Process for the preparation of an alkaline-resistant chromatography membrane and product

Abstract

The application discloses a preparation method of a chromatography membrane, comprising the following steps: (1) a polymer crosslinking coating is prepared by a nitrogen-containing polymer with a branched structure and a positive compound in the presence of a crosslinking agent; and (2) the polymer crosslinking coating is coated on a porous base membrane, and post-treatment is conducted to obtain the chromatography membrane. By adding the polymer with a certain degree of branching and loading positive charges, the net structure is formed between the molecules in the polymer crosslinking coating, the combination degree of the base material (carrier) is higher, the mechanical strength of the chromatography membrane is improved, the capture efficiency is higher, and the alkali resistance is better.

Description

Technical Field

[0001] This invention belongs to the field of chromatography technology, specifically relating to a method for preparing an alkali-resistant chromatography membrane and the product thereof. Background Technology

[0002] Chromatographic membranes primarily utilize the adsorption of positive ions present on the membrane to remove negatively charged large impurities, such as endotoxins, viruses, nucleic acids, and host cell proteins (HCPs). Existing preparation processes generally involve cross-linking a hydrophilic porous membrane (e.g., PES) with a positively charged polymer, followed by curing to obtain a positively charged chromatographic membrane.

[0003] Patent document CN101474552A discloses a porous adsorption medium comprising a substrate having a first outer side and a second outer side, both sides being porous and having a porous thickness in between, the substrate being hydrophilic, and having an adsorption material substantially covering the solid matrix of the substrate and the first and second outer surfaces, the adsorption material comprising a crosslinked polymer having quaternary ammonium ion functional groups linked by nonpolar linking groups, wherein the substrate comprises a microporous membrane, and the crosslinked polymer is modified with a charge modifier, the charge modifier comprising an organic compound having quaternary ammonium ion groups linked by the nonpolar linking groups to portions capable of reacting with the crosslinked polymer.

[0004] Existing chromatography membranes do not include alkali resistance evaluation, and the pH during elution in the application of chromatography membrane products on the market is limited, with a maximum of 0.5M NaOH. Summary of the Invention

[0005] This invention provides a highly branched alkali-resistant chromatography membrane, which has a wider range of applications and can withstand more demanding environments.

[0006] A method for preparing a chromatography membrane includes the following steps:

[0007] (1) A polymer crosslinking coating is prepared by mixing a nitrogen-containing polymer with a certain degree of branching with a positively charged compound in the presence of a crosslinking agent;

[0008] (2) The polymer crosslinking coating is applied (coated, impregnated or spin-coated) onto a porous base membrane, and then post-treated to obtain the chromatography membrane.

[0009] Preferably, the porous base membrane is a microfiltration membrane, and a hydrophilic porous base membrane can be selected. The hydrophilic porous base membrane includes, but is not limited to, hydrophilic polyethersulfone membranes, hydrophilic polytetrafluoroethylene membranes, cellulose acetate membranes, and other porous hydrophilic membranes, with hydrophilic polyethersulfone membranes being preferred. These membrane materials can be prepared by existing methods or commercially available products can be used. Generally, the pore size is between 0.1-20 μm, more preferably between 0.45-3 μm.

[0010] Preferably, the nitrogen-containing polymer has a branching degree of 10% (wt) or more. More preferably, the branching degree is 20% or more. Even more preferably, the nitrogen-containing polymer has a branching degree of 20-95%; even more preferably, the nitrogen-containing polymer has a branching degree of 30-95%.

[0011] Preferably, the nitrogen-containing polymer is selected from one or more of polyacrylamide (PAM), polyetheramine (PEA), polyoxyethylenediamine, polyoxyethyleneamine, polyethyleneimine (PEI), polyallylamine, and polyallylamine hydrochloride. More preferably, the nitrogen-containing polymer is selected from polyacrylamide and polyethyleneimine. The molecular weight of the polyacrylamide is 300w to 2000w; more preferably 500w to 1500w; the molecular weight of the polyethyleneimine is 5 million to 1 million; more preferably 5 million to 800,000.

[0012] In this invention, the nitrogen-containing polymers can be commercially available products or prepared in-house.

[0013] Preferably, the positively charged compound is selected from one or more of the following: epoxy quaternary ammonium (2,3-epoxypropyltrimethylammonium chloride (glycidyltrimethylammonium chloride), (3-chloro-2-hydroxypropyl)trimethylammonium chloride, (2-aminoethyl)trimethylammonium chloride (hydrochloride)), halogen quaternary ammonium ((2-chloroethyl)trimethylammonium chloride), and aldehyde quaternary ammonium (formylmethyltrimethylammonium chloride).

[0014] In step (1), under the action of the coupling agent, nitrogen in the nitrogen-containing polymer undergoes a grafting reaction with a positively charged compound (epoxy ring opening, halogenated hydrocarbon coupling, and aldehyde group undergoing Schiff base reaction).

[0015] Preferably, the crosslinking agent is selected from epichlorohydrin or other polyfunctional epoxy compounds (epoxychloropropane), various brominated chemicals or other polyfunctional halides; formaldehyde and other polyfunctional aldehydes (glutaraldehyde, etc.), di(2-hydroxyethyl) sulfone, dimethyldichlorosilane, dihydroxymethylurea, dihydroxymethyl ethylidene urea, diisocyanate ester or polyisocyanate.

[0016] As a preferred option, the reaction conditions for step (1) are: a reaction temperature of 25-90℃ and a reaction time of 0.5-10 hours.

[0017] After the reaction in step (1) is completed, no additional processing is required to directly obtain the polymer crosslinking coating solution.

[0018] After completing the coating, impregnation or spin coating in step (2), wait 0.5-30 minutes to remove the excess polymer crosslinking coating, and then place it under a UV lamp for 2-5 hours to fully react. After the reaction is complete, place the film in a 30-100℃ water bath for 10-150 minutes to clean, and then place it in a 40-80℃ oven to dry to constant weight.

[0019] Preferably, based on a total weight of 100, the mass percentages of the nitrogen-containing polymer, the positively charged compound, and the crosslinking agent are 1-30%, 3-20%, and 0.1-5%, respectively; the remainder is water. More preferably, based on a total weight of 100, the mass percentages of the nitrogen-containing polymer, the positively charged compound, and the crosslinking agent are 2-15%, 5-15%, and 0.5-3%, respectively; the remainder is water.

[0020] As a further preferred embodiment, based on a total weight of 100, the mass percentages of the nitrogen-containing polymer, the positively charged compound, and the crosslinking agent are 2-10%, 5-8%, and 0.5-2%, respectively; the remainder is water.

[0021] Preferably, in step (1), a nitrogen-based polymer with a certain degree of branching and a positively charged compound are added to water, and then the mixed solution is heated in a water bath at 25-90°C for 0.5-10 hours. After the reaction is completed, the solution is cooled to room temperature, and then the added crosslinking agent is stirred at room temperature for 5-40 minutes to obtain a polymer crosslinking coating solution.

[0022] Before the reaction, the mixed solution has the following weight percentage composition: branched nitrogen polymers: 1-30%; positively charged compounds: 1-20%; and the remainder is water. More preferably, the mixed solution has the following weight percentage composition: branched nitrogen polymers: 2-15%; positively charged compounds: 5-15%; and the remainder is water.

[0023] The amount of crosslinking agent added is 0.1% to 5% of the total material amount; more preferably 0.5% to 2%.

[0024] The present invention also provides a chromatography membrane prepared by the chromatography membrane preparation method described in any of the above technical solutions.

[0025] The chromatographic membrane prepared by this invention has a dynamic BSA loading between 30 and 80 mg / mL. In practical use, the dynamic BSA loading can be adjusted as needed by controlling the type of nitrogen-containing polymer, the molecular weight of the nitrogen-containing polymer, the degree of branching of the nitrogen-containing polymer, and the total amount of positively charged compounds, to obtain a chromatographic membrane with the target performance.

[0026] Compared with the prior art, the beneficial effects of the present invention are reflected in:

[0027] This invention incorporates a polymer with a certain degree of branching and loads it with a positive charge, which enables the molecules in the polymer cross-linked coating to form a network structure, resulting in a higher degree of adhesion to the substrate (carrier), improved mechanical strength of the chromatography membrane, higher capture efficiency, and better alkali resistance. Detailed Implementation

[0028] Example 1

[0029] The total material amount was 250g. 10wt% of polyacrylamide (PAM, molecular weight 500w, 25g) with a branching degree of 60% and 10wt% of formylmethyltrimethylammonium chloride were added to 79.5wt% deionized water. The mixture was then heated in a 30℃ water bath for 10 hours. After the reaction, it was cooled to room temperature, and 0.5% glutaraldehyde was added. The mixture was stirred at room temperature for 30 minutes to obtain a polymer crosslinking coating solution. A hydrophilic PES membrane (average pore size 0.45-3μm, obtained by conventional methods, such as the method published in CN 104258745A (no hydrophilic nanoparticles and surfactants are required during preparation)) was immersed in the polymer crosslinking coating solution by impregnation. Excess solution was removed from the PES membrane surface. The membrane was then irradiated with a UV lamp for 3 hours, washed in a 30℃ water bath for 30 minutes, and dried at 40℃ to obtain a hyperbranched chromatographic membrane. The tested BSA load was 50 mg / mL.

[0030] The method for detecting BSA load is as follows (Reference: He D, Ulbricht M. Preparation and characterization of porous anion-exchange membrane adsorbers with high protein-binding capacity[J]. Journal of Membrane Science, 2008, 315(1-2):155-163.):

[0031] In actual testing, a BSA protein solution of a certain concentration is passed through... The disposable filter in the Pure system contacts the target material (analyte membrane) at a predetermined flow rate of 10 times the membrane volume per minute (mV / min) until the concentration at the filter outlet exceeds 10% of the loaded concentration. Protein concentration exhibits a linear relationship with UV absorbance, and concentration changes can be detected using a UV detection probe within the system; this technique is well-known to those skilled in the art. Taking into account the dead volume in the system and reservoir, the total amount of protein adsorbed onto the filter at 10% breakthrough is determined by analyzing the chromatogram in Unicorn software.

[0032] The calculation method for branching degree DB:

[0033]

[0034] In the formula, D, T, and L represent the proportions of atomic mass in the dendritic, terminal, and linear structures of the hyperbranched polymer molecule, respectively. The linear structure is selected from the chain segments with the most main chain atoms, and its value can be measured by NMR. For fully branched polymer molecules, its DB is 100.

[0035] Example 2-37

[0036] Following the method of Example 1, and referring to the materials and material ratios in Tables 1, 2, and 3, experiments were conducted, and the BSA loading test results are shown in Tables 1, 2, and 3:

[0037] Table 1

[0038]

[0039] Table 2

[0040]

[0041]

[0042] Table 3

[0043]

[0044]

[0045] Tables 1 and 2 show that when the molecular weight remains constant, the dynamic loading increases with the increase of the degree of branching. When the degree of branching is the same, the dynamic loading increases with the increase of the polymer molecular weight. This may be because, at the same degree of branching, polymers with higher molecular weights can provide more active sites, thus increasing the loading. Table 3 shows that different quaternary ammonium active groups result in comparable dynamic protein loading after quaternization of polyacrylamide with the same degree of branching. This indicates that quaternary ammonium groups with different active groups can quaternize nitrogen-containing polymers and have excellent protein capture capabilities.

[0046] Alkali resistance evaluation:

[0047] (1) Alkali resistance test at different concentrations:

[0048] Specific operating steps: The chromatography membrane prepared in Example 3 was placed in an alkaline solution (sodium hydroxide aqueous solution) of the concentration shown in Table 3 and soaked for 12 hours. The BSA loading was then measured, and the results are shown in Table 4 below.

[0049] Table 4: Polyacrylamide after soaking for 12 hours

[0050] Alkali concentration (mol / L) 0.1 0.5 1 BSA load (mg / mL) 43 43 43

[0051] As can be seen from the above alkali resistance results, the chromatography membrane obtained by the method of the present invention has good alkali resistance and good BSA loading in the range of 0.1 to 1 mol / L.

[0052] (2) Alkali resistance test at different elution times:

[0053] Specific operating steps: The chromatography membrane prepared in Example 16 was placed in a 1 mol / L NaOH aqueous solution (sodium hydroxide aqueous solution) and soaked for 0.5, 1, 3, 7, and 14 hours. The BSA loading was measured, and the results are shown in Table 5 below:

[0054] Table 5: Alkali Concentration 1M NaOH

[0055] Time (d) 0.5 1 3 7 14 BSA load (mg / mL) 62.0 60.3 61.2 58.9 46.6

[0056] As can be seen from the above alkali resistance test results, the chromatography membrane obtained by the method of the present invention can still maintain a high BSA loading after long-term alkali washing, which further proves that the chromatography membrane obtained by the present invention has good alkali resistance.

[0057] (3) Alkali resistance test of different chromatography membranes

[0058] Specific operating steps: The prepared chromatography membrane was placed in a 1 mol / L sodium hydroxide aqueous solution and soaked for 12 hours. The BSA loading was then measured, and the results are shown in Table 6 below.

[0059] Table 6: Soaking in 1 mol / L sodium hydroxide aqueous solution for 12 hours

[0060]

[0061] As can be seen from the test results in Table 6, the modified chromatographic membranes of the polymers with lower branching degree have significantly reduced alkali resistance, while the other hyperbranched modified chromatographic membranes all have good alkali resistance, which greatly expands the application field of the chromatographic membranes of this invention.

Claims

1.A method for preparing a chromatography membrane, comprising the following steps: (1) preparing a polymer cross-linking coating by mixing a nitrogen-containing polymer with a branched structure and a positively charged compound in the presence of a cross-linking agent, wherein the nitrogen-containing polymer has a branching degree of 65-85%; and (2) coating the polymer cross-linking coating on a porous base membrane and post-processing to obtain the chromatography membrane. 2.The method according to claim 1, wherein the porous base membrane is a hydrophilic porous base membrane. 3.The method according to claim 2, wherein the hydrophilic porous base membrane is selected from a hydrophilic polyether sulfone membrane, a hydrophilic polytetrafluoroethylene membrane, and a cellulose acetate membrane. 4.The method according to claim 1, wherein the nitrogen-containing polymer is selected from one or more of polyethyleneimine, polyacrylamide, polyetheramine, polyoxyethylene diamine, polyoxyethylene amine, poly (allylamine), and poly (allylamine hydrochloride). 5.The method according to claim 1, wherein the positively charged compound is selected from one or more of an epoxy quaternary ammonium salt, a halogen quaternary ammonium salt, and an aldehyde quaternary ammonium salt. 6.The method according to claim 1, wherein the cross-linking agent is selected from epichlorohydrin, epibromohydrin, formaldehyde, bis (2-hydroxyethyl) sulfone, dimethyldichlorosilane, dimethylol urea, dimethylol ethylene urea, diisocyanate, or polyisocyanate. 7.The method according to claim 1, wherein the reaction conditions of step (1) are as follows: the reaction temperature is 25-90 ℃, and the reaction time is 0.5-10 hours. 8.The method according to claim 1, wherein after the coating in step (2) is completed, the membrane is left for 0.5-30 minutes, and then is subjected to ultraviolet irradiation under a UV lamp for 2-5 hours for sufficient reaction; after the reaction is completed, the membrane is washed in a water bath at 30-100 ℃ for 10-150 minutes, and then is dried to constant weight in an oven at 40-80 ℃. 9.The method according to claim 1, wherein the mass percentages of the nitrogen-containing polymer, the positively charged compound, and the cross-linking agent are 1-30%, 3-20%, and 0.1-5%, respectively, based on a total weight of 100. 10.A chromatography membrane, prepared by the method according to any one of claims 1-9. ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​

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