Dendritic spacer arm-based strong hydrophobic chromatography medium and preparation method thereof

Abstract

The invention discloses a strong hydrophobic chromatography medium based on a dendritic spacer arm and a preparation method thereof, the chromatography medium takes an epoxy modified microsphere as a carrier, the carrier is grafted with the dendritic spacer arm, the tail end of the spacer arm is connected with a phenyl hydrophobic ligand, and the chromatography medium is in a three-dimensional net shape; the preparation method comprises the steps of epoxy modification of microspheres, grafting of spacer arms and introduction of hydrophobic ligands. The chromatography medium contains a highly branched dendritic structure and has high adsorption capacity; strong pi-pi conjugation exists between phenyl groups of a net structure, so that the effective action area of a hydrophobic ligand can be maximized, the hydrophobicity is remarkably enhanced, non-specific binding is effectively reduced, specific binding of recombinant albumin is facilitated, and the selectivity and the yield are improved; an ideal solution is provided for efficient and high-activity purification of biomacromolecules such as recombinant human serum albumin; the preparation method of the chromatography medium is simple and easy to implement, suitable raw materials are wide, and industrial production is facilitated.

Description

TECHNICAL FIELD

[0001] The present application relates to a strong hydrophobic chromatography medium and a preparation method thereof, in particular to a strong hydrophobic chromatography medium based on dendritic spacers and a preparation method thereof. BACKGROUND

[0002] Recombinant human serum albumin (rHSA) is an important drug widely used in clinical applications. Its molecular weight is large (about 66.5 kDa), its structure is complex, and its surface hydrophobic sites are unevenly distributed. In the purification process, not only host cell proteins, endotoxins and pigments and other impurities need to be removed efficiently, but also the high-order structure and biological activity of rHSA need to be ensured not to be damaged. In the purification of rHSA, the traditional hydrophobic chromatography medium often faces a dilemma: if a weak hydrophobic medium is selected, the loading capacity is low and the impurity removal effect is poor; if a strong hydrophobic medium is selected, there will be non-specific adsorption forces such as hydrogen bonds and ions, which will easily lead to irreversible adsorption or denaturation of rHSA, resulting in low recovery rate.

[0003] In the biopharmaceutical and biotechnology industries, the separation and purification of proteins is a crucial step, and the purity and activity of the proteins are directly related to the quality, safety and efficacy of the final product. Chromatography technology is the most mainstream and efficient method for protein purification at present, and can be divided into ion exchange chromatography, affinity chromatography, size exclusion chromatography and hydrophobic interaction chromatography according to its principle. Among them, hydrophobic interaction chromatography uses the difference in hydrophobic force between the surface hydrophobic region of the protein and the immobilized hydrophobic ligand on the chromatography medium for separation, which has the advantages of mild separation conditions, not easy to inactivate the protein, high loading capacity, relatively low cost, etc. Therefore, in the purification process of biological macromolecules such as antibodies, enzymes, recombinant proteins (such as recombinant human serum albumin), it is often used as a key step for fine purification.

[0004] In the prior art, the commonly used hydrophobic ligands for hydrophobic chromatography mainly fall into two categories: one is short-chain alkyl ligand, such as butyl, hexyl, octyl, etc.; the other is aromatic ring ligand, such as phenyl. The hydrophobicity of short-chain alkyl ligand is relatively weak, and the binding with protein is mild, so the protein is easy to elute, but the loading capacity is low, and the separation effect is poor for proteins with weak hydrophobicity. Due to the π-π interaction, the aromatic ring ligand (especially phenyl) can provide stronger hydrophobic interaction than the same carbon chain alkyl, and therefore shows higher selectivity and loading capacity in many applications. However, whether it is a traditional alkyl or a phenyl ligand, the structure is mostly a single-point connected linear or simple cyclic structure, and the exposed hydrophobic surface area is limited, and for some target proteins with complex structure and dispersed hydrophobic region (such as recombinant human blood albumin), there are often problems of insufficient binding force or low selectivity. Secondly, the traditional spacer is mostly a simple linear molecule, and although such linear spacer improves the accessibility of the ligand to a certain extent, its stretching ability and spatial conformation are limited. When the ligand density is high, the linear spacer is easy to entangle and collapse, resulting in partial shielding of the ligand and failure to form an effective three-dimensional hydrophobic interaction space, thereby limiting the loading capacity and separation efficiency of the chromatography medium. SUMMARY

[0005] The purpose of the present application is to provide a strong hydrophobic chromatography medium based on dendritic spacer, which has high loading capacity, high selectivity, high recovery rate, and can effectively shield non-specific effects, and a preparation method thereof.

[0006] Technical scheme: The strong hydrophobic chromatography medium based on dendritic spacer takes an epoxy-modified microsphere as a carrier, the carrier is grafted with a dendritic spacer, and the end of the spacer is connected with a phenyl hydrophobic ligand; the chromatography medium is in a three-dimensional network shape.

[0007] The microsphere is preferably one of polymethacrylate microspheres (PMMA), silica microspheres, polystyrene microspheres, polystyrene-divinylbenzene microspheres, agarose microspheres, dextran microspheres, chitosan microspheres, konjac sugar microspheres, cellulose microspheres, and polyvinyl alcohol microspheres. The surface of the microsphere has a hydroxyl group or can be hydroxylated. The particle size of the microsphere can be monodisperse or polydisperse, the microsphere is non-porous, or includes one or more pores. The most preferred is a monodisperse porous microsphere.

[0008] The spacer arm is preferably a multi-arm polymer of polyethylene glycol (PEG) or polypropylene glycol (PPG) with a polyol as the core, the ends of the polyethylene glycol or polypropylene glycol being amino groups. Each hydroxyl group of the polyol is connected to a PEG chain or a PPG chain, and the hydroxyl group at the end of the chain is replaced with an amino group. The polyol is preferably pentaerythritol. The spacer arm can be a polyethylene glycol dendrimer with pentaerythritol as the core, or a polypropylene glycol dendrimer with pentaerythritol as the core. The terminal amino group of the dendrimer is used to form a new chemical bond by ring-opening reaction with the epoxy end of the epoxy-modified microspheres, forming a dendritic spacer arm. The most preferred is tetra-arm-polyethylene glycol-amino (CAS: 169501-65-9), with a molecular weight of 1000-10000, and the structure is shown in the following formula.

[0009]

[0010] The phenyl-based hydrophobic ligand preferably has a tricarboxyl functional group, such as 2,4,6-tris(4-carboxyphenyl)-1,3,5-triazine (CAS: 61414-16-2) or 1,3,5-trimethyl-2,4,6-tris(4-carboxyphenyl)benzene (CAS: 1246562-60-6). The most preferred is 2,4,6-tris(4-carboxyphenyl)-1,3,5-triazine, and the structure is shown in the following formula.

[0011]

[0012] The phenyl-based hydrophobic ligand is bonded to the surface of the matrix by amide condensation reaction (-COOH+-NH2→-CONH-+H2O), forming a three-dimensional network structure. The phenyl ligand of the network structure enhances hydrophobicity through π-π conjugation, shields ions and hydrogen bonding forces, and specifically binds to the hydrophobic pocket of recombinant albumin (containing Trp214, Phe215, etc. aromatic residues). The shielding of ions and hydrogen bonding forces includes reducing ionic interactions (such as the interaction between the positive charge of the amino group and the negative residues of recombinant albumin), and shielding hydrogen bonds (the hydrogen bond between the carbonyl group of the amide and the hydroxyl group of recombinant albumin).

[0013] The mass ratio of the spacer arm to the microspheres is preferably 1-2:1, and the amount of the phenyl-based hydrophobic ligand is preferably 1-2 times the molar ratio of the spacer arm.

[0014] The structure of the chromatography medium is preferably:

[0015]

[0016] The preparation method of the chromatography medium comprises the following steps:

[0017] (1) modifying the microspheres: under a nitrogen atmosphere, the microspheres and the epoxy compound are added to a solvent to obtain epoxy-modified microspheres;

[0018] (2) Grafting spacer arm: to the epoxy-modified microspheres, water, inorganic base and multi-arm polymer containing polyethylene glycol or polypropylene glycol are added, the multi-arm polymer has polyol as core and amino as terminal; grafting to obtain dendritic spacer arm-modified microspheres;

[0019] (3) Introducing hydrophobic ligand: to the dendritic spacer arm-modified microspheres, organic solvent, condensing agent and phenyl hydrophobic ligand are added, and a three-dimensional network strong hydrophobic chromatographic medium is connected.

[0020] In step (1), the epoxy compound is preferably epoxy silane or bis-epoxy compound, such as 3-glycidyloxypropyltriethoxysilane (CAS: 2602-34-8), 3-(2,3-epoxypropoxy)propyltrimethoxysilane (CAS: 2530-83-8), 3-glycidyloxypropylmethyldiethoxysilane (CAS: 2897-60-1), diglycidyl ether (CAS: 2238-07-5), 1,4-butanediol diglycidyl ether (CAS: 2425-79-8), ethylene glycol diglycidyl ether (CAS: 2224-15-9), and most preferably 3-glycidyloxypropyltriethoxysilane or diglycidyl ether. When the epoxy compound is bis-epoxy structure, an inorganic base selected from one or more of sodium hydroxide, potassium hydroxide, and the like, preferably sodium hydroxide, is also added. The molar ratio of inorganic base to epoxy compound is 0.5-1:1.

[0021] In step (1), the solvent is preferably one or more of methanol, ethanol, acetonitrile, toluene, N,N-dimethylformamide, tetrahydrofuran, dimethyl sulfoxide, 1,4-dioxane, and water, and most preferably toluene or water.

[0022] In step (1), the mass ratio of microspheres:epoxy compound:solvent is preferably 1:1-5:5-10. The reaction temperature is preferably 50-80°C, and the reaction time is preferably 12-24 h.

[0023] In step (2), the inorganic base is preferably one or more of sodium hydroxide, potassium hydroxide, and most preferably sodium hydroxide. The molar ratio of base to multi-arm polymer is preferably 1-5:1.

[0024] In step (2), the mass ratio of multi-arm polymer to microspheres is 1-2:1.

[0025] In step (3), the organic solvent is preferably one or more of methanol, ethanol, acetonitrile, toluene, N,N-dimethylformamide, tetrahydrofuran, dimethyl sulfoxide, 1,4-dioxane, and most preferably dimethyl sulfoxide.

[0026] In step (3), the condensing agent is preferably one or more of carbodiimides or onium salts, such as 1-ethyl-(3-dimethylaminopropyl)carbodiimide (CAS: 25952-53-8), diisopropylcarbodiimide (CAS: 693-13-0), carbonyldiimidazole (CAS: 530-62-1), 2-(7-azabenzotriazol)-N,N,N',N'-tetramethyluronium hexafluorophosphate (CAS: 148893-10-1), O-benzotriazol-tetramethyluronium hexafluorophosphate (CAS: 94790-37-1), O-benzotriazol-N,N,N',N'-tetramethyluronium tetrafluoroborate (CAS: 125700-67-6), and the amount of the condensing agent is 3-9 times the molar ratio of the phenyl hydrophobic ligand.

[0027] When the condensing agent is carbodiimide, a catalyst is also needed, preferably one or more of 4-dimethylaminopyridine (CAS: 1122-58-3), 4-pyrrolidinopyridine (CAS: 2456-81-7), N-hydroxysuccinimide (CAS: 6066-82-6), 1-hydroxybenzotriazole (CAS: 2592-95-2), 1-hydroxy-7-azabenzotriazole (CAS: 39968-33-7), and the amount of the catalyst is 3-9 times the molar ratio of the phenyl hydrophobic ligand.

[0028] When the condensing agent is onium salt, an organic base is also needed, preferably one or more of triethylamine, N,N-diisopropylethylamine, N-methylmorpholine, and the amount of the organic base is 3-9 times the molar ratio of the phenyl hydrophobic ligand; generally, onium salt condensing agents have high activity, and additional catalysts are often not needed, and the reaction is usually carried out under basic conditions.

[0029] In step (3), the phenyl hydrophobic ligand is preferably a phenyl hydrophobic ligand with a tricarboxyl functional group, such as 2,4,6-tris(4-carboxyphenyl)-1,3,5-triazine or 1,3,5-trimethyl-2,4,6-tris(4-carboxyphenyl)benzene; the amount of the phenyl hydrophobic ligand is 1-2 times the molar ratio of the multi-arm polymer.

[0030] Advantages: Compared with the prior art, the present application has the following significant advantages:

[0031] The chromatography medium contains highly branched dendritic structures, which provide more binding sites and have high adsorption capacity (BSA loading capacity up to 50 mg / mL). The chromatography medium has a unique three-dimensional structure, and there is strong π-π conjugation between the phenyl groups of the network structure, which not only maximizes the effective area of the hydrophobic ligand, significantly enhances the hydrophobicity, but also avoids non-specific binding such as ionic interaction and hydrogen bond by forming amide bonds, which is conducive to specific binding of recombinant albumin, improves selectivity (purity ≥ 99%) and yield (yield ≥ 95%), and provides an ideal solution for efficient and high-activity purification of biological macromolecules such as recombinant human blood albumin. The preparation method of the chromatography medium is simple and easy to operate, and is suitable for a wide range of raw materials, which is conducive to industrial production. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 Structure diagram of the dendritic spacer-based strong hydrophobic chromatography medium of Example 1;

[0033] Figure 2 BSA loading capacity spectrum of the medium of Example 1;

[0034] Figure 3 HPLC spectrum of recombinant albumin before purification of the hydrophobic chromatography medium;

[0035] Figure 4 HPLC spectrum of recombinant albumin after purification using the medium of Example 1. DETAILED DESCRIPTION

[0036] The technical solutions of the present application will be further described below in combination with the drawings.

[0037] Example 1

[0038] This embodiment provides a dendritic spacer-based strong hydrophobic chromatography medium, as shown in Figure 1 The chromatography medium uses epoxy-modified microspheres as carriers, the carriers are grafted with dendritic spacers, the ends of the spacers are connected with phenyl-based hydrophobic ligands, and the chromatography medium has a three-dimensional network structure.

[0039] The preparation method of the chromatography medium is as follows.

[0040] Step (1): Modify the microspheres: under a nitrogen atmosphere, add the microspheres and the epoxy compound to the solvent, and react to obtain epoxy-modified microspheres. The specific process is as follows.

[0041] Epoxy-silane coating modification: 10 g of polymethyl methacrylate (PMMA, particle size 45 μm) microspheres were dispersed in 50 g of toluene, 10 g of 3-glycidyloxypropyltriethoxysilane was added, stirring was performed at 120 r / min under a nitrogen atmosphere at 50 °C for 12 h, and then the PMMA microspheres were washed with ethanol and water in sequence until neutralization was achieved.

[0042] Step (2): grafting of a spacer arm: water, an inorganic base, and a multi-arm polymer containing polyethylene glycol or polypropylene glycol were added to the epoxy-modified microspheres, the multi-arm polymer had a polyol core and an amino group as a terminal end; and the microspheres were grafted to obtain dendritic spacer arm-modified microspheres. Details are as follows.

[0043] Dendritic spacer arm grafting: 20 mmol of four-arm-polyethylene glycol-amino (molecular weight 1000) was dispersed in 100 g of pure water, 3.2 g of sodium hydroxide was added in sequence, the mixture was uniformly dispersed, and then 10 g of epoxy-modified PMMA microspheres were added, stirring was performed at 120 r / min at 55 °C for 18 h, and then the microspheres were washed with pure water until neutralization was achieved, and the grafted dendritic spacer arm-modified PMMA microspheres were obtained after washing.

[0044] Step (3): introduction of a hydrophobic ligand: an organic solvent, a condensing agent, and a phenyl hydrophobic ligand were added to the dendritic spacer arm-modified microspheres, and a three-dimensional network strong hydrophobic chromatographic medium was obtained. Details are as follows.

[0045] Network strong hydrophobic ligand introduction: 60 mmol of 1-ethyl-(3-dimethylaminopropyl) carbodiimide, 60 mmol of 1-hydroxybenzotriazole, and 20 mmol of 2,4,6-tris(4-carboxyphenyl)-1,3,5-triazine were added in sequence to 50 g of dimethyl sulfoxide, stirring was performed at room temperature for 30 min, then 10 g of dendritic spacer arm-modified PMMA microspheres were added, stirring was performed at 120 r / min at 50 °C for 16 h, and then the microspheres were washed with ethanol and water in sequence until neutralization was achieved, and a three-dimensional network phenyl-modified PMMA hydrophobic chromatographic medium was obtained.

[0046] The loading capacity of the chromatographic medium was detected.

[0047] Detection method: 3.0 mg / mL of BSA was used.

[0048] Loading capacity calculation method: the loading capacity DBC (Dynamic binding capacity, i.e., the time t corresponding to 10% of the highest absorbance value, the amount of absorbed protein was calculated) was calculated at 10% breakthrough time. The formula is as follows.

[0049] DBC = (0.001 x 1.0 x t x C) / (V x 0.001), (mg / mL).

[0050] Wherein, 0.001: 0.001 in the molecule is the multiple introduced by converting mg into g, and 0.001 in the denominator refers to the multiple introduced by converting ml into L; 1.0: flow rate of sample loading, mL / min (converted according to the specifications of the chromatographic column, linear flow rate 180 cm / h); V: volume of chromatographic packing, mL; t: time corresponding to 10% of the maximum absorbance value, min; C: concentration in the sample solution, mg / mL.

[0051] The detection result is shown in Table 1. Figure 2 As shown in Table 1, MAX = 304.9 mAU, T = 54.6 min, and DBC is about (0.001 x 54.6 x 1.0 x 3.0) / (3.3 x 0.001) = 49.6 mg / mL.

[0052] Example 2

[0053] The present embodiment provides a strong hydrophobic chromatographic medium based on dendritic spacers.

[0054] The preparation method of the chromatographic medium is basically the same as that of Example 1, and the differences of each step are as follows.

[0055] Step (1): The base ball is replaced from polymethacrylate microspheres to dextran microspheres (particle size 90 μm).

[0056] Step (2): 20 mmol of four-arm-polyethylene glycol-amino (molecular weight 1000) is replaced by 2 mmol of four-arm-polyethylene glycol-amino (molecular weight 10000), and the amount of sodium hydroxide is replaced by 1.6 g.

[0057] Step (3): The amount of hydrophobic ligand 2,4,6-tris(4-carboxyphenyl)-1,3,5-triazine is replaced by 4 mmol, the condensing agent is replaced by 24 mmol O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate, no catalyst 1-hydroxybenzotriazole is added, and 36 mmol of organic base N,N-diisopropylethylamine is added.

[0058] Example 3

[0059] The present embodiment provides a strong hydrophobic chromatographic medium based on dendritic spacers.

[0060] The preparation method of the chromatographic medium is basically the same as that of Example 1, and the differences of each step are as follows.

[0061] Step (1): The base ball is replaced by silica microspheres (particle size 10 μm) from polymethacrylate microspheres, the amount of epoxy compound is replaced by 20 g of diglycidyl ether, 3.1 g of sodium hydroxide is added, the solvent is replaced by 100 g of water, and the reaction temperature is replaced by 55 °C.

[0062] Step (2): 20 mmol of four-arm-polyethylene glycol-amino (molecular weight 1000) is replaced by 10 mmol of four-arm-polyethylene glycol-amino (molecular weight 2000).

[0063] Step (3): The amount of hydrophobic ligand 2,4,6-tris(4-carboxyphenyl)-1,3,5-triazine is replaced by 15 mmol, the condensing agent is replaced by 50 mmol of 2-(7-oxobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate from 1-ethyl-(3-dimethylaminopropyl) carbonyldiimide, no catalyst 1-hydroxybenzotriazole is added, and 100 mmol of organic base N,N-diisopropylethylamine is added.

[0064] Example 4

[0065] The present embodiment provides a strong hydrophobic chromatography medium based on dendritic spacers.

[0066] The preparation method of the chromatography medium is basically the same as that of example 1, and the following is the difference of each step.

[0067] Step (1): The base ball is replaced by silica microspheres (particle size 10 μm) from polymethacrylate microspheres, the amount of epoxy compound is replaced by 20 g of diglycidyl ether, 3.1 g of sodium hydroxide is added, the solvent is replaced by 100 g of water, and the reaction temperature is replaced by 55 °C.

[0068] Step (2): 20 mmol of four-arm-polyethylene glycol-amino (molecular weight 1000) is replaced by 10 mmol of four-arm-polyethylene glycol-amino (molecular weight 2000).

[0069] Step (3): The amount of hydrophobic ligand 2,4,6-tris(4-carboxyphenyl)-1,3,5-triazine is replaced by 10 mmol, the condensing agent is replaced by 90 mmol of 2-(7-oxobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate from 1-ethyl-(3-dimethylaminopropyl) carbonyldiimide, no catalyst 1-hydroxybenzotriazole is added, and 90 mmol of organic base N,N-diisopropylethylamine is added, and the solvent is replaced by N,N-dimethylformamide from dimethyl sulfoxide.

[0070] Example 5

[0071] The embodiment provides a strong hydrophobic chromatography medium based on dendritic spacers.

[0072] The preparation method of the chromatography medium is basically same as that in embodiment 1, and the following is the difference of each step.

[0073] Step (1): the base ball is replaced from polymethyl methacrylate to polystyrene-divinylbenzene microspheres (particle size 45 μm), and the epoxy compound is replaced from 3-glycidyl ether oxypropyl triethoxysilane to 15 g γ-glycidyl ether propyl trimethoxysilane.

[0074] Step (2): 20 mmol of four-arm-polyethylene glycol-amino (molecular weight 1000) is replaced by 4 mmol of four-arm-polyethylene glycol-amino (molecular weight 5000).

[0075] Step (3): the amount of hydrophobic ligand 2,4,6-tris (4-carboxyphenyl)-1,3,5-triazine is replaced by 8 mmol, the condensing agent is replaced by 25 mmol 2-(7-oxidized benzotriazole)-N,N,N',N'-tetramethyl urea hexafluorophosphate, no catalyst 1-hydroxybenzotriazole is added, 25 mmol of organic base N,N-diisopropylethylamine is added, and the solvent is replaced from dimethyl sulfoxide to tetrahydrofuran.

[0076] Comparative example 1

[0077] The comparative example provides a hydrophobic chromatography medium, which is different from the chromatography medium in embodiment 1 in that the type and proportion of the hydrophobic ligand.

[0078] The preparation method of the chromatography medium is basically same as that in embodiment 1, and the difference lies in that 20 mmol of 2,4,6-tris (4-carboxyphenyl)-1,3,5-triazine in step (3) is replaced by 60 mmol of benzoic acid.

[0079] The obtained chromatography medium does not have a network structure.

[0080] Comparative example 2

[0081] The comparative example provides a hydrophobic chromatography medium, which is different from the chromatography medium in embodiment 1 in that no dendritic spacer is grafted.

[0082] The preparation method of the chromatography medium refers to embodiment 1, and the difference is as follows.

[0083] Step (1): the epoxy compound is replaced from 3-glycidyl ether oxypropyl triethoxysilane to 3-aminopropyl triethoxysilane (CAS: 919-30-2).

[0084] Step (2): The epoxy-modified microspheres obtained in step (1) are directly subjected to condensation reaction with a hydrophobic ligand, and other conditions remain unchanged.

[0085] Recombinant albumin purification applications

[0086] This example compares the loadings of the chromatography media of Examples 1-5 and Comparative Examples 1-2, and the effects of using the same for purifying recombinant albumin.

[0087] The method for detecting the loading is described in Reference Example 1.

[0088] The recombinant human blood albumin (rHSA, fermentation broth concentration 1.0 mg / mL, volume 100 mL) is produced by fermentation according to the patent CN200510068187.X, and is subjected to heating treatment, high-salt cation chromatography separation and weak anion chromatography separation to obtain the rHSA filtrate preliminarily purified.

[0089] The chromatography media obtained in the examples and comparative examples are respectively loaded into a chromatography column to prepare a hydrophobic chromatography column with a specification of a diameter of 6.6 mm, a height of 10 cm, and a compression ratio of 1.1. The instrument is a protein purification instrument Sep SCG03011040200. The rHSA filtrate is subjected to hydrophobic fine purification by the following method.

[0090] (1) Equilibration: flush with 5 column volumes of equilibration buffer (50 mM phosphate buffer + 200 mM sodium chloride + 10 mM arginine, pH 7.0) at a flow rate of 0.68 mL / min.

[0091] (2) Sample loading: pass the rHSA filtrate preliminarily purified (100 mL) through the column bed at a flow rate of 0.68 mL / min, and the adsorption is complete (the rHSA concentration of the effluent is ≤0.01 mg / mL).

[0092] (3) Elution: flush with 5 column volumes of equilibration buffer (50 mM phosphate buffer + 200 mM sodium chloride + 10 mM arginine, pH 7.0) at a flow rate of 1.0 mL / min to wash away the impurities on the column.

[0093] (4) Elution: flush with 5 column volumes of 50 mM phosphate buffer + 10 mM arginine (pH 7.0) at a flow rate of 1.5 mL / min to collect the protein effluent.

[0094] (5) Impurity removal: flush with 3 column volumes of 0.5 M sodium hydroxide to wash away the impurities on the medium.

[0095] (6) Flush with 5 column volumes of equilibration buffer containing 1.0 M sodium chloride to restore the strong hydrophobic state of the medium.

[0096] (7) Water wash: rinse with 5 column volumes of water, and the conductivity returns to the original steady state of the packing.

[0097] HPLC profile of rHSA filtrate before and after polishing.

[0098] Detection condition: analytical column: Zenix SEC-150 (3 pm 150 A, 7.8*300 mm). Mobile phase: A: 150 mM PB buffer, pH 7.0. Gradient: 0-20 min 100% A. Flow rate: 1.0 mL / min. Detector: UV 280 nm. Column temperature: room temperature. Injection volume: 10 uL. Instrument: high performance liquid chromatograph Shimadzu VP21-A.

[0099] Detection result: before purification using hydrophobic chromatography medium, the HPLC profile of recombinant albumin is shown in Figure 3 ; after purification using the medium of Example 1, the HPLC profile of recombinant albumin is shown in Figure 4 .

[0100] Result analysis: as shown in Table 1, the adsorption capacity of the hydrophobic chromatography medium of Examples 1-5 is significantly higher than that of the comparative example, and the synergistic effect of the dendritic structure providing more binding sites and the reticular phenyl group improves the recovery rate, and the mild elution condition (low ionic strength + small amount of arginine) avoids protein denaturation.

[0101] .

Claims

1. A strongly hydrophobic chromatography medium based on dendritic spacer arms, characterized in that, The chromatography medium uses epoxy-modified microspheres as a carrier, the carrier is grafted with dendritic spacer arms, and the ends of the spacer arms are connected to phenyl hydrophobic ligands; the chromatography medium is in the form of a three-dimensional network.

2. The chromatography medium according to claim 1, characterized in that, The spacer arm is a multi-arm polymer containing polyethylene glycol or polypropylene glycol with a polyol as the core, and the end of the polyethylene glycol or polypropylene glycol is an amino group.

3. The chromatography medium according to claim 2, characterized in that, The polyol is pentaerythritol.

4. The chromatography medium according to claim 1, characterized in that, The phenyl-type hydrophobic ligand has a tricarboxylic acid functional group.

5. The chromatography medium according to claim 4, characterized in that, The phenyl hydrophobic ligand is 2,4,6-tris(4-carboxyphenyl)-1,3,5-triazine or 1,3,5-trimethyl-2,4,6-tris(4-carboxyphenyl)benzene.

6. The chromatography medium according to claim 1, characterized in that, The mass ratio of spacer arms to microspheres is 1-2:1, and the amount of phenyl hydrophobic ligands used is 1-2 times the molar ratio of the spacer arms.

7. The chromatography medium according to claim 1, characterized in that, The structure is as follows:

8. A method for preparing the chromatography medium as described in any one of claims 1-7, characterized in that, Includes the following steps: (1) Modified microspheres: Under a nitrogen atmosphere, microspheres and epoxy compounds are added to a solvent and reacted to obtain epoxy-modified microspheres; (2) Grafting spacer arms: Water, inorganic base and multi-arm polymer containing polyethylene glycol or polypropylene glycol are added to the epoxy-modified microspheres, wherein the multi-arm polymer has a polyol as the core and an amino group as the end; grafting yields dendritic spacer arm modified microspheres; (3) Introducing hydrophobic ligands: Add organic solvents, condensing agents and phenyl hydrophobic ligands to the microspheres modified with dendritic spacer arms to obtain a three-dimensional network of strongly hydrophobic chromatography media.

9. The preparation method according to claim 8, characterized in that, In step (1), when the epoxy compound has a double epoxy structure, an inorganic base also needs to be added.

10. The preparation method according to claim 8, characterized in that, In step (3), the condensing agent is a carbodiimide or an onium salt; when the condensing agent is a carbodiimide, a catalyst is also required; when the condensing agent is an onium salt, an organic base is also required.

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