Hybrid ligand, hybrid biomimetic chromedia and preparing method and use thereof

Abstract

This invention relates to a hybrid ligand, a hybrid biomimetic chromedia and a preparing method and a use thereof, wherein the hybrid biomimetic chromedia takes hydrophilic porous microsphere as a substrate in chromatography, activated with allyl bromide and undergoing bromo-alcoholization with N-bromosuccinimide, then coupled with the hybrid ligands. The sequence of the hybrid ligand is phenylalanine-tyrosine-glutamine-5-aminobenzimidazole. The hybrid biomimetic chromedia has both of the two functional groups of phenylalanine-tyrosine-glutamine tripeptide and aminobenzimidazole, while maintaining the high antibody selectivity of polypeptide ligand, hydrophobic electric charge inductive ligand is introduced to achieve more moderate elution requirement, realizing effective antibody separation.

Claims

1. A hybrid ligand, wherein a structural formula of the hybrid ligand is as follows: ##STR00009##

2. A hybrid biomimetic chromedia, comprising a substrate in chromatography and a hybrid ligand, wherein the substrate in chromatography is a hydrophilic porous microsphere comprising hydroxyl; a structural formula of the hybrid ligand is as follows: ##STR00010## and a structural formula of the hybrid biomimetic chromedia is as follows: ##STR00011##

3. The hybrid biomimetic chromedia according to claim 2, wherein the substrate in chromatography is sepharose gel or cellulose microsphere.

4. A method for preparing the hybrid biomimetic chromedia according to claim 2, comprising the following steps: 1) performing an activating reaction by subjecting the substrate in chromatography with allyl bromide to obtain an activated substrate in chromatography; 2) performing a bromo-alcoholization reaction by subjecting the activated substrate in chromatography with N-bromosuccinimide to obtain a bromo-alcoholized substrate; and 3) performing a coupled reaction by subjecting the bromo-alcoholized substrate and the hybrid ligand to obtain the hybrid biomimetic chromedia.

5. The method according to claim 4, wherein the activating reaction in Step 1) comprises: mixing the substrate in chromatography, a dimethyl sulfoxide solution, allyl bromide and sodium hydroxide, and conducting water bath reaction in a shaker, leaching and washing to obtain the activated substrate in chromatography.

6. The method according to claim 4, wherein the bromo-alcoholization reaction in Step 2) comprises: mixing the activated substrate in chromatography, acetone and N-bromosuccinimide, and conducting water bath reaction in a shaker, leaching and washing to obtain the bromo-alcoholized substrate.

7. The method according to claim 4, wherein the coupled reaction in Step 3) comprises: dissolving the bromo-alcoholized substrate and the hybrid ligand into dimethyl sulfoxide, adding sodium carbonate buffer for mixing, and then conducting water bath reaction in a shaker, leaching and washing to obtain the hybrid biomimetic chromedia; and a mass ratio of the bromo-alcoholized substrate to the hybrid ligand is 1:0.1-0.3.

8. The method according to claim 4, wherein the hybrid biomimetic chromedia in Step 3) continues to undergo a blocking reaction with an ethanolamine solution.

9. The method according to claim 8, wherein the blocking reaction comprises: adding the hybrid biomimetic chromedia into the ethanolamine solution to have a pH value of 8.0, and conducting water bath reaction in a shaker.

10. A method for preparing the hybrid biomimetic chromedia according to claim 3, comprising the following steps: 1) performing an activating reaction by subjecting the substrate in chromatography with allyl bromide to obtain an activated substrate in chromatography; 2) performing a bromo-alcoholization reaction by subjecting the activated substrate in chromatography with N-bromosuccinimide to obtain a bromo-alcoholized substrate; and 3) performing a coupled reaction by subjecting the bromo-alcoholized substrate and the hybrid ligand to obtain the hybrid biomimetic chromedia.

11. The method according to claim 10, wherein the activating reaction in Step 1) comprises: mixing the substrate in chromatography, a dimethyl sulfoxide solution, allyl bromide and sodium hydroxide, and conducting water bath reaction in a shaker, leaching and washing to obtain the activated substrate in chromatography.

12. The method according to claim 10, wherein the bromo-alcoholization reaction in Step 2) comprises: mixing the activated substrate in chromatography, acetone and N-bromosuccinimide, and conducting water bath reaction in a shaker, leaching and washing to obtain the bromo-alcoholized substrate.

13. The method according to claim 10, wherein the coupled reaction in Step 3) comprises: dissolving the bromo-alcoholized substrate and the hybrid ligand into dimethyl sulfoxide, adding sodium carbonate buffer for mixing, and then conducting water bath reaction in a shaker, leaching and washing to obtain the hybrid biomimetic chromedia; and a mass ratio of the bromo-alcoholized substrate to the hybrid ligand is 1:0.1-0.3.

14. The method according to claim 10, wherein the hybrid biomimetic chromedia in Step 3) continues to undergo a blocking reaction with an ethanolamine solution.

15. The method according to claim 14, wherein the blocking reaction comprises: adding the hybrid biomimetic chromedia into the ethanolamine solution to have a pH value of 8.0, and conducting water bath reaction in a shaker.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is the high performance liquid chromatography of the hybrid ligand in embodiment 1.

(2) FIG. 2 is the mass spectrum of the hybrid ligand in embodiment 1.

(3) FIG. 3 is the breakthrough curve comparison diagram between human IgG and human serum albumin (HSA) in use example 1.

(4) FIG. 4 is the high performance liquid chromatography of the mixed protein separation material and elution fractions in use example 2.

(5) FIG. 5 is the high performance liquid chromatography of the mixed protein separation material and elution fractions in use example 3.

(6) FIG. 6 is the dynamic loading capacity change diagram after using different cycles in use example 4.

DETAILED DESCRIPTION OF DISCLOSED EMBODIMENTS

(7) Below is further description of this invention in combination with specific embodiments.

Embodiment 1

Preparing Hybrid Ligand

(8) Through computer molecule simulation, key residue at the binding site of protein A and antibody Fc was analyzed and evaluated to screen and design the tripeptide-heterocyclic small molecule hybrid ligand. The sequence of the hybrid ligand is phenylalanine-tyrosine-glutamine-5-aminobenzimidazole.

(9) The hybrid ligand, of which the structural formula is as follows:

(10) ##STR00008##

(11) The chemical synthesis method in the prior art may be adopted to synthesize the hybrid ligand, and the hybrid ligand in this embodiment is prepared by Chinese Peptide Co., Ltd.

(12) High performance liquid chromatography and mass spectrum representation of the hybrid ligand in embodiment 1 is performed and is respectively as shown in FIG. 1 and FIG. 2.

Embodiment 2

Preparing Hybrid Biomimetic Chromedia

(13) Take 3.0 g drained sepharose gel, add 3.0 g dimethyl sulfoxide of 20% (v/v), 1.5 g allyl bromide and 0.6 g sodium hydroxide, conduct an activating reaction in a shaker at 30° C. at 150 rpm for 24 hours, leach, wash with deionized water to obtain an activated substrate in chromatography.

(14) Mix the activated substrate in chromatography, 6.0 g acetone of 50% (v/v) and 0.9 g N-bromosuccinimide to conduct a bromo-alcoholization reaction in a shaker at 30° C. at 150 rpm for 3 hours, leach, wash with deionized water to obtain a bromo-alcoholized substrate.

(15) Mix 1.5 g dimethyl sulfoxide with 3.0 g sodium carbonate buffer of 1M, add 0.3 g phenylalanine-tyrosine-glutamine-5-aminobenzimidazole ligand to dissolve fully, then add the bromo-alcoholized substrate in chromatography, conduct a reaction in a shaker at 30° C. at 150 rpm for 12 hours, and repeatedly leach and flush with deionized water, 0.1M HCl and 0.1M NaOH to obtain a ligand coupled medium.

(16) Finally, add the medium into 9.0 g ethanolamine solution of 1.0 M (pH 8.0), conduct a reaction in a shaker at 25° C. at 150 rpm for 4 hours, wash with deionized water to obtain a hybrid biomimetic chromedia.

(17) Through analyzing by high performance liquid chromatography, the content of the left ligand in the bulk solution after reaction is 0.228 g, indicating that 0.072 g ligand is coupled onto the medium.

(18) Through material balancing calculation, the medium ligand density is 42 μmol/g medium, and the saturated absorption capacity of human immunoglobulin is 85 mg/ml medium.

Embodiment 3

Preparing Hybrid Biomimetic Chromedia

(19) Take 3.0 g drained sepharose gel, add 1.5 g dimethyl sulfoxide of 20% (v/v), 0.3 g allyl bromide and 0.3 g sodium hydroxide, conduct an activating reaction in a shaker at 30° C. at 150 rpm for 24 hours, leach, wash with deionized water to obtain an activated substrate in chromatography.

(20) Mix the activated substrate in chromatography, 3.0 g acetone of 50% (v/v) and 0.3 g N-bromosuccinimide to conduct a bromo-alcoholization reaction in a shaker at 30° C. at 150 rpm for 1 hour, leach, wash with deionized water to obtain a bromo-alcoholized substrate.

(21) Mix 1.5 g dimethyl sulfoxide with 3.0 g sodium carbonate buffer of 1M, add 0.3 g phenylalanine-tyrosine-glutamine-5-aminobenzimidazole ligand to dissolve fully, then add the bromo-alcoholized substrate in chromatography, conduct a reaction in a shaker at 30° C. at 150 rpm for 8 hours, and repeatedly leach and flush with deionized water, 0.1M HCl and 0.1M NaOH to obtain a ligand coupled medium.

(22) Finally, add the medium into 3.0 g ethanolamine solution of 1.0 M (pH 8.0), conduct a reaction in a shaker at 25° C. at 150 rpm for 4 hours, wash with deionized water to obtain a hybrid biomimetic chromedia.

(23) Through analyzing by high performance liquid chromatography, the content of the left ligand in the mother solution after reaction is 0.259 g, indicating that 0.041 g ligand is coupled onto the medium.

(24) Through material balancing calculation, the medium ligand density is 24 μmol/g medium, and the saturated absorption capacity of human immunoglobulin is 65 mg/ml medium.

Embodiment 4

Preparing Hybrid Biomimetic Chromedia

(25) Take 3.0 g drained sepharose gel, add 4.5 g dimethyl sulfoxide of 20% (v/v), 3 g allyl bromide and 1.5 g sodium hydroxide, conduct an activating reaction in a shaker at 30° C. at 150 rpm for 48 hours, leach, wash with deionized water to obtain an activated substrate in chromatography.

(26) Mix the activated substrate in chromatography, 9.0 g acetone of 50% (v/v) and 0.9 g N-bromosuccinimide to conduct a bromo-alcoholization reaction in a shaker at 30° C. at 150 rpm for 3 hours, leach, wash with deionized water to obtain a bromo-alcoholized substrate.

(27) Mix 3.0 g dimethyl sulfoxide with 6.0 g sodium carbonate buffer of 1M, add 0.9 g phenylalanine-tyrosine-glutamine-5-aminobenzimidazole ligand to dissolve fully, then add the bromo-alcoholized substrate in chromatography, conduct a reaction in a shaker at 30° C. at 150 rpm for 12 hours, and repeatedly leach and flush with deionized water, 0.1M HCl and 0.1M NaOH to obtain a ligand coupled medium.

(28) Finally, add the medium into 15.0 g ethanolamine solution of 1.0 M (pH 8.0), conduct a reaction in a shaker at 25° C. at 150 rpm for 8 hours, wash with deionized water to obtain a hybrid biomimetic chromedia.

(29) Through analyzing by high performance liquid chromatography, the content of the left ligand in the mother solution after reaction is 0.775 g, indicating that 0.125 g ligand is coupled onto the medium.

(30) Through material balancing calculation, the medium ligand density is 73 μmol/g medium, and the saturated absorption capacity of human immunoglobulin is 92 mg/ml medium.

Embodiment 5

Preparing Hybrid Biomimetic Chromedia

(31) Take 3.0 g drained sepharose gel, add 3.0 g dimethyl sulfoxide of 20% (v/v), 1.5 g allyl bromide and 0.9 g sodium hydroxide, conduct an activating reaction in a shaker at 30° C. at 150 rpm for 36 hours, leach, wash with deionized water to obtain an activated substrate in chromatography.

(32) Mix the activated substrate in chromatography, 6.0 g acetone of 50% (v/v) and 0.6 g N-bromosuccinimide to conduct a bromo-alcoholization reaction in a shaker at 30° C. at 150 rpm for 2 hours, leach, wash with deionized water to obtain a bromo-alcoholized substrate.

(33) Mix 2.0 g dimethyl sulfoxide with 6.0 g sodium carbonate buffer of 1M, add 0.9 g phenylalanine-tyrosine-glutamine-5-aminobenzimidazole ligand to dissolve fully, then add the bromo-alcoholized substrate in chromatography, conduct a reaction in a shaker at 30° C. at 150 rpm for 10 hours, and repeatedly leach and flush with deionized water, 0.1M HCl and 0.1M NaOH to obtain a ligand coupled medium.

(34) Finally, add the medium into 9.0 g ethanolamine solution of 1.0 M (pH 8.0), conduct a reaction in a shaker at 25° C. at 150 rpm for 6 hours, wash with deionized water to obtain a hybrid biomimetic chromedia.

(35) Through analyzing by high performance liquid chromatography, the content of the left ligand in the mother solution after reaction is 0.816 g, indicating that 0.084 g ligand is coupled onto the medium.

(36) Through material balancing calculation, the medium ligand density is 49 μmol/g medium, and the saturated absorption capacity of human immunoglobulin is 88 mg/ml medium.

Embodiment 6

Preparing Hybrid Biomimetic Chromedia

(37) Take 3.0 g drained sepharose gel, add 1.5 g dimethyl sulfoxide of 20% (v/v), 0.3 g allyl bromide and 1.5 g sodium hydroxide, conduct an activating reaction in a shaker at 30° C. at 150 rpm for 24 hours, leach, wash with deionized water to obtain an activated substrate in chromatography.

(38) Mix the activated substrate in chromatography, 9.0 g acetone of 50% (v/v) and 0.9 g N-bromosuccinimide to conduct a bromo-alcoholization reaction in a shaker at 30° C. at 150 rpm for 1 hour, leach, wash with deionized water to obtain a bromo-alcoholized substrate.

(39) Mix 1.5 g dimethyl sulfoxide with 9.0 g sodium carbonate buffer of 1M, add 0.3 g phenylalanine-tyrosine-glutamine-5-aminobenzimidazole ligand to dissolve fully, then add the bromo-alcoholized substrate in chromatography, conduct a reaction in a shaker at 30° C. at 150 rpm for 12 hours, and repeatedly leach and flush with deionized water, 0.1M HCl and 0.1M NaOH to obtain a ligand coupled medium.

(40) Finally, add the medium into 9.0 g ethanolamine solution of 1.0 M (pH 8.0), conduct a reaction in shaker at 25° C. at 150 rpm for 4 hours, wash with deionized water to obtain a hybrid biomimetic chromedia.

(41) Through analyzing by high performance liquid chromatography, the content of the left ligand in the mother solution after reaction is 0.254 g, indicating that 0.046 g ligand is coupled onto the medium.

(42) Through material balancing calculation, the medium ligand density is 27 μmol/g medium, and the saturated absorption capacity of human immunoglobulin is 70 mg/ml medium.

Embodiment 7

Preparing Hybrid Biomimetic Chromedia

(43) Take 3.0 g cellulose microsphere, add 3.0 g dimethyl sulfoxide of 20% (v/v), 1.5 g allyl bromide and 0.6 g sodium hydroxide, conduct an activating reaction in a shaker at 30° C. at 150 rpm for 24 hours, leach, wash with deionized water to obtain an activated substrate in chromatography.

(44) Mix the activated substrate in chromatography, 6.0 g acetone of 50% (v/v) and 0.9 g N-bromosuccinimide to conduct a bromo-alcoholization reaction in a shaker at 30° C. at 150 rpm for 3 hours, leach, wash with deionized water to obtain a bromo-alcoholized substrate.

(45) Mix 1.5 g dimethyl sulfoxide with 3.0 g sodium carbonate buffer of 1M, add 0.3 g phenylalanine-tyrosine-glutamine-5-aminobenzimidazole ligand to dissolve fully, then add the bromo-alcoholized substrate in chromatography, conduct a reaction in a shaker at 30° C. at 150 rpm for 12 hours, and repeatedly leach and flush with deionized water, 0.1M HCl and 0.1M NaOH to obtain a ligand coupled medium.

(46) Finally, add the medium into 9.0 g ethanolamine solution of 1.0 M (pH 8.0), conduct a reaction in a shaker at 25° C. at 150 rpm for 4 hours, wash with deionized water to obtain a hybrid biomimetic chromedia.

(47) Through analyzing by high performance liquid chromatography, the content of the left ligand in the mother solution after reaction is 0.232 g, indicating that 0.068 g ligand is coupled onto the medium.

(48) Through material balancing calculation, the medium ligand density is 40 μmol/g medium, and the saturated absorption capacity of human immunoglobulin is 80 mg/ml medium.

Use Example 1

(49) Take the chromedia obtained from embodiment 2 and fill 1 ml of the same into the Tricorn 5/100 chromatographic column, determine the protein breakthrough curve using ÄKTA explorer 100 chromatographic system.

(50) Respectively prepare 2 mg/ml human IgG solution and 2 mg/ml HSA solution as the loading sample liquid, and adjust pH to 7.0. Take 20 mM phosphate buffer (pH 7.0) as equilibration buffer to fully equilibrate the bed, feed sample at 0.5 ml/min flow rate until 90% protein breakthrough, detect the protein concentration of the effluent at the point of 280 nm with UV detector, the result is as shown in FIG. 3. Based on the loading sample volume at 10% protein breakthrough, calculate the dynamic loading capacity, IgG dynamic loading capacity is 22 mg/ml, HSA dynamic loading capacity is 0.8 mg/ml. Elute IgG with acetic acid-sodium acetate buffer at pH4.0 until the yield reaches 90%.

Use Example 2

(51) Take the chromedia obtained from embodiment 2 and fill 1 ml of the same into the Tricorn 5/100 chromatographic column, determine the separating performance of the mixed protein using ÄKTA explorer 100 chromatographic system.

(52) Prepare mixed protein solution containing 1 mg/ml human IgG and 4 mg/ml HSA as the loading sample liquid, and adjust pH to 7.0. Take 20 mM phosphate buffer (pH 7.0) as equilibration buffer to fully equilibrate the bed, feed 5 ml mixed protein solution sample at 0.5 ml/min flow rate, after sample loading is completed, flush with 20 mM phosphate buffer (pH 7.0) to the base line, then elute with 20 mM acetate buffer (pH 4.0), detect the protein concentration of the effluent at the point of 280 nm with UV detector, collect the elution fractions. Conduct HPLC analysis of the collected fractions, and the result is as shown in FIG. 4. Through calculation, IgG purity is 99.0%, the yield is 91.5%.

Use Example 3

(53) Take the chromedia obtained from embodiment 7 and fill 1 ml of the same into the Tricorn 5/100 chromatographic column, determine the separating performance of the mixed protein using ÄKTA explorer 100 chromatographic system.

(54) Prepare mixed protein solution containing 1 mg/ml human IgG and 4 mg/ml HSA as the loading sample liquid, and adjust pH to 7.0. Take 20 mM phosphate buffer (pH 7.0) as equilibration buffer to fully equilibrate the bed, feed 5 ml mixed protein solution sample at 0.5 ml/min flow rate, after sample loading is completed, flush with 20 mM phosphate buffer (pH 7.0) to the base line, then elute with 20 mM acetate buffer (pH 4.0), detect the protein concentration of the effluent at the point of 280 nm with UV detector, collect the elution fractions. Conduct HPLC analysis of the collected fractions, and the result is as shown in FIG. 5. Through calculation, IgG purity is 89.1%, the yield is 92.8%.

Use Example 4

(55) Take the chromedia obtained from embodiment 2 and fill 1 ml of the same into the Tricorn 5/100 chromatographic column, determine the protein breakthrough curve using ÄKTA explorer 100 chromatographic system.

(56) Prepare 2 mg/ml human IgG solution as the loading sample liquid, and adjust pH to 7.0. Take 20 mM phosphate buffer (pH 7.0) as equilibration buffer to fully equilibrate the bed, feed sample at 0.5 ml/min flow rate until 90% protein breakthrough, detect the protein concentration of the effluent at the point of 280 nm with UV detector, based on the loading sample volume at 10% protein breakthrough, calculate the dynamic loading capacity at 10% breakthrough. After the medium goes through the loading sample-flush-elution-regeneration cycle for 20 times, 50 times and 100 times of use, repeat the above-described operation to measure IgG dynamic loading capacity. At 1, 20, 50 and 100 cycles of the medium, the IgG dynamic loading capacities are respectively 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 loading capacity decreased by 3.5% only, refer to FIG. 6 for specific change curve.