RU2614119C2 - Method of producing human immunoglobulin - Google Patents

Abstract

FIELD: biochemistry.

SUBSTANCE: invention relates to biochemistry. Group of inventions is described, which involves method of producing human immunoglobulin and preparation of human immunoglobulin, obtained using said method. Method includes stage of dissolving of Cohn component I+II+III or Cohn component II+III in water for injections; deposition with caprylic acid or caprylate; first stage of anion-exchange chromatography, involving bringing pH of filtrate obtained at step (2), to 5.2, cleaning by anion-exchange chromatography and obtaining eluating solution; deposition of IgM, including control of conductivity of eluating solution obtained at step (3), up to 500–1,000 µS/cm by means of water and further bringing pH to 6.0–7.3,with subsequent settling for 1–2 hours, filtration and filtrate collection; second stage of anion-exchange chromatography, involving bringing pH of filtrate obtained at step (4) to 5.6, subsequent purification by anion-exchange chromatography and collection of eluating solution; production of human immunoglobulin by dialysis of eluating solution obtained at step (5), by ultrafiltration, obtaining pharmaceutical form and inactivation of viruses.

EFFECT: invention extends range of methods for producing human immunoglobulin.

9 cl, 2 dwg, 3 tbl, 2 ex

Description

Technical field

The present invention relates to a method for producing blood products, and in particular to a method for producing human immunoglobulin.

State of the art

Immunoglobulin includes at least IgG, IgM, IgA, IgE and IgD, of which IgG is the most important, and the absence of IgG will reduce the body's defense. Immunoglobulin preparations can be divided mainly into three groups: human immunoglobulin for intramuscular injection, specific immunoglobulin and immunoglobulin for intravenous injection. Currently, immunoglobulin preparations are mainly isolated from plasma.

In the 1940s, Professor E.Yu. Cohn (E.J. Cohn) of Harvard University invented a method for separating plasma proteins with low temperature ethanol. After that, Nischmann and Kistler et al. (Nistchmann and Kistler, et. Al.) Upgraded Kohn's low-temperature ethanol method and proposed an improved method for separating plasma proteins with low-temperature ethanol, which is characterized by simplified steps and reduced production time. Nevertheless, the method of separation by low-temperature ethanol has certain limitations in the production of immunoglobulin, and the recovery of globulins ranges from 3.8 to 4.4 g / L. In addition, the IgA content of the immunoglobulin preparation obtained by the low-temperature ethanol method is relatively high, and thus, patients suffering from congenital selective IgA deficiency may experience side effects after injection.

In 1969, Steinbruch et al. (Steinbruch, et al.) Described a method for separating IgG from mammalian plasma by caprylic acid precipitation in combination with anion exchange chromatography. Under the influence of a weakly acidic medium with pH≥4.5, other impurity proteins can be precipitated, in addition to immunoglobulin. However, this method requires further purification of IgG using diethylaminoethyl cellulose (DEAE cellulose) as an anion exchange medium, and is characterized by the complexity of the process and high cost.

US Pat. No. 6,307,028 proposes a purification method by means of precipitation with caprylic acid in combination with two-stage anion exchange chromatography, and a high-purity immunoglobulin preparation with the same distribution of IgG subclasses as in normal plasma was obtained.

Nevertheless, the Chinese plasma IgM content is higher than that of non-Chinese (Chinese plasma IgM content is about 10% of the total protein content, while plasma IgM content in non-Chinese ranges from 1% to 3% of the total protein content). To obtain immunoglobulin with suitable purity in accordance with the previous method, a large volume chromatographic column is required to absorb a large amount of IgM, which entails increased costs. This factor makes it difficult to implement the method on an industrial scale.

SUMMARY OF THE INVENTION

To solve the aforementioned problem, the present invention provides a new method for producing human immunoglobulin.

First of all, the present invention relates to a method for producing human immunoglobulin, comprising the following steps:

(1) dissolution: dissolving the Cohn component I + II + III or the Cohn component II + III in water for injection;

(2) precipitation with caprylic acid: precipitation of impurities with caprylic acid or caprylate, filtering and obtaining a filtrate;

(3) the first stage of anion exchange chromatography: adjusting the pH of the filtrate obtained in step (2) to 5.2, purification by anion exchange chromatography and obtaining an eluting solution;

(4) IgM precipitation: adjusting the conductivity of the eluting solution obtained in step (3) to 500-1000 μS / cm with water and then adjusting the pH to 6.0-7.3, followed by aging for 1-2 hours filtering and obtaining filtrate;

(5) the second stage of anion exchange chromatography: adjusting the pH of the filtrate obtained in step (4) to 5.6, subsequent purification by anion exchange chromatography and obtaining an eluting solution;

(6) obtaining a human immunoglobulin product by ultrafiltration or dialysis, obtaining a pharmaceutical preparation and inactivating viruses.

In step (1), the Cohn component I + II + III or the Cohn component II + III is added to water for injection and stirred at a temperature of 2-8 ° C until dissolved to bring the pH to 3.8-4.9. Typically, the amount of water for injection is: the ratio of the volume to the mass of water for injection to the Cohn component I + II + III or Cohn component II + III 10: 1-15: 1.

In step (2), caprylic acid or caprylate is added at a concentration of 10 mM - 20 mM and the pH is adjusted to 5.0-5.3.

In step (3), Capto Q, Gigacap Q, or Unosphere Q is used as a carrier for the indicated anion exchange chromatography, the column volume for anion exchange chromatography being 1 / 105-1 / 50 of the sample volume.

In step (4), the preferred pH is 6.3-6.74, even more preferred 6.51-6.7, and even more preferred 6.51 or 6.7.

In step (5), Macrocap Q is used as the carrier for the indicated anion exchange chromatography, wherein the volume of the column for exchange chromatography is 1 / 50-1 / 35 of the volume of the sample.

The present invention also provides a human immunoglobulin obtained by the above method.

The present invention ultimately provides a pharmaceutical composition which is prepared from the aforementioned human immunoglobulin as an active ingredient together with a pharmaceutically acceptable adjuvant or adjuvant.

The production method proposed in the present invention includes an additional IgM precipitation step, added according to the invention, between the two steps of anion chromatography, and thus, the volume of the second step of anion exchange chromatography is obviously reduced, the production cost is reduced, and thus The method has promising prospects for use in the market.

Obviously, in accordance with the above description of the present invention, in combination with general technical knowledge and conventional technical means in this field, various replacements or changes or modifications can be made without deviating from the basic technical concept according to the present invention mentioned above.

The above description of the present invention is hereinafter described in more detail in separate embodiments as examples, which, however, should not be construed as limiting the scope of the present invention by the following examples. All implementation methods based on the above description fall within the scope of the present invention.

Description of drawings

In FIG. 1 schematically shows a method for producing a human immunoglobulin according to the present invention.

In FIG. Figure 2 shows the relationship between pH, amount of precipitated IgM, and IgG yield.

Implementation options

Example 1. Obtaining human immunoglobulin by the method according to the present invention

1. Experimental materials

Cohn component I + II + III or component II + III: obtained by the low-temperature ethanol precipitation method as described on page 1194 of Medical Biologicology, 2 ^nd edition (Peopleʹs Medical Publishing House Co., LTD).

Hydrochloric acid, caprylic acid, Capto Q carrier, Gigacap Q carrier, Unosphere Q carrier, Macrocap Q, diatomite and Beckman IMMAGE IgA detection kit are all commercially available.

2. The methodology of the experiments

As shown in FIG. 1, immunoglobulin was prepared as follows:

(1) Component of Cohn I + II + III: 4 kg of a precipitate of the Cohn component I + II + III was dissolved in 40 L of WFI (water for injection) at 5 ° C and stirred for 1 hour. The pH was adjusted to 4.20 with 0.5M hydrochloric acid and the resulting solution was heated and stirred in a water bath at 25 ° C for 2 hours;

(2) precipitation with caprylic acid: caprylic acid was directly added until a final concentration of 15 mM was reached, and the pH was adjusted to 5.20 with 0.5 M NaOH. The resulting solution was heated and stirred in a water bath at 25 ° C for 2 hours, and then left for 2 hours at 8 ° C;

(3) The first stage of anion exchange chromatography: the suspension obtained by the precipitation reaction with caprylic acid was filtered with a filter layer of a usual depth, and the pH of the filtrate was adjusted to 5.20 with 0.5 M hydrochloric acid or 0.5 M NaOH. Subsequently, the first step of anion exchange chromatography was carried out with a Capto Q support, at a pH of 5.20 and with a linear flow rate of 200 cm / h. The volume of the column for exchange chromatography was 1/75 of the volume of the sample;

(4) IgM precipitation: first, the conductivity of the chromatographic elution solution was adjusted to 700 μS / cm. Then the pH of the solution was adjusted to 6.3 with 0.5 M NaOH, followed by aging for 1 h;

(5) the second stage of anion exchange chromatography: filtration was carried out with the addition of diatomite, and during the second stage of anion exchange chromatography, the pH of the filtrate was adjusted to 5.6. A Macrocap Q support with a linear flow rate of 75 cm / h was used, and the volume of the exchange chromatography column was 1/40 of the volume of the sample;

(6) the chromatographic elution solution was collected and its pH was adjusted to 3.9-4.2 with 0.5M hydrochloric acid. The solution was then subjected to ultrafiltration concentration until a protein concentration of 20 mg / ml was reached. In order to filter viruses, the solution was passed through virus filters with pore sizes of 200-15 nm, such as DV50, DV20, Planova75, Planova35 and the like. After completing this step, the solution was concentrated to the protein concentration required for the final preparation, such as 5% or 10% (w / v). After that, suitable additives were added so that the osmotic pressure of the solution met the requirement for intravenous administration, and the additives could be sugars or amino acids. The pH of the solution was adjusted to 4.0-4.8. The solution was incubated at 25 ° C for 21 days and individually packaged in infusion bottles in accordance with the requirements for obtaining a pharmaceutical preparation.

3. Product Definition

(1) Method for determining

According to the methodology specified for execution in the Chinese Pharmacopoeia (2010), indices were appropriately determined, such as product purity, product monomer + dimer distribution (i.e. molecule size), product subclass distribution, and others.

The IgA content in the product was determined on a Beckman IMMAGE plasma protein analyzer using an immune turbidimetry method. In addition, the determination was carried out using a Beckman IMMAGE IgA determination kit, a specific determination method is indicated on the instructions of said determination kit.

The method for determining the yield: after concentration by ultrafiltration, the protein concentration of the drug was determined by the method of determination of Kjeldahl (which is the first method specified in Appendix VI B in the Pharmacopoeia of China 2010), while

yield = protein concentration × composition volume / plasma volume.

(2) Definition result

The result of the determination is shown in table 1:

According to table 1, all indicators of human immunoglobulin obtained in accordance with the method according to the present invention meet the requirements set forth in the Pharmacopoeia of China. Compared with the traditional method using low-temperature ethanol, the method according to the present invention can increase the purity of the drug, reduce the content of multimer and IgA, and further improve safety in case of infusion of high doses. In addition, the method for producing human immunoglobulin according to the present application has a high yield, which is increased by more than 10% compared to the existing method using low-temperature ethanol.

Example 2. Experiments to optimize parameters

The pH values and conductivity during IgM precipitation were determined in step (4) of the method of the present invention:

(1) Conductivity Optimization Experiment

The experimental procedure: under constant other conditions, the conductivity and pH were changed in step (4) of Example 1 to determine the relationship between the conductivity and the amount of IgM precipitate and IgG yield.

Experimental result: through many experiments, it was found that when the conductivity of the chromatographic elution solution was higher than 1 μS / cm, even if the pH of the solution was raised, there was no IgM precipitation. When the conductivity of the chromatographic elution solution was between 500 and 1000 μS / cm, the pH was adjusted to 6.0 and higher, and IgM apparently precipitated.

(2) pH Optimization Experiment

Experimental procedure: under constant other conditions, the pH was changed in step (4) of Example 1 to determine the relationship between pH, amount of IgM precipitate and IgG yield.

Experiment result: through many experiments it was found that the IgM removal rate increased with increasing pH, but an excessively high pH led to an increase in IgG loss.

In Table 2 and in FIG. 1 shows:

According to Table 2 and FIG. 1, at a pH between 6.2 and 7.3, IgM obviously precipitates. At a pH between 6.3 and 6.74, IgM precipitates to a high degree and the yield of IgG is also high. At pH between 6.51 and 6.7, both IgM precipitation and IgG yield are better manifested.

In general, to balance the rate of IgM removal and IgG yield, in step (4) of the method according to the present invention, the conductivity was adjusted to 500-1000 μS / cm and the pH adjusted to 6.0-7.0.

In order to more thoroughly study the advantages of the method according to the present invention, the following comparative experiment was carried out:

1. Obtaining human immunoglobulin

(1) The production method according to the present invention: as described in Example 1;

(2) Comparative production method:

1) dissolution of the Cohn component I + II + III: 4 kg of the precipitate of the Cohn component I + II + III was dissolved in 40 l of VDI (water for injection) at 5 ° C and stirred for 1 hour. The pH was adjusted to 4.20 with 0.5M hydrochloric acid and the resulting solution was heated and stirred in a water bath at 25 ° C for 2 hours;

2) precipitation with caprylic acid: caprylic acid was directly added until a final concentration of 15 mM was reached, and the pH was adjusted to 5.20 with 0.5 M NaOH. The resulting solution was heated and stirred in a water bath at 25 ° C for 2 hours, and then left for 2 hours at 8 ° C;

3) the first stage of anion exchange chromatography: the suspension of the precipitation reaction of caprylic acid was filtered using a filter layer of usual depth, the pH of the filtrate was adjusted to 5.20 with 0.5M hydrochloric acid or 0.5M NaOH. Subsequently, the first step of anion exchange chromatography was carried out with a Capto Q support, at a pH of 5.20 and with a linear flow rate of 200 cm / h;

4) the second stage of anion exchange chromatography: the chromatographic elution solution was collected, the filtration was carried out with the addition of diatomite; during the second stage of anion exchange chromatography, the pH of the filtrate was adjusted to 5.6. A Macrocap Q support with a linear flow rate of 75 cm / h was used, and the volume of the exchange chromatography column was 1/19 of the volume of the sample;

5) the chromatographic elution solution was collected, its pH was adjusted to 3.9-4.2 with 0.5M hydrochloric acid. The solution was then subjected to ultrafiltration concentration until a protein concentration of 20 mg / ml was reached. In order to filter viruses, the solution was passed through virus filters with pore sizes of 200-15 nm, such as DV50, DV20, Planova75, Planova35 and the like. After completing this step, the solution was concentrated to the protein concentration required for the final composition, such as 5% or 10% (w / v). After that, suitable additives were added so that the osmotic pressure of the solution met the requirement for intravenous administration, and the additives could be sugars or amino acids. The pH of the solution was adjusted to 4.0-4.8. The solution was incubated at 25 ° C for 21 days and individually packaged in infusion bottles in accordance with the requirement for preparation.

2. Product Definition

(1) Method for determining

The method of determination is the same as in Example 1.

(2) Definition result

The result of the determination is shown in Table 3:

As shown in table 3, the quality indicators of immunoglobulins obtained by the two methods have no significant difference, and their yield is 5.5-6.0 g / l of plasma and 5.7 g / l of plasma, respectively, also without any or significant differences.

In the second step of anion exchange chromatography, the volume of the exchange chromatography column was 1/40 of the volume of the sample in the method of the present invention, while in the comparative method it was 1/19.

The experiments proved that to obtain immunoglobulins with appropriate quality indicators at the second stage of anion exchange chromatography, the method of the present invention is able to significantly reduce the required volume of the chromatographic column compared to the comparative method.

Thus, the method proposed in the present invention, can successfully solve the problem of the high cost of production of human immunoglobulin caused by an excessively high content of IgM in Chinese plasma; significantly reduce the cost of production, makes possible the industrial production of human immunoglobulins on a large scale and has promising prospects for use on the market.

Claims (15)

1. The method of obtaining human immunoglobulin, comprising the following steps: (1) dissolving the Cohn component I + II + III or the Cohn component II + III in water for injection; (2) precipitation with caprylic acid or caprylate, including precipitation of impurities with caprylic acid or caprylate, filtration and filtrate collection; (3) the first step of anion exchange chromatography, including adjusting the pH of the filtrate obtained in step (2) to 5.2, purification by anion exchange chromatography and obtaining an eluting solution; (4) IgM precipitation, including controlling the conductivity of the elution solution obtained in step (3) to 500-1000 μS / cm with water and then adjusting the pH to 6.0-7.3, followed by 1-2 h, filtration and filtrate collection; (5) the second stage of anion exchange chromatography, including adjusting the pH of the filtrate obtained in step (4) to 5.6, subsequent purification by anion exchange chromatography and collecting the eluting solution; (6) obtaining human immunoglobulin by dialysis of the elution solution obtained in step (5) by ultrafiltration, obtaining a pharmaceutical form and inactivation of viruses. 2. The method according to p. 1, characterized in that in step (1) the component of Cohn I + II + III or the component of Cohn II + III is added to water for injection and stirred at a temperature of 2-8 ° C until dissolved to bring the pH to 3.8-4.9. 3. The method according to p. 1, characterized in that in step (2) add caprylic acid or caprylate at a concentration of 10 mm - 20 mm and adjust the pH to 5.0-5.3. 4. The method according to p. 1, characterized in that the carrier for the specified anion exchange chromatography in step (3) is a Capto Q, Gigacap Q or Unosphere Q. 5. The method according to p. 1, characterized in that the pH in step (4) is 6.3-6.74. 6. The method according to p. 5, characterized in that the pH in step (4) is 6.51-6.7. 7. The method according to p. 6, characterized in that the pH in step (4) is 6.51 or 6.7. 8. The method according to p. 1, characterized in that the carrier for the specified anion exchange chromatography in step (5) is a Macrocap Q. 9. The preparation of human immunoglobulin obtained according to the method according to any one of paragraphs. 1-8, while this drug is characterized by increased purity and a reduced content of multimers and IgA, while the purity of the drug is from 99.3% to 99.9%, the indicated content of multimers does not exceed 0.8% and the indicated content of IgA does not exceed 18 1 mg / l.

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