WO2013123889A1

WO2013123889A1 - Method for preparing human immunoglobulin

Info

Publication number: WO2013123889A1
Application number: PCT/CN2013/071754
Authority: WO
Inventors: 杨汇川; 吕家成; 梁洪; 王焰; 武鹏; 李泽林; 兰学渊
Original assignee: 成都蓉生药业有限责任公司
Priority date: 2012-02-22
Filing date: 2013-02-22
Publication date: 2013-08-29
Also published as: IN2014DN07228A; RU2014135665A; BR112014020734B1; CO7141451A2; PH12014501909B1; PH12014501909A1; CN102532307A; RU2614119C9; BR112014020734A2; RU2614119C2; CN102532307B

Abstract

The present invention provides a method for preparing human immunoglobulin, comprising such steps as: dissolution of Cohn component I+ΙΙ+ΙII or Cohn component II+ΙII, caprylic acid precipitation, first-step anion exchange chromatography, IgM precipitation, second-step anion exchange chromatography, ultrafiltration or dialysis, preparation, and inactivation of virus; as a result, high-purity human immunoglobulin is obtained through the preparation. Also provided is a human immunoglobulin prepared through the method and a pharmaceutical combination. The preparation method of the present invention is simple, has a low cost, and has a good industrial application prospect.

Description

Method for preparing human immunoglobulin

The present invention relates to a method of preparing a blood product, and more particularly to a method of preparing a human immunoglobulin.

Background technique

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. There are three main types of immunoglobulin preparations: intramuscular immunoglobulin, specific immunoglobulin, and intravenous immunoglobulin. Currently, immunoglobulin preparations are mainly separated from blood plasma.

In 1940s, Professor E丄Cohn of Harvard University invented a process for the separation of blood protein from low-temperature ethanol. Later, Nistchmann and Kistler et al. improved on the basis of Cohn's low-temperature ethanol method, and proposed improved low-temperature ethanol separation. The paddle protein method simplifies the steps and shortens the production cycle. However, the low-temperature ethanol separation process also has certain limitations in the production of immunoglobulin, and the recovery of globulin stays between 3.8 and 4.4 g/L. In addition, the content of IgA in immunoglobulin preparations prepared by low-temperature ethanol process is relatively high, and patients with congenital selective IgA deficiency are prone to adverse reactions after infusion.

In 1969, Steinbruch et al. reported the use of octanoic acid precipitation combined with anion exchange chromatography to separate IgG from mammalian blood plasma. Under weakly acidic conditions not lower than pH 4.5, heteroproteins other than immunoglobulins can be precipitated. However, this method still requires further purification of IgG using DEAE-cellulose as an anion exchange medium, which is complicated and costly.

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.

However, compared with foreign countries, Chinese people have higher IgM content in blood paddles (IgM content in Chinese blood paddles accounts for about 10% of total protein content, and IgM content in foreign blood plasmas accounts for 1%-3% of total protein content. According to the above method, 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.

Summary of the invention

In order to solve the above problems, the present invention provides a novel method for preparing human immunoglobulin. First, the present invention provides a method for preparing human immunoglobulin, which comprises the steps of: (1) dissolving: dissolving Cohn component I + Π + ΠΙ or Cohn component II + ΙΠ in water for injection; (2) octanoic acid precipitation: Precipitate impurities with octanoic acid or octanoate, and filter to obtain a filtrate;

(3) 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;

(4) 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;

(5) The second step of anion exchange chromatography: the pH of the filtrate obtained in the step (4) is adjusted to 5.6, and purified by anion exchange chromatography to obtain a flow through solution;

(6) After ultrafiltration or dialysis, preparation, virus inactivation, that is, the human immunoglobulin finished product. Wherein the step (1) is: taking Cohn component I + II + ΙΠ 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. In general, the amount of water for injection is: The volume ratio of water for injection to Cohn component I + Π + ΠΙ or Cohn component Π + ΙΠ is 10: 1-15: 1.

Wherein, 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.

Wherein, 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.

Wherein, 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.

It is apparent that various other modifications, substitutions and changes can be made in the form of the above-described embodiments of the present invention without departing from the spirit and scope of the invention.

The above content of the present invention will be further described in detail below by way of specific embodiments in the form of embodiments. However, the scope of the above-mentioned subject matter of the present invention should not be construed as being limited to the following examples. Any technique implemented based on the above description of the present invention is within the scope of the present invention.

DRAWINGS

Figure 1 Schematic diagram of the preparation method of the human immunoglobulin of the present invention

Figure 2 Relationship between pH and IgM precipitation and IgG yield detailed description

Example 1 Preparation of human immunoglobulin by the method of the present invention

1. Experimental materials

Cohn component Ι + Π + ΠΙ or component Π + ΠΙ: 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.

2, the experimental method

As shown in Figure 1, preparation of human immunoglobulin:

(1) Cohn component I + II +III: Take 4kg Cohn component Ι + Π + ΠΙ 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;

(3) 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;

(4) Precipitation IgM: The chromatographic flow is first adjusted to 70 (^s/cm, then adjusted to pH 6.3 with 0.5M NaOH, and allowed to stand for lh;

(5) 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;

(6) Collect the chromatographic flow through the solution, adjust the pH to 3.9-4.2 with 0.5 M hydrochloric acid, and then concentrate the solution to a protein concentration of 20 mg/ml by ultrafiltration. To filter the virus, the solution was filtered through a virus filter having a pore size of 200-15 nm, such as DV50, DV20, Planova75, Planova35 and the like. After the end of the step, concentrate the solution to the protein concentration required for the final preparation, such as 5% or 10% (W/V). Add the appropriate additive to the osmotic pressure value of the solution to meet the requirements of intravenous injection. The additive can be used. Sugar or amino acid, adjust the pH of the solution to 4.0-4.8 again, incubate at 25 °C for 21 days, and then dispense into the infusion bottle according to the requirements of the preparation.

3, product inspection

(1) Detection method

According to the Chinese Pharmacopoeia (2010 edition), the product purity test, product monomer + dimer, ie molecular size distribution, product sub-class distribution and other test indicators were tested.

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. For specific assay methods, refer to the test kit instructions.

Yield detection method: After the product is concentrated by ultrafiltration, the protein concentration of the product is determined by Kjeldahl method (Chinese Pharmacopoeia 2010 edition, Appendix VI B first method), yield = protein concentration * product volume / blood paddle volume.

(2) Test results

The test results are shown in Table 1:

Table 1 Comparison of quality indexes of immunoglobulin preparations prepared by the present invention and products obtained by the existing low temperature ethanol method

As can be seen from Table 1, the indicators of human immunoglobulin prepared by the process of the present invention all meet the requirements of the pharmacopoeia. Compared with the conventional low-temperature ethanol process, 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. Example 2 Parameter Optimization Experiment

Steps of the method of the invention (4) Screening of conductivity and pH in precipitated IgM for screening:

(1) Conductivity optimization experiment

Experimental method: The other conditions were constant, and the relationship between the conductivity and the amount of IgM precipitated and the IgG yield was examined by changing the conductivity and pH in the step (4) of Example 1.

Experimental results: It has been observed through many experiments that when the conductivity of the chromatographic flowthrough is higher than lms/cm, even if the pH of the solution is raised, there is no precipitation of IgM. When the conductivity of the chromatographic flowthrough is between 500 and 1000 us/cm, the pH is adjusted to 6.0 or higher, and IgM is precipitated.

(2) pH optimization experiment

Experimental method: The other conditions were constant, the pH in the step (4) of Example 1 was changed, and the relationship between the pH and the amount of IgM precipitated, and the IgG yield was examined.

Experimental results: It has been observed through many experiments that the removal rate of IgM increases with the increase of pH, but Excessive pH causes an increase in IgG loss.

As shown in Table 2 and Figure 1:

Table 2 IgM precipitation

It can be seen from Table 2 and Figure 1. When the pH is between 6.2 and 7.3, the precipitation of IgM is obviously precipitated. When the pH is between 6.3 and 6.74, the precipitation of IgM is large, and the IgG yield is high. When the pH is between 6.51 and 6.7, IgM Both the precipitation amount and the IgG yield were superior.

In summary, 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. To further illustrate the beneficial effects of the method of the invention, the following comparative experiments are provided:

1. Preparation of human immunoglobulin

(1), the preparation method of the present invention: as described in Example 1;

(2), comparison preparation method:

1) Cohn component I + II + ΠΙ dissolution: Take 4kg Cohn component Ι + Π + ΠΙ 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, heat and stir for 2 hours in a 25 ° C water bath, and let stand at 8 ° C for 2 h;

3) 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 _;

4) 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;

5) Collect the chromatographic flow through the solution, adjust the pH to 3.9-4.2 with 0.5 M hydrochloric acid, and then concentrate the solution to a protein concentration of 20 mg/ml by ultrafiltration. To filter the virus, the solution was filtered through a virus filter having a pore size of 200-15 nm, such as DV50, DV20, Planova75, Planova35 and the like. After the end of the step, the solution is concentrated to the protein concentration required for the final preparation, such as 5% or 10% (W/V). After concentration, the appropriate osmotic pressure of the complete additive solution is suitable for intravenous injection. The additive can be used. Sugar or amino acid, adjust the pH of the solution to 4.0-4.8 again, incubate at 25 °C for 21 days, and then dispense into the infusion bottle according to the requirements of the preparation.

2, product inspection

(1) Detection method

The detection method is the same as in the first embodiment.

(2) Test results

The test results are shown in Table 3:

Table 3 Comparison of quality of human immunoglobulin prepared by the method of the present invention and comparative method

As shown in Table 3, there were no significant differences in the immunoglobulin quality indexes prepared by the two methods, and the yields were 5.5-6.0 g/L blood plasma and 5.7 g/L blood plasma, respectively, and there was no significant difference.

In the second step of anion exchange chromatography, the volume of the exchange chromatography column of the preparation method of the present invention is 1/40 of the sample volume, and the volume of the exchange chromatography column of the comparison method is 1/19 of the sample volume.

Experiments have shown that human immunoglobulins of comparable quality are prepared, and 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. In summary, 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.

Claims

Claim

A method for preparing a human immunoglobulin, comprising: the following steps:

(1) Dissolving: Dissolving Cohn component I +Π +ΠΙ or Cohn component II +ΙΠ in water for injection;

(2) octanoic acid precipitation: Precipitate impurities with octanoic acid or octanoate, and filter to obtain a filtrate;

(4) Precipitating IgM: The conductivity of the flow through solution obtained in step (3) is adjusted to 500-1000 us/cm with water, then the pH is adjusted to 6.0-7.3, allowed to stand for l-2 h, and filtered to obtain a filtrate;

(6) After ultrafiltration or dialysis, preparation, virus inactivation, that is, the human immunoglobulin finished product.

The method according to claim 1, characterized in that the step (1) is: taking Cohn component Ι + Π + ΠΙ or Cohn component Π + ΠΙ, adding water for injection, 2-8 ° C Stir until it dissolves and adjust the pH to 3.8-4.9.

3. The method according to claim 1, wherein the step (2) is: adding octanoic acid or octoate at a concentration of 10 mM to 20 mM to adjust the pH to 5.0 to 5.3.

4. The method according to claim 1, wherein: (3) the filler for the anion exchange chromatography is Capto Q, Gigacap Q or Unosphere Q.

The method according to claim 1, wherein the pH of the step (4) is 6.3 to 6.74.

The method according to claim 5, wherein the step (4) is 6.51 to 6.7.

The method according to claim 6, wherein the pH of the step (4) is 6.51 or 6.7.

8. The method according to claim 1, wherein: (5) the filler of the anion exchange chromatography is Macrocap Q.

9. A human immunoglobulin prepared by the method of any of claims 1-8.

A pharmaceutical composition which is prepared by using the human immunoglobulin of claim 9 as an active ingredient together with a pharmaceutically acceptable adjuvant or auxiliary ingredient.