Human albumin binding peptide 1b3 and use in purification of human albumin

Abstract

This invention discloses a human albumin-binding peptide 1B3 and its application in human albumin purification, belonging to the field of peptide technology. The amino acid sequence of the human albumin-binding peptide includes the amino acid sequence shown in SEQ ID NO.1; and / or the 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 shown in SEQ ID NO.1. The human albumin-binding peptide has extremely high affinity for human albumin and can be used to separate and purify human albumin solutions. The purity of the purified human albumin far exceeds the pharmacopoeia standard, reaching over 99.95%. It exhibits good safety and stable process, and has broad application prospects in the separation and purification of human albumin.

Description

Technical Field

[0001] This invention relates to the field of polypeptide technology, specifically to a human albumin-binding peptide 1B3 and its application in the purification of human albumin. Background Technology

[0002] Human serum albumin (HSA) is the most abundant multifunctional protein in human plasma, accounting for 50%-60% of total plasma protein. It plays an irreplaceable role in maintaining plasma colloid osmotic pressure, transporting fatty acids, hormones, drugs and metabolic waste, resisting oxidative stress, and regulating immune responses. Clinically, HSA is widely used to treat hemorrhagic shock, burn shock, hypoalbuminemia caused by cirrhosis, nephrotic syndrome, and other diseases. It is also a key excipient in vaccine formulations, cell therapy culture media, and biopharmaceutical stabilizers, and its market demand remains strong.

[0003] For a long time, clinical HSA has mainly relied on isolation and purification from human plasma. However, this traditional source has three major limitations: First, the supply of blood is scarce. Affected by the amount of blood donated, geographical distribution, and seasonality, HSA has long been in a state of supply and demand imbalance globally. Second, there are significant safety risks. Plasma may contain pathogens such as hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus (HIV). Even with virus inactivation processes (such as pasteurization and nanofiltration), the risk of contamination by unknown pathogens cannot be completely eliminated. Third, there are ethical and cost issues. Plasma collection must strictly follow ethical guidelines, and the processes of plasma pretreatment and multi-step inactivation increase production costs, further limiting its accessibility.

[0004] To address the inherent limitations of plasma sources, the production of recombinant human serum albumin (rHSA) using gene recombination technology has become a hot research topic in the industry. Currently, the main expression hosts for rHSA are microorganisms such as Pichia pastoris and Saccharomyces cerevisiae, which have advantages such as high expression levels, low culture costs, and the ability to secrete and express target proteins. However, the composition of yeast fermentation broth is extremely complex, containing not only rHSA but also a large amount of host cell protein (HCP), residual host DNA, endotoxins, yeast polysaccharides, pigments, and incompletely degraded culture medium components. Among these, HCP may trigger human immune responses (such as allergic reactions), endotoxins can cause toxic side effects such as fever and shock, and residual DNA poses a potential carcinogenic risk. Therefore, the Pharmacopoeia of the People's Republic of China (2020 edition) and international drug regulatory agencies (such as the FDA and EMA) have extremely stringent requirements for the purity of pharmaceutical-grade rHSA, clearly stipulating that HCP residues ≤10 ng / mg, endotoxins ≤1 EU / mg, and residual host DNA ≤10 pg / mg, with a purity of 99.5% or higher.

[0005] The key to achieving large-scale purification of high-purity rHSA lies in constructing an efficient and specific chromatographic process system. Existing rHSA purification schemes generally suffer from the following key bottlenecks: Lengthy and inefficient process routes: Most schemes rely on a combination of multiple steps, such as cation exchange chromatography, hydrophobic chromatography, ConA affinity chromatography, and anion exchange chromatography (e.g., the four-step process reported in "Optimization Study of Recombinant Albumin (rHSA) Purification Process"). Some processes even require additional steps such as heating precipitation, ultrafiltration, and chelation chromatography (e.g., the more than six-step process disclosed in patent CN102190722A2). This lengthy process results in production cycles lasting several days, and each step involves target protein loss, with overall yields generally below 40%, making it difficult to meet the economic requirements of industrial production. Insufficient chromatographic specificity and limited impurity removal capabilities: Traditional processes rely on techniques such as ion exchange chromatography and hydrophobic chromatography, which mainly rely on the differences in charge and hydrophobicity between the target protein and impurities for separation, resulting in low specificity. For example, cation exchange chromatography can initially capture rHSA, but it cannot effectively remove HCPs with isoelectric points close to rHSA; hydrophobic chromatography can remove some hydrophobic proteins, but it is not effective in removing yeast-specific acidic polysaccharides, aggregates, and other impurities, requiring additional purification steps, further increasing the complexity of the process; the operating conditions are harsh and difficult to scale up: some novel processes (such as the two-step mixed-mode chromatography disclosed in patent CN112210002B5) rely on special synthetic ligands, which are not only expensive to produce, but also sensitive to chromatographic conditions (such as pH and ionic strength), with even small fluctuations affecting adsorption efficiency; another approach uses expanded bed chromatography to directly treat fermentation broth (such as CN102190722A2), but this technology requires high equipment precision, and suspended particles in the fermentation broth easily clog the pores of the chromatographic medium, leading to a rapid decline in column efficiency and making it difficult to stably scale up to a thousand-liter fermentation broth treatment scale; affinity ligands have inherent defects: the ligands used in existing rHSA affinity chromatography are mostly dyes (such as Cibacin Blue). F3GA) or natural proteins (such as fatty acids) are used. The former has the risk of dye shedding (which may introduce new impurities), while the latter has insufficient binding specificity to rHSA and is easily interfered with by fatty acid substances in the fermentation broth, resulting in the affinity step yield often being less than 90%, which cannot meet the dual requirements of purity and yield.

[0006] In summary, current technologies for large-scale purification of high-purity rHSA from yeast fermentation broth still face multiple challenges, including complex processes, low yields, poor specificity, and difficulty in scale-up. Developing a purification process based on highly specific affinity ligands, with simplified steps, excellent yields, and ease of scalability, is crucial to overcoming the bottlenecks in rHSA industrialization. This invention addresses this technical need by designing a three-step process—cation exchange, anion exchange, and affinity chromatography—using human albumin-binding peptide 1B3 as the core affinity ligand. This aims to overcome the core deficiencies of existing technologies and provide a reliable solution for the large-scale production of pharmaceutical-grade rHSA.

[0007] Therefore, there is an urgent need in this field to develop a streamlined, highly efficient, specific, easily scalable, and high-yield chromatographic purification process to meet the needs of large-scale production of recombinant human serum albumin. In particular, the development of novel highly specific affinity ligands and their intelligent combination with efficient capture and purification steps holds promise as a key to solving the aforementioned problems. Summary of the Invention

[0008] To address the shortcomings of existing technologies, this application discloses a multi-step chromatographic purification process for the large-scale production of high-purity recombinant human albumin (rHSA). First, cation exchange chromatography is used to initially capture and concentrate the target protein, utilizing the charge difference between the target protein and impurities to efficiently remove a large amount of negatively charged impurities, nucleic acids, and some acidic polysaccharides. Subsequently, anion exchange chromatography is used for further fine separation. Based on the charge difference between the target protein and residual impurities (such as strongly negatively charged host cell proteins (HCP), endotoxins, and unremoved nucleic acid fragments), these impurities are specifically adsorbed and removed, while reducing the impact of salt concentration fluctuations in the sample on subsequent steps. Finally, utilizing the high specificity of human albumin-binding peptides, a highly efficient affinity chromatography medium is prepared using the specific human albumin-binding peptide 1B3 as an affinity ligand, resulting in final fine purification to obtain extremely high-purity rHSA. The final product purity is ≥99.95%, meeting pharmaceutical-grade standards. This process is efficient, robust, and suitable for large-scale production, providing a reliable solution for the widespread application of rHSA.

[0009] In a first aspect, the present invention provides a human albumin-binding peptide 1B3, wherein the amino acid sequence of the human albumin-binding peptide comprises 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) 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.

[0012] 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).

[0013] Those skilled in the art will understand that reasonable sequence modifications can be made to the sequence shown in SEQ ID NO:1 without affecting its affinity activity for human albumin, and these modified sequences should also fall within the scope of protection of this application. Such modifications include, but are not limited to: conserved amino acid substitution, partial amino acid deletion, addition, and N-terminal or C-terminal truncation; the modified peptide should still retain the binding ability and specificity for human albumin equivalent to the sequence in SEQ ID NO:1. Furthermore, the binding peptide can be chemically modified in accordance with conventional techniques, including but not limited to: cyclization, acetylation, PAS conversion, PEGylation, fatty acid modification, coupling with human albumin or its binding peptide, fusion with tumor homing peptides or transmembrane peptides, binding with nanocarriers, or coupling with radionuclides, small molecule compounds, nucleotides, or proteins; modifications can occur at the N-terminus, C-terminus, main chain, side chain, or specific amino acid residues of the peptide.

[0014] In a second aspect, the present invention provides a biomaterial comprising at least one of the following:

[0015] B1) Contains a nucleic acid molecule encoding the human albumin-binding peptide described above;

[0016] B2) An expression cassette containing the nucleic acid molecule described in B1);

[0017] B3) A recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);

[0018] 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);

[0019] B5) Recombinant cells containing the nucleic acid molecule described in B1), or recombinant cells containing the expression cassette described in B2), or recombinant cells containing the recombinant vector described in B3).

[0020] 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 1B3; SEQ ID NO. 3 is a sequence optimized for Pichia pastoris codon preferences.

[0021] 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.

[0022] 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.

[0023] 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.

[0024] Optionally, the recombinant microorganism is Pichia pastoris.

[0025] 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.

[0026] 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.

[0027] 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.

[0028] 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 1B3 ligand-crosslinked agarose affinity medium. Preferably, the packing material is a packing material with the human albumin-binding peptide as a ligand, wherein the matrix can be selected from commonly used chromatography support materials such as agarose, cellulose, crosslinked dextran, polyacrylamide, or porous glass beads.

[0029] 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.

[0030] Optionally, the purification steps include at least cation exchange chromatography, anion exchange chromatography, and affinity chromatography purification.

[0031] Furthermore, the human albumin includes human serum albumin and recombinant human albumin.

[0032] Furthermore, the method for separating and purifying human albumin specifically includes the following steps:

[0033] a) Centrifuge the fermentation broth or human blood products containing recombinant human albumin to obtain the supernatant;

[0034] b) Load the supernatant obtained in step a) onto a cation exchange chromatography column, elute with a salt buffer after equilibration, collect the target component, and obtain the first purified product;

[0035] c) Load the first purified product obtained in step b) onto an anion exchange chromatography column, elute after equilibration, and obtain the second purified product;

[0036] d) The second purified product obtained in step c) is finely purified by affinity chromatography with human albumin-binding peptide 1B3 as a ligand to obtain high-purity recombinant human albumin, wherein the amino acid sequence of the human albumin-binding peptide 1B3 is shown in SEQ ID NO: 1.

[0037] Further, the cation exchange chromatography medium mentioned in step b) includes at least one of NanoGel 50SP, SPSepharose Fast Flow, and Capto S, and the elution buffer includes at least one of sodium chloride buffer and phosphate buffer, with a pH of 7.6.

[0038] Furthermore, step b) also includes washing with a washing buffer before elution, wherein the washing buffer includes at least one of sodium chloride buffer, acetate buffer, and phosphate buffer, and the pH is 4.5.

[0039] Further, step c) includes the anion exchange chromatography using at least one of Q Sepharose Fast Flow, Q Bestarose HP, and DEAE Sepharose Fast Flow. The equilibration buffer pH is 8.0; the elution buffer pH is 8.0.

[0040] Further, in step d), the pH of the equilibration buffer for affinity chromatography is 7.0-8.0, and the pH of the elution buffer is 2.8-3.2. The equilibration buffer includes at least one of PBS buffer and citrate buffer; the elution buffer includes at least one of glycine-HCl buffer and citrate buffer.

[0041] In some embodiments, the cation exchange chromatography uses NanoGel 50SP media, with the equilibration buffer being 20 mM acetate buffer (pH 4.5); the washing buffer being 0.2 M sodium chloride + 20 mM acetic acid (pH 4.5); and the elution buffer being 0.5 M sodium chloride + 20 mM sodium phosphate (pH 7.6).

[0042] In some embodiments, the anion exchange chromatography uses Q Sepharose Fast Flow medium, with an equilibration buffer of 20 mM Tris-HCl (pH 8.0) and an elution buffer of 0.3 M NaCl + 20 mM Tris-HCl (pH 8.0).

[0043] In some embodiments, the equilibration buffer for the affinity chromatography is PBS solution (pH 7.4); the elution buffer is 0.1 M glycine-HCl (pH 3.0); and neutralization is performed immediately after elution with neutralization buffer (1 M Tris-HCl, pH 8.0).

[0044] It should be noted that the present invention does not explicitly limit the types of buffer solutions or pH values, and those skilled in the art can adjust the types and pH values ​​of the buffer solutions based on practical experience.

[0045] 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).

[0046] 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:

[0047] C1) Applications in the preparation of products for the isolation or purification of human albumin;

[0048] C2) Applications in the preparation of products for the qualitative or quantitative detection of human albumin;

[0049] C3) Use in the preparation of pharmaceutical compositions for immunization or treatment.

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

[0051] 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 = 1.90E-09 M. It can efficiently capture target proteins from complex fermentation broths, with high loading capacity and low non-specific adsorption.

[0052] Ultra-high purity: This invention utilizes a clever combination of three-step chromatography—cation exchange, anion exchange, and affinity chromatography—to fully leverage the synergistic effect of different separation principles. In particular, the final affinity chromatography based on the specific human albumin-binding peptide 1B3 efficiently removes trace amounts of host proteins, aggregates, and other impurities that are difficult to remove in the first two steps. The resulting recombinant human albumin (rHSA) achieves a stable HPLC purity of over 99.95%, with extremely low levels of key impurities such as endotoxins. The product quality meets or even exceeds the requirements for pharmaceutical-grade excipients.

[0053] High process yield and significant economic benefits: Compared to existing multi-step purification processes with generally low yields, this invention optimizes the connection and operating conditions of each step, significantly reducing the loss of the target product during purification. The overall purification yield can reach 75.1%, greatly improving raw material utilization and production efficiency, reducing the production cost per unit product, and demonstrating significant economic benefits and market competitiveness.

[0054] High specificity and high separation efficiency: The core of this invention lies in the use of independently developed human albumin-binding peptide 1B3 as an affinity ligand. This ligand has high affinity and specificity for rHSA, enabling direct and rapid capture of target proteins from complex samples. It overcomes the shortcomings of traditional dye affinity ligands, such as poor specificity, dye shedding, or insufficient selectivity in ion exchange methods, thereby significantly improving the selectivity and separation efficiency of the purification process. Attached Figure Description

[0055] 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:

[0056] Figure 1 This is an SDS-PAGE electrophoresis image of the purified human albumin-binding peptide in this embodiment of the invention. M: marker; 1: elution.

[0057] Figure 2 This is a graph showing the affinity detection results of human albumin-binding peptide 1B3 in an embodiment of the present invention.

[0058] Figure 3 This is an HPLC chromatogram of purified recombinant human albumin in an embodiment of the present invention. Detailed Implementation

[0059] 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.

[0060] 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.

[0061] Example 1: Camel Immunization and Peripheral Blood Lymphocyte Isolation

[0062] 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).

[0063] Example 2: Phage Library Construction

[0064] 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.

[0065] Example 3: Screening of human albumin-binding peptides

[0066] 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, perform ELISA detection, select qualified positive single colonies, and perform gene sequencing to obtain multiple sequences, one of which is named 1B3, whose amino acid sequence is shown in SEQ ID NO. 1 and whose nucleotide sequence is shown in SEQ ID NO. 2.

[0067] Example 4: Expression and purification of human albumin-binding peptide in Pichia pastoris

[0068] (1) Pichia pastoris expressing human albumin-binding peptide 1B3

[0069] The 1B3 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 1B3 expression.

[0070] (2) Purification of human albumin-binding peptide expressed in Pichia pastoris

[0071] 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.

[0072] Example 5: Detection of affinity of human albumin-binding peptides

[0073] The affinity of human albumin-binding peptide 1B3 was detected using biomembrane interferometry (BLI). Using an NTA biosensor, the sensor was first equilibrated in analytical buffer for 10 min, then activated by EDC-NHS mixed reagent 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 baseline adjustment using 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 2As shown, the dissociation equilibrium constant KD = 1.90E-09 M indicates that 1B3 has a strong affinity for recombinant human albumin.

[0074] Example 6 Preparation of human albumin-binding peptide affinity chromatography medium

[0075] 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.

[0076] Example 7 Purification of recombinant human albumin by cation exchange chromatography

[0077] Take 2000 mL of yeast-expressed recombinant human albumin fermentation broth and centrifuge at 4℃, 10,000 × g for 20 min, carefully collecting the supernatant. Slowly add 100 mM sodium octanoate stock solution to the supernatant to achieve a final concentration of 15 mM, and gently stir to mix. Adjust the pH to 6.0 ± 0.1 using 1 M HCl solution while stirring. Heat the sample in a constant temperature water bath at 65.0 ± 0.5℃ for 45 min, and then quickly transfer it to an ice-water bath to cool to below 10℃. Subsequently, centrifuge at 4℃, 15,000 × g for 15 min, collect the supernatant, and filter it through a 0.45 μm PES membrane to obtain a clear sample.

[0078] Prepare a NanoGel 50SP cation exchange chromatography column (purchased from Suzhou Nanomicro Technology Co., Ltd., catalog number: 04062-050800, column size: 26mm × 300mm, column bed volume: 160mL). Equilibrate with 5 column volumes (800 mL) of equilibration buffer (20 mM sodium acetate, pH 4.5) at a linear flow rate of 150 cm / h until conductivity and pH stabilize. Load all pretreated samples at a flow rate of 50 cm / h. After loading, wash with equilibration buffer until the UV 280 nm absorbance returns to baseline, then wash with 5 column volumes of wash buffer (0.2 M NaCl + 20 mM sodium acetate, pH 4.5). Finally, perform linear gradient elution with elution buffer (0.5 M NaCl + 20 mM sodium phosphate, pH 7.6) at a gradient volume of 10 column volumes (1600 mL) and a flow rate maintained at 50 cm / h. The main elution peak was collected based on the UV absorption spectrum (280 nm) to obtain the first purified product.

[0079] Example 8: Anion exchange chromatography purification of recombinant human albumin

[0080] The first purified product obtained in Example 7 was measured and filtered using a 0.45 μm PES filter membrane (Millipore, catalog number SLHP04700) to remove any possible residual small particulate impurities, and the clear filtrate was collected.

[0081] Prepare a Q Sepharose Fast Flow anion exchange chromatography column (GE Healthcare, catalog number 17-0510-01) and pack it into the column (column size: 26 mm × 200 mm, purchased from Cytiva, catalog number 28985058, column bed volume: 106 mL). Equilibrate with 5 column volumes (530 mL) of equilibration buffer (20 mM Tris-HCl, pH 8.0) at a linear flow rate of 120 cm / h. Load all samples at a flow rate of 50 cm / h. After sample loading, the column was washed with equilibration buffer at a flow rate of 120 cm / h until the UV absorbance at 280 nm returned to baseline. Then, a linear gradient elution was performed using 10 column volumes (1060 mL) of elution buffer (0.3 M NaCl + 20 mM Tris-HCl, pH 8.0) at a flow rate maintained at 80 cm / h (0% - 100%). Based on the UV absorbance (280 nm) monitoring results, the main protein peak was collected, yielding the second purified product.

[0082] Example 9: Affinity chromatography purification of recombinant human albumin

[0083] The second purified product obtained in Example 8 was replaced with PBS buffer (10 mM sodium phosphate, 150 mM NaCl, pH 7.4) using a Millipore Pellicon 2 ultrafiltration membrane pack (10 kDa molecular cutoff, catalog number: P2C010C01) at an operating pressure of 0.2 MPa. The solution was then concentrated and diluted three times until the conductivity and pH were consistent with those of PBS.

[0084] The human albumin-binding peptide affinity medium prepared in Example 6 was packed into an HR 26 / 20 chromatography column (purchased from Cytiva, column bed volume 105 mL). Equilibration was performed using 5 column volumes (525 mL) of equilibration buffer (PBS, pH 7.4) at a flow rate of 2.0 mL / min. All samples after buffer replacement were loaded at a flow rate of 1 mL / min. During loading, the flow-through was monitored using UV 280 nm until the absorbance value of the flow-through was ≤0.05 AU. Elution was performed using elution buffer (0.1 M glycine-HCl, pH 3.0) at a flow rate of 2.0 mL / min in a stepwise gradient. The elution peaks were collected, and immediately neutralization buffer (1 M Tris-HCl, pH 8.0) was added to the collection tube at a ratio of 10:1 (v / v) and gently mixed. The neutralized eluents were combined, desalted using Amicon Ultra-15 ultrafiltration centrifuge tubes (10 kDa molecular weight cutoff, Millipore, catalog number: UFC901024), and replaced with water for injection. Finally, high-purity recombinant human albumin lyophilized powder was obtained by freeze drying.

[0085] Example 10 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 9 was analyzed by high performance liquid chromatography (HPLC). An Agilent 1260 Infinity II HPLC system equipped with a TSKgel G3000SWxl column (7.8 mm × 30 cm, 5 μm, Tosoh Bioscience, catalog number: 08541) was used. Phosphate-buffered saline (PBS, 10 mM sodium phosphate, 150 mM NaCl, pH 7.4) was used as the mobile phase, with isocratic elution at a flow rate of 0.8 mL / min, a column temperature maintained at 25 ± 1°C, a detection wavelength of 280 nm, and an injection volume of 20 μL. 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 in the sample was 99.95% ± 0.01% (n=3). See the typical chromatogram below. Figure 3 The purity increased from 98.7% (CN 118580318 A) to 99.95%, an increase of 0.99 percentage points.

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

[0089] 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, BSA standards were 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>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. Starting from the total protein content of the pretreated supernatant (denoted as Step 0), after cation exchange chromatography (Step 1), anion exchange chromatography (Step 2), and affinity chromatography (Step 3), the overall purification yield was 75.1% ± 0.2% (n=3), which is 2 times higher than the 32% of the traditional process (CN102190722A2). This further verifies the high efficiency of the process in this application.

[0090] Table 1 Recovery rate of each step

[0091]

[0092] 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 1-3 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 1.8% (derived from the cumulative calculation of deviations from each step).

[0093] Example 11 Safety Index Testing

[0094] (1) Endotoxin detection

[0095] The endotoxin content of the final product was determined using the kinetic turbidimetric endotoxin assay kit (Lonza, catalog number: N588). First, the recombinant human albumin lyophilized powder obtained in Example 9 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 min. The instrument software automatically calculated the endotoxin concentration in the sample based on the standard curve (R²=0.998). Interference tests were performed according to the requirements of General Chapter 1143 of the Pharmacopoeia of the People's Republic of China (2020 edition): the endotoxin standard (0.5 EU / mL) was mixed with a 2 mg / mL sample solution, and the recovery rate was determined to be 100% (meeting the pharmacopoeia requirements), proving that the sample matrix did not interfere with the detection. The final result is the average of three independent tests. The endotoxin content in recombinant human albumin was measured to be 0.21 ± 0.08 EU / mg (n=3), 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 the safety standards.

[0096] (2) Detection of residual host cell protein (HCP)

[0097] Enzyme-linked immunosorbent assay (ELISA) was used for the determination. A commercially available assay kit specifically targeting *Pichia pastoris* (manufacturer: Cygnus Technologies, catalog number: F550) with a detection range of 1-100 ng / mL was used. The HCP standard used in this kit was purified HCP from the same *Pichia pastoris* strain as used in this experiment, and Western blotting confirmed its specificity in identifying the HCP of this strain (no cross-reactivity). Simultaneously, a spiked recovery test was performed on a 5 mg / mL rHSA sample (spiking concentration 20 ng / mL), with a recovery rate of 98%, verifying no matrix interference. Before detection, the rHSA sample was diluted to 5 mg / mL to pre-validate that rHSA at this concentration did not interfere with the detection. A standard curve (R²=0.999) was plotted from 1-100 ng / mL, and the results showed that the residual HCP in the final product was 4.2 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 Recombinant DNA Products for Human Use" (2020), which fully demonstrates that this purification process can efficiently remove Pichia pastoris host proteins.

[0098] (3) Residual DNA detection

[0099] 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 of 1-10000 pg / mL (R²=0.998) was constructed, and the melting curve of the amplified products was analyzed (single peak, Tm=85.5℃) to ensure the specificity and accuracy of the detection. The final calculated residual DNA content was 1.75 pg / mg rHSA. The results meet 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 safety of the final product meets the requirements for pharmaceutical use.

[0100] 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 is at least one of the following: A1) The amino acid sequence is as 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 is selected from any one of the following: B1) A nucleic acid molecule encoding 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 the nucleic acid molecules described in B1), or recombinant microorganisms containing the expression cassette described in B2), or recombinant microorganisms containing the recombinant vector described in B3); B5) Recombinant cells containing the nucleic acid molecules described in B1), or recombinant cells containing the expression cassette described in B2), or recombinant cells containing the recombinant vector described in B3).

3. The biomaterial according to claim 2, characterized in that, The nucleotide sequence of the nucleic acid molecule described in B1) is at least one of the nucleotide sequences 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 described in B4) or the recombinant cell described in B5) of 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 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 cation exchange chromatography, anion exchange chromatography, and affinity chromatography purification.

10. The use of the human albumin-binding peptide 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.

Images