Nanometer antibody 4C12 and application thereof in promoting purification of human albumin

Abstract

The invention discloses a nano antibody 4C12 and application thereof in promoting purification of human albumin, and belongs to the technical field of polypeptides. The amino acid sequence of the nano antibody comprises an amino acid sequence as shown in SEQ ID NO. 1; and / or an amino acid sequence of a fusion protein with the same function, which is obtained by connecting tag protein to the N terminal and / or C terminal of the amino acid sequence as shown in SEQ ID NO.1. The nano antibody has extremely high affinity with the human albumin, the convective mass transfer advantages of the nano antibody 4C12 and affinity membrane chromatography are combined, and gel filtration is combined, so that efficient, rapid and large-scale preparation of the human albumin is realized.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of polypeptides, in particular to a nanobody 4C12 and application of the same in promoting human albumin purification. BACKGROUND

[0002] Nanobodies, as a kind of single-domain antibody fragments derived from heavy chain antibodies of camelids, have shown great application potential in the fields of biological diagnosis and treatment in recent years due to their small molecular weight (only about 15 kDa), high stability (tolerance to extreme pH, high temperature and organic solvents), strong affinity and excellent penetration, etc. For example, in the fields of tumor targeted therapy, intracellular immunological imaging and virus infection detection, nanobodies and their fusion proteins (such as fusion with recombinant human serum albumin rHSA to prolong half-life) have become research hotspots.

[0003] Recombinant human albumin (rHSA) as a key raw material for clinical treatment, vaccine stabilization and cell culture has strict requirements on purity (≥99.9%), impurity residues (endotoxin ≤1 EU / mg, host cell protein HCP ≤10 ng / mg, residual DNA ≤10 pg / mg) and production efficiency. Traditional rHSA purification relies on “multi-step particle column chromatography” (such as ion exchange, hydrophobic interaction chromatography), and the sample flow rate is usually limited to 1-2 ml / min, resulting in a long processing time of 30 h for 2000 ml of fermentation broth and a total batch processing time of more than 70 h (referring to patent CN102190722A), a low total yield (about 32-65%), and difficulty in meeting the industrialization needs (referring to patent CN118580318A).

[0004] The large-scale production of nanobodies and their fusion proteins is severely dependent on efficient and robust downstream purification processes. At present, the capture step widely used in the industry mainly relies on affinity chromatography technology based on particle media (such as agarose gel, polymer microspheres). Such methods usually use ligands (such as Protein A, specific antigens or tag antibodies) fixed on the surface of porous particles to specifically bind to target proteins, so as to achieve the purpose of separation and purification.

[0005] Although this technology has the advantage of high selectivity, its separation mechanism is fundamentally dependent on the diffusion mass transfer principle: target molecules must pass through the tortuous pore structure inside the particles by slow diffusion process to combine with the ligand on the inner wall of the pore. This mass transfer mechanism has a series of inherent defects: inherent contradiction between flow rate and pressure drop: to improve the diffusion efficiency, small particle size media is often used, but it will cause the column bed resistance to increase significantly, the system back pressure to rise, and thus the operating flow rate to be reduced. Low process efficiency: long cycle time not only increases the time cost of the whole batch, but also increases the risk of degradation or inactivation of target proteins during long processing time. Significant challenges in scale-up: in large-scale production, to maintain a reasonable linear flow rate and residence time, the column bed diameter and height must be increased significantly, resulting in large equipment volume, high column packing difficulty, expensive packing cost, and dramatic increase in plant floor space and supporting facility investment. Limited economic efficiency: low flow rate, long time consumption, and expensive media cost together push up the production cost per unit dose, which is not conducive to the competitiveness and accessibility of the product in the market. In contrast, membrane chromatography technology can fundamentally overcome this bottleneck by relying on convective mass transfer mechanism.

[0006] To solve the mass transfer bottleneck of column chromatography, membrane chromatography technology was introduced into the field of biological macromolecule purification by relying on the advantage of "convective mass transfer" (without relying on molecular diffusion). Its flow rate can reach 5-10 times that of traditional column chromatography, and the equipment occupies small floor space and has low scale-up difficulty. In recent years, nanobodies have been widely used in the field of biological macromolecule purification due to their small molecular weight (about 15 kDa), high affinity (dissociation equilibrium constant KD usually ≤ 10 -9M) and strong stability (pH 2.8-8.5), making it a preferred membrane chromatography ligand. However, the following key gaps still exist in the application of existing technologies in rHSA purification: Insufficient optimization of membrane material pore size and ligand density coordination, rHSA mass transfer requires a membrane substrate with a pore size of ≥0.2 μm to avoid clogging, but existing research (such as Nb9 nanobody immobilized on epoxy-activated sepharose membrane at a density of 12 mg / mL, DOI: 10.1016 / j.pep.2024.106638) has not been optimized for rHSA. Lack of buffer coordination of nanobody conformation and rHSA stability: Nanobody activity depends on specific conformation (such as Q90 site), with activity ≥80% at pH 5.0-8.0, but rHSA is prone to aggregation at pH<6.0, and existing technologies have not addressed the buffer condition conflict between the two. Low economic efficiency of elution and regeneration strategies: For example, BC2-nb membrane can only withstand 5 mM NaOH regeneration for 10-15 times, while AAV8 purification membrane can support 10 mM NaOH treatment for 20 times, but it has not optimized the non-specific adsorption caused by HCP residues in rHSA purification. Insufficient connection between pretreatment and chromatography steps: Conventional pretreatment (such as centrifugation + 0.45 μm filtration) cannot remove 0.1-0.45 μm colloidal particles and sticky HCPs, resulting in a ≥30% decrease in membrane flux. Although new technologies (such as ProteinMPNN + AlphaFold nanobody design) provide support to improve stability, they lack integration with rHSA membrane chromatography scenarios. Therefore, developing an adaptive solution covering the whole chain of membrane material-antibody coupling-process parameters-regeneration strategies (see patent CN101768206B) is the key to breaking through the bottleneck of rHSA purification.

[0007] Therefore, to solve the above major challenges faced by traditional particle medium affinity chromatography in the large-scale purification of nanobodies, there is an urgent need in the art to develop a new chromatography technology platform that can break through the diffusion mass transfer limitation, significantly improve the purification efficiency, and be easily scaled up for industrialization. Such technological innovation will directly facilitate the faster transformation of nanobody drugs from the laboratory to commercial production, meeting the huge demand for such new therapeutic and diagnostic products in the future market. SUMMARY

[0008] To address the aforementioned issues, this application provides a multi-step chromatographic purification process and its application for the large-scale production of high-purity human albumin. This application utilizes 4C12 nanobodies that specifically bind to human albumin to prepare nanobody affinity membranes. Leveraging the "convective mass transfer" mechanism of membrane chromatography, the target protein is rapidly captured, efficiently enriched, and initially purified, significantly improving the chromatography rate and throughput. Subsequently, gel filtration chromatography is used for fine purification of the affinity elution products, effectively removing residual trace protein aggregates, degradation fragments, and small molecule impurities, ultimately obtaining a human albumin product with a purity exceeding 99.9%. This invention combines the high selectivity and high mass transfer efficiency of affinity membrane chromatography with the excellent molecular fractionation capabilities of gel filtration chromatography, successfully constructing a highly efficient purification process route characterized by short chromatography time, high recovery rate, process stability, and easy linear scale-up, significantly improving the overall quality of the product and the efficiency of large-scale production. The albumin includes serum albumin and recombinant albumin; optionally, the serum albumin is human serum albumin; optionally, the recombinant albumin is recombinant human albumin.

[0009] In a first aspect, the present invention provides a nanobody 4C12, wherein the amino acid sequence of the nanobody includes at least one of the following: A1) The amino acid sequence includes the amino acid sequence shown in SEQ ID NO.1; A2) is an amino acid sequence obtained by substituting and / or deleting and / or adding amino acid residues from the amino acid sequence of the nanobody described in A1), which has more than 95% identity with the amino acid sequence of the nanobody described in A1. A3) An amino acid sequence of a fusion protein with the same function obtained by attaching a tag protein to the N-terminus and / or C-terminus of the amino acid sequence described in A1) or A2).

[0010] As will be understood by those skilled in the art, the sequence of SEQ ID NO. 1 of this application can be simply modified, for example, by replacing or deleting some amino acid sites, but the binding characteristics and / or binding affinity of SEQ ID NO. 1 to human albumin in this application are still retained. It is understood that sequences that retain the same human albumin affinity function of the nanobody of this application by simply modifying the sequence of SEQ ID NO. 1 of this application should also be within the protection scope of this application.

[0011] Furthermore, those skilled in the art can also truncate or derive the nanobody based on the SEQ ID NO.1 sequence disclosed in this application to obtain a nanobody with the same affinity for human albumin. This is something that those skilled in the art can reasonably predict, for example, by removing one or more amino acids from the N-terminus or C-terminus of the polypeptide to obtain a polypeptide.

[0012] It should be noted that those skilled in the art can chemically modify the nanobody. The chemical modification can be any one of the following: cyclization modification, acetylation modification, PAS modification, PEG modification, fatty acid modification, albumin modification, nanobody conjugation, tumor homing peptide conjugation, membrane-penetrating peptide conjugation, nanocarrier conjugation, radionuclide conjugation, small molecule compound conjugation, nucleotide conjugation, and protein conjugation. The modification sites include, but are not limited to, N-terminal modification, C-terminal modification, backbone modification, side chain modification, and amino acid modification.

[0013] In a second aspect, the present invention provides a biomaterial comprising at least any one of the following: B1) Contains a nucleic acid molecule encoding the nanobody described above; B2) An expression cassette containing the nucleic acid molecule described in B1); B3) A recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2); B4) Recombinant microorganisms containing nucleic acid molecules of B1), or recombinant microorganisms containing recombinant vectors of expression cassettes of B2), or recombinant microorganisms containing recombinant vectors of B3); B5) Recombinant cells, wherein the recombinant cells contain the nucleic acid molecules of B1), or the recombinant cells contain the expression cassette of B2), or the recombinant cells contain the recombinant vector of B3).

[0014] Further, the nucleic acid molecule described in B1) includes a publicly disclosed nucleic acid molecule encoding the nanobody and / or an optimized nucleic acid molecule according to actual needs. Optionally, the nucleic acid molecule described in B1) includes at least one of the nucleotide sequences shown in SEQ ID NO. 2 or SEQ ID NO. 3. SEQ ID NO. 2 is the nucleotide sequence obtained after sequencing the 4C12 nanobody; SEQ ID NO. 3 is a sequence optimized for Pichia pastoris codon preferences.

[0015] It should be understood that this application does not limit the nucleotide sequence encoding the amino acid sequence of SEQ ID NO. 1. Those skilled in the art will understand that codon optimization for different host cells is a standard practice in the field when expressing and producing the amino acid sequence of SEQ ID NO. 1, and does not affect the affinity of human albumin for the amino acid sequence of SEQ ID NO. 1. Furthermore, even with simple modifications to the amino acid sequence of SEQ ID NO. 1, those skilled in the art can design corresponding nucleotide sequences based on the corresponding amino acid sequences.

[0016] Those skilled in the art will understand that they can also use the isolated nucleic acid molecules described above to prepare corresponding kits, such as kits containing a vector encoding the nucleotide sequence of SEQ ID NO. 1, for preparing host cells expressing the above-mentioned nanobodies. It should be noted that the isolated nucleic acid molecules described above are not limited to this, but are merely illustrative examples and do not constitute a limitation of this application.

[0017] Further, the recombinant vector described in B3) includes at least one of the following: pET series vectors, pBAD vectors, pGEX series vectors, pCAl-n / pCAl-pelB vectors, pPOW3.0 vectors, pPIC series vectors, and pYES2 vectors.

[0018] Furthermore, the recombinant microorganisms described in B4) or the recombinant cells described in B5) include at least one of hamster ovary cells, silkworm ovary cells, Pichia pastoris, Saccharomyces cerevisiae, Escherichia coli, and Bacillus subtilis.

[0019] Optionally, the recombinant microorganism is Pichia pastoris.

[0020] In a third aspect, the present invention provides a method for preparing the nanobody, the method comprising culturing the recombinant microorganism (B4) or the recombinant cell (B5) under suitable culture conditions, and isolating the nanobody or the fusion protein.

[0021] It should be noted that the present invention does not limit the recombinant microorganisms or recombinant cells mentioned above. Any cell that can express exogenous genes through recombinant engineering technology is protected by the present invention.

[0022] In a fourth aspect, the present invention provides a nanobody affinity membrane, wherein the nanobody is coupled to the nanobody.

[0023] Furthermore, the matrix of the nanoantibody affinity membrane includes at least one of an organic polymer membrane, an inorganic / organic composite membrane, and a hydrogel composite membrane.

[0024] Optionally, the organic polymer membrane is made of at least one of regenerated cellulose (RC), polysulfone (PS), polyethersulfone (PES), polypropylene (PP), and nylon; the inorganic / organic composite membrane is made of at least one of cellulose / silicone composite membrane and polymer / ceramic composite membrane; and the hydrogel composite is made of at least one of polyethylene glycol (PEG) and polyacrylamide (PAAm) hydrogel-infused porous membrane.

[0025] Furthermore, the medium that cooperates with the ligand can be selected from materials commonly used by those skilled in the art or other feasible materials, such as agarose, cellulose, cross-linked dextran, polyacrylamide, porous glass beads, etc.

[0026] In one embodiment of the present invention, the matrix is ​​an epoxy-activated polyethersulfone membrane (preferably with a pore size of 0.2-0.45 μm) or an epoxy-activated agarose composite membrane.

[0027] In a fifth aspect, the present invention provides a method for efficiently separating and purifying human albumin, the method comprising the step of purification using the aforementioned nanobody affinity membrane.

[0028] Optionally, the purification steps include at least affinity membrane chromatography and gel filtration chromatography.

[0029] Furthermore, the human albumin includes human serum albumin and recombinant human albumin.

[0030] Furthermore, the method for separating and purifying human albumin specifically includes the following steps: a) Centrifuge the fermentation broth or human blood products containing recombinant human albumin to obtain the supernatant; b) Load the supernatant obtained in step a) onto an affinity membrane with 4C12 nanobody as the ligand, wash with equilibration buffer, elute with elution buffer, and then add neutralization buffer to obtain the first purified product; c) Load the first purified product onto a gel filtration chromatography column, elute with isocratic buffer, collect the recombinant human albumin monomer peak, and obtain the high-purity human albumin final product.

[0031] Further, the amino acid sequence of the nanobody 4C12 described in step b) is shown in SEQ ID NO: 1. In step b), the pH of the equilibration buffer used for affinity chromatography is 7.0–8.0, the pH of the elution buffer is 2.8–3.2, and the pH of the neutralization buffer is 8.0–9.0.

[0032] Further, in step c), the gel filtration chromatography uses Sephadex, Superdex or Sephacryl series chromatography media, and the pH of the isocratic buffer is 7.5–8.5.

[0033] The equilibration buffer is a PBS solution, the elution buffer is a glycine solution, and the neutralization buffer is a Tris-HCl solution.

[0034] Furthermore, the method also includes a step of detecting the purity of purified human albumin, optionally, the detection of human albumin purity is performed by high performance liquid chromatography (HPLC).

[0035] In a sixth aspect, the present invention provides the use of the nanobody, the biomaterial, or the nanobody affinity membrane in any of the following aspects: C1) Applications in the preparation of products for the isolation or purification of human albumin; C2) Applications in the preparation of products for the qualitative or quantitative detection of human albumin; C3) Use in the preparation of pharmaceutical compositions for immunization or treatment.

[0036] The beneficial effects of the present invention include, but are not limited to: The purification efficiency is significantly improved: the loading flow rate of the nano-antibody affinity membrane reaches 8-10 ml / min, and the processing time of 2000 ml of fermentation broth is shortened from 19 h of traditional column chromatography to 2.5 h, with a processing efficiency improvement of 7.6 times. Moreover, when scaling up, only parallel membrane columns are required, and there is no need to adjust the flow rate parameters. High specificity capture capability: This invention utilizes the 4C12 nanobody, which specifically binds to human albumin, as an affinity ligand. This peptide can precisely bind to human albumin, maintaining high affinity and stable dissociation equilibrium even after conserved amino acid substitutions, terminal truncation, or chemical modifications. Affinity chromatography media based on this ligand can directionally capture human albumin from fermentation broth, effectively avoiding non-specific adsorption of host proteins (HCPs), nucleic acids, and other impurities. The purity of human albumin in the preliminary purified product can reach over 95%.

[0037] High process compatibility: The pretreatment and subsequent gel filtration steps can be directly integrated with existing human albumin purification processes without the need for special equipment, facilitating technology transfer and industrial application. Attached Figure Description

[0038] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this invention, illustrate exemplary embodiments of the invention and are used to explain the invention, but do not constitute an undue limitation of the invention. In the drawings: Figure 1 This is an SDS-PAGE electrophoresis image of the purified nanobody in this embodiment of the invention. M: marker; 1: elution.

[0039] Figure 2 This is a graph showing the affinity detection results of the 4C12 nanobody in an embodiment of the present invention.

[0040] Figure 3 This is a ligand density-DBC curve from an embodiment of the present invention; the horizontal axis represents the ligand density (mg / mL) of the nanobody 4C12, and the vertical axis represents the 10% penetration dynamic binding capacity (DBC). 10 %, mg / mL).

[0041] Figure 4 This is a dynamic penetration curve of the 4C12 nanobody affinity membrane in an embodiment of the present invention; the horizontal axis is the sample loading volume (mL), and the vertical axis is the UV280 absorbance value (mAU).

[0042] Figure 5 This is an HPLC chromatogram of purified recombinant human albumin in an embodiment of the present invention. Detailed Implementation

[0043] The present application is described in detail below with reference to the embodiments, but the present application is not limited to these embodiments. Unless otherwise specified, the raw materials and catalysts used in the embodiments of the present application were purchased commercially. Experimental methods without specific conditions are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's recommendations.

[0044] In this application, the fermentation broth containing recombinant human albumin can be obtained by conventional techniques in the art, such as fermentation by genetically engineered bacteria capable of secreting and expressing recombinant human albumin, or by commercial purchase. In some embodiments of the present invention, the fermentation broth is a Pichia pastoris fermentation broth containing recombinant human albumin, prepared with reference to the method in patent CN202410295109.6.

[0045] Example 1: Camel Immunization and Peripheral Blood Lymphocyte Isolation Recombinant human albumin was mixed with Freund's adjuvant and administered to camels via subcutaneous injection at multiple sites in the neck (0.2 mL per site, for a total of 10 sites). Each immunization was administered 2 weeks apart, for a total of 5 immunizations. Blood samples were collected before each immunization, before the 4th immunization, before the 5th immunization, and 2 weeks after the 5th immunization. Serum was obtained after centrifugation and the antibody titer was measured using ELISA (enzyme-linked immunosorbent assay). Successful immunization was defined as a serum titer ≥1:500,000 after the 5th immunization. After the 5th immunization, 100 mL of blood was collected from the jugular vein per animal. PBMCs were separated using the Ficoll-Paque PLUS density gradient centrifugation method (manufacturer: GE Healthcare, catalog number: 17-1440-02): the blood was diluted with an equal volume of sterile PBS and slowly added to the surface of the Ficoll solution (volume ratio 2:1). The mixture was centrifuged at 400×g and 20℃ for 30 min. The middle white membrane layer was aspirated and washed three times with PBS to obtain peripheral blood mononuclear cells (PBMCs).

[0046] Example 2: Phage Library Construction Total RNA was extracted from PBMCs using the Trizol method, and cDNA was synthesized via reverse transcription. Single-domain antibody fragments were amplified by two PCR cycles, digested with restriction endonucleases, and ligated into a phage plasmid. The fragments were then transformed into *E. coli* TG1 competent cells (manufacturer: TransGen, catalog number: CD201-01) using electroporation. The library volume was determined to be 5.15 × 10⁻⁶ cells using a serial dilution method. 9 Forty single clones were randomly selected for PCR identification, and the positive rate was 39 / 40 (97.5%), proving that the library was successfully constructed.

[0047] Example 3: Nanobody Panning Add streptavidin magnetic beads (Thermo Fisher, catalog number: 11205D) and enzymatically hydrolyzed casein (Solarbio, catalog number: C8210-100) to EP tubes, and block with shaking at 37°C for 1 h (100 rpm). Remove the blocking solution. Add recombinant human albumin, and incubate with shaking at 37°C for 1 h to coat the tubes, removing unbound recombinant human albumin. After blocking again with enzymatically hydrolyzed casein, add a phage library for binding. Wash 9 times with PBST (10X PBS (1.37 M NaCl, 27 mM KCl, 100 mM Na2HPO4, 18 mM KH2PO4, pH 7.4) diluted to 1X, then add 0.1% Tween-20, and wash once with PBS (pH 7.4) to thoroughly remove non-specifically bound phages. Add trypsin (manufacturer: Sigma, catalog number: T4799, diluted with PBS, 500 μL), elute at 37°C with shaking for 15 min, and immediately terminate the elution with enzymatic hydrolysis of casein to obtain the elution product. Infect TG1 *E. coli* with the elution product, plate it, and incubate overnight at 37°C. Infect all colonies of *E. coli* in the logarithmic growth phase with M13K07 to expand the culture. Perform three rounds of cyclic screening to enrich the library. Plate the final selected strains on ampicillin-resistant plates, pick single colonies on sterile cell culture plates, and perform ELISA detection. Select qualified positive single colonies and perform gene sequencing to obtain multiple sequences, one of which is named 4C12, whose amino acid sequence is shown in SEQ ID NO. 1 and whose nucleotide sequence is shown in SEQ ID NO. 2.

[0048] Example 4 Expression and purification of nanobodies in Pichia pastoris (1) Pichia pastoris expression of nanobody 4C12 The 4C12 gene was cloned into the yeast vector pPICZαA (purchased from Hunan Fenghui Biotechnology Co., Ltd.) after codon optimization in Pichia pastoris. The optimized nucleotide sequence is shown in SEQ ID NO. 3. Sac I After linearization by enzyme digestion, the cells were electroporated into the X-33 yeast strain (purchased from Thermo Fisher Scientific, catalog number C18000), and single colonies were screened on Zeocin-resistant plates. Secretory expression was induced by routine inoculation, with methanol added every 24 h to a final concentration of 0.5%. After 72 h of induction, the supernatant was collected by centrifugation. SDS-PAGE analysis confirmed correct 4C12 expression.

[0049] (2) Purification of nanobodies expressed in Pichia pastoris The yeast fermentation supernatant was added to a final concentration of 25 mM imidazole. After dissolution, it was filtered through a 0.45 μm filter membrane for loading. The nickel column was equilibrated with loading buffer (50 mM NaH₂PO₄, 300 mM NaCl, 10 mM imidazole, pH adjusted to 8.0 with NaOH). Once the column reached baseline, the sample was loaded, and flow-through was collected. The column was further washed with loading buffer until the baseline returned to level. Elution buffer was then used, and the eluent was collected. The desalting column was equilibrated to four column volumes with desalting buffer. All the eluent was loaded onto the desalting column, and the first peak observed was collected as the final nanobody solution. If the protein concentration was low after desalting, it was concentrated using ultrafiltration. The purity of the collected solutions at each purification stage was determined. SDS-PAGE results are shown below. Figure 1 As shown in the figure, the main band of the purified target protein was clear, and no obvious impurities were observed.

[0050] Example 5: Nanobody Affinity Detection The affinity of the 4C12 nanobody was detected using biomembrane interferometry (BLI). Using an NTA biosensor, the sensor was first equilibrated in analytical buffer for 10 min, then activated in an EDC-NHS mixture for 5 min. The activated sensor was then incubated in nanobody dilution buffer (100 nM) for 10 min and blocked with ethanolamine (1 M, pH 8.5). The blocked sensor was then zeroed by baseline adjustment in buffer solution. Next, the sensor was sequentially immersed in gradient concentrations of recombinant human albumin solutions (7.8 nM, 15.625 nM, 31.25 nM, 62.5 nM, 125 nM, 250 nM, 500 nM) for 5 min to bind, and complete binding curves were generated. Finally, the sensor was transferred to PBS buffer for dissociation for 5 min. Kinetic analysis was performed using a 1:1 binding model, and the results are as follows: Figure 2As shown, the dissociation equilibrium constant KD = 3.67E-10 M indicates that 4C12 has a strong affinity for recombinant human albumin.

[0051] Example 6 Preparation of Nanobody Affinity Membranes Epoxy-activated modified polyethersulfone (PES) membranes (pore size 0.2-0.45 μm, porosity 75%, epoxy group density 20 μmol / g, column size 20 ml, dimensions 25 mm × 40 mm) were used as the solid-phase matrix. First, the membrane column was pretreated: 20 ml of the column was flushed with 1 mM HCl solution pre-cooled at 4°C at a flow rate of 5 ml / min, for a total of 240 ml (12 ml / ml membrane medium), for 48 min. Then, nanobody conjugation was performed: the human albumin-specific nanobody 4C12 obtained in Example 5 (amino acid sequence as shown in SEQ ID NO:1) was dissolved in a conjugation buffer of 0.1 M NaHCO3 + 0.5 M NaCl (pH 8.3) to prepare 200 ml of a 10 mg / ml solution. This solution was circulated through the membrane column at a flow rate of 5 ml / min and reacted with shaking at 4°C and 120 rpm for 16 h. The system was then blocked by circulation with 1 M ethanolamine (pH 8.5) at a flow rate of 3 ml / min for 2 h. Finally, the system was washed alternately for 5 cycles with wash buffer I (0.1 M Acetic Acid + 0.5 M NaCl, pH 4.0) and wash buffer II (0.1 M Tris-HCl + 0.5 M NaCl, pH 8.0) at 100 ml / cycle. The system was then equilibrated with equilibration buffer (10 mM PBS containing 150 mM NaCl, pH 7.4) until the UV280 baseline was stable. The ligand density-DBC curve results are shown below. Figure 3 As shown, Figure 3 The DBC showed that when the ligand density increased from 5 mg / mL to 10.8 mg / mL, 10 When the concentration of DBC increased from 22.1 mg / mL to 52.3 mg / mL, and further increased the ligand density (>12 mg / mL), the concentration of DBC decreased. 10 The concentration tended to stabilize at 53.1 mg / mL, therefore 10.8 mg / mL was the optimal ligand density. The dynamic penetration curve results of the 4C12 nanobody affinity membrane are shown below. Figure 4 As shown, Figure 4 When the sample volume reaches 1800 mL, the UV280 absorbance rises to 10% of the baseline (breakthrough point), corresponding to DBC. 10 The percentage was 52.3 mg / mL; when the sample volume reached 2200 mL, the UV280 absorbance reached 90% of the baseline (saturation point), and the saturated binding capacity was 68.7 mg / mL.

[0052] Example 7: Affinity membrane chromatography purification of recombinant human albumin Take 2000 ml of Pichia pastoris recombinant human serum albumin (rHSA) fermentation broth, centrifuge at 8000 rpm for 20 min at 4℃, and collect 1850 ml of supernatant; add 27.75 ml of 100 mM sodium octanoate stock solution to the supernatant to make a final concentration of 15 mM, stir well, adjust the pH to 6.0 with 1 M HCl, heat in a 65℃ water bath for 45 min, and then quickly cool to 22℃ in an ice bath; centrifuge the treated solution at 12000 rpm for 15 min at 4℃, and collect 1780 ml of supernatant. Repeat the above heat denaturation-centrifugation operation once more to finally obtain 1720 ml of supernatant; finally, filter in series under constant pressure of 0.1 MPa at 4℃ using a 1.2 μm glass fiber pre-filtration membrane (Millipore FG120) and a 0.22 μm PES depth filtration membrane (Pall DFF02210) to obtain a clear sample.

[0053] The nanobody affinity membrane prepared in Example 6 was packed into an XK50 / 60 chromatography column (Cytiva, column bed size: 26 mm × 200 mm, bed volume approximately 107 ml). First, the column was equilibrated with equilibration buffer (10 mM PBS containing 150 mM NaCl, pH 7.4) at a flow rate of 10 ml / min for 30 min until the UV280 absorption baseline stabilized (approximately 0.2 mAU). Then, 1700 ml of pretreated clarified filtrate was loaded at a flow rate of 9 ml / min for a total loading time of 189 min, during which the UV280 peak reached 12.5 mAU. After loading, the column was washed with equilibration buffer at the same flow rate for 40 min until the UV280 signal returned to baseline. Elution was performed using elution buffer (0.1 M glycine-HCl, containing 0.5 M NaCl, pH 2.8) at a flow rate of 5 ml / min. A total of 420 ml of eluent was collected in fractions, and 200 μl of neutralization buffer (1 M Tris-HCl, pH 8.0) was immediately added to each fraction and gently mixed to restore the pH to neutral. The fractions were then combined to obtain the first purified product.

[0054] After elution, the membrane column was immediately flushed with regeneration buffer 1 (10 mM NaOH) at a flow rate of 3 ml / min for 30 min to thoroughly remove residual impurities. Subsequently, it was flushed with equilibration buffer (10 mM PBS containing 150 mM NaCl, pH 7.4) until the pH of the effluent stabilized at 7.4, completing the regeneration process. The membrane column could then be directly used for the next batch of purification. This regeneration process is stable and reliable. After 20 consecutive cycles, the membrane column's dynamic binding capacity (DBC) remained stable. 10The concentration (%) remained at 48.5 mg / mL, indicating good reusability and stability.

[0055] Example 8: Gel filtration chromatography purification of recombinant human albumin The first purified product obtained in Example 7 was concentrated by centrifugation at 4°C and 4000×g using an ultrafiltration centrifuge tube (molecular cutoff 10 kDa, purchased from Millipore, catalog number: UFC901024). The volume and protein concentration were monitored every 15 min until the protein concentration stabilized at 50±2 mg / mL (concentrated volume approximately 103 mL). During concentration, a small amount of 50 mM Tris-HCl + 0.1 M NaCl buffer (pH 7.6) was added to prevent localized high concentrations that could lead to protein denaturation. After concentration, the sample was filtered using a 0.22 μm PVDF membrane (purchased from Millipore, catalog number: SLGV033RB) in a sterile environment. The clear filtrate was collected and stored at 4°C for future loading.

[0056] This embodiment uses Sephadex G100 gel permeation media (purchased from Cytiva, catalog number: 17001001) to pack a glass chromatography column (column size: 50 mm × 520 mm, column bed volume approximately 1021 ml). Using Superdex 75 series media can further reduce elution time by 15% and improve monomer peak resolution to 1.8. The chromatography column was connected to an AKTA pure250 system (Cytiva), with the detection wavelength set to 280 nm, conductivity monitoring range 0-30 mS / cm, and column pressure limit 0.15 MPa. At least three column volumes (3063 ml) were equilibrated using 50 mM Tris-HCl + 0.1 M NaCl buffer (pH 7.6) at a linear flow rate of 20 cm / h. The concentrated sample was loaded at 5% of the column volume (approximately 51 ml), followed by constant flow elution with the same buffer at a maintained flow rate of 20 cm / h. Based on the ultraviolet absorption (280 nm) monitoring results, the main peak eluted fraction was collected.

[0057] The collected components were combined, desalted using an ultrafiltration membrane (10 kDa cutoff), and replaced with water for injection. Subsequently, they were freeze-dried to obtain high-purity recombinant human albumin lyophilized powder.

[0058] Example 9 Purity and Yield Analysis (1) Purity analysis of recombinant human albumin The purity of the recombinant human albumin lyophilized powder obtained in Example 8 was analyzed by high performance liquid chromatography (HPLC). An Agilent 1260 Infinity II HPLC system equipped with a TSKgel G3000SWxl column (7.8 mm × 30 cm, 5 μm, Tosoh Bioscience, catalog number: 08541) was used. Phosphate-buffered saline (PBS, 10 mM sodium phosphate, 150 mM NaCl, pH 7.4) was used as the mobile phase, with isocratic elution at a flow rate of 0.8 mL / min, a column temperature maintained at 25 ± 1°C, a detection wavelength of 280 nm, an injection volume of 20 μL, and a run time of 30 min. The sample was reconstituted with ultrapure water to a concentration of approximately 5 mg / mL, filtered through a 0.22 μm microporous membrane, and then injected. Data acquisition and analysis were performed using Agilent OpenLab CDS software, and purity was calculated using the external standard peak area normalization method. After three independent and repeated determinations, the average percentage of the main peak area of ​​the sample was 99.92% ± 0.02% (n=3), with an RSD of 0.02%, indicating repeatability. A typical chromatogram can be found in [reference needed]. Figure 5 .

[0059] (2) Calculation of recombinant human albumin yield The protein concentration at each purification step was quantified using the BCA (Bicinchoninic Acid) method, and the overall yield was calculated accordingly. The Pierce™ BCA protein assay kit (Thermo Fisher Scientific, catalog number: 23225) was used. The procedure was strictly followed according to the instructions: First, the BSA standard was diluted with PBS buffer to a series of concentrations of 0, 125, 250, 500, 750, 1000, and 1500 μg / mL to construct a standard curve (R²). 2 >0.9992). All test samples were appropriately diluted to ensure their concentration fell within the linear range of the standard curve. Each sample was tested in triplicate, with 25 μL of standard or diluted sample added to each well, followed by 200 μL of BCA working solution. After incubating the 96-well plate at 37°C for 30 min, the absorbance was measured at 562 nm using a BioTek Synergy H1 microplate reader. The sample concentration was calculated based on the standard curve. The results are shown in Table 1. Starting from the total protein content of the pretreated supernatant (Step 0), the overall purification yield after affinity chromatography (Step 1) and gel chromatography (Step 2) was 79.6% ± 1% (n=3), a 2.49-fold increase compared to the traditional process of 32% (CN102190722A2), further validating the high efficiency of the proposed process.

[0060] Table 1 Recovery rate of each step

[0061] Note: Total yield = Step0 × Step1 × Step2 = 90% × 94.3% × 93.8% ≈ 80.1%, actual value 79.6% ± 1%. The deviation is due to trace losses during ultrafiltration concentration and sample transfer, which is within the normal fluctuation range of industrial production.

[0062] Step 0 represents the recovery rate of the total protein in the supernatant after centrifugation and filtration of the initial fermentation broth relative to the total protein in the fermentation broth before centrifugation. Steps 1 and 2 represent the recovery rates of the total target protein in the eluent of each chromatography step relative to the total target protein before loading in the previous step. The total protein in all steps was calculated as concentration (measured by BCA method) × actual sample volume, and volume correction was performed during sample dilution. The yields in the table are the average of three independent experiments (n=3). No SD is indicated because the SD of each step is <1.5%, and the overall yield SD is 1.1% (derived from the cumulative calculation of deviations from each step).

[0063] Example 10 Safety Index Testing (1) Endotoxin detection The endotoxin content of the final product was determined using the dynamic turbidimetric endotoxin assay. A dynamic turbidimetric endotoxin assay kit (Lonza, catalog number: N588) was used. First, the recombinant human albumin lyophilized powder obtained in Example 8 was dissolved and diluted to a concentration of 2 mg / mL using water for endotoxin testing (BET water, Lonza, catalog number: W50-100). The endotoxin working standard (CSE, Lonza, catalog number: E0005) was diluted to a series of concentrations of 0.005, 0.05, 0.5, and 5 EU / mL using the same bottle of BET water to prepare a standard curve. In a pyrogen-free 96-well plate, 100 μL of the standard, sample solution, or negative control (BET water) was added to each well, with each sample performed in duplicate. Subsequently, 100 μL of Limulus Amebocyte Lysate (LAL) reagent was added to each well, and after gentle shaking to mix, the mixture was immediately placed in a rapid microbial detection system (or an ELISA reader with dynamic turbidimetric function, such as the Charles River Endosafe® Endotoxin Detection System). The absorbance of the reaction system at 405 nm was continuously monitored at 37.0°C ± 0.2°C for 70 minutes. The instrument software automatically calculated the absorbance based on the standard curve (R...). 2The endotoxin concentration in the sample was calculated using the formula (=0.9985). An interference test was performed according to the requirements of General Chapter 1143 of the Pharmacopoeia of the People's Republic of China (2020 edition): the endotoxin standard (0.5 EU / mL) was mixed with a 2 mg / mL sample solution, and the recovery rate was determined to be 100% (meeting the pharmacopoeia requirements), proving that the sample matrix did not interfere with the detection. The final result was the average of three independent tests, and the endotoxin content in recombinant human albumin was measured to be 0.35 ± 0.04 EU / mg (n=3, RSD=5.2%), which is far below the limit requirement of "less than 1 EU / mg" stipulated in General Chapter 1143 of the Pharmacopoeia of the People's Republic of China (2020 edition), proving that the final product meets safety standards.

[0064] (2) Detection of residual host cell protein (HCP) Enzyme-linked immunosorbent assay (ELISA) was used for the determination. A commercially available assay kit specifically targeting *Pichia pastoris* (manufacturer: Cygnus Technologies, catalog number: F550) with a detection range of 1-100 ng / mL was used. The HCP standard used in this kit was purified HCP from the same *Pichia pastoris* strain as used in this experiment, and Western blotting confirmed its specificity in identifying the HCP of this strain (no cross-reactivity). Simultaneously, a spiked recovery test was performed on a 5 mg / mL rHSA sample (spiking concentration 20 ng / mL), with a recovery rate of 99.2%, verifying no matrix interference. Before detection, the rHSA sample was diluted to 5 mg / mL to pre-verify that rHSA at this concentration did not interfere with the detection. A standard curve (R0) was plotted for 1-100 ng / mL. 2 The calculation using (=0.9978) showed that the residual HCP in the final product was 3.5 ± 0.3 ng / mg rHSA (n=3, RSD=6.8%). This value is significantly lower than the limit of "HCP residue ≤10 ng / mg" stipulated in the National Medical Products Administration's "Technical Guidelines for Quality Control of Human Recombinant DNA Products" (2020), fully demonstrating that this purification process can efficiently remove Pichia pastoris host proteins.

[0065] (3) Residual DNA detection Real-time quantitative PCR (qPCR) was used to target the conserved GAPDH gene in the Pichia pastoris genome. First, DNA enrichment and purification of 100 mg of the final product sample was performed using a DNA extraction kit (Qiagen, catalog number 51304). The sample was dissolved in 10 mL of BET water, and 1 mL was used for DNA extraction. The extraction efficiency was verified by a spiked assay: adding 10 pg of Pichia pastoris genomic DNA to the sample solution resulted in a qPCR recovery rate of 94.5%, and the final residual DNA content was corrected for extraction efficiency. Amplification was then performed using a specific qPCR detection kit (Thermo Fisher Scientific, catalog number A24554). A standard curve (R0) was constructed for 1-10000 pg / mL. 2 =0.9992), and the melting curve of the amplified product was analyzed (single peak, Tm=85.5℃) to ensure the specificity and accuracy of the detection. The final calculated residual DNA content was 3.2 ± 0.3 pg / mg rHSA (n=3, RSD=7.1%). This result meets the standard of "residual DNA ≤10 pg / mg" in General Chapter 3407 of the Pharmacopoeia of the People's Republic of China (2020 Edition), further verifying the effectiveness of this process in removing nucleic acid impurities, and the final product's safety meets pharmaceutical requirements. Comparative Example 1: Preparation of Nanobody Affinity Chromatography Medium The human albumin-specific nanobody 4C12 obtained in Example 5 was dissolved in a coupling solution (0.1M NaHCO3 + 0.5M NaCl, pH 8.3) to prepare a nanobody solution with a concentration of 6 mg / ml. 4 ml of CNBr-activated Bestarose 4B medium (purchased from BorgLyn (Shanghai) Biotechnology Co., Ltd.) was placed in a sintered glass funnel and washed with pre-cooled 1 mM HCl at 4°C for 30 min, using approximately 240 ml in multiple washes. The washed medium was diluted to 6 ml with 1 mM HCl, and an equal volume was mixed with the nanobody solution. The mixture was incubated overnight at 4°C on a shaker. The coupling supernatant was removed, and blocking buffer (0.1M Tris-HCl, pH 8.0) was added. The mixture was blocked at room temperature for 2 h. Wash with washing buffer 1 (0.1M HAC + 0.5M NaCl, pH 4.0) and washing buffer 2 (0.1M Tris-HCl + 0.5M NaCl, pH 8.0) for 5 cycles, each time using 5 times the volume of the medium. After washing with PBS (pH 7.4), store at 4°C for later use.

[0066] Comparative Example 2: Affinity chromatography purification of recombinant human albumin Take 2000 ml of yeast-expressed recombinant human albumin fermentation broth and centrifuge at 8000 rpm (approximately 10,000 × g) for 20 minutes at 4℃, and collect the supernatant. Add 100 mM sodium octanoate stock solution to the supernatant to achieve a final concentration of 15 mM, and slowly stir to mix. Adjust the pH to 6.0 ± 0.1 with 1 M HCl solution, then heat in a water bath at 65.0 ± 0.5℃ for 45 minutes, and quickly cool to below 25℃ in an ice bath. Centrifuge at 12000 rpm (approximately 20,000 × g) for 15 minutes at 4℃, collect the supernatant, and repeat the heat denaturation and centrifugation process once. Filter the final supernatant through a 0.45 μm PES membrane, and collect 1750 ml of the clarified sample after pretreatment.

[0067] The nanobody affinity chromatography medium prepared in Comparative Example 1 was packed into an XK50 / 60 column (purchased from Cytiva, column bed size: 26 mm × 200 mm, bed volume: 107 ml) and operated using an AKTA pure 150 system. At least 3 column volumes (321 ml) were equilibrated with equilibration buffer (10 mM sodium phosphate, 150 mM NaCl, pH 7.4) at a flow rate of 1.5 ml / min until the UV absorption baseline (280 nm) stabilized. All pretreated samples were loaded at a flow rate of 1.5 ml / min. After loading, the sample was washed with equilibration buffer until the UV280 signal returned to baseline. Elution buffer (0.1 M glycine-HCl, pH 2.8) was used to elute 4 column volumes (428 ml) at a flow rate of 1.0 ml / min, and 2 ml of eluent was collected in fractions. Immediately add 200 μl of neutralization buffer (1 M Tris-HCl, pH 8.0) to each elution fraction, mix gently to restore pH to neutral, and combine to obtain the first purified product.

[0068] Comparative Example 3: Gel filtration chromatography purification of recombinant human albumin The first purified product obtained from Comparative Example 2 was concentrated by centrifugation at 4000×g in an ultrafiltration centrifuge tube (molecular cutoff 10 kDa, purchased from Millipore, catalog number: UFC901024) at 4℃. The volume and protein concentration were monitored every 15 min until the protein concentration stabilized at 50±2 mg / mL (the concentrated volume was approximately 103 mL). During this period, a small amount of 50 mM Tris-HCl + 0.1 M NaCl buffer (pH 7.6) was added to avoid local high concentrations that could lead to protein denaturation. After concentration, the sample was filtered through a 0.22 μm PVDF membrane (purchased from Millipore, catalog number: SLGV033RB) in a sterile environment. The clear filtrate was collected and stored at 4℃ for later loading.

[0069] A glass chromatography column (50 mm × 520 mm, approximately 1021 ml bed volume) was packed with Sephadex G100 gel permeate media (Cytiva, catalog number 17001001). The column was connected to an AKTA pure 250 system (Cytiva), with the detection wavelength set to 280 nm, conductivity monitoring range of 0–30 mS / cm, and column pressure limit of 0.15 MPa. At least three column volumes (3063 ml) were equilibrated with 50 mM Tris-HCl + 0.1 M NaCl buffer (pH 7.6) at a linear flow rate of 20 cm / h. The concentrated sample was loaded at 5% of the column volume (approximately 51 ml), followed by constant flow elution with the same buffer at a rate of 20 cm / h. The main peak eluted fraction was collected based on UV absorption (280 nm) monitoring.

[0070] The collected components were combined, desalted using an ultrafiltration membrane (10 kDa cutoff), and replaced with water for injection. Subsequently, they were freeze-dried to obtain high-purity recombinant human albumin lyophilized powder. The test results are shown in Table 2.

[0071] Table 2 Comparative Recombinant Human Albumin Detection Data

[0072] Note: To ensure data comparability, the methods, instruments, reagents, standard curve preparation, and calculation methods used for the detection of purity, yield, and various safety indicators (endotoxin, HCP, residual DNA) of the comparative sample are completely consistent with those described in Examples 9 and 10 of this invention.

[0073] By comparison, the nanobody affinity membrane chromatography method, through the convective mass transfer advantages of membrane materials, shortens the longest "affinity chromatography stage" from 31.53 hours to 3.88 hours, reducing the overall production cycle by nearly 40%. Furthermore, during scale-up, membrane chromatography can maintain a high flow rate through "parallel membrane columns," while particle columns require larger column diameters, leading to a decrease in flow rate. The cycle difference between the two methods becomes more significant, making it more suitable for the needs of continuous industrial production. Specific data are shown in Table 3.

[0074] Table 3 Comparison of purification time between the examples and the comparative examples.

[0075] The above description is merely an embodiment of the present invention, and the scope of protection of the present invention is not limited to these specific embodiments, but is determined by the claims of the present invention. For those skilled in the art, the present invention can have various modifications and variations. Any modifications, equivalent substitutions, improvements, etc., made within the technical concept and principle of the present invention should be included within the scope of protection of the present invention.

Claims

1. A nanobody, characterized in that, The nanobody is a human albumin-specific nanobody, and the amino acid sequence of the nanobody includes at least one of the following: A1) The amino acid sequence includes the amino acid sequence shown in SEQ ID NO.1; A2) An amino acid sequence of a fusion protein with the same function obtained by linking a tag protein to the N-terminus and / or C-terminus of the amino acid sequence described in A1).

2. A biomaterial, characterized in that, The biomaterial includes at least one of the following: B1) Contains a nucleic acid molecule encoding the nanobody of claim 1; B2) An expression cassette containing the nucleic acid molecule described in B1); B3) A recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2); B4) Recombinant microorganisms containing nucleic acid molecules of B1), or recombinant microorganisms containing recombinant vectors of expression cassettes of B2), or recombinant microorganisms containing recombinant vectors of B3); B5) Recombinant cells, wherein the recombinant cells contain the nucleic acid molecules of B1), or the recombinant cells contain the expression cassette of B2), or the recombinant cells contain the recombinant vector of B3).

3. The biomaterial according to claim 2, characterized in that, The nucleotide sequence of the nucleic acid molecule described in B1) includes at least the nucleotide sequence shown in SEQ ID NO.2 or SEQ ID NO.

3.

4. The biomaterial according to claim 2, characterized in that, The recombinant microorganisms described in B4) or the recombinant cells described in B5) include at least one of hamster ovary cells, silkworm ovary cells, Pichia pastoris, Saccharomyces cerevisiae, Escherichia coli, and Bacillus subtilis.

5. A method for preparing the nanobody according to claim 1, characterized in that, The method includes the steps of culturing the recombinant microorganism (B4) or the recombinant cell (B5) in the biomaterial of claim 2 under suitable culture conditions, and isolating the nanobody.

6. A nanobody affinity membrane, characterized in that, The nanobody affinity membrane is coupled with the nanobody of claim 1.

7. The nanobody affinity membrane according to claim 6, characterized in that, The matrix of the nanoantibody affinity membrane includes at least one of organic polymer membranes, inorganic / organic composite membranes, and hydrogel composite membranes.

8. A method for efficiently separating and purifying human albumin, characterized in that, The method includes a purification step using the nanobody of claim 1 or the nanobody affinity membrane of claim 6 or 7.

9. The method according to claim 8, characterized in that, The purification steps include at least affinity membrane chromatography and gel filtration chromatography.

10. The use of the nanobody of claim 1, or the biomaterial of any one of claims 2-4, or the nanobody affinity membrane of claim 6 or 7, in any of the following aspects: C1) Applications in the preparation of products for the isolation or purification of human albumin; C2) Applications in the preparation of products for the qualitative or quantitative detection of human albumin; C3) Use in the preparation of pharmaceutical compositions for immunization or treatment.

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