WO2019127686A1 - 组合型配基、组合型仿生层析介质及其制备方法和应用 - Google Patents

组合型配基、组合型仿生层析介质及其制备方法和应用 Download PDF

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WO2019127686A1
WO2019127686A1 PCT/CN2018/072780 CN2018072780W WO2019127686A1 WO 2019127686 A1 WO2019127686 A1 WO 2019127686A1 CN 2018072780 W CN2018072780 W CN 2018072780W WO 2019127686 A1 WO2019127686 A1 WO 2019127686A1
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combined
chromatography
ligand
biomimetic
chromatography medium
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French (fr)
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姚善泾
林东强
张其磊
邹徐俊
卢慧丽
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浙江大学
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    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/32—Bonded phase chromatography
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    • B01D—SEPARATION
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    • B01D15/08—Selective adsorption, e.g. chromatography
    • B—PERFORMING OPERATIONS; TRANSPORTING
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    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 - B01D15/36
    • B01D15/3804—Affinity chromatography
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    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 - B01D15/36
    • B01D15/3804—Affinity chromatography
    • B01D15/3809—Affinity chromatography of the antigen-antibody type, e.g. protein A, G, L chromatography
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    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 - B01D15/36
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    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
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    • B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
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    • B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
    • B01J20/3244—Non-macromolecular compounds
    • B01J20/3246—Non-macromolecular compounds having a well defined chemical structure
    • B01J20/3248—Non-macromolecular compounds having a well defined chemical structure the functional group or the linking, spacer or anchoring group as a whole comprising at least one type of heteroatom selected from a nitrogen, oxygen or sulfur, these atoms not being part of the carrier as such
    • B01J20/3251—Non-macromolecular compounds having a well defined chemical structure the functional group or the linking, spacer or anchoring group as a whole comprising at least one type of heteroatom selected from a nitrogen, oxygen or sulfur, these atoms not being part of the carrier as such comprising at least two different types of heteroatoms selected from nitrogen, oxygen or sulphur
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    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
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    • B01J20/3248—Non-macromolecular compounds having a well defined chemical structure the functional group or the linking, spacer or anchoring group as a whole comprising at least one type of heteroatom selected from a nitrogen, oxygen or sulfur, these atoms not being part of the carrier as such
    • B01J20/3253—Non-macromolecular compounds having a well defined chemical structure the functional group or the linking, spacer or anchoring group as a whole comprising at least one type of heteroatom selected from a nitrogen, oxygen or sulfur, these atoms not being part of the carrier as such comprising a cyclic structure not containing any of the heteroatoms nitrogen, oxygen or sulfur, e.g. aromatic structures
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    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
    • B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
    • B01J20/3244—Non-macromolecular compounds
    • B01J20/3246—Non-macromolecular compounds having a well defined chemical structure
    • B01J20/3248—Non-macromolecular compounds having a well defined chemical structure the functional group or the linking, spacer or anchoring group as a whole comprising at least one type of heteroatom selected from a nitrogen, oxygen or sulfur, these atoms not being part of the carrier as such
    • B01J20/3255—Non-macromolecular compounds having a well defined chemical structure the functional group or the linking, spacer or anchoring group as a whole comprising at least one type of heteroatom selected from a nitrogen, oxygen or sulfur, these atoms not being part of the carrier as such comprising a cyclic structure containing at least one of the heteroatoms nitrogen, oxygen or sulfur, e.g. heterocyclic or heteroaromatic structures
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
    • B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
    • B01J20/3285—Coating or impregnation layers comprising different type of functional groups or interactions, e.g. different ligands in various parts of the sorbent, mixed mode, dual zone, bimodal, multimodal, ionic or hydrophobic, cationic or anionic, hydrophilic or hydrophobic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
    • C07K1/1072—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups
    • C07K1/1077—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0802—Tripeptides with the first amino acid being neutral
    • C07K5/0812—Tripeptides with the first amino acid being neutral and aromatic or cycloaliphatic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man

Definitions

  • the invention relates to the field of biomimetic chromatography, in particular to a combined ligand, a combined biomimetic chromatography medium, a preparation method and application thereof.
  • the antibody has high targeting and high biocompatibility, and has great potential for development into drugs for treating cancer.
  • the upstream antibody expression and preparation scale are continuously improved, and the development of efficient downstream separation and purification technology is the key to the development of the antibody industry.
  • Protein A affinity chromatography is currently the most commonly used antibody capture method with high selectivity, but the medium is expensive, the elution conditions are harsh, and there is a risk of ligand shedding. Therefore, the development of alternative technologies has become a hot spot. Short peptide biomimetic chromatography and Hydrophobic resistance induction chromatography (HCIC) are two important ones.
  • Short peptide biomimetic chromatography is a novel biomimetic affinity chromatography method using a short peptide compound as a ligand.
  • the short peptide ligand is designed based on the target protein, and has high selectivity, stability and biocompatibility.
  • U.S. Patent No. 7,408,030 B2 discloses a hexapeptide ligand HWRGWV for the isolation of antibodies in serum, ascites, cell culture fluids and milk. However, the charged arginine in the ligand will adsorb the serum albumin and reduce the selectivity of the ligand. Only at higher salt concentration or sodium octanoate can be obtained to obtain higher purity IgG (J.Chromatogr.
  • the Chinese invention patent (CN 104645949 A) discloses a tetrapeptide ligand YFRH with high IgG adsorption capacity and salt tolerance, and mild elution conditions.
  • charged arginine is also present in the ligand, and the adsorption amount of serum albumin is also large under the condition of pH greater than 4, which affects the selectivity of the medium to IgG (Biochem. Eng. J., 114: 191–201, 2016). Therefore, the polypeptide ligands need to be further optimized to maintain both good IgG binding selectivity and mild elution conditions.
  • HCIC was proposed by Burton and Harding (J. Chromatogr. A, 71:81, 1998).
  • the ligand has both hydrophobic and ionized groups.
  • the protein is bound by hydrophobic interaction under neutral pH conditions, and the pH of the solution is adjusted to make the protein and ligand. Electrostatic repulsion occurs to achieve elution.
  • the HCIC ligands have been reported to include a ruthenium compound (CN 101036877 A), an imidazole compound (CN 101185882 A), a compound consisting of imidazole and benzene (CN 101185881 A), etc., which have strong salt-tolerant adsorption characteristics and elution conditions.
  • HCIC medium for antibody separation with aminobenzimidazole as a functional ligand, which has high antibody binding ability and non-salt-dependent adsorption performance.
  • HCIC ligands also have some defects, the ligand structure is relatively simple, and the selectivity to antibodies is not high. For antibodies of different origins, a large amount of process optimization is required, and it is difficult to separate high-purity antibodies from complex feed liquids.
  • the object of the present invention is to provide a combined ligand with the phenylalanine-tyrosine-glutamine tripeptide and aminobenzimidazole functional groups, while retaining the polypeptide, in view of the deficiencies of the prior art.
  • the high selectivity of the ligand to the antibody introduces a hydrophobic charge-inducing ligand, which makes the elution conditions milder and enables efficient antibody isolation.
  • the combination ligand of the present invention comprises a tripeptide and a heterocyclic small molecule, and the key residues of the protein A and antibody Fc binding sites are analyzed and evaluated by means of computer molecular modeling, and the tripeptide-heterocyclic ring is screened and designed.
  • a tripeptide-heterocyclic small molecule can be synthesized by a chemical synthesis method in the prior art, and the sequence is phenylalanine-tyrosine-glutamine-5-aminobenzimidazole.
  • the present invention also provides a combined biomimetic chromatography medium comprising a chromatography matrix and a combined ligand, wherein the chromatography matrix is a hydrophilic porous microsphere with a hydroxyl group; the sequence of the combination ligand is benzene Alanine-tyrosine-glutamine-5-aminobenzimidazole;
  • the chromatography matrix is a hydrophilic microsphere having a porous structure and a surface hydroxyl group, and the structural formula is as follows:
  • the present invention obtains a combined biomimetic chromatography medium by coupling a combination ligand to a chromatography substrate, wherein the combination ligand has both a phenylalanine-tyrosine-glutamine tripeptide and an aminobenzimidazole.
  • a functional group mimics the key residues that specifically bind to the protein A ligand and the antibody Fc fragment by molecular simulation, and optimizes the design of the phenylalanine-tyrosine-glutamine tripeptide to make the ligands more High antibody selectivity; on the other hand, the introduction of hydrophobic charge-inducing ligand-5-aminobenzimidazole enhances hydrophobic interaction and assists protein dissociation by electrostatic repulsion by adjusting the pH of the solution, reducing elution difficulty and improving washing Remove the condition.
  • the chromatography matrix is an agarose gel or cellulose microspheres.
  • the invention also provides a preparation method of the combined bionic chromatography medium as described above, comprising the following steps:
  • the chromatography substrate is activated by allyl bromide to obtain an activated chromatography matrix
  • the reaction process in step 1) is as follows:
  • the reaction process in step 2) is as follows:
  • step 3 The reaction process in step 3) is as follows:
  • the activation reaction in the step 1) comprises: mixing the chromatography substrate, the dimethyl sulfoxide solution, the allyl bromide and the sodium hydroxide, reacting in a water bath in a shaker, suction filtration, and washing to obtain activated chromatography. Matrix.
  • the activation reaction in the step 1) comprises: after the chromatography substrate is drained, adding 0.5 to 1.5 times the mass of the matrix substrate of 18-22% (v/v) dimethyl sulfoxide solution, 0.1-1.0 times Chromatographic substrate mass of allyl bromide and 0.1-0.5 times chromatographic substrate mass of sodium hydroxide, 28-32 ° C water bath, 140-160 rpm shaking in a shaker for 24-48 hours, suction filtration, washing with deionized water , an activated chromatography matrix is obtained.
  • the bromination reaction in the step 2) comprises: mixing the activated chromatography matrix, acetone and N-bromosuccinimide, reacting in a water bath in a shaker, suction filtration, washing to obtain bromohydrinization.
  • the matrix is selected from the group consisting of: acetone and N-bromosuccinimide, reacting in a water bath in a shaker, suction filtration, washing to obtain bromohydrinization.
  • the bromination reaction in the step 2) comprises: taking an activation chromatography matrix, adding 1.0-3.0 times the matrix mass of 45-55% (v/v) acetone and 0.1-0.3 times the matrix mass of N-bromine.
  • the butyl succinimide was reacted in a 28-32 ° C water bath at 140-160 rpm on a shaker for 1-3 hours, suction filtered, and washed with deionized water to give a bromolated base.
  • the coupling reaction in the step 3) comprises: dissolving the bromohydrinated substrate and the combined ligand in dimethyl sulfoxide, adding the sodium carbonate buffer, mixing, and reacting in a water bath in a shaker. Filtration and washing to obtain a combined biomimetic chromatography medium; the mass ratio of the bromo-alcoholized substrate to the combined ligand is 1:0.1-0.3.
  • the coupling reaction in the step 3) comprises: taking a brominated alcoholic substrate in the reactor, and weighing 0.1-0.3 times the matrix mass of the phenylalanine-tyrosine-glutamine-5-amino group.
  • the benzimidazole combination ligand is dissolved in 0.5-1.0 times the matrix mass of dimethyl sulfoxide, mixed with 1.0-3.0 times 0.8-1.2M sodium carbonate buffer, and then added to the reactor, 28-32 ° C water bath, The reaction was carried out for 8-12 h in a shaker at 140-160 rpm, suction filtered, and repeatedly filtered with ionized water, 0.08-0.12 M HCl, 0.08-0.12 M NaOH to obtain a combined bionic chromatography medium.
  • the combined type biomimetic chromatography medium in the step 3) is continuously subjected to a blocking reaction using an aqueous ethanolamine solution.
  • the blocking reaction comprises: adding a combined biomimetic chromatography medium to an aqueous ethanolamine solution, controlling pH 8.0, and reacting in a water bath in a shaker.
  • the blocking reaction comprises: adding the combined biomimetic chromatography medium to a 0.8-1.2 M aqueous ethanolamine solution (pH 8.0) containing 1.0-5.0 times the mass of the medium, a water bath at 20-30 ° C, and a shaker at 140-160 rpm. The reaction was carried out for 4-8 hours, washed with deionized water and stored in 18-22% (v/v) ethanol solution.
  • aqueous ethanolamine solution pH 8.0
  • the invention also provides the use of a combined biomimetic chromatography medium as described above for isolating antibodies.
  • the density of the combined ligand is controllable, and by adjusting the mass ratio of the bromohydrinated substrate to the combined ligand, different ligand density media can be prepared, up to 70 ⁇ mol/g medium.
  • the combined bionic chromatography medium of the present invention has high antibody affinity, large adsorption capacity, static adsorption capacity of 80 mg/g or more, and dynamic loading of 20 mg/ml or more.
  • the combined bionic chromatography medium of the present invention has high antibody selectivity and extremely low adsorption amount of serum albumin.
  • the combined bionic chromatography medium of the present invention has a mild elution condition, and the pH of the solution is adjusted to 4.0-5.0.
  • the electrostatic repulsion between the ligand and the protein efficient elution of the protein can be achieved, and the peracid is avoided. Structure and activity have an adverse effect.
  • the combined bionic chromatography medium of the present invention has stable performance, convenient cleaning and regeneration, and can be reused more than 100 times.
  • Example 1 is a high performance liquid chromatogram of the combination type ligand in Example 1;
  • Example 2 is a mass spectrum of the combination type ligand in Example 1;
  • FIG. 3 is a graph showing a comparison of penetration curves of human IgG and human serum albumin (HSA) in Application Example 1;
  • Figure 5 is a high performance liquid chromatogram of the mixed protein separation material and elution component in Application Example 3;
  • Fig. 6 is a graph showing changes in dynamic load after application of different number of cycles in Application Example 4.
  • the key residues of protein A and antibody Fc binding sites were analyzed and evaluated by means of computer molecular modeling.
  • the combined ligands of tripeptide-heterocyclic small molecules were screened and designed, and the sequence was phenylalanine-tyrosine. Acid-glutamine-5-aminobenzimidazole.
  • the combined ligand can be synthesized by a chemical synthesis method in the prior art, and the combined ligand in the present embodiment is commissioned by Peptide Biochemical Co., Ltd.
  • the activated chromatography matrix 6.0 g of 50% (v/v) acetone and 0.9 g of N-bromosuccinimide were mixed for bromohydration, shaken at 150 rpm for 3 h at 30 ° C, suction filtered, deionized Wash with water to give a bromo-alcoholized substrate.
  • the medium was added to 9.0 g of a 1.0 M aqueous solution of ethanolamine (pH 8.0), reacted at 25 ° C for 45 hours in a shaker at 150 rpm, and washed with deionized water to obtain a combined biomimetic chromatography medium.
  • the remaining ligand content in the mother liquor after the reaction was analyzed by high performance liquid chromatography to be 0.228 g, indicating that 0.072 g of the ligand was coupled to the medium.
  • the medium ligand density was calculated to be 42 ⁇ mol/g medium by mass balance calculation, and the saturated adsorption capacity of human immunoglobulin was 85 mg/ml medium.
  • the activated chromatography substrate 3.0 g of 50% (v/v) acetone and 0.3 g of N-bromosuccinimide were mixed for bromohydration, shaken at 150 rpm for 1 h at 30 ° C, suction filtered, deionized Wash with water to give a bromo-alcoholized substrate.
  • the medium was added to 3.0 g of a 1.0 M aqueous solution of ethanolamine (pH 8.0), and reacted at 25 ° C for 45 hours in a shaker at 150 rpm, and washed with deionized water to obtain a combined type of bionic chromatography medium.
  • the remaining ligand content in the mother liquor after the reaction was analyzed by high performance liquid chromatography to be 0.259 g, indicating that 0.041 g of the ligand was coupled to the medium.
  • the medium ligand density was calculated to be 24 ⁇ mol/g medium by the material balance calculation, and the saturated adsorption capacity of human immunoglobulin was 65 mg/ml medium.
  • the activated chromatography substrate 9.0 g of 50% (v/v) acetone and 0.9 g of N-bromosuccinimide were mixed for bromohydration, shaken at 150 rpm for 3 h at 30 ° C, suction filtered, deionized Wash with water to give a bromo-alcoholized substrate.
  • the medium was added to 15.0 g of a 1.0 M aqueous solution of ethanolamine (pH 8.0), and reacted at 25 ° C for 15 hours in a shaker at 150 rpm, and washed with deionized water to obtain a combined type of bionic chromatography medium.
  • the residual ligand content in the mother liquor after the reaction was analyzed by high performance liquid chromatography to be 0.775 g, indicating that 0.125 g of the ligand was coupled to the medium.
  • the medium ligand density was calculated to be 73 ⁇ mol/g medium by the material balance calculation, and the saturated adsorption capacity of human immunoglobulin was 92 mg/ml medium.
  • the activated chromatography substrate 6.0 g of 50% (v/v) acetone and 0.6 g of N-bromosuccinimide were mixed for bromohydration, shaken at 150 rpm for 2 h at 30 ° C, suction filtered, deionized Wash with water to give a bromo-alcoholized substrate.
  • the medium was added to 9.0 g of a 1.0 M aqueous solution of ethanolamine (pH 8.0), and reacted for 6 hours at 25 ° C in a 150 rpm shaker, and washed with deionized water to obtain a combined type of bionic chromatography medium.
  • the residual ligand content in the mother liquor after the reaction was analyzed by high performance liquid chromatography to be 0.816 g, indicating that 0.084 g of the ligand was coupled to the medium.
  • the medium ligand density was calculated to be 49 ⁇ mol/g medium by mass balance calculation, and the saturated adsorption capacity of human immunoglobulin was 88 mg/ml medium.
  • the activated chromatography matrix 9.0 g of 50% (v/v) acetone and 0.9 g of N-bromosuccinimide were mixed for bromohydration, shaken at 150 rpm for 1 h at 30 ° C, suction filtered, deionized Wash with water to give a bromo-alcoholized substrate.
  • the medium was added to 9.0 g of a 1.0 M aqueous solution of ethanolamine (pH 8.0), reacted at 25 ° C for 45 hours in a shaker at 150 rpm, and washed with deionized water to obtain a combined biomimetic chromatography medium.
  • the remaining ligand content in the mother liquor after the reaction was analyzed by high performance liquid chromatography to be 0.254 g, indicating that 0.046 g of the ligand was coupled to the medium.
  • the medium ligand density was calculated by material balance to be 27 ⁇ mol/g medium, and the saturated adsorption capacity of human immunoglobulin was 70 mg/ml medium.
  • the activated chromatography matrix 6.0 g of 50% (v/v) acetone and 0.9 g of N-bromosuccinimide were mixed for bromohydration, shaken at 150 rpm for 3 h at 30 ° C, suction filtered, deionized Wash with water to give a bromo-alcoholized substrate.
  • the medium was added to 9.0 g of a 1.0 M aqueous solution of ethanolamine (pH 8.0), reacted at 25 ° C for 45 hours in a shaker at 150 rpm, and washed with deionized water to obtain a combined biomimetic chromatography medium.
  • the remaining ligand content in the mother liquor after the reaction was analyzed by high performance liquid chromatography to be 0.232 g, indicating that 0.068 g of the ligand was coupled to the medium.
  • the medium ligand density was calculated to be 40 ⁇ mol/g medium by the material balance calculation, and the saturated adsorption capacity of human immunoglobulin was 80 mg/ml medium.
  • the chromatographic medium obtained in Example 2 was taken and filled with 1 ml of medium in a Tricorn 5/100 column.
  • the explorer 100 chromatography system measures the protein breakthrough curve.
  • the chromatographic medium obtained in Example 2 was taken up, and 1 ml of the medium was loaded on a Tricorn 5/100 column.
  • the explorer 100 chromatography system measures the separation ability of mixed proteins.
  • a mixed protein solution containing 1 mg/ml of human immunoglobulin IgG and 4 mg/ml of human serum albumin (HSA) was prepared as a sample solution, and the pH was adjusted to 7.0. After 20 mM phosphate buffer (pH 7.0) was used as the equilibration buffer, the bed was fully equilibrated, and 5 ml of the mixed protein solution was applied at a flow rate of 0.5 ml/min. After the sample was completed, it was washed with 20 mM phosphate buffer (pH 7.0).
  • the chromatographic medium obtained in Example 7 was taken and filled with 1 ml of medium in a Tricorn 5/100 column.
  • the explorer 100 chromatography system measures the separation ability of mixed proteins.
  • a mixed protein solution containing 1 mg/ml of human immunoglobulin IgG and 4 mg/ml of human serum albumin (HSA) was prepared as a sample solution, and the pH was adjusted to 7.0. After 20 mM phosphate buffer (pH 7.0) was used as the equilibration buffer, the bed was fully equilibrated, and 5 ml of the mixed protein solution was applied at a flow rate of 0.5 ml/min. After the completion of the sample, rinse with 20 mM phosphate buffer (pH 7.0).
  • the baseline was then eluted with 20 mM acetate buffer (pH 4.0) and the protein concentration of the effluent was detected at 280 nm using a UV detector and the eluted fractions were collected.
  • the collected fractions were subjected to HPLC analysis, and the results are shown in Fig. 5. By calculation, the purity of IgG was 89.1%, and the yield was 92.8%.
  • the chromatographic medium obtained in Example 2 was taken up, and 1 ml of the medium was loaded on a Tricorn 5/100 column.
  • the explorer 100 chromatography system measures the protein breakthrough curve.
  • a 2 mg/ml human immunoglobulin IgG solution was prepared as a sample solution, and the pH was adjusted to 7.0. After 20 mM phosphate buffer (pH 7.0) was used as the equilibration buffer, the bed was fully equilibrated and loaded at a flow rate of 0.5 ml/min to 90% penetration of the protein. The protein concentration of the effluent was detected by ultraviolet detector at 280 nm. The dynamic loading of 10% penetration was calculated based on the loading volume at which the protein penetrated 10%. The medium was subjected to a loading-rinsing-elution-regeneration cycle of 20 times, 50 times and 100 times, and the above operation was repeated to determine the dynamic loading of IgG.
  • the dynamic loadings of IgG at 1, 20, 50, and 100 cycles were 22.68 mg/ml medium, 22.31 mg/ml medium, 22.21 mg/ml medium, and 21.89 mg/ml medium, after 100 cycles of use. The amount is only reduced by 3.5%, and the specific curve is shown in Figure 6.

Abstract

本发明涉及一种组合型配基、组合型仿生层析介质及其制备方法和应用，其中组合型仿生层析介质以亲水性多孔微球为层析基质，采用烯丙基溴活化，N-溴代丁二酰亚胺溴代醇化，然后偶联组合型配基。组合型配基的序列为苯丙氨酸-酪氨酸-谷氨酰胺-5-氨基苯并咪唑。该组合型仿生层析介质同时具有苯丙氨酸-酪氨酸-谷氨酰胺三肽和氨基苯并咪唑两种功能基团，在保留多肽配基对抗体选择性高的特点上，引入疏水电荷诱导配基，使得洗脱条件更加温和，能实现有效地抗体分离。

Description

组合型配基、组合型仿生层析介质及其制备方法和应用 技术领域
本发明涉及仿生层析领域，具体涉及一种组合型配基、组合型仿生层析介质及其制备方法和应用。
背景技术
抗体的靶向性强、生物相容性高，具有开发成治疗癌症等药物的极大潜力。随着抗体工程地不断发展，上游抗体表达和制备规模不断提高，开发高效的下游分离纯化技术是抗体产业发展的关键。
目前蛋白A亲和层析是最为常用的抗体捕获方法，具有很高的选择性，但是介质价格昂贵，洗脱条件苛刻，且存在配基脱落的风险。因此，开发替代技术成为当前的热点，短肽仿生层析和疏水电荷诱导层析(Hydrophobic charge induction chromatography,HCIC)是其中重要的两种。
短肽仿生层析是以短肽化合物作为配基的一种新型仿生亲和层析方法，短肽配基基于目标蛋白而设计，具有较高的选择性、稳定性和生物相容性好。美国专利(US 7408030 B2)公开了六肽配基HWRGWV，用于血清、腹水、细胞培养液和牛奶等料液中分离抗体。但是，配基中带有电荷的精氨酸会对血清白蛋白产生吸附，降低配基的选择性，只有在较高盐浓度或者添加辛酸钠时才能得到纯度较高的IgG(J.Chromatogr.A,1218:1691-1700,2011)。随着计算机分子模拟技术的引入，加速了多肽配基的筛选和优化设计。基于蛋白A亲和模型，中国发明专利(CN 103014880 A)公开了八肽配基FYWHCLDE，体现出良好的IgG分离性能。不过，相关机理研究发现IgG结合主要依靠静电 相互作用，需要通过添加NaCl进行洗脱(J.Chromatogr.A,1359:100-111,2014)。基于蛋白A的Fc片段结合位点的分子模拟，中国发明专利(CN 104645949 A)公开了四肽配基YFRH，具有较高的IgG吸附容量和耐盐特性，洗脱条件温和。但是，该配基中也存在带电的精氨酸，在pH大于4的条件下对血清白蛋白的吸附量也较大，影响了介质对IgG的选择性(Biochem.Eng.J.,114:191–201,2016)。因此，多肽配基还需要进一步优化设计，既保持良好的IgG结合选择性，又需要具有温和的洗脱条件。
HCIC由Burton和Harding提出(J.Chromatogr.A,71:81,1998)，配基兼具疏水和离子化基团，中性pH条件下通过疏水作用结合蛋白，调节溶液pH使得蛋白和配基间产生静电排斥作用，从而实现洗脱。美国专利(US 5652348 B2,US 7144743 B2)描述了HCIC介质的制备方法，指出在低盐和高盐条件下均能实现蛋白的有效结合。已报道的HCIC配基包括吲哚化合物(CN 101036877 A)、咪唑化合物(CN 101185882 A)、咪唑和苯组成的化合物(CN 101185881 A)等，具有较强的耐盐吸附特性，洗脱条件也较温和。中国发明专利(CN 104096544 A)报道了一种用于抗体分离的HCIC介质，以氨基苯并咪唑为功能配基，具有较高的抗体结合能力和非盐依赖吸附性能。但是，HCIC配基也存在一些缺陷，配基结构较单一，对抗体的选择性不高，对于不同来源的抗体，需要大量的过程优化，从复杂料液中分离得到高纯度的抗体较为困难。
综上所述，短肽仿生层析和HCIC各有优缺点。中国发明专利(CN 104117345 A)提出了色氨酸和氨基苯并咪唑组成的双功能基团介质，在保留氨基苯并咪唑的HCIC特点的同时，引入色氨酸后，在一定程度上提高了对抗体的选择性，不过效果比较有限，血清白蛋白仍会部分吸附(J.Chromatogr.A,258:264,2016)。
发明内容
本发明的目的在于针对现有技术的不足，提供一种组合型配基，同时具有苯丙氨酸-酪氨酸-谷氨酰胺三肽和氨基苯并咪唑两种功能基团，在保留多肽配基对抗体选择性高的特点上，引入疏水电荷诱导配基，使得洗脱条件更加温和，能实现有效地抗体分离。
本发明所提供的技术方案为：
一种组合型配基，结构式如下：
Figure PCTCN2018072780-appb-000001
本发明中的组合型配基包括三肽和一个杂环小分子，借助计算机分子模拟的手段，对蛋白A和抗体Fc结合位点的关键残基进行分析评估，筛选和设计三肽-杂环小分子的组合型配基，可以采用现有技术中的化学合成方法合成三肽-杂环小分子，序列为苯丙氨酸-酪氨酸-谷氨酰胺-5-氨基苯并咪唑。
本发明还提供一种组合型仿生层析介质，包括层析基质和组合型配基，所述层析基质为带有羟基的亲水性多孔微球；所述组合型配基的序列为苯丙氨酸-酪氨酸-谷氨酰胺-5-氨基苯并咪唑；
所述组合型配基的结构式如下：
Figure PCTCN2018072780-appb-000002
所述组合型仿生层析介质的结构式如下：
Figure PCTCN2018072780-appb-000003
本发明中组合型仿生层析介质的结构式中仅给出一个组合型配基基团，仅仅是示例性说明，层析基质的表面和内部孔道表面具有大量的组合型配基基团。
本发明中层析基质为具有多孔结构和表面羟基的亲水性微球，结构式如下：
Figure PCTCN2018072780-appb-000004
结构式仅给出一个-OH，仅仅是示例性说明，其表面具有大量的-OH。
本发明通过将组合型配基偶联到层析基质上得到组合型仿生层析介质，其中组合型配基同时具有苯丙氨酸-酪氨酸-谷氨酰胺三肽和氨基苯并咪唑两种功能基团，一方面通过分子模拟来模仿蛋白A配基与抗体Fc片段特异性结合的关键残基，优化设计苯丙氨酸-酪氨酸-谷氨酰胺三肽，使配基具有较高的 抗体选择性；另一方面引入疏水性电荷诱导配基-5-氨基苯并咪唑，强化疏水作用，且通过调节溶液pH，利用静电排斥作用协助蛋白解离，降低洗脱难度，改善洗脱条件。
作为优选，所述层析基质为琼脂糖凝胶或纤维素微球。
本发明还提供一种如上述的组合型仿生层析介质的制备方法，包括如下步骤：
1)层析基质采用烯丙基溴进行活化反应，得到活化层析基质；
步骤1)中反应过程如下：
Figure PCTCN2018072780-appb-000005
2)活化层析基质采用N-溴代丁二酰亚胺进行溴代醇化反应，得到溴代醇化的基质；
步骤2)中反应过程如下：
Figure PCTCN2018072780-appb-000006
3)将溴代醇化的基质与组合型配基进行偶联反应，得到组合型仿生层析介质；
步骤3)中反应过程如下：
Figure PCTCN2018072780-appb-000007
作为优选，所述步骤1)中活化反应包括：将层析基质、二甲基亚砜溶液、烯丙基溴和氢氧化钠混合，在摇床中水浴反应，抽滤，洗涤得到活化层析基质。
进一步优选，所述步骤1)中活化反应包括：层析基质抽干后，加入0.5-1.5倍层析基质质量的18-22％(v/v)二甲基亚砜溶液、0.1-1.0倍层析基质质量的烯丙基溴和0.1-0.5倍层析基质质量的氢氧化钠，28-32℃水浴，140-160rpm转速摇床中反应24-48小时，抽滤，用去离子水洗涤，得到活化层析基质。
作为优选，所述步骤2)中溴代醇化反应包括：将活化层析基质、丙酮和N-溴代丁二酰亚胺混合，在摇床中水浴反应，抽滤，洗涤，得到溴代醇化的基质。
进一步优选，所述步骤2)中溴代醇化反应包括：取活化层析基质，加入1.0-3.0倍基质质量的45-55％(v/v)丙酮和0.1-0.3倍基质质量的N-溴代丁二酰亚胺，28-32℃水浴，140-160rpm转速摇床中反应1-3小时，抽滤，用去离 子水洗涤，得到溴代醇化的基质。
作为优选，所述步骤3)中偶联反应包括：将溴代醇化的基质与组合型配基溶解于二甲亚砜中，再加入碳酸钠缓冲液混合后，在摇床中水浴反应，抽滤，洗涤，得到组合型仿生层析介质；所述溴代醇化的基质与组合型配基的质量比为1:0.1-0.3。
进一步优选，所述步骤3)中偶联反应包括：取溴代醇化的基质于反应器中，称取0.1-0.3倍基质质量的苯丙氨酸-酪氨酸-谷氨酰胺-5-氨基苯并咪唑组合型配基，溶解于0.5-1.0倍基质质量的二甲亚砜中，再与1.0-3.0倍0.8-1.2M的碳酸钠缓冲液混合后加入反应器，28-32℃水浴，140-160rpm转速摇床中反应8-12h，抽滤，用离子水、0.08-0.12M HCl、0.08-0.12M NaOH反复抽滤冲洗，得到组合型仿生层析介质。
作为优选，所述步骤3)中组合型仿生层析介质继续采用乙醇胺水溶液进行封闭反应。
作为优选，所述封闭反应包括：将组合型仿生层析介质加入到乙醇胺水溶液中，控制pH8.0，在摇床中水浴反应。
进一步优选，所述封闭反应包括：将组合型仿生层析介质加入到含有1.0-5.0倍介质质量的0.8-1.2M乙醇胺水溶液(pH 8.0)中，20-30℃水浴，140-160rpm摇床中反应4-8小时，去离子水洗涤，并保存在18-22％(v/v)乙醇溶液中。
本发明还提供一种如上述的组合型仿生层析介质在分离抗体中的应用。
同现有技术相比，本发明的有益效果体现在：
(1)本发明中组合型配基的密度可控，通过调节溴代醇化的基质与组合型配基的质量比，可制备不同配基密度介质，最高可达到70μmol/g介质以上。
(2)本发明的组合型仿生层析介质的抗体亲和力高，吸附容量大，静态 吸附容量达到80mg/g介质以上，动态载量达到20mg/ml介质以上。
(3)本发明的组合型仿生层析介质的抗体选择性强，血清白蛋白的吸附量极低。
(4)本发明的组合型仿生层析介质洗脱条件温和，调节溶液pH至4.0-5.0，借助配基-蛋白间的静电排斥力，就可实现蛋白的高效洗脱，避免过酸对抗体结构和活性产生不良影响。
(5)本发明的组合型仿生层析介质的性能稳定，清洗再生方便，可以重复使用100次以上。
附图说明
图1为实施例1中组合型配基的高效液相色谱图；
图2为实施例1中组合型配基的质谱图；
图3为应用例1中人IgG和人血清白蛋白(HSA)的穿透曲线比较图；
图4为应用例2中混和蛋白分离原料和洗脱组分的高效液相色谱图；
图5为应用例3中混和蛋白分离原料和洗脱组分的高效液相色谱图；
图6为应用例4中使用不同循环次数后的动态载量变化图。
具体实施方式
下面结合具体的实施例对本发明作进一步说明。
实施例1：组合型配基的制备
借助计算机分子模拟的手段，对蛋白A和抗体Fc结合位点的关键残基进行分析评估，筛选和设计三肽-杂环小分子的组合型配基，其序列为苯丙氨酸-酪氨酸-谷氨酰胺-5-氨基苯并咪唑。
组合型配基，结构式如下：
Figure PCTCN2018072780-appb-000008
组合型配基可以采用现有技术中的化学合成方法合成，本实施例中的组合型配基委托中肽生化有限公司制备。
针对实施例1中的组合型配基进行高效液相色谱和质谱表征，分别如图1和2所示。
实施例2：组合型仿生层析介质的制备
取抽干琼脂糖凝胶3.0g，加入3.0g 20％(v/v)二甲基亚砜、1.5g烯丙基溴和0.6g氢氧化钠，30℃下150rpm摇床活化24小时，抽滤，用去离子水洗涤得到活化的层析基质。
将活化的层析基质、6.0g 50％(v/v)丙酮和0.9g N-溴代丁二酰亚胺混合进行溴代醇化，30℃下150rpm摇床反应3h，抽滤，用去离子水洗涤，得到溴代醇化的基质。
1.5g二甲基亚砜和3.0g 1M碳酸钠缓冲液混合，加入0.3g苯丙氨酸-酪氨酸-谷氨酰胺-5-氨基苯并咪唑配基，充分溶解，再加入溴代醇化的层析基质，30℃下150rpm摇床反应12小时，用去离子水、0.1M HCl、0.1M NaOH反复抽滤冲洗，得到配基偶联的介质。
最后将介质加入到9.0g 1.0M乙醇胺水溶液(pH 8.0)中，25℃下150rpm摇床中反应4小时，去离子水洗涤，得到组合型仿生层析介质。
采用高效液相色谱分析反应后母液中的剩余配基含量0.228g，说明有 0.072g配基偶联到介质上。
通过物料平衡计算得到介质配基密度为42μmol/g介质，人免疫球蛋白的饱和吸附容量为85mg/ml介质。
实施例3：组合型仿生层析介质的制备
取抽干琼脂糖凝胶3.0g，加入1.5g 20％(v/v)二甲基亚砜、0.3g烯丙基溴和0.3g氢氧化钠，30℃下150rpm摇床活化24小时，抽滤，用去离子水洗涤得到活化的层析基质。
将活化的层析基质、3.0g 50％(v/v)丙酮和0.3g N-溴代丁二酰亚胺混合进行溴代醇化，30℃下150rpm摇床反应1h，抽滤，用去离子水洗涤，得到溴代醇化的基质。
1.5g二甲基亚砜和3.0g 1M碳酸钠缓冲液混合，加入0.3g苯丙氨酸-酪氨酸-谷氨酰胺-5-氨基苯并咪唑配基，充分溶解，再加入溴代醇化的层析基质，30℃下150rpm摇床反应8小时，用去离子水、0.1M HCl、0.1M NaOH反复抽滤冲洗，得到配基偶联的介质。
最后将介质加入到3.0g 1.0M乙醇胺水溶液(pH 8.0)中，25℃下150rpm摇床中反应4小时，去离子水洗涤，得到组合型仿生层析介质。
采用高效液相色谱分析反应后母液中的剩余配基含量0.259g，说明有0.041g配基偶联到介质上。
通过物料平衡计算得到介质配基密度为24μmol/g介质，人免疫球蛋白的饱和吸附容量为65mg/ml介质。
实施例4：组合型仿生层析介质的制备
取抽干琼脂糖凝胶3.0g，加入4.5g 20％(v/v)二甲基亚砜、3.0g烯丙基 溴和1.5g氢氧化钠，30℃下150rpm摇床活化48小时，抽滤，用去离子水洗涤得到活化的层析基质。
将活化的层析基质、9.0g 50％(v/v)丙酮和0.9g N-溴代丁二酰亚胺混合进行溴代醇化，30℃下150rpm摇床反应3h，抽滤，用去离子水洗涤，得到溴代醇化的基质。
3.0g二甲基亚砜和6.0g 1M碳酸钠缓冲液混合，加入0.9g苯丙氨酸-酪氨酸-谷氨酰胺-5-氨基苯并咪唑配基，充分溶解，再加入溴代醇化的层析基质，30℃下150rpm摇床反应12小时，用去离子水、0.1M HCl、0.1M NaOH反复抽滤冲洗，得到配基偶联的介质。
最后将介质加入到15.0g 1.0M乙醇胺水溶液(pH 8.0)中，25℃下150rpm摇床中反应8小时，去离子水洗涤，得到得到组合型仿生层析介质。
采用高效液相色谱分析反应后母液中的剩余配基含量0.775g，说明有0.125g配基偶联到介质上。
通过物料平衡计算得到介质配基密度为73μmol/g介质，人免疫球蛋白的饱和吸附容量为92mg/ml介质。
实施例5：组合型仿生层析介质的制备
取抽干琼脂糖凝胶3.0g，加入3.0g 20％(v/v)二甲基亚砜、1.5g烯丙基溴和0.9g氢氧化钠，30℃下150rpm摇床活化36小时，抽滤，用去离子水洗涤得到活化的层析基质。
将活化的层析基质、6.0g 50％(v/v)丙酮和0.6g N-溴代丁二酰亚胺混合进行溴代醇化，30℃下150rpm摇床反应2h，抽滤，用去离子水洗涤，得到溴代醇化的基质。
2.0g二甲基亚砜和6.0g 1M碳酸钠缓冲液混合，加入0.9g苯丙氨酸-酪氨 酸-谷氨酰胺-5-氨基苯并咪唑配基，充分溶解，再加入溴代醇化的层析基质，30℃下150rpm摇床反应10小时，用去离子水、0.1M HCl、0.1M NaOH反复抽滤冲洗，得到配基偶联的介质。
最后将介质加入到9.0g 1.0M乙醇胺水溶液(pH 8.0)中，25℃下150rpm摇床中反应6小时，去离子水洗涤，得到组合型仿生层析介质。
采用高效液相色谱分析反应后母液中的剩余配基含量0.816g，说明有0.084g配基偶联到介质上。
通过物料平衡计算得到介质配基密度为49μmol/g介质，人免疫球蛋白的饱和吸附容量为88mg/ml介质。
实施例6：组合型仿生层析介质的制备
取抽干琼脂糖凝胶3.0g，加入1.5g 20％(v/v)二甲基亚砜、0.3g烯丙基溴和1.5g氢氧化钠，30℃下150rpm摇床活化24小时，抽滤，用去离子水洗涤得到活化的层析基质。
将活化的层析基质、9.0g 50％(v/v)丙酮和0.9g N-溴代丁二酰亚胺混合进行溴代醇化，30℃下150rpm摇床反应1h，抽滤，用去离子水洗涤，得到溴代醇化的基质。
1.5g二甲基亚砜和9.0g 1M碳酸钠缓冲液混合，加入0.3g苯丙氨酸-酪氨酸-谷氨酰胺-5-氨基苯并咪唑配基，充分溶解，再加入溴代醇化的层析基质，30℃下150rpm摇床反应12小时，用去离子水、0.1M HCl、0.1M NaOH反复抽滤冲洗，得到配基偶联的介质。
最后将介质加入到9.0g 1.0M乙醇胺水溶液(pH 8.0)中，25℃下150rpm摇床中反应4小时，去离子水洗涤，得到组合型仿生层析介质。
采用高效液相色谱分析反应后母液中的剩余配基含量0.254g，说明有 0.046g配基偶联到介质上。
通过物料平衡计算得到介质配基密度为27μmol/g介质，人免疫球蛋白的饱和吸附容量为70mg/ml介质。
实施例7：组合型仿生层析介质的制备
取纤维素微球3.0g，加入3.0g 20％(v/v)二甲基亚砜、1.5g烯丙基溴和0.6g氢氧化钠，30℃下150rpm摇床活化24小时，抽滤，用去离子水洗涤得到活化的层析基质。
将活化的层析基质、6.0g 50％(v/v)丙酮和0.9g N-溴代丁二酰亚胺混合进行溴代醇化，30℃下150rpm摇床反应3h，抽滤，用去离子水洗涤，得到溴代醇化的基质。
1.5g二甲基亚砜和3.0g 1M碳酸钠缓冲液混合，加入0.3g苯丙氨酸-酪氨酸-谷氨酰胺-5-氨基苯并咪唑配基，充分溶解，再加入溴代醇化的层析基质，30℃下150rpm摇床反应12小时，用去离子水、0.1M HCl、0.1M NaOH反复抽滤冲洗，得到配基偶联的介质。
最后将介质加入到9.0g 1.0M乙醇胺水溶液(pH 8.0)中，25℃下150rpm摇床中反应4小时，去离子水洗涤，得到组合型仿生层析介质。
采用高效液相色谱分析反应后母液中的剩余配基含量0.232g，说明有0.068g配基偶联到介质上。
通过物料平衡计算得到介质配基密度为40μmol/g介质，人免疫球蛋白的饱和吸附容量为80mg/ml介质。
应用例1
取实施例2得到的层析介质，装填1ml介质于Tricorn 5/100层析柱中， 采用
Figure PCTCN2018072780-appb-000009
explorer 100层析系统测定蛋白穿透曲线。
分别配制2mg/ml的人免疫球蛋白IgG和人血清白蛋白(HSA)溶液作为上样料液，调节pH至7.0。以20mM磷酸盐缓冲液(pH 7.0)作为平衡缓冲液，充分平衡床层后，以0.5ml/min流速上样至蛋白90％穿透，流出液的蛋白浓度用紫外检测器在280nm处检测，结果见附图3。依据蛋白10％穿透时的上样体积，计算动态附载量，IgG的动态载量为22mg/ml，HSA的动态载量仅为0.8mg/ml。以pH4.0的醋酸-醋酸钠缓冲液洗脱IgG，收率达90％。
应用例2
取实施例2得到的层析介质，装填1ml介质于Tricorn 5/100层析柱中，采用
Figure PCTCN2018072780-appb-000010
explorer 100层析系统测定混合蛋白的分离能力。
配制含有1mg/ml的人免疫球蛋白IgG和4mg/ml人血清白蛋白(HSA)的混合蛋白溶液作为上样料液，调节pH至7.0。以20mM磷酸盐缓冲液(pH7.0)作为平衡缓冲液，充分平衡床层后，以0.5ml/min流速上样5ml混合蛋白溶液，上样完成后用20mM磷酸盐缓冲液(pH 7.0)冲洗至基线，然后用20mM醋酸盐缓冲液(pH 4.0)进行洗脱，流出液的蛋白浓度用紫外检测器在280nm处检测，收集洗脱组分。对收集到的组分进行HPLC分析，结果见附图4。通过计算得到，IgG的纯度为99.0％，收率为91.5％。
应用例3
取实施例7得到的层析介质，装填1ml介质于Tricorn 5/100层析柱中，采用
Figure PCTCN2018072780-appb-000011
explorer 100层析系统测定混合蛋白的分离能力。
配制含有1mg/ml的人免疫球蛋白IgG和4mg/ml人血清白蛋白(HSA)的混合蛋白溶液作为上样料液，调节pH至7.0。以20mM磷酸盐缓冲液(pH 7.0)作为平衡缓冲液，充分平衡床层后，以0.5ml/min流速上样5ml混合蛋白溶液，上样完成后用20mM磷酸盐缓冲液(pH 7.0)冲洗至基线，然后用20mM醋酸盐缓冲液(pH 4.0)进行洗脱，流出液的蛋白浓度用紫外检测器在280nm处检测，收集洗脱组分。对收集到的组分进行HPLC分析，结果见附图5。通过计算得到，IgG的纯度为89.1％，收率为92.8％。
应用例4
取实施例2得到的层析介质，装填1ml介质于Tricorn 5/100层析柱中，采用
Figure PCTCN2018072780-appb-000012
explorer 100层析系统测定蛋白穿透曲线。
配制2mg/ml的人免疫球蛋白IgG溶液作为上样料液，调节pH至7.0。以20mM磷酸盐缓冲液(pH 7.0)作为平衡缓冲液，充分平衡床层后，以0.5ml/min流速上样至蛋白90％穿透，流出液的蛋白浓度用紫外检测器在280nm处检测，依据蛋白10％穿透时的上样体积，计算10％穿透的动态附载量。介质经上样-冲洗-洗脱-再生循环使用20次，50次和100次后重复上述操作，测定IgG的动态附载量。介质在1、20、50和100个循环时IgG的动态载量分别为22.68mg/ml介质、22.31mg/ml介质、22.21mg/ml介质和21.89mg/ml介质，经100次循环使用后载量仅下降3.5％，具体变化曲线见附图6。

Claims (10)

  1. 组合型配基，其特征在于，结构式如下：
    Figure PCTCN2018072780-appb-100001
  2. 组合型仿生层析介质，其特征在于，包括层析基质和组合型配基，所述层析基质为带有羟基的亲水性多孔微球；所述组合型配基的序列为苯丙氨酸-酪氨酸-谷氨酰胺-5-氨基苯并咪唑；
    所述组合型配基的结构式如下：
    Figure PCTCN2018072780-appb-100002
    所述组合型仿生层析介质的结构式如下：
    Figure PCTCN2018072780-appb-100003
  3. 根据权利要求2所述的组合型仿生层析介质，其特征在于，所述层析基质为琼脂糖凝胶或纤维素微球。
  4. 如权利要求2或3所述的组合型仿生层析介质的制备方法，其特征在于，包括如下步骤：
    1)层析基质采用烯丙基溴进行活化反应，得到活化层析基质；
    2)活化层析基质采用N-溴代丁二酰亚胺进行溴代醇化反应，得到溴代醇化的基质；
    3)将溴代醇化的基质与组合型配基进行偶联反应，得到组合型仿生层析介质。
  5. 根据权利要求4所述的组合型仿生层析介质的制备方法，其特征在于，所述步骤1)中活化反应包括：将层析基质、二甲基亚砜溶液、烯丙基溴和氢氧化钠混合，在摇床中水浴反应，抽滤，洗涤得到活化层析基质。
  6. 根据权利要求4所述的组合型仿生层析介质的制备方法，其特征在于，所述步骤2)中溴代醇化反应包括：将活化层析基质、丙酮和N-溴代丁二酰亚胺混合，在摇床中水浴反应，抽滤，洗涤，得到溴代醇化的基质。
  7. 根据权利要求4所述的组合型仿生层析介质的制备方法，其特征在于，所述步骤3)中偶联反应包括：将溴代醇化的基质与组合型配基溶解于二甲亚砜中，再加入碳酸钠缓冲液混合后，在摇床中水浴反应，抽滤，洗涤，得到组合型仿生层析介质；所述溴代醇化的基质与组合型配基的质量比为1:0.1-0.3。
  8. 根据权利要求4所述的组合型仿生层析介质的制备方法，其特征在于，所述步骤3)中组合型仿生层析介质继续采用乙醇胺水溶液进行封闭反应。
  9. 根据权利要求8所述的组合型仿生层析介质的制备方法，其特征在于，所述封闭反应包括：将组合型仿生层析介质加入到乙醇胺水溶液中，控制 pH8.0，在摇床中水浴反应。
  10. 如权利要求2或3所述的组合型仿生层析介质在分离抗体中的应用。
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