Filler containing human serum albumin as well as construction method and application of filler

Abstract

The invention relates to a coupling filler for purifying a multivalent nano antibody capable of being specifically bound with human serum albumin as well as a preparation method and application of the coupling filler. The invention specifically provides a filler containing human serum albumin and a preparation method thereof, the filler is washed by a specific method, then the filler is mixed and coupled with a human serum albumin protein solution, and the following steps of sealing by a sealing liquid are carried out. The invention also provides a method for purifying the multivalent nano antibody by using the filler, and the method has the advantages of high specificity, mild operation conditions, high purity and high recovery rate of the nano antibody, and is beneficial to commercialized large-scale production.

Description

Technical field

[0001] The present invention belongs to the field of protein separation technology, and specifically relates to a human serum albumin coupling filler, a preparation method, affinity purification nano-antibody process and its use. Background technology

[0002] Nanobody (Nb) is a single domain antibody (sdAb) composed of only heavy chain variable region fragments (VHH) of heavy chain antibodies (single domain antibody) that is composed of heavy chain variable regions, i.e. heavy chain single domain antibody; its relative molecular weight is about 15kDa, which is only about 1 / 10 of that of ordinary antibodies. Nanobody is naturally found in the blood of alpacas and sharks. Although nano-antibody is small, compared with ordinary antibodies, nano-antibody has only three complementary determination regions (CDRs), it has good specific antigen binding ability and high affinity, and is the smallest unit antibody with complete biological functions. This special structure not only has the advantages of ordinary nano-antibody, but also has some uniqueness. First, nanoantibodies have only one domain and no Fc segment of ordinary antibodies, and there will be no complement effect caused by the Fc segment. Secondly, some hydrophobic groups in the VHH of the nano-antibody are replaced by hydrophilic groups, which greatly increases the water solubility of the nano-antibody and greatly improves the utilization rate as a drug. Nanobody can recognize unique constructed epitopes and have a broader antigen recognition ability than conventional antibodies. At the same time, nanobody has a longer CDR3, which can form a stable exposed convex ring structure that can penetrate deep into the antigen to better bind to the antigen, thereby improving its antigen specificity and affinity. Therefore, the nanobody VHH single domain has a broader antigen binding force. Finally, because the nanobody molecules are small and the nanobody is highly homologous to the VH sequence of human antibodies, the nanobody has weak immunogenicity to the human body. Repeated administration of nanobody generally does not cause any humoral and cellular immune responses.

[0003] Nanobody has strong and fast tissue penetration ability and can enter solid tumors and play a role. Nanobody can also pass through the blood-brain barrier and develop a series of developers and new drugs for diagnosing and treating brain diseases. Nanobody has a small molecular weight, a simple structure, and is encoded by a single gene. It is easy to express in large quantities in microorganisms such as E. coli and yeast. Its production cost is low, simple and convenient, and functional antibody preparations can be obtained in a short period of time. However, because the nano-antibody has a relatively small molecular weight, only 15 kDa, it is easily filtered by glomerular filtration and excreted from the body during circulation in vivo. It has a short half-life of serum and poor bioavailability. It requires frequent and high dose administration during clinical use, which undoubtedly increases the economic burden and inconvenience of use of the patient, limits the clinical application of these drugs, and urgently needs to be re-engineered. Long-term transformation of such drugs is a measure to improve pharmacokinetics, and it is also the current development trend of upgrading protein and polypeptide drugs. Long-acting strategy: Fusion of therapeutic nano-antibody to nano-antibody targeting human serum albumin is a common method.

[0004] In recent years, VHHs have favored therapeutic targeting because of their small size as monovalent structures or as multivalent or multispecific structures, and their possibility of extending the half-life of therapeutic proteins by fusion with VHHs targeting serum albumin antigens. Another example of serum albumin-binding VHH is provided by Ablynx N.V. (see Patent Documents WO2004 / 041865A2, Publication Date 2004-05-21 and WO2006 / 122787A1, Publication Date 2006-11-23), which describes nano-antibodies against serum albumin and, in particular, human serum albumin, which can be linked to other (therapeutic) proteins (such as one or more other targets for the desired target) to increase the half-life of the (therapeutic) protein in a patient. Thus, in summary, serum albumin-bound VHH will be of interest to increase the half-life of therapeutic proteins (also possibly VHH, as a fusion partner of serum albumin-bound VHH). However, there are also some common problems in the purification technology of nano-antibody, such as difficulty in purification of multivalent nano-antibody, low recovery rate in the purification process of nano-antibody leads to higher production costs. Therefore, developing some fillers for the purification of nanoantibodies targeted by human serum albumin has high practical significance for reducing the purification cost of nanoantibodies. The prior art discloses different nanoantibodies purification methods, such as patent document CN110204612A (publication date 2019-09-06) discloses a method for purifying nanoantibodies using Protein A affinity chromatography. However, the purity of the nanoantibodies obtained by this method is only about 80%. Patent Document CN114539351B (publication date 2023-03-03) discloses a method of cation chromatography and anion chromatography sequentially, collecting anion chromatography eluent, and purifying the nano-antibody. However, this method is relatively cumbersome and requires the management of two-step ion chromatography. It can be seen that the prior art nano-antibody has defects such as low purity and cumbersome steps in the target product. At present, there has been no research on the purification of nano-antibody using human serum albumin-coupled fillers. Whether it can be applied to the field of protein purification, how to optimize the purification steps, how to use human serum albumin to couple to improve the effect of protein purification, and how to use the obtained human serum albumin-coupled fillers to purify nano-antibody containing specifically binding to human serum albumin. These are technical problems that need to be solved urgently in this field. Contents of invention

[0005] In response to the above problems and shortcomings in the prior art, the purpose of this application is to provide a human serum albumin (HSA)-conjugated affinity chromatography filler and its preparation method, as well as an affinity purification process for nano-antibody specifically bound to HSA, which aims to improve the technical problems such as the low yield and purity of existing nano-antibody targeting human serum albumin and the recombinant protein and multivalent nano-antibody targeting human serum albumin during purification.

[0006] The first aspect of the present application provides a method for preparing an affinity chromatographic filler based on human serum albumin (HSA), characterized in that it comprises the following steps: (1) Activated agarose gel filler; preferably, the filler is a CNBr Focus 4FF pre-activated agarose gel; (2) Couple the activated filler with a ligand, the ligand comprises human serum albumin; the coupling comprises dissolving a human serum albumin sample at a concentration of 0.5-10 mg / ml in 0.1 M NaHCO 3 , 0.5 M NaCl, pH 8.3 solution, stir at room temperature for 4 hours; among which, the filler before coupling is diluted with 1 mM HCl, and 0.5-1 mL HCl is added per 1 mL of filler, and the final concentration after the addition of human serum albumin at the concentration of 0.5-10 mg / ml during coupling is 1 mg / ml; (3) The sealing liquid sealing the filler after coupling; preferably, the sealing step includes adding 2 times the volume of medium 0.1M sodium acetate, 0.5M NaCl, pH 3.0, blocking liquid, stirring evenly and draining.

[0007] Further, the step (1) activates the agarose filler comprises the following steps: drain the agarose filler, add pre-cooled HCl solution and stir evenly before draining.

[0008] Further, the step (1) activate the agarose filler includes the following steps: drain the required amount of agarose filler medium, add 2 times the volume of the medium to a pre-cooled 1 mM HCl solution, stir evenly and drain, and repeat the HCl solution treatment step twice.

[0009] Further, the step (2) further comprises steps (A)-(C): The step (A) includes draining the coupled filler medium, adding 0.1M Tris-HCl, pH 8.0 solution in 2 times the volume of the medium, and then draining, repeating twice; Step (B) includes adding 0.1M Tris-HCl, pH 8.0 solution in 2 times the volume of the medium, and gently stirring for 4 h at room temperature; Step (C) includes pumping the filler medium, adding 0.1M Tris-HCl, 0.5M NaCl, pH 9.0 solution in 2 times the volume of the medium, and then draining; Further, the step (3) is followed by steps (D)-(F): Step (D) includes adding 2 times the volume of 0.1 M sodium acetate, 0.5 M NaCl, pH 3.0 as a solution, stirring evenly and draining; Step (E) includes repeating Step (3) and Step (D) for a total of two times; Step (F) includes 20% ethanol treatment and storage.

[0010] Further, the preparation method comprises the following steps: (1) Take the required amount of agarose filler and drain it into the corresponding sand core funnel; (2) Add 2 times the volume of medium to pre-cool 1 mM HCl solution, stir evenly and drain; repeat twice; (3) Transfer the medium to the reactor of the corresponding specifications; (4) Dissolve the human serum albumin sample at a concentration of 0.5-10 mg / ml in 0.1 M NaHCO3, 0.5 MNaCl, pH 8.3 as a solution, and stir gently for 4 h at room temperature; (5) Transfer the medium to the sand core funnel and drain it, and store the coupled solution in the collection container; (6) Add 0.1M Tris-HCl in a volume of 2 times the medium volume, pH 8.0 solution, stir evenly and drain; repeat twice; (7) Transfer the medium to the reactor of the corresponding specifications, add 2 times the volume of the medium 0.1 M Tris-HCl, pH 8.0 solution, and stir gently for 4 h at room temperature; (8) Transfer the medium to the sand core funnel to drain; (9) Add 0.1 M Tris-HCl and 0.5 M NaCl in 2 times the volume of the medium, pH 9.0 as the solution, stir evenly and then drain; (10) Add 2 times the volume of medium 0.1 M sodium acetate and 0.5 M NaCl, pH 3.0 as the solution, stir evenly and then drain; (11) Repeat steps (9)-(10) 2 times in total; (12) Add 2 times the volume of 20% ethanol in the medium, stir evenly and drain, and repeat 2 times; (13) Transfer the medium to a container of the corresponding specifications, add an equal volume of 20% ethanol and save for later use.

[0011] The second aspect of the present application provides a preparation method to prepare the obtained affinity chromatography filler based on human serum albumin (HSA).

[0012] The third aspect of the present application provides a purification method for multivalent nanoantibodies using the human serum albumin (HSA)-based affinity chromatography filler, characterized in that the method comprises the steps of: (1) Column cleaning: Use 0.1 M NaOH cleaning solution; (2) Balance: Use 10 mM PBS with a balanced liquid with pH=7.4; (3) Loading: Loading samples containing multivalent nano-antibody; (4) Column cleaning: Use 10 mm PBS, pH=7.4 cleaning solution; (5) Eelution of the target product: elution is carried out using 20 mm sodium acetate, pH=3.5; (6) Column cleaning: Use 0.1 M NaOH cleaning solution; (7) Prepare before lowering the column: Clean 20% ethanol and store the affinity chromatography column.

[0013] Further, the multivalent nanobody comprises human serum albumin; preferably, the multivalent nanobody comprises trivalent nanobody; more preferably, the trivalent nanobody comprises Anti-TNFα&Albumin Nanobody, Anti-EGFR&Albumin Nanobody; Most preferably, the amino acid sequence of the Anti-TNFα&Albumin Nanobody is shown in SEQ ID NO:1, and the amino acid sequence of the Anti-EGFR&Albumin Nanobody is shown in SEQ ID NO:3.

[0014] Further, the purification method comprises the following steps: (1) Column cleaning stage: 0.1 M NaOH is cleaned at a speed of 1ml / min until A280 drops to near the constant value; (2) Equilibrium stage: 10 mM PBS, pH=7.4 is cleaned at a speed of 1 ml / min until A280 drops to near the constant value and ends the equilibrium; (3) Loading stage: The sample is loaded at a speed of 1 ml / min, and the sample retention time is 5 min until A280 drops to near the constant value and finish loading; pay attention to collecting the flow-through sample for testing; (4) Column cleaning stage: 10 mm PBS, pH=7.4 is cleaned at a speed of 1ml / min until A280 drops to near the constant value and ends the cleaning; (5) Elution stage: 20 mm sodium acetate, pH=3.5 elution is carried out at a rate of 1 ml / min until A280 drops to near the constant value and ends the elution; collect the eluted sample for detection; (6) Column cleaning stage: 0.1 M NaOH is cleaned at a speed of 1 ml / min until A280 drops to near the constant value and ends CIP. Pay attention to collecting CIP samples for testing; (7) Preparation before column: 20% ethanol was washed at 1 ml / min, cycle 10CV, and place an affinity chromatography column containing human serum albumin filler at 4°C for storage.

[0015] Further, the multivalent nanoantibodies are obtained by fermentation, which includes the preparation steps of primary seed culture, secondary seed culture, fermentation culture, and fermentation supernatant.

[0016] Further, the fermentation comprises the following steps: (1) First-level seed culture: The preserved recombinant Pichia cerevisiae was activated and cultured at 25-32°C, 100-250 rpm for 16-18 h; (2) Secondary seed culture: The primary seed liquid is inoculated into the seed culture medium at a volume ratio of 1-5%, and cultured to OD at 25-32°C, 100-300 rpm. 600 =6-8; (3) Fermentation and culture: Inoculate the seed liquid into the fermentation medium, cultivate the fermentation tank, and control the DO to 10%-50%, preferably, control the DO to 30%, stir the rotation speed 200-800 rpm, the ventilation volume 0.5-2.5 vvm, and add a certain concentration of pure oxygen to maintain the fermentation broth DO within the appropriate range; (4) Preparation of fermentation supernatant: After fermentation for at least 76 hours, take the fermentation broth and centrifuge to remove the bacteria. The supernatant is filtered by vacuum, and collects it to obtain the fermentation broth.

[0017] Further, the maintenance broth DO is within an appropriate range of 10%-50%.

[0018] Further, the method further comprises step (5) protein purity detection, including detecting the purity of the multivalent nanoantibodies obtained after step (4) purification; preferably, the detection method is SDS-PAGE.

[0019] The fourth aspect of the present application provides the use of the affinity chromatography filler preparation method, the affinity chromatography filler based on human serum albumin (HSA), or the purification method in the isolation and purification of human serum albumin monovalent nanobody or the isolation and purification of human serum albumin multivalent nanobody; preferably, the multivalent nanobody comprises trivalent nanobody; more preferably, the trivalent nanobody comprises Anti-TNFα&Albumin Nanobody, Anti-EGFR&Albumin Nanobody; most preferably, the amino acid sequence of the Anti-TNFα&Albumin Nanobody is shown in SEQ ID NO:1, and the amino acid sequence of the Anti-EGFR&Albumin Nanobody is shown in SEQ ID NO:3.

[0020] The HSA coupling filler method provided by the present invention and the use of the separation and purification of nano-antibody has the following excellent technical effects: 1. The present invention does not rely on purification labels for the purification of multivalent nano-antibodies, especially trivalent nano-antibodies. The obtained trivalent nano-antibodies can be directly used as a recombinant protein of the pharmaceutical grade.

[0021] 2. The purification process of the purification separation method of the present invention is simple and efficient. It can obtain nano-antibody with high recovery and high purity by reducing the pretreatment fermentation broth and eluting the acidic sodium acetate solution. Examples 3-4 of the present invention were obtained based on the purification of HSA coupling filler. The purity of the trivalent nano-antibodies Anti-TNFα&Albumin Nanobody and Anti-EGFR&Albumin Nanobody obtained by the purification of HSA coupling filler reached more than 90%. Comparative Examples 5-6 purified the trivalent nano-antibodies Anti-TNFα&Albumin Nanobody and Anti-EGFR&Albumin Nanobody based on the anion filler 50QHC. The results showed that the ion filler had no obvious capture effect on the trivalent nano-antibodies Anti-TNFα&Albumin Nanobody and Anti-EGFR&Albumin Nanobody.

[0022] 3. The present invention realizes the rapid and efficient purification of multivalent nano-antibody, especially trivalent nano-antibody, from the fermentation broth, solves the problem that the previous difficult purification of multivalent nano-antibody fermentation broth, and realizes the purification of rapid trivalent nano-antibody fermentation broth. The separation and purification method of the present invention has high specificity, mild operating conditions, simple steps, good purification effect, high recovery rate, low cost and fast speed, which is conducive to commercial large-scale production. Attached description of the drawings

[0023] Figure 1 It is the growth curve of bacterial organisms in different time periods of the present invention.

[0024] Figure 2 It is the electrophoresis result of the expression amount of trivalent nano-antibody in the fermentation broth after the fermentation of the present invention.

[0025] Figure 3 The chromatography purification program map of the Anti-TNFα & Albumin Nanobody fermentation broth chromatography based on HSA coupling filler of the present invention.

[0026] Figure 4 The peak protein content SDS-PAGE electrophoresis results were obtained for the chromatography purification procedure of Anti-TNFα & Albumin Nanobody fermentation broth based on HSA coupling filler of the present invention.

[0027] Figure 5 The chromatogram of Anti-EGFR & Albumin Nanobody fermentation broth chromatography for the present invention is based on HSA coupling filler.

[0028] Figure 6 The peak protein content SDS-PAGE electrophoresis results were obtained for the chromatography purification procedure of Anti-EGFR & Albumin Nanobody fermentation broth based on HSA coupling filler of the present invention.

[0029] Figure 7 The chromatography purification program map of the Anti-TNFα & Albumin Nanobody fermentation broth chromatography based on the anionic filler 50QHC of the present invention.

[0030] Figure 8 The peak protein content SDS-PAGE electrophoresis results were obtained for the chromatography purification procedure of the Anti-TNFα & Albumin Nanobody fermentation broth based on the anionic filler 50QHC of the present invention.

[0031] Figure 9 Chromatography purification program map of the Anti-EGFR & Albumin Nanobody fermentation broth based on anionic filler 50QHC of the present invention.

[0032] Figure 10 The peak protein content SDS-PAGE electrophoresis results were obtained for the chromatography purification procedure of the Anti-EGFR & Albumin Nanobody fermentation broth based on the anionic filler 50QHC of the present invention. Specific implementation methods

[0033] The present invention will be further described in detail below in conjunction with the accompanying drawings and implementation examples, but the scope of protection of the present invention is not limited to the above; conventional molecular biology and protein engineering methods are mainly used in the implementation examples, which are well known to those who are familiar with ordinary biotechnical skills in the art. The specific reagents, plasmids, culture media, instruments, etc. used in the examples are commercially available in the art.

[0034] Example 1 Preparation of high-expression nanoantibodies fermentation broth The preparation of fermentation broth includes the following steps: Step 1: Remove the stored glycerol tubes of recombinant high-expression of X33 / pPICZα A-target nanobody gene from an ultra-low temperature refrigerator of -80°C. The target nanobody is Anti-TNFα&Albumin Nanobody, Anti-EGFR&Albumin Nanobody, respectively. The amino acid sequence of Anti-TNFα & Albumin Nanobody is shown in SEQ ID NO:1 and the gene DNA sequence is shown in SEQ ID NO:2. The amino acid sequence of Anti-EGFR & Albumin Nanobody is shown in SEQ ID NO:3, and the gene DNA sequence is shown in SEQ ID NO:4. Inoculate 400 μL of bacterial seed solution into 200 mL of seed medium (BMGY) in the biosafety cabinet, seal and shake well, then place it in a constant temperature shaker for culture at a temperature of 30℃ and a speed of 250 rpm. After 24 hours of culture, use an ultraviolet spectrophotometer to measure the OD600 value between 2-6.

[0035] Step 2: The dissolved oxygen is 100% at the beginning of fermentation, and as the fermentation progresses, the dissolved oxygen will gradually be lower. When the DO is less than 20%, DO>20% is maintained by increasing the air intake rate (maximum intake volume <2 vvm) and increasing the oxygen ratio in turn (DO<40% after regulation); at the end of batch fermentation, glycerin is depleted, and DO gradually rises to close to 100%. At the end of the feeding confirmation, the glycerin content can be directly detected through a biochemical analyzer, or appropriately add a part of glycerin to observe whether the DO value decreases. The decrease indicates that glycerin is depleted and the next stage of culture can be carried out.

[0036] Step 3: Glycerol feed stage. Glycerol (18.15 mL / hr / L initial fermentation volume) was added at 0.2 rpm. When the wet weight of the bacteria reaches more than 200 g / L, the fermentation of this stage is completed, that is, the feeding is stopped, and the bacteria are hungry until the DO is close to 100%, which means that the glycerol is depleted and the bacteria are in a hungry state. You can start the next stage of culture (when the wet weight is approaching, the sampling interval is appropriately reduced, and only the feeding is stopped at the end, and other settings remain unchanged).

[0037] Step 4: Methanol feed stage. Add methanol feed solution at 6 mL / h (0.2 rpm, 3 L culture volume). During the initial stage of 2-3 hours, the bacteria are adapting to the methanol environment and the DO value fluctuates greatly, and will eventually return to stability. When the DO is less than 20%, the DO>20% is maintained by increasing the air intake rate (maximum intake volume ≤ 2 vvm) and increasing the oxygen ratio in turn (DO<40% after regulation); every 1 h, the methanol content can be directly detected through a biochemical analyzer, or the DO value decreases in the methanol flow rate, or the DO value rises rapidly in a short time (within 3 min) and increases the flow rate by 0.1 rpm. After adjusting the flow rate, repeat this step until the flow rate is adjusted to 1.5 rpm. Meanwhile, 10 mL of the fermentation broth was sampled. Used to measure pH and OD 600 , wet weight, methanol content, protein expression, and the growth curve of its strains is as follows: Figure 1 As shown, where 221109-R11 is Anti-TNFα&Albumin Nanobody, and 221109-R12 is Anti-EGFR&Albumin Nanobody. The electrophoresis results of the expression of nano-antibody in the fermentation broth after fermentation are as follows Figure 2 Shown ( Figure 2 The distribution of different lanes of the electrophoresis plot is lane 1; protein marker, lane 2: 0.5 μg of bovine serum albumin; lane 3: 1 μL of anti-TNFα&Albumin Nanobody fermentation end sample loading, lane 4: 3 μL of anti-TNFα&Albumin Nanobody fermentation end sample loading, lane 5: 5 μL of anti-TNFα&Albumin Nanobody fermentation end sample loading, lane 6: 7 μL of anti-TNFα&Albumin Nanobody fermentation end sample loading, lane 7: 10 μL of anti-TNFα&Albumin Nanobody fermentation end sample loading, lane 8: Anti-EGFR&Albumin Nanobody fermentation end sample loading 1 μL, Lane 9: Anti-EGFR & Albumin Nanobody fermentation end sample loading 3 μL, Lane 10: Anti-EGFR & Albumin Nanobody fermentation end sample loading 5 μL, Lane 11: Anti-EGFR & Albumin Nanobody fermentation end sample loading 7 μL, Lane 12: Anti-EGFR & Albumin Nanobody fermentation end sample loading 10 μL). The fermentation broth was placed in the tank, and the fermentation broth was collected. It was centrifuged in a floor-mounted centrifuge for 15 minutes. The precipitation was discarded and the supernatant was collected. The aliquoted part of the supernatant is stored at 4°C and set aside; the remaining supernatant stock solution is stored at -20°C.

[0038] Table 1 below shows the amino acid sequence and DNA sequence of the trivalent nanobody obtained from the fermentation broth expression.

[0039] Table 1 Sequence information of trivalent nanoantibodies of the present invention Sequence information Anti-TNFα&Albumin Nanobody amino acid sequence EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCARSPSGFNRGQGTLVTVSSGGGSGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTL YADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCARSPSGFNRGQGTLVTVSS (SEQ ID NO:1) Anti-TNFα&Albumin Nanobody DNA sequence GAAGTGCAGCTAGTCGAAAGTGGCGGAGCCTAGTCCAGCCAGGAGGCTCACTACGTTTATCATGTGCTGCTTCCGGTTTCACATTTAGTGACTATTGGATGTACTGGGTGCGTCAAGCCCCCAGGAAAGGGATTGGA ATGGGTGTCTGAGATTAATACGAATGGACTAATCACAAAATACCCCAGATTCAGTGAAGGGCAGATTCACGATCTCCCAGAGACAATGCTAAGAACATTATATCTTCAAATGAACTCATTAAGACCCGAGGATACAG CAGTATATTACTGCGCACGTTCCCCCATCCGGCTTCAATCGTGGTCAGGCACCTTAGTAACGGTCAGTTCAGGCGGTGGCTCAGGCGGTGGTTCCGAGGTCCAACTAGTCGAATCTGGAGGTGGTCTTGTACAG CCCGGCAACTCCTTGAGATTATCATGCGCCGCTTCTGGCTTTACGTTCTCATCCTTTGGAATGTCATGGGTTAGGCAGGCTCCCAGGTAAAGGTTTAGAGTGGGTATCCTCATCCATTTCAGGTAGTGGCTCCGACACGCT GTATGCAGATTCTGTGAAAGGAAGATTTACTATCTCCGTGACAATGCTAAGACTACTATACCTCAAATGAACTCCCTAAGACTCCCTAAGACCCGAAGATACAGCTGTCTATTACTGCACTATAGGTGGCTCACTAAGTAGTAGT CTAGTCAGGGTACGCTTGTTACTGTTTCCTCCGGCGGCGGCGGTAGTGGAGGAGCTCAGAAGTTCAACTAGTGGAATCTGGTGGTGGCCTGGTGCAACCTGGTGGAAGTCTAAGACTATCCTGTGCTGCTTCCGGA TTCACCTTCTCCGATTACTGGATGTATTGGGTGAGACAAGCCCCCAGGCAAAGGTCTTGAGTGGGTTCAGAGATTAACACCAATGGTCTGATTACTAAGTACCCAGACTCCGTAAAAGGACGTTTCACGATCAGTAG GGACAACGCAAAAAATACGCTTTATTTGCAGATGAACTCTCTAAGGCCAGAAGATACAGCCGTGTACTACTGCGCCAGGTCACCCAGTGGCTTTAACAGGGTCAGGCACTCTTGTTACCGTCAAGTCAAGTCAAGGGTCAGGCACTCTTGTTACCGTGTCAAGTTAA (SEQ ID NO:2) Anti-EGFR & Albumin Nanobody amino acid sequence QVKLEESGGGSVQTGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTTSRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSAWYGTLYEYDYWGQGTQVTVSSGGGSGGGSEVQLVESGGGSVQTGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTG YADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSAWYGTLYEYDYWGQGTQVTVSSGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS (SEQ ID NO:3) Anti-EGFR&Albumin Nanobody DNA sequence CAGGTGAAACTTGAGGAGTCTGGCGTGGATCCGTTCAAACAGGAGCTCTCTTAGATTAACTTGCGCAGCCTCTGGAAGGACTTCTAGGTCATATGGTATGGGCTGGTTCAGGCAAGCTCCCCGGAAAAGAGAGAATTTGT CTCCGGCATTTCATGGAGAGGCGACTCAACTGGATACGCTGACAGTGTCAAAGGCAGATTCACCTCTCGTGATAACGCCAAAAACACTGTCGATTTACAGATGAACTCATTGAAACCCGAGGACACAGCCATTTACT GCGCCGCTGCTGCCGGCTCAGCTTGGTATGGCACCCTATATGAGTATGATTATTGGGCCAGGGCACAGGTGACGGTTAGTTCCGGCGGAGGTGGTAGTGGAGGTGGTTCTCAGGTTAAATTAGAGGAGTGGCGGGA CTAGTTCAAGCAGGCGGTTCATTGAGGTTGTCCTGCGTGGCAAGTGGTAGGACATTTAGTAGAACAGCTAACGCATGGTTTAGACAGGCACCAGGTAAAGAGAGAATTCGTGGCTACTATTACCTGGAACAACG CGTTATGCAGATTCAGTTAAGGGTAGGTTCTTTATATCAAAGGATTCTGCAAAGAATACGATATATCTTGAAATGAATTCACTTGAACCAGAGGATACAGCCGTGTACTATTGTGCCGCAACCGCAGCCGCAGTCATCACACC CACCCGTGGTTATTATAATTACTGGGGTCAAGGCACGCAAGTGACTGTGTCTAGTGGTGGTGGCGGCTCTGGCGGCTCCGAGGTCCAGCTAGTTGAAAGTGGTGGCGGCTTGGTGCAGCCAGGCAATTCCTTACGTCTGTC ATGCGCAGCATCAGGTTTTACGTTCTCCTTCGGCATGTCCTGGGTGCGTCAGGCCCCCAGGAAAGGGCTTGGAATGGGTAAGTAGTATCTCCGGATCTGGATCTGACGTTATACGCAGATTCCGTCAAGGGACGTTTCA CGATCTCCCAGGGACAATGCTAAGACCACGCTGTACCTGCAGATGAATAGTCTAAGACCCGAGGATACGGCAGTCTATTATTGTACTATTGGAGGATCACTTAGTCGTTCCTCACAGGGAACGCTTGTGACGGTAAGTTCCC (SEQ ID NO:4) Example 2 Preparation of affinity purification filler for purified trivalent nanoantibodies The present invention provides a preparation method of HSA (human serum albumin) coupling filler, including the following steps: 1. Take the required amount of agarose filler and drain it into the corresponding sand core funnel.

[0040] 2. Add 2 times the volume of medium to pre-cool 1 mM HCl solution, stir evenly and drain; repeat 2 times.

[0041] 3. Transfer the medium to the reactor of the corresponding specifications.

[0042] 4. Dissolve the human serum albumin sample at a concentration of 0.5-10 mg / ml in 0.1 M NaHCO 3 , 0.5 MNaCl, pH 8.3 was added to the reactor, and the mixture was stirred gently at room temperature for about 4 h.

[0043] 5. Transfer the medium to the sand core funnel and drain it and store the coupled solution in the collection container.

[0044] 6. Add 2 times the volume of the medium 0.1M Tris-HCl, pH 8.0 solution, stir evenly and drain; repeat twice.

[0045] 7. Transfer the medium to the reactor of the corresponding specifications, add 2 times the volume of the medium 0.1 M Tris-HCl, pH 8.0 solution, and stir gently for about 4 h at room temperature.

[0046] 8. Transfer the medium to the sand core funnel and drain.

[0047] 9. Add 2 times the volume of 0.1 M Tris-HCl and 0.5 M NaCl, pH 9.0 as the solution, stir evenly and then drain.

[0048] 10. Add 2 times the volume of medium 0.1 M sodium acetate and 0.5 M NaCl, pH 3.0 as the solution, stir evenly and then drain.

[0049] 11. Repeat steps 9-10 in total 2 times.

[0050] 12. Add 2 times the volume of 20% ethanol in the medium, stir evenly and drain; repeat 2 times.

[0051] 13. Transfer the medium to a container of the corresponding specifications, add an equal volume of 20% ethanol and save for later use.

[0052] Example 3 Development of purification process of trivalent nanobody (Anti-TNFα & Albumin Nanobody) based on HSA coupling filler The present invention provides a general method for purifying nano-antibody of HSA-coupled filler, including the following steps: Step 1: Sample pretreatment. Before purification of the sample (the fermentation broth supernatant involving the target product Anti-TNFα & Albumin Nanobody in Example 1) was used to filter out the insoluble impurities in the sample using a 0.45 μm filter (manufacturer: Sedolis); if there are too many impurities in the sample and the filter membrane is significantly blocked, deep filtration (Claricap deep filter element, manufacturer: Kebat) can be used to remove impurities. At the same time, take 100 mL of the filtered fermentation broth to prepare for subsequent operations.

[0053] Step 2: Column cleaning stage: 0.1 M NaOH is cleaned at 1 ml / min until A280 drops to near the constant value; Equilibrium stage: 10 mM PBS (pH=7.4) is cleaned at 1 ml / min until A280 drops to near the constant value and ends with equilibrium; loading stage: Sample loading at 1 ml / min, the sample retention time is 5 min until A280 drops to near the constant value and ends with loading; pay attention to collecting the flow-through sample for detection; Column cleaning stage: 10 mM PBS (pH=7.4) is cleaned at 1 ml / min until A280 drops to near the constant value and ends with cleaning; elution stage: 20 mM sodium acetate (pH=3.5) was elution at 1 ml / min until A280 dropped to near a constant value and ended elution; the eluted samples were collected for inspection of the entire chromatography program map. Figure 3 Shown. Column cleaning stage: 0.1 M NaOH is cleaned at 1 ml / min until A280 drops to near a constant value and ends CIP (cleaning of chromatography medium). Pay attention to collecting CIP samples for detection; Preparation before column: 20% ethanol is cleaned at 1 ml / min, cycle 10CV (column volume), and place the affinity chromatography column containing human serum albumin filler at 4 °C for storage.

[0054] Step 3: Purify the sample SDS-PAGE detection. Mix the sample with the protein Loading Buffer (SDS, DTT and bromophenol blue) in a 5:1 ratio so that the protein Loading Buffer concentration of the final sample is not less than 1×, 98°C or denaturate for 10 minutes. Then, install the electrophoresis tank and rubber plate (de-sealing tape), pour in the electrophoresis buffer, the inner tank should be filled and overflowed, and the liquid level of the outer tank should be higher than the electrode wire. Slowly remove the pre-made gel comb, take the syringe and absorb the buffer, and put it into the glue holes one by one to blow away the glycerin. Spot the sample, close the slot cover, turn on the power, and electrophorese at 80 V for 40 minutes until the sample is compressed into a line. Increase the voltage value of 120 V Continue electrophoresis to the indicator band close to or to the bottom of the gel. The gels that were electrophoretic were stained and decolorized using a protein stainer. The setting procedure was Stain3 min; Destain 2 min; Destain 1 min. Transfer the gel to the gel imaging system, image, modify the image, set up the lanes, label the lanes, and perform corresponding analysis. The results of SDS-PAGE content of peak protein were collected in different time periods, such as Figure 4 The conjugated filler has a significant capture effect on trivalent nanobody (Anti-TNFα & Albumin Nanobody), and the purity of the nanobody captured was 93.3% purity of the nanobody captured nanobody.

[0055] Example 4 Development of purification process of trivalent nanobody (Anti-EGFR & Albumin Nanobody) based on HSA coupling filler The present invention provides a general method for purifying nano-antibody with HSA-coupled filler, including the following steps: Step 1: Sample pretreatment. Before purification, the sample (the fermentation broth supernatant involving the target product Anti-EGFR & Albumin Nanobody in Example 1) was used to filter out the insoluble impurities in the sample using a 0.45 μm filter (manufacturer: Sedolis); if there are too many impurities in the sample and the filter membrane is significantly blocked, deep filtration (Claricap deep filter element, manufacturer: Kebat) can be used to remove impurities. At the same time, take 100 mL of the filtered fermentation broth to prepare for subsequent operations.

[0056] Step 2: Column cleaning stage: 0.1 M NaOH is cleaned at 1 ml / min until A280 drops to near the constant value; Equilibrium stage: 10 mM PBS (pH=7.4) is cleaned at 1 ml / min until A280 drops to near the constant value and ends with equilibrium; loading stage: Sample loading at 1 ml / min, the sample retention time is 5 min until A280 drops to near the constant value and ends with loading; pay attention to collecting the flow-through sample for detection; Column cleaning stage: 10 mM PBS (pH=7.4) is cleaned at 1 ml / min until A280 drops to near the constant value and ends with cleaning; elution stage: 20 mM sodium acetate (pH=3.5) was elution at 1 ml / min until A280 dropped to near a constant value and ended elution; the eluted samples were collected for inspection of the entire chromatography program map. Figure 5 Shown. Column cleaning stage: 0.1 M NaOH was washed at a rate of 1 ml / min until A280 dropped to a constant value and ended CIP. Pay attention to collecting the CIP sample for detection; Preparation before column: 20% ethanol was washed at a rate of 1 ml / min, cycle 10CV, and place the affinity chromatography column containing human serum albumin filler at 4°C for storage.

[0057] Step 3: Purify the sample SDS-PAGE detection. Mix the sample with the protein Loading Buffer (SDS, DTT and bromophenol blue) in a 5:1 ratio so that the protein Loading Buffer concentration of the final sample is not less than 1×, 98°C or denaturate for 10 minutes. Then, install the electrophoresis tank and rubber plate (de-sealing tape), pour in the electrophoresis buffer, the inner tank should be filled and overflowed, and the liquid level of the outer tank should be higher than the electrode wire. Slowly remove the pre-made gel comb, take the syringe and absorb the buffer, and put it into the glue holes one by one to blow away the glycerin. Spot the sample, close the slot cover, turn on the power, and electrophorese at 80 V for 40 minutes until the sample is compressed into a line. Increase the voltage value of 120 V Continue electrophoresis to the indicator band close to or to the bottom of the gel. The gels that were electrophoretic were stained and decolorized using a protein stainer. The setting procedure was Stain3 min; Destain 2 min; Destain 1 min. Transfer the gel to the gel imaging system, image, modify the image, set up the lanes, label the lanes, and perform corresponding analysis. The results of SDS-PAGE content of peak protein were collected in different time periods, such as Figure 6 The coupled filler has a significant capture effect on trivalent nanobody (Anti-EGFR & Albumin Nanobody), and the purity of the nanobody captured was 90.2%.

[0058] Example 5 Development of purification process of trivalent nanobody (Anti-TNFα & Albumin Nanobody) based on anionic filler 50QHC Step 1: Sample pretreatment. Before purification of the sample (the fermentation broth supernatant involving the target product Anti-TNFα & Albumin Nanobody in Example 1) was used to filter out the insoluble impurities in the sample using a 0.45 μm filter (manufacturer: Sedolis); if there are too many impurities in the sample and the filter membrane is significantly blocked, deep filtration (Claricap deep filter element, manufacturer: Kebat) can be used to remove impurities. At the same time, take 100 mL of the filtered fermentation broth to prepare for subsequent operations. Subsequently, the anionic filler NanoGel-50Q HC (manufacturer: Suzhou Nawei Technology Co., Ltd., item number: 04064-050150-2030) column purification was used.

[0059] Step 2: Column cleaning stage: 0.5 M NaOH is cleaned at 1 ml / min until A280 drops to near constant value; Equilibration stage: 20 mM Na 3 PO 4 , pH 8.0 was cleaned at a rate of 1 ml / min until A280 dropped to near a constant value and ended with equilibrium; loading stage: the sample was loaded at a rate of 1 ml / min, and the sample retention time was 5 min until A280 dropped to near a constant value and ended with loading; pay attention to collecting the flow-through sample for detection; column cleaning stage: 20 mMNa 3 PO 4 , pH 8.0 was cleaned at 1 ml / min until A280 dropped to near constant value and ended with cleaning; elution phase: 20 mMNa 3 PO 4 +1 M NaCl, pH 8.0 linear elution at 1 ml / min until A280 drops to near constant value ends; collect the eluted samples for inspection for the entire chromatography program map as Figure 7 Shown. Column cleaning stage: 0.5 M NaOH is cleaned at a rate of 1 ml / min until A280 drops to a constant value and ends CIP. Pay attention to collecting the CIP sample for detection; Preparation before column: 20% ethanol is cleaned at a rate of 1 ml / min, cycle 10CV, and place the chromatography column of ionic filler at 4°C for storage.

[0060] Step 3: Purify the sample SDS-PAGE detection. Mix the sample with the protein Loading Buffer (SDS, DTT and bromophenol blue) in a 5:1 ratio so that the protein Loading Buffer concentration of the final sample is not less than 1×, 98°C or denaturate for 10 minutes. Then, install the electrophoresis tank and rubber plate (de-sealing tape), pour in the electrophoresis buffer, the inner tank should be filled and overflowed, and the liquid level of the outer tank should be higher than the electrode wire. Slowly remove the pre-made gel comb, take the syringe and absorb the buffer, and put it into the glue holes one by one to blow away the glycerin. Spot the sample, close the slot cover, turn on the power, and electrophorese at 80 V for 40 minutes until the sample is compressed into a line. Increase the voltage value of 120 V Continue electrophoresis to the indicator band close to or to the bottom of the gel. The gels that were electrophoretic were stained and decolorized using a protein stainer. The setting procedure was Stain3 min; Destain 2 min; Destain 1 min. Transfer the gel to the gel imaging system, image, modify the image, set up the lanes, label the lanes, and perform corresponding analysis. The results of SDS-PAGE content of peak protein were collected in different time periods, such as Figure 8 Shown (lane 1: Anti-TNFα & Albumin Nanobody fermentation broth stock solution, lane 2: Anti-TNFα & Albumin Nanobody loading flow through liquid 1, lane 3: Anti-TNFα & Albumin Nanobody loading flow through liquid 2, lane 4: Anti-TNFα & Albumin Nanobody loading flow through liquid 3, lane 5; Anti-TNFα & Albumin Nanobody loading flow through liquid 4, lane 6; Anti-TNFα & Albumin Nanobody loading flow through liquid 5, lane 7; Anti-TNFα & Albumin Nanobody loading flow through liquid 5, lane 7; Anti-TNFα & Albumin Nanobody loading flow through liquid 4, lane 6; Anti-TNFα & Albumin Nanobody loading flow through liquid 5, lane 7; Anti-TNFα & Albumin Nanobody eluent, lane 8; protein marker), ion filler has no obvious capture effect on trivalent nanobody (Anti-TNFα & Albumin Nanobody).

[0061] Example 6 Development of Anti-EGFR & Albumin Nanobody Purification Process Based on Anion Filler 50QHC Step 1: Sample pretreatment. Before purification of the sample (the fermentation broth supernatant involving the target product Anti-EGFR & Albumin Nanobody in Example 1) was used to filter out the insoluble impurities in the sample using a 0.45 μm filter (Sedolis). If there are too many impurities in the sample and the filter membrane is significantly blocked, deep filtration (Claricap deep filter element, Kebat) can be used to remove impurities. At the same time, take 100 mL of the filtered fermentation broth to prepare for subsequent operations. Subsequently, the anionic filler NanoGel-50Q HC (manufacturer: Suzhou Nawei Technology Co., Ltd., item number: 04064-050150-2030) column was used to purify the column.

[0062] Step 2: Column cleaning stage: 0.5 M NaOH is cleaned at 1 ml / min until A280 drops to near constant value; Equilibration stage: 20 mM Na 3 PO 4 , pH 8.0 was cleaned at a rate of 1 ml / min until A280 dropped to near a constant value and ended with equilibrium; loading stage: the sample was loaded at a rate of 1 ml / min, and the sample retention time was 5 min until A280 dropped to near a constant value and ended with loading; pay attention to collecting the flow-through sample for detection; column cleaning stage: 20 mMNa 3 PO 4 , pH 8.0 was cleaned at 1 ml / min until A280 dropped to near constant value and ended with cleaning; elution phase: 20 mMNa 3 PO 4 +1 M NaCl, pH 8.0 linear elution at 1 ml / min until A280 drops to near constant value ends; collect the eluted samples for inspection for the entire chromatography program map as Figure 9 Shown. Column cleaning stage: 0.5 M NaOH is cleaned at a rate of 1 ml / min until A280 drops to a constant value and ends CIP. Pay attention to collecting the CIP sample for detection; Preparation before column: 20% ethanol is cleaned at a rate of 1 ml / min, cycle 10CV, and place the chromatography column of ionic filler at 4°C for storage.

[0063] Step 3: Purify the sample SDS-PAGE detection. Mix the sample with the protein Loading Buffer (SDS, DTT and bromophenol blue) in a 5:1 ratio so that the protein Loading Buffer concentration of the final sample is not less than 1×, 98°C or denaturate for 10 minutes. Then, install the electrophoresis tank and rubber plate (de-sealing tape), pour in the electrophoresis buffer, the inner tank should be filled and overflowed, and the liquid level of the outer tank should be higher than the electrode wire. Slowly remove the pre-made gel comb, take the syringe and absorb the buffer, and put it into the glue holes one by one to blow away the glycerin. Spot the sample, close the slot cover, turn on the power, and electrophorese at 80 V for 40 minutes until the sample is compressed into a line. Increase the voltage value of 120 V Continue electrophoresis to the indicator band close to or to the bottom of the gel. The gels that were electrophoretic were stained and decolorized using a protein stainer. The setting procedure was Stain 3 min; Destain 2 min; Destain 1 min. Transfer the gel to the gel imaging system, image, modify the image, set up the lanes, label the lanes, and perform corresponding analysis. The results of SDS-PAGE content of peak protein were collected in different time periods, such as Figure 10 Shown (lane 1: protein Marker, lane 2: Anti-TNFα & Albumin Nanobody fermentation broth stock solution, lane 3: Anti-EGFR & Albumin Nanobody loading flow through liquid 1, lane 4: Anti-EGFR & Albumin Nanobody loading flow through liquid 2, lane 5: Anti-EGFR & Albumin Nanobody loading flow through liquid 3, lane 6; Anti-EGFR & Albumin Nanobody loading flow through liquid 4, lane 7; Anti-EGFR & Albumin Nanobody loading flow through liquid 5, lane 8; Anti-EGFR & Albumin Nanobody loading flow through liquid 5, lane 8; Anti-EGFR & Albumin Nanobody loading flow through liquid 4, lane 7; Anti-EGFR & Albumin Nanobody loading flow through liquid 5, lane 8; Anti-EGFR & Albumin Nanobody eluent), ionic fillers have no obvious capture effect on trivalent nanobody (Anti-EGFR & Albumin Nanobody).

[0064] The above-mentioned embodiments of the present disclosure are merely examples for the sake of clarity of the present disclosure, and are not limiting the embodiments of the present disclosure. For those skilled in the art, other different forms of changes or changes can be made based on the above description. All implementation methods are not required and cannot be exhaustive here. Any modification, equivalent substitution and improvement, etc., made within the spirit and principles of this disclosure shall be included within the scope of the protection of the claims of this disclosure.

Claims

1. A method for preparing an affinity chromatography filler based on human serum albumin (HSA), characterized in that: The steps include: Activated agarose gel filler; preferably, the filler is CNBr Focurose 4FF pre-activated agarose gel; The activated filler is coupled to a ligand, wherein the ligand comprises human serum albumin; the coupling comprises dissolving a human serum albumin sample having a concentration of 0.5-10 mg / ml in a solution of 0.1 M NaHCO3, 0.5 M NaCl, pH 8.3, and stirring for 4 hours at room temperature; wherein the filler is diluted with 1 mM HCl before coupling, and 0.5-1 mL HCl is added per 1 mL of filler, and during coupling, the final concentration of the human serum albumin having a concentration of 0.5-10 mg / ml after addition is 1 mg / ml; The sealing solution seals the coupled filler; preferably, the sealing step comprises adding 2 times the volume of the medium with 0.1 M Tris-HCl, 0.5 M NaCl, pH 8.0 of the sealing solution, stirring evenly and then draining.

2. The preparation method according to claim 1, characterized in that: The step (1) activating the agarose filler comprises the following steps: draining the agarose filler, adding a pre-cooled HCl solution, stirring evenly, and then draining; preferably, the step (1) activating the agarose filler comprises the following steps: draining the required amount of agarose filler medium, adding a pre-cooled 1 mM HCl solution with a volume twice that of the medium, stirring evenly, and then draining, and repeating the HCl solution treatment step twice.

3. The preparation method according to claim 1 or 2, characterized in that: The step (2) further comprises steps (A)-(C): the step (A) comprises draining the packing medium after coupling, adding 0.1M Tris-HCl, pH 8.0 solution with a volume twice that of the medium, stirring evenly and draining, and repeating twice; the step (B) comprises adding 0.1M Tris-HCl, pH 8.0 solution with a volume twice that of the medium, and gently stirring for 4 hours at room temperature; the step (C) comprises draining the packing medium, adding 0.1M Tris-HCl, 0.5M NaCl, pH 9.0 solution with a volume twice that of the medium, stirring evenly and draining; the step (3) further comprises steps (D)-(F): the step (D) comprises adding 0.1M sodium acetate, 0.5M NaCl, pH 3.0 solution with a volume twice that of the medium, stirring evenly and draining; the step (E) comprises repeating step (3) and step (D) twice in total; and the step (F) comprises treating with 20% ethanol and then storing.

4. The preparation method according to claim 1, characterized in that: The preparation method comprises the following steps: (1) Take the required amount of agarose filler and drain it into a sand core funnel of corresponding specifications; (2) Add 2 times the volume of medium to pre-cooled 1 mM HCl solution, stir evenly and drain; Repeat 2 times; (3) Transfer the medium to a reactor of corresponding specifications; (4) Dissolve the human serum albumin sample with a concentration of 0.5-10 mg / ml in 0.1 M NaHCO3, 0.5 M NaCl, pH 8.3, add it to the reactor, and stir gently at room temperature for 4 h; (5) Transfer the medium to a sand core funnel and drain it, saving the coupled solution in the collection container; (6) Add 2 times the volume of medium 0.1 M Tris-HCl, pH 8.0 solution, stir evenly and drain; Repeat 2 times; (7) Transfer the medium to a reactor of corresponding specifications, add 0.1 M Tris-HCl, pH 8.0 solution (2 times the volume of the medium), and stir gently at room temperature for 4 h; (8) Transfer the medium to a sand core funnel and drain it; (9) Add 2 times the volume of the medium to form a solution of 0.1 M Tris-HCl, 0.5 M NaCl, pH 9.0, stir well, and drain; (10) Add 2 times the volume of medium to a solution of 0.1 M sodium acetate, 0.5 M NaCl, pH 3.0, stir well, and drain; (11) Repeat steps (9)-(10) twice in total; (12) Add 20% ethanol in a volume twice that of the medium, stir evenly and drain, repeat twice; (13) Transfer the medium to a container of appropriate specifications, add an equal volume of 20% ethanol, and store for later use.

5. An affinity chromatography filler based on human serum albumin (HSA) prepared by the preparation method according to any one of claims 1 to 4.

6. A method for purifying multivalent nanobodies using the affinity chromatography filler based on human serum albumin (HSA) according to claim 5, characterized in that: The method comprises the following steps: Column cleaning: Use 0.1 M NaOH cleaning solution for cleaning; Balance: Use 10 mM PBS, pH = 7.4 balance solution; Loading: Loading samples containing multivalent nanobodies; Column washing: washing with 10 mM PBS, pH=7.4; Elution of target product: 20 mM sodium acetate, pH = 3.5 eluent; Column cleaning: Use 0.1 M NaOH cleaning solution for cleaning; Preparation before column loading: Wash with 20% ethanol and store the affinity chromatography column.

7. The purification method according to claim 6, characterized in that The multivalent Nanobody comprises human serum albumin; preferably, the multivalent Nanobody comprises a trivalent Nanobody; more preferably, the trivalent Nanobody comprises Anti-TNFα&Albumin Nanobody and Anti-EGFR&Albumin Nanobody; most preferably, the amino acid sequence of the Anti-TNFα&Albumin Nanobody is as shown in SEQ ID NO:1, and the amino acid sequence of the Anti-EGFR&Albumin Nanobody is as shown in SEQ ID NO:

3.

8. The purification method according to claim 6 or 7, characterized in that: The purification method comprises the following steps: (1) Column cleaning stage: 0.1 M NaOH was used for cleaning at a rate of 1 ml / min until A280 dropped to a constant value; (2) Equilibrium stage: 10 mM PBS, pH = 7.4, washing at a rate of 1 ml / min until A280 drops to a constant value and equilibrium is completed; (3) Sample loading stage: The sample is loaded at a rate of 1 ml / min, and the sample retention time is 5 min. The sample loading is completed until the A280 drops to a constant value. Pay attention to collecting the flow-through sample for detection. (4) Column cleaning stage: 10 mM PBS, pH = 7.4, at a rate of 1 ml / min until A280 drops to a constant value; (5) Elution stage: 20 mM sodium acetate, pH = 3.5, elution at a rate of 1 ml / min until A280 drops to a constant value; collect the eluted samples for detection; (6) Column cleaning stage: 0.1 M NaOH was used for cleaning at a rate of 1 ml / min until A280 dropped to a constant value, and the CIP was terminated. The CIP samples were collected for testing. (7) Preparation before column loading: Wash with 20% ethanol at a rate of 1 ml / min for 10 CV and store the affinity chromatography column containing human serum albumin filler at 4°C.

9. The purification method according to claim 6 or 7, characterized in that: The multivalent nanobody is obtained by fermentation, and the fermentation includes the steps of primary seed culture, secondary seed culture, fermentation culture, and preparation of fermentation supernatant.

10. The purification method according to claim 9, characterized in that The fermentation comprises the following steps: (1) Primary seed culture: activating the stored recombinant Pichia pastoris and culturing at 25-32° C. and 100-250 rpm for 16-18 h; (2) Secondary seed culture: Inoculate the primary seed solution into the seed culture medium at a volume ratio of 1-5%, and culture at 25-32°C and 100-300 rpm until the OD 600 =6-8; (3) Fermentation culture: inoculating the seed liquid into the fermentation medium, culturing in a fermenter, controlling the DO at 10%-50%, preferably, controlling the DO at 30%, stirring speed at 200-800 rpm, ventilation volume at 0.5-2.5 vvm, and adding a certain concentration of pure oxygen to maintain the DO of the fermentation liquid within an appropriate range; (4) Preparation of fermentation supernatant: After fermentation for at least 76 h, the fermentation broth was centrifuged to remove the bacteria, and the supernatant was vacuum filtered and collected to obtain the fermentation broth.

11. Use of the method for preparing the affinity chromatography filler according to any one of claims 1 to 4, the affinity chromatography filler based on human serum albumin (HSA) according to claim 5, or the purification method according to any one of claims 6 to 10 in the separation and purification of human serum albumin monovalent nanobodies or human serum albumin multivalent nanobodies; preferably, the multivalent nanobody comprises a trivalent nanobody; more preferably, the trivalent nanobody comprises Anti-TNFα&Albumin Nanobody and Anti-EGFR&Albumin Nanobody; most preferably, the amino acid sequence of the Anti-TNFα&Albumin Nanobody is as shown in SEQ ID NO: 1, and the amino acid sequence of the Anti-EGFR&Albumin Nanobody is as shown in SEQ ID NO: 3.

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