WO2022088220A1

WO2022088220A1 - Pmma matrix-based protein a affinity chromatography medium and preparation method and application thereof

Info

Publication number: WO2022088220A1
Application number: PCT/CN2020/126691
Authority: WO
Inventors: 江必旺; 程雷; 王永云; 林生跃
Original assignee: 苏州纳微科技股份有限公司
Priority date: 2020-10-28
Filing date: 2020-11-05
Publication date: 2022-05-05
Also published as: CN112341663A; CN112341663B

Abstract

Provided are a polymethyl methacrylate (PMMA) matrix-based Protein A affinity chromatography medium and preparation method and application thereof. Comprised are the following steps: performing hydrophilization of polymethyl methacrylate microspheres to prepare hydrophilic polymethyl methacrylate microspheres; mixing and reacting the hydrophilic polymethyl methacrylate microspheres, a base, and an epoxy compound to prepare epoxidized polymethyl methacrylate microspheres; mixing and reacting the epoxidized polymethyl methacrylate microspheres, Protein A, ethylenediaminetetraacetic acid, mercapto glycerol, and auxiliary agent to prepare a PMMA matrix-based Protein A affinity chromatography medium, having the advantages of good hydrophilicity, strong alkali resistance, strong acid resistance, smaller particle size, uniform particle size, and high mechanical strength; the invention has good separation performance, can be used at high flow rates, increases production efficiency, is low in cost, and is suitable for rapid separation and purification of biological macromolecules and for continuous flow chromatography.

Description

Protein A affinity chromatography medium based on PMMA matrix and its preparation method and application

technical field

The invention relates to the field of chemistry, in particular to a PMMA matrix-based Protein A affinity chromatography medium and a preparation method and application thereof.

Background technique

Affinity chromatography is a liquid chromatography method that utilizes the specific affinity between biologically active substances to separate and purify the target product, and can be applied to any two biological macromolecules that interact specifically. Because the interaction between antibodies and antigens is highly specific, and the affinity is extremely strong, affinity chromatography technology has the advantages of high yield, high purity, and the ability to maintain the natural state of biological macromolecules, so it is widely used in biological macromolecules. separation and purification.

Using Protein A as the affinity ligand is the most widely used affinity medium at present. In the actual production of antibody purification, one-step affinity chromatography can remove most of the host cell proteins, DNA and pigments and other impurities, and the purity can reach 95%. %above. However, Protein A affinity chromatography medium also has some limitations, such as high price, limited capacity, low yield, etc., it is difficult to meet the huge antibody market demand.

Continuous flow chromatography is a new chromatographic separation mode based on the concept of simulated moving bed. In the traditional batch chromatography process, in order to prevent the loss of protein, low-penetration point loading is usually adopted, and the utilization rate of the medium is low, while continuous flow chromatography is loaded through multiple columns in series, and the elution regeneration is performed alternately, which greatly improves the efficiency of the flow chromatography. It greatly improves the medium utilization, reduces the protein retention time in the chromatography column, and greatly increases the process yield. Due to the high price of Protein A, increasing production capacity and reducing costs have become the key to the transformation of the downstream process of antibody production, and the realization of continuous flow chromatography media is one of the important trends. The ideal protein A affinity chromatography medium for continuous flow chromatography has small particle size and pore size. Because the smaller particle size can shorten the mass transfer path and improve the mass transfer in the pores. The smaller pore size can effectively increase the specific surface area of the medium, provide more ligand coupling sites, facilitate protein binding, and have a relatively high capacity. However, the smaller the particle size, the greater the pressure drop in the bed.

It is generally believed that the matrix of the medium and its structure have an important influence on the properties of the medium, such as mechanical strength, specific surface area, and mass transfer performance. The matrix of antibody affinity media mainly includes natural polysaccharides (such as agarose, dextran, etc.), high molecular polymers (such as polystyrene, polyacrylic acid, etc.) and inorganic materials (such as porous glass and silica gel).

Among them, agarose microspheres have the advantages of good biocompatibility and easy derivatization of functional groups, and the introduction of cross-linking technology partially overcomes the shortcomings of low mechanical properties and small pore size of agarose microspheres, which can be used as a pressure resistance index. The increase to 0.3MP greatly broadens its application field, but its pressure resistance is still difficult to meet the requirements of high flow rate and large-scale chromatography at the same time. At present, domestic antibody production mostly uses imported Protein A affinity chromatography media, such as GE's MabSelect SuRe, whose matrix is highly cross-linked agarose microspheres, with the advantages of excellent hydrophilicity, good biocompatibility, and very good It is suitable for the separation and purification of antibody biological functional groups; but the disadvantages are that its structure is soft, the particle size distribution is wide, and there are problems such as poor column packing repeatability and low mechanical strength in the actual use process.

Prosep Ultra Plus from Millipore is also a widely used protein A affinity chromatography medium. However, the defect of the glass bead structure is its strong hydrophobicity, which is prone to non-specific binding with host proteins. In addition, it is not resistant to high concentrations of NaOH, and the impurities bound to the column are not easily removed, thus limiting its application in large-scale biological samples.

SUMMARY OF THE INVENTION

Based on this, it is necessary to provide a preparation method with good hydrophilicity, strong alkali resistance, strong acid resistance, small particle size, uniform particle size, and high mechanical strength, which can be used at high flow rate, good separation effect, and low cost. The method of ProteinA affinity chromatography medium based on polymethyl methacrylate (PMMA) is suitable for the rapid separation and purification of biological macromolecules (such as antibodies) and continuous flow chromatography, which can improve production efficiency and improve medium utilization. , Reduce the amount of buffer used.

The technical solution is as follows:

A kind of preparation method of the Protein A affinity chromatography medium of PMMA matrix, comprises the steps:

Hydrophilization treatment: mixing polymethyl methacrylate microspheres with the structure shown in formula (1), alkali 1, epichlorohydrin, water and dextran to prepare the structure shown in formula (2) The hydrophilic polymethyl methacrylate microspheres;

Epoxidation treatment: mixing the hydrophilic polymethyl methacrylate microspheres, alkali 2 and the epoxy compound represented by formula (2-1), and reacting to prepare the structure represented by formula (3) epoxidized polymethyl methacrylate microspheres;

Described epoxidized polymethyl methacrylate microspheres, Protein A, EDTA, mercaptoglycerol, auxiliary agent are mixed, react, prepare the described PMMA matrix with the structure shown in formula (I). Protein A affinity chromatography medium;

The synthetic route is as follows:

R is selected from halogen or

* indicates the attachment site;

The cross-linking degree of the polymethyl methacrylate microspheres is 5%-80%;

The particle size of the polymethyl methacrylate microspheres is 10 μm˜70 μm;

The particle size distribution variation coefficient of the polymethyl methacrylate microspheres is less than 5%.

In one embodiment, the cross-linking degree of the polymethyl methacrylate microspheres is 10%-70%;

In one embodiment, the polydispersity coefficient of the polymethyl methacrylate microspheres is 1.04-1.18.

In one embodiment, the particle size of the polymethyl methacrylate microspheres ranges from 20 μm to 60 μm.

In one of the more preferred embodiments, the coefficient of variation of the particle size distribution of the polymethyl methacrylate microspheres is less than 3%.

In one of the more preferred embodiments, the polymethyl methacrylate microspheres have a porous structure, and the pore size of each hole in the porous structure is

In one embodiment, the Protein A is obtained by splicing the E, C and Z-domains of Staphylococcus protein A.

In one of the embodiments, the preparation method of the hydrophilic polymethyl methacrylate microspheres comprises the following steps:

Mix the polymethyl methacrylate microspheres, part of the base 1, and epichlorohydrin to prepare an intermediate;

The intermediate, dextran and remaining base 1 were mixed to prepare the hydrophilic polymethylmethacrylate microspheres.

In one embodiment, the mass ratio of the polymethyl methacrylate microspheres and dextran is 1:0.1-1:0.4.

In one of the embodiments, the base 1 and the base 2 are independently selected from one or more of sodium hydroxide, potassium hydroxide, sodium methoxide, potassium methoxide and potassium tert-butoxide.

In one embodiment, the mass ratio of the polymethyl methacrylate microspheres and the alkali 1 is 1:0.1-1:0.4.

In one of the embodiments, the mass ratio of the hydrophilic polymethyl methacrylate microspheres and alkali 2 is 1:2-1:4.

In one embodiment, the mass ratio of the hydrophilic polymethyl methacrylate microspheres to the epoxy compound is 1:0.8-1:1.5.

In one of the embodiments, the reaction time for preparing epoxidized polymethyl methacrylate microspheres is 2h-20h, and the reaction temperature is 10°C-40°C.

In one embodiment, the mass ratio of the epoxidized polymethyl methacrylate microspheres to Protein A is 1:2 to 1:3.

In one embodiment, the mass ratio of the epoxidized polymethyl methacrylate microspheres to EDTA is 2.0:1-2.5:1.

In one embodiment, the mass ratio of the epoxidized polymethyl methacrylate microspheres to mercaptoglycerol is 2.0:1-2.5:1.

In one embodiment, the adjuvant includes one or more of a coupling accelerator, a PB buffer or a tailing buffer.

In one embodiment, the coupling accelerator is selected from sodium sulfate; and/or, the tail-capping buffer is selected from sodium carbonate or sodium bicarbonate.

The present invention also provides the Protein A affinity chromatography medium of the PMMA matrix prepared according to the above-mentioned preparation method.

The present invention also provides the application of the above-mentioned PMMA-based Protein A affinity chromatography medium in separating and purifying biological macromolecules.

Compared with the prior art, the present invention has the following beneficial effects:

The PMMA matrix Protein A affinity chromatography medium provided by the present invention contains Protein A, highly cross-linked PMMA microspheres and a large number of hydroxyl groups in its chemical structure. Among them, the use of highly cross-linked PMMA microspheres as a solid matrix can endow the affinity chromatography medium with excellent mechanical properties, making it exhibit excellent pressure resistance, which can not only withstand high flow rates, improve separation efficiency, but also improve the packaging efficiency. Column repeatability and medium utilization, reduce buffer consumption, save costs in many aspects, and solve the problems of high cost and low yield of Protein A affinity chromatography medium, which are difficult to meet market demand. At the same time, the surface of PMMA microspheres contains a large number of hydroxyl groups, which endows the affinity chromatography medium with excellent hydrophilicity, so that it will not produce non-specific binding with host proteins. At the same time, the protein A is connected to the solid matrix in the form of bonding through the chain ether spacer, so that the protein A affinity chromatography medium is dendritic, which improves the stability of the protein A affinity chromatography medium and has strong acid resistance. , The alkali cleaning resistance is enhanced, and the protein A ligand is less shed when it is washed with alkali, and it can be reused.

In addition, the use of a monodisperse microsphere structure makes the particle size and distribution of the affinity chromatography medium more controllable. The connection of Protein A and PMMA microspheres is conducive to the preparation of PMMA matrix with smaller particle size and uniform particle size. Protein A affinity chromatography medium. In the present invention, the particle size of the polymethyl methacrylate microspheres is controlled to be 10 μm to 70 μm, and the variation coefficient of particle size distribution is set to be less than 5%, which can not only shorten the mass transfer path, improve the mass transfer efficiency, but also prevent the bed lamination. If the drop is increased too much, the separation effect will be poor.

It can be seen that the Protein A affinity chromatography medium of the PMMA matrix of the present invention has excellent comprehensive performance, and is especially suitable for the rapid separation and purification of biological macromolecules (such as antibodies) and continuous flow chromatography. The preparation method is simple and efficient, and is suitable for industrial production. ,with broadly application foreground.

Description of drawings

Fig. 1 is the mechanical property and pressure resistance test result of the Protein A affinity chromatography medium of the PMMA matrix of embodiment 1;

Fig. 2 is the alkali resistance test result of the Protein A affinity chromatography medium of the PMMA matrix of embodiment 1;

Fig. 3 is the chromatogram that adopts the Protein A affinity chromatography medium of the PMMA matrix of embodiment 1 to separate IgG1;

Fig. 4 is the chromatogram that adopts the Protein A affinity chromatography medium of the PMMA matrix of embodiment 4 to separate IgG1;

Fig. 5 is the chromatogram that adopts the Protein A affinity chromatography medium of the PMMA matrix of embodiment 5 to separate IgG1;

Fig. 6 is the chromatogram that adopts the Protein A affinity chromatography medium of the PMMA matrix of embodiment 6 to separate IgG1;

Fig. 7 is the chromatogram that adopts the Protein A affinity chromatography medium of the PMMA matrix of comparative example 2 to separate IgG1;

Fig. 8 is the chromatogram that adopts the Protein A affinity chromatography medium of the PMMA matrix of Comparative Example 3 to separate IgG1;

Figure 9 is a chromatogram of the separation of IgG1 using the Protein A affinity chromatography medium of the PMMA matrix of Comparative Example 4.

Detailed ways

The present invention will be further described in detail below in conjunction with specific embodiments. The present invention may be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that a thorough and complete understanding of the present disclosure is provided.

Unless otherwise defined, 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 terms used herein in the description of the present invention are for the purpose of describing specific embodiments only, and are not intended to limit the present invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

Protein A affinity chromatography medium based on highly cross-linked agarose microspheres has excellent hydrophilicity and good biocompatibility, which is very suitable for the separation and purification of antibody biological functional groups; The distribution is wide, and there are problems such as poor column packing repeatability and low mechanical strength in the actual use process. The Protein A affinity chromatography medium based on the glass bead structure with controllable pore channels has the characteristics of pressure resistance and high flow rate. However, due to its strong hydrophobicity, it is prone to non-specific binding with host proteins; moreover, it is not resistant to high concentrations of NaOH, and the impurities bound to the column are not easily removed, thus limiting its application in large-scale biological samples.

In this regard, the present invention provides a kind of good hydrophilicity, strong alkali resistance, strong acid resistance, small particle size, uniform particle size, high mechanical strength, can be used under high flow rate, good separation effect and low cost The polymethyl methacrylate (PMMA)-based ProteinA affinity chromatography medium is suitable for the rapid separation and purification of biological macromolecules (such as antibodies) and continuous flow chromatography, which can improve production efficiency, improve medium utilization, reduce Buffer usage.

The Protein A affinity chromatography medium of the PMMA matrix of the present invention has the structure shown in formula (I):

Indicates polymethyl methacrylate microspheres.

In the present invention, the degree of crosslinking of PMMA refers to the mass ratio of the crosslinking agent to the MMA monomer when preparing PMMA.

Coefficient of variation of particle size distribution: The coefficient of variation of particle size distribution of a standard substance is used to express the degree of particle size dispersion of the standard substance. It is usually expressed as a percentage of the standard deviation or the ratio of the standard deviation to the average particle size of the standard substance, also known as the degree of dispersion.

The technical scheme of the present invention is as follows:

The synthetic route is as follows:

R is selected from halogen or

Preferably, R is selected from chlorine; * denotes the attachment site;

The cross-linking degree of the polymethyl methacrylate microspheres is 5% to 80%;

The PMMA matrix Protein A affinity chromatography medium provided by the present invention contains Protein A, highly cross-linked PMMA microspheres and a large number of hydroxyl groups in its chemical structure. Among them, the use of highly cross-linked PMMA microspheres as a solid matrix can endow the affinity chromatography medium with excellent mechanical properties, making it exhibit excellent pressure resistance, which can not only withstand high flow rates, improve separation efficiency, but also improve the packaging efficiency. Column repeatability and medium utilization, reduce buffer consumption, save costs in many aspects, and solve the problems of high cost and low yield of Protein A affinity chromatography medium, which are difficult to meet market demand. At the same time, the surface of PMMA microspheres contains a large number of hydroxyl groups, which endows the affinity chromatography medium with excellent hydrophilicity, so that it will not produce non-specific binding with host proteins. At the same time, the protein A is connected to the solid matrix in the form of bonding through the chain ether spacer, so that the protein A affinity chromatography medium is dendritic, which improves the stability of the protein A affinity chromatography medium and is resistant to alkali cleaning. The property is enhanced, and the amount of protein A ligand shedding is small when rinsed with alkali, which can be reused. In addition, the particle size and distribution of the microsphere structure are more controllable. The connection of Protein A and PMMA microspheres is conducive to the preparation of PMMA-based Protein A affinity chromatography media with smaller particle size and uniform particle size. . In the present invention, the particle size of the polymethyl methacrylate microspheres is controlled to be 10 μm to 70 μm, and the variation coefficient of particle size distribution is set to be less than 5%, which can not only shorten the mass transfer path, improve the mass transfer efficiency, but also prevent the bed lamination. If the drop is increased too much, the separation effect will be poor.

Preferably, the preparation method of the Protein A affinity chromatography medium of above-mentioned PMMA matrix is as follows:

(1) Preparation

It comprises the following steps: mixing the polymethyl methacrylate microspheres with the structure shown in formula (1), alkali 1, epichlorohydrin, water and dextran to prepare the polymethyl methacrylate microspheres with the structure shown in formula (2) Hydrophilic polymethyl methacrylate microspheres. That is, the PMMA microspheres are subjected to hydrophilization treatment to make the surface of the PMMA microspheres contain a large number of hydrophilic hydroxyl groups.

Preferably, the preparation method of the hydrophilic polymethyl methacrylate microspheres comprises the following steps:

In one embodiment, the base 1 is selected from one or more of sodium hydroxide, potassium hydroxide, sodium methoxide, potassium methoxide and potassium tert-butoxide.

Post-processing steps: suction filtration and washing with water until neutral, vacuum drying at 30 ℃ ~ 60 ℃ for 2h ~ 10h.

(2) Preparation

Include the following steps:

The base 2 and the epoxy compound represented by the formula (2-1) are mixed and reacted.

That is, under the action of alkali (catalyst),

Substitution reaction with halogenated epoxy group takes place to connect the chain ether spacer and epoxy group on the hydroxyl group on the surface of PMMA microspheres for the next step of ring opening.

Preferably, first

Mix with base 2 and react for 5min～30min to prepare intermediate A;

The intermediate A and the epoxy compound are then mixed to continue the reaction.

In one of the embodiments, the

And the mass ratio of epoxy compound is 1:0.8-1:1.5.

In one embodiment, the base 2 is selected from one or more of sodium hydroxide, potassium hydroxide, potassium tert-butoxide, potassium methoxide and sodium methoxide. Preferably, the alkali 2 is selected from sodium hydroxide.

In one of the embodiments, the

The mass ratio of base 2 is 1:2-1:4.

(3) Preparation

Will

Protein A, ethylenediaminetetraacetic acid, mercaptoglycerin, and additives are mixed and reacted.

The epoxy group ring-opening on the spacer arm is about to be coupled with the sulfhydryl group in the Protein A ligand to generate the Protein A affinity chromatography medium.

In one embodiment, the Protein A is obtained by splicing the E, C and Z-domains of Staphylococcus protein A (see Chinese Patent No. 202010747812.8 for details).

In one embodiment, the adjuvant includes one or more of a coupling accelerator, a PB buffer or a tailing buffer. Preferably, the mass ratio of coupling accelerator, PB buffer or tail-capping buffer to the epoxidized polymethyl methacrylate microspheres is 1.5:1, 2.0:1 and 1.5:1, respectively.

In one of the more preferred embodiments, the cross-linking degree of the polymethyl methacrylate microspheres is 10% to 70%.

In one of the more preferred embodiments, the particle size of the polymethyl methacrylate microspheres is 20 μm˜60 μm. Using polymethyl methacrylate microspheres in this particle size range as the matrix can shorten the mass transfer path and improve the mass transfer in the pores. More preferably, the particle size of the polymethyl methacrylate microspheres is 40 μm˜50 μm. Using polymethyl methacrylate microspheres in this particle size range as the matrix can not only shorten the mass transfer path, improve the mass transfer in the pores, but also have little effect on the pressure drop of the bed, with high production efficiency and pressure resistance. Strong sex and good separation effect.

In one of the more preferred embodiments, the coefficient of variation of the particle size distribution of the polymethyl methacrylate microspheres is less than 3%, which is more conducive to the preparation of a Protein A affinity chromatography medium with a uniform particle size PMMA matrix, and improves the separation efficiency. Effect.

It can be understood that the polymethyl methacrylate microspheres of the present invention may have a non-porous structure, or may be microspheres with a porous structure. The porous structure is conducive to increasing the specific surface area of the medium, providing more ligand coupling sites, which is conducive to protein binding and has a relatively high capacity.

Preferably, the polymethyl methacrylate microspheres of the present invention have a porous structure, and the pore size of each hole in the porous structure is

The present invention also provides the Protein A affinity chromatography medium prepared according to the above-mentioned preparation method to obtain the PMMA matrix.

The following is the specific embodiment part.

Unless otherwise specified, the raw materials used in the examples and comparative examples are all commercially available products.

Example 1

The present embodiment provides a PMMA matrix-based Protein A affinity chromatography medium and a preparation method thereof.

(1) Preparation of hydrophilic PMMA microspheres 1

Under nitrogen protection, measure 10g of PMMA microspheres 1 and place them in an Erlenmeyer flask, add 30mL of 2mol/L NaOH aqueous solution to it, put the Erlenmeyer flask containing the above solution into a shaker and shake for 10min, then add 5g epoxy resin Chloropropane (alalddin, purity 99.7%) was continuously shaken at 25° C. for 5 h. After the reaction was completed, suction filtration and washing with water until neutral. The above microspheres were placed in an Erlenmeyer flask, and 30mL of deionized water and 2g of dextran (molecular weight 100KDa) were added to it, and the Erlenmeyer flask filled with the above solution was placed in a shaker to shake for 10min, and then 1g of sodium hydroxide was added. , continue to shake at 35°C for 16h, after the reaction is completed, suction filter and wash with water until neutral, and dry in vacuum at 30°C for 4h to obtain hydrophilic PMMA microspheres 1.

The PMMA microspheres 1 in this embodiment are purchased from Suzhou Nano Micro Technology Co., Ltd., the product model is UniPMMA-40, the particle size is 40 μm, and the aperture is

The CV was 2.5%, the degree of crosslinking was 60%, and the polydispersity coefficient was 1.10.

(2) Preparation of epoxidized PMMA microspheres 1:

Under nitrogen protection, measure 10g of hydrophilic PMMA microspheres 1 and place them in a conical flask, and add 50mL 2mol/L NaOH aqueous solution to it, put the conical flask containing the above solution into a shaker and shake for 10min, Then add 10g epichlorohydrin (purchased from alalddin, purity 99.7%) and continue to shake at 25°C for 5h. After the reaction is completed, filter with suction and wash with water until neutral, and then vacuum dry at 50°C for 6h to obtain epoxidized PMMA microspheres. 1.

(3) Preparation of PMMA-based Protein A affinity chromatography medium 1:

Under nitrogen protection, 1.5 g of epoxidized PMMA microspheres 1 were placed in a 15 mL EP tube, to which was added 3.75 mL of 10 mg/mL Protein A, 5 mL of 1.3 M Na ₂ SO ₄ , 7.5 mL of 50 mM PB, 2.25 mL of 1 mM EDTA solution. After sealing with parafilm, nitrogen was passed for 5 min, sealed, placed in a shaker, and shaken at 37°C for 24h. The protein solution after the reaction was filtered with a syringe filter to remove the protein solution, and the OD value after the reaction was measured and recorded.

When the OD value is less than 0.8, add 0.5 mL of mercaptoglycerol and 4.5 mL of Na ₂ CO ₃ solution with a pH value of 10.0 to the EP tube, pass nitrogen for 5 min, seal with a parafilm and place it on a shaker for 4 h, and the reaction temperature is 37 ° C. After completion of the reaction, suction filtration and washing with water until neutral, drying at 50° C. for 6 h under vacuum to obtain PMMA-based Protein A affinity chromatography medium 1.

Example 2

The preparation method is basically the same as that of Example 1, except that the PMMA microspheres 1 are replaced with PMMA microspheres 2 to obtain the Protein A affinity chromatography medium 2 of the PMMA matrix.

PMMA microspheres 2 were purchased from Suzhou Nano Micro Technology Co., Ltd., the product model is UniPMMA-50, the particle size is 50 μm, and the pore size is

Example 3

The preparation method is basically the same as that in Example 1, except that the PMMA microspheres 1 are replaced with PMMA microspheres 3 to obtain the Protein A affinity chromatography medium 3 of the PMMA matrix.

PMMA microspheres 3 were purchased from Suzhou Nano Micro Technology Co., Ltd., the product model is UniPMMA-40, the particle size is 40 μm, and the pore size is

Example 4

The preparation method is basically the same as that in Example 1, except that the PMMA microspheres 1 are replaced with PMMA microspheres 4 to obtain the Protein A affinity chromatography medium 4 of the PMMA matrix.

PMMA microspheres 4 were purchased from Suzhou Nano Micro Technology Co., Ltd., the product model is UniPMMA-40, the particle size is 40 μm, and the pore size is

The CV was 2.5%, the degree of crosslinking was 80%, and the polydispersity coefficient was 1.10.

Example 5

The preparation method is basically the same as that of Example 1, except that the PMMA microspheres 1 are replaced with PMMA microspheres 5 to obtain the Protein A affinity chromatography medium 5 of the PMMA matrix.

PMMA microspheres 5 were purchased from Suzhou Nano Micro Technology Co., Ltd., the product model is UniPMMA-10, the particle size is 10 μm, and the pore size is

Example 6

The preparation method is basically the same as that of Example 1, except that the PMMA microspheres 1 are replaced with PMMA microspheres 6 to obtain the Protein A affinity chromatography medium 6 of the PMMA matrix.

PMMA microspheres 6 were purchased from Suzhou Nano Micro Technology Co., Ltd., the product model is UniPMMA-40, the particle size is 40 μm, and the pore size is

The CV was 4.5%, the degree of crosslinking was 60%, and the polydispersity coefficient was 1.10.

Example 7

The preparation method is basically the same as that of Example 1, except that the PMMA microspheres 1 are replaced with PMMA microspheres 7 to obtain the Protein A affinity chromatography medium 7 of the PMMA matrix.

PMMA microspheres 7 were purchased from Suzhou Nano Micro Technology Co., Ltd., non-porous, with a particle size of 40 μm, a CV of 2.5%, a degree of cross-linking of 60%, and a polydispersity coefficient of 1.10.

Comparative Example 1

This comparative example provides a preparation method of an agarose-based Protein A affinity chromatography medium.

Product model MabSelect SuRe LX, manufacturer GE, matrix agarose, particle size 60μm ~ 165μm,

Comparative Example 2

The preparation method is basically the same as that of Example 1, except that the PMMA microspheres 1 are replaced with PMMA microspheres 8 to obtain the Protein A affinity chromatography medium 8 of the PMMA matrix.

PMMA microspheres 8 were purchased from Suzhou Nano Micro Technology Co., Ltd., the product model is UniPMMA-40, the particle size is 40 μm, and the pore size is

The CV was 2.5%, the degree of crosslinking was 2%, and the polydispersity coefficient was 1.10.

Comparative Example 3

This comparative example provides a PMMA matrix-based Protein A affinity chromatography medium and a preparation method thereof.

The preparation method is basically the same as that of Example 1, except that the PMMA microspheres 1 are replaced with PMMA microspheres 9 to obtain the Protein A affinity chromatography medium 9 of the PMMA matrix.

PMMA microspheres 9 were purchased from Suzhou Nano Micro Technology Co., Ltd., with a particle size of 30 μm to 150 μm and a pore size of

CV>5%, the degree of crosslinking is 60%, and the polydispersity coefficient is 1.10.

Comparative Example 4

The preparation method is basically the same as that of Example 1, except that the PMMA microspheres 1 are replaced with PMMA microspheres 10 to obtain the Protein A affinity chromatography medium 10 of the PMMA matrix.

PMMA microspheres 8 were purchased from Suzhou Nano Micro Technology Co., Ltd., with a particle size of 60-90 μm and a pore size of

Test example

(1) Mechanical strength test:

Chromatography column: Pack the XK16-250mm chromatography column with the Protein A affinity chromatography medium prepared in Example 1.

Instrument: AKTA purifier (Suzhou Sepu Instrument Co., Ltd.)

Mobile phase: 0.15M NaCl solution

Column temperature: 25℃

Test conditions: equilibrated with deionized water until the baseline returned to zero, and rinsed with 0.5M sodium chloride equilibrated solution. Increase the flow rate and observe the pressure change.

The test results are shown in Figure 1. The pressure can withstand more than 0.8MPa, and the column pressure and flow rate maintain a linear relationship, indicating that the mechanical strength is good. As the column height increases, the pressure it can withstand increases. It can run at a faster flow rate above 30cm column height, and can withstand a maximum pressure of 1MPa.

The mechanical properties and column pressure of the Protein A affinity chromatography media of Examples 2 to 7 and Comparative Examples 1 to 4 were tested by the same method, and the results are shown in Table 1.

Table 1

(2) Alkali resistance test

Chromatography column: take the Protein A affinity chromatography medium prepared in Example 1 and pack a 1mm PP column (7*25mm);

Sample: Monoclonal antibody clarified fermentation broth, 3.6 mg/mL;

Test conditions: use 10 column volumes of 20 mM PBS buffer, 150 mM NaCl, pH 7.0; elution: 6 column volumes of 20 mM citric acid pH 3.0; equilibration: 3 column volumes of PBS buffer; 5 column volumes of CIP: 0.5M NaOH (contact time 15 min per round); re-equilibration: 10 column volumes of PBS buffer; flow rate: 1 mL/min (150 cm/h). Repeat the cycle 120 times.

The test results are shown in Figure 2. The results show that the 10% flow-through dynamic load is not lower than 90% of the initial flow-through dynamic load, indicating that Protein A affinity chromatography medium 1 has strong alkali cleaning resistance and less ligand shedding. .

Adopt the same method to test the acid and alkali resistance stability of the Protein A affinity chromatography media of Examples 2 to 7 and Comparative Examples 1 to 4, and the results are shown in Table 2.

Table 2

As can be seen from Table 2, the Protein A affinity chromatography media prepared in Examples 1 to 7 and Comparative Examples 1 to 4 of the present application are stable to acid and alkali.

(3) Purification and application of monoclonal antibodies

Chromatography column: get the Protein A affinity chromatography medium prepared in Example 1 and pack a 10mm PP column (height 25mm);

Equipment: AKTA pure (NM-BD-004) (Suzhou Sepu Instrument Co., Ltd.)

Test sample: IgG1 fermentation broth 2.7mg/mL;

Test conditions: use 15 column volumes of 10mM PBS buffer, pH 6.0; elution: 10 column volumes of 20mM citric acid pH 3.4; equilibration: 15 column volumes of 10mM PBS buffer; retention time 5min, flow rate 800cm/ h.

The test results are shown in Figure 3, the IgG1 elution peak is sharp, and some variants are also separated, indicating that it is suitable for mAb separation.

The prepared Protein A affinity chromatography media of Examples 4 to 7 and Comparative Examples 1 to 4 were tested in the same manner.

Figure 4 is the chromatogram of the separation of IgG1 using the Protein A affinity chromatography medium of the PMMA matrix of Example 4; it can be seen from Figure 4 that the IgG1 elution peaks are slightly less compatible and have a little tail, indicating that they can be used for mAb separation, the separation effect is poor.

Fig. 5 is the chromatogram of the separation of IgG1 using the Protein A affinity chromatography medium of the PMMA matrix of Example 5; it can be seen from Fig. 5 that the IgG1 elution peak is a sharp peak, and compared with Fig. 3, the retention time between impurities and impurities The elution peak is not single, indicating that it can be used for mAb separation, and the separation effect is poor.

Fig. 6 is the chromatogram of the separation of IgG1 using the Protein A affinity chromatography medium of the PMMA matrix of Example 6. It can be seen from Fig. 6 that the IgG1 elution peak is a sharp peak. Compared with Fig. 3, the retention time between impurities and impurities The elution peak is not single, indicating that it can be used for mAb separation, and the separation effect is poor.

IgG1 was separated using the Protein A affinity chromatography medium of PMMA matrix of Comparative Example 1, tail collapsed.

Figure 7 is the chromatogram of the separation of IgG1 using the Protein A affinity chromatography medium of the PMMA matrix of Comparative Example 2. It can be seen from Figure 7 that the IgG1 elution peaks are poorly matched, the tailing is obvious, and the elution peaks are not single, indicating that It is not suitable for mAb isolation.

Figure 8 is the chromatogram of the separation of IgG1 using the Protein A affinity chromatography medium of the PMMA matrix of Comparative Example 3. It can be seen from Figure 8 that the IgG1 elution peaks are poorly matched, the tailing is obvious, and the elution peaks are not single, indicating that It is not suitable for mAb isolation.

Fig. 9 is the chromatogram of separating IgG1 using the Protein A affinity chromatography medium of the PMMA matrix of Comparative Example 4. It can be seen from Fig. 9 that the IgG1 elution peaks are poorly matched, the tailing is obvious, and the elution peaks are not single, indicating that It is not suitable for mAb isolation.

The separation effects of the Protein A affinity chromatography media of Examples 1 to 7 and Comparative Examples 1 to 4 in continuous flow were characterized, and the methods were as follows:

Application of Continuous Flow Chromatography for Separation of IgG1

Chromatography column: Pack 4.7mL PP column with the Protein A affinity chromatography medium prepared in Examples 1 to 7;

Instrument: four-column circulating continuous flow chromatography system);

Sample: IgG1 (50mg/mL);

Working conditions:

The column was first equilibrated with 5CV of equilibration buffer, loaded, rinsed with 5CV of equilibration buffer, then rinsed with 5CV of high-salt buffer (20mM PBS buffer, pH 6.0, 1.0M NaCl, then rinsed with 5CV of equilibration buffer, Then 5CV of citrate buffer (0.1M, pH 3.0) was used for elution, 5CV of acetic acid solution 1.0M was fully washed, 5CV of equilibration buffer was used to equilibrate the column, and 0.1M NaOH was used to regenerate the column for 15min. Equilibrate with 5CV of equilibration buffer. The retention time of sample loading during the separation process is 1.5min, the retention time of the washing step is consistent with the sample loading, the retention time of the regeneration step is fixed at 4min, and other steps (including equilibration, rinsing, elution, etc.) The retention time was 1.5 min.

Table 3 Separation effect of different chromatographic media in continuous flow

In the application of continuous flow chromatography system, compared with the medium of agarose matrix, the protein A affinity chromatography medium of the PMMA matrix prepared by the present invention has higher yield and medium utilization rate, and less buffer consumption. . With the increase of the particle size and average particle size of the PMMA matrix, the yield, medium utilization rate, buffer consumption, etc. have corresponding differences. The smaller the particle size, the more uniform, the medium with high mechanical strength, the faster the flow rate, the higher the medium utilization rate, which can effectively reduce the production cost.

The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity, all possible combinations of the technical features in the above-described embodiments are not described. However, as long as there is no contradiction between the combinations of these technical features, All should be regarded as the scope described in this specification.

The above-mentioned embodiments only represent several embodiments of the present invention, and the descriptions thereof are specific and detailed, but should not be construed as a limitation on the scope of the invention patent. It should be pointed out that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can be made, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention should be subject to the appended claims.

Claims

A kind of preparation method of the Protein A affinity chromatography medium of PMMA matrix, is characterized in that, comprises the steps:

Hydrophilization treatment: mixing polymethyl methacrylate microspheres with the structure shown in formula (1), alkali 1, epichlorohydrin, water and dextran to prepare the structure shown in formula (2) The hydrophilic polymethyl methacrylate microspheres;

Epoxidation treatment: mixing the hydrophilic polymethyl methacrylate microspheres, alkali 2 and the epoxy compound represented by formula (2-1), and reacting to prepare the structure represented by formula (3) epoxidized polymethyl methacrylate microspheres;

Described epoxidized polymethyl methacrylate microspheres, Protein A, EDTA, mercaptoglycerol, auxiliary agent are mixed, react, prepare the described PMMA matrix with the structure shown in formula (I). Protein A affinity chromatography medium;

The synthetic route is as follows:

R is selected from halogen or
* indicates the attachment site;

The cross-linking degree of the polymethyl methacrylate microspheres is 5% to 80%;

The particle size of the polymethyl methacrylate microspheres is 10 μm˜70 μm;

The particle size distribution variation coefficient of the polymethyl methacrylate microspheres is less than 5%.
The method for preparing a PMMA-based Protein A affinity chromatography medium according to claim 1, wherein the cross-linking degree of the polymethyl methacrylate microspheres is 10% to 70%; and/or, The particle size of the polymethyl methacrylate microspheres is 20 μm˜60 μm.
The preparation method of the Protein A affinity chromatography medium of PMMA matrix according to claim 1, is characterized in that, the polydispersity coefficient of described polymethyl methacrylate microspheres is 1.04～1.18.
The PMMA-based Protein A affinity chromatography medium according to any one of claims 1 to 3, wherein the polymethyl methacrylate microspheres have a porous structure, and each hole in the porous structure has a The aperture is
The Protein A affinity chromatography medium of PMMA matrix according to any one of claims 1 to 3, wherein the Protein A is obtained by splicing E, C and Z-domains of Staphylococcus protein A.
The preparation method of the Protein A affinity chromatography medium of PMMA matrix according to claim 1, is characterized in that, the preparation method of described hydrophilic polymethyl methacrylate microspheres comprises the following steps:

Mix the polymethyl methacrylate microspheres, part of the base 1, and epichlorohydrin to prepare an intermediate;

The intermediate, dextran and remaining base 1 were mixed to prepare the hydrophilic polymethylmethacrylate microspheres.
The method for preparing a PMMA matrix-based Protein A affinity chromatography medium according to claim 1 or 6, wherein the mass ratio of the polymethyl methacrylate microspheres to the dextran is 1:0.1-1 : 0.4.
The preparation method of the Protein A affinity chromatography medium of PMMA matrix according to claim 1 or 6, is characterized in that, described alkali 1 and alkali 2 are respectively independently selected from sodium hydroxide, potassium hydroxide, sodium methoxide , one or more of potassium methoxide and potassium tert-butoxide.
The method for preparing a PMMA matrix-based Protein A affinity chromatography medium according to claim 8, wherein the mass ratio of the polymethyl methacrylate microspheres to the alkali 1 is 1:0.1 to 1:0.4; and / or,

The mass ratio of the hydrophilic polymethyl methacrylate microspheres and alkali 2 is 1:2-1:4.
The preparation method of the Protein A affinity chromatography medium of PMMA matrix according to any one of claims 1 to 3, characterized in that, the quality of the hydrophilic polymethyl methacrylate microspheres and epoxy compounds The ratio is 1:0.8～1:1.5.
The preparation method of the Protein A affinity chromatography medium of PMMA matrix according to claim 1, is characterized in that, the mass ratio of described epoxidized polymethyl methacrylate microspheres to Protein A is 1:2～1: 3; and/or,

The mass ratio of the epoxidized polymethyl methacrylate microspheres to EDTA is 2.0:1 to 2.5:1; and/or,

The mass ratio of the epoxidized polymethyl methacrylate microspheres and mercaptoglycerol is 2.0:1-2.5:1.
The PMMA matrix-based Protein A affinity chromatography medium according to any one of claims 1 to 3, wherein the auxiliary agent comprises one of a coupling accelerator, a PB buffer or a tail-capping buffer or variety.
The PMMA-based Protein A affinity chromatography medium according to claim 12, wherein the coupling accelerator is selected from sodium sulfate; and/or, the tail-capping buffer is selected from sodium carbonate or bicarbonate sodium.
A Protein A affinity chromatography medium of a PMMA matrix prepared by a method for preparing a Protein A affinity chromatography medium of a PMMA matrix according to any one of claims 1 to 13.
The application of the Protein A affinity chromatography medium of the PMMA matrix of claim 14 in separating and purifying biological macromolecules.