CN1314447C - A kind of preparation method of the medicine with protease inhibitory effect - Google Patents

Abstract

The invention relates to a preparation method of a medicine with protease inhibition effect, which comprises the following steps: subjecting an aqueous solution containing an alpha 1-protease inhibitor to virus inactivation treatment; adsorbing and eluting with ion exchange resin. The preparation method of the invention effectively realizes high yield, ensures high purity and high activity of the product, simplifies the process and reduces the cost.

Description

A kind of preparation method of the medicine with protease inhibitory effect

【Technical field】

The invention belongs to the field of protein purification, and in particular relates to a preparation method of a drug capable of inhibiting protease.

【Background technique】

α1-protease inhibitor (also known as α1-antitrypsin, alpha-1 Antitrypsin, α1-PI for short) is a glycoprotein with a molecular weight of about 53,000 Daltons, which contains an average of 1.5 grams per liter of plasma. The protein mainly inhibits the decomposition of elastase and other serine proteases in the body. When the concentration of active α1-PI in the body is significantly lower than the concentration of serine proteases, as occurs in people with a genetic defect of α1-PI, the lung tissue will be damaged by serine proteases and cause chronic diseases, such as pneumothorax swell. Patients with emphysema can use α1-PI as a long-term replacement therapy. However, the supply of α1-PI products on the market is in short supply. Therefore, the development of a process that can industrially produce high-quality α1-PI can meet the market demand.

In the past there are various published methods that can be used for the industrial production of drugs with protease inhibitory effect. Glaser et al. (Anal.Biochem.1982,124:364-371) used the ammonium sulfate precipitation method to obtain α1-PI with a purity of about 70% in one step from Cohn's fraction IV-1 of human plasma, and then through anion exchange Chromatography (DEAE-cellulose) refined, but the yield is low, only about 40%.

Coan et al. (US Pat. No. 4,379,087; Vox Sang. 1985, 48:333-342) purified α1-PI from Cohn's Fraction IV-1 of human plasma using polyethylene glycol precipitation and anion exchange chromatography, The yield is about 50%, and the purity is only about 60%. This method is the prototype of the α1-PI product Prolastin( ^R ) approved by the U.S. Bayer Corporation (Bayer Corporation) in 1988, and the active α1-PI in this preparation only accounts for ≥ 35% of the total protein (see its product description, 14- 7601-001 (Rev. Jan. 2002)).

Burnouf et al. (Vox Sang.1987, 52:291-297) used anion exchange (DEAE Sepharose CL6B FF) and gel filtration (Sephacryl S-200) chromatography from human plasma supernatant A (equivalent to Cohn's fraction α1-PI with a purity of 80-90% was isolated in II+III), and the yield was 65-75%.

None of the above-mentioned methods for purifying α1-PI can guarantee the high purity and high activity of the product while achieving high yield. Moreover, the process of the above-mentioned method is relatively complicated and the cost is relatively high. In practice, proteins whose molecular size and isoelectric point are close to α1-PI, such as albumin, which is the most abundant in plasma, make it difficult to separate high-purity α1-PI; and combining inactive α1-PI with active Separation of active α1-PI is a greater challenge. After repeated attempts on ion exchange chromatography conditions, the present invention integrates a simple two-step chromatography method into a preparation method for high-purity and high-activity α1-PI that can be used as a therapeutic drug.

Existing α1-PI detection techniques usually use specific antibodies to quantify α1-PI, including radioimmunodiffusion, rocket immunoelectrophoresis and ELISA. However, the existing technologies using specific antibodies cannot distinguish the active α1-PI from the inactive α1-PI, and the cost of these existing technologies is relatively high.

【Content of invention】

[Technical problem to be solved]

In order to achieve high yield, ensure the high purity and high activity of the product, simplify the process and reduce the cost at the same time, the present invention provides a preparation method of a drug capable of inhibiting protease. situation, the present invention provides a simple and highly sensitive detection method for protease-inhibiting drugs.

[Technical solutions]

In order to achieve high yield, ensure the high purity and high activity of the product, simplify the process and reduce the cost at the same time, the present invention provides a preparation method of a drug capable of inhibiting protease. The method includes the following steps: virus inactivation treatment containing α1- PI aqueous solution, ion exchange resin adsorption and elution.

The aqueous solution containing α1-PI is subjected to virus inactivation treatment. Firstly, the anion exchange resin is used to absorb α1-PI, and the active α1-PI is selectively eluted from the anion exchange resin; and then the cation exchange resin is obtained. Drugs that inhibit the action of proteases. The aqueous solution containing α1-PI includes: human plasma or plasma components, microorganism or cell culture fluid containing genetically recombined α1-PI, and animal body fluids containing α1-PI transgene including milk and plasma. Normal human plasma contains 0.83-4.0 grams of α1-PI per liter, and a considerable part of it is enriched in certain plasma components during the industrial production of plasma proteins. The research by Wright et al. found that in the milk of transgenic sheep, the content of α1-PI can be as high as 60 grams per liter (Biotechnology, 1991, 9: 830-834). In microorganisms such as Escherichia coli (Casolaro et al, 1987, J.Appl.Physiol., 63:2015-2023) and yeast (Hubbard et al, 1991, Proc.Natl.Acad.Sci., 86:680-684), The application of gene recombination technology can produce a large amount of α1-PI. A preferred α1-PI-containing material is Cohn's Fraction IV of human plasma, as it is a waste product of routine industrial plasma protein purification using low temperature ethanol precipitation (Cohn's method). To increase the safety of the α1-PI preparation, the solubilized Cohn's Fraction IV can be combined to remove/inactivate the virus by one or more methods before processing by anion exchange chromatography. Virus removal/inactivation methods include: pasteurization (pasteurization), dry heat, low pH incubation, organic solvent/detergent (S/D) treatment, octanoic acid or caprylate treatment, Ultrafiltration virus removal, nanofiltration, etc.

There are more than 100 different molecular variants (variants) of α1-PI in human plasma, and the isoelectric point of several variants with high content is between 4.3-4.6. When the pH of the solution is higher than its isoelectric point, α1-PI is negatively charged and can bind to the anion exchange resin. The more the pH of the solution is higher than its isoelectric point, the greater the binding force of α1-PI to the anion exchange resin. The anions in the solution compete with α1-PI for binding sites on the anion exchange resin, therefore, low ionic strength is favorable for α1-PI to be adsorbed to the anion exchange chromatography column. Adjust the pH of the aqueous solution containing α1-PI treated by virus removal/inactivation to ≥4.8 and ≤12.0, and adjust its ionic strength to ≥0.1mS/cm and ≤10.0mS/cm, so that it flows through the anion Exchange the column. Useful anion exchange resins include strong and weak anion exchange resins, eg, Q Sepharose Fast Flow and DEAESepharose Fast Flow from Amersham. The target protein α1-PI is bound to the anion exchange resin, and part of the impurity protein can be removed without being adsorbed. Increase the salt concentration of the washing liquid of the chromatography column or adjust the pH of the washing liquid to further elute some impurity proteins, and continue to increase the salt concentration of the washing liquid or adjust the pH of the washing liquid to selectively elute the adsorbed α1-PI . The eluate from the anion exchange chromatography column containing α1-PI was subsequently purified by cation exchange chromatography. Adjust the pH of the dialysis-treated anion-exchange chromatography column eluate containing α1-PI to ≥ 4.8, and ≤ 13.0, and adjust its ionic strength to ≥ 0.1mS/cm, and ≤ 10.0mS/cm, so that it flows through a cation exchange chromatography column. Useful cation exchange resins include strong cation exchange resins and weak cation exchange resins, for example, Amersham's SP Sepharose Fast Flow and CM Sepharose FastFlow. The target protein α1-PI flows through the cation exchange resin, and most of the remaining impurity proteins are adsorbed and removed. The effluent of the cation exchange chromatography column is collected, treated by ultrafiltration or dialysis, sterilized and filtered after formulating, then bottled, freeze-dried and sealed for storage, as a purified drug with the effect of inhibiting protease.

The above-mentioned method of the same concept can be changed as follows: the preparation method of a drug that inhibits protease action is characterized in that: after the virus inactivation treatment is carried out in the aqueous solution containing α1-PI, at first the α1-PI is made to flow through the cation exchange resin, Then it is adsorbed by anion exchange resin, and the active α1-PI is selectively eluted from the anion exchange resin to obtain a drug with the effect of inhibiting protease. The above two methods have basically the same effect.

The virus inactivation treatment of the aqueous solution containing α1-PI is completed by adding chemical reagents with virus inactivation effect for incubation. Chemical reagents with virus inactivation effects are added to the aqueous solution containing α1-PI, and incubated between 0°C and 50°C for more than one hour. Corresponding stabilizers can be added before virus inactivation treatment.

Chemical agents with virus inactivation are organic solvents and detergents (S/D). The detergent used therein is non-ionic. Chemical agents with virus inactivation effects also include: octanoic acid and caprylic acid salt, etc. Stabilizers commonly used in the virus inactivation process are 2%-50% sucrose and 0.001M-0.50M sodium citrate.

The most commonly used organic solvent is tributyl phosphate, and the most commonly used detergent is TritonX-100. In addition, Tween 80 (Tween 80) is also a commonly used detergent. The final concentration of tributyl phosphate added is between 0.01% and 10.0%, while the final concentration of Triton X-100 added is between 0.01% and 10.0%.

The detection method of the protease-inhibiting drug is to use serine protease to react with α1-PI to cause the change of light absorption of corresponding wavelength, so as to quantitatively measure the activity of α1-PI. The reaction between the serine protease and the substrate causes a change in the light absorption of the corresponding wavelength, and α1-PI changes the light absorption change of the wavelength by inhibiting the serine protease. The specific method includes the following steps: 1) using the serine protease to react with the α1-protease inhibitor; 2 ) Determining the change of light absorption caused by the corresponding wavelength; 3) Measuring the activity of the quantitative α1-protease inhibitor according to the change of light absorption.

The final concentration of the serine protease added was between 0.1 ppm and 100 ppm, and five times the weight of the enzyme corresponding substrate was added simultaneously. The activity of α1-PI can be quantitatively determined by measuring the change in light absorption at this wavelength over a certain period of time. The method for detecting α1-PI activity using serine protease is called, serine protease inhibition assay. Each unit of serine protease inhibition was defined as the activity that changed the light absorption of the corresponding wavelength by 1 unit within one minute compared to the control.

The most commonly used serine proteases are trypsin or elastase. Other useful serine proteases include: chymotrypsin, thrombin, plasmin and collagenase, among others. High-purity trypsin is relatively easy to obtain, so it is most commonly used for the determination of α1-PI activity. The method of detecting α1-PI activity using trypsin is called trypsin inhibition assay. In the absence of inhibitors, the degradation products of trypsin increase the light absorption of the corresponding wavelength per minute. Each unit of trypsin inhibition was defined as the activity that reduced the light absorption of the corresponding wavelength by 1 unit within one minute compared to the control. The common substrate of trypsin is ethyl benzoyl arginate, and the common light wavelength for the determination of trypsin inhibition is 253nm.

[beneficial effect]

The preparation method provided by the invention effectively realizes high yield, ensures high purity and high activity of the product, simultaneously simplifies the process and reduces the cost; the detection method is simple and easy, and has high sensitivity.

Apply the method provided by the invention and use anion exchange (DEAE Sepharose CL6BFF) and gel filtration (Sephacryl S-200) to purify α1-PI, detect with conventional ELISA method at last, the result shows that the method provided by the invention effectively realizes high yield The rate ensures the high purity and high activity of the product.

Existing α1-PI detection techniques usually use specific antibodies to quantify α1-PI, including radioimmunodiffusion, rocket immunoelectrophoresis and ELISA. However, the existing technologies using specific antibodies cannot distinguish the active α1-PI from the inactive α1-PI, and the cost of these existing technologies is relatively high. The detection method provided by the present invention uses simple serine protease and the reaction of corresponding substrate to quantitatively measure the active drug with the effect of inhibiting protease, eliminates the inactive α1-PI, and improves the accuracy; at the same time, the operation steps are simple and the cost is corresponding Reduced.

【Detailed ways】

The present invention designs following reaction condition altogether:

Table 1. Reaction conditions anion exchange resin cation exchange resin pH 5.0 6.5 8.0 9.5 11.0 12.0 4.8 5.4 7.0 9.6 11.0 12.6 Ionic strength (mS/cm) 0.5 2.5 4.5 6.5 8.5 10.0 0.5 1.5 3.0 6.0 8.0 10.0

The following examples are intended to further illustrate the present invention, but not to limit the invention to the exemplified examples.

Example 1

After Cohn's Component IV was dissolved with 20 times the weight of purified water, 37% sucrose and 0.38M sodium citrate were added as stabilizers for virus inactivation treatment. Then add tributyl phosphate at a final concentration of 0.3% and Triton X-100 at a final concentration of 1.0% and incubate at 30°C for 4 hours. After virus inactivation, adjust the pH to 6.5, reduce the ionic strength to 2.5mS/cm with water, and add to the DEAE Sepharose Fast Flow chromatography column pre-balanced at pH 6.5. The target protein α1-PI was selectively eluted with 20mM sodium dihydrogen phosphate and 100mM sodium chloride solution. Increasing the concentration of sodium chloride can elute strongly adsorbed foreign proteins, including ceruloplasmin, from the ion-exchange chromatography column. DEAE Sepharose Fast Flow resin can be reused after being sterilized by 0.5M sodium hydroxide.

The eluate containing α1-PI was passed through a 10kD ultrafiltration membrane bag (Pall Corporation) to remove salt. First reduce the ionic strength to 2.5mS/cm with water, then perform solution exchange with 5mM sodium citrate (pH 5.4). The treated solution containing α1-PI is added to the pH 5.4 pre-equilibrated CM Sepharose Fast Flow chromatography column, and the active α1-PI is purified by flowing through the ion exchange resin. In this example, 45.3 mg of α1-PI with a purity of 95.8% was purified from 4.18 g of fraction IV. The yield of active α1-PI was as high as 63.4%.

In this example, trypsin was used to quantitatively measure the activity of α1-PI. In the absence of inhibitors, 33.3 ppm of trypsin degrades 166.7 ppm of benzoyl arginine ethyl ester per minute to produce products that increase the light absorption at 253 nm by 0.1-0.2 units. Trypsin inhibition per unit was defined as the activity that reduced light absorption at 253 nm by 1 unit in one minute compared to the control. Table 2 is the experimental data of this example.

Table 2. α1-PI activity Total protein (mg) α1-PI activity (yield) Component IV 1695.2 612.0U (100%) DEAE chromatography 108.6 432.6U (70.7%) CM chromatography 45.3 388.1U (63.4%)

Example 2

The dissolved Cohn’s fraction IV was adjusted to pH 5.4 with 1M hydrochloric acid, and the ionic strength was adjusted to 1.5mS/cm with water, and then added to a CM Sepharose Fast Flow chromatography column pre-equilibrated with 5mM sodium citrate (pH 5.4). Collect the effluent from the ion exchange resin, adjust the pH to 6.5, adjust the ionic strength to 2.5mS/cm, and add it to a DEAE Sepharose Fast Flow chromatography column pre-balanced with 20mM sodium dihydrogen phosphate solution (pH 6.5). Active α1-PI was selectively eluted using 20 mM sodium dihydrogen phosphate and 100 mM sodium chloride solution. In this example, 58.9 mg of α1-PI with a purity of 92.5% was purified from 5.34 g of fraction IV. The yield of active α1-PI was as high as 81.2%. Table 3 is the experimental data of this example.

Table 3. α1-PI activity Total protein (mg) α1-PI activity (yield) Component IV 1191.2 448.7U (100%) CM chromatography 255.3 408.8U (91.1%) DEAE chromatography 58.9 364.5U (81.2%)

All the other are identical with embodiment 1.

Claims (5)

1, a kind of preparation has the method for the medicine of Profilin enzyme effect, it is characterized in that the aqueous solution that will contain α 1-protease inhibitor is after viral inactivation treatment, through ion exchange resin absorption, eluting, purification α 1-protease inhibitor, wherein ion exchange resin absorption, eluting are:

By anion exchange resin absorption α 1-protease inhibitor, and from the anion exchange resin the activated α 1-of eluting protease inhibitor selectively, then through a cation exchange resin;

Or filter by cation exchange resin, then by an anion exchange resin absorption, and from the anion exchange resin the activated α 1-of eluting protease inhibitor selectively.

2, preparation method according to claim 1, the aqueous solution that wherein contains α 1-protease inhibitor is: people's blood plasma or plasma component, contain gene recombined alpha 1-protease inhibitor microorganism or cell culture fluid, contain the genetically modified animal body fluid of α 1-protease inhibitor and comprise CohnShi component I V or IV-1 in milk and blood plasma and the conventional industrialization plasma protein purge process.

3, preparation method according to claim 1 is characterized in that the aqueous solution that viral inactivation treatment contains α 1-protease inhibitor is to have the chemical reagent of viral deactivation to incubate to put by apparatus to finish.

4, preparation method according to claim 3, the chemical reagent that it is characterized in that having in this method the inactivation of virus effect is sad or caprylate.

5, the preparation method of medicine according to claim 3, the chemical reagent that it is characterized in that having in this method the inactivation of virus effect is organic solvent and detergent, and wherein organic solvent is a tributyl phosphate, and detergent is Triton X-100 or Tween 80.