Human albumin binding peptide 1e3 and use for facilitating purification of human albumin

Abstract

The application discloses a human albumin binding peptide 1E3 and application of the human albumin binding peptide 1E3 in promoting purification of human albumin, and belongs to the technical field of polypeptides. The human albumin binding peptide comprises an amino acid sequence as shown in SEQ ID NO. 1, and / or an amino acid sequence of a fusion protein with the same function 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 with 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 a pharmacopoeia standard and can reach more than 99.99%. The human albumin binding peptide has good safety, stable process, and wide application prospect in the aspect of isolating and purifying human albumin.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of polypeptides, and particularly relates to a human albumin binding peptide 1E3 and application of the human albumin binding peptide 1E3 in promoting purification of human albumin. BACKGROUND

[0002] Human serum albumin (HSA) is the most abundant protein in human plasma, and its main functions include maintaining plasma osmotic pressure, transporting endogenous and exogenous substances, and scavenging free radicals. In clinical practice, albumin is widely used in the treatment of shock, burns, hypoproteinemia, etc., and as a stabilizer for vaccines, cell culture medium and therapeutic protein drugs. Traditional human serum albumin is extracted from plasma, which not only faces the problem of tight supply of plasma, but also has the risk of pathogen contamination (such as hepatitis virus, HIV). Therefore, the production of recombinant human albumin (rHSA) using recombinant DNA technology has become an important alternative, and its expression system has been expanded from Escherichia coli to yeast (such as Pichia pastoris), plants and transgenic animals, etc.

[0003] However, the purification of rHSA faces many challenges. There are a large number of host cell proteins (HCPs), nucleic acids, endotoxins, pigments, albumin degradation products (such as 45 kDa fragments) and aggregates in the yeast fermentation broth. Due to the large dose for clinical use, the purity requirement of pharmaceutical grade rHSA is extremely high (≥99.9%), and any trace amount of impurities may cause safety problems. In addition, the traditional purification process has many steps, low yield (about 32%), and high cost, which is difficult to meet the needs of industrial production.

[0004] Early purification strategies mainly rely on the combination of ion exchange chromatography, hydrophobic interaction chromatography (HIC) and metal chelate affinity chromatography (IMAC). For example, patent US5521287 uses a multi-step process of cation exchange, hydrophobic chromatography and metal chelate affinity chromatography; patent CN112210002B also uses a similar multi-step chromatography strategy. These methods can obtain rHSA with a certain purity, but the process is long and the yield is low. The application of expanded bed adsorption technology (EBA) improves the efficiency of preliminary purification, such as patent CN102190722A directly loading the fermentation broth into an anion exchange expanded bed, but subsequent multi-step processing is still required, and the process is complex. In terms of affinity chromatography, dye affinity chromatography (such as Cibacron blue filler) can be used to remove degradation fragments, while immunochromatography has high selectivity, but is expensive and has poor stability, which is not suitable for large-scale production

[0005] In recent years, new technologies such as mixed mode chromatography (MMC) and affinity peptide ligands have provided new ideas for rHSA purification. MMC ligands combine multiple forces, have the advantages of large adsorption capacity, high selectivity and good salt tolerance, for example, patent CN116693659A uses two-step mixed mode chromatography, only two steps of purification can obtain rHSA with purity greater than 95%, and the yield is more than 80%. Compared with traditional antibodies, affinity peptide ligands (such as albumin binding peptides) are easy to synthesize, have good stability, low cost and no toxicity concerns, and have shown great potential. In addition, integrated purification strategies (such as patent CN112210002B combines multiple chromatography technologies) have made progress in balancing purity and cost. SUMMARY

[0006] In view of the shortcomings of the prior art, a multi-step chromatography combination process for large-scale production of high-purity albumin is developed. The core innovation is to use specific albumin binding peptide 1E3 as an affinity ligand to efficiently capture the target protein directly from the fermentation broth; optimize the combination of anion exchange chromatography and gel filtration chromatography to deeply remove impurities; the final product purity is ≥99.99%, meeting the pharmaceutical grade standard. The process is efficient and robust, suitable for large-scale production, and provides a reliable solution for the widespread application of albumin. The albumin includes serum albumin and recombinant albumin, optionally, the serum albumin is human serum albumin; optionally, the recombinant albumin is recombinant human albumin.

[0007] In a first aspect of the present application, a human albumin binding peptide 1E3 is provided, the amino acid sequence of the human albumin binding peptide includes at least one of the following:

[0008] A1) the amino acid sequence includes the amino acid sequence shown as SEQ ID NO. 1;

[0009] A2) is an amino acid sequence having 95% or more identity with the amino acid sequence of the human albumin binding peptide of A1) obtained by substitution and / or deletion and / or addition of amino acid residues of the amino acid sequence of the human albumin binding peptide of A1);

[0010] A3) the amino acid sequence of a fusion protein with the same function obtained by connecting a tag protein to the N-terminus and / or C-terminus of the amino acid sequence of A1) or A2).

[0011] In a second aspect of the present application, a biological material is provided, the biological material includes at least any of the following:

[0012] B1) contains a nucleic acid molecule encoding the human albumin binding peptide;

[0013] B2) contains an expression cassette of B1) the nucleic acid molecule;

[0014] B3) a recombinant vector comprising the nucleic acid molecule of B1), or a recombinant vector comprising the expression cassette of B2);

[0015] B4) a recombinant microorganism comprising the nucleic acid molecule of B1), or a recombinant microorganism comprising the recombinant vector of B2), or a recombinant microorganism comprising the recombinant vector of B3);

[0016] B5) a recombinant cell comprising the nucleic acid molecule of B1), or a recombinant cell comprising the recombinant vector of B2), or a recombinant cell comprising the recombinant vector of B3).

[0017] Further, the nucleic acid molecule in B1) comprises 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) comprises 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 the human albumin binding peptide 1E3; and SEQ ID NO. 3 is a sequence optimized for Pichia pastoris codon bias.

[0018] 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.

[0019] Further, the recombinant vector in B3) comprises 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.

[0020] Further, the recombinant microorganism in B4) or the recombinant cell in B5) comprises at least one of hamster ovary cells, silkworm ovary cells, Pichia pastoris, Saccharomyces cerevisiae, Escherichia coli, and Bacillus subtilis.

[0021] Optionally, the recombinant microorganism is Pichia pastoris.

[0022] 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 or the fusion protein.

[0023] 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 scope of the present application.

[0024] In a fourth aspect, the present application provides a chromatography medium for separating and purifying human albumin, wherein the chromatography medium comprises the human albumin binding peptide.

[0025] 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 1E3 ligand-crosslinked agarose affinity medium.

[0026] In a fifth aspect, the present application provides a method for separating and purifying human albumin with high efficiency, wherein the method comprises a purification step using the chromatography medium.

[0027] Optionally, the purification step comprises at least affinity chromatography, anion exchange chromatography, and gel filtration chromatography.

[0028] Further, the human albumin comprises human serum albumin and recombinant human albumin.

[0029] Further, the method for separating and purifying human albumin comprises the following operations:

[0030] a) centrifuging a fermentation broth containing recombinant human albumin or a human blood product to obtain a supernatant;

[0031] b) loading the supernatant obtained in step a) into an affinity chromatography column with human albumin binding peptide 1E3 as the ligand, washing with an equilibration buffer, eluting with an elution buffer, and immediately neutralizing the elution fraction with a neutralization buffer to obtain a first purified product;

[0032] c) loading the first purified product into an anion exchange chromatography column, washing with a salt-containing buffer after equilibration, and collecting the target fraction to obtain a second purified product;

[0033] d) loading the second purified product into a gel filtration chromatography column, eluting with an isocratic or gradient buffer, and collecting the recombinant human albumin monomer peak to obtain a high-purity recombinant human albumin final product.

[0034] Further, the amino acid sequence of the human albumin binding peptide 1E3 in step b) is shown in SEQ ID NO: 1. The equilibration buffer is a PBS solution with a pH value of 7.0-8.0; the elution buffer is a glycine-HCl solution with a pH value of 2.8-3.2; and the neutralization buffer is a Tris-HCl solution with a pH value of 8.0-8.5.

[0035] Further, the anion exchange chromatography medium in step c) is Streamline Q, Streamline Q XL or Streamline DEAE; and the equilibration buffer is 20-50 mM sodium phosphate buffer, pH 7.0-8.0.

[0036] Further, the gel filtration chromatography medium in step d) is Sephadex, Superdex or Sephacryl series medium; and the mobile phase buffer is 50 mM Tris-HCl + 0.1 M NaCl, or phosphate buffer, pH 7.0-8.0.

[0037] Further, the method further comprises a step of detecting the purity of the purified human albumin, and optionally, the detection of the purity of the human albumin is by high performance liquid chromatography (HPLC).

[0038] In a sixth aspect of the present application, the human albumin binding peptide or the biomaterial or the chromatography medium is used in any of the following aspects:

[0039] C1) for preparing a product for separating or purifying human albumin;

[0040] C2) for preparing a product for qualitatively or quantitatively detecting human albumin;

[0041] C3) for preparing a product for an immunological or therapeutic pharmaceutical composition.

[0042] The beneficial effects of the present application include, but are not limited to:

[0043] High specificity and high efficiency: the human albumin binding peptide disclosed in the present application has extremely high affinity with human albumin, with a dissociation equilibrium constant KD= 5.80E-10 M. It can efficiently capture the target protein from complex fermentation broth in one step, with high loading capacity and low non-specific adsorption.

[0044] Ultra-high purity: the present application also discloses a chromatography medium for separation and purification, which is combined with a three-step chromatography process in a clever way and optimized, forming an efficient impurity removal process (affinity chromatography removes most impurities, anion exchange removes HCP / nucleic acid, and molecular sieve removes aggregates), and the final product purity far exceeds the pharmacopoeia standard, reaching more than 99.99%.

[0045] Good safety: compared with dye ligands, peptide ligands have no risk of foreign matter shedding, and are easy to clean and verify, thus having higher product safety

[0046] The process is stable and easy to scale up: the purification process steps are clear, the conditions are mild, the recovery rate of each step is high, the overall yield is considerable, and all chromatography techniques are very mature and easy to scale up for production, which has high industrial application value. BRIEF DESCRIPTION OF DRAWINGS

[0047] 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:

[0048] Figure 1 is an SDS-PAGE electrophoretogram of the purified human albumin-binding peptide in the embodiments of the present application, M: marker; 1: elution; 2: before loading.

[0049] Figure 2 is a graph of the affinity detection results of the human albumin-binding peptide 1E3 in the embodiments of the present application.

[0050] Figure 3 is an HPLC chromatogram of the purified recombinant human albumin in the embodiments of the present application. DETAILED DESCRIPTION

[0051] The present application will be described in detail below with reference to examples, but the present application is not limited to these examples. Unless otherwise specified, the raw materials and catalysts in the examples of the present application are 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.

[0052] In the present application, the fermentation broth containing recombinant human albumin can be obtained by conventional technical means in the art, such as by fermentation of genetically engineered bacteria capable of secreting expression of recombinant human albumin, or by commercial purchase. In some embodiments of the present application, the fermentation broth is a Pichia pastoris fermentation broth containing recombinant human albumin, which is prepared by referring to the method in patent CN202410295109.6.

[0053] Example 1 Camel immunization and peripheral blood lymphocyte separation

[0054] 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).

[0055] Example 2: Construction of phage library

[0056] 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.

[0057] Example 3: Human albumin binding peptide panning

[0058] 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. After blocking with enzyme hydrolyzed casein again, add phage library for binding, 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 1 time, 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 1E3, 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.

[0059] Example 4 Expression and purification of human albumin binding peptide 1E3 in Pichia pastoris

[0060] (1) Expression of human albumin binding peptide 1E3 in Pichia pastoris

[0061] The 1E3 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 was used for induction of secretory expression, and 0.5% methanol was added every 24 h, and the sample was collected after 72 h of induction and expression, and the supernatant was collected by centrifugation. The expression of 1E3 was correct as determined by SDS-PAGE.

[0062] (2) Purification of human albumin binding peptide expressed in Pichia pastoris

[0063] The yeast-expressed fermentation supernatant was added to a final concentration of 25 mM imidazole, dissolved, and filtered with a 0.45 μm filter membrane for loading; the nickel column was equilibrated with loading buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0 adjusted with NaOH), loaded, and the flow-through was collected; the column was washed with loading buffer until the baseline was again level, and eluted with elution buffer, and the eluate was collected; the desalting column was equilibrated with desalting buffer for 4 column volumes, and the eluate was loaded onto the desalting column, and the first peak to appear was collected as 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 impurity bands were observed. Figure 1

[0064] Example 5 Affinity detection of human albumin-binding peptide

[0065] The affinity of human albumin-binding peptide 1E3 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 sequentially 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= 5.80E-10 M, indicating that 1E3 has a strong affinity for recombinant human albumin. Figure 2

[0066] Example 6 Preparation of human albumin-binding peptide affinity chromatography medium

[0067] ​​The human albumin binding peptide lyophilized powder was dissolved in the coupling solution (0.1 M NaHCO3+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 4°C pre-cooled 1 mM HCl for 30 min, and the amount of washing was about 240 ml. The washed medium was diluted to 6 ml with 1 mM HCl, 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 the blocking was carried out at room temperature for 2 h. The medium was washed with washing solution 1 (0.1 M HAC+0.5 M NaCl, pH 4.0) and washing solution 2 (0.1 M Tris-HCl+0.5 M NaCl, pH 8.0) alternately for 5 cycles, and each time the medium was washed with 5 times the volume of liquid. After washing with PBS (pH 7.4), it was stored at 4°C for standby.

[0068] Example 7 Affinity chromatography purification of recombinant human albumin

[0069] Take 2000 ml of yeast-expressed recombinant human albumin fermentation broth, centrifuge at 4°C, 8000 rpm (centrifugal force about 10,000 x g) for 20 min, and collect the supernatant. Add 100 mM sodium caprylate stock solution to the supernatant to make the final concentration 15 mM, and mix slowly. Adjust the pH to 6.0 ± 0.1 with 1 M HCl solution, and then heat treat at 65.0 ± 0.5°C water bath for 45 min, and quickly cool to below 25°C in ice bath. Centrifuge at 4°C, 12000 rpm (centrifugal force about 20,000 x g) for 15 min, collect the supernatant, and repeat the heat denaturation and centrifugation operation once. The final supernatant was filtered through a 0.45 μm PES membrane to obtain a clear sample.

[0070] The human albumin binding peptide affinity chromatography media prepared in Example 6 was packed into an XK50 / 60 chromatography column (purchased from Cytiva, column bed size: 26 mm x 200 mm, bed volume: 107 ml) and operated using an AKTA pure 150 system. The column was equilibrated with equilibration buffer (10 mM sodium phosphate, 150 mM NaCl, pH 7.4) at a flow rate of 1.5 ml / min for at least 3 column volumes (321 ml) until the UV absorbance baseline (280 nm) was stable. The pretreated sample was loaded at a flow rate of 1.5 ml / min. After loading, the column was washed with equilibration buffer until the UV280 signal returned to baseline. Elution was performed with elution buffer (0.1 M glycine-HCl, pH 2.8) at a flow rate of 1.0 ml / min for 4 column volumes (428 ml), and the eluate was collected in 2 ml fractions. Immediately, 200 μl of neutralization buffer (1 M Tris-HCl, pH 8.0) was added to each fraction, mixed gently to restore the pH to neutral, and the first purified product was obtained by pooling the fractions.

[0071] Example 8 Purification of recombinant human albumin by anion exchange chromatography

[0072] The first purified product from Example 7 was exchanged into 20 mM sodium phosphate buffer (pH 7.4) using an ultrafiltration membrane cassette (molecular cut-off 10 kDa, purchased from Millipore) at an operating pressure of 0.2 MPa. The sample was concentrated and diluted repeatedly until the conductivity and pH were consistent with the target buffer.

[0073] A Streamline QXL anion exchange chromatography column (column specifications: purchased from Cytiva, 26 mm x 150 mm, gel volume 76 ml) was prepared and equilibrated with 20 mM sodium phosphate buffer (pH 7.4) at a linear flow rate of 60 cm / h for at least 5 column volumes (380 ml) until the conductivity and pH were stable. The sample after exchange was loaded at a flow rate of 40 cm / h. After loading, the column was washed with equilibration buffer until the UV baseline was stable, and then a linear gradient elution was performed using 20 mM sodium phosphate buffer (pH 7.4) containing 1.0 M NaCl, with a gradient volume of 10 column volumes (760 ml) and a flow rate of 60 cm / h. The main elution peak was collected according to the UV absorbance profile (280 nm), and the second purified product was obtained.

[0074] Example 9 Purification of recombinant human albumin by gel filtration chromatography

[0075] The second purified product obtained from Example 8 was concentrated by ultrafiltration centrifuge tube (molecular weight cut-off 10 kDa, purchased from Millipore Corporation, item number: UFC901024) at 4°C, 4000xg, and monitored the volume and protein concentration every 15 min until the protein concentration was stabilized at 50±2 mg / mL (the volume after concentration was about 25.9 mL), and a small amount of 50 mM Tris-HCl + 0.1 M NaCl buffer (pH 7.6) was added during the concentration to avoid local high concentration leading to protein denaturation. After the concentration was completed, the sample was filtered in a sterile operation table using a 0.22 μm PVDF filter membrane (purchased from Millipore Corporation, item number: SLGV033RB), and the clear filtrate was collected and stored at 4°C for loading.

[0076] A glass chromatography column (column specifications: 40 mm x 410 mm, column bed volume about 523 ml) was filled with Sephadex G100 gel filtration medium (purchased from Cytiva Corporation, item number: 17001001). The chromatography column was connected to the AKTA pure 250 system (Cytiva Corporation), and the detection wavelength was set to 280 nm, the conductivity monitoring range was 0-50 mS / cm, and the upper limit of column pressure was 0.15 MPa. At least 3 times the column volume (1569 ml) was equilibrated with 50 mM Tris-HCl + 0.1 M NaCl buffer (pH 7.6) at a linear flow rate of 20 cm / h. The concentrated sample was loaded at 5% of the column volume (about 26 ml), and then eluted at a constant flow rate using the same buffer, and the flow rate was maintained at 20 cm / h. According to the monitoring results of ultraviolet absorption (280 nm), the main peak elution components were collected.

[0077] The collected components were combined, desalted using an ultrafiltration membrane bag (10 kDa cut-off), and then replaced with water for injection, followed by freeze-drying to obtain a high-purity recombinant human albumin freeze-dried powder.

[0078] Example 10: Purity and yield analysis

[0079] (1) Analysis of the purity of recombinant human albumin

[0080] The purity of the rHSA end product (lyophilized powder) in Example 9 was analyzed using high performance liquid chromatography (HPLC). The mobile phase was dissolved and diluted to a concentration of 10 mg / mL, filtered through a 0.22 μm filter membrane, and then loaded. A TSKgel G3000SWxl chromatographic column was used with PBS as the mobile phase, a flow rate of 0.8 ml / min, and a detection wavelength of 280 nm. The final HPLC chromatogram (Figure 3) showed that the rHSA main peak had a retention time of 13.425 min, the peak shape was symmetrical, and there was no obvious tailing. The main peak area accounted for 99.99% of the total peak area, the aggregate peak (retention time of about 10.5 min) accounted for 0.008%, and the small molecule impurity peak (retention time of about 18.2 min) accounted for 0.002%, all of which were far lower than the requirements of the "Purity ≥ 99.9%, aggregate content ≤ 0.1%" for pharmaceutical rHSA in the "People's Republic of China Pharmacopoeia" (2020 edition).

[0081] (2) Recombinant human albumin yield calculation

[0082] The total yield was calculated by determining the protein concentration at each step using the BCA method. The Pierce™ BCA Protein Assay Kit (Manufacturer: Thermo Fisher Scientific, Catalog Number: 23225) was used to quantify the protein concentration at each purification step using the BCA (Bicinchoninic Acid) method, and the total yield was calculated accordingly. The Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, Catalog Number: 23225) was used. Strictly follow the instructions: first, dilute the BSA standard to a series of concentrations of 0, 125, 250, 500, 750, 1000, 1500 μg / mL with PBS buffer to make a standard curve (R 2>0.99). Each sample was diluted to fall within the linear range of the standard curve. Each sample was set up in triplicate, and 25 μL of the 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 using an enzyme-labeled instrument (BioTek Synergy H1). The sample concentration was calculated according to the standard curve. The yield of each step was calculated from the supernatant after pretreatment. The total amount of protein in all steps was calculated by "concentration (determined by BCA method) x actual sample volume", and the volume was corrected when the sample was diluted. The yield in the table is the average of three independent experiments (n = 3), and no SD is labeled because the SD of each step is <1.2% (the SD of the pretreatment step is 1.0%~1.2%, the SD of anion exchange chromatography, gel filtration chromatography is 0.5%~0.8%, and the SD of affinity chromatography is 0.3%~0.6%, and the SD of the total yield is 1.7% (resulting from the cumulative calculation of the deviation of each step).

[0083] Table 1 Recovery rate of each step

[0084]

[0085] The total yield reached 76.7%, which was 2.4 times higher than the traditional process of 32% (CN102190722A2). Further verification of the efficiency of the process of the present application.

[0086] Example 11 Safety index detection

[0087] (1) Endotoxin detection

[0088] The endotoxin was detected using a dynamic turbidity method endotoxin detection kit (manufacturer: Lonza, item number: N588). The recombinant human albumin sample obtained in Example 9 was dissolved and diluted with endotoxin test water (BET water) (manufacturer: Lonza, item number: W50-100) to a value within the standard curve range. The standard curve was prepared: the endotoxin working standard (manufacturer: Lonza, item number: E0005) was diluted with BET water to form a series of solutions with 5 concentration points. According to the kit instructions, 100 μL of the standard, sample or negative control (BET water) was added to a pyrogen-free reaction tube or 96-well plate. 100 μL of limulus amebocyte lysate (LAL) was added to each well, and immediately placed into a dynamic turbidity method microbial rapid detection system or an enzyme-labeled instrument with this function. The absorbance change was continuously monitored at 37 °C, and the reaction time was 70 min. The instrument software calculated the endotoxin value according to the standard curve (R 2= 0.998) to be 0.23 EU / mg rHSA, which is less than the limit of 0.5 EU / mg specified in the “People's Republic of China Pharmacopoeia” (2020 edition), proving that the final product meets the safety standards.

[0089] (2) Host cell protein (HCP) residual detection

[0090] Enzyme-linked immunosorbent assay (ELISA) was used for determination. A commercial detection kit specific to Pichia pastoris (manufacturer: Cygnus Technologies, item number: F550) was used, with a detection range of 1-100 ng / mL. Before detection, the rHSA sample was diluted to 5 mg / mL, and it was pre-verified that rHSA at this concentration had no interference with the detection. The results were calculated by drawing a standard curve of 1-100 ng / mL (R 2 = 0.997), and the results showed that the HCP residual amount in the final product was only 0.35 ng / mg rHSA. This value is significantly lower than the limit of “HCP residual ≤ 10 ng / mg” specified in the “Guiding Principles for Quality Control of Recombinant DNA Products for Human Use” (2020) of the National Medical Products Administration, fully proving that the purification process can efficiently remove Pichia pastoris host proteins.

[0091] (3) Residual DNA detection

[0092] Real-time fluorescent quantitative PCR (qPCR) was used to target the conserved GAPDH gene of the Pichia pastoris genome. First, a DNA extraction kit (manufacturer: Qiagen, item number: 51304) was used to enrich and purify DNA from 100 mg of the final product sample, and 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, and analyzing the melting curve of the amplification product (single peak, Tm=85.2°C), the specificity and accuracy of the detection were ensured. The final calculation showed that the residual DNA content was 0.12 pg / mg rHSA. This result far exceeds the standard of “residual DNA ≤ 10 pg / mg” in the “People's Republic of China Pharmacopoeia” (2020 edition) general rule 3407, further verifying the excellent performance of the process in removing nucleic acid impurities, and the safety of the final product meets the pharmaceutical requirements.

[0093] The above merely describes the embodiments of the present application, and the protection scope of the present application is not limited by these specific embodiments, but determined by the claims of the present application. The present application can have various changes and variations for those skilled in the art. Any modification, equivalent replacement, improvement, etc. within the technical thought 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) the amino acid sequence of a fusion protein with the same function obtained by connecting a tag protein to the N-terminus and / or C-terminus of the amino acid sequence of A1).

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

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

3.

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

5. A method of preparing the human albumin-binding peptide of claim 1, characterized by, The method includes the step of culturing the recombinant microorganism of B4) or the recombinant cell of B5) in the biological material of claim 2 under suitable culture conditions, and isolating the human albumin binding peptide.

6. A chromatography medium for the isolation and purification of human albumin, characterized in that, The chromatography medium includes 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 a pre-packed column, a packing material, and a magnetic bead.

8. A method for isolating and purifying human albumin, characterized by, The method includes the step of purifying using the human albumin binding peptide of claim 1 or the chromatography medium of claim 6 or 7.

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

10. Use of a human albumin binding peptide according to claim 1 or a biomaterial according to any of claims 2-4 or a chromatography medium according to claim 6 or 7, c h a ra cte ri zed in that, The application includes at least one of the following: C1) application for preparing a product for isolating or purifying human albumin; C2) application for preparing a product for qualitatively or quantitatively detecting human albumin.

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