CN115990464A

CN115990464A - Hydrophilic modified hydrophobic polymer matrix Protein A affinity chromatography media

Info

Publication number: CN115990464A
Application number: CN202111223760.5A
Authority: CN
Inventors: 马杰
Original assignee: Individual
Current assignee: Individual
Priority date: 2021-10-18
Filing date: 2021-10-18
Publication date: 2023-04-21

Abstract

The invention relates to a preparation method and application of a hydrophilic modified hydrophobic polymer matrix Protein A affinity chromatography medium. The preparation method comprises the following steps: 1) Preparing porous hydrophobic polymer microspheres with uniform size distribution and high crosslinking; 2) Impregnating the porous microspheres prepared in step 1 with a diluted aqueous solution containing moderately hydrophobically modified agarose or a similar substance or mixture; 3) Crosslinking reaction is carried out on the modified components covered on the surface of the modified porous microsphere and the surface of the inner hole; 4) Carrying out epoxy functional group grafting reaction on the porous microspheres subjected to surface crosslinking; 5) Finally, a Protein A grafting reaction is carried out. The Protein A affinity chromatography medium prepared by the method has the advantages of alkali resistance, acid resistance, high mechanical strength, low nonspecific adsorption, high rapid separation continuous chromatography efficiency on biological macromolecules, long service life and wide application prospect.

Description

Hydrophilic modified hydrophobic polymer matrix Protein A affinity chromatography media

Technical Field

The invention relates to a preparation and application of a hydrophilic modified hydrophobic polymer matrix Protein A affinity chromatography medium, which has the characteristics of excellent acid and alkali resistance, excellent biocompatibility, hydrophilicity, low nonspecific adsorption, high mechanical strength, high specific adsorption efficiency and the like, and is suitable for high-efficiency purification of biomacromolecule proteins, in particular antibodies.

Background

The affinity chromatography medium with the surface modified Protein A is a preferred separation and purification means for separating biological macromolecular proteins such as antibodies due to the excellent specific adsorption function. Continuous flow chromatography is often used to increase separation efficiency. In the chromatography process, the liquid containing the target molecules flows through a filling column containing the chromatography medium at a certain flow rate under a certain pressure, is obtained due to the efficient adsorption of Protein A on the target molecules, is eluted, is washed with an alkaline aqueous solution for multiple times, and is reused. Under the working condition, the chromatography medium is required to have the characteristics of sufficient mechanical strength, alkali resistance, uniform size distribution, good surface biocompatibility, low nonspecific adsorption and the like.

The technical scheme commonly used at present comprises: the surface of the crosslinked agarose particles is combined with Protein A (CN 00574840C), the surface of the crosslinked methacrylate copolymer particles is combined with Protein A (US 20130085199A1, CN 112341663A), and the surface of the crosslinked polyvinyl alcohol copolymer is combined with Protein A (EP 338971A 1).

The affinity chromatography medium which is introduced in the patent CN00574840C and takes the cross-linked agarose particles as the matrix and is combined with Protein A has the advantages of good biocompatibility, low nonspecific adsorption, hydrophilic surface and high specific adsorption efficiency. However, agarose has insufficient strength and is easily deformed under pressure, so that the pressure drop is increased and the separation efficiency is reduced.

The Protein A affinity chromatography media described in the patent US20130085199A1 and the patent CN112341663A are based on cross-linked methacrylate copolymer particles, and the alkali resistance of the cross-linked methacrylate copolymer particles needs to be further improved due to the existence of ester bonds, and the mechanical strength of the cross-linked methacrylate copolymer particles also needs to be improved due to the limitation of the cross-linking degree. The chromatographic medium described in patent US20130085199A1 is not completely covered with hydroxyl groups on the surface and nonspecific adsorption is to be improved.

Patent EP338971A1 describes a polyvinyl alcohol copolymer particle based on crosslinking as matrix, which, due to the insufficient degree of crosslinking of the mechanism, requires a further improvement in mechanical strength, alkali resistance and surface is not completely covered with hydroxyl groups, has a hydrophobic region and is more nonspecifically adsorbed.

Disclosure of Invention

Based on this, the present invention provides a method of preparing a highly crosslinked porous polymer particle having strong hydrophobicity, and coating a biocompatible substance such as agarose on the surface of the particle and performing chemical crosslinking to improve adhesion strength, and then grafting Protein A. The microsphere prepared in this way optimizes alkali resistance, mechanical strength and surface biocompatibility, reduces nonspecific adsorption, improves alkali resistance of Protein A, and improves specific adsorption efficiency.

The preparation method of the Protein A affinity chromatography medium comprises the following steps: 1) Preparing highly cross-linked hydrophobic porous polymer microspheres with uniform size distribution; 2) Impregnating the porous microspheres prepared in the step 1 with diluted mixed aqueous solution containing moderately hydrophobically modified agarose or dextran or similar structural substances or two or more kinds of mixed aqueous solution to ensure that the surfaces of the porous microspheres and the surfaces of inner holes in the step 1 are covered with rich hydroxyl groups; 3) Crosslinking reaction is carried out on the modified hydroxyl-rich component covered on the surface of the modified porous microsphere and the surface of the inner hole; 4) Carrying out epoxy functional group grafting reaction on the porous microspheres subjected to surface crosslinking; 5) Finally, a Protein A grafting reaction is carried out.

In step 1, in order to prepare porous microspheres with high crosslinking, uniform size distribution and strong hydrophobicity, the synthesis method, synthesis formula, process and reaction conditions need to be optimized to obtain porous polymer matrix microspheres with moderate particle size, uniform size distribution, inner pore volume and size and alkali resistance meeting the requirements.

The porous microsphere synthesis can be performed by using traditional suspension polymerization, dispersion polymerization, emulsion suspension polymerization (a pre-emulsion prepared by using a high-speed emulsifying head, which is uniformly dispersed), seed swelling polymerization (CN 1038756C), seed multi-step swelling polymerization (CN 101186661A), and the like. In the embodiment of the invention, a high-speed emulsifying device is used to disperse the reaction liquid to obtain reaction liquid drops with uniform size distribution, and then polymerization is carried out. In order to obtain the porous microsphere with strong alkali resistance and high mechanical strength, the reaction liquid contains alkali-resistant monomer, cross-linking agent, pore-forming agent, aqueous phase polymerization inhibitor, stabilizer and the like. In general, the initiator added during polymerization is mostly oil-soluble.

Typical suitable monomers are generally styryl monomers, which may be mono-and polyfunctional monomers, such as divinylbenzene DVB, divinylbenzene, and the like. These polyfunctional monomers may be used singly or in combination of two or more. To obtain excellent acid and alkali resistance, mechanical strength, divinylbenzene DVB is preferred. In addition to the polyfunctional monomers, suitable amounts of monofunctional monomers, such as, for example, styrene and derivatives thereof, may also be added. However, it is desirable to ensure that the mass content of the polyfunctional monomer is greater than 50wt%, preferably greater than 60wt%, and more preferably greater than 70wt% in the formulation.

The pore-forming agent is typically an aliphatic or aromatic hydrocarbon, ester, ketone, ether, or alcohol solvent. The monomers in an amount of 0 to 200wt% may be used singly or in combination. The size, shape and size distribution of the inner holes of the porous microspheres can be optimized by controlling the content and the type of the pore-forming agent. The solvent of the general reaction is water, which can also be used as pore-forming agent in practice, the pore-forming mechanism is: a portion of the oil-soluble surfactant is dissolved in the monomer and then water is absorbed to effect pore formation. These oil-soluble surfactants include: sorbitan monoesters of C16-C24 branched fatty acids, sorbitan monoesters of C16-C22 linear unsaturated fatty acids, sorbitan monoesters of C12-C14 segmented saturated fatty acids, sorbitan laurate (trademark: SPAN 20), sorbitan oleate (SAPN 80), and the like. These surfactants are used in amounts of 5% to 80% by weight, relative to the mass of the monomers.

The polymerization is generally carried out in a water-mediated system, which may also contain some amount of a co-soluble organic solvent such as ethanol. The water may also contain a surfactant, or a water-soluble macromolecular stabilizer, or a mixture of one or more surfactants and macromolecular stabilizers. The surfactant is typically a nonionic, anionic, or amphoteric surfactant commonly used in water. The macromolecular water-soluble polymer is preferably polyvinyl alcohol or hydroxyethyl cellulose or the like. The stabilizers may be used alone or in combination, and should generally be present in an amount of from 0.3 to 15% by weight, preferably from 1 to 10% by weight, based on the total amount of the monomers.

The initiator for polymerization should be oil-soluble to prevent polymerization in water to form small particles, typically representing organic peroxides such as benzoyl peroxide, lauroyl peroxide, and the like; azo compounds such as azobisisobutyronitrile and the like. The content thereof should generally be 0.1 to 7% by weight, preferably 0.1 to 5% by weight, based on the total amount of the monomers.

In order to prevent the formation of small particles, it is generally also necessary to add soluble inhibitors such as nitrates, sulphates, iodides, hydroquinone, vitamin C, citric acid, polyphenols etc. to the water. The amount added is generally such that it reaches its saturated solubility in water at the reaction temperature.

The polymerization temperature is generally 25 to 100℃and preferably 50 to 100 ℃.

The average particle size obtained by the polymerization is generally from 10 to 300. Mu.m, preferably from 30 to 100. Mu.m. The particle size distribution coefficient is generally less than 1.3, preferably less than 1.2, more preferably less than 1.1. The volume fraction of the particle pores is preferably 30 to 70%, more preferably 40 to 70%. The average pore size is preferably 100 to 500nm, more preferably 200 to 500nm. The specific surface area of the particles is preferably greater than 30m ² /g, more preferably 40m ² The upper limit of the ratio/g is sufficient to satisfy the mechanical strength of the particles, and is generally less than 100m ² /g。

In the step 2, the hydrophobic porous polymer microsphere obtained in the step 1 is modified by using a macromolecular material containing abundant hydroxyl groups, and the main purpose is to improve the hydrophilicity and biocompatibility of the surface of the hydrophobic porous polymer microsphere, so that the working pressure is reduced and the non-specific adsorption is reduced.

The above-described macromolecular material containing hydroxyl groups preferably contains two or more hydroxyl groups in one molecule, and the material itself is a hydrophilic material such as polysaccharide, polyvinyl alcohol, or the like. Preferred polysaccharides include: agarose, dextran, cellulose, chitosan, and the like. Generally suitable weight average molecular weights Mw are between 10000 and 200000.

Since the hydrophilic substance is to cover the surface of the porous microsphere polymerized by the hydrophobic monomer, the hydrophilic hydroxyl-rich macromolecular material needs to be moderately hydrophobically modified in order to achieve better uniform coating and modification. Hydrophobic groups that may be used for hydrophobic modification include: C1-C6 alkyl groups such as methyl, ethyl, propyl, C6-C10 aryl groups such as phenyl, naphthyl. To perform this property of the hydrophobic groups, the hydrophilic macromolecular material is generally modified by selecting a molecule containing the hydrophobic groups together with another group reactive with hydroxyl groups, such as a molecule containing an epoxy group and a phenyl group such as glycidyl phenyl ether. The proportion of hydrophobically modified should ensure a sufficient amount of adsorption without affecting the hydrophilicity used. In order to effectively adsorb the modified hydrophilic macromolecular material on the surface of the hydrophobic porous microsphere, firstly, the hydrophobic modified macromolecular material is dissolved in a solvent, preferably water is used as the solvent, and the concentration is generally controlled to be 5-20mg/mL. Then adding a proper amount of polymer porous microspheres into the aqueous solution, standing for a certain time, stirring properly to reach physical adsorption balance, and washing the microspheres as necessary to elute the non-adsorbed modified macromolecular material. In order to achieve more ideal surface modification, the surface modified hydrophilic macromolecular material is also required to be crosslinked, so that the use resistance of the polymer microsphere can be further improved.

In step 3, surface crosslinking is generally carried out using molecules containing at least two functional groups reactive with hydroxyl groups, such as ethylene glycol diglycidyl ether. If molecules containing epoxide functions are chosen, catalysts like sodium hydroxide are also used. The porous polymer microsphere with the surface physically adsorbed and modified macromolecular material is firstly dispersed in a proper medium such as water, then a proper amount of surface cross-linking agent such as glycol diglycidyl ether is added, a proper amount of catalyst such as sodium hydroxide is added, and then the mixture is heated to a proper reaction temperature for a proper time. The adding amount of the cross-linking agent is based on improving the surface strength but not obviously influencing the hydrophilicity, the amount of the catalyst and the reaction temperature are based on a reaction system, and the reaction time is 1-10 hours. After the reaction, the microspheres are filtered and washed with water or water containing a cosolvent, and the obtained microspheres generally contain 30-400mg of modified hydrophilic macromolecules per gram of microspheres after surface cross-linking.

In step 4, the microsphere surface having been covered with hydroxyl rich groups obtained in step 3 is bonded with suitable functional groups or Protein a according to the separation application requirements. In the present invention, protein A is the target bond. In order to bond Protein a, it is also necessary to attach a functional group, such as an epoxy group, capable of reacting with the amino group at the end of Protein a to the surface of the microsphere. The bonding reaction is realized by substitution reaction of residual hydroxyl and halogenated epoxy groups on the surface of the microsphere under the catalysis of alkali. For example, in the examples, epoxy groups are introduced using substitution reactions of epichlorohydrin with hydroxyl groups.

In step 5, protein A can be effectively bonded by ring-opening reaction of epoxy groups on the microsphere surface with amino groups at the end of Protein A. In general, the bonding reaction is carried out in a system containing a suitable buffer, water being the preferred reaction medium, the isoelectric point ([ EP) of the buffer being required to be close to the IEP of the desired protein. Some salts such as sodium chloride, sodium sulfate and the like are also added into the general reaction system. The surface of the microsphere after binding Protein A preferably contains 10-200mg Protein A/g microsphere, more preferably 25-100mg microsphere. The content should not be construed as a limitation of the requirements of the present invention, but an appropriate content is determined according to actual use requirements.

In addition to the above steps, there may be other steps including, but not limited to, ring opening capping reactions of the residual epoxy groups, the main purpose being to eliminate the residual epoxy groups and react to form hydroxyl groups, which improves both the storage stability of the medium and the hydrophilicity. The capping agent employed may contain a sulfhydryl group such as mercaptoethanol, mercaptoglycerol, or an amino group such as monoethanolamine. Mercaptoglycerol of low toxicity is preferred.

Detailed Description

Example 1

Preparation of porous microspheres

20g of Divinylbenzene (DVB) monomer (purity about 96%), 7.3g of SPAN80,0.8g of benzoyl peroxide were added to 500ml of an aqueous solution of polyvinyl alcohol having a concentration of 0.5% by weight. Emulsification was then carried out using Micro Process Server (Hitachi, ltd) to obtain uniformly dispersed monomer droplets ready for subsequent synthesis of polymer microspheres of uniform particle size. Transferring the dispersed pre-emulsion into a container with stirring, controlling the stirring speed to be 50-200rpm, heating to 80 ℃ in a water bath kettle, and reacting for 8 hours. After the reaction was completed, the synthesized polymer microspheres were filtered, and then the obtained microspheres were washed with acetone to prepare hydrophobic porous polymer microspheres 1.

Microsphere surface modification

Glycidyl phenyl ether modified agarose is selected as a coating layer. Firstly, carrying out hydrophobic modification on agarose: 5g NaOH and 0.5g glycidylphenyl ether were added to 100m agarose aqueous solution (2 wt%). The mixed aqueous solution was heated to 70℃and reacted for 12 hours. The modified agarose was obtained by extraction 3 times with isopropanol and then rinsed.

10g of microsphere 1 was added to 700mL of modified agarose aqueous solution (concentration 20 mg/mL) and stirred at 55℃for 24 hours. The agarose modified in this way can be adsorbed to the inner pores of the microsphere 1 as well as to the outer surface of the microsphere. And then filtering and washing with hot water to obtain the porous microsphere 2 with the surface adsorbed with the hydrophobically modified agarose.

Surface cross-linking

First, 10g of microspheres 2 were dispersed in a 0.4M aqueous NaOH solution, and then 39g of ethylene glycol diglycidyl ether was added thereto, followed by heating to 30℃and reacting for 24 hours. Then washed with 2wt% aqueous Sodium Dodecyl Sulfate (SDS) to prepare porous polymer microspheres 3 having surfaces covered with crosslinked hydrophobically modified agarose.

Grafting of epoxy functional groups

10g of microspheres 3 were taken in a vessel under nitrogen protection, and 50mL of 2M aqueous NaOH solution was added thereto, followed by stirring and dispersion. Then, 8.5g of epichlorohydrin was added, followed by stirring at 25℃for 5 hours, suction filtration and washing with water to neutrality after completion of the reaction, and drying at 50℃for 6 hours, to obtain porous microspheres 4 having both inner and outer surfaces grafted with epoxy groups.

Protein A grafting&Epoxy ring opening capping

Under nitrogen, 1.5g of microsphere 4 was mixed with 3.5mL of 10mg/mL Protein A,4.5mL of 1.3M Na ₂ SO ₄ After 7mL of 50mM phosphate buffer PBS was mixed, the mixture was turned and mixed at 10℃for 24 hours, and then suction filtration was performed, and the mixture was further mixed with 25mL of 1M aqueous mercaptoglycerol solution and heated to 30℃for 4 hours. Thus, the residual epoxy groups are subjected to ring opening end capping reaction, thereby improvingStability and hydrophilicity are improved. Finally, washing with water to neutrality, thus finally obtaining the hydrophilic modified hydrophobic polymer matrix Protein A affinity chromatography medium.

Comparative example 1

Commercial product MabSelect SuRe LX Protein A affinity chromatography media manufactured by Cytiva corporation was used as a comparative example. The product matrix is a crosslinked agarose microsphere with an average particle size (d 50 v) of about 80 μm, the surface of which is grafted with Protein A from E.Coli alkali resistance through epoxy groups.

Particle size and polydispersity measurement

Particle size testing used a Mastersizer 3000 in Markov, the particle size of this device was in the range of 0.1 μm to 3000 μm, the test medium could be water, an organic solvent, or a dry test kit, and the particle size was tested using a dry method. It is conventional to use water as a medium.

Particle pore volume and specific surface area measurement

When analyzing the pore size and pore volume of macropores (greater than 50 nm), it is generally considered that the method of gas adsorption test is inaccurate, and mercury porosimetry is often used. The mercury porosimetry test pore size may range from 3nm to millimeter. The more common importation equipment for mercury porosimetry is PoreMaster 60 from Quantachrome, U.S. Pat. No. Kang Da and Autopore 9500 from Micromeritics.

Measurement of mechanical strength of particles

The mechanical strength of the particles was measured and calculated by the following method. The particles were subjected to room temperature compression testing using a miniature compression tester (Fisher Scientific co.) with a compression head as a tetrahedral pyramid-shaped ram and a bottom dimension of 50 μmx50 μm. The pressing speed was 1mN/s. Mechanical strength is defined as the corresponding pressure at which the maximum displacement change occurs.

DBC (dynamic binding capacity dynamic load) measurement

First, using a separation column with a separation microsphere pre-loading capacity of 4mL (diameter 5mmX length 200 mM) according to the method of the present invention, the apparatus was AKTA prime plus (GE Healthcare Bioscience Co.) at a flow rate of 300 cm/hr, and the sample was 20mM phosphate buffer (pH 7.5) diluted to 5mg/mL of human IgG antibody. The adsorption of IgG at 10% flow-through was defined as DBC.

Nonspecific adsorption measurement

0.5g of the isolated microspheres prepared in example 1 was added to 50mL of phosphate buffer PBS (pH 7.5) containing BSA (bovine serum albumin) at a concentration of 20mg/mL. Then stirred slowly at room temperature for 24 hours. The supernatant was separated by centrifugation and the concentration of BSV in the supernatant was determined by the corresponding spectral absorption intensity of 280nm, the difference from the original BSV concentration being the nonspecific adsorption.

Alkali resistance measurement

The separation microspheres prepared in example 1 were preloaded with 1mL (diameter 5mmX length 50 mM) separation column, connected to AKTA prime plus (GE Healthcare Bioscience co.) instrument, washed first with 20mL PBS (ph 7.5) buffer, then neutralized with 75mM glycine-hcl buffer (ph 3.2) at a flow rate of 0.2 mL/min until ph6.5 was reached, and then the required glycine-hcl buffer volume was recorded. The separation column was then rinsed with 0.3N NaOH aqueous solution and immersed at room temperature for 15 hours. And repeating the experiment on the separation column after alkali liquor treatment, and recording the new required glycine-hydrochloric acid buffer solution volume, wherein the difference between the new volume and the old volume indirectly indicates alkali resistance, and the smaller the difference is, the better the alkali resistance is.

All test data are summarized in the following table. The examples are only intended to explain the content of the claims of the invention and should not be interpreted as limiting the scope.

TABLE 1

Claims

1. A preparation method of a hydrophilic modified hydrophobic polymer matrix Protein A affinity chromatography medium comprises the following preparation steps: 1) Preparing highly cross-linked hydrophobic porous polymer microspheres with uniform size distribution; 2) Impregnating the porous microspheres prepared in the step 1 with diluted mixed aqueous solution containing moderately hydrophobically modified agarose or dextran or similar structural substances or two or more kinds of mixed aqueous solution to ensure that the surfaces of the porous microspheres and the surfaces of inner holes in the step 1 are covered with rich hydroxyl groups; 3) Crosslinking reaction is carried out on the modified hydroxyl-rich component covered on the surface of the modified porous microsphere and the surface of the inner hole; 4) Carrying out epoxy functional group grafting reaction on the porous microspheres subjected to surface crosslinking; 5) Finally, a Protein A grafting reaction is carried out.

2. The method for preparing the porous hydrophobic polymer microsphere in the step 1 in the claim 1 is preferably an emulsion suspension polymerization method: and (3) using an emulsifying machine to efficiently emulsify a polymerization monomer, a stabilizer, a pore-forming agent and a polymerization inhibitor in water to prepare a stable pre-emulsion with uniform particle size, and then adding a hydrophobic initiator to initiate polymerization to obtain the porous polymer microsphere. Of these, the preferred monomers are based on divinylbenzene.

3. The divinylbenzene content of claim 2 is preferably greater than 50% by weight.

4. The hydroxyl group-enriched material preferred in step 2 of claim 1 is agarose ester.

5. The hydrophobically modified material of step 2 of claim 1 is preferably a chemical material containing phenyl and epoxy groups such as phenyl glycidyl ether.

6. The preferred crosslinker in step 3 of claim 1 is ethylene glycol diglycidyl ether.

7. The preferred epoxide function in step 4 of claim 1 is derived from epichlorohydrin.

8. Protein A of step 5 of claim 1 comprises a terminal group which is reactive with an epoxide group and which comprises a region which is alkali-resistant and which is capable of specifically adsorbing immunoglobulins.

9. A further capping reaction is also preferred after step 5 in claim 1, the purpose of which is to ring-open the residual epoxide groups to give hydroxyl groups.

10. In claim 9, it is preferred that the capping agent is mercaptoglycerol of low toxicity.

11. The method of claim 1, wherein the chromatographic medium has a particle size of 10 to 100 microns.

12. In claim 11, the particle size of the chromatographic medium is further preferably in the range of 35-80 microns.

13. In claim 1, the chromatography media particle size polydispersity is preferably less than 1.2.

14. In claim 13, the chromatography media particle size polydispersity is further preferably less than 1.1.

15. An immunoglobulin separation procedure using the chromatographic medium of claim 1, comprising: a first step of adsorbing immunoglobulins using the chromatographic medium of claim 1; second, eluting the adsorbed immunoglobulin; and thirdly, washing the chromatographic medium by using alkali liquor.