Human albumin binding peptide 1E3 and application of human albumin binding peptide 1E3 in promoting purification of human albumin

Abstract

The invention discloses a human albumin binding peptide 1E3 and application of the human albumin binding peptide 1E3 in promoting purification of human albumin, and belongs to the technical field of polypeptides. The human albumin binding peptide 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 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 99.99% or above. The method is good in safety and stable in process, and has a wide application prospect in the aspect of separation and purification of the human albumin.

Description

Technical Field

[0001] This invention relates to the field of polypeptide technology, specifically to a human albumin-binding peptide 1E3 and its application in promoting the purification of human albumin. Background Technology

[0002] Human serum albumin (HSA) is the most abundant protein in human blood plasma, and its main functions include maintaining plasma osmotic pressure, transporting endogenous and exogenous substances, and scavenging free radicals. Clinically, albumin is widely used to treat shock, burns, and hypoalbuminemia, and serves as a stabilizer in vaccines, cell culture media, and therapeutic protein drugs. Traditional extraction of human serum albumin from plasma not only faces the problem of plasma supply shortages but also carries the risk of pathogen contamination (such as hepatitis viruses and HIV). Therefore, the production of recombinant human albumin (rHSA) using recombinant DNA technology has become an important alternative, and its expression systems have expanded from Escherichia coli to yeast (such as Pichia pastoris), plants, and transgenic animals.

[0003] However, the purification of rHSA faces numerous challenges. Yeast fermentation broth contains a large number of impurities, including host cell proteins (HCP), nucleic acids, endotoxins, pigments, albumin degradation products (such as 45 kDa fragments), and aggregates. Due to the large doses used in clinical applications, pharmaceutical-grade rHSA requires extremely high purity (≥99.9%), as even trace impurities can cause safety issues. Furthermore, traditional purification processes involve numerous steps, have low yields (approximately 32%), and are costly, making them unsuitable for industrial-scale production.

[0004] Early purification strategies primarily relied on combinations of ion exchange chromatography, hydrophobic interaction chromatography (HIC), and metal chelate affinity chromatography (IMAC). For example, patent US5521287 employs a multi-step process combining cation exchange, hydrophobic chromatography, and metal chelate affinity chromatography; patent CN112210002B also uses a similar multi-step chromatographic strategy. While these methods can achieve a certain purity of rHSA, the processes are lengthy and yields are low. The application of expanded bed adsorption (EBA) technology has improved initial purification efficiency; for example, patent CN102190722A directly loads the fermentation broth onto an anion exchange expanded bed, but subsequent processing still requires multiple steps, making the process complex. Regarding affinity chromatography, dye affinity chromatography (such as Cibacon blue filler) can be used to remove degradation fragments, while immunoaffinity chromatography, although highly selective, is expensive and has poor stability, making it unsuitable for large-scale production. In recent years, new technologies such as mixed-mode chromatography (MMC) and affinity peptide ligands have provided new approaches for rHSA purification. MMC ligands combine multiple interactions, offering advantages such as high adsorption capacity, high selectivity, and good salt tolerance. For example, patent CN116693659A employs a two-step mixed-mode chromatography method, requiring only two purification steps to obtain rHSA with a purity greater than 95% and a yield exceeding 80%. Affinity peptide ligands (such as albumin-binding peptides) demonstrate great potential compared to traditional antibodies due to their simpler synthesis, better stability, lower cost, and lack of toxicity concerns. Furthermore, integrated purification strategies (such as patent CN112210002B, which combines multiple chromatographic techniques) have made progress in balancing purity and cost. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this application develops a multi-step chromatographic combination process for the large-scale production of high-purity albumin. Its core innovation lies in employing the specific albumin-binding peptide 1E3 as an affinity ligand to efficiently capture the target protein directly from the fermentation broth; optimizing the combination of anion exchange chromatography and gel filtration chromatography to deeply remove impurities; and achieving a final product purity ≥99.99%, meeting pharmaceutical-grade standards. This process is efficient, robust, and suitable for large-scale production, providing a reliable solution for the widespread application of albumin. The albumin includes serum albumin and recombinant albumin; optionally, the serum albumin is human serum albumin; optionally, the recombinant albumin is recombinant human albumin.

[0006] In a first aspect, the present invention provides a human albumin-binding peptide 1E3, wherein the amino acid sequence of the human albumin-binding peptide 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 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).

[0007] 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 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).

[0008] Further, the nucleic acid molecule described in B1) includes publicly disclosed nucleic acid molecules encoding the human albumin-binding peptide and / or those optimized 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 1E3; SEQ ID NO. 3 is a sequence optimized for Pichia pastoris codon preferences.

[0009] 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.

[0010] 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.

[0011] 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.

[0012] Optionally, the recombinant microorganism is Pichia pastoris.

[0013] In a third aspect, the present invention provides a method for preparing the human albumin-binding peptide, the method comprising culturing the recombinant microorganism (B4) or the recombinant cell (B5) under suitable culture conditions, and isolating the human albumin-binding peptide or the fusion protein.

[0014] 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.

[0015] 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 described above.

[0016] 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. Preferably, the chromatography medium is a recombinant human albumin-binding peptide 1E3 ligand-crosslinked agarose affinity medium.

[0017] 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.

[0018] Optionally, the purification steps include at least affinity chromatography, anion exchange chromatography, and gel filtration chromatography.

[0019] Furthermore, the human albumin includes human serum albumin and recombinant human albumin.

[0020] Furthermore, the method for separating and purifying human albumin specifically includes the following steps: a) Centrifuge the fermentation broth or human blood products containing recombinant human albumin to obtain the supernatant; b) Load the supernatant obtained in step a) onto an affinity chromatography column with human albumin-binding peptide 1E3 as the ligand, wash with equilibration buffer, elute with elution buffer, and immediately neutralize the eluted fraction with neutralization buffer to obtain the first purified product. c) Load the first purified product onto an anion exchange chromatography column, elute with a salt buffer after equilibration, collect the target component, and obtain the second purified product; d) Load the second purified product onto a gel filtration chromatography column, elute with isocratic or gradient buffer, collect the recombinant human albumin monomer peak, and obtain the high-purity recombinant human albumin final product.

[0021] Further, the amino acid sequence of the human albumin-binding peptide 1E3 described in step b) is shown in SEQ ID NO: 1. The equilibration buffer is a PBS solution with a pH of 7.0–8.0; the elution buffer is a glycine-HCl solution with a pH of 2.8–3.2; and the neutralization buffer is a Tris-HCl solution with a pH of 8.0–8.5.

[0022] Further, the anion exchange chromatography medium in step c) is Streamline Q, Streamline Q XL, or Streamline DEAE; the equilibration buffer is 20–50 mM sodium phosphate buffer with a pH of 7.0–8.0.

[0023] Further, the gel filtration chromatography medium in step d) is Sephadex, Superdex or Sephacryl series media; the mobile phase buffer is 50 mM Tris-HCl + 0.1 M NaCl, or phosphate buffer, with a pH of 7.0–8.0.

[0024] 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).

[0025] In a sixth aspect, the present invention provides the use of the human albumin-binding peptide, 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.

[0026] The beneficial effects of the present invention include, but are not limited to: High specificity and high efficiency: The human albumin-binding peptide disclosed in this invention has an extremely high affinity for human albumin, with a dissociation equilibrium constant KD = 5.80E-10 M. It can efficiently capture target proteins in a single step from complex fermentation broths, with high loading capacity and low non-specific adsorption.

[0027] Ultra-high purity: This invention also discloses a chromatography medium for separation and purification. The chromatography medium, combined with a clever combination and optimization of a three-step chromatography process, forms an efficient impurity removal process (affinity chromatography removes most impurities, anion exchange removes HCP / nucleic acid, and molecular sieve removes aggregates). The purity of the final product far exceeds the pharmacopoeia standard, reaching over 99.99%.

[0028] Good safety profile: Compared to dye ligands, peptide ligands pose no risk of foreign matter shedding, and are easier to clean and verify, resulting in higher product safety. The process is stable and easy to scale up: The purification process has clear steps, mild conditions, high recovery rates in each step, and a considerable overall yield. Moreover, all chromatography techniques are very mature and easy to scale up for production, making it highly valuable for industrial applications. Attached Figure Description

[0029] 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 1This is an SDS-PAGE electrophoresis image of the purified human albumin-binding peptide in this embodiment of the invention. M: marker; 1: elution; 2: before loading.

[0030] Figure 2 This is a graph showing the affinity detection results of human albumin-binding peptide 1E3 in an embodiment of the present invention.

[0031] Figure 3 This is an HPLC chromatogram of purified recombinant human albumin in an embodiment of the present invention. Detailed Implementation

[0032] 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.

[0033] 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.

[0034] 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).

[0035] Example 2: Phage Library Construction Total RNA was extracted from PBMCs using the Trizol method, and cDNA was synthesized via reverse transcription. Single-domain antibody fragments were amplified by two PCR cycles, digested with restriction endonucleases, and ligated into a phage plasmid. The fragments were then transformed into *E. coli* TG1 competent cells (manufacturer: TransGen, catalog number: CD201-01) using electroporation. The library volume was determined to be 5.15 × 10⁻⁶ cells using a serial dilution method. 9 Forty single clones were randomly selected for PCR identification, and the positive rate was 39 / 40 (97.5%), proving that the library was successfully constructed.

[0036] Example 3: Screening of human albumin-binding peptides Add streptavidin magnetic beads (Thermo Fisher, catalog number: 11205D) and enzymatically hydrolyzed casein (Solarbio, catalog number: C8210-100) to EP tubes, and block with shaking at 37°C for 1 h (100 rpm). Remove the blocking solution. Add recombinant human albumin, and incubate with shaking at 37°C for 1 h for coating, 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 by 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, perform ELISA detection, select qualified positive single colonies, and perform gene sequencing to obtain multiple sequences, one of which is named 1E3, whose amino acid sequence is shown in SEQ ID NO. 1 and whose nucleotide sequence is shown in SEQ ID NO. 2.

[0037] Example 4 Expression and purification of human albumin-binding peptide in Pichia pastoris (1) Pichia pastoris expressing human albumin-binding peptide 1E3 The 1E3 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 Thermo Fisher Scientific, catalog number C18000), and single colonies were screened on Zeocin-resistant plates. Secretory expression was induced by routine inoculation, with methanol added every 24 h to a final concentration of 0.5%. After 72 h of induction, the supernatant was collected by centrifugation. SDS-PAGE analysis confirmed correct 1E3 expression.

[0038] (2) Purification of human albumin-binding peptide 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 solution. If the protein concentration was low after desalting, it was concentrated using an ultrafiltration tube. The purity of the collected solutions at each stage of purification 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.

[0039] Example 5: Detection of affinity of human albumin-binding peptides The affinity of human albumin-binding peptide 1E3 was detected using biomembrane interferometry (BLI). Using an NTA biosensor, the sensor was first equilibrated in analytical buffer for 10 min, then activated by immersing it in an EDC-NHS mixture for 5 min. The activated sensor was then incubated in human albumin-binding peptide dilution buffer (100 nM) for 10 min and blocked with ethanolamine (1 M, pH 8.5). The blocked sensor was then zeroed by immersing it in buffer solution. Next, the sensor was sequentially immersed in gradient concentrations of recombinant human albumin solutions (7.8 nM, 15.625 nM, 31.25 nM, 62.5 nM, 125 nM, 250 nM, 500 nM) for 5 min to bind, and complete binding curves were generated. Finally, the sensor was transferred to PBS buffer for dissociation for 5 min. Kinetic analysis was performed using a 1:1 binding model, and the results are as follows: Figure 2 As shown, the dissociation equilibrium constant KD = 5.80E-10 M indicates that 1E3 has a strong affinity for recombinant human albumin.

[0040] Example 6 Preparation of human albumin-binding peptide affinity chromatography medium Human albumin-binding peptide 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 mixed with an equal volume of 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. Five cycles were performed, alternating between washing buffer 1 (0.1 M HAC + 0.5 M NaCl, pH 4.0) and washing buffer 2 (0.1 M Tris-HCl + 0.5 M NaCl, pH 8.0), each cycle using 5 times the volume of the medium. After washing with PBS (pH 7.4), store at 4°C for later use.

[0041] 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 min 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 min, and quickly cool to below 25℃ in an ice bath. Centrifuge at 12000 rpm (approximately 20,000 × g) for 15 min 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.

[0042] The human albumin-binding peptide 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. Elution buffer (0.1 M glycine-HCl, pH 2.8) was used to elute 4 column volumes (428 ml) at a flow rate of 1.0 ml / min, and 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.

[0043] Example 8: Anion exchange chromatography purification of recombinant human albumin The first purified product obtained in Example 7 was replaced with an ultrafiltration membrane pack (10 kDa molecular cutoff, purchased from Millipore) at an operating pressure of 0.2 MPa, and replaced with 20 mM sodium phosphate buffer (pH 7.4). The concentration and dilution were repeated 3 times until the conductivity and pH were consistent with the target buffer.

[0044] Prepare a Streamline QXL anion exchange chromatography column (Cytiva, 26 mm × 150 mm, gel volume 76 ml). Equilibrate at least 5 column volumes (380 ml) with 20 mM sodium phosphate buffer (pH 7.4) at a linear flow rate of 60 cm / h until conductivity and pH stabilize. Load the equilibrated sample at a flow rate of 40 cm / h. After loading, wash with equilibration buffer until the UV baseline is stable. Then, perform linear gradient elution with 20 mM sodium phosphate buffer (pH 7.4) containing 1.0 M NaCl, with a gradient volume of 10 column volumes (760 ml) and a flow rate maintained at 60 cm / h. Collect the main elution peak based on the UV absorption spectrum (280 nm) to obtain the second purified product.

[0045] Example 9: Gel filtration chromatography purification of recombinant human albumin The second purified product obtained in Example 8 was concentrated by centrifugation at 4°C and 4000×g using an ultrafiltration centrifuge tube (molecular cutoff 10 kDa, purchased from Millipore, catalog number: UFC901024). The volume and protein concentration were monitored every 15 min until the protein concentration stabilized at 50±2 mg / mL (concentrated volume approximately 25.9 mL). During concentration, a small amount of 50 mM Tris-HCl + 0.1 M NaCl buffer (pH 7.6) was added to prevent localized high concentrations that could lead to protein denaturation. After concentration, the sample was filtered using a 0.22 μm PVDF membrane (purchased from Millipore, catalog number: SLGV033RB) in a sterile environment. The clear filtrate was collected and stored at 4°C for future loading.

[0046] A glass chromatography column (40 mm × 410 mm, bed volume approximately 523 ml) was packed with Sephadex G100 gel permeate media (Cytiva, catalog number 17001001). The column was connected to an AKTA pure 250 system (Cytiva), with the detection wavelength set to 280 nm, conductivity monitoring range of 0–50 mS / cm, and column pressure limit of 0.15 MPa. At least three column volumes (1569 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 26 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 (10 kDa cutoff), and replaced with water for injection. Subsequently, they were freeze-dried to obtain high-purity recombinant human albumin lyophilized powder.

[0048] Example 10 Purity and Yield Analysis (1) Purity analysis of recombinant human albumin The purity of the final rHSA product (lyophilized powder) in Example 9 was analyzed using high performance liquid chromatography (HPLC). The product was dissolved and diluted to a concentration of 10 mg / mL with the mobile phase, filtered through a 0.22 μm filter, and loaded onto a TSKgel G3000SWxl column with PBS as the mobile phase at a flow rate of 0.8 ml / min and a detection wavelength of 280 nm. The final HPLC chromatogram (Figure 3) showed that the retention time of the rHSA main peak was 13.425 min, with a symmetrical peak shape and no obvious tailing. The main peak area accounted for 99.99% of the total peak area, with the aggregate peak (retention time approximately 10.5 min) accounting for 0.008% and the small molecule impurity peak (retention time approximately 18.2 min) accounting for 0.002%. Both values ​​are far below the requirements of the Pharmacopoeia of the People's Republic of China (2020 edition) for pharmaceutical-grade rHSA: "purity ≥ 99.9% and aggregate content ≤ 0.1%".

[0049] (2) Calculation of recombinant human albumin yield Protein concentrations at each step were determined using the BCA method, and the overall yield was calculated. The Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, catalog number: 23225) was used to quantify the protein concentration at each purification step 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>0.99). All test samples were appropriately diluted to ensure their concentration fell within the linear range of the standard curve. Each sample was tested in triplicate, with 25 μL of standard or diluted sample added to each well, followed by 200 μL of BCA working solution. After incubating the 96-well plate at 37°C for 30 min, the absorbance was measured at 562 nm using a microplate reader (BioTek Synergy H1). The sample concentration was calculated based on the standard curve. The yields of each step were calculated starting from the supernatant after pretreatment, as shown in Table 1. The total protein content in all steps was calculated as “concentration (BCA method determination) × 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 <1.2% (the SD of the pretreatment step is 1.0%~1.2%, the SD of anion exchange chromatography and gel filtration chromatography is 0.5%~0.8%, and the SD of affinity chromatography is 0.3%~0.6%). The SD of the overall yield is 1.7% (derived from the cumulative calculation of the deviations of each step).

[0050] Table 1 Recovery rate of each step

[0051] The overall yield reached 76.7%, which is 2.4 times higher than the 32% of the traditional process (CN102190722A2). This further verifies the high efficiency of the process in this application.

[0052] Example 11 Safety Index Testing (1) Endotoxin detection Endotoxin was detected using a dynamic turbidimetric endotoxin assay kit (manufacturer: Lonza, catalog number: N588). The recombinant human albumin sample obtained in Example 9 was dissolved and diluted with water for endotoxin testing (BET water) (manufacturer: Lonza, catalog number: W50-100) until its expected endotoxin value was within the range of the standard curve. Standard curve preparation: The endotoxin working standard (manufacturer: Lonza, catalog number: E0005) was diluted with BET water to prepare a series of solutions with 5 concentration points. Following the kit instructions, 100 μL of the standard, sample, or negative control (BET water) was added to pyrogen-free reaction tubes or 96-well plates. 100 μL of Limulus Amebocyte Lysate (LAL) reagent was added to each well, and the plate was immediately placed in a dynamic turbidimetric rapid microbial detection system or a microplate reader with this function. The absorbance was continuously monitored at 37°C for 70 min. The instrument software was programmed according to the standard curve (R... 2 =0.998) The automatic calculation showed that the endotoxin concentration in recombinant human albumin was 0.23 EU / mg rHSA, which is less than the limit of 0.5 EU / mg specified in the Pharmacopoeia of the People's Republic of China (2020 edition), proving that the final product meets the safety standards.

[0053] (2) Detection of residual host cell protein (HCP) Enzyme-linked immunosorbent assay (ELISA) was used for the determination. A commercially available assay kit specifically targeting Pichia pastoris (manufacturer: Cygnus Technologies, catalog number: F550) with a detection range of 1-100 ng / mL was used. Before the assay, the rHSA sample was diluted to 5 mg / mL to pre-validate that rHSA at this concentration would not interfere with the detection. A standard curve (R0) was plotted for 1-100 ng / mL. 2 Calculations were performed using a ratio of 0.997, and the results showed that the residual HCP in the final product was only 0.35 ng / mg rHSA. 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 genome of Pichia pastoris. GAPDH Gene. First, DNA enrichment and purification were performed on a 100 mg final product sample using a DNA extraction kit (Qiagen, catalog number: 51304). Amplification was then performed using a specific qPCR detection kit (Thermo Fisher Scientific, catalog number: A24554). A standard curve of 1-10000 pg / mL was constructed, and the melting curve of the amplified products was analyzed (single peak, Tm = 85.2℃) to ensure the specificity and accuracy of the detection. The final calculated residual DNA content was 0.12 pg / mg rHSA. This result far exceeds 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 validating the superior efficiency of this process in removing nucleic acid impurities, and ensuring that the final product meets pharmaceutical safety requirements.

[0055] 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, characterized in that, The amino acid sequence of the human albumin-binding peptide includes at least one of the following: A1) 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 of claim 1; B2) An expression cassette containing the nucleic acid molecule described in B1); B3) A recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2); B4) Recombinant microorganisms containing nucleic acid molecules of B1), or recombinant microorganisms containing recombinant vectors of expression cassettes of B2), or recombinant microorganisms containing recombinant vectors of B3); B5) Recombinant cells, wherein the recombinant cells contain the nucleic acid molecules of B1), or the recombinant cells contain the expression cassette of B2), or the recombinant cells contain the recombinant vector of B3).

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

3.

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

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

6. A chromatography medium for separating and purifying human albumin, characterized in that, The chromatography medium comprises the human albumin-binding peptide of 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 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, anion exchange chromatography, and gel filtration chromatography.

10. The application of the human albumin-binding peptide as described in claim 1, or the biomaterial as described in any one of claims 2-4, or the chromatography medium as described in claim 6 or 7, characterized in that, The application includes at least one of the following: 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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