Nanobody 4c12 and use in facilitating purification of human albumin

Abstract

The application discloses a kind of nanobody 4C12 and the application of promoting human albumin purification, belong to polypeptide technical field.The amino acid sequence of the nanobody includes the amino acid sequence as shown in SEQ ID NO.1;And / or the amino acid sequence of the fusion protein with same function obtained by connecting tag protein at the N terminal and / or C terminal of the amino acid sequence as shown in SEQ ID NO.1.The nanobody has very high affinity with human albumin, the nanobody 4C12 of the application is combined with the countercurrent mass transfer advantage of affinity membrane chromatography, and then combined with gel filtration, to realize the efficient, rapid, large-scale preparation of human albumin.

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 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 force the operating flow rate to decrease. Low process efficiency: long cycle time not only increases the time cost of the entire 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 significantly increased, 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 a small floor area 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] In order to solve the above problems, a multi-step chromatography purification process and application for large-scale production of high-purity human albumin are provided. In the application, a nanobody 4C12 specifically binding to human albumin is used to prepare a nanobody affinity membrane. The membrane chromatography realizes rapid capture, efficient enrichment and preliminary purification of the target protein by virtue of the "counter-current mass transfer" mechanism, significantly improving the chromatography rate and processing flux. Subsequently, gel filtration chromatography is used to finely purify the affinity elution product, effectively removing residual trace protein aggregates, degradation fragments and small molecule impurities, and finally obtaining a human albumin product with a purity higher than 99.9%. The application combines the high selectivity and high mass transfer efficiency of affinity membrane chromatography with the excellent molecular classification ability of gel filtration chromatography, successfully constructing an efficient purification process route with short chromatography time, high recovery rate, stable process and easy linear amplification, significantly improving the overall quality and large-scale production efficiency of the product. The albumin includes serum albumin and recombinant albumin, and the serum albumin is human serum albumin, and the recombinant albumin is recombinant human albumin.

[0009] In the first aspect of the application, a nanobody 4C12 is provided, and the amino acid sequence of the nanobody includes at least one of the following:

[0010] A1) the amino acid sequence includes the amino acid sequence shown in SEQ ID NO. 1;

[0011] A2) an amino acid sequence obtained by substitution, deletion and / or addition of amino acid residues in the amino acid sequence of the nanobody of A1), and having 95% or more identity with the amino acid sequence of the nanobody of A1);

[0012] A3) an amino acid sequence of a fusion protein with the same function obtained by connecting a tag protein to the N-terminus and / or C-terminus of the amino acid sequence of A1) or A2).

[0013] For those skilled in the art, it can be understood that simple transformations can be made to the sequence of SEQ ID NO. 1 in the application scheme, such as replacing or deleting part of the amino acid sites, but still retaining the properties and / or binding affinity of SEQ ID NO. 1 in the application scheme to bind to human albumin. It can be understood that the sequence obtained by simple transformation based on the sequence of SEQ ID NO. 1 in the application and retaining the same human albumin affinity function of the nanobody in the application should also be within the protection scope of the application scheme.

[0014] In addition, for those skilled in the art, the nanobodies based on the SEQ ID NO. 1 sequence disclosed in the application scheme can also be truncated or derived to obtain nanobodies with the same human albumin affinity effect, which can be reasonably predicted by those skilled in the art, for example: polypeptides obtained by cutting one or more amino acids from the N- or C-terminus of the polypeptide.

[0015] It should be noted that those skilled in the art can chemically modify the nanobodies, which are any one of cyclization modification, acetylation modification, PAS modification, PEG modification, fatty acid modification, albumin modification, nanobody coupling, tumor homing peptide coupling, transmembrane peptide coupling, nanocarrier coupling, radionuclide coupling, small molecule compound coupling, nucleotide coupling, protein coupling, modification sites include but are not limited to N-terminal modification, C-terminal modification, backbone modification, side chain modification, amino acid modification, etc.

[0016] In a second aspect of the application, a biological material is provided, which comprises at least any one of the following:

[0017] B1) contains a nucleic acid molecule encoding the nanobody;

[0018] B2) contains an expression cassette of B1) the nucleic acid molecule;

[0019] B3) contains a recombinant vector of B1) the nucleic acid molecule, or a recombinant vector containing B2) the expression cassette;

[0020] B4) contains a recombinant microorganism containing B1) the nucleic acid molecule, or a recombinant microorganism containing B2) the expression cassette of the recombinant vector, or a recombinant microorganism containing B3) the recombinant vector;

[0021] B5) recombinant cells containing B1) nucleic acid molecules, or recombinant cells containing B2) the expression cassette of the recombinant vector, or recombinant cells containing B3) the recombinant vector.

[0022] Further, the nucleic acid molecule in B1) includes the nucleic acid molecule encoding the nanobody disclosed and / or optimized according to actual needs, and optionally, the nucleic acid molecule in B1) includes at least one of the nucleotide sequences shown as SEQ ID NO. 2 or SEQ ID NO. 3. Wherein, SEQ ID NO. 2 is a nucleotide sequence obtained after sequencing the nanobody 4C12; SEQ ID NO. 3 is a sequence optimized for Pichia pastoris codon bias.

[0023] It should be understood that the nucleotide sequence encoding the amino acid sequence of SEQ ID NO. 1 is not limited in the present application, and those skilled in the art can understand that, in the expression of the amino acid sequence of SEQ ID NO. 1, codon optimization for different host cells is a routine operation in the art, and does not affect the human albumin affinity effect of the amino acid sequence of SEQ ID NO. 1. In addition, for the case of simple change of the amino acid sequence of SEQ ID NO. 1, those skilled in the art can also design the corresponding nucleotide sequence according to the corresponding amino acid sequence.

[0024] Those skilled in the art can understand that those skilled in the art can also use the above-mentioned isolated nucleic acid molecules to make corresponding kits, for example, the kit contains a vector containing the nucleotide sequence encoding SEQ ID NO. 1, which is used to prepare host cells expressing the above-mentioned nanobodies. It should be noted that the above-mentioned isolated nucleic acid molecules are not limited to this, but are only exemplary examples, and do not constitute the limitation of the present application.

[0025] Further, B3) the recombinant vector comprises at least one of pET series vectors, pBAD vectors, pGEX series vectors, pCAl-n / pCAl-pelB vectors, pPOW3.0 vectors, pPIC series vectors, pYES2 vectors.

[0026] Further, the recombinant microorganism in B4) or the recombinant cell in B5) comprises at least one of hamster ovary cells, silkworm ovary cells, Pichia pastoris, Saccharomyces cerevisiae, Escherichia coli, Bacillus subtilis.

[0027] Optionally, the recombinant microorganism is Pichia pastoris.

[0028] In a third aspect of the present application, a method for preparing the nanobody is provided, which comprises the steps of culturing the recombinant microorganism in B4) or the recombinant cell in B5) under suitable culture conditions, and isolating the nanobody or the fusion protein.

[0029] It should be noted that the present application does not limit the recombinant microorganism or the recombinant cell, and any cell that can express foreign genes through recombinant engineering technology is within the protection scope of the present application.

[0030] In a fourth aspect of the present application, a nanobody affinity membrane is provided, wherein the nanobody affinity membrane is coupled with the nanobody.

[0031] Further, the matrix of the nanobody affinity membrane comprises at least one of an organic polymer membrane, an inorganic / organic composite membrane, and a hydrogel composite membrane.

[0032] Optionally, the material of the organic polymer membrane comprises at least one of regenerated cellulose (RC), polysulfone (PS), polyethersulfone (PES), polypropylene (PP), and nylon; the material of the inorganic / organic composite membrane comprises at least one of cellulose / silica gel composite membrane and polymer / ceramic composite membrane; and the hydrogel composite comprises at least one of polyethylene glycol (PEG) and polyacrylamide (PAAm) hydrogel perfused porous membrane.

[0033] Further, the ligand-combined medium 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.

[0034] In an embodiment of the present application, 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.

[0035] In a fifth aspect of the present application, a method for efficiently separating and purifying human albumin is provided, which comprises the step of purifying using the nanobody affinity membrane.

[0036] Optionally, the purification step comprises at least affinity membrane chromatography and gel filtration chromatography.

[0037] Further, the human albumin comprises human serum albumin and recombinant human albumin.

[0038] Further, the method for separating and purifying human albumin specifically comprises the following operations:

[0039] a) centrifuging a fermentation broth containing recombinant human albumin or a human blood product to obtain a supernatant;

[0040] b) loading the supernatant obtained in step a) onto an affinity membrane with nanobody 4C12 as the ligand, washing with an equilibration buffer, eluting with an elution buffer, and then adding a neutralization buffer to obtain a first purified product;

[0041] c) loading the first purified product onto a gel filtration chromatography column, eluting with an isocratic buffer, and collecting a recombinant human albumin monomer peak to obtain a high-purity human albumin final product.

[0042] Further, the amino acid sequence of the nanobody 4C12 in step b) is shown in SEQ ID NO: 1. In step b), the pH of the equilibration buffer used in the 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.

[0043] Further, in step c), the gel filtration chromatography employs Sephadex, Superdex or Sephacryl series chromatography medium, and the pH of the isocratic buffer is 7.5-8.5.

[0044] The equilibrium buffer is a PBS solution, the elution buffer is a glycine solution, and the neutralization buffer is a Tris-HCl solution.

[0045] Further, the method further comprises a step of detecting the purity of the purified human albumin, and optionally, the detection of the purity of the human albumin is by high performance liquid chromatography (HPLC).

[0046] In a sixth aspect of the present application, the nanobody or the biomaterial or the nanobody affinity membrane is applied in any of the following aspects:

[0047] C1) application in the aspect of preparing a product for separating or purifying human albumin;

[0048] C2) application in the aspect of preparing a product for qualitatively or quantitatively detecting human albumin;

[0049] C3) application in the aspect of preparing a product of a pharmaceutical composition for immunization or treatment.

[0050] The beneficial effects of the present application include but are not limited to:

[0051] Significant improvement in purification efficiency: the loading flow rate of the nanobody affinity membrane is 8-10 ml / min, the processing time of 2000 ml of fermentation broth is shortened from 19 h in the traditional column chromatography to 2.5 h, the processing efficiency is improved by 7.6 times, and when scaled up, only membrane columns need to be connected in parallel, without the need to adjust the flow rate parameters;

[0052] High specific capture capacity: the present application uses the nanobody 4C12 which specifically binds to human albumin as the affinity ligand, and this peptide segment can precisely bind to human albumin, and even after conservative amino acid substitution, terminal truncation or chemical modification, it still maintains high affinity and stable dissociation equilibrium. The affinity chromatography medium based on this ligand can directionally capture human albumin from fermentation broth, effectively avoiding the non-specific adsorption of host proteins (HCP), nucleic acids and other impurities. The purity of human albumin in the preliminary purified product can be more than 95%.

[0053] High process compatibility: the pretreatment and subsequent gel filtration steps can be directly connected to the existing human albumin purification process without the need to introduce special equipment, facilitating technology transfer and industrial application. BRIEF DESCRIPTION OF DRAWINGS

[0054] The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this application, illustrate embodiments of the application and together with the description serve to explain the application. In the drawings:

[0055] Figure 1 Figure 4 is an SDS-PAGE electrophoretogram of the purified nanobody in the embodiment of the present application, M: marker; 1: elution.

[0056] Figure 2 Figure 5 is a graph of the affinity detection results of the nanobody 4C12 in the embodiment of the present application.

[0057] Figure 3 Figure 6 is a graph of the ligand density-DBC curve in the embodiment of the present application; the abscissa is the ligand density (mg / mL) of the nanobody 4C12, and the ordinate is the 10% dynamic binding capacity (DBC 10 %, mg / mL).

[0058] Figure 4 Figure 7 is a graph of the dynamic penetration curve of the affinity membrane of the nanobody 4C12 in the embodiment of the present application; the abscissa is the loading volume (mL), and the ordinate is the UV280 absorbance (mAU).

[0059] Figure 5 Figure 8 is an HPLC chromatogram of the purified recombinant human albumin in the embodiment of the present application. DETAILED DESCRIPTION

[0060] The present application will be described in detail below with reference to examples, but the present application is not limited to these examples. Unless otherwise specified, the raw materials and catalysts in the examples of the present application are all purchased through commercial channels. The experimental methods not specified with specific conditions are usually performed according to the conventional conditions, for example, the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or the conditions recommended by the manufacturer.

[0061] In the present application, the fermentation broth containing recombinant human albumin can be obtained by conventional technical means in the art, for example, by fermentation of genetically engineered bacteria capable of secreting expression of recombinant human albumin, or by commercial channels. In some embodiments of the present application, the fermentation broth is a Pichia pastoris fermentation broth containing recombinant human albumin, which is prepared by referring to the method in the patent CN202410295109.6.

[0062] Example 1 Camel immunization and peripheral blood lymphocyte separation

[0063] The recombinant human albumin was mixed with Freund's adjuvant, and then the camel was immunized by subcutaneous injection of multiple points (0.2 mL per point, a total of 10 points) in the neck, with an interval of 2 weeks between each immunization, and a total of 5 immunizations. Blood was collected before immunization, before the fourth immunization, before the fifth immunization, and 2 weeks after the fifth immunization, and serum was obtained after standing and centrifugation. The ELISA (enzymelinked immunosorbent assay) method was used to detect the immune antibody titer, and the serum titer after the fifth immunization was all ≥1:500,000, which was determined to be successful. After the fifth immunization, 100 mL of blood was collected from the jugular vein, and PBMC was separated by Ficoll-Paque PLUS density gradient centrifugation (manufacturer: GE Healthcare, catalog number: 17-1440-02). The blood was diluted with an equal volume of sterile PBS, and then slowly added to the Ficoll liquid surface (volume ratio 2:1). Centrifugation was performed at 400xg and 20°C for 30 min. The middle white membrane layer was aspirated, and washed with PBS for 3 times to obtain peripheral blood mononuclear cells (PBMC).

[0064] Example 2 Construction of phage library

[0065] Total RNA was extracted from PBMC by Trizol method, and cDNA was synthesized by reverse transcription. The single-domain antibody fragment was amplified by two PCR, and then connected into phage plasmid after restriction enzyme digestion. Electroporation was used to transform into E. coli TG1 competent cells (manufacturer: TransGen, catalog number: CD201-01). The library capacity was determined by gradient dilution method to be 5.15x10 9 . 40 monoclonal antibodies were randomly selected for PCR identification, and the positive rate was 39 / 40 (97.5%), which proved that the library construction was successful.

[0066] Example 3 Nanobody screening

[0067] Add streptavidin magnetic beads (manufacturer: Thermo Fisher, item number: 11205D) and enzyme hydrolyzed casein (manufacturer: Solarbio, item number: C8210-100) casein in an EP tube, 37°C shaking blocking for 1 h (rotation speed 100 rpm), remove the blocking solution. Add recombinant human albumin, 37°C shaking incubation for 1 h for coating, remove the unbound recombinant human albumin. Add phage library after blocking with enzyme hydrolyzed casein again, PBST (10X PBS (1.37 M NaCl, 27 mM KCl, 100 mM Na2HPO4, 18 mM KH2PO4, pH 7.4) diluted to 1X, and then add 0.1% Tween-20. ) wash 9 times, PBS (pH 7.4) wash once, completely remove the non-specific binding phage. Add trypsin (manufacturer: Sigma, item number: T4799, diluted with PBS, 500 μL), 37°C shaking elution for 15 min, and immediately terminate with enzyme hydrolyzed casein, and the elution product is obtained. Infect TG1 E. coli with the elution product, plate, and incubate at 37°C overnight. Infect the E. coli cultured from all the colonies in the logarithmic phase with M13K07, and expand the culture. Cycle the panning for 3 rounds, and enrich the library. Plate the last screening strain 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, the amino acid sequence of which is shown as SEQ ID NO. 1, and the nucleotide sequence of which is shown as SEQ ID NO. 2.

[0068] Example 4 Expression and purification of nanobody in Pichia pastoris

[0069] (1) Expression of nanobody 4C12 in Pichia pastoris

[0070] The 4C12 gene was cloned into the yeast vector pPICZαA (purchased from Hunan Fenghui Biotechnology Co., Ltd.) after codon optimization in Pichia pastoris, and the optimized nucleotide sequence is shown as SEQ ID NO. 3, Sac I After linearization by enzyme digestion, it was electroporated into X-33 yeast strain (purchased from Thermo Fisher scientific company, item number C18000), and single colonies were screened on Zeocin-resistant plates. Routine inoculation was used for induction of secretory expression, and 0.5% methanol was added every 24 h, and the sample was collected after 72 h of induction and expression. The supernatant was collected by centrifugation. The expression of 4C12 was correct as determined by SDS-PAGE.

[0071] (2) Purification of nanobody expressed in Pichia pastoris

[0072] The yeast-expressed fermentation supernatant was added to a final concentration of 25 mM imidazole, dissolved, and filtered with 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 8.0 adjusted with NaOH), loaded, and the flow-through was collected; the column was washed with loading buffer until the baseline was again level, and eluted with elution buffer, and the eluate was collected; the desalting column was equilibrated with desalting buffer for 4 column volumes, and the eluate was loaded onto the desalting column, and the first peak to appear was the final nanobody solution. If the protein concentration was low after desalting, the solution was concentrated using an ultrafiltration tube. The collected solutions at each stage of purification were tested for purity, and the results of SDS-PAGE are shown in Figure 1 Figure 6, which shows that the main band of the target protein is clear after purification, and no obvious bands are present.

[0073] Example 5 Affinity detection of nanobody

[0074] The affinity of nanobody 4C12 was detected using the biofilm interference technology (BLI method). An NTA biosensor was used, and first the sensor was immersed in the analysis buffer for 10 min, and then the equilibrated sensor was immersed in the EDC-NHS mixed reagent for 5 min for activation; the activated sensor was immersed in the nanobody diluent (100 nM) for 10 min for incubation and blocked with ethanolamine (1 M, pH 8.5); the blocked sensor was immersed in the buffer for baseline zeroing; then the sensor was sequentially immersed in gradient concentrations of recombinant human albumin solution (7.8 nM, 15.625 nM, 31.25 nM, 62.5 nM, 125 nM, 250 nM, 500 nM) for 5 min for binding, and a complete binding curve was obtained; then the sensor was transferred to PBS buffer for dissociation for 5 min. The 1:1 binding model was used for kinetic analysis, and the results are shown in Figure 2 Figure 7, which shows that the dissociation equilibrium constant KD= 3.67E-10 M, indicating that 4C12 has strong affinity for recombinant human albumin.

[0075] Example 6 Preparation of nanobody affinity membrane

[0076] 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.

[0077] Example 7: Affinity membrane chromatography purification of recombinant human albumin

[0078] Take 2000 ml of Pichia pastoris recombinant human serum albumin (rHSA) fermentation broth, centrifuge at 4°C, 8000 rpm for 20 min, collect 1850 ml of supernatant; add 100 mM sodium caprylate stock solution 27.75 ml to the supernatant, so that the final concentration is 15 mM, stir uniformly, then adjust the pH to 6.0 with 1 M HC1, heat treat in a 65°C water bath for 45 min, then quickly ice bath cool to 22°C; centrifuge the treated liquid at 4°C, 12000 rpm for 15 min, collect 1780 ml of supernatant, repeat the above heat denaturation-centrifugation operation once again, finally obtain 1720 ml of supernatant; finally, under the condition of 4°C, 0.1 MPa constant pressure, use 1.2 μm glass fiber pre-filter membrane (Millipore FG120) and 0.22 μm PES depth filtration membrane (Pall DFF02210) in series to obtain a clear sample.

[0079] The nanobody affinity membrane prepared in Example 6 was packed in an XK50 / 60 chromatography column (Cytiva, column bed size: 26 mm x 200 mm, bed volume about 107 ml). First, the membrane 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 was stable (about 0.2 mAU). Then 1700 ml of pretreated clear filtrate was loaded at a flow rate of 9 ml / min, and the loading process took a total of 189 min, with a UV280 peak value of 12.5 mAU. After the loading was completed, the equilibration buffer was continued to be washed at the same flow rate for 40 min until the UV280 signal returned to the baseline. Elution was performed with elution buffer (0.1 M glycine-HCl containing 0.5 M NaCl, pH 2.8) at a flow rate of 5 ml / min, and the eluate was collected in 420 ml fractions, and immediately each tube was added with 200 μl of neutralization buffer (1 M Tris-HCl, pH 8.0) and mixed gently to restore the pH to neutral, and the combined product was the first purified product.

[0080] After elution, the membrane column was immediately washed with regeneration solution 1 (10 mM NaOH) at a flow rate of 3 ml / min for 30 min to completely remove residual impurities; then the equilibration buffer (10 mM PBS containing 150 mM NaCl, pH 7.4) was used to wash until the pH of the effluent was stable at 7.4, and the regeneration operation was completed, and the membrane column could be directly used for the next batch of purification. This regeneration process is stable and reliable, and after 20 cycles of continuous use, the dynamic binding capacity (DBC 10 %) of the membrane column still remained at 48.5 mg / mL, indicating that it has good reusability and stability.

[0081] Example 8 Purification of recombinant human albumin by gel filtration chromatography

[0082] The first purified product obtained in Example 7 was concentrated by centrifugation with an ultrafiltration centrifuge tube (molecular cut-off 10 kDa, purchased from Millipore, item number: UFC901024) at 4°C and 4000xg, monitoring the volume and protein concentration every 15 min until the protein concentration was stabilized at 50±2 mg / mL (the volume after concentration was about 103 mL), and a small amount of 50 mM Tris-HCl + 0.1 M NaCl buffer (pH 7.6) was added during the concentration to avoid local over-concentration and protein denaturation. After the concentration was completed, the sample was filtered in a sterile operation table using a 0.22 μm PVDF filter membrane (purchased from Millipore, item number: SLGV033RB), and the clear filtrate was collected and stored at 4°C for loading.

[0083] In this example, Sephadex G100 gel filtration medium (purchased from Cytiva, item number: 17001001) was used to pack a glass chromatography column (column specifications: 50 mm x 520 mm, column bed volume about 1021 ml), and if Superdex 75 series medium was used, the elution time could be further shortened by 15%, and the monomer peak separation degree could be increased to 1.8. The chromatography column was connected to an AKTA pure250 system (Cytiva), and the detection wavelength was set to 280 nm, the conductivity monitoring range was 0-30 mS / cm, and the upper limit of column pressure was 0.15 MPa. The column was equilibrated with 50 mM Tris-HCl + 0.1 M NaCl buffer (pH 7.6) at a linear flow rate of 20 cm / h for at least 3 column volumes (3063 ml). The concentrated sample was loaded at 5% of the column volume (about 51 ml), and then eluted at a constant flow rate using the same buffer, and the flow rate was maintained at 20 cm / h. According to the monitoring results of ultraviolet absorption (280 nm), the main peak elution components were collected.

[0084] The collected components were combined, desalted using an ultrafiltration membrane bag (10 kDa cut-off), and then replaced with water for injection, followed by freeze-drying to obtain high-purity recombinant human albumin freeze-dried powder.

[0085] Example 9 Purity and yield analysis

[0086] (1) Purity analysis of recombinant human albumin

[0087] 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 was used, equipped with a TSKgel G3000SWxl chromatographic column (7.8 mm x 30 cm, 5 μm, Tosoh Bioscience, item number: 08541). Phosphate buffer (PBS, 10 mM sodium phosphate, 150 mM NaCl, pH 7.4) was used as the mobile phase for isocratic elution, with a flow rate set at 0.8 mL / min, the column temperature maintained at 25 ± 1°C, the detection wavelength at 280 nm, the injection volume at 20 μL, and the run time at 30 min. The sample was reconstituted to a concentration of about 5 mg / mL with ultrapure water and filtered through a 0.22 μm microporous filter before injection. The Agilent OpenLab CDS software was used for data acquisition and analysis, and the purity was calculated by the external standard peak area normalization method. After three independent repeated determinations, the average value of the area percentage of the main peak of the sample was 99.92% ± 0.02% (n = 3), with an RSD of 0.02%, which was reproducible. See Figure 1 for a typical chromatogram. Figure 5 .

[0088] (2) Calculation of the yield of recombinant human albumin

[0089] The protein concentration of each purification step was quantified by the BCA (Bicinchoninic Acid) method, and the total yield was calculated accordingly. The Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, item number: 23225) was used. Strictly follow the instructions: first, dilute the BSA standard with PBS buffer to a series of concentrations of 0, 125, 250, 500, 750, 1000, 1500 μg / mL to make a standard curve (R 2 >0.9992). The test samples were appropriately diluted so that their concentrations fell within the linear range of the standard curve. Each sample was set up in triplicate, with 25 μL of standard or diluted sample added to each well, followed by 200 μL of BCA working solution. After incubation at 37°C for 30 min, the absorbance value was measured at 562 nm wavelength using an enzyme-labeled instrument (BioTek Synergy H1). The sample concentration was calculated according to the standard curve. The results are shown in Table 1. Starting from the total protein amount of the supernatant after pretreatment (denoted as Step 0), the total yield of the purification after affinity membrane chromatography (Step 1) and gel chromatography (Step 2) was 79.6% ± 1% (n = 3), which was 2.49 times higher than the traditional process of 32% (CN102190722A2), further verifying the high efficiency of the process of the present application.

[0090] Table 1 Recovery rate of each step

[0091]

[0092] Note: Total yield = Step0 x Step1 x Step2 = 90% x 94.3% x 93.8% ≈ 80.1%, measured value 79.6% ± 1%, deviation from the source of ultrafiltration concentration and trace loss in sample transfer, in line with the conventional fluctuations in industrial production range.

[0093] Step 0 is the recovery rate of the total protein amount of the supernatant after centrifugation and filtration pretreatment of the initial fermentation broth relative to the total protein amount before centrifugation of the fermentation broth; Step 1 and Step 2 are the recovery rates of the total amount of target protein in the eluate of each chromatography step relative to the total amount of target protein before sample loading in the previous step. The total protein amount of all steps is calculated by 'concentration (determined by BCA method) x actual sample volume', and volume correction is made when the sample is diluted. The yield in the table is the average value of three independent experiments (n = 3), and there is no SD annotation because the SD of each step is <1.5%, and the SD of the total yield is 1.1% (derived from the cumulative calculation of the deviation of each step).

[0094] Example 10 Safety index detection

[0095] (1) Endotoxin detection

[0096] The endotoxin content of the final product was detected by dynamic turbidity method limulus test. Dynamic turbidity method endotoxin detection kit (Lonza, item number: N588) was used. First, the recombinant human albumin freeze-dried powder obtained in Example 8 was dissolved and diluted to a concentration of 2 mg / mL with endotoxin check water (BET water, Lonza, item number: W50-100). The endotoxin working standard (CSE, Lonza, item number: E0005) was diluted to a series of concentrations of 0.005, 0.05, 0.5, 5 EU / mL with the same bottle of BET water to prepare a standard curve. In a pyrogen-free 96-well plate, 100 μL of standard, sample solution or negative control (BET water) was added to each well, and each sample was made in duplicate. Then, 100 μL of limulus reagent (LAL) was added to each well, mixed gently, and immediately placed in a microbial rapid detection system (or an enzyme-labeled instrument with dynamic turbidity method function, such as Charles River Endosafe® Endotoxin Detection System) to continuously monitor the change in absorbance at 405 nm wavelength of the reaction system at 37.0°C ± 0.2°C, and the reaction time was 70 minutes. The instrument software automatically calculates the endotoxin concentration of the sample according to the standard curve (R 2The endotoxin concentration in the sample was calculated. According to the requirements of the Pharmacopoeia of the People's Republic of China (2020 edition) 1143 General Principles, the interference test was carried out: the endotoxin standard (0.5 EU / mL) was mixed with the sample solution of 2 mg / mL, and the recovery rate was 100% (meeting the requirements of the Pharmacopoeia), which proved that the sample matrix had no interference with the detection. The final result is the average value of three independent detections, and the endotoxin content in the recombinant human albumin is 0.35 ± 0.04 EU / mg (n=3, RSD=5.2%), which is much lower than the limit requirement of "less than 1 EU / mg" in the Pharmacopoeia of the People's Republic of China (2020 edition) 1143 General Principles, which proves that the safety of the final product meets the standards.

[0097] (2) Host cell protein (HCP) residual detection

[0098] Determination was carried out by enzyme-linked immunosorbent assay (ELISA). A commercial detection kit specific to Pichia pastoris (manufacturer: Cygnus Technologies, product number: F550) was used, with a detection range of 1-100 ng / mL. The HCP standard used in this kit was the purified HCP of the same Pichia pastoris strain as the experiment, which was verified by Western Blot to specifically recognize the HCP of the strain (no cross-reaction); at the same time, a spiked recovery test was carried out on the rHSA sample of 5 mg / mL (spiked concentration 20 ng / mL), with a recovery rate of 99.2%, verifying that the detection had no matrix interference. Before detection, the rHSA sample was diluted to 5 mg / mL, and pre-verification showed that rHSA at this concentration had no interference with the detection. The standard curve (R 2 =0.9978) was calculated, and the results showed that the HCP residual amount 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 value of "HCP residual ≤10 ng / mg" specified in the Quality Control Technical Guidelines for Human Recombinant DNA Products (2020) of the National Medical Products Administration, fully proving that the purification process can efficiently remove Pichia pastoris host proteins.

[0099] (3) Residual DNA detection

[0100] Real-time quantitative PCR (qPCR) was used to target the conserved GAPDH gene in the Pichia pastoris genome. First, DNA enrichment and purification were performed on 100 mg of the final product sample using a DNA extraction kit (manufacturer: 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 spiking test: 10 pg of Pichia pastoris genomic DNA was added to the sample solution, and the recovery rate of the qPCR detection after extraction was 94.5%. The final residual DNA content was corrected according to the extraction efficiency. Subsequently, specific qPCR detection kits (manufacturer: Thermo Fisher Scientific, catalog number: A24554) were used for amplification. By constructing a standard curve of 1-10000 pg / mL (R 2 =0.9992), and analyzing the melting curve of the amplification product (single peak, Tm=85.5°C), the specificity and accuracy of the detection were ensured. The final calculation showed that the 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 the "Chinese Pharmacopoeia" (2020 edition) general rule 3407, further verifying the effectiveness of this process in removing nucleic acid impurities, and the safety of the final product meets the pharmaceutical requirements.

[0101] Preparation of nanobody affinity chromatography medium

[0102] The human albumin-specific nanobody 4C12 obtained in Example 5 was dissolved in a coupling solution (0.1M NaHCO3+0.5M NaCl, pH8.3) to prepare a nanobody solution with a concentration of 6mg / ml. 4ml of CNBr-activated Bestarose 4B medium (purchased from Bogu (Shanghai) Biotech Co., Ltd.) was placed in a sand core funnel and washed with 4℃ pre-cooled 1mM HCl for 30min, with a volume of about 240ml. The washed medium was diluted to 6ml with 1mM HCl, mixed with an equal volume of nanobody solution, and incubated at 4℃ overnight on a shaker. The coupling supernatant was removed and a blocking solution (0.1M Tris-HCl, pH8.0) was added, and the blocking was carried out at room temperature for 2h. Wash with washing solution 1 (0.1M HAC+0.5M NaCl, pH4.0) and washing solution 2 (0.1M Tris-HCl+0.5M NaCl, pH8.0) alternately for 5 cycles, each time with 5 times the medium volume of liquid washing. After washing with PBS (pH7.4), it was stored at 4℃ for standby.

[0103] Affinity chromatography purification of recombinant human albumin

[0104] Take 2000 ml of the recombinant human albumin fermentation broth expressed by yeast, centrifuge at 4°C, 8000 rpm (centrifugal force about 10,000 x g) for 20 minutes, and collect the supernatant. Add 100 mM sodium caprylate stock solution to the supernatant to make the final concentration 15 mM, and mix well under slow stirring. Adjust the pH to 6.0 ± 0.1 with 1 M HC1 solution, and then heat treat in a 65.0 ± 0.5°C water bath for 45 minutes, and quickly cool to below 25°C in an ice bath. Centrifuge at 4°C, 12000 rpm (centrifugal force about 20,000 x g) for 15 min, collect the supernatant, and repeat the heat denaturation and centrifugation operation once. The final supernatant is filtered through a 0.45 μm PES membrane, and 1750 ml of clear sample is collected after pretreatment.

[0105] Take the nanobody affinity chromatography medium prepared in Comparative Example 1, load it into an XK50 / 60 chromatography column (purchased from Cytiva, column bed size: 26 mm x 200 mm, bed volume: 107 ml), and use an AKTA pure 150 system for operation. First, use the equilibration buffer (10 mM sodium phosphate, 150 mM NaCl, pH 7.4) to equilibrate at least 3 times the column volume (321 ml) at a flow rate of 1.5 ml / min until the ultraviolet absorption baseline (280 nm) is stable. Load the pretreated sample at a flow rate of 1.5 ml / min. After the loading is completed, use the equilibration buffer to rinse until the UV280 signal returns to the baseline. Use the elution buffer (0.1 M glycine-HCl, pH 2.8) to elute 4 times the column volume (428 ml) at a flow rate of 1.0 ml / min, and collect the eluate in 2 ml per tube. Immediately add 200 μl of neutralization buffer (1 M Tris-HCl, pH 8.0) to each tube of eluate, mix gently to restore the pH to neutral, and combine to obtain the first purified product.

[0106] Comparative Example 3 Gel filtration chromatography purification of recombinant human albumin

[0107] The first purified product obtained from Comparative Example 2 was concentrated by centrifugation with an ultrafiltration centrifuge tube (molecular cut-off 10 kDa, purchased from Millipore, item number: UFC901024) at 4°C, 4000xg, and the volume and protein concentration were monitored every 15 min until the protein concentration was stabilized at 50±2 mg / mL (the volume after concentration was about 103 mL), and a small amount of 50 mM Tris-HCl + 0.1 M NaCl buffer (pH 7.6) was added during the concentration to avoid local concentration too high to cause protein denaturation. After the concentration was completed, the sample was filtered in a sterile operation table using a 0.22 μm PVDF filter membrane (purchased from Millipore, item number: SLGV033RB), and the clear filtrate was collected and stored at 4°C before loading.

[0108] A glass chromatography column (column specifications: 50 mm x 520 mm, column bed volume about 1021 ml) was filled with Sephadex G100 gel filtration medium (purchased from Cytiva, item number: 17001001). The chromatography column was connected to an AKTA pure 250 system (Cytiva), and the detection wavelength was set to 280 nm, the conductivity monitoring range was 0-30 mS / cm, and the upper limit of column pressure was 0.15 MPa. At least 3 times the column volume (3063 ml) was 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 (about 51 ml), and then eluted at a constant flow rate with the same buffer, and the flow rate was maintained at 20 cm / h. According to the monitoring results of ultraviolet absorption (280 nm), the main peak elution components were collected.

[0109] The collected components were combined, desalted with an ultrafiltration membrane bag (10 kDa cut-off) and exchanged into water for injection, and then freeze-dried to obtain a high-purity recombinant human albumin freeze-dried powder. The detection results are shown in Table 2.

[0110] Table 2 Detection data table of recombinant human albumin in comparative example

[0111]

[0112] Note: In order to ensure the comparability of the data, the purity, yield and safety indicators (endotoxin, HCP, residual DNA) of the sample in this comparative example were detected using the same methods, instruments, reagents, standard curve preparation and calculation methods as described in Examples 9 and 10 of the present application.

[0113] By comparison, the nanobody affinity membrane chromatography method shortens the longest "affinity chromatography stage" from 31.53 h to 3.88 h through the convection mass transfer advantage of the membrane material, shortens the overall production cycle by nearly 40%, and when scaled up, the membrane chromatography can further maintain a high flow rate through "parallel membrane columns", while the particle column needs to increase the column diameter, resulting in a decrease in flow rate, and the cycle difference between the two will be more significant, more suitable for industrial continuous production needs. The specific data is shown in Table 3.

[0114] Table 3 Comparison of purification time of examples and comparative examples

[0115]

[0116] The above merely illustrates the embodiments of the present application, and the protection scope of the present application is not limited by these specific embodiments, but is determined by the claims of the present application. The present application can have various modifications and changes for those skilled in the art. Any modification, equivalent replacement, improvement, etc. within the technical idea and principle of the present application shall be included in the protection scope of the present application.

Claims

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

2. A biomaterial, characterized by, The biological material at least includes any of the following: B1) a nucleic acid molecule encoding the nanobody of claim 1; B2) an expression cassette containing the nucleic acid molecule of B1); B3) a recombinant vector containing the nucleic acid molecule of B1) or an expression cassette of B2); B4) a recombinant microorganism containing the nucleic acid molecule of B1), or an expression cassette of B2), or a recombinant vector of B3); B5) a recombinant cell, which is a recombinant cell containing the nucleic acid molecule of B1), or an expression cassette of B2), or a recombinant vector of B3).

3. The biomaterial of claim 2, wherein, The nucleotide sequence of the nucleic acid molecule in B1) at least includes the nucleotide sequence as shown in SEQ ID NO. 2 or SEQ ID NO.

3.

4. The biomaterial of claim 2, wherein, The recombinant microorganism in B4) or the recombinant cell in B5) includes at least one of hamster ovary cells, silkworm ovary cells, Pichia pastoris, Saccharomyces cerevisiae, Escherichia coli, and Bacillus subtilis.

5. A method of preparing the Nanobody of claim 1, characterized in that, The method includes the step of culturing the recombinant microorganism of B4) or the recombinant cell of B5) in the biological material 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, wherein The matrix of the nanobody affinity membrane includes at least one of an organic polymer membrane, an inorganic / organic composite membrane, and a hydrogel composite membrane.

8. A method for isolating and purifying human albumin, characterized by, The method includes the step of using the nanobody of claim 1 or the nanobody affinity membrane of claim 6 or 7 for purification.

9. The method of claim 8, wherein, The purification step at least includes affinity membrane chromatography and gel filtration chromatography.

10. The nanobody of claim 1 or the biological material of any one of claims 2-4 or the nanobody affinity membrane of claim 6 or 7 is used in any of the following aspects: C1) for preparing a product for separating or purifying human albumin; C2) for preparing a product for qualitatively or quantitatively detecting human albumin.

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