CN112830967B

CN112830967B - Affinity chromatography purification method of anti-HSA single domain antibody and fusion protein thereof

Info

Publication number: CN112830967B
Application number: CN202110168479.XA
Authority: CN
Inventors: 万云超; 郜鹏; 周加义; 何志娟; 郭树华
Original assignee: Reyoung Suzhou Biology Science & Technology Co ltd
Current assignee: Reyoung Suzhou Biology Science & Technology Co ltd
Priority date: 2019-01-10
Filing date: 2019-01-10
Publication date: 2022-06-24
Anticipated expiration: 2039-01-10
Also published as: CN111423509B; CN112830967A; CN111423509A

Abstract

The invention belongs to the field of protein purification, and particularly relates to an affinity chromatography purification method of an anti-Human Serum Albumin (HSA) single-domain antibody and a fusion protein thereof. It includes: mixing HSA with an affinity medium, and performing coupling reaction to prepare an HSA affinity chromatography medium; passing a sample to be purified through an HSA affinity chromatography medium which is well balanced by an equilibrium buffer solution in advance; then washing the affinity chromatography medium combined with the sample by using a washing buffer solution; eluting the anti-HSA single domain antibody or the fusion protein thereof from the chromatographic medium by using an elution buffer solution, and collecting an elution peak to obtain an elution sample; and finally, adjusting the eluted sample to be neutral by using a neutralization buffer solution to obtain the final product. The purity of the anti-HSA single domain antibody and the fusion protein thereof obtained by the method of the invention reaches more than 95%, and the single domain antibody and the fusion protein thereof can keep good drug forming property and stability.

Description

Affinity chromatography purification method of anti-HSA single-domain antibody and fusion protein thereof

The application is a divisional application of Chinese patent application No. 201910024941.1, having application date of 2019, month 01 and 10 and having the name of "affinity chromatography purification method of anti-HSA single domain antibody and fusion protein thereof".

Technical Field

The invention belongs to the field of protein purification, and particularly relates to an affinity chromatography purification method of an anti-HSA single domain antibody and a fusion protein thereof.

Background

Human Serum Albumin (HSA) is used as the largest single component protein in human blood, and has a half-life of up to 20 days and no enzymatic and immunological activities, and is widely applied to carriers for improving long-acting of protein drugs in recent years. However, with the intensive research of the serum albumin fusion technology, a series of defects of low yield, poor stability, large molecular weight and the like of the fusion protein, which further influence the biological activity of the fusion protein, appear. Today, with the rapid development of protein engineering technology, the trend is to find more suitable long-acting protein platform carriers.

The anti-HSA single domain antibody not only has the advantages of high affinity, good thermal stability, small molecular weight, strong specificity and the like of a common single domain antibody, but also has the advantages of prolonging the half-life period of protein after being combined with HSA, not influencing the space group position and biological activity of fusion protein and the like, and has good and wide application prospect when being used as a long-acting protein platform carrier. The current conventional technical means for prolonging the half-life of the target protein are PEG modification, Fc fusion, HSA fusion, other macromolecular protein or protein chaperone fusion and the like. Although the methods prolong the stability of the target protein in the body to a certain extent, the respective disadvantages are particularly prominent, for example, PEG modification causes poor molecular uniformity and large molecular weight after modification due to different modification degrees; the Fc fusion can bring a series of problems of large molecular weight, toxic and side effects, reduced activity and the like due to glycosylation modification of Fc, Fc mediated cytotoxicity and complement activation, possible influence of Fc on the activity of target proteins, dimer existence form and the like; the HSA fusion can reduce the activity of the target protein, and the degradation and polymerization phenomena of the HSA can occur in the fermentation process, so that the defects of reduced biological activity of the target protein, large molecular weight, poor stability of a fusion protein sample, toxic and side effects and the like are caused; other macromolecular proteins or protein chaperones also have the defects of influence on the biological activity of target proteins, poor stability and the like. The fusion with the anti-HSA single domain antibody is adopted, so that the half-life of the target protein can be prolonged by combining the anti-HSA single domain antibody with HSA; the single-domain antibody has small molecular weight, does not cause large fusion protein molecules after fusion, has good sample stability, does not influence the biological activity of target protein, and does not bring toxic or side effect; moreover, the single-domain antibody fusion protein has wider selectable expression system and easier production.

The purification process of the existing conventional anti-HSA single domain antibody fusion protein relies on a tag peptide segment, usually by adding a histidine tag to facilitate the later purification of the anti-HSA single domain antibody fusion protein by nickel column affinity chromatography. However, the existence of the tag peptide segment not only influences the druggability of the anti-HSA single domain antibody fusion protein, but also introduces heavy metals such as Ni and the like in the purification process, increases the difficulty of the process and analysis, further influences the druggability of the antibody fusion protein and limits the application of the protein; the purification of the protein without the tag can be realized only by multi-step purification, and the operation period of the whole purification experiment can be prolonged, so that the method has certain challenge on the physical and chemical stability of the protein, the final yield is low, and the complexity of the process and the production cost are increased. Therefore, the problem to be solved urgently in the field at present is to find a purification method which has good specificity, high product purity and large sample adsorption capacity and does not influence the property of the single-domain antibody fusion protein. Based on the affinity chromatography medium, the affinity chromatography medium with HSA as the ligand is developed, the purification process is improved, and the affinity chromatography medium is applied to the purification preparation of the anti-HSA single-domain antibody and the fusion protein thereof, so that a good effect is achieved.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides an affinity chromatography purification method of an anti-HSA single domain antibody and fusion protein thereof, which takes HSA as a ligand and utilizes the specific combination of the HSA-anti-HSA single domain antibody to carry out affinity chromatography, thereby getting rid of the dependence on other purification labels and the difficulty brought by the purification of non-label protein, improving the purification process, obviously improving the purity of the protein and ensuring the druggability and the stability of the single domain antibody and the fusion protein thereof in the process of affinity chromatography.

As used herein, unless otherwise specified, "about" means within ± 10%.

Specifically, the affinity chromatography purification method of the anti-HSA single domain antibody and the fusion protein thereof of the present invention comprises the following steps:

step 1, preparation of affinity chromatography media:

(1) activation: washing the activated affinity medium with an activation buffer;

(2) coupling: mixing HSA with an affinity medium, and performing coupling reaction to prepare an HSA affinity chromatography medium;

(3) washing I: washing unconjugated HSA with a coupling buffer;

(4) and (3) sealing: blocking the HSA affinity chromatography medium with a blocking buffer;

(5) washing II: washing the non-specifically bound HSA with washing buffer a;

(6) washing III: washing the non-specifically bound HSA with washing buffer B;

further, the pH value of the activation buffer solution is 2.5-9.0; preferably a buffer comprising HCl or NaOH at pH 2.5-9.0; preferably a buffer comprising HCl at pH 2.5-3.5 or a buffer comprising NaOH at pH 7.0-9.0; preferably a buffer containing about 1mM HCl at a pH of 2.8-3.3; more preferably a buffer containing about 1mM HCl at pH 3.0;

further, the affinity medium is selected from cross-linked agarose, cellulose, nitrocellulose, acrylamide polymers, polyethersulfone or chitosan; preferably agarose 4B or cellulose;

further, the pH of the coupling buffer solution is 7.0-9.0; preferably pH 7.5-9.0, comprising NaHCO₃Or a phosphate buffer; preferably pH 7.5-9.0, comprising 0.1M NaHCO₃Buffer of 0.5M NaCl or 0.1M PB, EDTA; preferably, the pH is 7.8-8.3 and the NaHCO content is about 0.1M₃Buffer solution of 0.5M NaCl; more preferably, the pH is 8.0 and the aqueous solution contains about 0.1M NaHCO₃Buffer solution of 0.5M NaCl;

further, the pH value of the blocking buffer is 7.0-9.0, and the blocking condition is as follows: sealing at room temperature for 2h or sealing at 4 ℃ overnight; preferably a buffer comprising Tris or ethanolamine at pH 7.0-8.5; more preferably a buffer comprising about 0.1M Tris-HCl or 1M ethanolamine at a pH of 7.0-8.5; preferably a buffer comprising about 0.1M Tris-HCl or 1M ethanolamine at pH 7.7-8.3; more preferably a buffer containing about 0.1M Tris-HCl or 1M ethanolamine at pH 8.0;

further, the pH value of the washing buffer solution A is 3.0-6.0; preferably pH 3.5-4.5, buffer comprising about 0.1M HAc/NaAc, 0.5M NaCl; preferably a buffer at pH 3.8-4.3 comprising about 0.1M HAc/NaAc, 0.5M NaCl; more preferably a buffer comprising about 0.1M HAc/NaAc, 0.5M NaCl at pH 4.0;

further, the pH value of the washing buffer solution B is 7.0-9.0; preferably a buffer comprising about 0.1M Tris-HCl, 0.5M NaCl at pH 7.5-8.5; preferably a buffer comprising about 0.1M Tris-HCl, 0.5M NaCl at pH 7.7-8.3; more preferably a buffer comprising about 0.1M Tris-HCl, 0.5M NaCl at pH 8.0.

Step 2, purifying the sample to be purified by using an affinity chromatography medium

(1) Balancing: balancing and filling the HSA affinity chromatography medium prepared in the step 1 with an equilibrium buffer solution;

(2) sampling: passing the sample to be purified through a well-balanced affinity chromatography medium to combine the sample with the affinity chromatography medium;

(3) cleaning: washing the sample-bound affinity chromatography medium with a wash buffer;

(4) and (3) elution: eluting the anti-HSA single domain antibody or the fusion protein thereof from the affinity chromatography medium by using an elution buffer solution, and collecting an elution peak to obtain an elution sample;

(5) neutralizing: adjusting the eluted sample to be neutral by using a neutralization buffer solution to obtain the final product;

further, the equilibration buffer is a buffer containing phosphate or Tris; further, the equilibration buffer is a buffer containing phosphate or Tris-HCl with the pH of 6.0-8.5; preferably a buffer comprising phosphate or Tris-HCl at pH 7.0-8.5; preferably a buffer comprising PB at pH 7.3-8.5; more preferably a buffer comprising about 10mM PB, 130mM NaCl at pH 7.3;

further, the washing buffer is a buffer containing phosphate, Tris or boric acid; further, the washing buffer is a buffer containing phosphate, Tris or boric acid with pH of 6.0-8.5; preferably a buffer comprising phosphate or Tris-HCl at pH 6.6-8.4; more preferably a buffer comprising about 20mM PB, 1M NaCl at pH 8.3;

further, the elution buffer is a buffer containing phosphate, sodium salt, potassium salt, calcium salt, magnesium salt or ammonium salt; further, the elution buffer is a buffer containing phosphate, sodium salt, potassium salt, calcium salt, magnesium salt or ammonium salt with the pH of 2.5-9.0; further, the elution buffer may be a buffer comprising HAc/NaAc, citric acid-sodium hydrogen phosphate or citric acid-sodium citrate at pH 2.5-3.5; preferably a buffer at pH 2.6-3.3 containing about 20mM HAc/NaAc; more preferably a buffer containing about 20mM HAc/NaAc at pH 3.0; further, the elution buffer solution can also be a high pH salt eluent with pH of 5.0-9.0; preferably a monovalent salt of not less than 1M or a multivalent salt of 3-5M; more preferably an elution buffer comprising about 2M magnesium salt or about 4-5M sodium salt;

further, the neutralization buffer is a buffer containing Tris or arginine; further, the neutralization buffer is a buffer containing Tris or arginine with pH 6.5-9.0; preferably a buffer comprising Tris-HCl at pH 7.0-9.0; preferably a buffer comprising about 1M Tris-HCl at pH 7.2-8.3; more preferably a buffer comprising about 1M Tris-HCl at pH 8.0;

further, the affinity chromatography purification according to the present invention may be carried out in an apparatus conventional in the art; preferably in a chromatography column;

further, the balancing method comprises the following steps: equilibrating the column containing the HSA affinity chromatography media prepared in step 1 with an equilibration buffer at pH 6.0-8.5, equilibrating 3-15 column volumes at a flow rate of 20-60 cm/h;

further, the loading method comprises the following steps: passing 20-80mL of a sample to be purified through a well-balanced affinity chromatography column at a flow rate of 20-60 cm/h;

further, the cleaning method comprises the following steps: washing the chromatographic column with pH 6.0-8.5 washing buffer solution in the flow rate of 20-60 cm/hr and in the amount of 3-15 times the volume of the chromatographic column;

further, the elution method comprises the following steps: eluting the anti-HSA single domain antibody or the fusion protein thereof from the chromatographic column by using an elution buffer solution with the pH of 2.5-9.0 at the flow rate of 10-30cm/h, and collecting an elution peak to obtain an elution sample, wherein the dosage of the elution buffer solution is 3-15 times of the volume of the chromatographic column;

further, the neutralization method comprises the following steps: adjusting the eluted sample to be neutral by using a neutralization buffer solution with the pH of 6.5-9.0 to obtain the final product;

further, the sample to be purified is a cell or a culture solution expressing an anti-HSA single domain antibody or a fusion protein comprising an anti-HSA single domain antibody;

further, the method for preparing the cell or the culture solution expressing the fusion protein comprising the anti-HSA single domain antibody is as follows:

connecting a polypeptide molecule with a therapeutic function and an anti-HSA single domain antibody in series through a flexible linker, inserting the polypeptide molecule and the anti-HSA single domain antibody into a vector to obtain a recombinant plasmid, and introducing the recombinant plasmid into an expression host cell for expression to obtain a cell or a culture solution which expresses a fusion protein containing the anti-HSA single domain antibody;

further, a polypeptide molecule having a therapeutic function is connected in series with an anti-HSA single domain antibody via a (GnS) m flexible linker, (note: n-0, 1,2,3,4, … …; m-0, 1,2,3,4, … …);

further, the host cell is a bacterium, yeast, filamentous fungus, insect cell, mammalian cell or plant cell;

further, the expression conditions may be optimized according to the chosen host;

further, the heavy chain variable region of the anti-HSA single domain antibody comprises an amino acid sequence of SEQ ID NO: 1-3 or the HCDR shown in SEQ ID NO: HCDR shown as 4-6;

further, the amino acid sequence of the heavy chain variable region is SEQ ID NO: 7. SEQ ID NO: 8. SEQ ID NO: 9 or SEQ ID NO: 10 is shown in the figure;

further, the anti-HSA single domain antibody is "the same applicant filed on 2018, 11, 6, with patent application No. CN2018113141932, entitled anti-human serum albumin single domain antibody preparation and use thereof; and the patent application number of the same applicant is CN2018113141773 applied in 2018, 11 and 6, and the name is a novel anti-human serum albumin antibody fragment, a preparation method and a single-domain antibody described in the patent application;

further, the various buffer solutions are prepared according to the methods described in molecular cloning, second edition, appendix B: preparing reagents and buffers used in molecular cloning; shanghai science publishers, 1993;

further, the purity of the purified anti-HSA single domain antibody or the fusion protein thereof is analyzed by SDS-PAGE and SEC-HPLC, and the conditions for detection and analysis are conventional.

Compared with the prior art, the invention has the beneficial effects that:

(1) the invention provides an affinity chromatography purification method of an anti-HSA single domain antibody and a fusion protein thereof, which reduces the introduction of factors influencing the molecular pharmaceutical property and simplifies the process steps by improving the purification process, increases the final yield of the target protein and obtains a high-purity protein sample.

(2) Reduces the production cost and the process risk, ensures the druggability of the single-domain antibody and the fusion protein thereof and the stability in the affinity chromatography process, and is beneficial to the beneficial effect of the protein biological medicine in treatment.

Drawings

FIG. 1 is a SDS-PAGE analysis of the purified samples of example 1, wherein "M" is a protein molecular weight marker, "S" is an expression sample supernatant, "FT" is a sample run-through, and "E" is a purified protein sample.

FIG. 2 is a SDS-PAGE analysis of the purified samples of example 2, wherein "M" is a protein molecular weight marker, "S" is an expression sample supernatant, "FT" is a sample run-through, and "E" is a purified protein sample.

FIG. 3 is a SDS-PAGE analysis of the purified samples of example 3, wherein "M" is a protein molecular weight marker, "S" is an expression sample supernatant, "FT" is a sample run-through, and "E1-E4" is a purified protein sample.

FIG. 4 is a SDS-PAGE analysis of the purified sample in comparative example 1, wherein "M" is a protein molecular weight marker, "S" is an expression sample supernatant, "FT" is a sample flow-through, and "E1-E3" is a purified protein sample.

FIG. 5 is a SDS-PAGE analysis of the purified sample in comparative example 2, wherein "M" is a protein molecular weight marker, "S" is an expression sample supernatant, "FT" is a sample run-through, and "E1-E2" is a purified protein sample.

Detailed Description

The following non-limiting examples are presented to enable those of ordinary skill in the art to more fully understand the present invention and are not intended to limit the invention in any way. The following is merely an exemplary illustration of the scope of the invention as claimed, and various changes and modifications of the invention of the present application may be made by those skilled in the art based on the disclosure, which also fall within the scope of the invention as claimed.

The present invention will be further described below by way of specific examples. The various chemicals used in the examples of the present invention were obtained by conventional commercial routes unless otherwise specified.

Example 1 purification of anti-HSA Single Domain antibodies Using the methods of the present invention

(1) Preparation of affinity chromatography media

Activating: weighing a certain amount of Sepharose 4B dry powder, adding a certain volume of 1mM HCl and a buffer solution with pH of 3.0 according to a ratio of 1:20(w/v), uniformly mixing, centrifuging at 400x g room temperature for 5min, and removing supernatant; repeating the swelling experiment step once; at this time, the volume of the medium is swelled to 3.5 times of the weighed amount of the dry powder;

② coupling: to the above medium was added 0.1M NaHCO in a ratio of 1:5(w/v) of dry powder₃0.5M NaCl and buffer solution with pH of 8.3, then adding HSA according to the proportion of 5-10mg HSA/mL medium, and incubating at room temperature for 1h or incubating at 4 ℃ overnight at low rotation speed; centrifuging at 400x g room temperature for 5min, and removing supernatant;

washing I: with 5-10 medium volumes of 0.1M NaHCO₃Washing unbound HSA protein with 0.5M NaCl, pH 8.3 buffer; centrifuging at 400x g room temperature for 5min, and removing supernatant;

sealing: adding 0.1M Tris-HCl with the volume 5-10 times of that of the medium and pH 8.0 buffer solution, and sealing for 2h at room temperature or overnight at 4 ℃; centrifuging at 400x g room temperature for 5min, and removing supernatant;

washing II: washing with 5-10 times of medium volume of 0.1M HAc/NaAc, 0.5M NaCl, pH 4.0 buffer solution, centrifuging at 400x g room temperature for 5min, and discarding supernatant;

sixth, wash III: washing with 5-10 times medium volume of 0.1M Tris-HCl, 0.5M NaCl, pH 8.0 buffer solution, centrifuging at 400x g room temperature for 5min, and removing supernatant;

dd H in 5-10 medium volumes₂Washing with O for 3 times, centrifuging at 400x g room temperature for 5min, and removing supernatant; the filling is stored in 20% ethanol (4 ℃) for later use.

(2) Preparation of anti-HSA Single Domain antibodies

The preparation method of the single domain antibody for resisting HSA comprises the following steps:

constructing an anti-HSA single domain antibody library: mixing HSA and Freund's adjuvant, and immunizing two Xinjiang bactrian camels for seven times each week. And after the immunization is finished, extracting camel peripheral blood cells, separating lymphocytes, freezing the lymphocytes by dry ice, and sending the lymphocytes to Nanjing Jacksie Biotech limited for extracting and separating the single-domain antibody fragments. The single domain antibody fragment extraction and separation steps are as follows: extracting total RNA, reverse transcribing to synthesize cDNA, amplifying single-domain antibody fragment by using nested PCR, digesting with restriction enzyme, connecting to phage display carrier, and electrically transferring to competent cell. Two independent anti-HSA single domain antibody phage display libraries were successfully constructed. The library capacity was determined by counting the number of single clones coated on a plate by gradient dilution of the library, and both libraries were about 10 in size⁸. 24 single clones are randomly picked from each library for colony PCR detection, and the load rate of the built libraries is less than 5%.

② the screening process of the anti-HSA single domain antibody: coupling HSA on an enzyme label plate for coating overnight, adding a phage library after sealing a sealing solution, washing PBST for multiple times, dissociating the phage specifically combined with HSA by using TEA eluent, infecting Escherichia coli cells in logarithmic growth phase, and carrying out amplification culture on the phage for the next round of screening. After three rounds of Bio-panning, each library is enriched by more than 500 times, so that the aim of screening the antibody library by using a phage display technology to combine the HSA specific antibody is achieved.

Screening specific single positive clone by enzyme linked immunosorbent assay (ELISA) of phage: randomly selecting 600 single colonies from the two libraries, inoculating and culturing, after growing to a logarithmic phase, carrying out IPTG induced expression, centrifugally collecting thalli, obtaining crude antibodies from periplasm by using a penetration impact method, adding the crude antibodies into an enzyme label plate coated with HSA for incubation, washing the plate by PBST, then respectively taking a mouse Anti-HAtag antibody as a primary antibody and a goat Anti-mouse alkaline phosphatase labeled antibody (goat Anti-mouse alkaline phosphatase labeled antibody) as a secondary antibody for binding, adding alkaline phosphatase for color development and reading an absorbance value, and judging a sample well with an OD value more than 3 times larger than that of a control well as a positive control well. Culturing all positive clones, extracting plasmids and sequencing to obtain a plurality of single-domain antibody sequences, wherein one of the single-domain antibody sequences is named as NB22, and the amino acid sequence of the single-domain antibody sequences is shown as SEQ ID in a sequence table: shown at 9.

Expression of the single domain antibody in host bacteria escherichia coli: according to the amino acid sequence of the single domain antibody NB22 obtained above, a gene fragment is synthesized after codon optimization of escherichia coli and is connected into a pET-22 expression vector, and the plasmid is transformed into a BL-21 competent strain (Bio-Rad) for expression. The transformed BL-21 positive clone strain was inoculated into LB medium and cultured until OD600 reached about 0.7, followed by induction expression with the addition of IPTG (final concentration of 1mM) at 220rpm at 28 ℃ overnight. The bacterial cells were collected by centrifugation (8000 Xg, 15min, 4 ℃) to obtain a sample of the anti-HSA single domain antibody NB22 to be purified.

(3) Affinity chromatography purification of anti-HSA single domain antibodies

Affinity chromatography media: (1) sepharose 4B coupled with HSA: HSA-conjugated Sepharose 4B

Sample to be purified: (2) the collected bacterial cells expressing the anti-HSA single domain antibody NB22

The sample to be purified was purified by affinity chromatography according to the following steps:

balancing: connecting the affinity chromatography column to a purification system, equilibrating the column containing HSA with an equilibration buffer pH 7.0 containing 10mM PB, 130mM NaCl, equilibrating 5 column volumes at a flow rate of 30 cm/h;

sample loading: 30mL of a sample to be purified is passed through a well-balanced affinity chromatography column at a flow rate of 30 cm/h;

thirdly, cleaning: washing the chromatographic column with a pH 7.5 washing buffer solution containing 20mM PB, 1M NaCl at a flow rate of 30cm/h, in an amount of 5 times the volume of the chromatographic column;

and fourthly, elution: eluting the anti-HSA single domain antibody NB22 from the chromatographic column with an elution buffer solution containing 20mM HAc-NaAc at pH 2.8 at a flow rate of 10cm/h, and collecting the elution peak to obtain an elution sample, wherein the amount of the elution buffer solution is 5 times of the volume of the chromatographic column;

neutralizing: adjusting the eluted sample to neutrality by using a neutralization buffer solution with the pH of 7.0 and containing 1M Tris-HCl, centrifuging for 10min at the temperature of 400x g4 ℃, and taking the supernatant to obtain purified protein;

(4) detecting the purified protein sample by SDS-PAGE and SEC-HPLC

The purity of the protein sample before and after purification in (3) was identified by SDS-PAGE and SEC-HPLC, and the experimental results are shown in FIG. 1, wherein "M" is the protein molecular weight marker, "S" is the expression sample supernatant, "FT" is the sample flow-through, and "E" is the purified protein sample. The purity of the purified protein sample can reach more than 95% by SEC confirmation. The results show that: the purification method provided by the invention can be used for effectively purifying the anti-HSA single-domain antibody.

Example 2 purification of fusion proteins comprising anti-HSA Single Domain antibodies Using the methods of the invention (1) preparation of affinity chromatography media

Activating: weighing a certain amount of cellulose microspheres (PGMA-OH), adding a certain volume of acetone and a certain volume of epichlorohydrin according to the proportion of 1:1.6(w/v) and the proportion of 1:0.8(w/v), stirring at 120rpm for 0.5h, and then dropwise adding 120mL of 1mol/L NaOH into the system at a constant speed. And (5) carrying out constant-temperature water bath, keeping the reaction temperature constant at 30 ℃, and reacting for 5 hours at a stirring speed of 120 rpm. After the reaction is finished, transferring the feed liquid into a sand core funnel, draining, washing the sand core funnel to be neutral by using 95% ethanol and deionized water respectively, draining the product, washing the product for three times by using a buffer solution (0.1M PB +1mM EDTA) with the pH value of 8.5, and draining for later use;

② coupling: adding 0.1M PB, 0.1M EDTA and pH 8.5 buffer solution into the medium according to the ratio of dry powder 1:2(w/v), adding HSA according to the ratio of 5-10mg HSA/mL medium, mixing uniformly, placing in a three-port reactor, and introducing N in the whole process₂Reacting at 37 ℃ and 120rpm for 24h, and draining;

washing I: washing unbound HSA protein with 5-10 times of medium volume of 0.1M PB, 0.1M EDTA, and pH 8.5 buffer solution, and draining;

sealing: adding 1M ethanolamine with the volume 5-10 times of the medium volume and a buffer solution with the pH value of 8.0, reacting for 4 hours at 37 ℃ at the stirring speed of 120rpm, and draining;

washing II: washing with 5-10 times of medium volume of 0.1HAc/NaAc, 0.5M NaCl and pH 4.0 buffer solution, and draining;

sixthly, washing III: washing with 5-10 times of medium volume of 0.1M boric acid-sodium tetraborate, 0.5M NaCl and pH 8.0 buffer solution, and draining from a 4-sand core funnel;

dd H in 5-10 medium volumes₂Washing for 3 times by using O, and draining; the filling is stored in 20% ethanol (4 ℃) for later use.

(2) Construction and expression of fusion proteins

Connecting human glucagon-like peptide-1 (7-37) (GLP-1, 7-37) and anti-HSA single domain antibody NB22 (prepared according to the method described in example 1) in series by a (G4S)2 flexible linker, synthesizing a recombinant protein gene fragment after optimizing a sequence by a yeast codon, connecting the recombinant protein gene fragment into a Pichia pastoris expression vector pPIC9K, and electrically transforming a recombinant plasmid into a Pichia pastoris GS115 strain for expression of the recombinant protein, wherein the specific method comprises the following steps: after the pPIC9K vector containing the exogenous gene is linearized by Sal I restriction enzyme, the vector is mixed with Pichia pastoris GS115 competent cells in proportion and is electrically transformed by an electrotransformation machine, the transformed cells are coated on an MD plate and are cultured for 3 days at 30 ℃; and (3) washing colonies on the MD plate by using sterile water, transferring the colonies to YPD plates with G418 concentrations of 0.5, 1.0, 2.0 and 4.0mg/mL respectively, carrying out high copy screening, culturing at 30 ℃ for 3 days, selecting the colonies on the plates with different G418 concentrations respectively for expression and identification, and selecting the strain with the highest expression level for amplification culture expression to obtain the somatic cells expressing the fusion protein GLP-1-NB 22.

(3) Affinity chromatography purification of GLP-1-NB22 fusion protein

Affinity chromatography media: (1) PGMA-OH coupled with HSA: HSA-conjugated PGMA-OH

Sample to be purified: (2) bacterial cells in which fusion protein GLP-1-NB22 is expressed

The GLP-1-NB22 fusion protein was purified by affinity chromatography using the following steps:

balancing: connecting the affinity chromatography column to a purification system, equilibrating the column containing HSA with an equilibration buffer pH 7.5 containing 10mM Tri-HCl, 130mM NaCl, equilibrating 10 column volumes at a flow rate of 50 cm/h;

sample loading: 50mL of a sample to be purified was passed through a well-balanced affinity chromatography column at a flow rate of 50 cm/h;

thirdly, cleaning: washing the chromatographic column with a washing buffer solution containing 20mM PBS and 1M NaCl at pH 8.0 at a flow rate of 50cm/h, wherein the amount of the washing buffer solution is 10 times of the volume of the chromatographic column;

and fourthly, elution: eluting the fusion protein GLP-1-NB22 from the chromatographic column with an elution buffer solution containing 20mM citric acid-sodium citrate at pH 3.0 at a flow rate of 20cm/h, and collecting an elution peak to obtain an elution sample, wherein the dosage of the elution buffer solution is 10 times of the volume of the chromatographic column;

neutralizing: adjusting the eluted sample to neutrality by using a neutralization buffer solution with the pH value of 8.0 and containing 1M Tris-HCl, centrifuging for 10min at the temperature of 400x g4 ℃, and taking the supernatant to obtain purified protein;

(4) detection of purified protein samples by SDS-PAGE and SEC-HPLC

The purity of the protein sample before and after purification in (3) was identified by SDS-PAGE and SEC-HPLC, and the experimental results are shown in FIG. 1, wherein "M" is the protein molecular weight marker, "S" is the expression sample supernatant, "FT" is the sample flow-through, and "E" is the purified protein sample. The purity of the purified protein sample can reach more than 95% by SEC confirmation. The results show that: the purification method provided by the invention can be used for effectively purifying the fusion protein containing the anti-HSA single-domain antibody.

Example 3 purification of fusion proteins comprising anti-HSA Single Domain antibodies Using the methods of the invention

(1) Preparation of affinity chromatography media

Activating: weighing a certain amount of PGMA-OH, adding a certain volume of acetone and a certain volume of epichlorohydrin according to the proportion of 1:1.6(w/v) and the proportion of 1:0.8(w/v), stirring at 120rpm for 0.5h, and then dropwise adding 120mL of 1mol/L NaOH into the system at a constant speed. And (5) carrying out constant-temperature water bath, keeping the reaction temperature constant at 30 ℃, and reacting for 5 hours at a stirring speed of 120 rpm. After the reaction is finished, transferring the feed liquid into a sand core funnel, draining, washing the sand core funnel to be neutral by using 95% ethanol and deionized water respectively, draining the product, washing the product for three times by using a buffer solution (0.1mPB +1mm EDTA) with the pH value of 8.5, and draining for later use;

② coupling: adding 0.1M PB, 0.1MEDTA and pH 8.5 buffer solution into the above medium at a ratio of dry powder 1:2(w/v), adding HSA at a ratio of 5-10mg HSA/ml medium, mixing, placing in a three-port reactor, and introducing N in the whole process₂Reacting at 37 ℃ and 120rpm for 24h, and draining;

sealing: add 5-10 medium volumes of 1M ethanolamine, pH 8.0 buffer. The stirring speed is 120rpm, the reaction is carried out for 4 hours at the temperature of 37 ℃, and the sand core funnel is drained.

Washing II: washing with 5-10 times of medium volume of 0.1HAc/NaAc, 0.5M NaCl, pH 4.0 buffer solution, and draining;

sixthly, washing III: washing with 5-10 times of medium volume of 0.1M boric acid-sodium tetraborate, 0.5M NaCl and pH 8.0 buffer solution, and draining;

dd H in 5-10 medium volumes₂Washing for 3 times, and pumping out from a sand core funnel; the filling is stored in 20% ethanol (4 ℃) for later use.

(2) Construction and expression of fusion proteins

Connecting human glucagon-like peptide-1 (7-37) (GLP-1, 7-37) and anti-HSA single domain antibody NB22 (prepared according to the method described in example 1) in series by a (G4S)2 flexible linker, synthesizing a recombinant protein gene fragment after optimizing a sequence by a yeast codon, connecting the recombinant protein gene fragment into a Pichia pastoris expression vector pPIC9K, and electrically transforming a recombinant plasmid into a Pichia pastoris GS115 strain for expression of the recombinant protein, wherein the specific method comprises the following steps: after linearization is carried out on pPIC9K vector containing exogenous genes by Sal I restriction endonuclease, the vector is mixed with Pichia pastoris GS115 competent cells in proportion and then is subjected to electric transformation by using an electric transformation instrument, the transformed cells are coated on an MD flat plate and are cultured for 3 days at 30 ℃; and (3) washing colonies on the MD plate by using sterile water, transferring the colonies to YPD plates with G418 concentrations of 0.5, 1.0, 2.0 and 4.0mg/mL respectively, carrying out high copy screening, culturing at 30 ℃ for 3 days, selecting the colonies on the plates with different G418 concentrations respectively for expression and identification, and selecting the strain with the highest expression level for amplification culture expression to obtain the somatic cells expressing the fusion protein GLP-1-NB 22.

(3) Affinity chromatography purification of GLP-1-NB22 fusion protein

balancing: connecting the affinity chromatography column to a purification system, equilibrating the column containing HSA with an equilibration buffer pH 8.0 containing 10mM PB, 130mM NaCl, equilibrating 12 column volumes at a flow rate of 60 cm/h;

sample loading: 60mL of a sample to be purified was passed through a well-balanced affinity chromatography column at a flow rate of 50 cm/h;

thirdly, cleaning: washing the column with a washing buffer solution containing 10mM PB and 130mM NaCl at pH 8.0 at a flow rate of 60cm/h, in an amount of 15 times the volume of the column;

and fourthly, elution: eluting the fusion protein GLP-1-NB22 from the chromatographic column by using an elution buffer solution with pH 6.0, 10mM PB and 1.5M NaCl at the flow rate of 30cm/h, and collecting an elution sample according to peak tubes, wherein the dosage of the elution buffer solution is 12 times of the volume of the chromatographic column;

neutralizing: adjusting the eluted sample to neutrality by using a neutralization buffer solution with the pH of 8.5 and containing 1M Tris-HCl, centrifuging for 10min at the temperature of 400x g4 ℃, and taking the supernatant to obtain purified protein;

(4) detecting the purified protein sample by SDS-PAGE and SEC-HPLC

The purity of the purified protein sample in (3) was identified by SDS-PAGE and SEC-HPLC, and the experimental results are shown in FIG. 1, wherein "M" is a protein molecular weight marker, "S" is an expression sample supernatant, "FT" is a sample flow-through, and "E1-E4" are purified protein samples of different collection tubes. The purity of the purified protein sample can reach more than 95% by SEC confirmation. The results show that: the fusion protein containing the anti-HSA single domain antibody can be effectively purified by adopting the purification method provided by the invention.

Comparative example 1 purification of fusion protein comprising anti-HSA single domain antibody using existing methods

(1) Preparation of samples

The human glucagon-like peptide-1 (7-37) (GLP-1, 7-37) and anti-HSA single domain antibody NB22 (prepared according to the method described in example 1) are connected in series by a (G4S)2 flexible linker, a 6X histidine tag is added at the C terminal, a fusion protein gene fragment is synthesized, the fusion protein gene fragment is subcloned into a pPIC9K expression vector, and the recombinant plasmid is electrically transformed into a Pichia pastoris GS115 strain for expression of recombinant protein, and the specific method is as follows: the pPIC9K vector containing the exogenous gene is linearized by Sac I restriction endonuclease, mixed with Pichia pastoris GS115 competent cells and subjected to electrical transformation by using an electrotransfer instrument, the transformed cells are coated on an MD (MD) flat plate and cultured at 30 ℃ for 3 days, a plurality of monoclonals are selected to be placed in 3mL YPD (YPD) culture medium, when OD2-6 is reached, centrifugation is carried out, 3mL BMMY is added after supernatant is discarded, 1% methanol is added for induction, 1% methanol is added every 24h for total induction for 72h, and supernatant of fermentation liquor is collected. Yeast strains with expression were screened for high copy using G418 antibiotic. And (3) washing colonies on the MD plate by using sterile water, transferring the colonies to YPD plates with the G418 concentrations of 0.5, 1.0, 2.0 and 4.0mg/mL respectively, culturing for 3 days at 30 ℃, selecting the colonies on the plate with the highest concentration for expression identification, and selecting the strain with the highest expression level for amplification culture expression to obtain the thallus cells expressing the fusion protein GLP-1-NB 22.

(2) Purification of fusion proteins by nickel column affinity chromatography

The bacterial cells expressing the fusion protein GLP-1-NB22 were treated by osmotic pressure to extract periplasmic proteins, and a crude antibody extract was obtained and stored at 4 ℃. The crude periplasmic protein extract was filtered through a 0.22 μm filter and loaded onto a nickel column equilibrated with PBS buffer (laboratory load), the column was washed with PBS + 5% elution buffer (300mM imidazole), and the elution peak was collected. Adding a loading buffer solution into a sample after IPTG induced expression, and treating for 5min in a boiling water bath.

And (3) detection: the purity of the purified protein sample is identified by SDS-PAGE and SEC-HPLC, and the experimental result is shown in FIG. 4, wherein "M" is a protein molecular weight marker, "S" is an expression sample supernatant, "FT" is an upper sample flow-through, "E1-E3" is the purified protein sample, and the purity is confirmed to reach 90% by SEC.

Comparative example 2 purification of fusion protein comprising anti-HSA Single Domain antibody Using existing methods

Sample to be purified: example 2 preparation of bacterial cells expressing GLP-1-NB22 fusion protein

A purification step: purifying the sample by cation purification method

Balancing: connecting the cation chromatographic column into a purification system, balancing the chromatographic column filled with Sepharose with an equilibration buffer solution containing 10mM PBS at pH 6.0, and balancing 12 column volumes at a flow rate of 60 cm/h;

sample loading: 60mL of a sample to be purified is passed through a well-balanced cation chromatography column at a flow rate of 50 cm/h;

thirdly, cleaning: washing the column with a washing buffer solution containing 20mM PB at pH 6.0 at a flow rate of 60cm/h in an amount of 12 times the volume of the column;

linear elution: linearly eluting GLP-1-NB22 fusion protein by using an elution buffer solution containing 20mM PB and 1M NaCl at the pH of 6.0 as a solution B at the flow rate of 30cm/h and the column volume of 0-100% of the solution B, and collecting an elution peak to obtain a purified protein E1-E3;

and (3) detection: the purity of the purified protein sample was identified by SDS-PAGE and SEC-HPLC, and the experimental results are shown in FIG. 5, wherein "M" is the protein molecular weight marker, "S" is the expression sample supernatant, "FT" is the sample flow-through, "E1-E2" is the purified protein sample, and the purity was confirmed to be 90% by SEC.

In conclusion, the purification method provided by the invention can obviously improve the purity of the anti-HSA single-domain antibody or the fusion protein sample thereof, and ensure the druggability (without label segment) and the stability of the single-domain antibody and the fusion protein thereof in the affinity chromatography process, thereby being beneficial to the beneficial effect of the protein biological drug in treatment.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Sequence listing

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Claims

1. A method for affinity chromatography purification of an anti-HSA single domain antibody or a fusion protein thereof, comprising the steps of:

step 1, preparation of affinity chromatography media:

mixing HSA with an affinity medium, and performing coupling reaction to prepare an HSA affinity chromatography medium;

and 2, purifying the sample to be purified by using the HSA affinity chromatography medium prepared in the step 1:

balancing: balancing the HSA affinity chromatography medium prepared in the step 1 by using a balance buffer solution;

loading: passing the sample to be purified through a well-balanced affinity chromatography medium to combine the sample with the affinity chromatography medium;

cleaning: washing the sample-bound affinity chromatography medium with a wash buffer;

and (3) elution: eluting the anti-HSA single domain antibody or the fusion protein thereof from the affinity chromatography medium by using an elution buffer solution, and collecting an elution peak to obtain an elution sample;

neutralizing: adjusting the eluted sample to be neutral by using a neutralization buffer solution to obtain the product;

the heavy chain variable region of the anti-HSA single domain antibody comprises an amino acid sequence of SEQ ID NO: 1-3.

2. The affinity chromatography purification method according to claim 1, wherein the sample to be purified in step 2 is a cell or culture fluid expressing an anti-HSA single domain antibody or a fusion protein comprising an anti-HSA single domain antibody.

3. The affinity chromatographic purification method according to claim 1, wherein the affinity medium in step 1 is selected from cross-linked agarose, cellulose, nitrocellulose, acrylamide polymer, polyethersulfone or chitosan.

4. The affinity chromatography purification process of claim 1, wherein the equilibration buffer is a buffer comprising phosphate or Tris; the washing buffer solution is a buffer solution containing phosphate, Tris or boric acid; the elution buffer solution is a buffer solution containing phosphate, sodium salt, potassium salt, calcium salt, magnesium salt or ammonium salt; the neutralization buffer is a buffer containing Tris or arginine.

5. The affinity chromatography purification method according to claim 4, wherein the equilibration buffer is a buffer comprising PB and NaCl; the washing buffer solution is a buffer solution containing PB and NaCl; the elution buffer solution is a buffer solution containing acetic acid-sodium acetate; the neutralization buffer is a buffer containing Tris-HCl.

6. The affinity chromatographic purification method according to claim 4, wherein the pH value of the equilibration buffer is 6.0-8.5; the pH value of the washing buffer solution is 6.0-8.5; the pH value of the elution buffer solution is 2.5-9.0; the pH value of the neutralization buffer solution is 6.5-9.0.

7. The affinity chromatography purification method according to claim 2, wherein the cell or culture fluid expressing the fusion protein comprising the anti-HSA single domain antibody is prepared by:

the polypeptide molecule with the therapeutic function and the anti-HSA single domain antibody are connected in series through a flexible linker and inserted into a vector to obtain a recombinant plasmid, and the recombinant plasmid is introduced into an expression host cell to be expressed to obtain a cell or culture solution expressing the fusion protein containing the anti-HSA single domain antibody.

8. The affinity chromatography purification process of claim 7, wherein the host cell is a bacterium, yeast, filamentous fungus, insect cell, mammalian cell, or plant cell.

9. The affinity chromatography purification method of claim 1, wherein the amino acid sequence of the heavy chain variable region is SEQ ID NO: 7 or SEQ ID NO: 8.