Chromatographic medium for adenovirus purification and preparation method thereof

Abstract

The invention provides a chromatography medium for adenovirus purification and a preparation method thereof, the chromatography medium for adenovirus purification has a structure as shown in formula (I); wherein the microspheres are one of dextran microspheres, agarose microspheres and polymethacrylate microspheres. The chromatography medium for adenovirus purification is high in adenovirus recovery rate, good in protein impurity removal effect and high in resolution ratio.

Description

Technical Field

[0001] The invention relates to the technical field of chromatography separation, in particular to a chromatography medium for adenovirus purification and a preparation method thereof. Background Art

[0002] Adenovirus is a non-enveloped particle with a diameter of 70-90nm, composed of 252 capsomeres arranged in an icosahedron, and the capsid contains a linear double-stranded DNA molecule. The isolation and purification of adenovirus plays an important role in the fields of vaccines and cell therapy.

[0003] At present, chromatography purification has become the preferred method for large-scale production of adenovirus. During chromatography, adenovirus is generally separated from other impurities by exclusion, ion exchange or hydrophobic effect. Among them, exclusion is to use the difference in molecular weight between the substances to be separated, and the different resistance they encounter in the chromatographic medium, so that they flow out in order according to their weight. Ion exchange or hydrophobic effect is based on the reversible interaction between the substance to be separated and the chromatographic matrix to separate them. Exclusion has little effect on viral activity, but it has high requirements for sample purity and low processing efficiency. Improving the processing efficiency will lead to poor resolution. Ion exchange or hydrophobic effect has high processing efficiency, but it is easy to bind to a certain amount of adenovirus, resulting in large adenovirus loss, low adenovirus recovery rate, poor protein impurity removal effect, and less than ideal resolution. Summary of the invention

[0004] The purpose of the present invention is to provide a chromatographic medium for adenovirus purification, so as to solve the problems of low adenovirus recovery rate, poor protein impurity removal effect and low resolution in adenovirus purification in the prior art.

[0005] In order to solve the above technical problems, the technical solution of the present invention is as follows:

[0006] A chromatographic medium for adenovirus purification has a structure as shown in formula (I):

[0007]

[0008] The microspheres are selected from the group consisting of dextran microspheres, agarose microspheres, and polymethacrylate microspheres.

[0009] Preferably, the particle size of the microspheres is 40-150 μm.

[0010] Preferably, the microspheres are activated with allyl glycidyl ether, then brominated with bromine, and then reacted with 4-(4-isobutylphenyl)butan-1-amine to obtain the microspheres.

[0011] The present invention also provides a method for preparing a chromatographic medium for adenovirus purification, comprising the following steps:

[0012] (1) taking microspheres, adding allyl glycidyl ether, and reacting to obtain allylated microspheres;

[0013] (2) adding bromine to the allylated microspheres obtained in step (1) to react to obtain brominated microspheres;

[0014] (3) Add 4-(4-isobutylphenyl)butan-1-amine to the brominated microspheres obtained in step (2), and react to obtain a chromatographic medium for adenovirus purification.

[0015] Wherein, the CAS number of the 4-(4-isobutylphenyl)butan-1-amine is 1216170-51-2.

[0016] Preferably, step (1) specifically comprises: taking microspheres and sodium hydroxide, reacting at a temperature of 40-50° C. and a stirring speed of 110-150 rpm for 1-2 hours to obtain a first reactant; then adding allyl glycidyl ether thereto, reacting at a temperature of 40-50° C. and a stirring speed of 110-150 rpm for 16-20 hours to obtain allylated microspheres.

[0017] Preferably, in step (1), the weight ratio of the microspheres, sodium hydroxide, and allyl glycidyl ether is 1000:50:(100-300);

[0018] Optionally, the reaction in step (1) uses water as the reaction solvent.

[0019] Preferably, step (2) specifically comprises: adding sodium acetate to the allylated microspheres obtained in step (1) and mixing them evenly; then adding bromine thereto, reacting for 1-2 hours at a temperature of 20-35° C. and a stirring speed of 110-150 rpm; then, adding sodium formate thereto and reacting to obtain brominated microspheres.

[0020] Preferably, in step (2), the weight ratio of the allylated microspheres to sodium acetate is 1000:(2-8);

[0021] Optionally, the weight ratio of the allylated microspheres to the bromine is 1000:(5-10); the weight ratio of the allylated microspheres to the sodium formate is 1000:(10-20);

[0022] Optionally, the reaction in step (2) uses water as the reaction solvent.

[0023] Preferably, step (3) specifically comprises: adding 4-(4-isobutylphenyl)butan-1-amine to the brominated microspheres obtained in step (2), adjusting the pH value to 9-11, reacting for 16-20 hours at a temperature of 40-60° C. and a stirring speed of 110-150 rpm to obtain a chromatographic medium for adenovirus purification.

[0024] Preferably, in step (3), the weight ratio of the brominated microspheres to the 4-(4-isobutylphenyl)butan-1-amine is 1000:(10-20);

[0025] Optionally, the reaction in step (3) uses water as the reaction solvent.

[0026] The above solution of the present invention includes at least the following beneficial effects:

[0027] The chromatographic medium for adenovirus purification of the present invention has a structure as shown in formula (I). The microspheres are selected from dextran microspheres, agarose microspheres, and polymethacrylate microspheres. The chromatographic medium for adenovirus purification of the present invention has a high recovery rate for adenovirus, good protein impurity removal effect, and high resolution.

[0028] When the adenovirus sample passes through the chromatographic medium, it cannot enter the interior of the microspheres due to the exclusion effect and can only flow through the gaps between the microspheres. However, the protein impurities in the adenovirus sample can enter the interior of the microspheres and be adsorbed through the ionic effect of the amino groups on the surface and internal pores of the microspheres and the hydrophobic effect of the isobutylphenyl groups. Compared with the chromatographic medium that only utilizes the exclusion effect, the processing efficiency is greatly improved, and at least can achieve the same or similar processing efficiency as that of ion exchange chromatography medium and / or hydrophobic chromatography medium.

[0029] In particular, as a hydrophobic group, isobutylphenyl shows good selectivity for the separation of adenovirus and protein impurities. This may be because the icosahedral symmetric structure and fibrous protrusions of the adenovirus capsid are difficult to fully approach and interact with isobutylphenyl in space, and the adenovirus is not easy to bind to the chromatographic medium, which is conducive to the flow of adenovirus and improves the recovery rate of adenovirus. At the same time, the benzene ring and branch structure of isobutylphenyl are relatively limited in flexibility compared to the chain structure, and there is a certain spatial hindrance, which can limit the pore space of the microspheres to a certain extent, so that the residence time of impurity proteins on the surface of the microspheres and in the internal pores is prolonged, thereby improving the adsorption efficiency of protein impurities and enhancing the selectivity and resolution of separation. DETAILED DESCRIPTION

[0030] In the embodiments of the present invention, if the specific conditions are not specified, the conventional conditions or the conditions recommended by the manufacturer are followed. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially. Raw materials of different manufacturers and models do not affect the implementation of the technical solution of the present invention and the realization of the technical effects.

[0031] The chromatographic medium for adenovirus purification in the following embodiments of the present invention has a structure as shown in formula (I):

[0032]

[0033] The microspheres are selected from the group consisting of dextran microspheres, agarose microspheres and polymethacrylate microspheres, and have a particle size of 40-150 μm.

[0034] The chromatographic medium for adenovirus purification described in this example is obtained by activating the microspheres with allyl glycidyl ether, then brominating them with bromine, and then reacting them with 4-(4-isobutylphenyl)butan-1-amine. The specific preparation method is as follows:

[0035] (1) taking microspheres, adding allyl glycidyl ether, and reacting to obtain allylated microspheres;

[0036]

[0037] (2) adding bromine to the allylated microspheres obtained in step (1) to react to obtain brominated microspheres;

[0038]

[0039] (3) Add 4-(4-isobutylphenyl)butan-1-amine to the brominated microspheres obtained in step (2), and react to obtain a chromatographic medium for adenovirus purification.

[0040]

[0041] It should be noted that the exclusion limit of the microspheres is sufficient to ensure that adenovirus cannot enter the microspheres. For example, Focurose 4B agarose microspheres can be used, and the exclusion limit of the microspheres is 60-20000 kDa.

[0042] Example 1

[0043] The chromatographic medium used for adenovirus purification in this embodiment has a structure as shown in formula (I), wherein the microspheres are dextran microspheres with an average particle size of 50 μm.

[0044] The chromatographic medium for adenovirus purification described in this example is obtained by activating the microspheres with allyl glycidyl ether, then brominating them with bromine, and then reacting them with 4-(4-isobutylphenyl)butan-1-amine. The specific preparation method is as follows:

[0045] (1) Take microspheres and sodium hydroxide, react at a temperature of 40° C. and a stirring speed of 110 rpm for 1 hour to obtain a first reactant; then add allyl glycidyl ether thereto, react at a temperature of 40° C. and a stirring speed of 130 rpm for 16 hours to obtain allylated microspheres.

[0046] The weight ratio of the microspheres, sodium hydroxide and allyl glycidyl ether is 1000:50:200; water is used as the reaction solvent in the above reaction. When preparing the first reactant, the weight ratio of water to the sodium hydroxide is 1:1. When preparing the allylated microspheres, the weight ratio of water to the allyl glycidyl ether is 3:2.

[0047] As a preferred implementation of this embodiment, the step of post-treating the allylated microspheres is further included: the obtained reaction mixture containing the allylated microspheres is filtered, and the filtrate is washed with 5 volumes of deionized water, 5 volumes of 95V / V% ethanol, 2 volumes of deionized water, 2 volumes of 0.2mol / L acetic acid, and 5 volumes of deionized water in sequence, and then dried to obtain allylated microspheres.

[0048] (2) Add sodium acetate and water to the allylated microspheres obtained in step (1) and mix well; then add bromine to the allylated microspheres and react at a temperature of 20° C. and a stirring speed of 110 rpm for 2 h; then add sodium formate to the allylated microspheres to obtain brominated microspheres.

[0049] The weight ratio of the allylated microspheres to sodium acetate is 1000:2; the weight ratio of the allylated microspheres to the bromine is 1000:7; the weight ratio of the allylated microspheres to the sodium formate is 1000:10; the above reaction uses water as the reaction solvent, and the weight ratio of water to the allylated microspheres is 3:1.

[0050] As a preferred implementation of this embodiment, the method further includes post-treating the brominated microspheres: filtering the obtained reaction mixture containing the brominated microspheres, washing the filtrate with deionized water, and drying the filtrate to obtain the brominated microspheres.

[0051] (3) Add 4-(4-isobutylphenyl)butan-1-amine and water to the brominated microspheres obtained in step (2), adjust the pH value to 10 with NaOH, react for 18 hours at a temperature of 40° C. and a stirring speed of 130 rpm to obtain a chromatographic medium for adenovirus purification.

[0052] The weight ratio of the brominated microspheres to the 4-(4-isobutylphenyl)butan-1-amine is 1000:10; the reaction uses water as a reaction solvent, and the weight ratio of water to the brominated microspheres is 3:1.

[0053] As a preferred implementation method of this embodiment, it also includes the step of post-treating the obtained chromatographic medium for adenovirus purification: filtering the reaction mixture of the obtained chromatographic medium for adenovirus purification, washing the filtrate with deionized water, and drying it.

[0054] Example 2

[0055] The chromatographic medium used for adenovirus purification in this embodiment has a structure as shown in formula (I), wherein the microspheres are polymethacrylate microspheres, and the average particle size of the microspheres is 150 μm.

[0056] The chromatographic medium for adenovirus purification described in this example is obtained by activating the microspheres with allyl glycidyl ether, then brominating them with bromine, and then reacting them with 4-(4-isobutylphenyl)butan-1-amine. The specific preparation method is as follows:

[0057] (1) Take microspheres and sodium hydroxide, react at a temperature of 50° C. and a stirring speed of 130 rpm for 2 hours to obtain a first reactant; then add allyl glycidyl ether thereto, react at a temperature of 45° C. and a stirring speed of 150 rpm for 20 hours to obtain allylated microspheres.

[0058] The weight ratio of the microspheres, sodium hydroxide and allyl glycidyl ether is 1000:50:300; water is used as the reaction solvent in the above reaction. When preparing the first reactant, the weight ratio of water to the sodium hydroxide is 1:1. When preparing the allylated microspheres, the weight ratio of water to the allyl glycidyl ether is 3:2.

[0059] As a preferred implementation of this embodiment, the step of post-treating the allylated microspheres is further included: the obtained reaction mixture containing the allylated microspheres is filtered, and the filtrate is washed with 5 volumes of deionized water, 5 volumes of 95V / V% ethanol, 2 volumes of deionized water, 2 volumes of 0.2mol / L acetic acid, and 5 volumes of deionized water in sequence, and then dried to obtain allylated microspheres.

[0060] (2) Add sodium acetate and water to the allylated microspheres obtained in step (1) and mix well; then add bromine to the allylated microspheres and react at a temperature of 35° C. and a stirring speed of 130 rpm for 1 hour; then add sodium formate to the allylated microspheres to obtain brominated microspheres.

[0061] The weight ratio of the allylated microspheres to sodium acetate is 1000:4; the weight ratio of the allylated microspheres to the bromine is 1000:10; the weight ratio of the allylated microspheres to the sodium formate is 1000:20; the above reaction uses water as the reaction solvent, and the weight ratio of water to the allylated microspheres is 3:1.

[0062] As a preferred implementation of this embodiment, the method further includes post-treating the brominated microspheres: filtering the obtained reaction mixture containing the brominated microspheres, washing the filtrate with deionized water, and drying the filtrate to obtain the brominated microspheres.

[0063] (3) Add 4-(4-isobutylphenyl)butan-1-amine and water to the brominated microspheres obtained in step (2), adjust the pH value to 11 with NaOH, react at a temperature of 60° C. and a stirring speed of 150 rpm for 20 hours to obtain a chromatographic medium for adenovirus purification.

[0064] The weight ratio of the brominated microspheres to the 4-(4-isobutylphenyl)butan-1-amine is 1000:20; the reaction uses water as a reaction solvent, and the weight ratio of water to the brominated microspheres is 3:1.

[0065] As a preferred implementation method of this embodiment, it also includes the step of post-treating the obtained chromatographic medium for adenovirus purification: filtering the reaction mixture of the obtained chromatographic medium for adenovirus purification, washing the filtrate with deionized water, and drying it.

[0066] Example 3

[0067] The chromatographic medium used for adenovirus purification in this embodiment has a structure as shown in formula (I), wherein the microspheres are agarose microspheres, and the average particle size of the microspheres is 65 μm.

[0068] The chromatographic medium for adenovirus purification described in this example is obtained by activating the microspheres with allyl glycidyl ether, then brominating them with bromine, and then reacting them with 4-(4-isobutylphenyl)butan-1-amine. The specific preparation method is as follows:

[0069] (1) Take microspheres and sodium hydroxide, react at a temperature of 45° C. and a stirring speed of 150 rpm for 1 hour to obtain a first reactant; then add allyl glycidyl ether thereto, react at a temperature of 50° C. and a stirring speed of 110 rpm for 18 hours to obtain allylated microspheres.

[0070] The weight ratio of the microspheres, sodium hydroxide and allyl glycidyl ether is 1000:50:100; water is used as the reaction solvent in the above reaction. When preparing the first reactant, the weight ratio of water to the sodium hydroxide is 1:1. When preparing the allylated microspheres, the weight ratio of water to the allyl glycidyl ether is 3:2.

[0071] As a preferred implementation of this embodiment, the step of post-treating the allylated microspheres is further included: the obtained reaction mixture containing the allylated microspheres is filtered, and the filtrate is washed with 5 volumes of deionized water, 5 volumes of 95V / V% ethanol, 2 volumes of deionized water, 2 volumes of 0.2mol / L acetic acid, and 5 volumes of deionized water in sequence, and then dried to obtain allylated microspheres.

[0072] (2) Add sodium acetate and water to the allylated microspheres obtained in step (1) and mix them evenly; then add bromine to the allylated microspheres and react them at a temperature of 25° C. and a stirring speed of 150 rpm for 2 hours; then add sodium formate to the allylated microspheres to obtain brominated microspheres.

[0073] Among them, the weight ratio of the allylated microspheres to sodium acetate is 1000:5; the weight ratio of the allylated microspheres to the bromine is 1000:5; the weight ratio of the allylated microspheres to the sodium formate is 1000:15; the above reaction uses water as the reaction solvent, and the weight ratio of water to the allylated microspheres is 3:1.

[0074] As a preferred implementation of this embodiment, the method further includes post-treating the brominated microspheres: filtering the obtained reaction mixture containing the brominated microspheres, washing the filtrate with deionized water, and drying the filtrate to obtain the brominated microspheres.

[0075] (3) Add 4-(4-isobutylphenyl)butan-1-amine and water to the brominated microspheres obtained in step (2), adjust the pH value to 9 with NaOH, react for 16 hours at a temperature of 50° C. and a stirring speed of 110 rpm to obtain a chromatographic medium for adenovirus purification.

[0076] The weight ratio of the brominated microspheres to the 4-(4-isobutylphenyl)butan-1-amine is 1000:15; the reaction uses water as a reaction solvent, and the weight ratio of water to the brominated microspheres is 3:1.

[0077] As a preferred implementation method of this embodiment, it also includes the step of post-treating the obtained chromatographic medium for adenovirus purification: filtering the reaction mixture of the obtained chromatographic medium for adenovirus purification, washing the filtrate with deionized water, and drying it.

[0078] Example 4

[0079] The chromatographic medium used for adenovirus purification in this embodiment has a structure as shown in formula (I), wherein the microspheres are agarose microspheres, and the average particle size of the microspheres is 95 μm.

[0080] The chromatographic medium for adenovirus purification described in this example is obtained by activating the microspheres with allyl glycidyl ether, then brominating them with bromine, and then reacting them with 4-(4-isobutylphenyl)butan-1-amine. The specific preparation method is as follows:

[0081] (1) Take microspheres and sodium hydroxide, react at a temperature of 40° C. and a stirring speed of 120 rpm for 1 hour to obtain a first reactant; then add allyl glycidyl ether thereto, react at a temperature of 40° C. and a stirring speed of 120 rpm for 20 hours to obtain allylated microspheres.

[0082] The weight ratio of the microspheres, sodium hydroxide and allyl glycidyl ether is 1000:50:240; water is used as the reaction solvent in the above reaction. When preparing the first reactant, the weight ratio of water to the sodium hydroxide is 1:1. When preparing the allylated microspheres, the weight ratio of water to the allyl glycidyl ether is 3:2.

[0083] As a preferred implementation of this embodiment, the step of post-treating the allylated microspheres is further included: the obtained reaction mixture containing the allylated microspheres is filtered, and the filtrate is washed with 5 volumes of deionized water, 5 volumes of 95V / V% ethanol, 2 volumes of deionized water, 2 volumes of 0.2mol / L acetic acid, and 5 volumes of deionized water in sequence, and then dried to obtain allylated microspheres.

[0084] (2) Add sodium acetate and water to the allylated microspheres obtained in step (1) and mix them evenly; then add bromine to the allylated microspheres and react them at a temperature of 30° C. and a stirring speed of 120 rpm for 1 hour; then add sodium formate to the allylated microspheres to obtain brominated microspheres.

[0085] The weight ratio of the allylated microspheres to sodium acetate is 1000:6; the weight ratio of the allylated microspheres to the bromine is 1000:8; the weight ratio of the allylated microspheres to the sodium formate is 1000:18; the above reaction uses water as the reaction solvent, and the weight ratio of water to the allylated microspheres is 3:1.

[0086] As a preferred implementation of this embodiment, the method further includes post-treating the brominated microspheres: filtering the obtained reaction mixture containing the brominated microspheres, washing the filtrate with deionized water, and drying the filtrate to obtain the brominated microspheres.

[0087] (3) Add 4-(4-isobutylphenyl)butan-1-amine and water to the brominated microspheres obtained in step (2), adjust the pH value to 10 with NaOH, react for 20 hours at a temperature of 50° C. and a stirring speed of 120 rpm to obtain a chromatographic medium for adenovirus purification.

[0088] The weight ratio of the brominated microspheres to the 4-(4-isobutylphenyl)butan-1-amine is 1000:18; the reaction uses water as a reaction solvent, and the weight ratio of water to the brominated microspheres is 3:1.

[0089] As a preferred implementation method of this embodiment, it also includes the step of post-treating the obtained chromatographic medium for adenovirus purification: filtering the reaction mixture of the obtained chromatographic medium for adenovirus purification, washing the filtrate with deionized water, and drying it.

[0090] Comparative Example 1

[0091] This comparative example is the same as the chromatographic medium for adenovirus purification in Example 4 and is prepared by the same method, with the only difference being that in step (3), 4-(4-isobutylphenyl)butan-1-amine is replaced by an equal molar amount of octylamine.

[0092] Comparative Example 2

[0093] This comparative example is the same as the chromatographic medium for adenovirus purification in Example 4 and is prepared by the same method, with the only difference being that in step (3), 4-(4-isobutylphenyl)butan-1-amine is replaced by an equimolar amount of nonylamine.

[0094] Comparative Example 3

[0095] This comparative example is the same as the chromatographic medium for adenovirus purification in Example 4 and is prepared by the same method, with the only difference being that in step (3), 4-(4-isobutylphenyl)butan-1-amine is replaced by an equimolar amount of isononylamine.

[0096] Comparative Example 4

[0097] This comparative example is the same as the chromatographic medium for adenovirus purification in Example 4 and is prepared by the same method, with the only difference being that in step (3), 4-(4-isobutylphenyl)butan-1-amine is replaced with an equal molar amount of 4-phenylbutan-1-amine.

[0098] The 4-phenylbutan-1-amine has a structure as shown in formula (II):

[0099]

[0100] Comparative Example 5

[0101] This comparative example is the same as the chromatographic medium for adenovirus purification in Example 4 and is prepared by the same method, except that: in step (3), 4-(4-isobutylphenyl)butan-1-amine is replaced with an equal molar amount of 4-(4-ethylphenyl)butan-1-amine;

[0102] The 4-(4-ethylphenyl)butan-1-amine has a structure as shown in formula (IIIIV):

[0103]

[0104] Comparative Example 6

[0105] The chromatographic medium for adenovirus purification in this comparative example is the same as that in Example 4 and is prepared by the same method, with the only difference being that in step (1), the weight ratio of the microspheres to the allyl glycidyl ether is 1000:50.

[0106] Comparative Example 7

[0107] This comparative example is the same as the chromatographic medium for adenovirus purification of Example 4 and is prepared by the same method, except that: in step (1), the weight ratio of the microspheres to the allyl glycidyl ether is 1000:500. In step (2), the weight ratio of the allylated microspheres to the bromine is 1000:50. In step (3), the weight ratio of the brominated microspheres to the 4-(4-isobutylphenyl)butan-1-amine is 1000:50.

[0108] Effect experiment example

[0109] In order to verify the technical effect of the chromatographic medium for adenovirus purification described in the present invention, the following experiments were performed:

[0110] The crude adenovirus product was taken and filtered through a 0.45 μm filter membrane, and then the column was loaded with the chromatography media in Examples 1-4 and Comparative Examples 1-7, respectively, and purified. After loading, the flow-through was collected. The adenovirus titer before and after purification was determined by fluorescence quantitative PCR, and the adenovirus recovery rate was calculated; the purity of the adenovirus in the purified flow-through was determined by high performance liquid chromatography.

[0111] Wherein, adenovirus recovery rate = (titer of sample after purification × volume) / (titer of sample before purification × volume).

[0112] The specific steps of purification using the chromatography media in Examples 1-4 and Comparative Examples 1-7 are as follows:

[0113] First, use 5 column volumes of equilibration solution (50mM Tris-HCl + 0.5M NaCl, pH 7.5) at a flow rate of 1.0ml / min for equilibration, then load the sample at a flow rate of 0.3ml / min, with the sample volume being 1 column volume, and then collect the flow-through liquid in the flow-through mode. Then use 20 column volumes of equilibration solution at a flow rate of 0.3ml / min for washing, and finally, use the eluent (50mMTris-HCl + 2.0M NaCl) for elution.

[0114] After experimentation, the results are as follows:

[0115]

[0116]

[0117] According to the results of Examples 1-4 and Comparative Examples 1-7, the chromatographic medium for adenovirus purification of the present invention has a high recovery rate for adenovirus, high adenovirus purity, good protein impurity removal effect and high resolution.

[0118] According to the results of Example 4 and Comparative Examples 1-3, the chromatographic medium uses a chain hydrophobic group, and the adenovirus recovery rate decreases to varying degrees during the adenovirus purification process, while the purity of the adenovirus decreases significantly; the chain hydrophobic group is relatively flexible in the mobile phase, and the resolution of separating adenovirus from protein impurities is poor. The isononylamine in Comparative Example 3 has a short side chain, and its adenovirus purity is improved compared to Comparative Examples 1-2, but the adenovirus recovery rate is relatively low.

[0119] According to the results of Example 4 and Comparative Examples 4-5, the chromatographic medium uses a hydrophobic group containing a benzene ring and has no isobutyl group at the end, and its resolution for separating adenovirus and protein impurities is still not ideal.

[0120] According to the results of Example 4 and Comparative Examples 6-7, it can be seen that the density of hydrophobic groups in the chromatographic medium is too low, the binding sites of protein impurities are few, and although the adenovirus recovery rate is high, it cannot be effectively separated from the protein impurities, and the adenovirus purity is low. If the density of hydrophobic groups in the chromatographic medium is too high, the adenovirus purity does not change much, but the adenovirus recovery rate will drop significantly.

[0121] It is known from common technical knowledge that the present invention can be implemented by other embodiments that do not deviate from its spirit or essential features. Therefore, the above disclosed embodiments are only illustrative in all respects and are not exclusive. All changes within the scope of the present invention or within the scope equivalent to the present invention are encompassed by the present invention.

Claims

1. A chromatographic medium for adenovirus purification, characterized in that: It has a structure as shown in formula (I): The microspheres are selected from the group consisting of dextran microspheres, agarose microspheres, and polymethacrylate microspheres.

2. The chromatographic medium for adenovirus purification according to claim 1, characterized in that The particle size of the microspheres is 40-150 μm.

3. The chromatographic medium for adenovirus purification according to claim 1, characterized in that The microspheres are activated by using allyl glycidyl ether, then brominated by using bromine, and then reacted with 4-(4-isobutylphenyl)butan-1-amine to obtain the microspheres.

4. A method for preparing a chromatographic medium for adenovirus purification, characterized in that: The steps include: (1) taking microspheres, adding allyl glycidyl ether, and reacting to obtain allylated microspheres; (2) adding bromine to the allylated microspheres obtained in step (1) to react to obtain brominated microspheres; (3) Add 4-(4-isobutylphenyl)butan-1-amine to the brominated microspheres obtained in step (2), and react to obtain a chromatographic medium for adenovirus purification.

5. The method for preparing a chromatographic medium for adenovirus purification according to claim 4, characterized in that: Step (1) specifically comprises: taking microspheres and sodium hydroxide, reacting at a temperature of 40-50° C. and a stirring speed of 110-150 rpm for 1-2 hours to obtain a first reactant; then adding allyl glycidyl ether thereto, reacting at a temperature of 40-50° C. and a stirring speed of 110-150 rpm for 16-20 hours to obtain allylated microspheres.

6. The method for preparing a chromatographic medium for adenovirus purification according to claim 5, characterized in that: In step (1), the weight ratio of the microspheres, sodium hydroxide, and allyl glycidyl ether is 1000:50:(100-300); Optionally, the reaction in step (1) uses water as the reaction solvent.

7. The method for preparing a chromatographic medium for adenovirus purification according to claim 4, characterized in that: Step (2) specifically comprises: adding sodium acetate to the allylated microspheres obtained in step (1) and mixing them evenly; then adding bromine thereto, reacting for 1-2 hours at a temperature of 20-35° C. and a stirring speed of 110-150 rpm; then adding sodium formate thereto and reacting to obtain brominated microspheres.

8. The method for preparing a chromatographic medium for adenovirus purification according to claim 7, characterized in that: In step (2), the weight ratio of the allylated microspheres to sodium acetate is 1000:(2-8); Optionally, the weight ratio of the allylated microspheres to the bromine is 1000:(5-10); the weight ratio of the allylated microspheres to the sodium formate is 1000:(10-20); Optionally, the reaction in step (2) uses water as the reaction solvent.

9. The method for preparing a chromatographic medium for adenovirus purification according to claim 4, characterized in that: Step (3) specifically includes: adding 4-(4-isobutylphenyl)butan-1-amine to the brominated microspheres obtained in step (2), adjusting the pH value to 9-11, reacting for 16-20 hours at a temperature of 40-60° C. and a stirring speed of 110-150 rpm to obtain a chromatographic medium for adenovirus purification.

10. The method for preparing a chromatographic medium for adenovirus purification according to claim 9, characterized in that: In step (3), the weight ratio of the brominated microspheres to the 4-(4-isobutylphenyl)butan-1-amine is 1000:(10-20); Optionally, the reaction in step (3) uses water as the reaction solvent.

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