验室 亲和层析基质
CN103554253A (zh) 2013-11-15 2014-02-05 同路生物制药股份有限公司 一种静注人免疫球蛋白的制备方法
CN106103473A (zh) * 2014-03-11 2016-11-09 法国血液分割暨生化制品实验室 制备人血浆蛋白的方法
CN105254754A (zh) * 2015-11-28 2016-01-20 上海洲跃生物科技有限公司 一种静注人免疫球蛋白（ph4）的制备方法
CN108101981A (zh) * 2018-01-15 2018-06-01 四川远大蜀阳药业股份有限公司 一种静注免疫球蛋白的生产工艺

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of EP3741775A4 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3741775A4 (en) * 2018-01-15 2021-10-27 Sichuan Yuanda Shuyang Pharmaceutical Co., Ltd. PROCESS FOR MANUFACTURING IMMUNGLOBULIN FOR INTRAVENOUS INJECTION
WO2022008658A1 (en) * 2020-07-10 2022-01-13 Grifols Worldwide Operations Limited Method for obtaining a composition comprising human plasma-derived immunoglobulin m

Also Published As

Publication number Publication date
CN108101981B (zh) 2019-06-04
CN108101981A (zh) 2018-06-01
EP3741775A1 (en) 2020-11-25
EP3741775A4 (en) 2021-10-27
RU2742655C1 (ru) 2021-02-09

Similar Documents

Publication Publication Date Title
WO2019137435A1 (zh) 一种静注免疫球蛋白的生产工艺
JP7142915B2 (ja) ラクトフェリン／アルブミン融合タンパク質及びその製造方法
RU2266914C2 (ru) Способ получения igg
EP3491025B1 (en) Fcrn antibodies and methods of use thereof
CA1206412A (en) Process for the production of blood-clotting factors and preparation of factors ii and vii, prepared by this process
EP1225180B1 (en) Process for the production of virus-inactivated human gammaglobulin G
JP2644946B2 (ja) ＩｇＧ− モノクロナール抗体の精製方法及びその使用方法
KR20070009995A (ko) 바이러스 안전성 면역글로불린을 제조하는 방법
TW311884B (zh)
WO2013123889A1 (zh) 一种制备人免疫球蛋白的方法
CN104245730A (zh) 源自血浆的免疫球蛋白的级分i-iv-1沉淀
ITFI20090273A1 (it) Nuovo processo di purificazione su scala industriale di gammaglobuline da plasma umano per uso industriale.
CN107964044B (zh) 从乳样中纯化抗cd20单克隆抗体的方法
US20130172536A1 (en) Intravenous Cytomegalovirus Human Immune Globulin and Manufacturing Method Thereof
NO300041B1 (no) Fremgangsmåte for fremstilling av renset albumin
US5851524A (en) Human monoclonal anti-rhesus (D) antibodies and cell lines producing same
US10358462B2 (en) Method for the preparation of immunoglobulins
JPH0365327B2 (zh)
JPS6034521B2 (ja) 血清アルブミンの製法
AU2016231646B2 (en) Method for the preparation of immunoglobulins
BR112012004054B1 (pt) Método para produzir um anticorpo anti-her2
ZA200110168B (en) Manufacturing method for intravenous immune globulin and resultant product.
AU2013337346A1 (en) Method of isolating Synagis in the absence of benzonase
CA2943328C (en) Method for the preparation of immunoglobulins
JP6370853B2 (ja) 免疫グロブリンの調製方法

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 19738798

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2019738798

Country of ref document: EP

Effective date: 20200817