CN111825757A

CN111825757A - Method for removing heat source in recombinant IL-18 and product prepared by method

Info

Publication number: CN111825757A
Application number: CN202010771259.1A
Authority: CN
Inventors: 张朔; 李雪娇; 冯铃; 刘辉; 赵航; 吴骁; 刘畅; 赵洪礼; 何向东; 何伟
Original assignee: Shenyang Eye Industry Technology Research Institute Co ltd; Shenyang Heshi Eye Industry Group Co ltd
Current assignee: Shenyang Eye Industry Technology Research Institute Co ltd; Shenyang Heshi Eye Industry Group Co ltd
Priority date: 2020-08-04
Filing date: 2020-08-04
Publication date: 2020-10-27

Abstract

The invention relates to the technical field of biology, in particular to a method for removing a heat source in recombinant IL-18 and a prepared product. The purification method provided by the invention adopts high performance liquid chromatography to purify to obtain the recombinant IL-18, the chromatographic conditions are reasonable, thereby realizing the good separation of the recombinant IL-18 and a heat source, and the recombinant IL-18 which meets the requirement of an injection product and is removed from the heat source is obtained by recovering the chromatographic peak of the recombinant IL-18. Experiments show that the concentration of the recombinant IL-18 which is purified by the method and is obtained by removing the heat source is 15.1mg/ml, the recovery rate is 85.32%, the content of the heat source meets the requirement, and the physiological activity of the recombinant IL-18 which is removed from the heat source is not influenced.

Description

Method for removing heat source in recombinant IL-18 and product prepared by method

Technical Field

The invention relates to the technical field of biology, in particular to a method for removing a heat source in recombinant IL-18 and a prepared product.

Background

Diabetic fundus vasculopathy (DR) and age-related macular degeneration (AMD) are the common major factors affecting vision and even blindness in the advanced stages of diabetes and the elderly. Since DR and AMD are long-term pathological processes, the abnormal growth of their new blood vessels and metabolites block the subretinal space so that important nutrients cannot enter the retina from the blood, and the discharge of waste products is also hindered, which leads to the death of retinal cells and ultimately to permanent blindness. Thus, while inhibiting blood vessel growth, promoting blood vessel maturation, which allows more nutrients to enter the retina and eliminates existing metabolites, is the primary goal of DR and AMD treatment.

In recent years, research finds that Interleukin 18(Interleukin 18, IL-18) has a regulating effect on the growth of new blood vessels, can inhibit the overgrowth of the new blood vessels, promotes the maturation of the new blood vessels, and prevents and treats leakage. IL-18 is currently prepared by genetic engineering. Escherichia coli is the most common host in genetic engineering bacteria and is mostly used in the biopharmaceutical industry. However, since escherichia coli is a typical gram-negative bacterium, and the cell wall thereof contains lipopolysaccharide, in a pharmaceutical method using escherichia coli as a host, a large amount of endotoxin (endotoxin is also called a heat source, main chemical components are lipopolysaccharide, and the molecular weight is different) is contained, and particularly, when the inclusion body is expressed intracellularly, the inclusion body needs to be obtained by crushing the bacterium, and the crushed bacterium causes a large amount of lipopolysaccharide in the cell wall to remain in a sample, thereby generating a large amount of heat source. Very trace amounts of endotoxin entering the body can cause fever and inflammatory reactions, which in severe cases can lead to death.

Biologics for intraocular injection must be characterized by high purity and very low pyrogen levels to ensure that they do not produce other inflammatory reactions when applied to the eye. The existence of a heat source in recombinant IL-18 results in a limited range of applications.

The main chemical component of the heat source is lipopolysaccharide, the molecular weight of the lipopolysaccharide is different, and the lipopolysaccharide is difficult to remove. At present, common methods for removing heat sources include activated carbon methods, acid-base methods, ultrafiltration methods, ion exchange methods, affinity chromatography methods and the like. The activated carbon method can adsorb a sample while adsorbing a heat source, so that the concentration of the sample is reduced; the acid-base method can destroy the activity of biological preparation; the ultrafiltration method is only suitable for biological preparation with smaller molecular weight, and the ultrafiltration condition is strict and is easy to be polluted; most of the ion exchange method and the affinity chromatography method can not adopt a process flow to remove the endotoxin to reach the standard. The above methods have limited effectiveness in removing heat sources and are not applicable to all samples. Especially, biological products, not only the concentration and purity of the sample, but also the biological activity of the sample should be ensured while removing the heat source.

Disclosure of Invention

In view of the above, the technical problem to be solved by the present invention is to provide a method for removing pyrogens from recombinant IL-18 and to provide a product obtained by the method, which can remove pyrogens well and ensure the concentration, purity and biological activity of the recombinant IL-18 from which pyrogens are removed.

In the method for removing the heat source in the recombinant IL-18, the recombinant IL-18 is purified by adopting a high performance liquid chromatography to obtain the recombinant IL-18 with the heat source removed, and the parameters comprise:

the chromatographic column is a C18 column; the temperature of the chromatographic column is 20-30 ℃;

the mobile phase A is an aqueous solution containing 0.05 vol% -0.2 vol% of trifluoroacetic acid, and the mobile phase B is an acetonitrile solution containing 0.05 vol% -0.2 vol% of trifluoroacetic acid;

the elution procedure included: 0-55 min, wherein the volume fraction of the phase A is from 100% to 30%;

the flow rate of the mobile phase is 5-30 mL/min.

The invention separates the recombinant IL-18 and the heat source substance contained in the recombinant IL-18 sufficiently through reasonable chromatographic conditions, thereby achieving good purification effect, and the activity of the purified recombinant IL-18 is not influenced and the yield and the purity are high. The invention adopts a two-phase elution mode to purify the recombinant IL-18, wherein, the flow relative separation effect plays a crucial role, and the composition of the mobile phase and the proportion of the two phases in the elution process are adjusted, thereby realizing the good separation of the recombinant IL-18 and the heat source. Thus, by collecting the chromatographic peak of recombinant IL-18, the heat source removal is achieved.

In some embodiments, mobile phase a is an aqueous solution containing 0.1 vol% trifluoroacetic acid and mobile phase B is an acetonitrile solution containing 0.1 vol% trifluoroacetic acid.

The preparation method of the mobile phase comprises the following steps:

mobile phase A: adding trifluoroacetic acid into non-heat source water until the concentration of the trifluoroacetic acid is 0.05 vol% -0.2 vol%; preferably, trifluoroacetic acid is added to a concentration of 0.1 vol%.

Mobile phase B: adding trifluoroacetic acid into acetonitrile until the concentration of the trifluoroacetic acid is 0.05 vol% -0.2 vol%; preferably, trifluoroacetic acid is added to a concentration of 0.1 vol%.

In the invention, the elution procedure is 0-55 min, and the volume fraction of the mobile phase A is continuously reduced from 100% to 30%.

For liquid chromatography, an increase in column temperature may accelerate the separation process, but may result in a decrease in resolution; while lowering the column temperature increases the resolution, but the separation process time is lengthened and may cause crystallization of impurities, thereby affecting the service life of the column. The temperature of the chromatographic column is 20-30 ℃. In some embodiments, the column temperature of the chromatography column is 25 ℃.

The detection wavelength should be selected in consideration of the recombinant IL-18 itself and the absorption wavelength of the heat source so that both the recombinant IL-18 and the heat source can absorb at the appropriate wavelength, and the detection wavelength of the liquid chromatography of the present invention is 280 nm.

The size of the column has a great influence on the separation effect, and for example, it has been reported that a decrease in the column diameter has a positive effect on the improvement of the separation effect, but the throughput of the sample is decreased. Increasing the column diameter, while increasing throughput, can result in uneven distribution of the sample in the column, resulting in poor separation. In other cases, it has been reported that longer columns generally have better separation performance, but in some cases, the separation performance is not positively correlated with the length of the column. In addition, decreasing the filler particle size increases the column pressure, and too fast a mobile phase flow rate can adversely affect the separation performance. The flow rate of the mobile phase is 5-30 mL/min. In some embodiments, the flow rate of the mobile phase is 10 mL/min. In the present invention, the specification of the column is 21.2mm × 250mm, and the particle size is 7 μm.

The sample solution of the recombinant IL-18 is a liquid sample collected by a gel chromatographic column of the recombinant IL-18 prepared under the application No. 201510455749. In the embodiment of the invention, the concentration of the recombinant IL-18 in the sample liquid of the recombinant IL-18 is 1-10 mg/ml. In some embodiments, the recombinant IL-18 sample solution concentration is 2.68 mg/mL. In the embodiment of the invention, the loading volume of the recombinant IL-18 is 100-500 mL. In some embodiments, the loading volume is 350 mL.

Under the chromatographic condition of the invention, the peak-off time of the sample peak of the recombinant IL-18 is 15.0 min-15.5 min. In some embodiments, the peak off time is 15.308 min.

The high performance liquid chromatography purification also comprises the steps of renaturation and acetonitrile removal. The steps of renaturation and acetonitrile removal comprise: carrying out rotary evaporation at the temperature of 32 ℃, the pressure of-100 kPa and the rotating speed of 35-45rpm for 20-30 min until 1/2 liquid remains; adding a renaturation solution until the pH value is 7.0-7.2, and dialyzing by adopting a 20mmol PB buffer solution with the volume of 100 times that of the renaturation solution; the renaturation solution is 20mmolPB buffer solution containing 20mM DTT, and the pH value is 8.0.

The invention also provides recombinant IL-18 produced by the methods of the invention.

The purification method provided by the invention adopts high performance liquid chromatography to purify to obtain the recombinant IL-18, the chromatographic conditions are reasonable, thereby realizing the good separation of the recombinant IL-18 and a heat source, and the recombinant IL-18 meeting the requirements of injection products is obtained by recovering the chromatographic peak of the recombinant IL-18. Experiments show that the concentration of the recombinant IL-18 which is purified by the method and is obtained by removing the heat source is 15.1mg/ml, the recovery rate is 85.32%, the content of the heat source meets the requirement, and the physiological activity of the recombinant IL-18 which is removed from the heat source is not influenced.

Drawings

FIG. 1 shows a graph of elution peaks of a liquid phase purified sample prepared in example 1;

FIG. 2 shows the electrophoretograms of samples before and after purification of the preparative liquid phase of example 1: the Marker is the molecular mass of protein, and the size of the Marker is respectively 97.4kD, 66.2kD, 43kD, 31kD, 22kD and 14.4 kD; 1, preparing a sample before liquid phase purification; 2 preparing a sample after liquid phase purification;

FIG. 3 is a graph showing the analysis of HUVEC cell lumen formation by the sample after purification in example 1;

fig. 4 shows the peak elution pattern of the liquid phase purified sample prepared in comparative example 1.

Detailed Description

The invention provides a method for removing a heat source in recombinant IL-18 and a product prepared by the method, and a person skilled in the art can realize the method by appropriately improving process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

The test materials adopted by the invention are all common commercial products and can be purchased in the market.

In the present invention, the sample used for sample loading and purification is a liquid sample collected by a gel chromatography column described in patent 201510455749. X.

For the lyophilized sample described in patent 201510455749.X, the components are recombinant IL-18, sodium dihydrogen phosphate and disodium hydrogen phosphate, and the lyophilized sample can be reconstituted and then purified if it is purified, and the optional solvent includes pyrogen-free water.

The invention is further illustrated by the following examples:

example 1

1. Preparative liquid phase purification of recombinant IL-18

Purifying the recombinant IL-18 by using a preparation liquid to remove endotoxin, wherein the sample is a sample disclosed as a patent number: 201510455749.X liquid sample collected on gel chromatography column of recombinant IL-18 prepared.

The chromatographic column medium is C18, the specification is 21.2mm multiplied by 250mm, and the particle size is 7 mu m.

Mobile phase A: a mixed solution of pyrogen-free water and 0.1% trifluoroacetic acid, for example: 1L pyrogen-free water was added with 1ml trifluoroacetic acid.

Mobile phase B: to a mixed solution of acetonitrile and 0.1% trifluoroacetic acid, for example, 1L of acetonitrile, 1ml of trifluoroacetic acid was added.

And (3) taking the mobile phase A and the mobile phase B as eluents, wherein the flow rate is 10mL/min, and eluting by using a gradient elution mode, wherein the column temperature is 25 ℃, and the detection wavelength is 280 nm.

Gradient elution procedure:

time (min)	Flow rate (ml/min)	A％	B％
				0	10	100	0
55	10	30	70

The chromatogram is shown in FIG. 1, and the peak of the sample with retention time of 15.308min is collected.

2. Removing acetonitrile and renaturation after liquid phase collection

1. Removing acetonitrile by a rotary evaporation method: a rotary evaporator is adopted, the temperature is 32 ℃, the pressure is-100 KPa, the rotating speed is 35-45rpm, and the time is 20-30 minutes. The volume after evaporation was about half that collected for the liquid phase.

2. Renaturation: and slowly adding the renaturation solution into the evaporated sample solution until the pH value of the sample solution is 7.0-7.2. The renaturation solution is 20mmol PB buffer solution (pH8.0), 20mmol DTT.

3. And (3) dialysis: the sample was dialyzed against 100-fold volume of 20mmol PB buffer (pH7.0-7.2) by changing the solution every 12 hours for 24 hours to remove the residual acetonitrile.

3. Protein content determination

And (3) measuring the protein content and calculating the recovery rate of the sample before and after purification.

Protein content measurement method protein content was measured according to appendix VII M of the three parts of pharmacopoeia of the people's republic of China, 2010 edition, fifth method Coomassie Brilliant blue method (Bradford method).

The sample concentration before this purification was 2.68mg/ml, the volume before purification was 350ml, the concentration after purification was 15.1mg/ml, the volume was 53ml, and the recovery was 85.32%.

4. SDS-polyacrylamide gel electrophoresis detection of purified sample

The measurement was carried out by SDS-polyacrylamide gel electrophoresis according to VF electrophoresis method, which is an appendix of the second part of the pharmacopoeia of the people's republic of China, 2010 edition. The concentration of the concentrated gel is 5 percent, the concentration of the separation gel is 12 percent, electrophoresis is carried out by adopting constant current 25V, electrophoresis is carried out for 1.5 hours, dyeing is carried out for 30 minutes, decoloring is carried out for 2 hours, photographing records are carried out, and the result is shown in figure 2, and the result shows that the purification effect is good.

5. Heat source detection analysis

The detection method of the endotoxin content comprises the following steps: the limulus reagent method is based on the three-part appendix of the pharmacopoeia of the people's republic of China (2010 edition)

XII E "bacterial endotoxin test (gel Limit test)".

Sample treatment: the sample before purification (concentration of 2.68mg/mL) and the sample after purification (concentration of 15.1mg/mL) are respectively diluted to the concentration of 10 mug/mL, 2ng/mL and 1ng/mL by pyrogen-free water, and the diluted samples are subjected to pyrogen detection analysis and comparison. The sensitivity of the limulus reagent selected for the analysis was 0.06EU/ml, and the results are shown in Table 1.

TABLE 1

Numbering	Concentration of sample	Sample before purification	Purified sample
				1	10μg/ml	Fail to be qualified	Qualified
2	2ng/ml	Fail to be qualified	Qualified
				3	1ng/ml	Qualified	Qualified

The results show that even if diluted more than one thousand times, the unpurified sample still contains a large amount of heat source and cannot meet the requirement of being used as an injection, and the purified sample can meet the requirement. It can be seen that the method for removing heat source described in this patent is effective. And the concentration of the sample diluted to 1ng/mL is too low to exceed the detection threshold value, so that the heat source cannot be detected.

6. Cell activity identification of purified sample

A blood vessel forming experiment is carried out by using Human Umbilical Vein Endothelial Cells (HUVEC), the influence of the treated HUVEC Cells on the formation of blood vessels on matrigel after VEGF and IL18 (purified by the method) is detected by using the lumen forming experiment, and the influence of VEGF medicaments on the formation of blood vessels on the matrigel after the HUVEC Cells are subjected to the treatment of the VEGF and IL18 is verified.

1) The HUVEC cell suspension was seeded in 6-well cell culture plates (20W cells/well) at 37 ℃ with 5% CO₂Culturing in an incubator.

2) The cells were treated with VEGF for 6h at the following concentrations in each group, and then with recombinant IL-18 at a concentration of 100ng/ml for 16h, grouped as follows:

blank control group (treatment with neither VEGF nor recombinant IL-18)

VEGF (12.5ng/ml) group

VEGF (25ng/ml) group

VEGF (50ng/ml) group

VEGF (100ng/ml) group

Recombinant IL-18+ VEGF (12.5ng/ml) panel

Recombinant IL-18+ VEGF (25ng/ml) panel

Recombinant IL-18+ VEGF (50ng/ml) group

Recombinant IL-18+ VEGF (100ng/ml) group

3) And observing the formation of blood vessels

A. After the medicine is treated, photographing and recording, digesting and centrifugally collecting cells;

B. spreading 50ul of culture medium with 5 cells per well into the well plate with matrix glue;

C. observing the formation of blood vessels after HUVEC cells are paved for 4 hours respectively, and photographing and recording;

D. the blood vessel formation picture is subjected to measurement analysis.

As can be seen from FIG. 3, the number of tubes in the HUVEC cell experiment treated with VEGF alone was greater than that in the HUVEC cell experiment group treated with both VEGF and IL18, thus demonstrating that the purified IL18 of the present invention has its corresponding biological activity and is not affected. In addition, earlier experiments show that the recombinant IL-18 sample before purification has adverse effect on cells due to heat sources.

Comparative example 1

1. Preparative liquid phase purification of recombinant IL-18

And purifying the biological product recombinant IL-18 by using a preparation solution to remove endotoxin, wherein the sample is a sample disclosed in the patent number: 201510455749.X, collecting a liquid sample from the prepared gel chromatography column of recombinant IL-18;

the chromatographic column medium is C18, the specification is 21.2mm multiplied by 250mm, and the grain diameter is 7 mu m

Mobile phase A: water without heat source

Mobile phase B: methanol

Gradient elution procedure

Time (min)	Flow rate (ml/min)	A％	B％
				0	10	100	0
30	10	30	70

The chromatogram is shown in FIG. 4, and the result shows that recombinant IL-18 does not have any effect on the chromatographic column, and a flow-through peak is formed at 1.537 min.

2. Heat source detection analysis of purified samples using the method described in example 1

Numbering	Concentration of sample	Sample before purification	Purified sample
				1	10μg/ml	Fail to be qualified	Fail to be qualified
2	2ng/ml	Fail to be qualified	Fail to be qualified
				3	1ng/ml	Qualified	Qualified

The results show that the sample was not purified, and the reason why dilution to 1ng/ml was acceptable was that the pyrogen substance could not be detected due to too low a concentration.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements should also be considered as the protection scope of the present invention.

Claims

1. The method for removing the pyrogen in the recombinant IL-18 is characterized in that the recombinant IL-18 is purified by adopting a high performance liquid chromatography method to obtain the recombinant IL-18 with the pyrogen removed, and the parameters comprise:

the mobile phase A is an aqueous solution containing 0.05 vol% -0.2 vol% of trifluoroacetic acid,

the mobile phase B is acetonitrile solution containing 0.05 vol% -0.2 vol% of trifluoroacetic acid;

the elution procedure included: the volume fraction of the mobile phase A is from 100% to 30% in 0-55 min;

the flow rate of the mobile phase is 5-30 mL/min.

2. The method of claim 1,

the mobile phase A is an aqueous solution containing 0.1 vol% of trifluoroacetic acid,

the mobile phase B is acetonitrile solution containing 0.1 vol% of trifluoroacetic acid;

the flow rate of the mobile phase was 10 mL/min.

3. The method according to claim 1, wherein the detection wavelength of the high performance liquid chromatography is 280 nm.

4. The method of claim 1, wherein the column temperature of the chromatography column is 25 ℃; the specification of the chromatographic column is 21.2mm multiplied by 250mm, 7 μm.

5. The method of claim 4, wherein the recombinant IL-18 is loaded at a volume of 100-500 mL and a concentration of 1-10 mg/mL.

6. The method according to any one of claims 1 to 5, wherein the peak-off time of the sample peak of the recombinant IL-18 is 15.0min to 15.5 min.

7. The method according to any one of claims 1 to 5, further comprising the steps of renaturation and removal of acetonitrile after the purification by high performance liquid chromatography.

8. The method of claim 7, wherein the steps of renaturing and removing acetonitrile comprise: carrying out rotary evaporation at the temperature of 32 ℃, the pressure of-100 kPa and the rotating speed of 35-45rpm for 20-30 min until 1/2 liquid remains; adding a renaturation solution until the pH value is 7.0-7.2, and dialyzing by adopting a 20mmol PB buffer solution with the volume of 100 times that of the renaturation solution; the renaturation solution is 20mmol PB buffer solution containing 20mmol DTT, and the pH value is 8.0.

9. The depyrogenated recombinant IL-18 prepared by the method of any one of claims 1 to 9.

10. Use of depyrogenated recombinant IL-18 according to claim 9 for the preparation of a formulation for the treatment of DR, AMD and/or tumors.