High-load benzamidine affinity filler and preparation method thereof

Abstract

The invention discloses a high-load benzamidine affinity filler and a preparation method thereof, and the benzamidine affinity filler belongs to a high polymer material and can be used as an affinity chromatography medium. The benzamidine affinity filler obtained by the invention has good performance of high loading capacity, the loading capacity of the benzamidine affinity filler to trypsin can reach 61.44 mg / mL at most, and the benzamidine affinity filler is beneficial to separation and purification of serine protease substances.

Description

Technical Field

[0001] The invention relates to an affinity filler and a preparation method thereof, in particular to a benzamidine affinity filler and a preparation method thereof, belonging to the technical field of polymer materials. Background Art

[0002] Benzamidine affinity fillers are broad-spectrum inhibitors of serine proteases and are widely used to purify serine proteases such as trypsin, thrombin, and urokinase. However, the loading capacity of existing benzamidine affinity fillers does not exceed 35 mg trypsin / mL. The low loading capacity seriously limits the application efficiency and economy of benzamidine affinity fillers in large-scale bioseparation processes. Summary of the invention

[0003] Purpose of the invention: The purpose of the present invention is to provide a high-load benzamidine affinity filler and a preparation method thereof.

[0004] Technical solution: The benzamidine affinity filler of the present invention has the following general structural formula:

[0005]

[0006] in: For the base ball; is a single carbon atom or a spacer arm.

[0007] The base ball may be a commonly used matrix in the art, such as agarose. Generally, the longer the length of the spacer arm, the more conducive it is to bind to serine protease substances with a larger molecular weight; the shorter the length of the spacer arm or the absence of the spacer arm, the more conducive it is to bind to serine protease substances with a smaller molecular weight.

[0008] Preferably, the spacer arm is or polymers.

[0009] Preferably, the polymer is cellulose, chitosan or dextran.

[0010] Preferably, the base ball has a pure water flow rate of >800 cm / h at a pressure of 3 bar. Selecting a base ball with good pressure resistance is more conducive to the filler obtaining properties such as strong stability, high loading capacity, and a large number of recycling times. Further, the base ball has a pure water flow rate of >1500 cm / h at a pressure of 3 bar. Further, the base ball has a pure water flow rate of >2400 cm / h at a pressure of 3 bar.

[0011] Preferably, the base sphere is an agarose base sphere.

[0012] The present invention also provides a preparation method of the benzamidine affinity filler. The surface of the base ball contains hydroxyl groups. The preparation method comprises: oxidizing the hydroxyl groups to aldehyde groups and then bonding the benzamidine functional groups to obtain the benzamidine affinity filler; or grafting a spacer arm on the hydroxyl groups and then bonding the benzamidine functional groups to obtain the benzamidine affinity filler.

[0013] Preferably, the bonding of the benzamidine functional group is achieved by adding p-aminobenzoic acid and sodium cyanoborohydride.

[0014] Preferably, the base ball is cleaned before preparation. Cleaning helps sterilize, remove microbial contamination, and improve synthesis yield.

[0015] Preferably, the cleaning uses pure water, 2M guanidine hydrochloride or 10mM PB+30mM NaCl.

[0016] Preferably, the benzamidine affinity filler is end-capped.

[0017] Preferably, sodium borohydride is used for end-capping.

[0018] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: the benzamidine affinity filler provided by the present invention has good performance of high loading capacity, and the loading capacity of trypsin can reach up to 61.44 mg / mL, which is beneficial to the separation and purification of serine protease substances. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the chromatogram of the trypsin purified by the benzamidine affinity filler obtained in Example 1;

[0020] Figure 2 This is the chromatogram of the trypsin purified by the benzamidine affinity filler obtained in Example 2;

[0021] Figure 3 The chromatogram of the trypsin purified by the benzamidine affinity filler obtained in Example 3;

[0022] Figure 4 The chromatogram of the trypsin purified by the benzamidine affinity filler obtained in Example 4;

[0023] Figure 5 The chromatogram of enterokinase purified by benzamidine affinity filler obtained in Example 1;

[0024] Figure 6 This is the chromatogram of thrombin purified by benzamidine affinity filler obtained in Example 1;

[0025] Figure 7 Standard curve of trypsin used for media performance testing. DETAILED DESCRIPTION

[0026] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0027] The embodiment uses agarose microspheres produced by Suzhou Saifen Technology Co., Ltd. as base spheres, with a particle size of 90 μm and hydroxyl groups on the surface.

[0028] Example 1: A high-load benzamidine affinity filler and preparation method thereof

[0029] Step 1: Weigh 1000mL of base ball with a measuring cylinder and wash it with 10 times the volume of pure water. Let it settle in the measuring cylinder for more than 24 hours to measure the volume. And adjust the volume of the filler to 50% (V / V) with pure water, and mix well to obtain a homogenate. Transfer the homogenate to a 5L reactor and add 21.389g of sodium periodate / 1L of homogenate. Stir the reaction at 60rpm and room temperature (20-30℃) for 1.5 hours.

[0030] After the reaction is completed, use a 5000mL sand core funnel to filter and wash, and then wash with 5 times the volume of pure water. Wash with 3 times the volume of 2M guanidine hydrochloride, and then stir evenly with 1L 2M guanidine hydrochloride and the filler in a 5L reactor for 2h, with a stirring speed of 1500rpm. After completion, wash with 10 times the volume of pure water. Wash with 3 times the volume of 10mM PB+30mMNaCl, pH9.0. Add 1L 10mM PB+30mM NaCl, pH9.0 to the filler, stir evenly, transfer to a 2L measuring cylinder and settle for more than 24h, and filter to obtain a clean and sterile filler intermediate containing aldehyde groups on the surface.

[0031] Step 2: The intermediate was transferred to a 2L reactor, 800mL of PBS solution was added, and mechanical stirring was performed at 150rpm for 10 minutes. After being dispersed evenly, 7.2g of p-aminobenzoic acid and 4.0g of NaBH3CN were added, and the mixture was stirred at 150rpm and room temperature for 16h to obtain the benzamidine affinity filler.

[0032] End-capping. The obtained benzamidine affinity filler was transferred to a 2L reactor with water, and 2g NaBH4 was slowly added to the reactor while stirring. The reaction was carried out at 150rpm and room temperature for 1h. After the reaction was completed, it was first filtered with a Buchner funnel, and then washed with 2L pure water until neutral, and then drained to complete the end-capping of the filler.

[0033] The reaction process is as follows (arrows correspond to steps):

[0034]

[0035] Example 2: A high-load benzamidine affinity filler and preparation method thereof

[0036] Step 1: Weigh 1000mL of base ball with a measuring cylinder, add 144g 50wt% NaOH and 1.28g NaBH4, wait until the base ball is evenly dispersed, slowly add 100mL of epichlorohydrin with a syringe pump, stir at room temperature for 2h, stirring speed: 150rpm. After the reaction is completed, filter with a Buchner funnel, wash with 2L ethanol and 2L pure water until neutral for use.

[0037] Step 2: Weigh 1000 mL of the intermediate with a measuring cylinder, add 1000 mL of 1 M NaOH solution, stir at 40°C for 2 h, stirring speed: 150 rpm. After the reaction is completed, filter with a Buchner funnel, wash with 2 L of pure water until neutral and set aside.

[0038] Step 3: Treat the material obtained in the previous step with the method of step 1 in example 1 (i.e. replace the base ball described in step 1 in example 1 with the material obtained in step 2 of this example. Subsequent descriptions are similar).

[0039] Step 4: Treat the material obtained in the previous step using the method of step 2 of Example 1.

[0040] End-capping: The material obtained in the previous step was treated with the end-capping method of Example 1.

[0041] The reaction process is as follows (arrows correspond to steps):

[0042]

[0043] Example 3: A high-load benzamidine affinity filler and its preparation method

[0044] Step 1: Weigh 1000mL of base ball with a measuring cylinder, settle 1000mL of base ball, filter, weigh, add water, (total volume of base ball and water 1200mL) and transfer to a 2000mL three-necked round-bottom flask. Set up the experimental device, place the reaction bottle in a 50℃ water bath, adjust the speed to 150rpm, and disperse the filler evenly. Add 2.57g of anhydrous sodium sulfate, 0.08g of sodium borohydride and 6.71g of 50wt.% sodium hydroxide to the bottle. Add 15.06g of 1,4-dioxane to the reaction bottle and stir for 5min to mix evenly. When the internal temperature of the reaction bottle reaches 50℃, use a syringe (pump) device (model: HSW syringe 20mL range, syringe needle gauge 18) to add allyl glycidyl ether (PMA013, 46.10mL, rate: 7.68mL / h), and drip continuously and evenly for 6h. After the addition was completed, the syringe feed pump was turned off, and the reaction was continued overnight at a rotation speed of 150 rpm and a reaction temperature of 50° C. for 16 hours.

[0045] The reaction flask should be allowed to gradually cool to room temperature, and 60 wt% glacial acetic acid should be gradually added to the reaction flask while stirring until the pH of the solution is about 7. After adjusting the pH, the filler is filtered, washed with ethanol 3 times, and washed with pure water 3 times until the pH of the filtrate is neutral for use.

[0046] Step 2: Transfer the above substrate to a 5L reactor, dilute it to 3L with pure water, add 166g of sodium acetate, stir mechanically at 150rpm for 10 minutes at room temperature to make it evenly dispersed, slowly add liquid bromine to the system until the solution turns dark yellow and does not change color for 10 minutes. Stir mechanically at 150rpm at room temperature for 0.5h.

[0047] After the reaction is completed, 60% sodium formate solution is added until the reaction solution turns milky white, and then washed with pure water three times until the pH of the filtrate is neutral for later use.

[0048] Step 3: Weigh 1000 mL of the intermediate using a measuring cylinder, add 1000 mL of 1 M NaOH solution, and stir the mixture at 40° C. for 2 h. The stirring speed is 150 rpm.

[0049] After the reaction is completed, filter with a Buchner funnel and wash with 2L of pure water until neutral for later use.

[0050] Step 4: Treat the material obtained in the previous step using the method of step 1 in Example 1.

[0051] Step 5: Treat the material obtained in the previous step using the method of step 2 of embodiment 1.

[0052] End-capping: The material obtained in the previous step was treated with the end-capping method of Example 1.

[0053] The reaction process is as follows (arrows correspond to steps):

[0054]

[0055] Example 4: A high-load benzamidine affinity filler and preparation method thereof

[0056] Step 1: Same as step 1 of Example 3.

[0057] Step 2: Same as step 2 of Example 3.

[0058] Step 3: Transfer the above substrate to a 5L reactor, add 2L of water, add 219g NaCl, 20g Na2CO3, and 2g NaBH4 in sequence, and react with mechanical stirring at 150rpm at room temperature for 10min. After being evenly dispersed, add 50g 6-aminohexanoic acid, and react with mechanical stirring at 150rpm at room temperature overnight.

[0059] After the reaction is completed, ethanol is added and the mixture is washed three times with pure water until the pH of the filtrate is neutral for later use.

[0060] Step 4: The above substrate was transferred to a 5L reactor, 1L of water and 1L of pyridine were added, and the mixture was mechanically stirred at 150rpm for 10min at room temperature. After being evenly dispersed, 50g of EDCI and 50g of p-aminobenzoic acid were added, and the mixture was mechanically stirred at 150rpm for overnight at room temperature.

[0061] After the reaction is completed, ethanol is added and the mixture is washed three times with pure water until the pH of the filtrate is neutral for later use.

[0062] End-capping: The material obtained in the previous step was treated with the end-capping method of Example 3.

[0063] The reaction process is as follows (arrows correspond to steps):

[0064]

[0065] Performance Testing

[0066] The loading capacity of the benzamidine affinity fillers prepared in Examples 1 to 4 was tested using three serine proteases (trypsin, enterokinase, and thrombin).

[0067] The standard curves of the three serine proteases were diluted in half with acetonitrile, and the concentrations of the standard curves were: 0, 0.15625, 0.3125, 0.625, 1.25, 2.5, 5, and 10 mmol / mL. The test equipment was HPLC Agilent 1260 (YZ-LC-01).

[0068] Standard curve test method: Chromatographic column: Zenix-C SEC-300 (4.6x 300mm). Column temperature: 25℃. Injection volume: 5μL. Mobile phase: 150mM PB, PH 7.0; Flow rate: 0.350mL / min. Detection wavelength 280nm.

[0069] Trypsin Figure 7 shown.

[0070] The benzamidine affinity filler prepared in Examples 1 to 4 was filled into a 1 mL chromatographic column and tested using the following purification method:

[0071]

[0072] The results are shown in Table 1 and Figures 1 to 6 (The black line indicates pH, the blue line indicates conductivity, and the red line indicates UV absorption.) The fillers obtained in Examples 1 to 4 have a high loading capacity, and the loading capacity for trypsin can reach up to 61.44 mg / mL, which is beneficial for the separation and purification of serine protease substances.

[0073] Table 1 Loading of fillers in Examples 1 to 4

[0074]

Claims

1. A high-load benzamidine affinity filler, characterized in that: The general structure is as follows: in: For the base ball; is a single carbon atom or a spacer arm.

2. The benzamidine affinity filler according to claim 1, characterized in that The spacer arm is or polymers.

3. The benzamidine affinity filler according to claim 2, characterized in that The polymer is cellulose, chitosan or dextran.

4. The benzamidine affinity filler according to claim 1, characterized in that The base ball is an agarose base ball.

5. A method for preparing the benzamidine affinity filler as claimed in claim 1, characterized in that: The surface of the base ball contains hydroxyl groups, and the preparation method is: oxidizing the hydroxyl groups to aldehyde groups, bonding with benzamidine functional groups to obtain the benzamidine affinity filler; or grafting a spacer arm on the hydroxyl groups, and then bonding with benzamidine functional groups to obtain the benzamidine affinity filler.

6. The preparation method according to claim 5, characterized in that: The bonding of the benzamidine functional group is achieved by adding p-aminobenzoic acid and sodium cyanoborohydride.

7. The preparation method according to claim 5, characterized in that: The base ball is cleaned before preparation.

8. The preparation method according to claim 7, characterized in that: The washing was performed using pure water, 2M guanidine hydrochloride or 10mM Pb+30mM NaCl.

9. The preparation method according to claim 5, characterized in that: The benzamidine affinity filler is end-capped.

10. The preparation method according to claim 9, characterized in that: Sodium borohydride was used for end-capping.

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