阅读说明：本技术 一种高浓度静注人免疫球蛋白的分离纯化方法 (Separation and purification method of high-concentration intravenous injection human immunoglobulin ) 是由 罗二华 梁小明 李雅文 陈婷婷 陈秀良 陈家啡 龚佳莉 于 2020-06-29 设计创作，主要内容包括：本发明涉及一种高浓度静注人免疫球蛋白的分离纯化方法,属于生物制药领域。在静注人免疫球蛋白制备工艺过程中,采用高浓度尿素溶液进行预沉淀,然后采用辛酸盐沉淀法处理静注人免疫球蛋白制备工艺中的组分Ⅱ+Ⅲ沉淀,去除组分Ⅱ+Ⅲ沉淀中的杂蛋白,再进行层析工艺。本发明与现有相比,通过高浓度尿素溶液预沉淀工艺可效地降低静注人免疫球蛋白中IgA、多聚体的含量,提高了免疫球蛋白的纯度。(The invention relates to a separation and purification method of high-concentration intravenous injection human immunoglobulin, belonging to the field of biological pharmacy. In the preparation process of the human immunoglobulin for intravenous injection, high-concentration urea solution is adopted for pre-precipitation, then component II + III precipitates in the preparation process of the human immunoglobulin for intravenous injection are treated by adopting an octanoate precipitation method, the hybrid proteins in the component II + III precipitates are removed, and then a chromatography process is carried out. Compared with the prior art, the method can effectively reduce the content of IgA and polymers in the human immunoglobulin for intravenous injection by a high-concentration urea solution pre-precipitation process, and improves the purity of the immunoglobulin.)

1. A separation and purification method of high-concentration intravenous injection human immunoglobulin adopts high-concentration urea solution pre-precipitation and octanoic acid precipitation methods to remove impurity proteins and coagulation factor substances, and then carries out chromatography technology, and is characterized in that:

the preparation process comprises the following steps:

(1) pre-precipitation of a high-concentration urea solution: centrifuging human blood plasma qualified in quarantine in a quarantine stage, wherein the centrifugation speed is not more than 4L/min/station, and the liquid outlet temperature is controlled at 0-4 ℃; separating and performing cold precipitation, wherein the cold precipitation is used for producing factor VIII, conveying the centrifugal supernatant protein liquid to a protein separation reaction tank, adding a urea solution with the mass fraction of 50% at normal temperature according to the mass multiple of 1-2, wherein the flow rate is not more than 1.0L/min, uniformly dispersing, adding 95% ethanol with the temperature of-15 ℃ or lower, the flow rate is not more than 1.5L/min, enabling the final concentration of the ethanol volume ratio to be 8%, and controlling the final temperature to be-1-3 ℃;

(2) stirring the product obtained in the step (1) for more than 30min, centrifuging, wherein the effluent flow rate is not more than 4L/min/station in the centrifuging process, the effluent temperature is controlled to be-1 to-3 ℃, and centrifuging to obtain supernatant protein liquid I;

(3) Precipitating caprylate, namely dripping 0.5mol/L NaOH into the supernatant protein liquid prepared in the step (2) to adjust the pH value to 4.3-4.5, adding a sodium caprylate solution with the mass fraction of 10% to the supernatant protein liquid, wherein the content of the sodium caprylate in the supernatant protein liquid is 18-22 mmol/L, then adjusting the pH value to 5.0-5.2, stirring for 2 hours, then performing filter pressing, and collecting the supernatant protein liquid;

(4) the chromatography process comprises the following steps: regulating the pH value of the supernatant protein liquid prepared in the step (3) to 5.20-5.60, adding 1 time of water for injection at the temperature of 2-8 ℃, dialyzing until the conductivity is 0.8-1.6 ms/cm, performing ultrafiltration concentration until the protein concentration is 1.5-3.5%, performing column chromatography by using a Macrocap-Q ion exchange column, and collecting flow-through liquid;

(5) adjusting the pH of the flow-through liquid prepared in the step (4) to 3.8-4.0 by using 1mol/L HCl, starting an ultrafiltration machine to adjust the protein concentration of the flow-through liquid to be more than 5%, dialyzing for 4-6 times by using water for injection with the same volume, concentrating the protein liquid to be more than 6%, adding maltose into the protein liquid, and adjusting the pH value by using 1mol/L HCl to ensure that the maltose content is 10 +/-1%, the pH value is 3.8-4.4 and the protein content is 5.5 +/-0.5%;

(6) filtering the product obtained in the step (5) by a 0.2-micron sterilizing filter element, placing the product in an incubation chamber, and incubating the product at 24 +/-1 ℃ for 21 days; after the incubation is finished, virus is removed by using a DV20 filter element and filtered; filtering and subpackaging by a 0.2-micron sterilizing filter element, inspecting the subpackaged products by a lamp, packaging after the inspection is qualified, and warehousing;

The percentages are, except for a limited number, the remaining percentages by mass.

2. The method for separating and purifying high-concentration intravenous human immunoglobulin according to claim 1, wherein the method comprises the following steps:

the preparation method of the high-concentration urea solution comprises the following steps: adding medicinal urea into purified water according to the mass ratio of 1:1 at the normal temperature of 25 ℃, stirring and dissolving completely, and filtering to obtain the urea.

3. The method for separating and purifying high-concentration intravenous human immunoglobulin according to claim 1, wherein the method comprises the following steps:

the Macrocap-Q ion exchange column chromatography process parameters are as follows:

the sample flow rate is not more than 100L/h/10L column volume, and the total protein is not more than 200g/L gel.

4. The method for separating and purifying high-concentration intravenous human immunoglobulin according to any one of claims 1 to 3, wherein:

the Macrocap-Q ion exchange column can be regenerated and reused, and the regeneration method comprises the following steps:

washing the chromatographic column with 1mol/L NaCL 2-4 times of the column volume to elute the foreign protein, wherein the flow rate is not more than 100L/h/10L of the column volume, and discarding the flow-through liquid;

washing and preserving the chromatographic column by using 0.5mol/L NaOH with 2-4 times of column volume, wherein the flow rate does not exceed 100L/h/10L of column volume, and discarding a flow-through solution;

When the column is not used for more than 1 month, the column is cleaned by 4 times of column volume of injection water, and then the column is washed and stored by 4 times of column volume of 20 percent ethanol, the flow rate does not exceed 100L/h/10L of column volume, and the flow-through liquid is discarded.

Technical Field

The invention relates to a separation and purification method of high-concentration intravenous injection human immunoglobulin, belonging to the field of biological pharmacy.

Background

The active component of the intravenous injection human immunoglobulin is protein, wherein more than 95 percent of the protein is the immunoglobulin. Is prepared from the blood plasma of healthy person through low-temp alcohol protein separation or caprylic acid deposition, removing anticomplementary activity and virus inactivation. The intravenous injection human immunoglobulin contains broad-spectrum IgG antibody resisting virus, bacteria or other pathogens, has double treatment effects of immunity substitution and immunity regulation, and is mainly used for primary immunoglobulin deficiency, secondary immunoglobulin deficiency and autoimmune diseases clinically. Clinical application of IVIG has become one of the fastest growing areas of immunotherapy over the last 20 years. To date, the FDA-approved IVIG indications have increased to 11, further supporting the use of IVIG as an immunomodulator and anti-inflammatory factor based on significant advances in the understanding of the effects and potential mechanisms of action for widespread clinical IVIG use.

Disclosure of Invention

The invention aims to provide a method for separating and purifying high-concentration intravenous injection human immunoglobulin, which can greatly reduce the content of IgA and polymers in the intravenous injection human immunoglobulin, improve the purity of the intravenous injection human immunoglobulin and reduce the use risk.

The technical scheme of the invention is as follows:

a separation and purification method of high-concentration intravenous injection human immunoglobulin adopts high-concentration urea solution pre-precipitation and octanoic acid precipitation methods to remove impurity proteins and coagulation factor substances, and then carries out chromatography technology, and the preparation technology is as follows:

(1) pre-precipitation of a high-concentration urea solution: centrifuging human blood plasma qualified in quarantine in a quarantine stage, wherein the centrifugation speed is not more than 4L/min/station, and the liquid outlet temperature is controlled at 0-4 ℃; separating and performing cold precipitation, wherein the cold precipitation is used for producing factor VIII, conveying the centrifugal supernatant protein liquid to a protein separation reaction tank, adding a urea solution with the mass fraction of 50% at normal temperature according to the mass multiple of 1-2, wherein the flow rate is not more than 1.0L/min, uniformly dispersing, adding 95% ethanol with the temperature of-15 ℃ or lower, the flow rate is not more than 1.5L/min, enabling the final concentration of the ethanol volume ratio to be 8%, and controlling the final temperature to be-1-3 ℃;

(2) stirring the product obtained in the step (1) for more than 30min, centrifuging, wherein the effluent flow rate is not more than 4L/min/station in the centrifuging process, the effluent temperature is controlled to be-1 to-3 ℃, and centrifuging to obtain supernatant protein liquid I;

(3) Precipitating caprylate, namely dripping 0.5mol/L NaOH into the supernatant protein liquid prepared in the step (2) to adjust the pH value to 4.3-4.5, adding a sodium caprylate solution with the mass fraction of 10% to the supernatant protein liquid, wherein the content of the sodium caprylate in the supernatant protein liquid is 18-22 mmol/L, then adjusting the pH value to 5.0-5.2, stirring for 2 hours, then performing filter pressing, and collecting the supernatant protein liquid;

(4) the chromatography process comprises the following steps: regulating the pH value of the supernatant protein liquid prepared in the step (3) to 5.20-5.60, adding 1 time of water for injection at the temperature of 2-8 ℃, dialyzing until the conductivity is 0.8-1.6 ms/cm, performing ultrafiltration concentration until the protein concentration is 1.5-3.5%, performing column chromatography by using a Macrocap-Q ion exchange column, and collecting flow-through liquid;

(5) adjusting the pH of the flow-through liquid prepared in the step (4) to 3.8-4.0 by using 1mol/L HCl, starting an ultrafiltration machine to adjust the protein concentration of the flow-through liquid to be more than 5%, dialyzing for 4-6 times by using water for injection with the same volume, concentrating the protein liquid to be more than 6%, adding maltose into the protein liquid, and adjusting the pH value by using 1mol/L HCl to ensure that the maltose content is 10 +/-1%, the pH value is 3.8-4.4 and the protein content is 5.5 +/-0.5%;

(6) filtering the product obtained in the step (5) by a 0.2-micron sterilizing filter element, placing the product in an incubation chamber, and incubating the product at 24 +/-1 ℃ for 21 days; after the incubation is finished, virus is removed by using a DV20 filter element and filtered; filtering and subpackaging by a 0.2-micron sterilizing filter element, inspecting the subpackaged products by a lamp, packaging after the inspection is qualified, and warehousing;

The percentages are, except for a limited number, the remaining percentages by mass.

Further, the preparation method of the high-concentration urea solution comprises the following steps: adding medicinal urea into purified water according to the mass ratio of 1:1 at the normal temperature of 25 ℃, stirring and dissolving completely, and filtering to obtain the urea.

Furthermore, the Macrocap-Q ion exchange column chromatography process parameters are as follows:

the sample flow rate is not more than 100L/h/10L column volume, and the total protein is not more than 200g/L gel.

Furthermore, the Macrocap-Q ion exchange column can be regenerated and reused, and the regeneration method comprises the following steps:

washing the chromatographic column with 1mol/L NaCL 2-4 times of the column volume to elute the foreign protein, wherein the flow rate is not more than 100L/h/10L of the column volume, and discarding the flow-through liquid;

washing and preserving the chromatographic column by using 0.5mol/L NaOH with 2-4 times of column volume, wherein the flow rate does not exceed 100L/h/10L of column volume, and discarding a flow-through solution;

when the column is not used for more than 1 month, the column is cleaned by 4 times of column volume of injection water, and then the column is washed and stored by 4 times of column volume of 20 percent ethanol, the flow rate does not exceed 100L/h/10L of column volume, and the flow-through liquid is discarded.

The invention has positive significance:

according to the invention, the impurity proteins and coagulation factor substances are removed by adopting a high-concentration urea solution pre-precipitation and caprylic acid precipitation method, then a chromatography process is carried out, and a process technology of crystallizing, separating out and adsorbing IgA and polymers at a low temperature by utilizing a high-concentration urea solution is utilized, so that the IgA and polymer removing efficiency is high, the prepared intravenous injection human immunoglobulin has higher purity, and the risk of anaphylaxis possibly caused during use is reduced.

The invention utilizes the characteristic that the adsorption capacity of urea on IgA and polymers is far higher than that of IgG in crystallization, and in the urea precipitate obtained by centrifugation in the step (2), the main components are the polymers and IgA, the removal rate of the polymers in the step is more than or equal to 98 percent, the removal rate of IgA is more than or equal to 80 percent, and the loss of IgG is less than 5 percent. Therefore, most polymers and IgA are removed, a high-quality pretreatment raw material is provided for the subsequent purification step, and the final quality of the product is improved.

Detailed Description

The present invention will be further described by way of the following examples, which, however, are not intended to limit the scope of the invention.