Human albumin binding peptide 1a3 and use in purification of human albumin

Abstract

The application discloses a human albumin binding peptide 1A3 and application thereof in human albumin purification, and belongs to the technical field of polypeptides. The amino acid sequence of the human albumin binding peptide comprises an amino acid sequence as shown in SEQ ID NO. 1, and / or the amino acid sequence of a fusion protein with the same function obtained by connecting a 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 very high affinity to human albumin, can be used for isolating and purifying a human albumin solution, and the purity of the human albumin after purification is far higher than the pharmacopoeia standard, and can reach more than 99.99%. The safety is good, the process is stable, and the human albumin binding peptide has a wide application prospect in the aspect of human albumin isolation and purification.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of polypeptides, in particular to a human albumin binding peptide A3 and its application in human albumin purification. BACKGROUND

[0002] Human serum albumin (HSA) is the most abundant protein in human plasma, which plays a vital physiological function in maintaining plasma osmotic pressure, binding and transporting endogenous and exogenous substances (such as fatty acids, hormones, drugs) and so on. In clinic, it is widely used for treating shock, burn, hypoproteinemia and other diseases, and is also commonly used as a stabilizer and excipient in vaccines, cell culture medium and other biological agents. At present, the clinical human serum albumin mainly depends on isolation and purification from human plasma. However, the shortage of blood source, the risk of contamination by potential pathogens (such as hepatitis B virus, HIV virus) and the complex ethical issues seriously limit its supply and safety guarantee.

[0003] The production of recombinant human serum albumin (rHSA) by using gene recombination technology is considered as a fundamental way to solve the above problems. However, the fermentation expression system of rHSA is complex, which contains a large amount of host cell proteins (HCP), nucleic acids, endotoxins, pigments and host polysaccharides in addition to the target product. These impurities, especially the heterologous host proteins and polysaccharides, may trigger immune response, so the purity requirement of rHSA is extremely strict. The traditional rHSA purification process mainly depends on the combination of multi-step chromatography technology, such as cation exchange chromatography, anion exchange chromatography, hydrophobic chromatography, hydroxyapatite chromatography and so on.

[0004] There are many purification schemes in the prior art. The article "Optimization of Purification Process of Recombinant Human Serum Albumin" introduces a multi-step process combining cation capture, hydrophobic purification, ConA affinity chromatography and anion chromatography. This process can effectively remove host proteins, residual sugars, DNA and other impurities, but the steps are numerous and the process is long. Patent CN102190722A2 discloses a method for capturing rHSA directly from fermentation broth using anion exchange expanded bed (such as Streamline series resin). This method integrates solid-liquid separation, concentration and preliminary purification, but still needs to go through six or more steps of fine purification such as heating, hydrophobic chromatography, cation exchange, ultrafiltration, chelation and precipitation. The whole process is complex, and the total yield is only 32.2%. In addition, the expanded bed operation requires high equipment and requirements, and when the fermentation broth is not treated directly, the complex nature of the feedstock may affect the stability of the adsorbent performance. Another patent CN112210002B5 attempts to use a new two-step mixed mode chromatography method to simplify the process and improve efficiency, but it relies on specific synthetic ligands, and the universality and scalability of the process still need to be verified.

[0005] In summary, the main challenges of current large-scale purification of high-purity rHSA from complex fermentation broth are: first, the process route is long, involving multiple buffer replacement and intermediate treatment, resulting in long production cycle and low target protein recovery (usually low); second, the dependent chromatography mode is classic but has limited specificity, in order to achieve the extremely high purity required by the pharmacopoeia, multiple chromatography steps must be added, increasing production cost and operation complexity; third, in some existing processes, in order to meet the sample conditions of chromatography medium (such as low conductivity), the fermentation broth needs to be diluted or adjusted, increasing the processing volume and subsequent load.

[0006] Therefore, there is an urgent need in the art to develop a chromatography purification process with simplified steps, high efficiency and specificity, easy to scale up and excellent yield, in order to meet the needs of large-scale production of recombinant human serum albumin. In particular, the development of new high-specificity affinity ligands and their intelligent combination with high-efficiency capture and fine purification steps is expected to be the key to solving the above problems. SUMMARY

[0007] In view of the deficiencies of the prior art, the present application discloses a multi-step chromatographic purification process for large-scale production of high-purity recombinant human albumin. First, cation exchange chromatography is used to initially capture and concentrate the target protein, and remove a large amount of impurities such as albumin, nucleic acids, etc. Then, hydrophobic chromatography is used to remove aggregates, strongly hydrophobic impurities and part of endotoxins. Finally, by using the high specificity of human albumin binding peptide, a specific human albumin binding peptide 1A3 is used as the affinity ligand to prepare a high-efficiency affinity chromatography medium, and finally refined to obtain rHSA with extremely high purity, the purity of the final product is ≥ 99.99%, which meets the medical grade standard. The process is efficient and robust, suitable for large-scale production, and provides a reliable solution for the wide application of rHSA.

[0008] In a first aspect of the present application, a human albumin binding peptide 1A3 is provided, and the amino acid sequence of the human albumin binding peptide comprises at least one of the following:

[0009] A1) the amino acid sequence comprises the amino acid sequence shown in SEQ ID NO. 1;

[0010] A2) an amino acid sequence obtained by substitution, deletion and / or addition of amino acid residues of the amino acid sequence of the human albumin binding peptide of A1), which has more than 95% identity with the amino acid sequence of the human albumin binding peptide of A1);

[0011] A3) an amino acid sequence of a fusion protein with the same function obtained by connecting a tag protein to the N-terminus and / or C-terminus of the amino acid sequence of A1) or A2).

[0012] Those skilled in the art can understand that reasonable sequence changes that do not affect the human albumin affinity activity of the sequence shown in SEQ ID NO: 1 can be made based on the sequence, and these changed sequences should also be within the protection scope of the present application. The changes include but are not limited to conservative amino acid substitution, partial amino acid deletion, addition, and N-terminal or C-terminal truncation; the changed polypeptide should still retain the binding ability and specificity to human albumin comparable to the sequence of SEQ ID NO: 1. In addition, the binding peptide can also be subjected to conventional chemical modification in the art, and the modification methods include but are not limited to cyclization, acetylation, PASylation, PEGylation, fatty acid modification, coupling with human albumin or its binding peptide, fusion with tumor homing peptide or transmembrane peptide, binding with nanocarrier, or coupling with radionuclide, small molecule compound, nucleotide or protein; the modification can occur at the N-terminus, C-terminus, main chain, side chain or specific amino acid residues of the polypeptide.

[0013] In a second aspect of the present application, a biomaterial is provided, which comprises at least any one of the following:

[0014] B1) contains a nucleic acid molecule encoding the human albumin binding peptide;

[0015] B2) an expression cassette containing the nucleic acid molecule of B1);

[0016] B3) a recombinant vector containing the nucleic acid molecule of B1), or a recombinant vector containing the expression cassette of B2);

[0017] B4) a recombinant microorganism containing the nucleic acid molecule of B1), or a recombinant microorganism containing the recombinant vector of B2), or a recombinant microorganism containing the recombinant vector of B3);

[0018] B5) a recombinant cell containing the nucleic acid molecule of B1), or a recombinant cell containing the recombinant vector of B2), or a recombinant cell containing the recombinant vector of B3).

[0019] Further, the nucleic acid molecule in B1) includes a nucleic acid molecule encoding the human albumin binding peptide which has been disclosed and / or optimized according to actual needs, and optionally, the nucleic acid molecule in B1) includes at least one of the nucleotide sequences as shown in SEQ ID NO. 2 or SEQ ID NO. 3. Wherein, SEQ ID NO. 2 is a nucleotide sequence obtained after sequencing of the human albumin binding peptide 1A3; and SEQ ID NO. 3 is a sequence optimized for Pichia pastoris codon bias.

[0020] It should be understood that the nucleic acid molecule encoding SEQ ID NO: 1 can be optimized by those skilled in the art according to different expression systems (such as other engineering bacteria), and such variants are within the scope of the present application.

[0021] Further, the recombinant vector in B3) includes at least one of pET series vectors, pBAD vectors, pGEX series vectors, pCAl-n / pCAl-pelB vectors, pPOW3.0 vectors, pPIC series vectors, and pYES2 vectors.

[0022] Further, the recombinant microorganism in B4) or the recombinant cell in B5) includes at least one of hamster ovary cells, silkworm ovary cells, Pichia pastoris, Saccharomyces cerevisiae, Escherichia coli, and Bacillus subtilis.

[0023] Optionally, the recombinant microorganism is Pichia pastoris.

[0024] In a third aspect, the present application provides a method for preparing the human albumin binding peptide, which comprises the steps of culturing the recombinant microorganism of B4) or the recombinant cell of B5) under suitable culture conditions, and isolating the human albumin binding peptide.

[0025] It should be noted that the present application does not limit the recombinant microorganism or the recombinant cell, and any cell capable of expressing an exogenous gene through recombinant engineering technology is within the protection scope of the present application.

[0026] In a fourth aspect of the present application, a chromatography medium for separating and purifying human albumin is provided, wherein the chromatography medium comprises the human albumin binding peptide.

[0027] Optionally, the chromatography medium comprises at least one of a pre-packed column, a packing material, and a magnetic bead. Preferably, the magnetic bead is a magnetic bead coupled with the human albumin binding peptide. Preferably, the chromatography medium is a recombinant human albumin binding peptide 1A3 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 common chromatography support materials such as agarose, cellulose, cross-linked dextran, polyacrylamide, or porous glass beads.

[0028] In a fifth aspect of the present application, a method for efficiently separating and purifying human albumin is provided, wherein the method comprises a purification step using the chromatography medium.

[0029] Optionally, the purification step comprises at least cation exchange chromatography, hydrophobic chromatography purification, and affinity chromatography purification.

[0030] Further, the human albumin comprises human serum albumin and recombinant human albumin.

[0031] Further, the method for separating and purifying human albumin specifically comprises the following operations:

[0032] a) centrifuging a fermentation broth containing recombinant human albumin or a human blood product to obtain a supernatant;

[0033] b) loading the supernatant obtained in step a) into a cation exchange chromatography column, equilibrating, and then eluting with a salt-containing buffer to collect the target component and obtain a first purified product;

[0034] c) loading the first purified product obtained in step b) into a hydrophobic chromatography column, equilibrating, and then eluting to obtain a second purified product;

[0035] d) finely purifying the second purified product obtained in step c) by affinity chromatography with human albumin binding peptide 1A3 as a ligand to obtain high-purity recombinant human albumin, wherein the amino acid sequence of the human albumin binding peptide 1A3 is shown in SEQ ID NO: 1.

[0036] Further, the cation exchange chromatography medium in step b) comprises at least one of NanoGel 50SP, SP Sepharose Fast Flow, Capto S, the elution buffer comprises at least one of sodium chloride buffer, phosphate buffer, and the pH is 7.6.

[0037] Further, step b) further comprises a step of washing with a washing buffer before elution, the washing buffer comprises at least one of sodium chloride buffer, acetic acid buffer, phosphate buffer, and the pH is 4.5.

[0038] Further, step c) comprises that the chromatography medium used in the hydrophobic chromatography comprises at least one of UniHR Butyl-80L, Phenyl Sepharose HP, Butyl Sepharose 4FF. The pH of the equilibration buffer is 6.0; and the pH of the elution buffer is 6.0.

[0039] Further, the pH of the equilibration buffer of the affinity chromatography in step d) is 7.0-8.0, and the pH of the elution buffer is 2.8-3.2. The equilibration buffer comprises at least one of PBS buffer, citric acid buffer; and the elution buffer comprises at least one of glycine-HCl buffer, citric acid buffer.

[0040] In some embodiments, the cation exchange chromatography uses NanoGel 50SP medium, the equilibration buffer is 20 mM acetic acid buffer (pH 4.5), the washing buffer is 0.2 M sodium chloride + 20 mM acetic acid (pH 4.5), and the elution buffer is 0.5 M sodium chloride + 20 mM sodium phosphate (pH 7.6).

[0041] In some embodiments, the hydrophobic chromatography uses UniHR Butyl-80L medium, the equilibration buffer is 0.3 M NaCl + 50 mM sodium phosphate (pH 6.0), and the elution buffer is 50 mM sodium phosphate (pH 6.0).

[0042] In some embodiments, the equilibration buffer of the affinity chromatography is PBS solution (pH 7.4), the elution buffer is 0.1 M glycine-HCl (pH 3.0), and the neutralization buffer (1 M Tris-HCl, pH 8.0) is used for neutralization immediately after elution.

[0043] It should be noted that the present application does not make explicit limitation on various buffers or pH values, and those skilled in the art can adjust the types of buffers and pH values according to actual experience.

[0044] Further, the method further comprises a step of detecting the purity of the purified human albumin, and the detection of the purity of the human albumin is performed by high performance liquid chromatography (HPLC).

[0045] In a sixth aspect, the present application provides the use of the human albumin binding peptide or the biomaterial or the chromatography medium in any of the following aspects:

[0046] C1) the use in the preparation of a product for separating or purifying human albumin;

[0047] C2) the use in the preparation of a product for qualitatively or quantitatively detecting human albumin;

[0048] C3) the use in the preparation of a product for an immunological or therapeutic pharmaceutical composition.

[0049] The beneficial effects of the present application include but are not limited to:

[0050] High specificity and high efficiency: the human albumin binding peptide disclosed in the present application has extremely high affinity for human albumin, with a dissociation equilibrium constant KD= 6.73E-10 M. It can efficiently capture target proteins from complex fermentation broth, with high loading capacity and low non-specific adsorption.

[0051] Ultra-high purity: the present application combines the three steps of "cation exchange-hydrophobic-affinity" chromatography in a clever way, fully exerting the synergistic effect of different separation principles. In particular, the final affinity chromatography based on the specific human albumin binding peptide 1A3 can efficiently remove trace amounts of host proteins, aggregates and other impurities that are difficult to remove in the first two steps. The HPLC purity of the final recombinant human albumin (rHSA) is stably above 99.99%, and the content of key impurities such as endotoxin is extremely low, and the product quality meets or even exceeds the requirements of pharmaceutical-grade excipients.

[0052] High process yield and significant economic benefit: compared with the multi-step purification process in the prior art, which generally has a low yield, the present application optimizes the connection and operating conditions of each step, significantly reducing the loss of target products during the purification process. The total yield of purification can reach 64.7%, greatly improving the utilization rate of raw materials and production efficiency, reducing the production cost per unit of product, and having significant economic benefits and market competitive advantages.

[0053] High specificity and high separation efficiency: the core of the present application is the use of the self-developed human albumin binding peptide 1A3 as the affinity ligand. This ligand has high affinity and specificity for rHSA, and can directly and quickly capture target proteins from complex samples, overcoming the shortcomings of traditional dye affinity ligands such as poor specificity, dye shedding or insufficient selectivity of ion exchange method, and greatly improving the selectivity and separation efficiency of the purification process. BRIEF DESCRIPTION OF DRAWINGS

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

[0055] Figure 1 is a SDS-PAGE electrophoretogram of purified human albumin-binding peptide in an embodiment of the application, M: marker; 1: elution; 2: before loading.

[0056] Figure 2 is a graph of affinity detection results of human albumin-binding peptide 1A3 in an embodiment of the application.

[0057] Figure 3 is a HPLC chromatogram of purified recombinant human albumin in an embodiment of the application. DETAILED DESCRIPTION

[0058] The application will be described in greater detail with reference to the following examples, but the application is not limited to these examples. Unless otherwise specified, the raw materials and catalysts in the examples of the application are all purchased through commercial channels. The experimental methods not specified with specific conditions are usually performed according to conventional conditions, for example, the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or the conditions recommended by the manufacturer.

[0059] In the application, the fermentation broth containing recombinant human albumin can be obtained by conventional technical means in the art, for example, by fermentation of genetically engineered bacteria capable of secreting expression of recombinant human albumin, or by commercial channels. In some embodiments of the application, the fermentation broth is a Pichia pastoris fermentation broth containing recombinant human albumin, which is prepared according to the method in patent CN202410295109.6.

[0060] Example 1 Camel immunization and peripheral blood lymphocyte separation

[0061] The recombinant human albumin was mixed with Freund's adjuvant and immunized to the camel by subcutaneous injection at multiple points on the neck (0.2 mL per point, a total of 10 points), with an interval of 2 weeks between each immunization, for a total of 5 immunizations. Blood was collected before immunization, before the fourth immunization, before the fifth immunization, and 2 weeks after the fifth immunization, and serum was obtained after standing and centrifugation. The ELISA (enzymelinked immunosorbent assay) method was used to detect the titer of the immune antibody, and the serum titer after the fifth immunization was all ≥1:500,000, indicating that the immunization was successful. After the fifth immunization, 100 mL of blood was collected from the jugular vein, and PBMCs were isolated by Ficoll-Paque PLUS density gradient centrifugation (manufacturer: GE Healthcare, catalog number: 17-1440-02). The blood was diluted with an equal volume of sterile PBS, and then slowly added to the Ficoll liquid surface (volume ratio 2:1). The mixture was centrifuged at 400 x g and 20°C for 30 min. The middle white membrane layer was aspirated and washed with PBS three times to obtain peripheral blood mononuclear cells (PBMCs).

[0062] Example 2: Construction of phage library

[0063] Total RNA was extracted from PBMCs using the Trizol method, and cDNA was synthesized by reverse transcription. The single-domain antibody fragment was amplified by two rounds of PCR, and then ligated into the phage plasmid after restriction enzyme digestion. The transformed E. coli TG1 competent cells (manufacturer: TransGen, catalog number: CD201-01) were obtained by electroporation. The library capacity was determined to be 5.15 x 10 9 . Forty monoclonal antibodies were randomly selected for PCR identification, and the positive rate was 39 / 40 (97.5%), indicating that the library construction was successful.

[0064] Example 3: Human albumin binding peptide panning

[0065] Add streptavidin magnetic beads (manufacturer: Thermo Fisher, item number: 11205D) and enzyme hydrolyzed casein (manufacturer: Solarbio, item number: C8210-100) casein in the EP tube, 37°C shaking blocking for 1 h (rotation speed 100 rpm), remove the blocking solution. Add recombinant human albumin, 37°C shaking incubation for 1 h for coating, remove the unbound recombinant human albumin. Add phage library after blocking with enzyme hydrolyzed casein again, PBST (10X PBS (1.37 M NaCl, 27 mM KCl, 100 mM Na2HPO4, 18 mM KH2PO4, pH 7.4) diluted to 1X, and then add 0.1% Tween-20. ) wash 9 times, PBS (pH 7.4) wash once, completely remove the non-specific binding phage. Add trypsin (manufacturer: Sigma, item number: T4799, diluted with PBS, 500 μL), 37°C shaking elution for 15 min, and immediately terminate with enzyme hydrolyzed casein, and the elution product is obtained. Infect TG1 E. coli with the elution product, plate, and incubate at 37°C overnight. Infect the E. coli cultured from all the colonies in the logarithmic phase with M13K07, and expand the culture. Cycle the panning for 3 rounds, and enrich the library. Plate the last screening strain on ampicillin-resistant plates, pick single colonies on sterile cell culture plates, and perform ELISA detection. Select qualified positive single colonies and perform gene sequencing to obtain multiple sequences, one of which is named 1A3, the amino acid sequence of which is shown as SEQ ID NO. 1, and the nucleotide sequence of which is shown as SEQ ID NO. 2.

[0066] Example 4 Expression and purification of human albumin binding peptide in Pichia pastoris

[0067] (1) Expression of human albumin binding peptide 1A3 in Pichia pastoris

[0068] The 1A3 gene was cloned into the yeast vector pPICZαA (purchased from Hunan Fenghui Biotechnology Co., Ltd.) after codon optimization in Pichia pastoris, and the optimized nucleotide sequence is shown as SEQ ID NO. 3, Sac I After enzyme digestion and linearization, it was electroporated into X-33 yeast strain (purchased from Thermo Fisher scientific company, item number C18000), and single colony was screened on Zeocin-resistant plates. Routine inoculation for induction of secretory expression, 0.5% methanol was added every 24 h, and the sample was collected after 72 h of induction and expression. The supernatant was collected by centrifugation. The expression of 1A3 was correct as determined by SDS-PAGE.

[0069] (2) Purification of human albumin binding peptide expressed in Pichia pastoris

[0070] The yeast-expressed fermentation supernatant was added to a final concentration of 25 mM imidazole, dissolved, and filtered with a 0.45 μm filter membrane for loading; the nickel column was equilibrated with loading buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0 adjusted with NaOH), loaded, and the flow-through collected; the column was washed with loading buffer until the baseline was again level, and eluted with elution buffer, and the eluate collected; the desalting column was equilibrated with desalting buffer for 4 column volumes, and the eluate was loaded onto the desalting column, and the first peak to appear was the final human albumin-binding peptide solution. If the protein concentration was low after desalting, the solution was concentrated using an ultrafiltration tube. The collected solutions at each stage of purification were tested for purity, and the results of SDS-PAGE are shown in Figure 1, which shows that the main band of the target protein was clear after purification, and no obvious bands were observed. Figure 1

[0071] Example 5 Affinity detection of human albumin-binding peptide

[0072] The affinity of human albumin-binding peptide 1A3 was detected using the biofilm interference technology (BLI method). An NTA biosensor was used, and the sensor was first immersed in an analysis buffer for 10 min, and then immersed in an EDC-NHS mixed reagent for 5 min for activation; the activated sensor was immersed in a human albumin-binding peptide diluent (100 nM) for 10 min and blocked with ethanolamine (1 M, pH 8.5); the blocked sensor was immersed in a buffer for baseline zeroing; the sensor was then immersed in a gradient concentration of recombinant human albumin solution (7.8 nM, 15.625 nM, 31.25 nM, 62.5 nM, 125 nM, 250 nM, 500 nM) for 5 min to run a complete binding curve; then the sensor was transferred to PBS buffer for 5 min of dissociation. A 1:1 binding model was used for kinetic analysis, and the results are shown in Figure 2, which shows that the dissociation equilibrium constant KD= 6.73E-10 M, indicating that 1A3 has a strong affinity for recombinant human albumin. Figure 2

[0073] Example 6 Preparation of human albumin-binding peptide affinity chromatography medium

[0074] ​​The human albumin binding peptide lyophilized powder was dissolved in coupling solution (0.1 M NaHC03+ 0.5 M NaCl, pH 8.3) to prepare a 6 mg / ml binding peptide solution. 4 ml of CNBr-activated Bestarose 4B medium (purchased from Boster Biological Technology Co., Ltd. (Shanghai)) was placed in a sand core funnel and washed with 1 mM HC1 at 4°C for 30 min, and the volume was about 240 ml. The washed medium was diluted to 6 ml with 1 mM HC1, mixed with an equal volume of the binding peptide solution, and incubated at 4°C overnight. The coupling supernatant was removed and blocking solution (0.1 M Tris-HCl, pH 8.0) was added, and blocked at room temperature for 2 h. Wash with wash solution 1 (0.1 M HAC + 0.5 M NaCl, pH 4.0) and wash solution 2 (0.1 M Tris-HCl + 0.5 M NaCl, pH 8.0) alternately for 5 cycles, each time with 5 times the medium volume of liquid washing. After washing with PBS (pH 7.4), store at 4°C for standby.

[0075] Example 7 Purification of recombinant human albumin by cation exchange chromatography

[0076] Take 2000 mL of yeast-expressed recombinant human albumin fermentation broth, centrifuge at 4°C, 10,000 x g for 20 min, and carefully collect the supernatant. Slowly add 100 mM sodium caprylate stock solution to the supernatant to make the final concentration 15 mM, and mix gently. Adjust the pH to 6.0 ± 0.1 using 1 M HC1 solution under stirring. Place the sample in a constant temperature water bath at 65.0 ± 0.5°C for heating treatment for 45 min, and then quickly transfer to an ice water bath to cool to below 10°C. Then centrifuge at 4°C, 15,000 x g for 15 min, collect the supernatant and filter through a 0.45 μm PES membrane to obtain a clear sample.

[0077] 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.

[0078] Example 8: Hydrophobic chromatography purification of recombinant human albumin

[0079] The first purified product obtained in Example 7 was measured and solid ammonium sulfate was gradually added under slow stirring until the final concentration reached 1.5 M. After ensuring complete dissolution, it was filtered through a 0.45 μm filter membrane.

[0080] Prepare a UniHR Butyl-80L hydrophobic chromatography column (purchased from Suzhou Nanomicro Technology Co., Ltd., catalog number: 04062-050100, column size: 26mm × 250mm, column bed volume: 130mL). Equilibrate using 5 column volumes (650 mL) of equilibration buffer (0.3 M NaCl + 50 mM sodium phosphate, pH 6.0) at a linear flow rate of 100 cm / h. Load all samples at a flow rate of 50 cm / h. After loading, wash with equilibration buffer until the baseline stabilizes, then perform a decreasing gradient elution using 10 column volumes (1300 mL) of equilibration buffer (from 100% equilibration buffer to 100% elution buffer), maintaining a flow rate of 80 cm / h. The elution buffer is 50 mM sodium phosphate, pH 6.0. Based on UV absorption (280 nm) monitoring results, collect the main protein peak to obtain the second purified product.

[0081] Example 9: Affinity chromatography purification of recombinant human albumin

[0082] The second purified product obtained from Example 8 was subjected to liquid exchange using Millipore Pellicon 2 ultrafiltration membrane bag (molecular weight cut-off 10 kDa, Cat. No. P2C010C01) at an operating pressure of 0.2 MPa, and was replaced with PBS buffer (10 mM sodium phosphate, 150 mM NaCl, pH 7.4). The concentration and dilution were repeated 3 times until the conductivity and pH were consistent with PBS.

[0083] The human albumin-binding peptide affinity medium prepared in Example 6 was packed into a HR 26 / 20 chromatography column (purchased from Cytiva, with a column bed volume of 105 mL). The column was equilibrated with 5 column volumes (525 mL) of equilibration buffer (PBS, pH 7.4) at a flow rate of 2.0 mL / min. The entire sample after liquid exchange was loaded at a flow rate of 1 mL / min. The flow-through was monitored by UV280nm during the loading process 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, and the elution peaks were collected and immediately mixed with neutralization buffer (1 M Tris-HCl, pH 8.0) at a ratio of 10:1 (v / v) in the collection tube. The neutralized eluate was combined, desalted using an Amicon Ultra-15 ultrafiltration centrifuge tube (10 kDa molecular weight cut-off, Millipore, Cat. No. UFC901024), and replaced with water for injection. Finally, a high-purity recombinant human albumin lyophilized powder was obtained by freeze-drying.

[0084] Example 10 Purity and yield analysis

[0085] (1) Analysis of the purity of recombinant human albumin

[0086] 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 was used, equipped with a TSKgel G3000SWxl chromatographic column (7.8 mm x 30 cm, 5 μm, Tosoh Bioscience, Catalog No.: 08541). Phosphate buffer (PBS, 10 mM sodium phosphate, 150 mM NaCl, pH 7.4) was used as the mobile phase for isocratic elution, with a flow rate set at 0.8 mL / min, the column temperature maintained at 25 ± 1°C, the detection wavelength at 280 nm, and the injection volume at 20 μL. The sample was reconstituted with ultrapure water to a concentration of about 5 mg / mL, filtered through a 0.22 μm microporous filter, and then injected. The Agilent OpenLab CDS software was used for data acquisition and analysis, and the purity was calculated by the external standard peak area normalization method. After three independent repeated determinations, the average value of the area percentage of the main peak of the sample was 99.99% ± 0.01% (n = 3), and a typical chromatogram is shown in Figure 3 . The purity was improved from 98.7% (CN 118580318 A) to 99.99%, an increase of 1.29 percentage points.

[0087] (2) Calculation of the yield of recombinant human albumin

[0088] The protein concentration of each purification step was quantified by BCA (Bicinchoninic Acid) method, and the total yield was calculated accordingly. The Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, Catalog No.: 23225) was used. Strictly follow the instructions: first, dilute the BSA standard with PBS buffer to a series of concentrations of 0, 125, 250, 500, 750, 1000, 1500 μg / mL to make a standard curve (R 2 >0.99). The sample to be tested was appropriately diluted so that its concentration fell within the linear range of the standard curve. Each sample was set up in triplicate, and 25 μL of standard or diluted sample was added to each well, followed by the addition of 200 μL of BCA working solution. After incubation at 37°C for 30 min, the absorbance value was measured at 562 nm wavelength using an enzyme-labeled instrument (BioTek SynergyH1). The sample concentration was calculated according to the standard curve. Starting from the total amount of protein in the supernatant after pretreatment (referred to as Step 0), after cation exchange chromatography (Step 1), hydrophobic chromatography (Step 2), and affinity chromatography (Step 3), the total yield of the purified product was 64.7% ± 1.2% (n = 3), which was 2 times higher than the traditional process of 32% (CN102190722A2). This further verifies the high efficiency of the process of the present application.

[0089] Table 1 Recovery rate of each step

[0090]

[0091] Step 0 is the recovery rate of the total amount of protein in the supernatant after centrifugation and filtration of the initial fermentation broth relative to the total amount of protein in the fermentation broth before centrifugation; Steps 1-3 are the recovery rates of the total amount of target protein in the eluate of each chromatography step relative to the total amount of target protein before sample loading in the previous step. The total amount of protein in all steps is calculated by the concentration (determined by BCA method) x actual sample volume, and the sample is diluted with volume correction. The recovery rate in the table is the average value of three independent experiments (n=3), and there is no SD label because the SD of each step is <1.5%, and the SD of the total recovery rate is 1.2% (derived from the cumulative calculation of the deviation of each step).

[0092] Example 11 Safety index detection

[0093] (1) Endotoxin detection

[0094] The endotoxin content of the final product was detected by dynamic turbidity method limulus test. Dynamic turbidity method endotoxin detection kit (Lonza, item number: N588) was used. First, the recombinant human albumin freeze-dried powder obtained in Example 9 was dissolved and diluted to a concentration of 2 mg / mL with endotoxin check water (BET water, Lonza, item number: W50-100). The endotoxin working standard (CSE, Lonza, item number: E0005) was diluted to a series of concentrations of 0.005, 0.05, 0.5, 5 EU / mL with the same bottle of BET water to prepare a standard curve. In a pyrogen-free 96-well plate, 100 μL of standard, sample solution or negative control (BET water) was added to each well, and each sample was made in duplicate. Then, 100 μL of limulus reagent (LAL) was added to each well, mixed gently, and immediately placed in a microbial rapid detection system (or an enzyme-labeled instrument with dynamic turbidity method function, such as Charles River Endosafe® Endotoxin Detection System) to continuously monitor the change in absorbance at 405 nm wavelength of the reaction system at 37.0°C ± 0.2°C, and the reaction time was 70 min. The instrument software automatically calculates the endotoxin concentration according to the standard curve (R 2The endotoxin concentration in the sample was calculated. According to the requirements of the Pharmacopoeia of the People's Republic of China (2020 edition) 1143 General Rules, the interference test was carried out: the endotoxin standard (0.5 EU / mL) was mixed with the sample solution of 2 mg / mL, and the recovery rate was 100% (meeting the requirements of the Pharmacopoeia), which proved that the sample matrix had no interference with the detection. The final result is the average value of three independent detections, and the endotoxin content in the recombinant human albumin is 0.25 ± 0.08 EU / mg (n=3), which is much lower than the limit requirement of "less than 1 EU / mg" in the Pharmacopoeia of the People's Republic of China (2020 edition) 1143 General Rules, which proves that the safety of the final product meets the standards.

[0095] (2) Host cell protein (HCP) residual detection

[0096] Determination was carried out by enzyme-linked immunosorbent assay (ELISA). A commercial detection kit specific to Pichia pastoris (manufacturer: Cygnus Technologies, product number: F550) was used, with a detection range of 1-100 ng / mL. The HCP standard used in the kit was purified HCP of the same Pichia pastoris strain as the experiment, which was verified by Western Blot to specifically recognize the HCP of the strain (no cross-reaction); at the same time, a 5 mg / mL rHSA sample was added with a standard recovery test (addition concentration 20 ng / mL), with a recovery rate of 98%, verifying that the detection had no matrix interference. Before detection, the rHSA sample was diluted to 5 mg / mL, and pre-verification showed that rHSA had no interference with the detection at this concentration. By drawing a standard curve of 1-100 ng / mL (R 2 =0.995), the results showed that the HCP residual amount in the final product was 0.45 ng / mg rHSA. This value is significantly lower than the limit value of "HCP residual ≤10 ng / mg" specified in the Quality Control Technical Guidelines for Human Recombinant DNA Products (2020) of the National Medical Products Administration, fully proving that the purification process can efficiently remove Pichia pastoris host proteins.

[0097] (3) Residual DNA detection

[0098] Real-time quantitative PCR (qPCR) was used to target the conserved GAPDH gene in the Pichia pastoris genome. First, the DNA extraction kit (manufacturer: Qiagen, item number: 51304) was used to purify the DNA of 100 mg of the final product sample. The sample was dissolved in 10 mL of BET water, and 1 mL was used for DNA extraction. The extraction efficiency was verified by a spiking test: 10 pg of Pichia pastoris genomic DNA was added to the sample solution, and the recovery rate of the qPCR detection after extraction was 92%. The final residual DNA content was corrected according to the extraction efficiency. Then, a specific qPCR detection kit (manufacturer: Thermo Fisher Scientific, item number: A24554) was used for amplification. By constructing a standard curve of 1-10000 pg / mL (R 2 =0.998), and analyzing the melting curve of the amplification product (single peak, Tm=85.5℃), the specificity and accuracy of the detection were ensured. The final calculation showed that the residual DNA content was 0.8 pg / mg rHSA. This result meets the standard of "residual DNA ≤10 pg / mg" in the "Chinese Pharmacopoeia" (2020 edition) general rule 3407, further verifying the effectiveness of this process in removing nucleic acid impurities, and the safety of the final product meets the pharmaceutical requirements.

[0099] The above only describes the embodiments of the present application, and the protection scope of the present application is not limited by these specific embodiments, but is determined by the claims of the present application. For those skilled in the art, the present application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. within the technical idea and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A 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 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 includes at least 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, wherein the recombinant cells are 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) 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 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, hydrophobic chromatography purification, 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