Process for removing bovine serum albumin and method thereof

Abstract

The present application relates to a bovine serum albumin removal process and method thereof, belonging to the technical field of protein separation and purification, comprising solid-phase synthesis of a functionalized modified polypeptide, activation of a solid-phase carrier, and covalent coupling of the two; the polypeptide is composed of a binding domain, a flexible linker, and a bonding domain in sequence; the activated carrier is modified from polysaccharide or resin microspheres, and has maleimide groups on the surface to dock with sulfhydryl groups; in the later purification, the affinity material is filled into a chromatography column, and the material liquid is separated through the steps of neutral liquid balancing, material liquid pumping, sample loading, and alkaline cleaning and elution. The flexible linker composed of a polyethylene glycol segment or a flexible peptide segment effectively reduces the steric hindrance, so that the ligand fully extends outward and is exposed.

Description

Technical Field

[0001] This invention relates to the field of protein separation and purification, specifically to the process and method for removing bovine serum albumin. Background Technology

[0002] In the purification and separation process of biopharmaceuticals, affinity chromatography is often used to remove bovine serum albumin impurities from materials. However, existing traditional antibody affinity ligands suffer from insufficient alkaline stability, making it difficult to meet the requirements of reusable processes with strict online cleaning. Conventional solid-phase carriers have significant steric hindrance on their surfaces, which reduces target recognition and contact efficiency. During coupling operations, multi-point binding and non-specific adsorption are prone to occur, resulting in the recovery rate and process stability of the final separation and purification system failing to reach the ideal state. Summary of the Invention

[0003] The purpose of this invention is to overcome the shortcomings of existing affinity ligands, such as poor base stability, steric hindrance, and easy non-specific impurity adsorption, and to provide a highly specific, strong-alkali-resistant, and highly regenerable bovine serum albumin removal process and method.

[0004] This invention constructs a novel affinity separation system. This system utilizes solid-phase synthesis to prepare functionalized modified peptides with specific configurations. The peptides are sequentially composed of a binding domain, a flexible linker arm, and a binding domain with reactive thiol groups. Simultaneously, a porous microsphere carrier is chemically activated, introducing maleimide groups onto its surface. The reactive thiol groups at the peptide ends undergo a directional addition reaction with the maleimide groups on the carrier surface to construct a stable, irreversible thioether cross-linked structure, thus preparing a bovine serum albumin affinity material. In the separation and purification stage, this affinity material is packed into a chromatography column, establishing a continuous extraction and separation system that includes neutral liquid equilibration, feed pumping and separation, and alkaline washing and elution.

[0005] Compared with the prior art, the present invention has the following advantages: 1. Flexible linkers composed of polyethylene glycol segments or flexible peptide segments effectively increase the spatial distance between the ligand and the carrier surface, allowing the affinity ligand to be fully exposed and improving the accuracy of target recognition and contact binding efficiency of bovine serum albumin. 2. The thioether anchor chain formed by the directional binding of a single reactive thiol group provided by a cysteine ​​residue and a maleimide group has a stable covalent bond force; this structure can effectively resist the high flow rate scouring and strong alkaline elution environment in subsequent processes, and prevent ligand detachment and leakage. 3. With the help of a specialized active group quenching and sealing mechanism, free impurities and unstable residues in the system are removed, and the non-specific blocking or dragging effect of recombinant components caused by the active gap in the later separation operation is eliminated, so as to achieve high purity and high yield extraction of the target liquid. Detailed Implementation

[0006] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments.

[0007] Example 1: A bovine serum albumin (BSA) removal process includes an initial synthesis step involving the preparation of a BSA affinity material, and a subsequent purification and separation step based on the BSA affinity material to separate the feed solution. The initial synthesis step includes: solid-phase immobilization synthesis of functionalized modified peptides for targeted adsorption of BSA, surface activation treatment of the solid-phase support, and covalent coupling between the functionalized modified peptides and the activated solid-phase support. The subsequent purification and separation process includes: loading the bovine serum albumin affinity material obtained by covalent coupling into a chromatography column with an outlet and an inlet to establish an extraction and separation system containing the chromatography column, and sequentially performing neutral liquid equilibration, sample loading and separation by pumping the feed solution in, and alkaline washing and elution steps. In the solid-phase immobilization synthesis of functionalized modified peptides, the functionalized modified peptides consist of a binding domain, a flexible linker arm, and a bonding domain covalently linked in sequence; wherein the binding domain is a selected affinity sequence that matches and binds to bovine serum albumin; the flexible linker arm is selected from polyethylene glycol segments with a degree of polymerization of 4 to 12, or has the structure (Gly4Ser). n The flexible peptide segment, where n is limited to 2 to 4; The bonding domain is a cysteine ​​residue located at the C-terminus or N-terminus of the polypeptide, and the cysteine ​​residue has a reactive thiol group; the activated solid support is obtained by chemically activating and modifying polysaccharide or resin microspheres with a crosslinking agent, and the surface of the activated solid support has maleimide groups for docking covalent coupling. The bovine serum albumin removal process uses functionalized modified peptides as recognition units, maleimide-activated microspheres as solid-phase carriers, and chromatography columns as carriers for subsequent purification and separation. The functionalized modified peptides are selected as linear structures with an N-terminal binding domain, a central flexible linker arm, and a C-terminal bonding domain. The binding domain is selected as the short peptide sequence B1, which has been screened in the early stage and can cooperate with the hydrophobic and charged microenvironment of the bovine serum albumin surface. The flexible linker arm is selected as PEG4, and the bonding domain is selected as the C-terminal cysteine ​​residue. The C-terminal carboxyl group of the binding domain is covalently linked to the amino group at one end of PEG4 through an amide bond, and the carboxyl group at the other end of PEG4 is covalently linked to the N-terminal amino group of the cysteine ​​residue in the bonding domain through an amide bond. In this structure, the binding domain is used for selective adsorption of bovine serum albumin. PEG4 separates the binding domain from the surface of the microspheres, reducing the influence of steric hindrance on the recognition contact. Cysteine ​​provides a single thiol reaction site, which facilitates the formation of directional thioether bonds with maleimide groups. The activated solid-phase support is made of polymethacrylate microspheres, which are then packed into a 5mL empty column after being made into an affinity material. An extraction and separation system is established with the inlet end, outlet end, peristaltic pump and buffer tank connected. Neutral liquid equilibration was performed using PBS buffer. The sample was pumped into the feed solution for separation using a mixture containing recombinant monoclonal antibody and bovine serum albumin. Alkaline washing and elution were performed using an aqueous sodium hydroxide solution. This bovine serum albumin removal process uses peptides instead of antibodies as affinity ligands, which improves alkali resistance while maintaining selectivity and is suitable for repeated separation processes that require in-situ washing.

[0008] The solid-phase immobilized synthesis of functionalized modified peptides includes the following steps: using Fmoc-RinkAmideMBHA resin with preloaded bonding domains as the starting support structure, loading it into the synthesizer, and using a mixed solution of piperidine and N,N-dimethylformamide with a volume concentration of 15%-25% as the deprotection solution for preprotection treatment. After deprotection treatment, a mixture containing the target amino acid, HATU and DIEA condensation system is added to the system. The mixture is allowed to stand at room temperature for 0.5-1.5 hours to perform a static coupling reaction to couple the required short chain substance. The target amino acid is introduced sequentially according to the sequence of flexible linker arms and binding domains to form the polypeptide backbone, and then the polypeptide resin-bound state carrying the protective shell is obtained. After coupling to generate the main chain, a cleavage solution prepared by mixing trifluoroacetic acid, triisopropylsilane and deionized water is added. The volume ratio of trifluoroacetic acid, triisopropylsilane and water is adjusted to within the range of 80-95:2.5-10:2.5-10. The peptide resin is subjected to continuous room temperature shaking reaction for 1.5-2.5 hours to achieve cleavage from the resin. The free liquid in the reaction tank is collected as crude peptide solution. After collecting the crude polypeptide solution, it was centrifuged and precipitated by injecting ice-cold ether detergent, and the precipitate was extracted to obtain the primary filter. The primary filter was purified by gradient elution using a reversed-phase high-performance liquid chromatography device and then transferred to a freeze dryer for freeze drying to generate solid functionalized modified polypeptide powder. Solid-phase propagation synthesis of functionalized modified peptides was carried out using Fmoc-RinkAmide MBHA resin as the starting support structure with a degree of substitution of 0.50 mmol / g and pre-loaded with cysteine ​​binding domain. The deprotection solution was a mixture of piperidine and N,N-dimethylformamide at a volume concentration of 15%, and the deprotection was carried out for 8 min each time, for a total of 2 times. The static coupling reaction used the HATU and DIEA system, with the target amino acid amount being 3 times the molar amount of resin loading, and the single coupling time being 0.5 h; the flexible linker used Fmoc-NH-PEG4-COOH, and the binding domain was coupled segment by segment according to the B1 sequence to obtain the peptide resin-bound state; the excision solution used trifluoroacetic acid, triisopropylsilane and water in a volume ratio of 95:2.5:2.5, and was shaken at room temperature for 2.0 h; The crude peptide solution was precipitated with 10 times the volume of ice-cold diethyl ether, centrifuged at 4000 rpm for 10 min, and washed three times. The precipitate was purified by C18 reversed-phase high-performance liquid chromatography with a water and acetonitrile gradient system containing 0.1% trifluoroacetic acid as the mobile phase. The main peak was collected and freeze-dried to obtain solid functionalized modified peptide powder. This step employs a resin-based initiating carrier structure and pre-loading of terminal cysteine ​​residues, which can stably retain the reactive thiol group at a single end position, reducing the risk of multi-point linkage in subsequent coupling. MALDI-TOFMS analysis showed that the measured molecular weight was consistent with the theoretical value, and the HPLC purity was 98.3%.

[0009] The surface activation treatment of the solid support includes the following steps: selecting polymethyl methacrylate microspheres with a supporting framework pore structure and hydroxylated surface as matrix microspheres, injecting epichlorohydrin mixed solution to immerse them for the first round of chemical ring-opening activation reaction, and then introducing an appropriate amount of ethylenediamine co-dissolving to initiate a secondary reaction operation to forcibly bond and introduce amino groups on the surface of the matrix microspheres; After the amino group is introduced, a mixture containing succinimide-4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid ester crosslinking reagent is added dropwise to the surface of the reaction tank to provide displacement driving energy. This transforms the surface into a modified surface carrying maleimide groups that can specifically react with thiol groups. Finally, the residue is washed away and filtered to form a dried and ready-to-use activated solid support. For the surface activation treatment of the solid-phase support, polymethacrylate microspheres were used as the matrix microspheres. The crosslinking degree of the microspheres was 6% and the average particle size was 90 μm. The matrix microspheres were soaked in an alkaline mixture containing epichlorohydrin for 4 h, washed, and then reacted with ethylenediamine aqueous solution for 6 h to introduce amino groups on the surface of the microspheres. The matrix microspheres with introduced amino groups were washed with phosphate buffer until neutral, and then reacted with a solution containing SMCC for 2 h to convert the amino groups into maleimide groups. The activated solid support was washed alternately with pure water and 20% ethanol, and then dehydrated under reduced pressure before use. The surface activation treatment adopts the route of first opening the ring to introduce amino groups and then converting maleimide, which can concentrate the covalent coupling sites on the surface of the matrix microspheres into single reaction sites sensitive to thiol groups, reduce side reactions with other amino acid residues in the peptide, and improve the consistency of coupling direction and the degree of ligand exposure.

[0010] In the covalent coupling step: the functionalized modified peptide powder obtained in the previous step is transferred into a neutral or slightly alkaline coupling buffer system with a pH of 7.0-7.4 for thorough dissolution and dispersion, and diluted to a fixed volume to establish a peptide solution with a concentration of 3-8 mg / mL. At the same time, a maintenance matrix containing tris(2-carboxyethyl)phosphonic acid hydrochloride with a concentration of 0.5-2.0 mM is added to the coupling buffer system. After establishing the peptide solution pool, the activated solid support is introduced into the liquid phase. The ratio of the sedimentation volume of the solid support to the liquid surface volume of the peptide solution is strictly controlled to be maintained at 1:1.5 to 1:2.5. During the feeding process, a light-proof incubation device is used to carry out physical rotation incubation for 3-5 hours. This induces the thiol sites of the functionalized modified peptides to contact and collide with the maleimide groups on the surface of the microspheres, and spontaneously form a fixed irreversible thioether cross-linking structure to complete the synthetic correlation binding. Functionalized modified peptide powder was dissolved in phosphate buffer at pH 7.0 to prepare a peptide solution of 3 mg / mL. 0.5 mM tris(2-carboxyethyl)phosphonic acid hydrochloride was added to the buffer to keep the cysteine ​​sulfhydryl groups in a reduced state. The volume ratio of the activated solid support to the peptide solution was controlled at 1:1.5, and the mixture was incubated at room temperature for 3 hours in a light-protected incubation device. This covalent coupling relies on the addition reaction between thiol groups and maleimide groups to form an irreversible thioether anchor chain. This irreversible coupling method differs from ion adsorption fixation, and ligand detachment is less likely to occur after alkaline washing. After coupling, the decrease in free thiol groups in the supernatant was measured, and the peptide coupling density was calculated to be 6.1 mg / mL resin.

[0011] After completing the main process of incubation in the dark, the reaction termination and quenching of free active groups targeting unclosed reaction sites are initiated: L-cysteine ​​termination blocking solution with a concentration in the range of 30-80 mM is slowly added to the reaction system, the inversion force is removed and the system is placed in a static state for incubation at room temperature for 0.5-1.5 hours. At the end of the incubation period, open the bottom drain valve and sequentially pump out sodium chloride high-salt brine solution with a molar concentration of 0.5-1.5M, deionized water, and low-boiling alcohol detergent solution with a volume concentration of 15%-25% to carry out an alternating countercurrent washing process for filtration and collection of purified affinity material. After the incubation was completed in the dark, 30 mL of cysteine-terminated blocking solution was added to the coupling system and allowed to stand at room temperature for 0.5 h to block unclosed reaction sites. The blocked affinity material was then washed alternately with 0.5 M sodium chloride high-salt aqueous solution, deionized pure water and 15% ethanol detergent solution, for 3 resin volumes each. The reaction termination and gap quenching blocking steps allow the residual maleimide sites on the microsphere surface to be occupied by low-molecular-weight blocking agents, which can reduce non-specific contact between free thiol or amine groups in the target protein and the carrier during subsequent loading; the treated bovine serum albumin affinity material is stored in 20% ethanol for later use.

[0012] The chromatography column included in the extraction and separation system is a high-pressure resistant empty chromatography column. In the neutral liquid equilibration step, the flow rate of the pretreatment solution pumped into the chromatography column is uniformly controlled within the range of 100-180 cm / h. In the feed solution pumping and sample loading separation step, the matching working flow rate is adjusted and limited to 80-120 cm / h. In the alkaline washing and elution step, the regeneration base solution selected is a sodium hydroxide strong alkaline elution system with a pH of 12.0-13.0 or a glycine-hydrochloric acid buffer with a pH of 2.2-2.8. The chromatography column used in the extraction and separation system was a high-pressure resistant empty chromatography column with a column bed volume of 5 mL; the pretreatment solution for neutral liquid equilibration was PBS buffer, with a pump flow rate of 100 cm / h; the working flow rate for sample loading and separation was 80 cm / h; the regeneration base solution for alkaline washing and elution was a strong alkaline solution of sodium hydroxide at pH 13.0, corresponding to a sodium hydroxide concentration of 0.05 M. This flow rate combination is beneficial for uniform wetting of the column bed and for full contact between bovine serum albumin and the binding domain. The regeneration stage uses a strong alkaline solution of sodium hydroxide at pH 13.0 to dissociate the binding and also to perform in-situ cleaning. The chromatography column was run 20 times under the above conditions without any abnormal increase in column pressure.

[0013] The procedure for using the bovine serum albumin removal process is as follows: a. System preparation flow path setup: Load bovine serum albumin affinity material into the chromatography column, close both ends of the chromatography column and connect it to the circulation flow path system, and perform degassing operation inside the chromatography column; b. Dynamic homogenization equilibrium: Neutral phosphate buffer with pH set in the range of 7.2-7.6 and without the introduction of exogenous factors is drawn by a peristaltic pump and delivered to the flow surface of the chromatography column. The volume of the buffer is limited to 4-8 column bed volumes. The flushing flow rate is set at 100-180 cm / h during the working period. The flushing and replacement treatment continues until the affinity material contained in the pipeline system reaches a physicochemical equilibrium state. c. Target Retention and Breakthrough Separation: Pump in the target solution containing bovine serum albumin impurities and the components to be refined and purified, and pass it through the chromatography column at a flow rate controlled at 80-120 cm / h; the bovine serum albumin is specifically retained by the affinity material, and the breakthrough liquid is collected to obtain the purified target solution. d. In-situ regeneration and elution: When the target feed solution supply is detected and confirmed to have reached the point of complete depletion, the feed valve is immediately interrupted, and a neutral phosphate buffer solution with a volume of 2-4 times the column bed volume is quickly introduced through the parallel backflush valve to wash away unbound impurities; After the first flush, 3-5 column bed volumes of sodium hydroxide eluent with a molar concentration of 0.05-0.15M are introduced to dissociate the retained bovine serum albumin and remove it with the liquid flow, thereby restoring the initial binding activity of the affinity material and completing one cycle of process operation. During the system preparation flow path setup, the bovine serum albumin affinity material prepared in Example 1 was wet-packed into a 5 mL shell chromatography column. Sealing mesh filters were installed at the top and bottom of the column. A peristaltic pump, injection vessel, equilibration buffer, and waste container were connected, and a 5-minute negative pressure static bubble degassing process was performed. Dynamic homogenization equilibration was achieved using a pH 7.2 neutral phosphate buffer solution, with a flow rate of 4 times the column bed volume and a flushing flow rate of 120 cm / h. The target intercept and penetration separation system used a recombinant monoclonal antibody solution in PBS matrix, with an antibody concentration of 2.0 mg / mL and a bovine serum albumin concentration of 50 μg / mL. The loading flow rate was 80 cm / h and the loading volume was 50 mL. For in-situ regeneration and elution, the washing volume of neutral phosphate buffer was 2 times the column bed volume, and the concentration of sodium hydroxide strong ion elution buffer was 0.05 M, with a loading volume of 3 times the column bed volume. The flow-through solution was analyzed by BSA-specific ELISA, and the bovine serum albumin concentration was 0.09 μg / mL with a removal rate of 99.82%; the antibody recovery rate was 96.1%. This method of use shows that bovine serum albumin is selectively retained on the surface of the affinity material, and the target recombinant pure feed solution can be obtained from the flow-through end. The separation path is simplified and suitable for continuous batch use.

[0014] Example 2: The bovine serum albumin removal process maintains the same extraction and separation system and separation sequence as in Example 1, only adjusting the flexible linker structure in the functionalized modified peptide; the functionalized modified peptide is composed of a binding domain B1, a flexible linker (Gly4Ser)2, and an N-terminal cysteine ​​residue; This design replaces the PEG segment with a flexible peptide segment for the flexible linker, improving compatibility with the aqueous system while maintaining the intermolecular distance. The activated solid support still uses maleimide-modified polymethacrylate microspheres. This bovine serum albumin removal process can maintain a low level of non-specific adsorption in protein-based solutions, making it suitable for product purification steps where ethylene glycol residues need to be controlled.

[0015] The solid-phase supported synthesis of functionalized modified peptides includes the following steps: using Fmoc-RinkAmide MBHA resin with preloaded bonding domains as the starting support structure, loading it into a synthesizer and using a 15%-25% (v / v) mixed solution of piperidine and N,N-dimethylformamide as the deprotection solution for preprotection treatment; after deprotection treatment, adding a mixed system containing the target amino acid, HATU, and DIEA condensation system into the system; The desired short-chain substance is coupled by static coupling reaction at room temperature for 0.5-1.5 hours. The target amino acid is introduced sequentially according to the sequence of flexible linker arms and binding domains to form the polypeptide backbone. Then, the polypeptide resin-bound state with a protective shell is obtained. After the coupling is completed and the backbone is generated, the excision solution prepared by mixing trifluoroacetic acid, triisopropylsilane and deionized water is added. The volume ratio of trifluoroacetic acid, triisopropylsilane, and water was adjusted to within the range of 80-95:2.5-10:2.5-10. The peptide resin was subjected to continuous room temperature shaking for 1.5-2.5 hours to achieve cleavage from the resin. The free liquid in the reaction tank was collected as the crude peptide solution. After collecting the crude peptide solution, ice-cold ether was injected for centrifugation and precipitation washing. The precipitate was extracted to obtain the initial filter. The initial filter was purified by gradient elution using reversed-phase high-performance liquid chromatography (RP-HPLC) and then transferred to a freeze dryer for freeze drying to generate a solid functionalized modified peptide powder. In the solid-phase propagation synthesis of functionalized modified peptides, the deprotection solution was a mixture of piperidine and N,N-dimethylformamide at a volume concentration of 20%, and the deprotection was carried out in one step for 10 min. The static coupling reaction used a mixture containing the target amino acid, HATU and DIEA condensing agent, wherein the amount of the target amino acid was 3 times the molar amount of the resin loading, the amount of HATU was 2.85 times the molar amount of the resin loading, and the amount of DIEA was 6 times the molar amount of the resin loading. The static coupling reaction time was set to 1.0 h. The excision solution consisted of trifluoroacetic acid, triisopropylsilane, and water in a volume ratio of 90:5:5. The mixture was shaken at room temperature for 1.5 hours, and the free liquid residue collected from the reaction chamber was the crude peptide solution. This crude peptide solution was precipitated with 10 times its volume of ice-cold diethyl ether, centrifuged at 4000 rpm for 10 minutes, and washed three times. The precipitate was extracted to obtain the initial filter. This initial filter removed most of the non-polar impurities and free protecting groups, effectively reducing the burden on subsequent purification. Subsequently, the initial filter was purified by C18 reversed-phase high-performance liquid chromatography to obtain a functionalized modified peptide powder with a purity of 97.9%. Compared to Example 1, when the flexible linker used (Gly4Ser)2, the peptide chain was more hydrophilic overall, resulting in better dispersibility in PBS after coupling.

[0016] In the covalent coupling step: the functionalized modified peptide powder obtained in the previous step is transferred into a neutral or slightly alkaline coupling buffer system with a pH of 7.0-7.4 for thorough dissolution and dispersion, and diluted to a fixed volume to establish a peptide solution with a concentration of 3-8 mg / mL. At the same time, a maintenance matrix containing tris(2-carboxyethyl)phosphonic acid hydrochloride with a concentration of 0.5-2.0 mM is added to the coupling buffer system. After establishing the peptide solution pool, the activated solid support is introduced into the liquid phase. The ratio of the sedimentation volume of the solid support to the liquid surface volume of the peptide solution is strictly controlled to be maintained at 1:1.5 to 1:2.5. During the feeding process, a light-proof incubation device is used to carry out physical rotation incubation for 3-5 hours. This induces the thiol sites of the functionalized modified peptides to contact and collide with the maleimide groups on the surface of the microspheres, and spontaneously form a fixed irreversible thioether cross-linking structure to complete the synthetic correlation binding. The functionalized modified peptide powder was dissolved in a coupling buffer aqueous system at pH 7.2 at a concentration of 5 mg / mL, and the concentration of tris(2-carboxyethyl)phosphine hydrochloride was set at 1.0 mM. The volume ratio of the solid support to the peptide solution was set at 1:2.0, and the incubation time was 4 h in the dark. The peptide coupling density was determined to be 6.8 mg / mL resin. Under these conditions, the ligand coverage on the microsphere surface was relatively uniform, and the BSA removal rate was 99.76% and the antibody recovery rate was 96.8% during separation.

[0017] Example 3: In this embodiment, the flexible linker of the functionalized modified peptide is selected with a degree of polymerization of [insert degree here]. The polyethylene glycol segment and the remaining peptide chain synthesis steps are the same as in Example 1; the surface activation treatment of the solid support includes the following steps: selecting polymethacrylate microspheres with a supporting framework pore structure and hydroxylated surface as matrix microspheres, injecting epichlorohydrin mixed solution to immerse them for the first round of chemical ring-opening activation reaction, and then introducing an appropriate amount of ethylenediamine co-dissolving to initiate a secondary reaction operation to forcibly bond and introduce amino groups on the surface of the matrix microspheres; After the amino group is introduced, a mixture containing succinimide-4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid ester crosslinking reagent is added dropwise to the surface of the reaction tank to provide displacement driving energy. This transforms the surface into a modified surface carrying maleimide groups that can specifically react with thiol groups. Finally, the residue is washed away and filtered to form a dried and ready-to-use activated solid support. The surface activation treatment of the solid support followed the process route in Example 1, except that the matrix microspheres were replaced with polymethacrylate microspheres with hydroxylation modification on the surface. The crosslinking degree of these microspheres was 10%, and the average particle size was 80 μm. The matrix microspheres were soaked in a 0.4 M sodium hydroxide alkaline mixture containing 20% ​​epichlorohydrin at a solid-liquid ratio of 1:1 for 5 hours. After washing, a 0.5 M ethylenediamine aqueous solution was added, and the ethylenediamine reaction time was 8 hours. The matrix microspheres with introduced amino groups were washed with buffer until neutral, and then a mixture of N,N-dimethylformamide and PBS containing 10 mg / mL SMCC was added. The SMCC conversion time was 2.5 h. Polymethacrylate microspheres were used because they have high mechanical strength and are suitable for high flow rate column chromatography. After washing, the surface maleimide group density of the activated solid support was measured to be 34 μmol / mL resin, which can meet the requirements of subsequent peptide coupling.

[0018] After completing the main process of incubation in the dark, the reaction termination and quenching of free active groups targeting unclosed reaction sites are initiated: L-cysteine ​​termination blocking solution with a concentration in the range of 30-80 mM is slowly added to the reaction system, the inversion force is removed and the system is placed in a static state for incubation at room temperature for 0.5-1.5 hours. At the end of the incubation period, open the bottom drain valve and sequentially pump out sodium chloride high-salt brine solution with a molar concentration of 0.5-1.5M, deionized water, and low-boiling alcohol detergent solution with a volume concentration of 15%-25% to carry out an alternating countercurrent washing process for filtration and collection of purified affinity material. The L-cysteine ​​blocking solution concentration was set at 80 mM, and the incubation was carried out at room temperature for 1.5 h. The washing sequence remained unchanged, with a high-salt sodium chloride solution concentration of 1.5 M and a low-boiling alcohol detergent solution of 25% ethanol by volume. The high-intensity blocking and washing conditions were beneficial for removing unbound peptides and weakly adsorbed organic residues. After soaking in 0.1 M NaOH for 24 h, no significant loss of coupling ligands was detected in the bovine serum albumin affinity material obtained by this treatment.

[0019] The chromatography column included in the extraction and separation system is a high-pressure resistant empty chromatography column. In the neutral liquid equilibration step, the flow rate of the pretreatment solution pumped into the chromatography column is uniformly controlled within the range of 100-180 cm / h. In the feed solution pumping and sample loading separation step, the matching working flow rate is adjusted and limited to 80-120 cm / h. In the alkaline washing and elution step, the regeneration base solution selected is a sodium hydroxide strong alkaline elution system with a pH of 12.0-13.0 or a glycine-hydrochloric acid buffer with a pH of 2.2-2.8. The flow rate of the pretreatment solution in neutral liquid equilibrium was set to 180 cm / h, and the working flow rate of the feed solution for sample loading and separation was set to 120 cm / h. The alkaline washing and elution step was replaced with a glycine-hydrochloric acid buffer at pH 2.2, with an elution volume of 4 column volumes. These values ​​were used to verify the availability of the affinity material under high flow rate and acid washing conditions. The results showed that the BSA removal rate was 99.58%, and the antibody recovery rate was 95.4%. Compared with Example 1, the contact time was shortened under high flow rate, and the removal rate decreased slightly, but it still remained at a high level, indicating that the combination of the activated solid-phase carrier and the functionalized modified peptide has stable availability in different regeneration systems.

[0020] Comparative Example 1: Comparative Example 1 is identical to Example 1 in terms of solid support, coupling method, chromatography column, and separation process. The difference is that the functionalized modified peptide does not have a flexible linker arm and directly uses the binding domain B1 to connect with the C-terminal cysteine ​​to form a simplified peptide. The other conditions are the same as in Example 1. This comparative example is used to evaluate the effect of the flexible linker arm on the degree of exposure of the binding domain and the removal performance. Test results show that without the flexible connecting arm, the steric hindrance on the surface of the microsphere increases, and both the BSA removal rate and antibody recovery rate decrease, indicating that the flexible connecting arm is not only a connecting structure, but also participates in improving the recognition contact efficiency.

[0021] Verification experiment: The validation experiment used the bovine serum albumin affinity materials prepared in Examples 1, 2, 3 and Comparative Example 1 for unified validation; the test standards included bovine serum albumin removal rate, target antibody recovery rate, ligand conjugation density and retention rate after alkali resistance; Bovine serum albumin quantification was performed using a BSA-specific ELISA kit, and the target antibody concentration was determined using a BCA protein quantification kit combined with A. 280 Correction was performed, and the ligand coupling density was calculated by measuring the difference in free thiol groups in the supernatant before and after coupling using the Ellman reagent method. The retention rate after alkali resistance was expressed as the ratio of the BSA removal rate obtained by repeated sampling after soaking in 0.1M NaOH for 24 hours to the initial BSA removal rate. The specific testing procedures are as follows: Each affinity material was packed into a 5 mL column; PBS was used as the equilibration buffer; a mixture containing 2.0 mg / mL recombinant monoclonal antibody and 50 μg / mL bovine serum albumin was used as the loading buffer; the loading volume was 50 mL. Example 1 was equilibrated at 100 cm / h, loaded at 80 cm / h, and regenerated with 0.05 M NaOH; Example 2 was equilibrated at 140 cm / h, loaded at 100 cm / h, and regenerated with 0.10 M NaOH; Example 3 was equilibrated at 180 cm / h, loaded at 120 cm / h, and regenerated with glycine-hydrochloric acid at pH 2.2; Comparative Example 1 used the same flow rate and regeneration conditions as Example 1. Table 1 Performance test data of Examples 1 to 3 and Comparative Example 1: Group Flexible connecting arm form Ligand coupling density (mg / mL resin) BSA removal rate antibody recovery rate Retention rate after alkali resistance Example 1 PEG4 6.1 99.82% 96.1% 99.3% Example 2 (Gly4Ser)2 6.8 99.76% 96.8% 98.9% Example 3 PEG12 6.5 99.58% 95.4% 98.5% Comparative Example 1 none 6.0 96.34% 91.2% 97.8% Examples 1 to 3 all showed high bovine serum albumin removal rates and high target antibody recovery rates, indicating that the combination of binding domain, flexible linker arm, and bonding domain can work together. In Example 2, the flexible peptide linker arm gave a high recovery rate under the test conditions, indicating that the hydrophilic linker structure helps to reduce the non-specific adsorption of the target antibody. Example 1 showed a high retention rate under alkaline regeneration conditions, indicating that the combination of the thioether anchor chain and the PEG4 linker has good cleaning resistance. Comparative Example 1, lacking the flexible linker, had lower BSA removal and antibody recovery rates than the examples, indicating that the flexible linker makes a practical contribution to the spatial extension and effective recognition of the binding domain. Furthermore, when the piperidine concentration in the deprotection solution is below 15%, incomplete deprotection leads to a decrease in the final peptide yield to 68%; above 25%, increased side reactions result in a decrease in purity to below 90%; when the trifluoroacetic acid ratio in the excision solution is below 80%, the efficiency of peptide cleavage from the resin is significantly reduced, with a yield of less than 60%; when the concentration of the L-cysteine ​​termination blocking solution is below 30 mM, incomplete blocking of unclosed reaction sites leads to increased non-specific adsorption, and the target antibody recovery rate decreases to 84.5%; above 80 mM, the blocking effect no longer significantly improves and reagent residues are difficult to wash off; by controlling the above process parameter ranges, an optimal balance between peptide yield and coupling efficiency is achieved.

[0022] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. A bovine serum albumin removal process, characterized in that, This includes an initial synthesis process involving the preparation of bovine serum albumin affinity materials, and a subsequent purification and separation process based on the bovine serum albumin affinity materials to separate the feed solution; The preliminary synthesis process includes: solid-phase immobilized synthesis of functionalized modified peptides for targeted adsorption of bovine serum albumin, surface activation treatment of the solid-phase support, and covalent coupling between the functionalized modified peptides and the activated solid-phase support; the subsequent purification and separation process includes: loading the bovine serum albumin affinity material obtained by covalent coupling into a chromatography column with an outlet and an inlet, establishing an extraction and separation system containing the chromatography column, and sequentially performing neutral liquid equilibration, sample loading and separation by pumping in the feed solution, and alkaline washing and elution steps; In the solid-phase immobilization synthesis of the functionalized modified peptide, the functionalized modified peptide is composed of a binding domain, a flexible linker arm, and a bonding domain covalently linked in sequence; wherein the binding domain is a selected affinity sequence that matches bovine serum albumin; the flexible linker arm is selected from polyethylene glycol segments with a degree of polymerization of 4 to 12, or has the structure (Gly4Ser). n The flexible peptide segment, where n is limited to 2 to 4; The bonding domain is a cysteine ​​residue located at the C-terminus or N-terminus of the polypeptide, and the cysteine ​​residue has a reactive thiol group; the activated solid support is obtained by chemically activating and modifying polysaccharide or resin microspheres with a crosslinking agent, and the surface of the activated solid support has maleimide groups for docking covalent coupling.

2. The bovine serum albumin removal process according to claim 1, characterized in that, The solid-phase immobilization synthesis of the functionalized modified peptide includes the following steps: Fmoc-RinkAmide MBHA resin preloaded with the bonding domain was used as a starting support structure, loaded into a synthesizer, and pre-deprotected using a mixed solution of piperidine and N,N-dimethylformamide with a volume concentration of 15%-25% as a deprotection solution. After deprotection treatment, a mixture containing the target amino acid, HATU and DIEA condensation system is added to the system. The mixture is allowed to stand at room temperature for 0.5-1.5 hours to perform a static coupling reaction to couple the desired short chain substance. The target amino acid is introduced sequentially according to the sequence of the flexible linker and the binding domain to form the polypeptide backbone, and then the polypeptide resin-bound state carrying the protective shell is obtained. After coupling to generate the main chain, a cleavage solution prepared by mixing trifluoroacetic acid, triisopropylsilane and deionized water is added. The volume ratio of trifluoroacetic acid, triisopropylsilane and water is adjusted to within the range of 80-95:2.5-10:2.5-10. The peptide resin-bound state is subjected to continuous room temperature shaking reaction for 1.5-2.5 hours to achieve cleavage from the resin. The free liquid in the reaction tank is collected as crude peptide solution. After collecting the crude polypeptide liquid, it is centrifuged and precipitated by injecting ice-cold ether detergent, and the precipitate is extracted to obtain the primary filter. The primary filter is then purified by gradient elution using a reversed-phase high-performance liquid chromatography device, and then transferred to a freeze dryer for freeze drying to generate solid functionalized modified polypeptide powder.

3. The bovine serum albumin removal process according to claim 2, characterized in that, The surface activation treatment of the solid support includes the following steps: selecting polymethyl methacrylate microspheres with a supporting pore structure and hydroxylated surface as matrix microspheres, injecting them into an epichlorohydrin mixed solution to perform a first round of chemical ring-opening activation reaction, and then introducing a solution with a concentration of... The secondary reaction was initiated by co-dissolving ethylenediamine in an aqueous solution to forcibly introduce amino groups onto the surface of the matrix microspheres; After the amino group is introduced, a mixture containing a crosslinking agent of succinimide-4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid ester is added dropwise to the surface of the reaction tank to provide displacement driving energy. This transforms the surface into a modified surface carrying maleimide groups that can specifically undergo addition reactions with thiol groups. The surface is then washed and filtered to remove the residue, forming a dried and ready-to-use activated solid support.

4. The bovine serum albumin removal process according to claim 3, characterized in that, In the step of the covalent coupling connection: The functionalized modified polypeptide powder obtained in the previous process is transferred into a neutral or slightly alkaline coupling buffer system with a pH of 7.0-7.4 for thorough dissolution and dispersion. The solution is then diluted to a volume of 3-8 mg / mL to establish a polypeptide solution. Simultaneously, a maintenance matrix containing tris(2-carboxyethyl)phosphonic acid hydrochloride at a concentration of 0.5-2.0 mM is added to the coupling buffer system. After establishing the peptide solution pool, the activated solid-phase support is introduced into the liquid phase. The ratio of the sedimentation volume of the solid-phase support to the liquid surface volume of the peptide solution is strictly controlled to be maintained at 1:1.5 to 1:2.

5. During the feeding process, a light-proof incubation device is used to perform physical rotation incubation for 3-5 hours. This induces the free thiol sites of the functionalized modified peptide to contact and collide with the maleimide groups on the surface of the microspheres, and spontaneously forms a fixed irreversible thioether cross-linked structure to complete the synthetic linkage.

5. The bovine serum albumin removal process according to claim 4, characterized in that, After the main process is completed through the light-protected incubation and flipping operation, the reaction termination and quenching of unclosed reaction sites are initiated: Slowly add L-cysteine ​​to the reaction system to terminate the blocking solution with a concentration in the range of 30-80 mM, remove the inverting force and let it stand at room temperature for 0.5-1.5 hours. At the end of the incubation period, open the bottom drain valve and sequentially pump out sodium chloride high-salt brine solution with a molar concentration of 0.5-1.5M, deionized water, and low-boiling alcohol detergent solution with a volume concentration of 15%-25% to perform an alternating countercurrent washing process for filtration and collection of purified affinity material.

6. The bovine serum albumin removal process according to any one of claims 1-5, characterized in that, The chromatography column included in the extraction and separation system is a high-pressure resistant empty chromatography column, and the flow rate of the pretreatment liquid pumped into the chromatography column during the neutral liquid equilibration step is uniformly controlled within the range of 100-180 cm / h. In the subsequent sample loading and separation step, the working flow rate is adjusted and limited to 80-120 cm / h; in the alkaline washing and elution step, the regeneration base solution selected is a sodium hydroxide strong alkaline elution system with a pH of 12.0-13.0 or a glycine-hydrochloric acid buffer solution with a pH of 2.2-2.

8.

7. The bovine serum albumin removal process according to any one of claims 1-6, characterized in that, The subsequent purification and separation process includes the following steps: a. System preparation flow path setup: The bovine serum albumin affinity material is loaded into the chromatography column, the two ends of the chromatography column are closed and connected to the circulation flow path system, and degassing is performed inside the chromatography column; b. Dynamic homogenization equilibrium: Neutral phosphate buffer with pH set in the range of 7.2-7.6 and without the introduction of exogenous factors is drawn by a peristaltic pump and delivered to the flow surface of the chromatography column. The volume of the buffer is limited to 4-8 column bed volumes. The flushing flow rate is set at 100-180 cm / h during the working period. The flushing and replacement treatment continues until the affinity material contained in the pipeline system reaches a physicochemical equilibrium state. c. Target Retention and Breakthrough Separation: The target feed solution containing bovine serum albumin impurities and the components to be refined and purified is pumped in and passed through the chromatography column at a flow rate controlled at 80-120 cm / h; the bovine serum albumin is specifically retained by the affinity material, and the breakthrough liquid is collected to obtain the purified target feed solution. d. In-situ regeneration and elution: When the target feed solution supply is detected and confirmed to have reached the point of complete depletion, the feed valve is immediately interrupted, and the neutral phosphate buffer solution with a volume of 2-4 times the column bed volume is quickly introduced through the parallel backflushing valve to wash away unbound impurities; after the first flush, 3-5 column bed volumes of sodium hydroxide eluent with a molar concentration of 0.05-0.15M are introduced to dissociate the retained bovine serum albumin and discharge it with the liquid flow, restoring the initial binding activity of the affinity material and completing one cycle of process operation.