Human albumin binding peptide 4F1 and method for purifying human albumin

Abstract

The invention discloses a human albumin binding peptide 4F1 and a method for purifying human albumin, and belongs to the technical field of polypeptides. The amino acid sequence of the human albumin binding peptide 4F1 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 human albumin binding peptide 4F1 has extremely high affinity with human albumin and can be used for separating and purifying a human albumin solution, and the purity of the purified human albumin far exceeds the pharmacopoeia standard and can reach 99.9% or above. According to the method, high-selectivity capture of affinity chromatography and strong impurity removal capacity of gel filtration chromatography are combined, a purification process route which is efficient, stable and easy to amplify is constructed, and the recovery rate and the production efficiency are remarkably improved.

Description

Technical Field

[0001] This invention relates to the field of polypeptide technology, specifically to a human albumin-binding peptide 4F1 and a method for purifying human albumin. Background Technology

[0002] Human serum albumin (HSA) is the most abundant protein in human blood plasma, playing vital physiological roles such as maintaining plasma osmotic pressure, transporting endogenous and exogenous substances, and scavenging free radicals. Clinically, it is widely used to treat shock, burns, and hypoalbuminemia, and can also serve as a vaccine adjuvant, a component of cell culture media, and a stabilizer for therapeutic protein drugs. Traditional human HSA production primarily relies on plasma extraction, which is limited by plasma supply shortages and carries the risk of contamination by pathogens such as hepatitis viruses and HIV. Therefore, the production of recombinant human serum albumin (rHSA) using recombinant DNA technology has become an important alternative. Expression systems include Escherichia coli, yeast (such as Pichia pastoris), plants, and transgenic animals.

[0003] However, rHSA purification still faces significant challenges. The fermentation broth produced by expression systems such as yeast is complex, containing a large amount of host cell proteins (HHP), nucleic acids, endotoxins, pigments, albumin degradation products (such as 45 kDa fragments), and aggregates. Due to the large doses used clinically, pharmaceutical-grade rHSA requires extremely high purity (≥99.9%); even trace impurities can cause safety issues. Furthermore, traditional purification processes often rely on multi-step chromatographic combinations, resulting in lengthy procedures, low yields (approximately 32%), and high costs, making large-scale production difficult.

[0004] Early rHSA purification often employed combined strategies such as ion exchange, hydrophobic interaction chromatography (HIC), and metal chelate affinity chromatography (IMAC). For example, US5521287 used a three-step method involving cation exchange, hydrophobic chromatography, and metal chelation, while CN112210002B also used a similar multi-step chromatographic method. Although these methods could yield products with a certain level of purity, they were cumbersome and had low recovery rates. Expanded bed adsorption (EBA) technology improved the initial purification efficiency to some extent; for example, CN102190722A directly loaded the fermentation broth onto an anion exchange expanded bed, but subsequent multi-step purification was still required, making the process complex. Dye affinity chromatography (such as Cibacon Blue) can be used to remove degradation fragments, while immunoaffinity chromatography has high selectivity but is expensive and has poor stability, making it unsuitable for large-scale applications.

[0005] In recent years, the emergence of new technologies such as mixed-mode chromatography (MMC) and affinity peptide ligands has brought breakthroughs in rHSA purification. MMC ligands, relying on multiple mechanisms of action, possess characteristics such as high adsorption capacity, good selectivity, and salt tolerance. For example, CN116693659A uses two-step MMC purification to obtain rHSA with a purity exceeding 95% and a yield greater than 80%. Affinity peptide ligands (such as albumin-binding peptides) show significant advantages due to their simple synthesis, high stability, low cost, and lack of toxic side effects. Integrated purification strategies, such as the multilayer chromatography combination in CN112210002B, have also made progress in improving product purity and controlling costs. Summary of the Invention

[0006] To address the problems of complex processes, low yields, difficulty in scale-up, and insufficient product purity in existing technologies, this application develops a combined affinity chromatography and gel filtration process based on the specific albumin-binding peptide 4F1 ligand. This process can efficiently capture rHSA from fermentation broth and deeply remove impurities, achieving a final product purity of 99.9%, meeting pharmaceutical-grade standards. This process features simple operation, high stability, and ease of scale-up, providing a reliable solution for the large-scale production and widespread application of rHSA. The albumin includes serum albumin and recombinant albumin; optionally, the serum albumin is human serum albumin; optionally, the recombinant albumin is recombinant human albumin.

[0007] In a first aspect, the present invention provides a human albumin-binding peptide 4F1, wherein the amino acid sequence of the human albumin-binding peptide 4F1 comprises 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 that has more than 95% identity with the amino acid sequence of the human albumin-binding peptide 4F1 described in A1) obtained by substituting and / or deleting and / or adding amino acid residues. 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).

[0008] 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 human albumin-binding peptide 4F1 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).

[0009] Further, the nucleic acid molecule described in B1) includes a publicly disclosed nucleic acid molecule encoding the human albumin-binding peptide 4F1 and / or an optimized nucleic acid molecule as needed. 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 human albumin-binding peptide 4F1; SEQ ID NO. 3 is a sequence optimized for Pichia pastoris codon preferences.

[0010] It should be understood that those skilled in the art can optimize the nucleic acid molecule encoding SEQ ID NO: 1 according to different expression systems (such as other engineered bacteria), and such variants are all within the scope of protection of this application.

[0011] 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.

[0012] 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.

[0013] Optionally, the recombinant microorganism is Pichia pastoris.

[0014] In a third aspect, the present invention provides a method for preparing the human albumin-binding peptide 4F1, the method comprising culturing the recombinant microorganism (B4) or the recombinant cell (B5) under suitable culture conditions, and isolating the human albumin-binding peptide 4F1 or the fusion protein.

[0015] 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.

[0016] In a fourth aspect, the present invention provides a chromatography medium for separating and purifying human albumin, the chromatography medium comprising the human albumin-binding peptide 4F1.

[0017] Optionally, the chromatography medium includes at least one of a pre-packed column, packing material, and magnetic beads. Preferably, the magnetic beads are magnetic beads coupled with the human albumin-binding peptide 4F1. Preferably, the chromatography medium is a recombinant human albumin-binding peptide 4F1 ligand-crosslinked agarose affinity medium.

[0018] 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 chromatography medium.

[0019] Optionally, the purification step includes at least affinity chromatography and gel filtration chromatography.

[0020] Furthermore, the human albumin includes human serum albumin and recombinant human albumin.

[0021] Furthermore, the method for separating and purifying human albumin specifically includes the following steps: a) The fermentation broth containing recombinant human albumin was pretreated by centrifugation; b) The fermentation broth, which has been pretreated by centrifugation, is loaded into an affinity chromatography column containing human albumin-binding peptide 4F1 as a ligand. After washing with equilibration buffer and elution buffer, neutralization buffer is added to obtain the first purified product. c) Pass the first purified product through a gel filtration chromatography column, elute with mobile phase buffer, collect the recombinant human albumin monomer peak, and obtain the high-purity recombinant human albumin final product.

[0022] It will be understood by those skilled in the art that the sequence of SEQ ID NO. 1 of this application can be simply modified, such as by replacing or deleting some amino acid sites, while still retaining the same human albumin affinity properties as SEQ ID NO. 1 in this application. It is understood that sequences that retain the same human albumin affinity function of the binding peptide based on simple modifications to the sequence of SEQ ID NO. 1 of this application should also be within the scope of protection of this application. Furthermore, it will be reasonable for those skilled in the art to truncate or derive the binding peptide based on the sequence of SEQ ID NO. 1 disclosed in this application to obtain binding peptides with the same human albumin affinity effect. 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.

[0023] It should be noted that those skilled in the art can chemically modify the binding peptide. The chemical modification can be any one of the following: cyclization modification, acetylation modification, PAS modification, PEG modification, fatty acid modification, albumin modification, albumin-binding peptide coupling, tumor homing peptide coupling, membrane-penetrating peptide coupling, nanocarrier coupling, radionuclide coupling, small molecule compound coupling, nucleotide coupling, and protein coupling. The modification sites include, but are not limited to, N-terminal modification, C-terminal modification, backbone modification, side chain modification, and amino acid modification.

[0024] Further, in step b), the equilibration buffer is a PBS solution, the elution buffer is a glycine solution, and the neutralization buffer is a Tris-HCl solution.

[0025] Further, the pH range of the PBS solution is 6.5 to 7.5; the pH range of the glycine solution is 2.8 to 3.5; the pH range of the Tris-HCl solution is 8.0 to 8.5; and the pH range of the equilibration buffer / mobile phase buffer Tris-HCl + NaCl solution is 7.5 to 8.5.

[0026] 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).

[0027] In a sixth aspect, the present invention provides the use of the human albumin-binding peptide 4F1, the biomaterial, or the chromatography medium 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.

[0028] The beneficial effects of the present invention include, but are not limited to: 1. High specificity capture capability: This invention uses the specific human albumin-binding peptide 4F1 as an affinity ligand. This peptide can precisely bind to recombinant human albumin (rHSA), 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 rHSA from fermentation broth, effectively avoiding non-specific adsorption of host proteins (HCP), nucleic acids, and other impurities. The purity of rHSA in the preliminary purified product can reach over 95%.

[0029] 2. Simple, stable, and easily scalable process: This invention achieves efficient purification through only two core processes: affinity chromatography and gel filtration, reducing the number of steps by more than 30%. The process parameters are clearly defined, the bound peptide 4F1 exhibits high stability, tolerates pH 2.8–8.5, and has no risk of detachment; the process has good reproducibility and is easily scaled up from the laboratory to industrial scale (100L–1000L), increasing production efficiency by more than 40%.

[0030] 3. High purity and excellent safety of the final product: A combined process of "affinity capture + gel filtration" achieves stepwise impurity removal, resulting in an rHSA purity exceeding 99.9%, superior to conventional processes (95%~98%). This process demonstrates outstanding safety; the levels of HCP, endotoxins, and aggregates in the final product are significantly lower than the limits set by the Chinese Pharmacopoeia (2020) and related guidelines. It can be directly used in clinical treatment, vaccine stabilizers, and cell culture, avoiding immunogenicity risks. Attached Figure Description

[0031] 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 human albumin-binding peptide 4F1 in this embodiment of the invention. M: marker; 1: elution.

[0032] Figure 2 This is a graph showing the results of the affinity test between human albumin and human albumin-binding peptide 4F1 in an embodiment of the present invention.

[0033] Figure 3 This is an HPLC chromatogram of purified recombinant human albumin in an embodiment of the present invention. Detailed Implementation

[0034] 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.

[0035] 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.

[0036] 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).

[0037] 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: Amid Biosciences, catalog number: ETG1-201) 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.

[0038] Example 3: Screening of human albumin-binding peptide 4F1 Add streptavidin magnetic beads (Thermo Fisher, catalog number: 65006D) 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 4F1, whose amino acid sequence is shown in SEQ ID NO. 1 and whose nucleotide sequence is shown in SEQ ID NO. 2.

[0039] Example 4: Expression and purification of human albumin-binding peptide 4F1 in Pichia pastoris (1) Pichia pastoris expressing human albumin-binding peptide 4F1 The 4F1 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 yeast strain X-33 (purchased from Shanghai Zeye Biotechnology Co., Ltd., catalog number ZY1027). 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 4F1 expression.

[0040] (2) Purification of human albumin-binding peptide 4F1 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 baseline was reached, the sample was loaded, and flow-through was collected. The column was further washed with loading buffer until the baseline was again reached. Elution was then performed with elution buffer, 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 that appeared was collected as the final human albumin-binding peptide 4F1 solution. If the protein concentration was low after desalting, it was concentrated using an ultrafiltration tube. The purity of the collected solutions at each purification stage was tested, and the 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.

[0041] Example 5: Detection of affinity for human albumin-binding peptide 4F1 The affinity of human albumin-binding peptide 4F1 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 a 100 nM human albumin-binding peptide 4F1 dilution buffer and blocked with ethanolamine (1 M, pH 8.5). The blocked sensor was then zeroed by baseline adjustment in buffer. 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 2 As shown, the dissociation equilibrium constant KD = 1.56E-09 M indicates that 4F1 has a strong affinity for recombinant human albumin.

[0042] Example 6 Preparation of affinity chromatography medium for human albumin-binding peptide 4F1 Human albumin-binding peptide 4F1 lyophilized powder was dissolved in coupling solution (0.1M NaHCO3 + 0.5M NaCl, pH 8.3) to prepare a 6 mg / ml binding peptide solution. 4 ml of CNBr-activated Bestarose 4B medium (purchased from Borglon Biotechnology (Shanghai) 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 binding peptide 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.

[0043] Example 7: 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. Finally, filter the supernatant through a 0.45 μm PES membrane to obtain a clear sample.

[0044] The human albumin-binding peptide 4F1 affinity chromatography medium prepared in Example 6 was packed into an XK50 / 60 column (purchased from Cytiva, column bed size: 26 mm × 200 mm, bed volume: 107 ml), and the operation was performed 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. Four column volumes (428 ml) were eluted with elution buffer (0.1 M glycine-HCl, pH 2.8) at a flow rate of 1.0 ml / min, and the eluent was collected fractionally in 2 ml tubes. 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.

[0045] 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 minutes 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 excessively high local 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°C for future loading.

[0046] A Superdex 200 gel permeation medium (Cytiva, catalog number 17-1043-01) was used to pack an XK50 / 100 glass chromatography column (50 mm × 520 mm, approximately 1021 ml bed volume). The column was connected to an AKTApure 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 maintained flow rate of 20 cm / h. The main peak eluted fraction was collected based on UV absorption (280 nm) monitoring.

[0047] The collected components were combined, desalted using an ultrafiltration membrane (purchased from Millipore, model: P2B010C01, 10 kDa cutoff) and replaced with water for injection, and then freeze-dried to obtain high-purity recombinant human albumin lyophilized powder.

[0048] 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 of 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 99.9% ± 0.02% (n=3), with an RSD of 0.02%, indicating repeatability. A typical chromatogram can be found in [reference needed]. Figure 3 The purity increased from 98.7% (CN118580318 A) to 99.9%, an increase of 1.2 percentage points.

[0049] (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 minutes, the absorbance was measured at 562 nm using a BioTek Synergy H1 microplate reader. The sample concentration was calculated based on the standard curve, and 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 77% ± 1.2% (n=3), a 2.4-fold increase compared to the traditional process of 32% (CN102190722A2), further validating the high efficiency of the proposed process.

[0050] Table 1 Recovery rate of each step

[0051] 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).

[0052] Example 10 Safety Index Testing (1) Endotoxin detection The endotoxin content of the final product was determined using the dynamic turbidimetric endotoxin assay kit (Lonza, catalog number: N588). 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...). 2 The 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): an 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 0.65 ± 0.08 EU / mg (n=3, RSD=7.7%), 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.

[0053] (2) Detection of residual host cell protein (HCP) Enzyme-linked immunosorbent assay (ELISA) was used for the assay. 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 (20 ng / mL), with a recovery rate of 98%, verifying no matrix interference. Before assay, 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 3.2 ± 0.3 ng / mg rHSA (n=3, RSD=9.4%). 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.

[0054] (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 92% recovery rate by qPCR. 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 from 1 to 10000 pg / mL. 2 =0.9983), 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 4.5 ± 0.4 pg / mg rHSA (n=3, RSD=8.9%). 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.

[0055] Comparative Example 1: Changing the affinity chromatography medium 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. Finally, filter the supernatant through a 0.45 μm PES membrane to obtain a clear sample.

[0056] Commercial Protein A affinity chromatography medium was packed into the chromatography column and equilibrated with 3 column volumes of equilibration buffer (PBS, pH 7.4). Sample was loaded at a loading of 15 mg / mL. The column was washed with equilibration buffer to the UV baseline level. Elution was performed with 4 column volumes of elution buffer (0.1 M Glycine, pH 2.8). The eluted fraction was collected and immediately added to neutralization buffer (1 M Tris-HCl, pH 8.0) at a ratio of 20:1 to obtain the first purified product.

[0057] The first purified product containing recombinant human albumin obtained above was concentrated and filtered through a 0.22 μm filter membrane. It was then pumped into a Superdex 200 column (column size: 50 mm × 520 mm, column volume: 1021 ml) equilibrated with twice the volume of 50 mM Tris-HCl + 0.1 M NaCl. Subsequently, it was eluted at low speed with the same 50 mM Tris-HCl + 0.1 M NaCl buffer, desalted, and lyophilized to obtain recombinant human albumin. The detection results are shown in Table 2.

[0058] Table 2 Comparative Example 1 Recombinant Human Albumin Detection Data

[0059] Protein A has low specificity for rHSA and adsorbs a large amount of HCP; Protein A has low binding capacity, resulting in significant flow-through loss and incomplete collection of the target protein.

[0060] Comparative Example 2: Changes in Affinity Chromatography Elution Buffer 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. Finally, filter the supernatant through a 0.45 μm PES membrane to obtain a clear sample.

[0061] The human albumin-binding peptide 4F1 affinity chromatography medium prepared in Example 6 was packed into a chromatography column. The column was equilibrated with 3 column volumes of equilibration buffer (PBS, pH 7.4), and the sample was loaded at a loading of 50 mg / ml. The column was washed with equilibration buffer to the UV baseline level. The column was eluted with 4 column volumes of elution buffer (0.05 M Glycine, pH 3.5). The eluted fraction was collected and immediately added to neutralization buffer (1 M Tris-HCl, pH 8.0) at a ratio of 20:1 to obtain the first purified product.

[0062] The first purified product containing recombinant human albumin obtained above was concentrated and filtered through a 0.22 μm filter membrane. It was then pumped into a Superdex 200 column (column size: 50 mm × 520 mm, column volume: 1021 ml) equilibrated with twice the volume of 50 mM Tris-HCl + 0.1 M NaCl. Subsequently, it was eluted at low speed with the same 50 mM Tris-HCl + 0.1 M NaCl buffer, desalted, and freeze-dried to obtain recombinant human albumin. The detection data for recombinant human albumin in Comparative Example 2 are shown in Table 3.

[0063] Table 3 Comparative Example 2 Recombinant Human Albumin Detection Data

[0064] The low concentration of 0.05M Glycine and pH 3.5 resulted in insufficient elution strength, leading to incomplete elution of rHSA and contaminating proteins. Insufficient elution buffer strength also resulted in residual rHSA in the media, reducing recovery rates.

[0065] Comparative Example 3: Changing the pH of the affinity chromatography equilibration buffer 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. Finally, filter the supernatant through a 0.45 μm PES membrane to obtain a clear sample.

[0066] The human albumin-binding peptide 4F1 affinity chromatography medium prepared in Example 6 was packed into a chromatography column. Three column volumes were equilibrated with equilibration buffer (PBS, pH 6.5). The sample was loaded at a loading of 50 mg / ml. The column was washed with equilibration buffer to the UV baseline level. Four column volumes were eluted with elution buffer (0.1 M Glycine, pH 2.8). The eluted fraction was collected and immediately added to neutralization buffer (1 M Tris-HCl, pH 8.0) at a ratio of 20:1 to obtain the first purified product.

[0067] The first purified product containing recombinant human albumin obtained above was concentrated and filtered through a 0.22 μm filter membrane. It was then pumped into a Superdex 200 column (column dimensions: 50 mm × 520 mm, column volume: 1021 ml) equilibrated with two volumes of 50 mM Tris-HCl + 0.1 M NaCl. Following this, it was eluted at low speed with a buffer solution of 50 mM Tris-HCl + 0.1 M NaCl, desalted, and lyophilized to obtain recombinant human albumin. The detection results are shown in Table 4.

[0068] Table 4. Comparative Example 3: Recombinant Human Albumin Detection Data

[0069] Using PBS at pH 6.5 reduces the binding affinity between the binding peptide 4F1 and rHSA, resulting in co-elution of other proteins; affinity chromatography with increased rHSA residue leads to a decrease in the recovery rate of the target protein.

[0070] Key Explanation All pretreatment steps (centrifugation, heat denaturation, filtration) of the comparative examples were completely consistent with those of Example 7, ensuring that the differences stemmed only from modifications to chromatography parameters and that the data comparisons were unique. The impurity detection methods (endotoxin: dynamic turbidity assay, HCP: ELISA, residual DNA: qPCR, aggregates: SEC-HPLC) are consistent with those in Example 11, and the data accuracy is traceable. The purity, yield, and impurity levels of the final products in all comparative examples were significantly worse than those in the examples, fully demonstrating the optimization of the parameters in Examples 7 and 8: “affinity chromatography equilibration buffer pH 7.4 + elution buffer 0.1M Glycine pH 2.8 + gel filtration 50mM Tris-HCl + 0.1M NaCl equilibration Superdex 200”.

[0071] 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 human albumin-binding peptide 4F1, characterized in that, The amino acid sequence of the human albumin-binding peptide 4F1 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) A nucleic acid molecule containing the human albumin-binding peptide 4F1 as described in 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 human albumin-binding peptide 4F1 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 biological material of claim 2 under suitable culture conditions, and isolating the human albumin-binding peptide 4F1.

6. A chromatography medium for separating and purifying human albumin, characterized in that, The chromatography medium comprises the human albumin-binding peptide 4F1 as described in claim 1.

7. The chromatography medium according to claim 6, characterized in that, The chromatography medium includes at least one of pre-packed columns, packing materials, and magnetic beads.

8. A method for efficiently separating and purifying human albumin, characterized in that, The method includes a purification step using the human albumin-binding peptide 4F1 of claim 1 or the chromatography medium of claim 6 or 7.

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

10. The use of the human albumin-binding peptide 4F1 of claim 1, or the biomaterial of any one of claims 2-4, or the chromatography medium 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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