Cyanogen bromide-activated agarose gel lyoprotectant, lyophilized powder and preparation and use thereof

Abstract

The present application relates to the technical field of bioengineering, and provides a cyanogen bromide activated agarose gel freeze-drying protective agent, a freeze-dried powder and a preparation and application thereof, the freeze-drying protective agent comprises: a solvent and first and second saccharide substances in the solvent; the first saccharide substance is selected from dextran; the second saccharide substance is selected from monosaccharide and / or disaccharide; and the ratio of the first saccharide substance to the second saccharide substance is (45-55) micromoles to (5-7) millimoles. The present application proposes to use the freeze-drying protective agent to realize the preparation of a freeze-dried powder of cyanogen bromide activated agarose gel by using a low-temperature vacuum freeze-drying method, and the method has the advantages of prolonging the storage period of cyanogen bromide activated agarose gel, facilitating the transportation of cyanogen bromide activated agarose gel and improving the quality of cyanogen bromide activated agarose gel.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of bioengineering technology, and particularly relates to a CNBr-activated agarose gel freeze-drying protective agent, a freeze-drying powder and preparation and application thereof. BACKGROUND

[0002] CNBr-activated agarose gel has a microspherical microstructure and is often used for separating proteins. It plays an important role in protein separation technology and has played a key role in many important scientific discoveries, such as helping to discover pesticides, being used for purifying a polyclonal antibody of yeast invertase sugar group, and being used for purifying related antibodies in an immune response of xenograft rejection.

[0003] However, CNBr-activated agarose gel, such as CNBr-activated Chromstar 4B as a commercial product, is generally stored in a toxic reagent acetone. During storage, it needs to be kept away from light and cooled to 2-8 DEG C. The transportation condition is harsh, so that it is limited to export to foreign countries. Moreover, CNBr-activated agarose gel can be degraded and the system can be yellow after long-term storage. The protective liquid acetone is a flammable and toxic item with moderate toxicity. Mild poisoning can irritate the eyes and upper respiratory mucosa. Severe poisoning can cause syncope, convulsions, and symptoms such as protein and red blood cells in urine. Long-term contact can cause harm to the human body.

[0004] In view of this, the present application is proposed. SUMMARY

[0005] The present application provides a CNBr-activated agarose gel freeze-drying protective agent, a freeze-drying powder and preparation and application thereof, to solve the defects caused by the harsh storage condition of CNBr-activated agarose gel in the prior art. The CNBr-activated agarose gel is obtained by activating agarose microspheres by a CNBr-ACN system. The microstate is still a microspherical structure, which can also be called CNBr-activated agarose gel microspheres.

[0006] Specifically, in a first aspect, the present application provides a CNBr-activated agarose gel freeze-drying protective agent, comprising: a solvent and first and second saccharide substances in the solvent.

[0007] The first saccharide substance is selected from dextran.

[0008] The second saccharide substance is selected from monosaccharides and / or disaccharides.

[0009] The ratio of the first saccharide substance to the second saccharide substance is (45-55) μmol:(5-7) mmol.

[0010] The research finds that the active group cyanate contained in cyanogen bromide activated agarose gel has harsh storage conditions. In order to avoid the use of toxic reagents such as acetone which is not conducive to the environment and human body, the present application first proposes to use the freeze-drying protective agent as described above to realize the method of low-temperature vacuum freeze-drying to prepare cyanogen bromide activated agarose gel into freeze-dried powder. The method has the advantages of prolonging the storage period of cyanogen bromide activated agarose gel, facilitating the transportation of cyanogen bromide activated agarose gel, and avoiding the deterioration of the quality of cyanogen bromide activated agarose gel.

[0011] In the freeze-drying protective agent, sugar substances are often used as important components to play a protective role. The main reason is to use the glass transition property of sugar substances, that is, in the freeze-drying process, sugar substances can form a glassy structure or produce hydrogen bonds. This structure or the original hydrogen bond force can stabilize the three-dimensional structure of proteins or other biological molecules, preventing them from denaturing or aggregating during the freeze-drying process.

[0012] Specifically in the present application, cyanogen bromide activated agarose gel microspheres are obtained by oil-water emulsification of agarose. Due to its low mechanical strength, the gel microspheres lose bound water and lose hydrogen bond force during freeze-drying, causing the collapse of the microsphere pores. According to the principle of similarity and the hydrogen bond replacement hypothesis, macromolecular sugars with similar structures can replace bound water and form a network structure inside the microsphere pores to provide support, thereby ensuring that the gel microspheres maintain their initial shape. Because the active group cyanate contained in cyanogen bromide activated agarose gel has high reactivity, it is difficult for general sugar substances and other high molecular compounds to protect cyanogen bromide activated agarose gel. The present application found in experiments that the second sugar substance can protect the active functional groups of cyanogen bromide activated agarose gel. By strictly controlling the ratio of the second sugar substance to dextran, cyanogen bromide activated agarose gel freeze-dried powder can be prepared, and the obtained cyanogen bromide activated agarose gel freeze-dried powder has rapid solubility recovery ability and can avoid the destruction of the original structure of agarose gel microspheres.

[0013] Compared with other types of monosaccharides or disaccharides, trehalose, glucose, lactose and galactose are more conducive to the shape maintenance and active group binding capacity maintenance of the cyanogen bromide activated agarose gel freeze-drying protective agent. Preferably, according to the cyanogen bromide activated agarose gel freeze-drying protective agent provided by the present application, the second sugar substance is selected from one or a combination of two or more of trehalose, glucose, lactose and galactose; the concentration of dextran in the freeze-drying protective agent is (45-55) μmol / 50 mL.

[0014] The CNBr-activated sepharose gel freeze-drying protective agent provided by the present application is an acidic solution with a pH value of 3; if the pH value is too low or too high, it is not conducive to the stable maintenance of the binding capacity of the CNBr-activated sepharose gel freeze-dried powder.

[0015] Preferably, the acidic solution is selected from an aqueous HCl solution or an aqueous citric acid solution.

[0016] In a second aspect, the present application also provides a preparation method of the CNBr-activated sepharose gel freeze-dried powder as described above, wherein the CNBr-activated sepharose gel freeze-drying protective agent as described above is mixed with CNBr-activated sepharose gel, and vacuum freeze-drying is performed to obtain the CNBr-activated sepharose gel freeze-dried powder.

[0017] The preparation method of the CNBr-activated sepharose gel freeze-dried powder provided by the present application has a mass-volume ratio of the CNBr-activated sepharose gel and the freeze-drying protective agent of about 1-3 g: 1-100 mL.

[0018] In the preparation method of the CNBr-activated sepharose gel freeze-dried powder provided by the present application, the pre-freezing temperature in the vacuum freeze-drying is preferably -100 to -60℃, considering that ultra-low temperature quick freezing can improve efficiency.

[0019] In the preparation method of the CNBr-activated sepharose gel freeze-dried powder provided by the present application, the drying time in the vacuum freeze-drying is 18-24 h.

[0020] In the preparation method of the CNBr-activated sepharose gel freeze-dried powder provided by the present application, too high vacuum degree can prolong the freeze-drying time and cause the product to be not powdery or have color change, and therefore, the vacuum degree in the vacuum freeze-drying is preferably -0.8 to -0.9 MPa.

[0021] In the preparation method of the CNBr-activated sepharose gel freeze-dried powder provided by the present application, the CNBr binding capacity of the CNBr-activated sepharose gel is 10-20 μmol / ml.

[0022] In a third aspect, the present application also provides the CNBr-activated sepharose gel freeze-dried powder prepared by the preparation method as described above, wherein the CNBr binding capacity of the CNBr-activated sepharose gel freeze-dried powder is 10-20 μmol / ml.

[0023] The CNBr binding capacity of the CNBr-activated sepharose gel freeze-dried powder does not change after the CNBr-activated sepharose gel freeze-dried powder is stored at 2-8℃ in the dark for 180 days;

[0024] After the CNBr-activated sepharose gel freeze-dried powder is swelled in an aqueous HCl solution, it is in a microspherical shape.

[0025] The cyanogen bromide-activated agarose gel freeze-dried powder provided by the application has a microsphere size of 45-165 μm after swelling in an HCL aqueous solution.

[0026] In a fourth aspect, the application further provides use of the cyanogen bromide-activated agarose gel freeze-dried powder as described above in at least one of the following (1)-(4):

[0027] (1) affinity chromatography;

[0028] (2) protein immobilization;

[0029] (3) drug carrier;

[0030] (4) antibody purification.

[0031] The cyanogen bromide-activated agarose gel freeze-dried protective agent, freeze-dried powder, and preparation and use thereof provided by the application can realize preparation of the cyanogen bromide-activated agarose gel freeze-dried powder by mixing specific saccharides and strictly controlling the proportion of different saccharide substances, and the cyanogen bromide-activated agarose gel freeze-dried powder has a high binding capacity of active groups and can maintain the original structure of agarose gel microspheres.

[0032] In addition, the cyanogen bromide-activated agarose gel freeze-dried powder of the application has the following advantages:

[0033] (1) protection of active groups from hydrolysis or oxidation, long-term preservation, and avoidance of product quality deterioration;

[0034] (2) convenient transportation, which greatly solves the trouble caused by long-distance logistics transportation;

[0035] (3) fast solubility recovery, which is not different from a new product;

[0036] (4) use of a freeze-dried protective agent harmless to the human body;

[0037] (5) compliance with green chemistry, which reduces damage to the environment and the human body; BRIEF DESCRIPTION OF DRAWINGS

[0038] In order to more clearly illustrate the technical solutions of the application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or prior art description. Obviously, the drawings in the following description are some embodiments of the application, and those skilled in the art can also obtain other drawings according to these drawings without creative labor.

[0039] Figure 1 is the initial micrograph of the cyanogen bromide-activated agarose gel of Example 1 provided by the application, wherein the microscope is configured with an objective lens of 40x and an eyepiece of 10x, and the magnification is 400 times.

[0040] Figure 2 is a micrograph of the swollen cyanogen bromide-activated sepharose gel lyophilized powder of Example 1 provided by the present application, wherein the microscope is configured with an objective lens of 40x and an eyepiece of 10x, and the magnification is 400 times.

[0041] Figure 3 is a micrograph of the swollen cyanogen bromide-activated sepharose gel lyophilized powder of Comparative Example 1 provided by the present application, wherein the microscope is configured with an objective lens of 40x and an eyepiece of 10x, and the magnification is 400 times.

[0042] Figure 4 is a micrograph of the swollen cyanogen bromide-activated sepharose gel lyophilized powder of Comparative Example 2 provided by the present application, wherein the microscope is configured with an objective lens of 40x and an eyepiece of 10x, and the magnification is 400 times.

[0043] Figure 5 is an appearance diagram of the cyanogen bromide-activated sepharose gel lyophilized powder of Comparative Example 5 provided by the present application. DETAILED DESCRIPTION

[0044] In order to make the objects, technical solutions and advantages of the present application clearer, the technical solutions in the present application will be described clearly and completely below in conjunction with the drawings in the present application. Obviously, the described embodiments are some embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all the other embodiments obtained by those of ordinary skill in the art without creative work fall within the protection scope of the present application.

[0045] The present application will be described below in conjunction with Figures 1-5 The present application provides a cyanogen bromide-activated sepharose gel lyophilized protective agent, a lyophilized powder and a preparation and application thereof.

[0046] Example 1

[0047] The present embodiment provides a lyophilized protective agent, which comprises 50 ml of an aqueous HCl solution with a pH value of 3, 50 μmol of dextran T500 and 6 mmol of trehalose.

[0048] The present embodiment also provides a method for preparing a cyanogen bromide-activated sepharose gel lyophilized powder by using the above lyophilized protective agent, and the steps are as follows:

[0049] (1) Preparation of uncrosslinked agarose microspheres: self-prepared or purchased uncrosslinked agarose microspheres (Chromstar 4B) with an agarose content of 4% were prepared by a traditional emulsification method, and the particle size of the microspheres was 45-165 μm. The traditional emulsification method can refer to the method in the literature “Jin Zijie. Preparation and performance of high-loading metal chelate affinity chromatography medium based on agarose microspheres [D]. Zhejiang University, 2023. DOI: 10.27461 / d.cnki.gzjdx.2023.00033.”.

[0050] (2) Preparation of CNBr-ACN system: 10 g of CNBr (cyanogen bromide) was added to 10 mL of ACN (acetonitrile), and the CNBr-ACN system was obtained after dissolution. It was ready for use.

[0051] (3) Coupling preparation of CNBr-Chromstar 4B: 100 g of Chromstar 4B was added to a 250 ml reactor, 100 ml of pure water was added, and the temperature was reduced to 3°C by ice bath. The CNBr-ACN system was added, and 3M NaOH was added dropwise to control the pH of the reaction system to 12.0-12.6. The reaction was carried out for 20 min.

[0052] After the reaction was completed, the reaction system was quickly added to a sand core funnel, and 3 times the volume of 5 mM HCl and 10 times the volume of 1 mM HCl were used for washing, respectively. The morphology of the agarose gel microspheres in the gel was shown in Figure 1 .

[0053] After washing, the gel was dried to a surface cracking state, and the CNBr-Chromstar 4B was obtained.

[0054] (4) The freeze-drying protectant was added to the CNBr-Chromstar 4B obtained in step (3), and the mass-volume ratio of cyanogen bromide activated agarose gel and freeze-drying protectant was 2 g:1 mL. Then the system was placed in a stainless steel tray (about 2.5 cm deep) and placed in a vacuum freeze-drying oven. The pre-freezing temperature was -80°C, the drying time was 24 h, and the vacuum degree was -0.8 to -0.9 MPa.

[0055] The CNBr-Chromstar 4B freeze-dried powder after vacuum freeze-drying was tested, and the CNBr binding capacity was 18 μmol / ml. The freeze-dried powder was stored in the dark at 2-8°C for 180 days, and the CNBr binding capacity was tested to be 18 μmol / ml.

[0056] The CNBr-activated agarose gel freeze-dried powder prepared by the above method has a rapid dissolving recovery ability. 1 g of the freeze-dried powder can swell to about 12-15 ml of the initial state of the gel in 1 mM HCL solution in 30 min, and the original structure of the agarose gel microspheres is maintained, as shown in Figure 2 The particle size is 45-165 μm.

[0057] Example 2

[0058] This example is basically the same as Example 1, except that the use amount of the substances involved in Example 1 is expanded to 10 times, i.e. 1000 g of CNBr-activated agarose gel is added to 500 ml of freeze-drying protectant for freeze-drying.

[0059] The CNBr-Chromstar 4B freeze-dried powder after vacuum freeze-drying is tested, and the CNBr binding capacity is 18 μmol / ml. The freeze-dried powder is stored at 2-8°C in the dark for 180 days, and the CNBr binding capacity is tested to be 18 μmol / ml.

[0060] The CNBr-activated agarose gel freeze-dried powder prepared by the above method has a rapid dissolving recovery ability. 1 g of the freeze-dried powder can swell to about 12-15 ml of the initial state of the gel in 1 mM HCL solution in 30 min, and the original structure of the agarose gel microspheres is maintained.

[0061] Example 3

[0062] This example is basically the same as Example 1, except that the use amount of the substances involved in Example 1 is expanded to 50 times, i.e. 5000 g of CNBr-activated agarose gel is added to 2500 ml of freeze-drying protectant for freeze-drying.

[0063] The CNBr-Chromstar 4B freeze-dried powder after vacuum freeze-drying is tested, and the CNBr binding capacity is 18 μmol / ml. The freeze-dried powder is stored at 2-8°C in the dark for 180 days, and the CNBr binding capacity is tested to be 18 μmol / ml.

[0064] The CNBr-activated agarose gel freeze-dried powder prepared by the above method has a rapid dissolving recovery ability. 1 g of the freeze-dried powder can swell to about 12-15 ml of the initial state of the gel in 1 mM HCL solution in 30 min, and the original structure of the agarose gel microspheres is maintained.

[0065] Example 4

[0066] This example is basically the same as Example 1, except that the isomolar amount of trehalose in the freeze-drying protectant is replaced by lactose.

[0067] The obtained freeze-dried powder was tested, and the results showed that the CNBr binding capacity thereof was 18 μmol / ml. The freeze-dried powder was stored in the dark at 2-8°C for 180 days, and then tested for CNBr binding capacity, which was 18 μmol / ml.

[0068] The freeze-dried cyanogen bromide-activated agarose gel powder prepared by the above method has a rapid dissolving and restoring ability. 1 g of the freeze-dried powder can swell to about 12-15 ml of the initial state of the gel in 1 mM HCL solution in 30 min, and the original structure of the agarose gel microspheres is maintained.

[0069] Example 5

[0070] This example is basically the same as Example 1, except that the equimolar amount of trehalose in the freeze-drying protective agent is replaced by galactose.

[0071] The obtained freeze-dried powder was tested, and the results showed that the CNBr binding capacity thereof was 18 μmol / ml. The freeze-dried powder was stored in the dark at 2-8°C for 180 days, and then tested for CNBr binding capacity, which was 18 μmol / ml.

[0072] The freeze-dried cyanogen bromide-activated agarose gel powder prepared by the above method has a rapid dissolving and restoring ability. 1 g of the freeze-dried powder can swell to about 12-15 ml of the initial state of the gel in 1 mM HCL solution in 30 min, and the original structure of the agarose gel microspheres is maintained.

[0073] Example 6

[0074] This example is basically the same as Example 1, except that the equimolar amount of trehalose in the freeze-drying protective agent is replaced by galactose.

[0075] The obtained freeze-dried powder was tested, and the results showed that the CNBr binding capacity thereof was 18 μmol / ml. The freeze-dried powder was stored in the dark at 2-8°C for 180 days, and then tested for CNBr binding capacity, which was 18 μmol / ml.

[0076] The freeze-dried cyanogen bromide-activated agarose gel powder prepared by the above method has a rapid dissolving and restoring ability. 1 g of the freeze-dried powder can swell to about 12-15 ml of the initial state of the gel in 1 mM HCL solution in 30 min, and the original structure of the agarose gel microspheres is maintained.

[0077] Example 7

[0078] This example is basically the same as Example 1, except that the equimolar amount of trehalose in the freeze-drying protective agent is replaced by galactose.

[0079] The obtained lyophilized powder was tested, and the results showed that the CNBr binding capacity was reduced to 3 μmol / ml compared with Example 1.

[0080] The lyophilized powder was stored at 2-8°C in the dark for 180 days, and then tested for CNBr binding capacity, which was 0 μmol / ml.

[0081] The CNBr-activated agarose gel lyophilized powder prepared by the above method has a rapid dissolution recovery capability, 1 g of the lyophilized powder can swell to about 12-15 ml of the initial state of the gel in 1 mM HCL solution in 30 min, and the original structure of the agarose gel microspheres is maintained.

[0082] Example 8

[0083] This example is basically the same as Example 1, except that the equal volume of pH 3 HCl aqueous solution is replaced by pH 2 HCl aqueous solution.

[0084] The obtained lyophilized powder was tested, and the results showed that the CNBr binding capacity was reduced to 6 μmol / ml compared with Example 1.

[0085] The lyophilized powder was stored at 2-8°C in the dark for 180 days, and then tested for CNBr binding capacity, which was 2 μmol / ml.

[0086] The CNBr-activated agarose gel lyophilized powder prepared by the above method has a rapid dissolution recovery capability, 1 g of the lyophilized powder can swell to about 12-15 ml of the initial state of the gel in 1 mM HCL solution in 30 min, and the original structure of the agarose gel microspheres is maintained.

[0087] Example 9

[0088] This example is basically the same as Example 1, except that the equal volume of pH 3 HCl aqueous solution is replaced by pH 4 HCl aqueous solution.

[0089] The obtained lyophilized powder was tested, and the results showed that the CNBr binding capacity was reduced to 15 μmol / ml compared with Example 1.

[0090] The lyophilized powder was stored at 2-8°C in the dark for 180 days, and then tested for CNBr binding capacity, which was 8 μmol / ml.

[0091] The CNBr-activated agarose gel lyophilized powder prepared by the above method has a rapid dissolution recovery capability, 1 g of the lyophilized powder can swell to about 12-15 ml of the initial state of the gel in 1 mM HCL solution in 30 min, and the original structure of the agarose gel microspheres is maintained.

[0092] Example 10

[0093] This example is basically the same as Example 1, except that the equal volume of aqueous HC1 solution of pH 3 is replaced by aqueous HC1 solution of pH 5.

[0094] The obtained lyophilized powder is tested, and it is found that the CNBr binding capacity thereof is reduced to 13 μmol / ml as compared with Example 1.

[0095] The lyophilized powder is stored at 2-8°C in the dark for 180 days, and then tested for CNBr binding capacity, which is 5 μmol / ml.

[0096] The CNBr-activated agarose gel lyophilized powder prepared by the above method has a rapid dissolving and restoring ability, 1 g of the lyophilized powder can swell to about 12-15 ml of the initial state of the gel in 1 mM HCL solution in 30 min, and the original structure of the agarose gel microspheres is maintained.

[0097] Example 11

[0098] This example is basically the same as Example 1, except that the equal volume of aqueous HC1 solution is replaced by water.

[0099] The obtained lyophilized powder is tested, and it is found that the CNBr binding capacity thereof is reduced to 9 μmol / ml as compared with Example 1.

[0100] The lyophilized powder is stored at 2-8°C in the dark for 180 days, and then tested for CNBr binding capacity, which is 0 μmol / ml.

[0101] The CNBr-activated agarose gel lyophilized powder prepared by the above method has a rapid dissolving and restoring ability, 1 g of the lyophilized powder can swell to about 12-15 ml of the initial state of the gel in 1 mM HCL solution in 30 min, and the original structure of the agarose gel microspheres is maintained.

[0102] Comparative Example 1

[0103] This comparative example is basically the same as Example 1, except that no dextran T500 is added to the lyoprotectant.

[0104] The obtained lyophilized powder is tested, and it is found that the microspheres are basically all deformed after swelling, as shown in Figure 3 .

[0105] Comparative Example 2

[0106] This comparative example is basically the same as Example 1, except that the dextran T500 in the lyoprotectant is replaced by an equal molar amount of hydroxyethyl starch.

[0107] The obtained lyophilized powder was tested, and it was found that the lyophilized powder was not spherical but deformed, as shown in Figure 4

[0108] Comparative Example 3

[0109] This comparative example is basically the same as Example 4, except that no lactose is added to the lyoprotectant.

[0110] The obtained lyophilized powder was tested, and it was found that the lyophilized powder without the addition of lactose as a lyoprotectant had a CNBr binding capacity of 0.

[0111] Comparative Example 4

[0112] This comparative example is basically the same as Example 1, except that no trehalose is added.

[0113] The obtained lyophilized powder was tested, and it was found that the lyophilized powder without the addition of trehalose as a lyoprotectant had a CNBr binding capacity of 0.

[0114] Comparative Example 5

[0115] This comparative example is basically the same as Example 1, except that the amount of dextran T500 is increased from 50 μmol to 200 μmol.

[0116] The obtained lyophilized powder was tested, and it was found that the lyophilized powder product appeared to have flocculation, not powder, which is not conducive to the later product because the product state is not uniform, leading to uneven dispensing, which ultimately indirectly leads to problems such as uneven swelling of the product, as shown in Figure 5

[0117] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present application, and are not limited thereto; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that they can still modify the technical solutions described in the foregoing examples, or make equivalent substitutions for part of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solution to deviate from the spirit and scope of the technical solutions of the embodiments of the present application.​​

Claims

1. A cyanogen bromide-activated sepharose gel lyophilized protectant characterized in that, The application relates to a freeze-drying protective agent, comprising: a solvent and a first saccharide and a second saccharide in the solvent; the first saccharide is selected from dextran; the second saccharide is selected from monosaccharide and / or disaccharide; the second saccharide is selected from one or a combination of two or more of trehalose, glucose, lactose and galactose; the ratio of the first saccharide to the second saccharide is 50 mu mol:6 mmol; the solvent is an acidic solution with a pH value of 3; the acidic solution is selected from an aqueous HCl solution or an aqueous citric acid solution; the concentration of dextran in the freeze-drying protective agent is 50 mu mol / 50 ml.

2. A process for the preparation of a lyophilized powder of cyanogen bromide-activated sepharose, characterized in that, The freeze-drying protective agent is mixed with cyanogen bromide-activated agarose gel to prepare a freeze-drying protective agent.

3. The process for the preparation of a lyophilized powder of cyanogen bromide-activated sepharose gel according to claim 2, characterized in that, The mass-volume ratio of the cyanogen bromide-activated agarose gel to the freeze-drying protective agent is 2 g:1 ml.

4. A process for the preparation of a lyophilized powder of cyanogen bromide-activated sepharose according to claim 2 or 3, characterized in that, In the vacuum freeze-drying, the pre-freezing temperature is -80 DEG C, the drying time is 24 h, and the vacuum degree is -0.8 to -0.9 MPa.

5. A process for the preparation of a lyophilized powder of cyanogen bromide-activated sepharose according to claim 2 or 3, characterized in that, The binding capacity of CNBr in the cyanogen bromide-activated agarose gel is 18 mu mol / ml.

6. The process for the preparation of a cyanogen bromide-activated agarose gel lyophilized powder according to any one of claims 2 to 5, characterized in that, The binding capacity of CNBr in the cyanogen bromide-activated agarose gel freeze-drying powder is 18 mu mol / ml. The binding capacity of CNBr in the cyanogen bromide-activated agarose gel freeze-drying powder does not change after the cyanogen bromide-activated agarose gel freeze-drying powder is stored in the dark at 2-8 DEG C for 180 days. The cyanogen bromide-activated agarose gel freeze-drying powder is in a microspherical shape after being swelled in an aqueous HCl solution.

7. The cyanogen bromide-activated agarose gel lyophilized powder according to claim 6, wherein, The particle size of the microspheres of the cyanogen bromide-activated agarose gel freeze-drying powder is 45-165 mu m after being swelled in an aqueous HCl solution.

8. The cyanogen bromide-activated agarose gel freeze-drying powder according to claim 6 or 7 is applied in at least one of the following (1)-(4): (1) affinity chromatography; (2) protein fixation; (3) drug carrier; (4) antibody purification.

Images