Preparation method of multi-mode cation exchange chromatography medium

Abstract

The invention relates to the technical field of ion exchange chromatography media, in particular to a multi-mode cation exchange chromatography medium and a preparation method thereof. Hydroxyl functionalized microspheres are used as a matrix, active groups are introduced through chemical modification, and then the hydroxyl functionalized microspheres are coupled with sulfur-containing amino acid and benzoic acid. The preparation method provided by the invention has the advantages of easily available raw materials, low cost and wide ligand selection range. The prepared multi-mode cation exchange chromatography medium contains carboxyl, hydrophobic groups of benzene rings and hydrogen bonds. Through the synergistic effect of hydrophobic-hydrophilic balance and hydrogen bonds, a high protein adsorption capacity can be maintained under a high salt concentration, an additional desalination step needed by a traditional medium is avoided, a purification process flow can be reduced, and the production cost is greatly reduced.

Description

Technical Field

[0001] This invention relates to the field of ion exchange chromatography media technology, specifically to a multimode cation exchange chromatography media and its preparation method. Background Technology

[0002] In downstream purification processes of biopharmaceuticals, cation exchange chromatography media serve as crucial separation tools, and their performance directly impacts the purity, recovery rate, and production efficiency of the target product. Traditional cation exchange media primarily rely on single functional groups such as carboxyl and sulfonic acid groups to achieve electrostatic adsorption, but they suffer from the following technical bottlenecks:

[0003] 1. Limitation of functional group singularity: Traditional media achieve separation through a single hydrophobic group (such as phenyl) or ion exchange group (such as sulfonic acid group), which makes it difficult to take into account both charge interaction and hydrophobic / hydrogen bond synergistic effect.

[0004] 2. Insufficient salt tolerance: Under high-salt loading conditions (e.g., 0.5M NaCl), traditional media significantly reduce adsorption capacity due to electrostatic shielding effects, requiring additional desalination steps, increasing process complexity and cost. While existing salt-tolerant media (such as sulfonic acid-modified media) can partially alleviate this problem, their functional groups are singular, failing to accommodate multi-mode interactions such as hydrogen bonding and hydrophobicity.

[0005] To address the aforementioned problems, this invention proposes a method for preparing a multimode cation exchange chromatography medium. Using hydroxyl-functionalized microspheres as a matrix, active groups are introduced through chemical modification, followed by coupling with sulfur-containing amino acids and benzoic acid to construct a composite ligand system exhibiting synergistic effects of charge, hydrophobicity, and hydrogen bonding. The preparation method provided by this invention utilizes readily available raw materials, has low cost, and allows for a wide range of ligand selection. The prepared multimode cation exchange chromatography medium maintains high protein adsorption capacity even at high salt concentrations, avoiding the additional desalting step required by traditional media, thus reducing purification process steps and significantly lowering production costs. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art. The core of this invention is to solve the above problems by constructing multi-mode functionalized microspheres through a targeted functionalization strategy, and to provide a multi-mode cation exchange chromatography medium and its preparation method.

[0007] The technical solution of the present invention to solve the above-mentioned technical problems is as follows:

[0008] This invention protects a method for preparing a multimode cation exchange chromatography medium, comprising:

[0009] Step 1: Using hydroxyl-functionalized microspheres as a matrix, hydroxyl groups are activated to introduce active groups, resulting in preliminarily modified microspheres;

[0010] Step 2: The preliminarily modified microspheres are coupled with sulfur-containing amino acids to obtain amino-functionalized microspheres.

[0011] Step 3: The above-mentioned amino-functionalized microspheres undergo a coupling reaction with benzoic acid under the action of a condensing agent and a buffer system to obtain a multimode cation exchange chromatography medium.

[0012] According to the present invention, the hydroxyl-functionalized microspheres in step 1 are one of agarose microspheres, cellulose microspheres, dextran microspheres, polyacrylamide microspheres, polymethacrylate microspheres, styrene microspheres, etc., preferably agarose microspheres.

[0013] According to the present invention, the method for activating the hydroxyl group in step 1 is one of allylation-bromination and epoxy activation, preferably allylation-bromination.

[0014] According to the present invention, the allylation-bromination is carried out in three steps, specifically pre-reaction, allylation, and bromination.

[0015] According to the present invention, the allylation reaction is carried out directly after the pre-reaction is completed without the need to separate the product. After allylation, the product needs to be separated and cleaned with a cleaning agent, which includes one or more of methanol, ethanol, acetone, and water.

[0016] According to the present invention, the pre-reaction needs to be carried out in an alkaline environment.

[0017] According to the present invention, the base selected for the pre-reaction is sodium hydroxide, which needs to be prepared as an aqueous solution for use.

[0018] According to the present invention, the pre-reaction requires the addition of sodium borohydride and anhydrous sodium sulfate.

[0019] According to the present invention, the volume ratio of the alkaline solution used in the pre-reaction to the hydroxyl-functionalized microspheres is 4:1 to 1:4.

[0020] According to the present invention, the alkaline solution used in the pre-reaction has a concentration of 20-60 wt%, preferably 25-50 wt%.

[0021] According to the present invention, the mass of sodium borohydride is 0.1-0.5 wt%, preferably 0.2-0.3 wt%, based on the mass of the hydroxyl-functionalized microspheres.

[0022] According to the present invention, the mass of anhydrous sodium sulfate is 10-35 wt%, preferably 15-25 wt%, based on the mass of the hydroxyl-functionalized microspheres.

[0023] According to the present invention, the reaction temperature of the pre-reaction is 25-60℃, the reaction time is 0.5-6h, and the stirring speed is 180-250rpm.

[0024] According to the present invention, the allylation reaction is carried out after the pre-reaction, and the temperature and rotation speed are the same for both.

[0025] According to the present invention, in the allylation process, the volume ratio of hydroxyl-functionalized microspheres to allyl glycidyl ether (AGE) is 4:1 to 1:1, and the reaction time is 16 to 24 h.

[0026] According to the present invention, the bromination reaction must be carried out in a sodium acetate solution system.

[0027] According to the present invention, the volume ratio of sodium acetate solution to allylated microspheres is 1:1 to 2:1.

[0028] According to the present invention, the concentration of sodium acetate solution is 2-20 wt%, preferably 5-10 wt%.

[0029] According to the present invention, in the bromination reaction, liquid bromine is selected as the brominating reagent.

[0030] According to the present invention, the mass of liquid bromine is 1-10 wt% based on the mass of the allylated microspheres, with the specific amount depending on the actual operation.

[0031] According to the present invention, the reaction temperature of the bromination reaction is 20-40℃, the reaction time is 10-60 min, and the stirring speed is 180-250 rpm.

[0032] According to the present invention, quenching is required after the bromination reaction.

[0033] According to the present invention, the reagent used in the quenching process is sodium formate.

[0034] According to the present invention, the mass of sodium formate is 1-10 wt% based on the mass of the allylated microspheres, with the specific amount depending on the actual operation.

[0035] According to the present invention, step 2 needs to be carried out in an alkaline buffer system.

[0036] According to the present invention, the alkaline buffer system is one or more of phosphate buffer and sodium carbonate buffer, preferably sodium carbonate-sodium bicarbonate buffer.

[0037] According to the present invention, the concentration of the sodium carbonate-sodium bicarbonate buffer solution is one or more of 0.05M, 0.1M, and 0.2M, preferably 0.1M.

[0038] According to the present invention, the pH value of the sodium carbonate-sodium bicarbonate buffer solution is 8.0-10.5, preferably 8.0-8.5.

[0039] According to the present invention, the volume ratio of sodium carbonate-sodium bicarbonate buffer solution to the pre-modified microspheres is 1:1-2:1.

[0040] According to the present invention, in step 2, the mass of the modified sulfur-containing amino acids is 5-20 wt%, based on the mass of the initially modified microspheres.

[0041] According to the present invention, the sulfur-containing amino acid in step 2 is one of DL-cysteine, DL-homocysteine, BOC-L-cysteine, Fmoc-L-cysteine, etc., preferably Fmoc-L-cysteine.

[0042] According to the present invention, Fmoc-L-cysteine ​​needs to be dissolved in dimethyl sulfoxide (DMSO).

[0043] According to the present invention, the volume ratio of DMSO to the pre-modified microspheres is 1:2 to 1:1.

[0044] According to the present invention, the reaction temperature in step 2 is 50-60℃, the reaction time is 8-24h, and the stirring speed is 180-250rpm.

[0045] According to the present invention, after coupling Fmoc-L-cysteine ​​in step 2, a deprotection step is required.

[0046] According to the present invention, the deprotection step is performed using a piperidine-DMF solution.

[0047] According to the present invention, the volume ratio of piperidine-DMF solution to pre-modified microspheres is 1:1 to 2:1.

[0048] According to the present invention, the concentration of piperidine in the piperidine-DMF solution is 5-30 wt%, preferably 10-20 wt%.

[0049] According to the present invention, the reaction temperature of the deprotection step is 20-40°C, the reaction time is 1-10 h, and the stirring speed is 180-250 rpm.

[0050] According to the present invention, the condensing agent in step 3 is one of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC), N,N'-dicyclohexylcarbodiimide (DCC), 1-hydroxybenzotriazole (HOBT), and benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate (HBTU), preferably EDC.

[0051] According to the present invention, the buffer system in step 3 is one of 4-morpholine ethanesulfonic acid buffer, phosphate buffer, and sodium carbonate buffer, preferably 4-morpholine ethanesulfonic acid buffer.

[0052] According to the present invention, the pH value of the buffer solution is 4.5-10.5, preferably 4.5-7.2.

[0053] According to the present invention, in step 3, the mass of the condensing agent used is 5-20 wt%, based on the mass of the amino-functionalized microspheres.

[0054] According to the present invention, in step 3, the mass of benzoic acid used is 2.5-10 wt%, based on the mass of the amino-functionalized microspheres.

[0055] According to the present invention, in step 3, the mass of water used is 50-200 wt%, based on the mass of the amino-functionalized microspheres.

[0056] According to the present invention, the reaction temperature in step 3 is 10-25℃, the reaction time is 8-24h, and the stirring speed is 180-250rpm.

[0057] The beneficial effects of this invention are as follows:

[0058] ① Hydroxyl-functionalized microspheres are used as the matrix. The hydrophilic surface of this matrix can significantly reduce protein adsorption impurities and improve the retention rate of biological activity.

[0059] ② The introduction of bifunctional groups enables synergistic capture via charge-hydrophobicity-hydrogen bonding, broadening the adsorption window. Simultaneously, the introduction of a benzene ring onto the ligand gives this type of medium some tolerance to salts, avoiding the additional desalination step required by traditional media, simplifying the process and reducing costs.

[0060] ③The preparation method provided by this invention uses readily available raw materials, has low cost, and allows for a wide range of ligand selection. Attached Figure Description

[0061] Figure 1 This is a synthetic route diagram for the preparation of the multimode cation exchange chromatography medium provided in Example 1 of the present invention;

[0062] Figure 2 The protein loading of the multi-mode cation exchange chromatography medium obtained in Example 1 was compared with that of conventional cation exchange chromatography medium and commercial Capto MMC under different salt concentration loading conditions. Detailed Implementation

[0063] The present invention will be further described in detail below with reference to embodiments, but is not limited thereto.

[0064] Example 1:

[0065] This embodiment provides a method for preparing a multimode cation exchange chromatography medium.

[0066] Step 1 - Allylation: Take 50 ml of dried agarose microspheres, add 0.1 g sodium borohydride, 8.3 g anhydrous sodium sulfate, and 25 ml of 50% sodium hydroxide. Stir at 180 rpm at 50°C for 1 hour. Then add 50 ml of allyl glycidyl ether and continue the reaction overnight for 16 hours. After the reaction is complete, wash with 20 column volumes of water to obtain allylated agarose microspheres.

[0067] Step 1 - Bromination: Take 50 ml of dried allylated microspheres and suspend them in 100 ml of 5% sodium acetate solution at room temperature. Add bromine water dropwise while stirring until the reaction solution turns yellow and does not fade for 2-3 minutes. Add sodium formate solution and stop adding sodium formate solution when the suspension becomes colorless. Wash thoroughly with pure water and dry to obtain the brominated microspheres.

[0068] Step 2: Take 50 ml of the dried brominated microspheres, add 50 ml of 0.1 M sodium carbonate-sodium bicarbonate buffer (pH = 8.5), 6.1 g of Fmoc-L-cysteine, and 25 ml of dimethyl sulfoxide (DMSO). Set the reaction temperature to 50 °C, the reaction time to 18 h, and the stirring speed to 180 rpm. After the reaction, wash thoroughly with pure water, add 50 ml of DMF solution containing 20% ​​piperidine, set the reaction temperature to 28 °C, the reaction time to 2 h, and the stirring speed to 180 rpm. After the reaction, wash thoroughly with DMF and pure water in sequence, and dry to obtain amino-functionalized microspheres.

[0069] Step 3: Add 4.79 g of EDC and 50 ml of 4-morpholine ethanesulfonic acid buffer (pH = 5.0) to 50 ml of 0.5 M benzoic acid solution. Add the prepared solution to 50 ml of functionalized microspheres, set the reaction temperature to 20 °C, and the reaction time to 18 h. After the reaction is complete, wash thoroughly with pure water and dry to obtain a multimode cation exchange medium with a protein loading of 62.1 mg BSA / ml.

[0070] Example 2:

[0071] This embodiment provides a method for preparing a multimode cation exchange chromatography medium, which is the same as in Example 1, except that in step 1-allylation, "50 ml allyl glycidyl ether" is replaced with "25 ml allyl glycidyl ether", resulting in a multimode cation exchange chromatography medium with a protein loading of 40.8 mg BSA / ml.

[0072] Example 3:

[0073] This embodiment provides a method for preparing a multimode cation exchange chromatography medium, which is the same as in Example 1, except that in step 1-allylation, "25 ml 50% sodium hydroxide" is replaced with "25 ml 20% sodium hydroxide", resulting in a multimode cation exchange chromatography medium with a protein loading of 48.4 mg BSA / ml.

[0074] Example 4:

[0075] This embodiment provides a method for preparing a multimode cation exchange chromatography medium.

[0076] Step 1: Take 50 ml of dried agarose microspheres, add 50 ml of pure water, 2 g of sodium hydroxide, 0.1 g of sodium borohydride, and 20 ml of epichlorohydrin. Stir at 180 rpm at a reaction temperature of 40°C for 1.5 h. After the reaction is complete, wash with water at a volume equivalent to 20 column volumes to obtain epoxidized agarose microspheres.

[0077] Step 2: Prepare 50 ml of 1M DL-homocysteine ​​solution, add 1M tetrabutylammonium fluoride, and stir at room temperature for 30 min to form thiol anions (RS-). Add 50 ml of the treated DL-homocysteine ​​solution to 50 ml of epoxidized agarose microspheres, and react at 40°C for 6 hours under nitrogen protection. The thiol anions attack the epoxy groups to form thioether bonds. After the reaction is complete, wash thoroughly with pure water.

[0078] Step 3 follows the procedure of Example 1 to obtain a multimode cation chromatography medium with a protein loading of 26.2 mg BSA / ml.

[0079] Example 5:

[0080] This embodiment provides a method for preparing a multimode cation exchange chromatography medium, which is the same as in Example 4, except that "epoxychloropropane" in step 1 is replaced with "1,4-butanediol diglycidyl ether", resulting in a multimode cation exchange chromatography medium with a protein loading of 34.6 mg BSA / ml.

[0081] A comparison of Examples 1-3 shows that increasing the amount of allyl glycidyl ether and increasing the NaOH concentration significantly improves the subsequent BSA loading. A comparison of Examples 1, 4, and 5 shows that the activation efficiency of allylated microspheres is significantly higher than that of epoxidized microspheres. Comparing the loading data from Examples 1-5, the preparation method of the multimode cation exchange chromatography medium in Example 1 is preferred.

[0082] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several modifications and improvements without departing from the inventive concept of the present invention, and these all fall within the protection scope of the present invention.

[0083] Test example:

[0084] This embodiment provides a multi-mode cation exchange chromatography medium (Example 1), a method for determining the dynamic binding loading of traditional cation exchange chromatography medium (CM Beads 6FF from Changzhou Tiandi Renhe Biotechnology Co., Ltd.) and commercial Capto MMC.

[0085] Using bovine serum albumin (BSA) at a concentration of 4.0 mg / ml as a protein sample, the protein was bound to multimode cation exchange chromatography media / conventional cation exchange chromatography media at different salt concentrations. The binding capacity of the media was calculated when 10% of the protein flowed through.

[0086] Experimental equipment and consumables

[0087]

[0088] Experimental reagents

[0089] Equilibration / washing solution 1: 50 mM CH3COONa, pH 4.75;

[0090] Equilibrium / wash solution 2: 50 mM CH3COONa, 50 mM NaCl, pH 4.75;

[0091] Equilibrium / wash solution 3: 50 mM CH3COONa, 125 mM NaCl, pH 4.75;

[0092] Equilibrium / wash solution 4: 50 mM CH3COONa, 250 mM NaCl, pH 4.75;

[0093] Equilibrium / wash solution 5: 50 mM CH3COONa, 500 mM NaCl, pH 4.75;

[0094] Eluent: 50 mM Tris-HCl, 1 M NaCl, pH 8.0;

[0095] Sample: 4.0 mg / ml BSA.

[0096] Column loading steps

[0097] Take 6 ml of packing material (denoted as V1), rinse it thoroughly with ultrapure water, and then fix the volume ratio at 65% ± 5%.

[0098] For column assembly, prepare two empty HiSelect medium-pressure chromatography columns. Wet the sieve plate with anhydrous ethanol beforehand. Insert a sieve plate into one end of the column, then press in the distribution plug, and screw on the sealing sleeve and lower plug.

[0099] Add a small amount of water to the bottom of the empty column tube. Stir the prepared packing material and pour it into the column tube slowly and evenly along the wall. Make up the remaining volume with 0.4M NaCl. Then connect the two column tubes through the connector, making sure there are no air bubbles in the column tubes.

[0100] Sedimentation and column compression: Connect the chromatography column to the chromatography system and allow it to settle at a flow rate of 0.5 ml / min for 30 min. Then, compress the column at a flow rate of 3.5 ml / min for 15 min until the gel surface no longer descends. Remove the connector and upper column tube. Remove the packing material from the lower column tube to a suitable height (collect the excess packing material and determine the packing volume, denoted as V2). After adding a small amount of deionized water, add a 7.8 mm sieve plate again, press in the distribution plug, and screw on the sealing sleeve and lower plug.

[0101] Dynamic combined load test

[0102] Connect the chromatography column to the chromatography system and set up the chromatography system flow path according to the following requirements.

[0103] A1 Inlet: Equilibration buffer; B1 Inlet: 150ml eluent; Superfluid Loop: 50ml sample.

[0104] For the system dead volume test, unscrew the Luer connector on the Position1 injection valve tubing, set the column position, and dry all tubing from the injection point to the column inlet. Connect any loop and fill it with sample, set the inject and flow rate to 1 ml / min, and execute until the first drop of liquid flows out of the inlet tubing. Record the volume from loading to the pause, denoted as V0.

[0105] The pump flushes the equilibration solution through a bypass path to the UV detector until a stable baseline is reached, at which point the system is automatically zeroed. Then, 7 ml of sample solution is passed through the bypass path to obtain a stable maximum UV absorbance value for the sample. max .

[0106] Equilibrate the column by connecting it to the chromatography system and adding 25 ml of equilibration buffer at a flow rate of 3.0 ml / min.

[0107] Load 50 ml of sample at a flow rate of 2.35 ml / min (retention time 2 min) and collect flow through.

[0108] Wash the column with 50 ml of equilibration buffer until UV light is applied. 280 The value tended to stabilize at a flow rate of 3.0 ml / min.

[0109] Elution: Elute the sample with 50 ml of eluent at a flow rate of 3.0 ml / min, and collect the eluent.

[0110] Dynamic calculation based on load results

[0111] Calculate dynamic load: Q dyn10％=(V 10％ -V0)×C0 / V volumn

[0112] Note: V 10％ = Sample loading volume (ml) when the sample reaches 10% of the maximum UV value.

[0113] V0 = System dead volume (ml)

[0114] C0 = BSA sample (mg / ml) V volumn = Column packing volume (ml)

[0115] Results Analysis

[0116]

[0117]

[0118] Compared to traditional cation exchange chromatography media, the multimode cation exchange chromatography media prepared in this patent exhibits significantly improved salt tolerance. The multimode cation exchange chromatography media prepared in this patent outperforms commercially available Capto MMC in terms of salt tolerance.

Claims

1. A method for preparing a multimode cation exchange chromatography medium, characterized in that, The preparation method includes the following steps: Step 1: Using hydroxyl-functionalized microspheres as a matrix, hydroxyl groups are activated to introduce active groups, resulting in preliminarily modified microspheres; Step 2: The preliminarily modified microspheres are coupled with sulfur-containing amino acids to obtain amino-functionalized microspheres. Step 3: The above-mentioned amino-functionalized microspheres undergo a coupling reaction with benzoic acid under the action of a condensing agent and a buffer system to obtain a multimode cation exchange chromatography medium.

2. The preparation method according to claim 1, characterized in that, The hydroxyl-functionalized microspheres are one of the following: agarose microspheres, cellulose microspheres, dextran microspheres, polyacrylamide microspheres, polymethacrylate microspheres, and styrene microspheres.

3. The preparation method according to claim 1, characterized in that, The activation method for the hydroxyl groups is one of allylation-bromination, epoxy activation, or 1,4-butanediol diglycidyl ether activation. The molar amount of the active group is 100-400 μmol / g, based on the mass of the initially modified microspheres.

4. The preparation method according to claim 1, characterized in that, The sulfur-containing amino acid is one of DL-cysteine, DL-homocysteine, BOC-L-cysteine, Fmoc-L-cysteine, etc. Based on the mass of the pre-modified microspheres, the modified sulfur-containing amino acids account for 5-20 wt%. The coupling reaction temperature is 50-60℃, the reaction time is 8-24h, and the stirring speed is 180-250rpm.

5. The preparation method according to claim 1, characterized in that, The condensing agent is one of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, N,N'-dicyclohexylcarbodiimide, 1-hydroxybenzotriazole, and benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate. The condensing agent has a mass of 5-20 wt%, based on the mass of the amino-functionalized microspheres.

6. The preparation method according to claim 1, characterized in that, The buffer system is one of 4-morpholine ethanesulfonic acid buffer, phosphate buffer, and sodium carbonate buffer. The pH of the buffer solution is between 4.5 and 10.

5.

7. The preparation method according to claim 1, characterized in that, The benzoic acid comprises 2.5-10 wt% of the amino-functionalized microspheres. The coupling reaction temperature is 10-25℃, the reaction time is 8-24h, and the stirring speed is 180-250rpm.

8. A chromatography medium, characterized in that, The chromatography medium includes the multimode cation exchange chromatography medium prepared by the preparation method according to any one of claims 1-7.

9. The application of the multimode cation exchange chromatography medium prepared by any one of claims 1-7 in protein purification.

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