阅读说明：本技术 分离介质及其制备方法和应用 (Separation medium, preparation method and application thereof ) 是由 吕小林 胡新妹 杨克 黄学英 于 2019-08-27 设计创作，主要内容包括：本发明涉及一种分离介质及其制备方法和应用。该分离介质具有如式I所示结构：<Image he="227" wi="700" file="DDA0002180825440000011.GIF" imgContent="drawing" imgFormat="GIF" orientation="portrait" inline="no"></Image>其中,SP为介质,R为间隔臂,rProtein A-cys为重组蛋白A。本发明提供的新的分离介质,其动态结合载量达到50mg/mL,分离效率高、分离效果好、成本低,适合工业化大规模分离纯化抗体及Fc融合蛋白。(The invention relates to a separation medium, a preparation method and application thereof. The separation medium has a structure as shown in formula I: wherein SP is a medium, R is a spacer arm, and rProtein A-cys is recombinant protein A. The novel separation medium provided by the invention has the advantages of dynamic binding capacity of 50mg/mL, high separation efficiency, good separation effect and low cost, and is suitable for large-scale industrialized separation and purification of the antibody and the Fc fusion protein.)

1. A separation medium having a structure according to formula I:

wherein SP is a medium, R is a spacer arm, and rProtein A-cys is recombinant protein A.

2. The separation medium of claim 1, wherein the SP is a polyMethacrylate microspheres, polyethylene-divinylbenzene microspheres or agarose microspheres; the structure of R is

3. A method of preparing a separation medium, comprising the steps of:

modifying amino on the surface of the basic medium to obtain an intermediate II,

SP-R-NH₂

II；

activating the amino group of the intermediate II by using iodoacetic acid or bromoacetic acid to obtain an intermediate III,

coupling the intermediate III with rProtein A-cys to obtain a separation medium shown as a formula I;

wherein SP is a medium, R is a spacer arm, and rProtein A-cys is recombinant protein A.

4. The method for preparing a separation medium according to claim 3, wherein the base medium is a microsphere having an epoxy group or a vicinal diol hydroxyl group on the surface, and the microsphere is a polymethacrylate microsphere, a polyethylene-divinylbenzene microsphere or an agarose microsphere.

5. The method for preparing the separation medium according to claim 3, wherein the modifying agent for modifying the amino group on the surface of the base medium is selected from the following organic amines: at least one of ethylenediamine, 1, 3-propylenediamine, 1, 6-hexamethylenediamine, and 3,3' -diaminodipropylamine.

6. The method for preparing a separation medium according to claim 5, wherein the step of modifying the surface of the base medium with an amino group comprises:

if the surface of the basic medium has an epoxy group, carrying out ring-opening reaction on the basic medium and the organic amine to obtain an intermediate II, wherein the mass ratio of the basic medium to the organic amine is 100 (20-80); alternatively, the first and second electrodes may be,

if the surface of the basic medium has the vicinal diol hydroxyl, oxidizing the vicinal diol hydroxyl on the surface of the basic medium into aldehyde group, then reacting with the organic amine to generate Schiff base, and then reducing the Schiff base to obtain an intermediate II, wherein the dosage ratio of the basic medium to the organic amine is 100g (0.1-0.3) mol.

7. The method for producing a separation medium according to claim 3, wherein in the step of activating the amino group of the intermediate II with iodoacetic acid or bromoacetic acid, the mass ratio of the iodoacetic acid or bromoacetic acid to 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and the intermediate II is (8-15): (10-20): 100.

8. The method for preparing a separation medium according to claim 3, wherein the step of coupling the intermediate III to rProtein A-cys comprises: from 7.1g to 17.8g of Na per 100g of said intermediate III₂SO₄Controlling the pH value to be 7.5-8.5, and reacting every 100g of the intermediate III with 0.5-5 g of rProtein A-cys in 100mL-200mL of phosphate buffer solution for 2h-16h, and retaining the solid product.

9. The method of claim 3, further comprising the step of adding a capping agent to the product of the coupling of the rProtein A-cys to effect an endcapping reaction.

10. Use of a separation medium according to claim 1 or 2 or a separation medium obtained by a method of preparing a separation medium according to any one of claims 3 to 9 for separation and purification.

Technical Field

The invention relates to the technical field of filler synthesis, in particular to a separation medium and a preparation method and application thereof.

Background

Protein A (Protein A) is a surface Protein present in the cell wall of Staphylococcus aureus and binds tightly to the Fc region of an antibody, and the native Protein A molecule has 5 homology domains (E, D, A, B, C) that bind to the Fc region and has a molecular weight of 42 kDa. In 1978, Protein A packing formed by immobilizing Protein A as an affinity ligand on a medium was first commercialized and used for antibody affinity chromatography. The first therapeutic antibody, Orthoclone, was approved for marketing in 1986 and used Protein A chromatography as a capture step in the purification process. With the development of antibody drugs, Protein a fillers used in the antibody capture phase have also been rapidly developed, and in the subsequent FDA application, 86% of antibodies used Protein a fillers in the capture step.

Currently, methods for immobilizing Protein a to a medium are classified into two types: one is realized by the covalent bonding of amino groups in lysine residues of Protein A molecules and a matrix, and the method is called multipoint bonding because of a plurality of lysines in the Protein A molecules; secondly, the Protein A molecule is subjected to mutation modification by utilizing genetic engineering, a cysteine (marked as rProtein A-cys) is added at the C end of a peptide chain, and then the immobilization of the Protein A molecule is realized through the covalent bond of a sulfhydryl group and a medium, and the method is called single-point bonding. Although both methods can achieve the fixation of Protein A molecules, multi-point bonding can firmly fix Protein A on a medium, and the binding of Protein A and antibody molecules is influenced, but single-point bonding cannot.

The immobilization of the rProtein A-cys on the medium surface by single-point bonding is generally realized by utilizing the reaction of epoxy groups and mercapto groups, and the reaction of the epoxy groups and the mercapto groups on the medium surface generally requires higher temperature (more than 37 ℃), which can cause adverse effect on the activity of the rProtein A-cyss ligand.

Disclosure of Invention

Based on the above, it is necessary to provide a separation medium, a preparation method and applications thereof, aiming at the problem that the activity of the rProtein A-cys ligand is adversely affected by the traditional method for fixing the rProtein A-cys on the surface of the medium.

A separation medium having a structure according to formula I:

wherein SP is a medium, R is a spacer arm, and rProtein A-cys is recombinant protein A.

In one embodiment, the SPs are polymethacrylate microspheres, polyethylene-divinylbenzene microspheres, or agarose microspheres; the structure of R isOr

The invention provides a preparation method of a separation medium, which comprises the following steps:

modifying amino on the surface of the basic medium to obtain an intermediate II,

activating the amino group of the intermediate II by using iodoacetic acid or bromoacetic acid to obtain an intermediate III,

coupling the intermediate III with rProtein A-cys to obtain a separation medium shown as a formula I;

wherein SP is a medium, R is a spacer arm, and rProtein A-cys is recombinant protein A.

In one embodiment, the base medium is a microsphere having an epoxy group or a vicinal diol hydroxyl group on the surface, and the microsphere is a polymethacrylate microsphere, a polyethylene-divinylbenzene microsphere or an agarose microsphere.

In one embodiment, the modifier used for modifying the amino group on the surface of the base medium is selected from the following organic amines: at least one of ethylenediamine, 1, 3-propylenediamine, 1, 6-hexamethylenediamine, and 3,3' -diaminodipropylamine.

In one embodiment, the step of modifying the surface of the base medium with an amino group is:

if the surface of the basic medium has an epoxy group, carrying out ring-opening reaction on the basic medium and the organic amine to obtain an intermediate II, wherein the mass ratio of the basic medium to the organic amine is 100 (20-80); or if the surface of the basic medium has the vicinal diol hydroxyl, oxidizing the vicinal diol hydroxyl on the surface of the basic medium into an aldehyde group, then reacting with the organic amine to generate Schiff base, and then reducing the Schiff base to obtain an intermediate II, wherein the dosage ratio of the basic medium to the organic amine is 100g (0.1-0.3) mol.

In one embodiment, in the step of activating the amino group of the intermediate II with iodoacetic acid or bromoacetic acid, the mass ratio of the iodoacetic acid or bromoacetic acid to the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and the intermediate II is (8-15): (10-20): 100.

In one embodiment, the specific steps for coupling the intermediate III to rProtein a-cys are: from 7.1g to 17.8g of Na per 100g of said intermediate III₂SO₄Controlling the pH value to be 7.5-8.5, and reacting every 100g of the intermediate III with 0.5-5 g of rProtein A-cys in 100mL-200mL of phosphate buffer solution for 2h-16h, and retaining the solid product.

In one embodiment, the method further comprises the step of adding a blocking agent into the product after the rProtein A-cys is coupled to carry out end group blocking reaction.

The invention provides an application of the separation medium or the separation medium prepared by the preparation method of any separation medium in separation and purification.

The invention provides a new separation medium, the dynamic binding capacity of which reaches 50mg/mL, the separation efficiency is high, the separation effect is good, the cost is low, and the separation medium is suitable for separating and purifying the antibody and the Fc fusion protein in an industrialized large scale.

The invention also provides a novel method for fixing the rProtein A-cys on the surface of the medium, which comprises the steps of firstly modifying amino on the basic medium, then activating the amino by using iodoacetic acid or bromoacetic acid to obtain an intermediate III, wherein the iodoacetyl or bromoacetyl on the intermediate III has higher activity than epoxy groups, so that the reaction efficiency of the iodoacetyl or bromoacetyl on the intermediate III and the sulfhydryl on the rProtein A-cys ligand is higher when the rProtein A-cys is coupled. And during coupling, the nitrogen atom on the intermediate III has positive charge, and the surface of the rProtein A-cys has negative charge, so that the intermediate III has the effect of enabling the rProtein A-cys to approach the surface of a medium, and the coupling efficiency is more than 85%. In addition, the coupling reaction is carried out at room temperature, so that the adverse effect on the activity of the rProtein A-cys ligand is greatly weakened, and the separation efficiency is improved.

Detailed Description

The present invention will be described in detail with reference to the accompanying drawings, which illustrate embodiments of the present invention. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

The invention provides a separation medium of an embodiment, which has a structure shown in a formula I:

wherein, SP is a medium, and the medium is polymethacrylate microspheres, polyethylene-divinylbenzene microspheres or agarose microspheres. R is a spacer arm, forconnecting-NH-with a medium SP, the structure of which is: the rProtein A-cys is recombinant Protein A, the rProtein A-cys is Protein A molecules which are subjected to mutation modification, and a cysteine is added at the C end of a peptide chain.

The invention also provides a preparation method of the separation medium, which comprises the following steps:

s10, modifying amino on the surface of the basic medium to obtain an intermediate II,

in one embodiment, the base medium is a microsphere having an epoxy group or a vicinal diol hydroxyl group on the surface, and the microsphere is a polymethacrylate microsphere, a polyethylene-divinylbenzene microsphere or an agarose microsphere.

In one embodiment, the modifier used for modifying the amino group on the surface of the basic medium is selected from the following organic amines: at least one of ethylenediamine, 1, 3-propylenediamine, 1, 6-hexamethylenediamine, and 3,3' -diaminodipropylamine.

In one embodiment, in the intermediate II obtained, SP is a medium, and the medium is polymethacrylate microspheres, polyethylene-divinylbenzene microspheres or agarose microspheres.

When the organic amine is adopted to modify amino on the surface of the basic medium, one of the amino of the organic amine reacts with the group on the surface of the basic medium, so that the organic amine is connected with the medium, the spacer arm is R, and the structure is The other amino group, i.e., the amino group modified on the surface of the base medium in this step, is used for the next reaction.

In one embodiment, the step of modifying the surface of the base medium with amino groups is:

if the surface of the basic medium has an epoxy group, carrying out ring-opening reaction on the basic medium and the organic amine to obtain an intermediate II, wherein the mass ratio of the basic medium to the organic amine is 100 (20-80);

the method comprises the following specific steps: every 100g of the basic medium is reacted with 100g of 20-20 wt-80 wt% organic amine solution for 4-16 h per 100g of the basic medium, the solid product is reserved, then 200ml of 0.4M-0.6M sulfuric acid solution is added, and the reaction is carried out for 2-5h at the temperature of 30-50 ℃, and the solid product is reserved. The solid product obtained from the above reaction is washed to neutrality with deionized water.

If the surface of the basic medium has the vicinal diol hydroxyl, oxidizing the vicinal diol hydroxyl on the surface of the basic medium into aldehyde group, then reacting with organic amine to generate Schiff base, and then reducing the Schiff base to obtain an intermediate II, wherein the dosage ratio of the basic medium to the organic amine is 100g (0.1-0.3) mol.

The method comprises the following specific steps: based on each 100g of the base medium, each 100g of the base medium is mixed with 100mL of 0.1M-0.3M NaIO₄The solution reacts for 1h to 3h, the solid product is reserved, and then organic amine containing 1M to 3M and NaCNBH containing 0.2g to 1g are added₃100mL of 0.1M phosphate buffer solution, reacting for 2h-5h, and retaining the solid product. Similarly, the solid product obtained from the reaction was washed to neutrality with deionized water.

S20, activating the amino group of the intermediate II by iodoacetic acid or bromoacetic acid to obtain an intermediate III,

in one embodiment, in the step of activating the amino group of intermediate II with iodoacetic acid or bromoacetic acid, the mass ratio of the iodoacetic acid or bromoacetic acid to 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and intermediate II is (8-15): 10-20): 100.

In one embodiment, the specific steps for activating the amino group of intermediate II with iodoacetic acid or bromoacetic acid are: mixing 100g of the intermediate II with 100mL (pH value is 4.7-5) of 0.1M2- (N-morpholine) ethanesulfonic acid buffer solution containing 8g-15g of iodoacetic acid, adding 10g-20g of EDC, reacting for 2h-5h at room temperature in a dark place, and retaining the solid product.

S30, coupling the intermediate III with rProtein A-cys to obtain a separation medium shown in the formula I;

in one embodiment, the specific steps for coupling intermediate III to rProtein a-cys are: based on 100g of intermediate III, in the range of 7.1g to 17.8g Na₂SO₄Controlling the pH value to be 7.5-8.5, and reacting every 100g of intermediate III with 0.5-5 g of rProtein A-cys in 100mL-200mL of phosphate buffer solution for 2h-16h, and retaining the solid product.

More specifically, per 100g of intermediate III, per 100g of the above intermediate III, 7.1g to 17.8g of Na₂SO₄And 100mL of 0.1M phosphate buffer solution containing 10mg/mL-25mg/mL rProtein A-cys and 8mM-10mM EDTA, and reacting for 2h-16h at room temperature in the dark, and keeping the solid product.

In the above embodiment, the solid product obtained by the reaction was washed with 0.1M phosphate buffer solution having a pH of 7.0.

In one embodiment, the method further comprises the step of adding a blocking agent into the product after the rProtein A-cys is coupled to carry out end group blocking reaction.

Specifically, the end group blocking reaction comprises the following steps: 100mL of 0.1M phosphate buffer solution (pH 8.5) containing 20mM to 70mM of a blocking agent was added per 100g of separation medium I, and the reaction was stirred at room temperature for 0.5h to 2h, and the solid product was retained and washed with 100mL of 0.1M phosphate buffer solution (pH 7.0), 100mL of 1M NaCl, and 100mL of 0.1M phosphate buffer solution (pH 7.0), in this order.

In one embodiment, the blocking agent is at least one of mercaptoethanol, mercaptoglycerol, and cysteine.

The invention also provides the application of the separation medium or the separation medium prepared by the preparation method of any one of the separation media in separation and purification.

The following are specific examples.

In the following examples, the test conditions for dynamic binding loading (DBC) were: 20mM PBS +150mM NaCl +2mg/mL human immunoglobulin, pH7.4, 4min binding time, 10% flow through.