A method for preparing a chromatographic purification medium for efficient separation of subunit viruses

Abstract

The application discloses a preparation method of a chromatography purification medium for separating subunit viruses efficiently. The method comprises the following steps: (1) preparing a base material; (2) adding a sodium dextran sulfate salt into the base material; (3) adding a phosphate buffer, a phase transfer catalyst and a crosslinking agent; (4) performing a reaction under a staged alkaline pH condition, and the pH of the second stage is higher than that of the first stage; and (5) after the reaction is completed, washing the chromatography purification medium with alkaline liquor and purified water for multiple times. The molecular weight of the sodium dextran sulfate salt is 25-35 kDa, and is preferably 30 kDa. The base material is agarose microspheres, the agarose microspheres are prepared from agarose with a concentration of 3.0%-3.5%, and the particle size of the agarose microspheres is 45-165 µm. The mass ratio of the agarose microspheres, the purified water and the sodium dextran sulfate salt is 5:1-1.5. The phosphate buffer is tri-potassium phosphate or sodium phosphate, and the concentration of the phosphate buffer is 0.005-0.02 mol / L, and is preferably 0.01 mol / L.

Description

Technical Field

[0001] This application belongs to the field of bioengineering technology and proposes a method for preparing chromatographic purification media for efficient separation of subunit viruses. Background Technology

[0002] Subunit virus vaccines are gradually becoming an important direction in viral disease prevention and control research due to their high safety and good immunogenicity. Subunit virus antigens are usually specific structural domains of viral surface glycoproteins, and the efficiency and purity of their isolation and purification directly affect the immunogenicity and industrialization level of vaccines.

[0003] Currently, commonly used methods for purifying subunit viral antigens include centrifugation, ultrafiltration, precipitation, and chromatography. Centrifugation is difficult to use for large-scale purification; ultrafiltration, while capable of partially enriching the target protein, has limited ability to remove impurities; precipitation suffers from poor selectivity and reproducibility. Chromatography, due to its high selectivity and good purification effect, is widely used, with affinity chromatography media being the core material for achieving subunit viral isolation and purification.

[0004] However, existing affinity chromatography media still have the following problems:

[0005] First, the limited types of commonly used ligands and their coupling methods result in insufficient binding density of ligands on the medium, thus causing a low binding load of the target antigen. Second, the unreasonable selection of the molecular weight range of ligands, when too low, the chain length is insufficient to cover the binding domain on the antigen surface, and when too high, chain coiling and steric exclusion effects are easily generated, both of which are not conducive to improving purification efficiency.

[0006] Furthermore, traditional crosslinking and coupling conditions are not precisely controlled, especially the single reaction stage under alkaline conditions, leading to insufficient ligand immobilization or unreasonable spatial structure, ultimately reducing the performance of the chromatography medium. In addition, existing medium preparation processes often lack effective phase transfer catalysts and ionic strength control methods, which further limits the coupling efficiency and effective binding capacity of ligands.

[0007] Therefore, a new method for preparing chromatographic purification media is needed to improve the coupling efficiency and effective binding capacity of ligands while ensuring separation purity, rationally control the molecular weight and cross-linking degree of ligands, and improve the dynamic binding capacity of the chromatographic media, thereby achieving efficient separation and purification of target protein subunit viral antigens. Summary of the Invention

[0008] This invention provides a method for preparing a chromatographic purification medium for the efficient separation of subunit viruses, comprising the following steps:

[0009] (1) Prepare the base frame materials;

[0010] (2) Add purified water and sodium dextran sulfate to the above-mentioned base material;

[0011] (3) Add phosphate buffer, phase transfer catalyst and crosslinking agent;

[0012] (4) The reaction is carried out under alkaline pH conditions in stages, and the pH of the second stage is higher than that of the first stage;

[0013] (5) After the reaction is completed, the medium is washed multiple times with alkaline solution and purified water to obtain the chromatography purification medium.

[0014] Furthermore, the molecular weight of the above-mentioned sodium dextran sulfate is 25~35kDa, preferably 30kDa.

[0015] Furthermore, the aforementioned substrate material is agarose microspheres, which are prepared from agarose at a concentration of 3.0% to 3.5% and have a particle size of 45 to 165 μm.

[0016] Furthermore, the mass ratio of the above-mentioned agarose microspheres to sodium dextran sulfate is 5:1 to 1.5.

[0017] Furthermore, the aforementioned staged alkaline pH conditions are specifically as follows: in the first stage, the reaction system is adjusted to alkaline pH1 and reacted for 0.5 to 2 hours; in the second stage, the reaction system is adjusted to alkaline pH2 and reacted for 15 to 20 hours, wherein pH2 is higher than pH1, pH1 is 10.5 to 11.5, and pH2 is 12.5 to 13.5.

[0018] Furthermore, the phosphate buffer solution is a tripotassium phosphate or sodium phosphate solution, and the concentration of the phosphate buffer solution is 0.005–0.02 mol / L, preferably 0.01 mol / L.

[0019] Furthermore, the aforementioned phase transfer catalyst is a quaternary ammonium salt, specifically tetrabutylammonium bromide (TBAB), tetrabutylammonium bisulfate (TBAHS), or benzyltriethylammonium chloride (BTEAC), with tetrabutylammonium bromide (TBAB) being the preferred choice.

[0020] Furthermore, the above-mentioned crosslinking agent is an epoxy crosslinking agent, wherein the epoxy crosslinking agent is selected from epichlorohydrin (ECH), 1,4-butanediol diglycidyl ether (BDDE), 1,6-hexanediol diglycidyl ether (HDDE), and preferably 1,4-butanediol diglycidyl ether (BDDE).

[0021] Furthermore, the amount of the crosslinking agent is 5% to 6% of the mass of the agarose microspheres, preferably 6%, and the reaction is carried out at 30°C for 0.5 to 2 hours; the amount of the phase transfer catalyst is 2% of the mass of the agarose microspheres.

[0022] Furthermore, a chromatography purification medium is prepared by the above method, wherein the sulfur content of the medium is 380~450 μmol / mL and the dynamic binding capacity is 75~85 mg Pre-F / mL.

[0023] Furthermore, the chromatography purification medium is used to separate and purify subunit viruses.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] This invention effectively improves the coupling efficiency and space utilization of ligands in the medium by synergistically optimizing process parameters such as ligand molecular weight and addition ratio, buffer system, phase transfer catalyst, crosslinking agent dosage, and staged alkaline pH conditions. This results in more complete coupling and more reasonable distribution of ligands in the medium, and keeps the sulfur content, dynamic binding capacity, and separation purity within the optimal range. It improves the effective binding capacity and separation and purification performance of the chromatography medium for subunit viral antigens, addresses the problem of insufficient binding capacity in existing technologies, and ensures the stability and applicability of the process, thus possessing good industrial application value. Detailed Implementation

[0026] The technical solutions in the embodiments of this application will be clearly described below with reference to the examples. Obviously, the described embodiments are only some, not all, of the embodiments of this application. All other embodiments obtained by those skilled in the art based on the embodiments of this application are within the scope of protection of this application.

[0027] The terms "first," "second," etc., used in the specification and claims of this application are used to distinguish similar objects and not to describe a specific order or sequence. It should be understood that such use of data can be interchanged where appropriate so that embodiments of this application can be implemented in orders other than those illustrated or described herein, and the objects distinguished by "first," "second," etc., are generally of the same class and the number of objects is not limited; for example, a first object can be one or more. Furthermore, in the specification and claims, "and / or" indicates at least one of the connected objects, and the character " / " generally indicates that the preceding and following objects are in an "or" relationship.

[0028] This invention relates to a method for preparing a chromatographic purification medium for the efficient separation of subunit viruses, comprising the following steps: preparing a substrate material; then adding sodium dextran sulfate to the substrate material; subsequently adding phosphate buffer, a phase transfer catalyst, and a crosslinking agent to establish a reaction system; then carrying out the reaction under staged alkaline pH conditions, wherein the pH of the second stage is higher than that of the first stage; after the reaction is completed, washing the medium multiple times with alkaline solution and purified water to obtain the target chromatographic purification medium.

[0029] In one embodiment, sodium dextran sulfate with different molecular weights is selected. The preferred molecular weight of the sodium dextran sulfate is 25-35 kDa, more preferably 30 kDa, to achieve spatial matching and charge complementarity with the binding domains on the surface of the subunit viral particles, thereby obtaining higher binding capacity and selectivity. If the molecular weight is too low, the chain segment length is insufficient to cover the positively charged binding domains on the antigen surface, resulting in insufficient binding force; if the molecular weight is too high, the chain segments are prone to spatial exclusion and coiled conformations in the pores, reducing the exposure of effective binding sites.

[0030] In one embodiment, the substrate material is agarose microspheres, but it can also be other polysaccharide or polymeric microspheres with a hydrophilic multihydroxyl backbone, such as dextran gel, agarose gel, or acrylamide-agarose copolymer, but is not limited thereto.

[0031] In one embodiment, the substrate material is preferably agarose microspheres, which are prepared from agarose at a concentration of 3.0% to 3.5% and have a particle size of 45 to 165 μm. This range is chosen because when the agarose concentration and particle size are controlled within the above range, it is possible to ensure that the medium has sufficient mechanical strength and permeability while taking into account separation resolution and dynamic binding capacity, so that the chromatography purification medium maintains excellent separation performance and structural stability.

[0032] In one embodiment, during the coupling reaction, the mass ratio of the agarose microspheres to sodium dextran sulfate is 5:1 to 1.5, preferably 5:1. This ratio ensures that the sodium dextran sulfate has a suitable distribution and reaction contact ratio on the carrier surface. On the one hand, it can promote the full coupling of ligands and their stable fixation on the surface of agarose microspheres. On the other hand, it avoids molecular aggregation or precipitation caused by excessive ligands, thereby ensuring the uniformity of the coupling reaction, coupling efficiency, and product repeatability.

[0033] Regarding reaction conditions, this invention employs staged alkaline pH control, i.e., gradually increasing the alkalinity of the reaction system. In one embodiment, in the first stage, the reaction system is adjusted to alkaline pH 1 (10.5~11.5) and reacted for 0.5~2 h to promote initial coupling; subsequently, in the second stage, the reaction system is further adjusted to alkaline pH 2 (12.5~13.5) and the reaction continues for 15~20 h to further improve the final coupling efficiency and binding stability of the ligand. Although the mechanism is not clear, it is speculated that the reason for using a staged reaction may be that the hydroxyl groups on the medium surface and the hydroxyl groups on the ligand molecule have different activities when they undergo ring-opening reactions with the linker arm. Under moderately alkaline pH conditions, the linker arm preferentially reacts with the hydroxyl groups of the medium, causing it to be quickly fixed to the carrier surface; while under higher alkaline pH conditions, the coupling reaction between the linker arm and the ligand is catalyzed, requiring a longer time to achieve sufficient cross-linking. This allows the two-stage reaction to proceed under their respective advantageous conditions, significantly improving the uniformity of coupling. The inventors unexpectedly discovered that this staged reaction can make the reaction processes at both ends of the connecting arm more coordinated, avoid chain segment curling and aggregation under highly alkaline conditions, and ensure the full progress of the later reaction, thus significantly improving the coupling effect.

[0034] In the coupling reaction, to further improve the ligand immobilization efficiency, the present invention preferably adds a buffer solution to the reaction system. The buffer solution can be a phosphate buffer, or other common biological buffer systems, such as a carbonate buffer or Tris buffer, with a preference for a phosphate buffer. The phosphate buffer solution can be a tripotassium phosphate or sodium phosphate solution, preferably with a concentration range of 0.005–0.02 mol / L, more preferably 0.01 mol / L.

[0035] The buffer solution serves to regulate the ionic strength of the reaction system. On one hand, it shields the electrostatic repulsion between the multi-anionic ligands and the agarose framework, keeping the ligand segments extended and facilitating their binding with the crosslinking agent. On the other hand, although the inventors are not fully aware of the mechanism, they hypothesize that moderately increasing the ionic strength can cause the ligands to shift from a fully extended to a partially coiled conformation, avoiding pore size changes caused by multi-site coupling and more effectively utilizing the near-surface space of the support to increase the binding capacity. If the ionic strength is too low, the electrostatic repulsion is significant, and the coupling efficiency decreases; if the ionic strength is too high, it will inhibit the effect of the reactive groups.

[0036] To further improve reaction efficiency, this invention adds a phase transfer catalyst to the reaction system. The phase transfer catalyst can be a quaternary ammonium salt, a quaternary phosphine salt, or a crown ether, preferably a quaternary ammonium salt. Suitable quaternary ammonium salts include, but are not limited to, tetrabutylammonium bromide (TBAB), tetrabutylammonium hydrogen sulfate (TBAHS), and benzyltriethylammonium chloride (BTEAC), with TBAB being more preferred. The phase transfer catalyst promotes the interaction between the hydrophilic ligand and the lipophilic crosslinking agent at the interface between the aqueous and organic phases, accelerating the reaction rate and thus significantly improving the coupling efficiency.

[0037] In one embodiment, the amount of phase transfer catalyst is preferably 2% of the mass of agarose microspheres. At this ratio, the interfacial reaction rate between the hydrophilic ligand and the organic crosslinking agent can be effectively promoted, while avoiding side reactions or phase separation caused by excessive catalyst, thereby obtaining higher coupling efficiency.

[0038] In one embodiment, the crosslinking agent is a commonly used crosslinking compound in crosslinking reactions, preferably an epoxy crosslinking agent, including but not limited to epichlorohydrin (ECH), 1,4-butanediol diglycidyl ether (BDDE), and 1,6-hexanediol diglycidyl ether (HDDE), more preferably 1,4-butanediol diglycidyl ether (BDDE). This crosslinking agent can not only maintain the pore size and mechanical stability of the medium, but also improve the coupling efficiency and binding stability of the ligands. The amount of crosslinking agent used is preferably 5% to 6% of the mass of the agarose microspheres, more preferably 6%.

[0039] In one embodiment, 1,4-butanediol diglycidyl ether is preferred as a crosslinking agent. With the assistance of tripotassium phosphate and phase transfer catalyst TBAB, the crosslinking agent is added dropwise under alkaline conditions. The reaction is first carried out at pH 11 for 1 hour, and then at pH 13 for 18 hours, thereby achieving stable coupling of the ligand.

[0040] The chromatography purification medium described in this invention is suitable for separating and purifying various target proteins, namely subunit viral antigen proteins, including but not limited to respiratory syncytial virus pre-fusion protein (Pre-F), influenza virus hemagglutinin (HA), adeno-associated virus (AAV) capsid protein, and other subunit viral antigens, and has strong versatility and application value.

[0041] To demonstrate the performance of the chromatographic purification medium of this invention, the prepared chromatographic purification medium was tested. The test indicators included the sulfur content, dynamic binding capacity (DBC), and separation purity of the medium. The sulfur content reflects the degree of ligand introduction and potential ligand density on the medium; the dynamic binding capacity characterizes the effective binding ability of the chromatographic purification medium to the target protein under flow conditions, reflecting the amount of target protein that can be bound and processed per unit volume during actual chromatography; and the separation purity characterizes the removal of impurities from the target protein obtained after separation by the chromatographic purification medium, reflecting the separation selectivity and application effect of the medium on the target protein.

[0042] It should be noted that separation purity (e.g., the purity percentage measured by SEC-HPLC in this invention) mainly reflects the proportion of the target protein relative to impurity proteins in the elution product. Its value is affected not only by the separation selectivity of the medium itself, but also by multiple factors such as the composition of the loading solution, the loading load, elution conditions, and detection parameters. Therefore, even if the separation purity values ​​are similar between different media, it does not necessarily mean that the media has the same effective binding capacity for the target protein or the same sample volume that can be processed per unit volume of medium. Therefore, this invention uses dynamic binding capacity as a key indicator reflecting the effective binding capacity of the medium under flow conditions, and combines it with separation purity to comprehensively evaluate the separation performance of the elution product, thereby more comprehensively reflecting the performance of the chromatographic purification medium in practical applications.

[0043] Furthermore, sulfur content is primarily used to characterize the degree of ligand introduction on the medium, thus reflecting the coupling level and density of the ligands. Generally, moderately increasing the ligand density is beneficial for increasing the number of potential binding sites, thereby improving the dynamic binding capacity of the medium. However, higher sulfur content is not always better; when it exceeds a fixed threshold, it can actually affect the dynamic binding capacity. Although the mechanism is not fully understood, it is speculated that overcrowded ligand molecules can create steric hindrance, potentially preventing target protein molecules from approaching the ligand's active site. Even if some proteins achieve binding, spatial stress may cause conformational deformation, preventing the formation of a stable, specific binding complex. Consequently, these proteins may prematurely detach during chromatography, further reducing the detected value of the dynamic binding capacity. Simultaneously, the binding specificity (the ability to distinguish the target protein from other proteins) decreases significantly, making it difficult to achieve the required purity for the purified product.

[0044] Furthermore, if the sulfur content is below 380 μmol / mL, the ligand density is insufficient, which may lead to insufficient binding sites and a decrease in dynamic binding capacity, failing to meet the needs of large-scale purification. If it is above 450 μmol / mL, the amount of ligand introduced increases, which may lead to excessive group density, which may cause non-specific adsorption (such as strong adsorption of impurity proteins), and the dynamic binding capacity and separation purity of the medium may decrease.

[0045] By comparing media prepared under different conditions, this invention demonstrates that through staged control of ligand molecular weight, mass ratio, alkaline pH, and the synergistic effect of phase-transfer catalysts and buffer systems, ligands achieve a more rational distribution on the media, thereby increasing dynamic binding capacity while maintaining good separation purity. In practical applications, a higher dynamic binding capacity means that more target proteins can be processed under the same media volume, which helps reduce the amount of chromatography media used, the volume of chromatography columns, and the number of sample loadings, thereby improving the overall efficiency of the separation and purification process and reducing production costs.

[0046] Through the above tests, this invention characterizes the degree of ligand introduction by combining sulfur content, and further combines the dynamic binding capacity and separation purity of the chromatographic purification medium as comprehensive performance evaluation indicators to compare chromatographic purification media prepared under different conditions, thereby demonstrating the improvement effect of this invention on the medium in terms of effective binding capacity and separation and purification performance.

[0047] The following examples further illustrate the proportions:

[0048] Example 1

[0049] Example 1 describes the preparation of a chromatography purification medium, and the specific steps are as follows.

[0050] (1) Preparation of agarose microspheres: Agarose microspheres were prepared using a traditional emulsification-gelation method. 3.0 g of agarose was dissolved in 97.0 g of water (purchased from Maclean's). After heating until completely dissolved, the solution was slowly added dropwise at 70–80 °C to a liquid paraffin oil phase containing 1% Span-80 emulsifier. A homogeneous emulsion was formed by stirring at 500 rpm. The emulsion was then cooled to 4 °C in an ice-water bath to allow the agarose droplets to gel into spheres. The obtained agarose microspheres were washed alternately with organic solvent and purified water to obtain agarose microspheres with a particle size of 45–165 μm (particle size was measured using a laser particle size analyzer, model Bettersize2600, purchased from Baker Analytical Instruments Co., Ltd.).

[0051] (2) Coupling reaction: Weigh 100 g of the above agarose microspheres, add 20 g of sodium dextran sulfate with a molecular weight of 30 kDa (purchased from Maclean Company) to the system, and add an appropriate amount of purified water to promote uniform dispersion. The mass ratio of agarose microspheres to sodium dextran sulfate is 5:1. Stir at 30°C for 30 min to make sodium dextran sulfate evenly distributed on the surface of agarose microspheres, and obtain the reaction system.

[0052] Subsequently, 0.18 g of tripotassium phosphate (0.01 mol / L, purchased from Maclean's) was added to the reaction system, the pH of the system was adjusted to 11.0, and 2 g of tetrabutylammonium bromide (TBAB, purchased from Maclean's, 99% purity) was added, the amount being 2% of the mass of the agarose microspheres.

[0053] Under stirring, 6 g of 1,4-butanediol diglycidyl ether (BDDE, purchased from Maclean's, 95% purity) was added dropwise at a rate of 0.2 mL / min, representing 6% of the mass of the agarose microspheres. The reaction was carried out at 30 °C for 1 h. The pH of the system was then adjusted to 13 with 50% NaOH solution, and the reaction was continued for 18 h.

[0054] (3) Washing and detection: After the reaction is completed, the system is placed into a G3 sintered glass funnel and washed 10 times with an equal volume of purified water (100 mL each time) until the conductivity of the washing solution is close to 0.1 mS / cm or less, and the chromatography purification medium is obtained.

[0055] The obtained chromatographic purification medium was tested using respiratory syncytial virus pre-fusion protein (Pre-F, a model antigen protein obtained by laboratory recombinant expression and affinity chromatography purification) as the model antigen protein. The detection indicators were sulfur content, dynamic binding capacity and separation purity of the chromatographic purification medium.

[0056] The method for detecting sulfur content is as follows:

[0057] The sulfur content was determined by an external laboratory using an Elementar Vario EL elemental analyzer. Before the determination, the wet chromatography purification medium sample was vacuum dried to constant weight at 60℃ and finely ground. Approximately 5 mg was weighed for analysis. The measured sulfur mass fraction was converted to sulfur content (μmol / mL) based on the wet gel volume, and the average value of three parallel experiments was used as the result.

[0058] The dynamic load detection method is as follows:

[0059] The prepared chromatographic purification medium was packed into a Chrom-Screen 4.7 mL column and connected to a protein chromatography system. Using ultrapure water containing 1% acetone or 0.2 mol / L NaCl as the mobile phase, the column was equilibrated at a flow rate of 1.0 mL / min until the UV280 and conductivity (Cond) baselines were stable. Subsequently, 100 μL of 1% acetone or 2 mol / L NaCl solution was injected to test column efficiency. A theoretical plate number N / m ≥ 2500 and peak symmetry As = 0.80–1.50 were considered acceptable; otherwise, the column was repacked.

[0060] All experimental solutions were filtered through a 0.45 μm filter and degassed by sonication. The buffer solution consisted of 20 mmol / L NaH₂PO₄ and 0.15 mol / L NaCl, pH 7.4. Equilibration was then carried out at the same buffer solution at a flow rate of 0.78 mL / min until the UV baseline stabilized.

[0061] Under the same buffer conditions, using a 3 mg / mL protein standard solution as the sample, the breakthrough curve was recorded under a UV-900 detector (280 nm). The dynamic binding capacity (DBC) was calculated based on the sample loading amount corresponding to the breakthrough value of 10% (100% breakthrough value). The results are expressed as mg protein / mL wet gel.

[0062] Dynamic combined load capacity is calculated using the following formula:

[0063]

[0064] in:

[0065] C represents the corrected standard protein concentration (mg / mL).

[0066] The loading volume (mL) required to reach the 10% breakthrough point;

[0067] The volume (mL) of wet gel packed into the chromatography column.

[0068] The method for detecting the purity of the separation is as follows:

[0069] The purity of the target protein in the chromatographic eluent was determined by size exclusion chromatography (SEC-HPLC). The specific steps are as follows.

[0070] A Waters Ultrahydrogel 250 column (7.8 mm × 300 mm) was used. The mobile phase was a mixture of 0.1 mol / L sodium phosphate buffer and 0.1 mol / L sodium sulfate, adjusted to pH 6.7. The mobile phase was filtered through a 0.22 μm or 0.45 μm filter and degassed by sonication for 15 min before use. The chromatographic conditions were set as follows: flow rate 0.8 mL / min, column temperature 25 °C, UV detector wavelength 280 nm; injection volume (protein content) 20 μg; single run time 20 min, ensuring complete elution of any aggregates, target protein monomers, and low-molecular-weight impurities present in the sample.

[0071] The column was equilibrated with the mobile phase at a rate of 0.8 mL / min for 60 min until the UV baseline and system pressure stabilized.

[0072] A mixture of protein standards was injected to conduct a system suitability test, evaluating indicators such as chromatographic resolution Rs, peak shape symmetry, and theoretical plate number; when the resolution met the requirement of Rs > 1.5 and the peak shape was normal, sample testing was performed.

[0073] Next, the sample to be tested (in this example, the target protein solution obtained by chromatographic elution) is prepared with a mobile phase or a buffer compatible with the mobile phase; before injection, it is filtered with a 0.22 μm centrifugal filter to remove particulate matter; the sample is injected and detected under the above conditions and the chromatogram is recorded.

[0074] According to the SEC principle, the components elute in descending order of apparent molecular volume, namely aggregate peak, target protein monomer peak, and fragment / small molecule impurity peak.

[0075] Separation purity is expressed as a percentage of the target protein monomer peak area relative to the total area of ​​all protein-related peaks in the sample (excluding solvent peaks / system peaks), calculated using the following formula:

[0076]

[0077] in, This represents the peak area of ​​the target protein monomer. This is the sum of the peak areas of all proteins in the sample. The separation purity is calculated as the percentage of the target protein monomer peak area relative to the total peak area of ​​all related proteins.

[0078] Example 2

[0079] In this embodiment, the molecular weight of the ligand was adjusted, and the molecular weight of the sodium dextran sulfate was 26 kDa. The other conditions were the same as in Example 1.

[0080] The specific procedure is as follows: Take 100 g of agarose medium, add 20 g of sodium dextran sulfate with a molecular weight of 26 kDa, and add an appropriate amount of purified water to promote uniform dispersion. Stir at 30°C for 30 min. The remaining feeding steps and reaction conditions are the same as in Example 1.

[0081] The target media were tested using respiratory syncytial virus pre-fusion protein (Pre-F) as the model antigen protein. The detection indicators included the sulfur content, dynamic binding capacity, and separation purity of the chromatographic purification medium. The detection method was the same as in Example 1.

[0082] Example 3

[0083] In this embodiment, the molecular weight of the ligand was adjusted, and the molecular weight of the sodium dextran sulfate was 35 kDa. The other conditions were the same as in Example 1.

[0084] The specific procedure is as follows: Take 100 g of agarose medium, add 20 g of sodium dextran sulfate with a molecular weight of 35 kDa, and add an appropriate amount of purified water to promote uniform dispersion. Stir at 30°C for 30 min. The remaining feeding steps and reaction conditions are the same as in Example 1.

[0085] The target media were tested using respiratory syncytial virus pre-fusion protein (Pre-F) as the model antigen protein. The detection indicators included the sulfur content, dynamic binding capacity, and separation purity of the chromatographic purification medium. The detection method was the same as in Example 1.

[0086] Example 4

[0087] This embodiment uses the same preparation method as Example 1, except that the phase transfer catalyst used is tetrabutylammonium bisulfate (TBAHS), which accounts for 2% of the mass of the agarose microspheres. The remaining experimental steps, conditions and detection methods are the same as in Example 1.

[0088] The target media were tested using respiratory syncytial virus pre-fusion protein (Pre-F) as the model antigen protein. The detection indicators included the sulfur content, dynamic binding capacity, and separation purity of the chromatographic purification medium. The detection method was the same as in Example 1.

[0089] Example 5

[0090] This embodiment uses the same preparation method as Example 1, except that the phase transfer catalyst used is benzyltriethylammonium chloride (BTEAC), which accounts for 2% of the mass of the agarose microspheres. The remaining experimental steps, conditions, and detection methods are the same as in Example 1.

[0091] The target media were tested using respiratory syncytial virus pre-fusion protein (Pre-F) as the model antigen protein. The detection indicators included the sulfur content, dynamic binding capacity, and separation purity of the chromatographic purification medium. The detection method was the same as in Example 1.

[0092] Example 6

[0093] The only difference between this embodiment and Example 1 is the buffer salt used; specifically, the phosphate buffer solution is replaced with sodium phosphate solution instead of tripotassium phosphate solution. All other conditions and steps are the same as in Example 1.

[0094] The target media were tested using respiratory syncytial virus pre-fusion protein (Pre-F) as the model antigen protein. The detection indicators included the sulfur content, dynamic binding capacity, and separation purity of the chromatographic purification medium. The detection method was the same as in Example 1.

[0095] Example 7

[0096] In this embodiment, the amount of ligand added was adjusted, and the mass ratio of medium to ligand was 5:1.25. The other conditions were the same as in Example 1.

[0097] The specific procedure is as follows: Take 100g of agarose medium, add 25g of sodium dextran sulfate with a molecular weight of 30kDa, and add an appropriate amount of purified water to promote uniform dispersion. Stir at 30°C for 30 minutes. The remaining feeding steps and reaction conditions are the same as in Example 1.

[0098] The target media were tested using respiratory syncytial virus pre-fusion protein (Pre-F) as the model antigen protein. The detection indicators included the sulfur content, dynamic binding capacity, and separation purity of the chromatographic purification medium. The detection method was the same as in Example 1.

[0099] Example 8

[0100] In this embodiment, the amount of ligand added was adjusted, and the mass ratio of medium to ligand was 5:1.5. The other conditions were the same as in Example 1.

[0101] The specific procedure is as follows: Take 100g of agarose medium, add 30g of sodium dextran sulfate with a molecular weight of 30kDa, and add an appropriate amount of purified water to promote uniform dispersion. Stir at 30°C for 30 minutes. The remaining feeding steps and reaction conditions are the same as in Example 1.

[0102] The target media were tested using respiratory syncytial virus pre-fusion protein (Pre-F) as the model antigen protein. The detection indicators included the sulfur content, dynamic binding capacity, and separation purity of the chromatographic purification medium. The detection method was the same as in Example 1.

[0103] Comparative Example 1

[0104] This comparative example adjusted the molecular weight of the ligand, and the sodium dextran sulfate had a molecular weight of 10 kDa. The other conditions were the same as in Example 1.

[0105] The specific procedure is as follows: Take 100 g of agarose medium, add 20 g of sodium dextran sulfate with a molecular weight of 10 kDa, and add an appropriate amount of purified water to promote uniform dispersion. Stir at 30°C for 30 min. The remaining feeding steps and reaction conditions are the same as in Example 1.

[0106] The target media were tested using respiratory syncytial virus pre-fusion protein (Pre-F) as the model antigen protein. The detection indicators included the sulfur content, dynamic binding capacity, and separation purity of the chromatographic purification medium. The detection method was the same as in Example 1.

[0107] Comparative Example 2

[0108] This comparative example adjusted the molecular weight of the ligand, and the sodium dextran sulfate had a molecular weight of 20 kDa. The other conditions were the same as in Example 1.

[0109] The specific procedure is as follows: Take 100 g of agarose medium, add 20 g of sodium dextran sulfate with a molecular weight of 20 kDa, and add an appropriate amount of purified water to promote uniform dispersion. Stir at 30°C for 30 min. The remaining feeding steps and reaction conditions are the same as in Example 1.

[0110] The target media were tested using respiratory syncytial virus pre-fusion protein (Pre-F) as the model antigen protein. The detection indicators included the sulfur content, dynamic binding capacity, and separation purity of the chromatographic purification medium. The detection method was the same as in Example 1.

[0111] Comparative Example 3

[0112] This comparative example adjusted the molecular weight of the ligand, and the sodium dextran sulfate had a molecular weight of 50 kDa. The other conditions were the same as in Example 1.

[0113] The specific procedure is as follows: Take 100 g of agarose medium, add 20 g of sodium dextran sulfate with a molecular weight of 50 kDa, and add an appropriate amount of purified water to promote uniform dispersion. Stir at 30°C for 30 min. The remaining feeding steps and reaction conditions are the same as in Example 1.

[0114] The target media were tested using respiratory syncytial virus pre-fusion protein (Pre-F) as the model antigen protein. The detection indicators included the sulfur content, dynamic binding capacity, and separation purity of the chromatographic purification medium. The detection method was the same as in Example 1.

[0115] Comparative Example 4

[0116] This comparative example has an adjusted molecular weight of the ligand, and the sodium dextran sulfate has a molecular weight of 100 kDa. The other conditions are the same as in Example 1.

[0117] The specific procedure is as follows: Take 100g of agarose medium, add 20g of sodium dextran sulfate with a molecular weight of 100kDa, and add an appropriate amount of purified water to promote uniform dispersion. Stir at 30°C for 30 minutes. The remaining steps and reaction conditions are the same as in Example 1.

[0118] The target media were tested using respiratory syncytial virus pre-fusion protein (Pre-F) as the model antigen protein. The detection indicators included the sulfur content, dynamic binding capacity, and separation purity of the chromatographic purification medium. The detection method was the same as in Example 1.

[0119] Comparative Example 5

[0120] The medium used in this comparative example is a commercially available product (Capto DeVirS manufactured by Cytiva), with the molecular weight of the ligand adjusted. The molecular weight of the sodium dextran sulfate is 500 kDa, and the other conditions are the same as in Example 1.

[0121] The specific procedure is as follows: Take 100g of agarose medium, add 20g of sodium dextran sulfate with a molecular weight of 500kDa, and add an appropriate amount of purified water to promote uniform dispersion. Stir at 30°C for 30 minutes. The remaining feeding steps and reaction conditions are the same as in Example 1.

[0122] The target media were tested using respiratory syncytial virus pre-fusion protein (Pre-F) as the model antigen protein. The detection indicators included the sulfur content, dynamic binding capacity, and separation purity of the chromatographic purification medium. The detection method was the same as in Example 1.

[0123] Comparative Example 6

[0124] In this comparative example, instead of using staged pH control, the reaction system was directly adjusted to pH 11 and maintained as a single-stage reaction, with the remaining conditions being the same as in Example 1.

[0125] The specific procedure is as follows: Take 100 g of agarose medium, add 20 g of sodium dextran sulfate with a molecular weight of 30 kDa, and add an appropriate amount of purified water to promote uniform dispersion. Stir at 30 °C for 30 min. The remaining feeding steps are the same as in Example 1, except that the reaction is maintained at pH 11 for 18 h.

[0126] The target media were tested using respiratory syncytial virus pre-fusion protein (Pre-F) as the model antigen protein. The detection indicators included the sulfur content, dynamic binding capacity, and separation purity of the chromatographic purification medium. The detection method was the same as in Example 1.

[0127] Comparative Example 7

[0128] In this comparative example, instead of using staged pH control, the reaction system was directly adjusted to pH 13 and maintained as a single-stage reaction, with the remaining conditions being the same as in Example 1.

[0129] The specific procedure is as follows: Take 100 g of agarose medium, add 20 g of sodium dextran sulfate with a molecular weight of 30 kDa, and add an appropriate amount of purified water to promote uniform dispersion. Stir at 30 °C for 30 min. The remaining feeding steps are the same as in Example 1, except that the reaction is maintained at pH 13 for 18 h.

[0130] The target media were tested using respiratory syncytial virus pre-fusion protein (Pre-F) as the model antigen protein. The detection indicators included the sulfur content, dynamic binding capacity, and separation purity of the chromatographic purification medium. The detection method was the same as in Example 1.

[0131] Comparative Example 8

[0132] This comparative example uses staged pH control, but the pH of the system is adjusted to 8 in the first stage and to 13 in the second stage. The other conditions are the same as in Example 1.

[0133] The specific procedure is as follows: Take 100 g of agarose medium, add 20 g of sodium dextran sulfate with a molecular weight of 30 kDa, and add an appropriate amount of purified water to promote uniform dispersion. Stir at 30 °C for 30 min. The remaining feeding steps are the same as in Example 1. The difference is that in this comparative example, the pH of the system was adjusted to 8 and reacted for 1 h in the first stage, and the pH of the system was raised to 13 and reacted for another 18 h in the second stage.

[0134] The target media were tested using respiratory syncytial virus pre-fusion protein (Pre-F) as the model antigen protein. The detection indicators included the sulfur content, dynamic binding capacity, and separation purity of the chromatographic purification medium. The detection method was the same as in Example 1.

[0135] Comparative Example 9

[0136] This comparative example uses a phased operation, but the pH of the system is adjusted to 4 in the first stage and to 8 in the second stage, while the other conditions are the same as in Example 1.

[0137] The specific procedure is as follows: Take 100 g of agarose medium, add 20 g of sodium dextran sulfate with a molecular weight of 30 kDa, and add an appropriate amount of purified water to promote uniform dispersion. Stir at 30 °C for 30 min. The remaining feeding steps are the same as in Example 1. The difference is that in this comparative example, the pH of the system is adjusted to 4 and reacted for 1 h in the first stage, and the pH of the system is raised to 8 and reacted for another 18 h in the second stage.

[0138] The target media were tested using respiratory syncytial virus pre-fusion protein (Pre-F) as the model antigen protein. The detection indicators included the sulfur content, dynamic binding capacity, and separation purity of the chromatographic purification medium. The detection method was the same as in Example 1.

[0139] Comparative Example 10

[0140] This comparative example uses reverse staged pH control, that is, the reaction is first carried out at pH 13 for 1 h, and then the pH is reduced to pH 11 for 18 h. The other conditions are the same as in Example 1.

[0141] The specific procedure is as follows: Take 100 g of agarose medium, add 20 g of sodium dextran sulfate with a molecular weight of 30 kDa, and add an appropriate amount of purified water to promote uniform dispersion. Stir at 30 °C for 30 min. The difference is that in the first stage, the pH of the reaction system is adjusted to 13, and the reaction is carried out for 1 h while maintaining stirring; in the second stage, under continuous stirring and pH monitoring, dilute hydrochloric acid is slowly added dropwise to lower the pH of the system to about 11, and then the reaction is continued for 18 h.

[0142] The target media were tested using respiratory syncytial virus pre-fusion protein (Pre-F) as the model antigen protein. The detection indicators included the sulfur content, dynamic binding capacity, and separation purity of the chromatographic purification medium. The detection method was the same as in Example 1.

[0143] Comparative Example 11

[0144] In this comparative example, the phase transfer catalyst TBAB was not added, and the other conditions were the same as in Example 1.

[0145] The specific procedure is as follows: Take 100g of agarose medium, add 20g of sodium dextran sulfate with a molecular weight of 30kDa, and add an appropriate amount of purified water to promote uniform dispersion. Stir at 30℃ for 30min. Add 0.18g of tripotassium phosphate (concentration 0.01mol / L), without adding TBAB. The remaining feeding steps and reaction conditions are the same as in Example 1.

[0146] The target media were tested using respiratory syncytial virus pre-fusion protein (Pre-F) as the model antigen protein. The detection indicators included the sulfur content, dynamic binding capacity, and separation purity of the chromatographic purification medium. The detection method was the same as in Example 1.

[0147] Comparative Example 12

[0148] The difference between this comparative example and Example 1 is that the amount of ligand used is reduced, and the mass ratio of agarose microspheres to sodium dextran sulfate is adjusted to 5:0.3. All other conditions and steps are the same as in Example 1.

[0149] The specific procedure is as follows: Weigh 100g of agarose medium, add 6g of sodium dextran sulfate with a molecular weight of 30kDa, and add an appropriate amount of purified water to promote uniform dispersion. Stir at 30℃ for 30 min. Then add 0.18g of tripotassium phosphate (0.01mol / L) to adjust the pH to approximately 11.0, and then add 2g of tetrabutylammonium bromide (TBAB, accounting for 2% of the medium mass). Under stirring conditions, add 6g of 1,4-butanediol diglycidyl ether (BDDE, accounting for 6% of the medium mass) dropwise at a rate of 0.2mL / min, and react at 30℃ for 1 h. Afterward, adjust the pH of the system to 13.0 with 50% NaOH solution and continue the reaction for 18 h.

[0150] After the reaction, the medium was washed according to the method in Example 1 to obtain the chromatographic purification medium. The target medium was then tested using respiratory syncytial virus pre-fusion protein (Pre-F) as the model antigen protein. The test indicators included the sulfur content, dynamic binding capacity, and separation purity of the chromatographic purification medium. The test method was the same as in Example 1.

[0151] Comparative Example 13

[0152] The difference between this comparative example and Example 1 is that the amount of ligand is increased, and the mass ratio of agarose microspheres to sodium dextran sulfate is adjusted to 5:2. All other conditions and steps are the same as in Example 1.

[0153] The specific procedure is as follows: Weigh 100g of agarose medium, add 40g of sodium dextran sulfate with a molecular weight of 30kDa, and add an appropriate amount of purified water to promote uniform dispersion. Stir at 30℃ for 30 min. Then add 0.18g of tripotassium phosphate (0.01mol / L) to adjust the pH to approximately 11.0, and then add 2g of tetrabutylammonium bromide (TBAB, accounting for 2% of the medium mass). Under stirring conditions, add 6g of 1,4-butanediol diglycidyl ether (BDDE, accounting for 6% of the medium mass) dropwise at a rate of 0.2mL / min, and react at 30℃ for 1 h. Afterward, adjust the pH of the system to 13.0 with 50% NaOH solution and continue the reaction for 18 h.

[0154] After the reaction, the medium was washed according to the method in Example 1 to obtain the chromatographic purification medium. The target medium was then tested using respiratory syncytial virus pre-fusion protein (Pre-F) as the model antigen protein. The test indicators included the sulfur content, dynamic binding capacity, and separation purity of the chromatographic purification medium. The test method was the same as in Example 1.

[0155] Comparative Example 14

[0156] The only difference between this comparative example and Example 1 is the crosslinking agent used. Specifically, the crosslinking agent is replaced with epichlorohydrin (ECH), and the amount added is the same as in Example 1, 6g. All other conditions and steps are the same as in Example 1.

[0157] The target media were tested using respiratory syncytial virus pre-fusion protein (Pre-F) as the model antigen protein. The detection indicators included the sulfur content, dynamic binding capacity, and separation purity of the chromatographic purification medium. The detection method was the same as in Example 1.

[0158] Comparative Example 15

[0159] The only difference between this comparative example and Example 1 is the crosslinking agent used. Specifically, the crosslinking agent is replaced with 1,6-hexanediol diglycidyl ether (HDDE), and the amount added is the same as in Example 1, 6g. All other conditions and steps are the same as in Example 1.

[0160] The target media were tested using respiratory syncytial virus pre-fusion protein (Pre-F) as the model antigen protein. The detection indicators included the sulfur content, dynamic binding capacity, and separation purity of the chromatographic purification medium. The detection method was the same as in Example 1.

[0161] Comparative Example 16

[0162] The conditions for the ligand in this comparative example have been adjusted. No phosphate is added in this example, and the other conditions are the same as in Example 1.

[0163] The specific operation is as follows: take 100 g of agarose medium, add 20 g of sodium dextran sulfate with a molecular weight of 30 kDa, and add an appropriate amount of purified water to promote uniform dispersion. Stir at 30°C for 30 min. Do not add phosphate. The remaining feeding steps and reaction conditions are the same as in Example 1.

[0164] The target media were tested using respiratory syncytial virus pre-fusion protein (Pre-F) as the model antigen protein. The detection indicators included the sulfur content, dynamic binding capacity, and separation purity of the chromatographic purification medium. The detection method was the same as in Example 1.

[0165] Comparative Example 17

[0166] This comparative example adjusted the amount of buffer salt and used excess tripotassium phosphate; the other conditions were the same as in Example 1.

[0167] The specific operation is as follows: take 100g of agarose medium, add 20g of sodium dextran sulfate with a molecular weight of 30kDa, and add an appropriate amount of purified water to promote uniform dispersion. Stir at 30℃ for 30min, add 0.03mol / L of tripotassium phosphate, and the remaining feeding steps and reaction conditions are the same as in Example 1.

[0168] The target media were tested using respiratory syncytial virus pre-fusion protein (Pre-F) as the model antigen protein. The detection indicators included the sulfur content, dynamic binding capacity, and separation purity of the chromatographic purification medium. The detection method was the same as in Example 1.

[0169] Table 1 Experimental variables set for each embodiment and comparative example

[0170]

[0171]

[0172]

[0173]

[0174] Table 2. Test results of each embodiment and comparative example.

[0175]

[0176]

[0177] As shown in Tables 1 and 2, the performance of the chromatographic purification media prepared in Examples 1-8 and Comparative Examples 1-17 was tested. The test indicators included sulfur content, dynamic binding capacity, and separation purity. The test results are shown in Table 2. The test results indicate that factors such as ligand molecular weight, media-to-ligand mass ratio, pH control method, crosslinking agent type, phase transfer catalyst, and buffer salt conditions all affect the degree of ligand introduction on the media and its binding and separation results in the actual chromatography process.

[0178] In this invention, sulfur content is mainly used to characterize the degree of ligand introduction and potential ligand density on the medium, while dynamic binding capacity and separation purity reflect the effective binding capacity of the medium to the target protein under flow conditions and the separation performance of the eluted products. As shown in Table 2, this invention preferably controls the sulfur content within the range of 380-450 μmol / mL to maintain a suitable ligand density, so that the medium obtains a stable and high dynamic binding capacity (75-85 mg Pre-F / mL) while maintaining a high level of separation purity.

[0179] It should be noted that separation purity (e.g., purity percentage measured by SEC-HPLC) is a characterization parameter of the composition of the eluted products, and its results are also affected by the composition of the loading solution, the loading level, and the elution conditions. Therefore, similar separation purity does not necessarily correspond to the same effective binding capacity between different media. Based on this, the present invention uses dynamic binding capacity to reflect the effective binding capacity of the medium, and combines separation purity with a comprehensive evaluation of the separation performance of the eluted products, thereby more comprehensively reflecting the performance of the chromatographic purification medium in practical applications.

[0180] First, regarding the influence of ligand molecular weight on media performance, when the molecular weight of sodium dextran sulfate is in the range of 25-35 kDa (Examples 2 and 3), the media exhibits a high dynamic binding capacity and stable separation purity (e.g., the separation purity of Examples 1-3 is around 93%). This indicates that this molecular weight range is conducive to the formation of suitable extended conformations of ligand segments on the media surface, thereby achieving effective multi-site binding under flow conditions and obtaining good elution separation performance. Among them, Example 1, with a molecular weight of 30 kDa, demonstrates a better overall balance between dynamic binding capacity and separation purity.

[0181] In contrast, when the molecular weight of the ligand is too low (e.g., 10 kDa in Comparative Example 1), the chain length is insufficient, resulting in limited spatial coverage of the effective binding sites. The dynamic binding capacity is lower than in Examples 1-3. Although the separation purity of Comparative Example 1 is relatively high, its dynamic binding capacity is significantly low, indicating that the medium's effective binding capacity to the target protein is insufficient under these conditions. Even if the elution product appears to have high purity, the amount of target protein that can be bound per unit volume of medium is still limited. When the molecular weight is slightly higher or lower than the preferred range (e.g., 20 kDa in Comparative Example 2 and 50 kDa in Comparative Example 3), although a certain degree of coupling structure can still be formed, the dynamic binding capacity is slightly lower than in Examples 1-3. This indicates that excessively short chains reduce spatial coverage, while excessively long chains are partially coiled or affected by exclusion in solution, both of which weaken the exposure of the effective binding sites. Comparative Example 2 still has relatively high separation purity, but Comparative Example 3 shows a decrease in separation purity (approximately 86.55%), indicating that deviations from the preferred range not only affect effective binding capacity but may also weaken separation selectivity. When the molecular weight was further increased to 100 kDa (Comparative Example 4), although a higher sulfur content was obtained, the dynamic binding capacity did not increase synchronously, and the separation purity decreased more significantly (e.g., approximately 76.55% in Comparative Example 4). This result indicates that when the molecular weight is too large, the spatial distribution of ligands and the utilization of effective binding sites are limited. Simply increasing the degree of ligand introduction does not bring about a corresponding increase in effective binding capacity; on the contrary, it may be detrimental to separation purity due to non-specific effects. The separation purity of Comparative Example 5 remained at a high level (approximately 91.92%), but its sulfur content and dynamic binding capacity were only 118 μmol / mL and 29 mg Pre-F / mL, respectively, which are significantly lower than the preferred range of the embodiments of the present invention, indicating that the effective capture capacity of this medium is limited. Therefore, it can be seen that controlling the molecular weight of the ligand within the range of 25-35 kDa can achieve a better balance between effective binding capacity and separation performance.

[0182] Furthermore, the mass ratio of medium to ligand also significantly affects the performance of the medium. In Example 1, when the mass ratio of agarose microspheres to ligand was 5:1, a good balance was achieved between the sulfur content, dynamic binding capacity, and separation purity of the resulting medium. In Examples 7 and 8, when the mass ratio was adjusted to 5:1.25 and 5:1.5, respectively, the sulfur content and dynamic binding capacity were further increased, while the separation purity remained at a high level. This indicates that appropriately increasing the ligand ratio within the above range is beneficial to improving the effective binding capacity without significantly impairing the separation performance. However, when the ligand dosage was too low (Comparative Example 12), the effective ligand concentration in the reaction system was insufficient, resulting in insufficient effective binding sites. The dynamic binding capacity decreased to 29 mg Pre-F / mL. Although the separation purity did not show an extreme decrease, it was still not superior compared to the examples, indicating that in the case of insufficient sites, the main limitation of the medium comes from the effective binding capacity rather than the level of separation purity. When an excessive amount of ligand was added (Comparative Example 13), although the sulfur content increased to 596 μmol / mL, the dynamic binding capacity decreased, and the separation purity dropped significantly (68.54%). This indicates that excessive ligand introduction may lead to segmental congestion and enhanced non-specific interactions, thereby reducing the effective binding capacity under flow conditions (decreased dynamic binding capacity) and increasing the proportion of impurities in the elution product (decreased separation purity). Therefore, a media-to-ligand mass ratio of 5:1 to 1.5 is the preferred range, achieving a good balance between the ligand introduction level, effective binding capacity, and separation performance.

[0183] Furthermore, the pH control method also plays a crucial role in the results of this invention. For example, in Example 1, a staged pH control (first pH 11 then increasing to pH 13) was used, which enabled the initial coupling of the linker arm on the medium at a lower alkalinity, and then promoted further coupling of the linker arm and ligand under high alkalinity conditions, thereby obtaining a higher ligand fixation amount and improving the effective binding capacity of the medium. In terms of separation purity, the SEC-HPLC purity of Example 1 was 93.76%, which is at a high level, indicating that good separation and purification performance can be maintained while improving the dynamic binding capacity. In contrast, Comparative Examples 6 and 7 used a single pH=11 or a single pH=13 reaction condition, respectively, and failed to distinguish the different activation levels required for the two-stage reaction, resulting in insufficient coupling or obvious chain aggregation. The resulting media had low dynamic binding capacity and only showed a general alkaline promoting effect. Furthermore, the separation purity could still be maintained at a high level under a single pH condition (Comparative Example 6: 94.00%; Comparative Example 7: 93.85%). This result indicates that the separation purity reflects the composition of the elution products more, while the dynamic binding capacity reflects the effective binding capacity of a unit volume of medium under a given loading concentration and flow conditions. Therefore, the advantage of staged pH control is mainly reflected in significantly improving the dynamic binding capacity and processing capacity, while maintaining a high separation purity.

[0184] Furthermore, Comparative Examples 8, 9, and 10 demonstrate that simply employing staged control without satisfying suitable alkaline conditions and the sequence of stages makes it difficult to obtain the efficient coupling required by this invention.

[0185] Specifically, in Comparative Example 8, the pH was only 8 in the first stage and increased to pH 13 in the second stage (8→13). The alkalinity in the first stage was insufficient, resulting in inadequate fixation of the linker arms on the medium. The sulfur content of the resulting medium was only 124 μmol / mL, and the dynamic binding capacity was 27 mg Pre-F / mL, significantly lower than that of Example 1. Although its separation purity was 94.68%, close to that of the example, this close purity does not mean that the medium processing capacity is the same. Comparative Example 8 mainly suffered from insufficient binding sites, leading to a significant reduction in the target protein that could be bound per unit volume of medium. In Comparative Example 9, the pH was 4 in the first stage and 8 in the second stage (4→8). Neither stage was within the effective alkalinity range. The final measured sulfur content, dynamic binding capacity, and separation purity were all 0, indicating that effective coupling could not be achieved under these conditions. In Comparative Example 10, a reverse phased control (13→11) was employed, meaning the reaction was first carried out under higher alkalinity conditions, followed by a decrease to lower alkalinity for subsequent reactions. While a certain sulfur content (205 μmol / mL) was obtained, the dynamic binding loading was only 34 mg Pre-F / mL, lower than in Example 1. This result further illustrates that the key to this invention lies in adopting a phased control approach within an appropriate alkalinity range, starting with lower alkalinity and gradually increasing it, to obtain more effective binding sites, thereby significantly improving the effective binding capacity per unit volume of medium while maintaining high separation purity.

[0186] Furthermore, the choice of crosslinking agent also has a significant impact on coupling efficiency. For example, Example 1 used 1,4-butanediol diglycidyl ether (BDDE) as the crosslinking agent; Comparative Examples 14 and 15 used epichlorohydrin (ECH) and 1,6-hexanediol diglycidyl ether (HDDE) respectively for comparison. The results showed that the linker arms formed by BDDE were of moderate length, while the linker arms formed by ECH were shorter, making it difficult to provide sufficient space and multi-site binding conditions between the medium surface and the ligand. The increased chain length of HDDE led to uneven distribution of ligand coupling, reduced exposure of binding sites, and a slight decrease in binding performance. This indicates that BDDE has a better balance between maintaining structural stability and reactivity, and the linker arms it forms help improve the binding efficiency and stability of the medium.

[0187] Furthermore, the use of phase transfer catalysts also plays a crucial role in the results. For example, in Example 1, the addition of 2% TBAB to the reaction system resulted in high levels of sulfur content and dynamic binding loading in the medium; while in Comparative Example 11, without the addition of TBAB, the reaction interface transfer was limited, the coupling reaction was difficult to proceed smoothly, and the performance significantly decreased. These results indicate that phase transfer catalysts can effectively promote the reaction mass transfer between organic and inorganic phases, improve coupling efficiency and the number of effective binding sites, thereby increasing the dynamic binding loading and maintaining high separation purity.

[0188] Furthermore, the choice and concentration of the buffer salt directly affect the experiments of this invention. For example, in Example 1, 0.01 mol / L tripotassium phosphate was used as the buffer salt, with a dynamic binding capacity of 81 mg Pre-F / mL and a separation purity of 93.76%. In Example 6, after replacing it with sodium phosphate, the dynamic binding capacity remained at 80 mg Pre-F / mL, and the separation purity was 92.95%, indicating that different phosphate systems are applicable and can maintain high separation purity. However, in Comparative Example 16 without buffer salt, the low ionic strength led to enhanced electrostatic repulsion and insufficient coupling, resulting in a sulfur content of 86 μmol / mL and a dynamic binding capacity of 12 mg Pre-F / mL. Although the separation purity was still 94.41%, the media processing capacity was significantly insufficient. In contrast, the excessive buffer salt in Comparative Example 17 led to an imbalance in the ionic strength of the system, with a dynamic binding capacity of only 21 mg Pre-F / mL. At the same time, the separation purity dropped to 75.20%. This indicates that excessively high ionic strength not only inhibits the formation of coupling and effective binding sites, but may also introduce more obvious non-specific effects, affecting separation selectivity and having an adverse impact on separation purity.

[0189] A comprehensive comparison of the experimental results shows that, for example, in Example 1, the mass ratio of agarose microspheres to sodium dextran sulfate was 5:1, the molecular weight of the ligand was 30 kDa, the buffer salt was 0.01 mol / L tripotassium phosphate, the amount of phase transfer catalyst TBAB was 2% of the media mass, the amount of crosslinking agent 1,4-butanediol diglycidyl ether was 6% of the media mass, and a staged pH control was used (first pH 11, then increasing to pH 13). Under these conditions, the dynamic binding capacity of the medium obtained was 81 mg Pre-F / mL, and the SEC-HPLC purity was 93.76%, demonstrating high effective binding capacity and good separation and purification results. Examples 2-8, while maintaining high SEC-HPLC purity (approximately 91%-95%), also showed a stable dynamic binding capacity of 75-85 mg Pre-F / mL, indicating that the combination of process parameters of the present invention has a wide range of applicability.

[0190] Compared with the comparative example, the chromatographic purification medium prepared by the present invention exhibits higher dynamic binding capacity and unit volume processing capacity while maintaining high separation purity. This allows for the binding and purification of more viral proteins under the same volume of chromatographic purification medium, or a significant reduction in the amount of chromatographic purification medium required to obtain the same viral protein yield, thereby improving overall purification efficiency and reducing process costs.

[0191] In summary, the technical solution of this invention solves the problems of insufficient binding capacity and unstable performance of chromatography media in the prior art by rationally selecting the medium, the ratio of purified water to ligand, the molecular weight range of ligand, the concentration of buffer salt, the amount of crosslinking agent and catalyst, and the staged pH control method, thus forming a method for preparing chromatography purification media that can efficiently separate subunit viral antigens.

[0192] The embodiments of this application have been described above, but this application is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of this application without departing from the spirit and scope of the claims, and all of these forms are within the protection scope of this application.

Claims

1. A method for preparing a chromatographic purification medium for efficient separation of subunit viruses, characterized in that, Includes the following steps: (1) Prepare the base frame materials; (2) Add sodium dextran sulfate to the base material; (3) Add phosphate buffer, phase transfer catalyst and crosslinking agent; (4) The reaction is carried out under alkaline pH conditions in stages, and the pH of the second stage is higher than that of the first stage; (5) After the reaction is complete, the medium is washed multiple times with alkaline solution and purified water to obtain the chromatographic purification medium. The molecular weight of the sodium dextran sulfate is 25-35 kDa. The substrate material is agarose microspheres, which are prepared from 3.0% to 3.5% agarose and have a particle size of 45 to 165 μm. The mass ratio of the agarose microspheres to sodium dextran sulfate is 5:1 to 1.

5. The specific phased alkaline pH conditions are as follows: in the first phase, the reaction system is adjusted to alkaline pH 1 and reacted for 0.5–2 hours; in the second phase, the reaction system is adjusted to alkaline pH 2 and reacted for 15–20 hours, wherein pH 2 is higher than pH 1, where pH 1 is 10.5–11.5 and pH 2 is 12.5–13.

5. The crosslinking agent is an epoxy crosslinking agent, and the epoxy crosslinking agent is 1,4-butanediol diglycidyl ether.

2. The preparation method according to claim 1, characterized in that, The sodium sulfate dextran salt has a molecular weight of 30 kDa.

3. The preparation method according to claim 1 or 2, characterized in that, The phosphate buffer solution is a tripotassium phosphate or sodium phosphate solution, and the concentration of the phosphate buffer solution is 0.005–0.02 mol / L.

4. The preparation method according to claim 1 or 2, characterized in that, The phase transfer catalyst is a quaternary ammonium salt, which is selected from tetrabutylammonium bromide, tetrabutylammonium hydrogen sulfate, and benzyltriethylammonium chloride.

5. The preparation method according to claim 4, characterized in that, The quaternary ammonium salt is tetrabutylammonium bromide.

6. The preparation method according to claim 1, characterized in that, The amount of the crosslinking agent is 5% to 6% of the mass of the agarose microspheres, and the reaction is carried out at 30°C for 0.5 to 2 hours; and / or the amount of the phase transfer catalyst is 2% of the mass of the agarose microspheres.

7. The preparation method according to claim 1, characterized in that, The sulfur content of the medium is 380~450 μmol / mL, and the dynamic binding loading is 75~85 mg Pre-F / mL.

8. A chromatography purification medium, characterized in that, It is prepared by the preparation method according to any one of claims 1 to 7, wherein the chromatography purification medium is used for the separation and purification of subunit viruses.

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