Gradient density purification membrane and method for purifying target protein

Abstract

The invention discloses a gradient density purification membrane and a method for purifying target protein, and relates to the technical field of biological protein purification. The invention comprises a three-layer structure: an outer layer, which has a pore diameter of 1-5 [mu] m and a thickness ratio of 55-65%, and is used as a support layer of a liquid inlet side; the aperture of the middle layer is 0.1-0.3 mu m, the thickness of the middle layer accounts for 25-35%, and the middle layer is formed by staggering nanofibers; the pore diameter of the inner layer is 0.01-0.05 mu m, the thickness of the inner layer accounts for 10-15%, the inner layer serves as a functional layer on the liquid outlet side, and a SpyCatcher ligand is fixed on the surface of the inner layer; the problems that in the prior art, efficiency is low, loading capacity is low, process steps are tedious, and large-scale application of synthetic biology and the like is limited are solved.

Description

Technical Field

[0001] The present invention relates to the technical field of biological protein purification, in particular to a gradient density purification membrane and a method for purifying target protein. Background Art

[0002] The current mainstream biopurification methods used are affinity chromatography membranes (such as Protein A / Ni-NTA media) and physical retention membranes (such as hollow fiber ultrafiltration membranes). These two methods exhibit inherent contradictions. Affinity chromatography membranes rely on specific ligand-target binding (e.g., antibody-Fc fragment). While offering high selectivity, these membranes suffer from low ligand immobilization density, high protein inactivation rates due to the strong deformation conditions required, and flux degradation due to pore clogging of the chromatographic packing. Physical retention membranes, on the other hand, rely on pore size screening (e.g., 100kDa ultrafiltration membranes). While offering high throughput, they suffer from insufficient precision and are unable to distinguish impurities of similar molecular weight (e.g., HCPs and target proteins). Furthermore, they suffer from large recovery fluctuations, particularly when used with large molecules such as monoclonal antibodies, where shear forces can lead to protein aggregation.

[0003] While improvements are currently focused on affinity tag systems and membrane integration, limitations remain. For example, traditional amino coupling methods lead to clustering of EBs, reducing the number of effective binding sites and resulting in uneven ligand distribution. Chemical elution, on the other hand, destroys ligand-protein bonds, necessitating reactivation of the membrane medium and increasing process complexity. Furthermore, increasing ligand density requires reducing pore size, but this decreases throughput, making it impossible to meet high-throughput requirements.

[0004] The rise of synthetic biology and continuous flow production urgently requires breakthrough purification technologies. Summary of the Invention

[0005] The purpose of the present invention is to provide a gradient density purification membrane and a method for purifying a target protein, so as to solve the problems of low efficiency, low loading capacity, complicated process steps and limitations in large-scale applications such as synthetic biology in the existing technology.

[0006] In order to solve the above problems, the present invention adopts the following technical means: A gradient density purification membrane comprises a three-layer structure: Outer layer: pore size is 1-5μm, thickness accounts for 55-65%, and serves as the support layer on the liquid inlet side; Middle layer: pore size is 0.1-0.3μm, thickness accounts for 25-35%, and is formed by interlaced nanofibers; Inner layer: The pore size is 0.01-0.05μm, and the thickness accounts for 10-15%. It serves as the functional layer on the liquid outlet side, and the SpyCatcher ligand is fixed on its surface.

[0007] Preferably, the inner layer fixes the SpyCatcher ligand via a sulfonylhydrazide bond.

[0008] Furthermore, the gradient density purification membrane is made of polyethersulfone material through RTIPS spinning process, wherein the RTIPS spinning process adopts a double-bath coagulation system, the first bath is a DMAC / H2O mixture, and the second bath is a Na2CO3 / EtOH mixture.

[0009] Furthermore, the inner layer surface is also provided with a bifunctional block polymer intermediary layer, which includes Pluronic F127-b-PGMA, wherein the hydrophobic segment of Pluronic F127 is anchored to the membrane surface, the hydrophilic segment of PGMA exposes the carboxyl group, and SpyCatcher is coupled to the carboxyl group of the PGMA chain by the carbodiimide method.

[0010] Furthermore, the molecular weight of Pluronic F127-b-PGMA in the bifunctional block polymer intermediary layer is 12,000-15,000 g / mol, wherein the molecular weight of the PGMA segment accounts for 30%-40%.

[0011] Furthermore, a method for purifying a target protein comprises the following steps: a) passing a solution of a target protein expressing a SpyTag tag through a gradient density purification membrane according to any one of claims 1 to 5 for clarification and filtration, so that the SpyTag binds to the SpyCatcher on the membrane, thereby capturing the target protein on the membrane; b) Wash the membrane with binding buffer to allow the target protein to bind to the membrane; c) removal of non-specifically bound impurities; d) Activate SpyTag self-cleavage under the synergistic conditions of pH 5.8-6.0 and 45-60°C, causing the target protein to fall off the membrane; e) Collect the detached target protein.

[0012] Furthermore, in step d), the pH is 5.9±0.05 and the temperature is 50±2°C.

[0013] Furthermore, in step a), the target protein solution includes human serum albumin or monoclonal antibody.

[0014] Furthermore, in step d), the recovery rate of the target protein is greater than 90%, and the activity retention rate is greater than 93%.

[0015] Furthermore, in step c), non-specifically bound impurities are removed by eluting with an eluent, wherein the eluent is a PBS buffer solution with a pH of 7.2-7.5.

[0016] The present invention has the following beneficial effects during practical application: Through a three-layer gradient membrane design (macroporous support layer, nano-transition layer, and dense functional layer), the SpyCatcher ligand density was increased to 15.2 mg / cm² while maintaining high flux (NWP>3000 LMH@bar); Utilizing the SpyTag / SpyCatcher bioorthogonal reaction, combined with pH-temperature synergistically triggered self-cleavage (pH 5.8-6.0 and 45-60°C), >98% protein release can be achieved under mild conditions. The double-bath coagulation spinning process and block polymer directional coupling technology overcome the problem of uneven ligand distribution, and the flux attenuation rate is controlled at <8%. DETAILED DESCRIPTION

[0017] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments.

[0018] Therefore, the following detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but rather merely represents selected embodiments of the present invention. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without creative effort are also within the scope of protection of the present invention.

[0019] It should be noted that, in the absence of conflict, the embodiments and features of the embodiments of the present invention can be combined with each other.

[0020] A gradient density purification membrane comprises a three-layer structure: Outer layer: pore size is 1-5μm, thickness accounts for 55-65%, and serves as the support layer on the liquid inlet side; Middle layer: pore size is 0.1-0.3μm, thickness accounts for 25-35%, and is formed by interlaced nanofibers; Inner layer: The pore size is 0.01-0.05μm, and the thickness accounts for 10-15%. It serves as the functional layer on the liquid outlet side, and the SpyCatcher ligand is fixed on its surface.

[0021] Preferably, the inner layer fixes the SpyCatcher ligand via a sulfonylhydrazide bond.

[0022] Furthermore, the gradient density purification membrane is made of polyethersulfone material through RTIPS spinning process, wherein the RTIPS spinning process adopts a double-bath coagulation system, the first bath is a DMAC / H2O mixture, and the second bath is a Na2CO3 / EtOH mixture.

[0023] Furthermore, the inner layer surface is also provided with a bifunctional block polymer intermediary layer, which includes Pluronic F127-b-PGMA, wherein the hydrophobic segment of Pluronic F127 is anchored to the membrane surface, the hydrophilic segment of PGMA exposes the carboxyl group, and SpyCatcher is coupled to the carboxyl group of the PGMA chain by the carbodiimide method.

[0024] Furthermore, the molecular weight of Pluronic F127-b-PGMA in the bifunctional block polymer intermediary layer is 12,000-15,000 g / mol, wherein the molecular weight of the PGMA segment accounts for 30%-40%.

[0025] Furthermore, a method for purifying a target protein comprises the following steps: a) passing a solution of a target protein expressing a SpyTag tag through a gradient density purification membrane according to any one of claims 1 to 5 for clarification and filtration, so that the SpyTag binds to the SpyCatcher on the membrane, thereby capturing the target protein on the membrane; b) Wash the membrane with binding buffer to allow the target protein to bind to the membrane; c) removal of non-specifically bound impurities; d) Activate SpyTag self-cleavage under the synergistic conditions of pH 5.8-6.0 and 45-60°C, causing the target protein to fall off the membrane; e) Collect the detached target protein.

[0026] Furthermore, in step d), the pH is 5.9±0.05 and the temperature is 50±2°C.

[0027] Furthermore, in step a), the target protein solution includes human serum albumin or monoclonal antibody.

[0028] Furthermore, in step d), the recovery rate of the target protein is greater than 90%, and the activity retention rate is greater than 93%.

[0029] Furthermore, in step c), non-specifically bound impurities are removed by elution using an eluent, wherein the eluent is a PBS buffer solution with a pH of 7.2-7.5.

[0030] The following is a detailed description of the effects of the present invention with reference to specific embodiments: Example 1 The SpyTag-HAS fusion protein expressed in yeast was selected as the protein sample, with an expression level of 5.8 g / L and a bacterial solid content of 25%.

[0031] The gradient density PES membrane was prepared by the RTIPS spinning process described above in this application, wherein the spinning speed was 20 L / h; the double-bath coagulation selected DMAC / H20 was 7:3, and 0.5 M Na2CO3 / EtOH was used.

[0032] The three-layer gradient pore sizes of the outer layer, middle layer and inner layer of the prepared purification membrane are 3.2±0.8μm, 0.22±0.05μm and 0.03±0.01μm respectively.

[0033] After the purification membrane is prepared, the membrane is functionalized by directional coupling, wherein 10 g / L SpyCatcher solution and 2 mM sulfosuccinimide cross-linker are pre-activated at 2-8°C.

[0034] The membrane fibers were then immersed in PBS containing F127-b-PGMA block polymer at pH 7.4 for carbodiimide coupling.

[0035] The final functionalized purified membrane ligand density reached 15.4 mg / cm2.

[0036] Purification was performed using the aforementioned method for purifying the target protein. Ultimately, under the conditions of the high surface area of ​​the inner layer of the gradient membrane, an ultra-high ligand density was supported, with a loading capacity of 82.3 g / L, an activity retention rate of 94.2%, an HCP removal rate greater than 99.8%, and a flux decay rate of 6.7% after 50 cycles.

[0037] Example 2 Experimental design Target protein: SpyTag-mAb expressed in CHO cells (titer 4.2 g / L) Release variables: Comparison of the effect of different pH / temperature combinations on activity.

[0038] Functionalized membrane: Gradient membrane as in Example 1 (ligand density 15.2 mg / cm²) Release optimization revealed the optimal release window for mAb: pH 5.8-5.9; 47-50°C (more sensitive than HSA) and the addition of 0.5 mM reducing agent (TCEP) to prevent disulfide bond cleavage.

[0039] Performance Results Electron microscopy confirmed that there was no protein residue on the inner surface of the membrane after release (the initial pore size was maintained).

[0040] Comparative Example Experimental setup Reference object, commercial His-tag purification membrane (Ni-NTA material, pore size 0.45μm) Sample: Same as HSA in Example 1 (with His-tag label instead).

[0041] Purification process The binding phase used PBS with 20 mM imidazole, pH = 8. The elution phase used PBS with 300 mM imidazole, pH = 7.4.

[0042] Performance Comparison The conventional membranes provided in the comparative examples had structural flaws. For example, the uniform pore size resulted in insufficient specific surface area, resulting in low loading capacity. Furthermore, the membranes lacked anti-fouling properties, resulting in pore clogging after 10 runs.

[0043] However, through chemical elution, high concentrations of imidazole will destroy the hydrophobic core of the protein, causing a significant decrease in activity.

[0044] In summary, for the technical solution of the present application, a three-layer gradient design is adopted to expand the binding sites and increase the load capacity; the nanofibers in the middle layer maintain a gap ratio of not less than 65%, ensuring the smooth flow channel and reducing the attenuation rate.

[0045] Moreover, by precisely controlling self-cleavage through pH-temperature triggering, the activity retention rate is greatly improved without the addition of denaturing reagents, and the applicability can also be enhanced.

[0046] Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A gradient density purification membrane, characterized in that It includes three-layer structure: a) Outer layer: pore size is 1-5μm, thickness accounts for 55-65%, and serves as the support layer on the liquid inlet side; b) Middle layer: pore size is 0.1-0.3 μm, thickness accounts for 25-35%, and is formed by interlaced nanofibers; c) Inner layer: The pore size is 0.01-0.05 μm, and the thickness accounts for 10-15%. It serves as the functional layer on the liquid outlet side, and the SpyCatcher ligand is fixed on its surface.

2. The gradient density purification membrane according to claim 1, characterized in that The inner layer immobilizes the SpyCatcher ligand via a sulfonylhydrazide bond.

3. The gradient density purification membrane according to claim 1 or 2, characterized in that The gradient density purification membrane is made of polyethersulfone material through RTIPS spinning process, wherein the RTIPS spinning process adopts a double-bath coagulation system, the first bath is a DMAC / H2O mixed solution, and the second bath is a Na2CO3 / EtOH mixed solution.

4. The gradient density purification membrane according to claim 1, characterized in that The inner layer surface is also provided with a bifunctional block polymer intermediary layer, which includes Pluronic F127-b-PGMA, wherein the hydrophobic segment of Pluronic F127 is anchored on the membrane surface, the hydrophilic segment of PGMA exposes the carboxyl group, and SpyCatcher is coupled to the carboxyl group of the PGMA chain through the carbodiimide method.

5. The gradient density purification membrane according to claim 4, characterized in that The molecular weight of Pluronic F127-b-PGMA in the bifunctional block polymer intermediary layer is 12,000-15,000 g / mol, of which the molecular weight of the PGMA segment accounts for 30%-40%.

6. A method for purifying a target protein, characterized in that: The following steps are involved: a) passing a solution of a target protein expressing a SpyTag tag through a gradient density purification membrane according to any one of claims 1 to 5 for clarification and filtration, so that the SpyTag binds to the SpyCatcher on the membrane, thereby capturing the target protein on the membrane; b) Wash the membrane with binding buffer to allow the target protein to bind to the membrane; c) removal of non-specifically bound impurities; d) Activate SpyTag self-cleavage under the synergistic conditions of pH 5.8-6.0 and 45-60°C, causing the target protein to fall off the membrane; e) Collect the detached target protein.

7. The method according to claim 6, characterized in that In step d), the pH is 5.9±0.05 and the temperature is 50±2°C.

8. The method according to claim 6, characterized in that In step a), the target protein solution includes human serum albumin or monoclonal antibody.

9. The method according to claim 6, characterized in that In step d), the recovery rate of the target protein is greater than 90%, and the activity retention rate is greater than 93%.

10. The method according to claim 6, characterized in that In step c), non-specifically bound impurities are removed by elution using an eluent, wherein the eluent is a PBS buffer solution with a pH of 7.2-7.5.

Images