Human albumin binding peptide 4f1 and methods for purifying human albumin

Abstract

The application discloses a human albumin binding peptide 4F1 and a human albumin purification method, and belongs to the technical field of polypeptides. The amino acid sequence of the human albumin binding peptide 4F1 comprises an amino acid sequence as shown in SEQ ID NO. 1, and / or 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 4F1 has extremely 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 the pharmacopoeia standard, and can reach more than 99.9%. The application combines the high selectivity capture of affinity chromatography with the strong impurity removal capacity of gel filtration chromatography, constructs a high-efficiency, stable and easy-to-scale purification process route, and significantly improves the recovery rate and production efficiency.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of polypeptides, in particular to a human albumin binding peptide 4F1 and a method for purifying human albumin. BACKGROUND

[0002] Human serum albumin (HSA) is the most abundant protein in human plasma, which has important physiological functions such as maintaining plasma osmotic pressure, transporting endogenous and exogenous substances, and scavenging free radicals. It is widely used in clinical treatment of shock, burns, and hypoproteinemia, and can be used as a vaccine adjuvant, a cell culture medium component, and a stabilizer for therapeutic protein drugs. Traditional human HSA is mainly extracted from plasma, which is not only limited by the shortage of plasma supply, but also has the risk of contamination by hepatitis virus, HIV and other pathogens. Therefore, the production of recombinant human serum albumin (rHSA) using recombinant DNA technology has become an important alternative approach, and its expression system includes Escherichia coli, yeast (such as Pichia pastoris), plants, and transgenic animals, etc.

[0003] However, rHSA purification still faces significant challenges. The fermentation broth produced by expression systems such as yeast is complex, containing a large amount of host cell proteins (HCP), nucleic acids, endotoxins, pigments, and albumin degradation products (such as 45 kDa fragments) and aggregates. Due to the large dose for clinical use, the purity requirement of pharmaceutical-grade rHSA is extremely high (≥99.9%), and trace impurities may cause safety problems. In addition, traditional purification processes mostly rely on multi-step chromatography combination, with long process, low yield (about 32%), and high cost, making it difficult to achieve large-scale production.

[0004] Early rHSA purification mostly used combination strategies such as ion exchange, hydrophobic interaction chromatography (HIC), and metal chelate affinity chromatography (IMAC). For example, US5521287 used three-step method of cation exchange, hydrophobic chromatography and metal chelation, and CN112210002B also used similar multi-step chromatography method, which can obtain products with certain purity, but the process is complicated and the recovery rate is low. Expanded bed adsorption technology (EBA) improves the efficiency of preliminary purification to some extent, such as CN102190722A directly samples the fermentation broth to anion exchange expanded bed, but still needs subsequent multi-step refining, and the process is complex. Dye affinity chromatography (such as Cibacron Blue) can be used to remove degradation fragments, and immunoaffinity chromatography has high selectivity but is expensive and has poor stability, which are not suitable for large-scale application.

[0005] In recent years, the emergence of mixed mode chromatography (MMC) and affinity peptide ligands has brought breakthroughs in rHSA purification. MMC ligands rely on multiple mechanisms, combining high adsorption capacity, good selectivity, and salt tolerance, etc. For example, CN116693659A uses a two-step MMC purification to obtain rHSA with a purity of more than 95% and a yield of more than 80%. Affinity peptide ligands (such as albumin binding peptides) show significant advantages due to their ease of synthesis, high stability, lower cost, and no toxic side effects. Integrated purification strategies, such as the combination of multiple chromatography techniques in CN112210002B, have also made progress in improving product purity and controlling costs. SUMMARY

[0006] To address the issues of process complexity, low yield, difficulty in scaling up, and insufficient product purity in the prior art, the present application develops an affinity chromatography and gel filtration combined process based on the specific albumin binding peptide 4F1 ligand, which can efficiently capture rHSA from fermentation broth and deeply remove impurities. The final product purity is 99.9%, meeting the pharmaceutical grade standard. This process is simple to operate, highly stable, and easy to scale up, providing a reliable solution for large-scale production and widespread application of rHSA. The albumin includes serum albumin and recombinant albumin, and the serum albumin is human serum albumin, and the recombinant albumin is recombinant human albumin.

[0007] In a first aspect of the present application, a human albumin binding peptide 4F1 is provided, and the amino acid sequence of the human albumin binding peptide 4F1 includes at least one of the following:

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

[0009] 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 4F1 of A1), which has more than 95% identity with the amino acid sequence of the human albumin binding peptide 4F1 of A1);

[0010] 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).

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

[0012] B1) contains a nucleic acid molecule encoding the human albumin binding peptide 4F1;

[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 4F1 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 4F1; 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 4F1, which comprises the steps of culturing the recombinant microorganism of B4) or the recombinant cell of B5) under suitable conditions for cultivation, and isolating the human albumin binding peptide 4F1 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 4F1.

[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 4F1. Preferably, the chromatography medium is a recombinant human albumin binding peptide 4F1 ligand-crosslinked agarose affinity medium.

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

[0027] Optionally, the purification step comprises at least affinity 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 specifically comprises the following operations:

[0030] a) centrifuging a fermentation broth containing recombinant human albumin for pretreatment;

[0031] b) loading the centrifugally pretreated fermentation broth into an affinity chromatography column with human albumin binding peptide 4F1 as the ligand, washing with an equilibration buffer, eluting with an elution buffer, and then adding a neutralization buffer to obtain a first purified product;

[0032] c) passing the first purified product through a gel filtration chromatography column, eluting with a mobile phase buffer, and collecting a recombinant human albumin monomer peak to obtain a high-purity recombinant human albumin final product.

[0033] It is understood by those skilled in the art that simple changes can be made to the sequence of SEQ ID NO. 1 of the present application, such as replacing or deleting part of the amino acid sites, but still retaining the same human albumin affinity properties of SEQ ID NO. 1 in the present application. It is understood that sequences based on the simple changes to SEQ ID NO. 1 of the present application while retaining the same human albumin affinity properties of the binding peptide of the present application should also be within the scope of protection of the present application. In addition, those skilled in the art can also truncate or derive the binding peptide based on the SEQ ID NO. 1 sequence disclosed in the present application to obtain a binding peptide with the same human albumin affinity effect, which can be reasonably predicted by those skilled in the art, for example: a polypeptide obtained by truncating one or more amino acids from the N-terminus or C-terminus of the polypeptide.

[0034] It should be noted that the skilled person in the art can perform chemical modification on the binding peptide, such as cyclization modification, acetylation modification, PAS modification, PEG modification, fatty acid modification, albumin modification, albumin binding peptide coupling, tumor homing peptide coupling, transmembrane peptide coupling, nanocarrier coupling, radionuclide coupling, small molecule compound coupling, nucleotide coupling, protein coupling, and the like, and the modification sites include but are not limited to N-terminal modification, C-terminal modification, backbone modification, side chain modification, amino acid modification, etc.

[0035] Further, the equilibrium buffer in step b) is a PBS solution, the elution buffer is a glycine solution, and the neutralization buffer is a Tris-HCl solution.

[0036] Further, the pH range of the PBS solution is 6.5 ~ 7.5; the pH range of the glycine solution is 2.8 ~ 3.5; the pH range of the Tris-HCl solution is 8.0 ~ 8.5; and the pH range of the equilibrium buffer / flow phase buffer Tris-HCl + NaCl solution is 7.5 ~ 8.5.

[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 4F1 or the biological material or the chromatography medium is applied 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] 1. High specific capture ability: the present application uses the specific human albumin binding peptide 4F1 as the affinity ligand, which can precisely bind to recombinant human albumin (rHSA), and even after conservative amino acid substitution, terminal truncation or chemical modification, it still maintains high affinity and stable dissociation equilibrium. The affinity chromatography medium based on the ligand can capture rHSA from the fermentation broth in a targeted manner, effectively avoiding the non-specific adsorption of host proteins (HCP), nucleic acids and other impurities. The purity of rHSA in the preliminary purified product can reach more than 95%.

[0044] 2. The process is simple, stable and easy to scale up: the present application realizes efficient purification through only two core processes of "affinity chromatography + gel filtration", which reduces more than 30% of steps. The process parameters are clear, and the combined peptide 4F1 has high stability and can tolerate pH 2.8-8.5 without the risk of falling off. The process has good repeatability and is easy to scale up from laboratory to industrial scale (100L-1000L), which improves the production efficiency by more than 40%.

[0045] 3. The final product has high purity and safety: the combined process of "affinity capture + gel filtration impurity removal" realizes step-by-step impurity removal, and the final rHSA purity is more than 99.9%, which is better than the conventional process (95%-98%). The process has outstanding safety, and the contents of HCP, endotoxin and aggregate in the final product are significantly lower than the limit values of Chinese Pharmacopoeia (2020) and related guidelines, which can be directly used in medical fields such as clinical treatment, vaccine stabilizer and cell culture, avoiding the risk of immunogenicity. BRIEF DESCRIPTION OF DRAWINGS

[0046] The drawings described herein are used to provide further understanding of the present application, and form a part of the present application. The illustrative embodiments of the present application and their descriptions are used to explain the present application, and do not constitute an improper limitation on the present application. In the drawings:

[0047] Figure 1 is the SDS-PAGE electrophoretogram of human albumin binding peptide 4F1 after purification in the embodiment of the present application, M: marker; 1: elution.

[0048] Figure 2 is the human albumin-human albumin binding peptide 4F1 affinity detection result graph in the embodiment of the present application.

[0049] Figure 3 is the HPLC chromatogram of recombinant human albumin after purification in the embodiment of the present application. DETAILED DESCRIPTION

[0050] The present application will be 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 in the embodiments of the present application are purchased through commercial channels. The experimental methods without specific conditions are usually carried out according to the 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.

[0051] In the present application, the fermentation broth containing recombinant human albumin can be obtained by means of conventional technical means in the art, such as by fermentation of genetically engineered bacteria capable of secreting expressed 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.

[0052] Example 1 Camel immunization and peripheral blood lymphocyte separation

[0053] The recombinant human albumin was mixed with Freund's adjuvant and then injected into the camel by subcutaneous injection at multiple points on the neck (0.2 mL per point, a total of 10 points). Each immunization was spaced 2 weeks apart, and a total of 5 immunizations were performed. Blood was collected before immunization, before the fourth immunization, before the fifth immunization, and 2 weeks after the fifth immunization. After standing and centrifugation, serum was obtained. The ELISA (enzyme-linked immunosorbent assay) method was used to detect the titer of the immune antibody. The serum titer after the fifth immunization was all ≥1:500,000, and the immunization was determined to be successful. After the fifth immunization, 100 mL of blood was collected from the jugular vein of each camel, and PBMCs were separated 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).

[0054] Example 2 Phage library construction

[0055] 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 a phage plasmid after restriction enzyme digestion. The resulting product was transformed into E. coli TG1 competent cells (manufacturer: Amid Biosciences, catalog number: ETG1-201) by electroporation. The library capacity was determined to be 5.15 x 1011 by gradient dilution method. 9 Forty single clones were randomly selected for PCR identification, and the positive rate was 39 / 40 (97.5%), indicating that the library construction was successful.

[0056] Example 3 Human albumin-binding peptide 4F1 panning

[0057] Add streptavidin magnetic beads (manufacturer: Thermo Fisher, item number: 65006D) and enzyme hydrolyzed casein (manufacturer: Solarbio, item number: C8210-100) casein in an 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 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 4F1, 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.

[0058] Example 4 Expression and purification of human albumin binding peptide 4F1 in Pichia pastoris

[0059] (1) Expression of human albumin binding peptide 4F1 in Pichia pastoris

[0060] The 4F1 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 Shanghai Zeye Biotechnology Co., Ltd., item number ZY1027), 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, and the supernatant was collected by centrifugation. The correct expression of 4F1 was determined by SDS-PAGE.

[0061] (2) Purification of human albumin binding peptide 4F1 expressed by Pichia pastoris

[0062] 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), and the baseline was leveled, the sample was loaded, and the flow-through was collected; the column was washed with loading buffer until the baseline was leveled again, and elution buffer was used for elution, 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 that appeared was collected as the final human albumin-binding peptide 4F1 solution. If the protein concentration was low after desalting, the sample was concentrated using an ultrafiltration tube. The samples collected at each stage of purification were tested for purity, and the results of SDS-PAGE are shown in Figure 1

[0063] Example 5 Affinity detection of human albumin-binding peptide 4F1

[0064] The affinity of human albumin-binding peptide 4F1 was detected using the biofilm interference technology (BLI method). An NTA biosensor was used, and the sensor was first immersed in the analysis buffer for 10 min, and then immersed in the EDC-NHS mixed reagent for 5 min for activation; the activated sensor was immersed in the human albumin-binding peptide 4F1 diluent (100 nM) for 10 min and blocked with ethanolamine (1 M, pH 8.5); the blocked sensor was immersed in the buffer for baseline zeroing; then the sensor was sequentially immersed in gradient concentrations 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. The 1:1 binding model was used for kinetic analysis, and the results are shown in Figure 2

[0065] Example 6 Preparation of human albumin-binding peptide 4F1 affinity chromatography medium

[0066] ​​The human albumin binding peptide 4F1 lyophilized powder was dissolved in 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 volume was about 240 ml. The washed medium was diluted to 6 ml with 1 mM HCl, and an equal volume of the binding peptide solution was mixed 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. 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) were used to wash 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.

[0067] Example 7 Affinity chromatography purification of recombinant human albumin

[0068] 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 minutes, 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 minutes, 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 minutes, 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.

[0069] The human albumin-binding peptide 4F1 affinity chromatography medium 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 the loading was completed, 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.

[0070] Example 8 Gel filtration chromatography purification of recombinant human albumin

[0071] The first purified product obtained in Example 7 was concentrated by centrifugation using an ultrafiltration centrifuge tube (molecular cut-off 10 kDa, purchased from Millipore, item number: UFC901024) at 4°C and 4000 x g, and the volume and protein concentration were monitored every 15 minutes until the protein concentration stabilized at 50 ± 2 mg / mL (the volume after concentration was about 103 mL), and a small amount of 50 mM Tris-HCl + 0.1 M NaCl buffer (pH 7.6) was added during the concentration to prevent local over-concentration from causing protein denaturation. After the concentration was completed, the sample was filtered using a 0.22 μm PVDF filter membrane (purchased from Millipore, item number: SLGV033RB) in a sterile operation table, and the clear filtrate was collected and stored at 4°C until loading.

[0072] A Superdex 200 gel filtration medium (purchased from Cytiva, Cat. No. 17-1043-01) was packed into a glass chromatography column XK50 / 100 (column size: 50 mm x 520 mm, column bed volume about 1021 ml). The chromatography column was connected to an AKTApure 250 system (Cytiva), and the detection wavelength was set to 280 nm, the conductance monitoring range was 0-30 mS / cm, and the upper limit of column pressure was 0.15 MPa. A 50 mM Tris-HCl + 0.1 M NaCl buffer (pH 7.6) was used to equilibrate at least 3 times the column volume (3063 ml) at a linear flow rate of 20 cm / h. The concentrated sample was loaded at 5% of the column volume (about 51 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.

[0073] The collected components were combined, desalted using an ultrafiltration membrane bag (purchased from Millipore, Model: P2B010C01, 10 kDa cutoff), and exchanged into water for injection, and then freeze-dried to obtain a high-purity recombinant human albumin freeze-dried powder.

[0074] Example 9: Purity and yield analysis

[0075] (1) Purity analysis of recombinant human albumin

[0076] The purity of the recombinant human albumin freeze-dried powder obtained in Example 8 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, Cat. No. 08541). The phosphate buffer (PBS, 10 mM sodium phosphate, 150 mM NaCl, pH 7.4) was used as the mobile phase for isocratic elution, the flow rate was set to 0.8 mL / min, the column temperature was maintained at 25 ± 1°C, the detection wavelength was 280 nm, the injection volume was 20 μL, and the run time was 30 min. 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 external standard peak area normalization. After three independent repeated determinations, the average value of the main peak area percentage of the sample was 99.9% ± 0.02% (n = 3), RSD = 0.02%, which was reproducible, and a typical chromatogram is shown in Figure 3 The purity was improved from 98.7% (CN118580318 A) to 99.9%, an increase of 1.2 percentage points.

[0077] (2) Recombinant human albumin yield calculation

[0078] The protein concentration of each purification step was quantified by BCA (Bicinchoninic Acid) method, and the total yield was calculated accordingly. Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, Cat 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 >0.9992). The sample to be tested is appropriately diluted so that its concentration falls within the linear range of the standard curve. Each sample is set up in triplicate, and 25 μL of standard or diluted sample is added to each well, followed by 200 μL of BCA working solution. After incubation at 37°C for 30 minutes, the absorbance value is measured at 562 nm wavelength using a microplate reader (BioTek Synergy H1). The sample concentration is calculated according to the standard curve, and the results are shown in Table 1. The total protein amount of the supernatant after pretreatment (denoted as Step 0) is used as the starting point for calculation. After affinity chromatography (Step 1) and gel chromatography (Step 2), the total yield of the purified product is 77% ± 1.2% (n=3), which is 2.4 times higher than the traditional process (32% (CN102190722A2)), further verifying the high efficiency of the process of the present application.

[0079] Table 1 Recovery rate of each step

[0080]

[0081] Step 0 is the recovery rate of the total protein amount of the supernatant after pretreatment of the initial fermentation broth by centrifugation and filtration relative to the total protein amount before centrifugation of the fermentation broth; Step 1 and Step 2 are the recovery rates of the total target protein amount in the eluate of each chromatography step relative to the total target protein amount before loading in the previous step. The total protein amount of all steps is calculated by 'concentration (determined by BCA method) x actual sample volume', and volume correction is made when the sample is diluted. The recovery rate in the table is the average value of three independent experiments (n=3), and there is no SD notation because the SD of each step is <1.5%, and the SD of the total recovery rate is 1.1% (resulting from the cumulative calculation of the deviation of each step).

[0082] Example 10 Safety index detection

[0083] (1) Endotoxin detection

[0084] The endotoxin content of the final product was detected by dynamic turbidity method limulus test. The dynamic turbidity method endotoxin detection kit (Lonza, item number: N588) was used. First, the recombinant human albumin lyophilized powder obtained in Example 8 was dissolved and diluted to a concentration of 2 mg / mL with endotoxin test 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 minutes. The instrument software automatically calculates the endotoxin concentration in the sample according to the standard curve (R 2 =0.9985). According to the requirements of the “People's Republic of China Pharmacopoeia” (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 0.65 ± 0.08 EU / mg (n=3, RSD=7.7%), which is much lower than the limit requirement of “less than 1 EU / mg” in the “People's Republic of China Pharmacopoeia” (2020 edition) 1143 general rules, which proves that the safety of the final product meets the standards.

[0085] (2) Host cell protein (HCP) residual detection

[0086] 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. The HCP standard used in the kit was the purified HCP of the same Pichia pastoris strain as in 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 to the recovery test (spiked concentration 20 ng / mL), with a recovery rate of 98%, verifying that there was no matrix interference in the detection. Before detection, the rHSA sample was diluted to 5 mg / mL, and pre-verification showed that rHSA at this concentration had no interference with the detection. The standard curve (R 2 =0.9978) was calculated, and the results showed that the HCP residual amount in the final product was 3.2 ± 0.3 ng / mg rHSA (n=3, RSD=9.4%). This value is significantly lower than the limit value of "HCP residual ≤10 ng / mg" specified in the "Guidelines for Quality Control of Recombinant DNA Products for Human Use" (2020) of the National Medical Products Administration, fully demonstrating that the purification process can efficiently remove Pichia pastoris host proteins.

[0087] (3) Residual DNA detection

[0088] Real-time fluorescent quantitative PCR (qPCR) was used, targeting the conserved GAPDH gene of the Pichia pastoris genome. First, a DNA extraction kit (manufacturer: Qiagen, item number: 51304) was used to purify 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 adding 10 pg of Pichia pastoris genomic DNA to the sample solution, and the recovery rate of qPCR detection after extraction was 92%. The final residual DNA content has been corrected according to the extraction efficiency. Then a specific qPCR detection kit (manufacturer: Thermo Fisher Scientific, item number: A24554) was used for amplification. A standard curve (R 2The specificity and accuracy of the detection were ensured by the fact that the amplification product was analyzed by agarose gel electrophoresis (0.9983) and by the analysis of the melting curve of the amplification product (single peak, Tm=85.5°C). The final calculation gave a residual DNA content of 4.5 ± 0.4 pg / mg rHSA (n=3, RSD=8.9%). This result meets the standard of "residual DNA ≤10 pg / mg" in the "Chinese Pharmacopoeia" (2020 edition) General 3407, further verifying the effectiveness of the process in removing nucleic acid impurities, and the safety of the final product meets the pharmaceutical requirements.

[0089] Comparative Example 1 Change of affinity chromatography medium

[0090] Take 2000 ml of yeast-expressed recombinant human albumin fermentation broth, centrifuge at 4°C, 8000 rpm (centrifugal force about 10,000 ×g) for 20 minutes, collect the supernatant. Add 100 mM sodium caprylate stock solution to the supernatant to make its final concentration reach 15 mM, mix slowly. Adjust the pH to 6.0 ± 0.1 with 1 M HCl solution, then heat treat in a 65.0 ± 0.5°C water bath for 45 minutes, quickly ice bath to below 25°C. Centrifuge at 4°C, 12000 rpm (centrifugal force about 20,000 × g) for 15 minutes, collect the supernatant, repeat the heat denaturation and centrifugation operation once. The final supernatant is filtered through a 0.45 μm PES membrane to obtain a clear sample.

[0091] Pack the commercial Protein A affinity chromatography medium in the chromatography column, equilibrate with 3 column volumes of equilibration buffer (PBS, pH 7.4), load with a loading capacity of 15 mg / ml, rinse the column to the UV baseline level with equilibration buffer, elute with 4 column volumes of elution buffer (0.1M Glycine, pH2.8), collect the elution fraction and immediately add neutralization buffer (1M Tris-HCl, pH8.0) at a ratio of 20:1 to obtain the first purified product.

[0092] Concentrate the first purified product containing recombinant human albumin obtained above, filter through a 0.22 μm filter, pump into a Superdex 200 (column size: 50 mm × 520 mm, column volume: 1021 ml) column equilibrated with 2 volumes of 50 mM Tris-HCl + 0.1 M NaCl, then elute at low speed with the same buffer, desalt, freeze-dry to obtain recombinant human albumin. The detection results are shown in Table 2.

[0093] Table 2 Detection data table of recombinant human albumin in comparative example 1

[0094]

[0095] Protein A has low specificity for rHSA and adsorbs a large amount of HCP; Protein A has low binding capacity, high flow-through loss, and incomplete collection of target protein.

[0096] Comparative Example 2: Change of elution buffer for affinity chromatography

[0097] Take 2000 ml of the fermentation broth of recombinant human albumin expressed by yeast, centrifuge at 8000 rpm (centrifugal force of about 10,000 x g) at 4°C for 20 minutes, and collect the supernatant. Add 100 mM sodium octanoate stock solution to the supernatant to make the final concentration 15 mM, and mix well under slow stirring. Adjust the pH to 6.0 ± 0.1 with 1 M HCl solution, and then heat treat in a water bath at 65.0 ± 0.5°C for 45 minutes, and quickly cool to below 25°C in an ice bath. Centrifuge at 12000 rpm (centrifugal force of about 20,000 x g) at 4°C for 15 minutes, collect the supernatant, and repeat the heat denaturation and centrifugation operation once. Filter the final supernatant through a 0.45 μm PES membrane to obtain a clear sample.

[0098] Pack the human albumin binding peptide 4F1 affinity chromatography medium prepared in Example 6 in a chromatography column, equilibrate with 3 column volumes of equilibration buffer (PBS, pH 7.4), load with a loading capacity of 50 mg / ml, rinse the column to the UV baseline level with equilibration buffer, elute with 4 column volumes of elution buffer (0.05 M glycine, pH 3.5), and collect the elution fractions, immediately add neutralization buffer (1 M Tris-HCl, pH 8.0) at a ratio of 20:1 to obtain the first purified product.

[0099] Concentrate the first purified product containing recombinant human albumin obtained above, filter through a 0.22 μm filter membrane, and pump into a Superdex 200 column (column specifications: 50 mm x 520 mm, column volume: 1021 ml) equilibrated with 2 column volumes of 50 mM Tris-HCl + 0.1 M NaCl, then elute at low speed with the same buffer of 50 mM Tris-HCl + 0.1 M NaCl, desalt, and freeze-dry to obtain recombinant human albumin. The detection data results of recombinant human albumin in Comparative Example 2 are shown in Table 3.

[0100] Table 3: Detection data table of recombinant human albumin in Comparative Example 2

[0101]

[0102] 0.05M Glycine concentration low, pH 3.5 insufficient elution strength, rHSA not completely eluted from impurities. Insufficient elution buffer strength, rHSA medium carryover, decreased recovery.

[0103] Comparative Example 3 Change affinity chromatography equilibration buffer pH

[0104] Take 2000 ml of the yeast-expressed recombinant human albumin fermentation broth, centrifuge at 8000 rpm (centrifugal force of about 10,000 x g) at 4°C for 20 minutes, and collect the supernatant. Add 100 mM sodium octanoate stock solution to the supernatant to make the final concentration 15 mM, and mix well under slow stirring. Adjust the pH to 6.0 ± 0.1 with 1 M HC1 solution, and then heat treat in a 65.0 ± 0.5°C water bath for 45 minutes, and quickly cool to below 25°C in an ice bath. Centrifuge at 12000 rpm (centrifugal force of about 20,000 x g) at 4°C for 15 minutes, collect the supernatant, and repeat the heat denaturation and centrifugation operation once. Filter the final supernatant through a 0.45 μm PES membrane to obtain a clear sample.

[0105] Pack the human albumin binding peptide 4F1 affinity chromatography medium prepared in Example 6 in a chromatography column, equilibrate with 3 column volumes of equilibration buffer (PBS, pH 6.5), load with a 50 mg / ml loading capacity, rinse the column to the UV baseline level with equilibration buffer, elute with 4 column volumes of elution buffer (0.1 M Glycine, pH 2.8), and collect the elution fractions, immediately add neutralization buffer (1 M Tris-HCl, pH 8.0) at a ratio of 20:1 to obtain the first purified product.

[0106] Concentrate the first purified product containing recombinant human albumin obtained above, filter through a 0.22 μm filter membrane, and pump into a Superdex 200 (column specifications: 50 mm x 520 mm, column volume: 1021 ml) column equilibrated with 2 column volumes of 50 mM Tris-HCl + 0.1 M NaCl, then elute at low speed with 50 mM Tris-HCl + 0.1 M NaCl buffer, desalt, and freeze-dry to obtain recombinant human albumin. The detection results are shown in Table 4.

[0107] Table 4 Detection data table of recombinant human albumin in Comparative Example 3

[0108]

[0109] PBS pH 6.5 reduces the binding of peptide 4F1 to rHSA, co-elution of impurities; affinity chromatography flow-through rHSA carryover increases, resulting in decreased recovery of target protein.

[0110] Key notes

[0111] All pretreatment steps (centrifugation, heat denaturation, filtration) of the comparative examples were fully consistent with Example 7, ensuring that the differences only come from the modification of the chromatography parameters, and the data comparison has uniqueness;

[0112] The impurity detection methods (endotoxin: dynamic turbidity method, HCP: ELISA, residual DNA: qPCR, aggregate: SEC-HPLC) are consistent with Example 11, and the data accuracy is traceable;

[0113] The purity, yield and impurity indicators of the final product of all comparative examples are significantly worse than those of the examples, fully proving the optimization of the parameters in Examples 7 and 8: "affinity chromatography equilibration buffer pH 7.4 + elution buffer 0.1M Glycine pH 2.8 + gel filtration 50mM Tris-HCl + 0.1M NaCl equilibration Superdex 200".

[0114] The above is only an embodiment 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. The present application can have various changes and variations for those skilled in the art. 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 4F1, characterized in that, The amino acid sequence of the human albumin-binding peptide 4F1 is selected from any 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 is selected from any one of the following: B1) A nucleic acid molecule encoding the human albumin-binding peptide 4F1 as described in claim 1; B2) An expression cassette containing the nucleic acid molecule described in B1); B3) A recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2); B4) Recombinant microorganisms containing 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 4F1 according to claim 1, characterized in that, The method includes the steps of culturing the recombinant microorganism (B4) or the recombinant cell (B5) in the biological material of claim 2 under suitable culture conditions, and isolating the human albumin-binding peptide 4F1.

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

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

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

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

10. The use of the human albumin-binding peptide 4F1 of claim 1, or the biomaterial of any one of claims 2-4, or the chromatography medium of claim 6 or 7, in any of the following aspects: C1) Applications in the preparation of products for the isolation or purification of human albumin; C2) Applications in the preparation of products for the qualitative or quantitative detection of human albumin.

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