A nano antibody against human IgG1-CH1 and a method for purifying IgG1 antibody or its fragments using the nano antibody

Abstract

The present invention belongs to the field of biomedicine, and specifically relates to a method for capturing a nano antibody containing a human IgG1-CH1 domain, and separating and purifying an IgG1 antibody, Fab or F(ab')2 using the nano antibody. Because VHH is a single-domain antibody that is very stable, it is expected to have very low immunogenicity compared to bacterial surface proteins such as protein A and protein G, and is independent of the IgG-Fc domain to capture the antibody, which can effectively solve the problem of light chain dropout. The antibody can specifically recognize the IgG1-CH1 domain with high affinity. The affinity chromatography medium with the anti-human IgG1-CH1 domain nano antibody of the present invention as a ligand can achieve the purification of IgG1 and its Fab or F(ab')2 fragments, and has a very broad application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to a nano antibody capable of capturing a human IgG1-CH1 domain, and a method for purifying an IgG1 antibody or a fragment thereof by using the nano antibody. Background Art

[0002] The content of IgG1 subtype in human IgG serum is the highest, about 60%. It was originally isolated from the serum of multiple myeloma patients. Its structure and function have been studied clearly, making it the first to be selected and applied in antibody development. Therapeutic antibodies based on IgG1 subtype are currently the most widely used antibodies, and are mainly used in tumors, infections and autoimmune diseases. Fab fragment (antigen-binding fragment) is the region in the antibody structure that can bind to the antigen. It consists of a complete light chain (variable region and constant region) and a partial heavy chain structure (variable region and a constant region fragment). Compared with the complete IgG, the Fab fragment lacks the Fc segment. Although it can selectively bind to the antigen, it will not precipitate. Due to its low autoimmunogenicity, it cannot be recognized by living immune cells, so it can significantly reduce the probability of hypersensitivity reactions and improve the safety of the product. Fab antibody fragments have the characteristics of small molecular weight, strong tissue distribution specificity, low immune prototype, and genetic engineering operation, making Fab one of the important members in the field of medical research. For example, the already marketed certolizumab ester (a Fab fragment) can effectively target rheumatoid arthritis.

[0003] With the emergence of new antibody drugs, the purification of IgG1 antibodies and Fab fragments has become a more critical and expensive step in antibody drug production, requiring high purity. In addition, such therapeutic antibodies must maintain their binding affinity and biological activity, including effector function. How to efficiently and quickly purify humanized IgG1 antibodies and their Fab fragments has become a topic worthy of in-depth study.

[0004] Conventional methods for purifying human IgG1 antibodies and their Fab fragments include anion / cation exchange, size exclusion gel filtration, precipitation, and application of specific affinity ligands. Commonly used affinity ligands on the market are bacterially derived protein A and protein G, which bind to one or more subclasses of humanized IgG. Although protein A and protein G bind to human IgG1 antibodies, they rely on the IgG-Fc domain and have low binding affinity or even no binding to Fab fragments. Therefore, a purification method that can specifically bind to human IgG1 antibodies and their Fab fragments is needed, such as using anti-human IgG1-CH1 domain nanobodies to separate and purify IgG1 antibodies and their Fab fragments.

[0005] Nanobodies are the smallest naturally occurring antigen-binding domains, usually obtained by phage display. Through appropriate screening, it is possible to ensure that the best nanobody fragments are selected. The advantage of using these nanobody fragments is that they are single-domain peptides, very stable even at higher temperatures, and have low immunogenicity. In addition, VHH has the advantages of small relative molecular mass, high affinity, high stability, good solubility, low immunogenicity, strong penetration, simple humanization, and the ability to be expressed in large quantities in Escherichia coli. The use of nanobodies to purify humanized IgG1 antibodies and Fab or F(ab')2 fragments has broad application prospects. Summary of the invention

[0006] The main purpose of the present invention is to provide a nano antibody against human IgG1-CH1 domain, which can specifically recognize the IgG1-CH1 domain. At the same time, a method for separating and purifying IgG1 antibody, Fab or F(ab')2 using the nano antibody against human IgG1-CH1 domain is also provided.

[0007] The technical solution of the present invention to solve the technical problem is as follows:

[0008] In a first aspect of the present invention, a method for purifying IgG1, Fab or F(ab')2 fragments is provided, the purification method comprising the following steps:

[0009] 1) coupling the anti-human IgG1-CH1 domain nanobody to a solid phase NHS-activated agarose filler to prepare an affinity medium, which is then packed into a prepacked column;

[0010] 2) using an equilibrium buffer to equilibrate the chromatography column coupled with the anti-human IgG1-CH1 VHH protein, and loading the sample solution onto the column;

[0011] 3) equilibrate the chromatography column using an equilibration buffer;

[0012] 4) washing with washing buffer 1 and washing buffer 2 in sequence;

[0013] 5) eluting with an elution buffer to collect and obtain a purified antibody solution;

[0014] The anti-human IgG1-CH1 domain Nanobody comprises a heavy chain variable region, wherein the heavy chain variable region comprises the following CDR regions: CDR1 having an amino acid sequence as shown in SEQ ID NO: 1, CDR2 having an amino acid sequence as shown in SEQ ID NO: 2, and CDR3 having an amino acid sequence as shown in SEQ ID NO: 3.

[0015] Preferably,

[0016] In the step 1), the mass volume ratio of the solid phase agarose filler to the human IgG1-CH1 VHH is 1 ml:10 mg.

[0017] In the steps 2) and 3), the equilibrium buffer is 1×PBS solution, the flow rate is 0.5-2 ml / min, and the volume of the column is 5-10 times.

[0018] In the step 4), the washing buffer 1 comprises 20 mM PB and 1 M NaCl, with a pH of 7.5, and the washing buffer 2 comprises 50 mM NaAC and 150 mM NaCl, with a pH of 5.5, a flow rate of 1-5 ml / min, and 3-10 times the column volume.

[0019] In the step 5), the elution buffer composition is 100 mM glycine-HCl, pH is 3.2-2.5, flow rate is 0.5-3 ml / min, and 3-5 times column volume.

[0020] In a second aspect of the present invention, there is provided a nanobody against the human IgG1-CH1 domain, wherein the nanobody can specifically bind to the Human IgG1-CH1 antigen; the nucleotide sequence of the Human IgG1-CH1 antigen is shown in SEQ ID NO:5, and the amino acid sequence is shown in SEQ ID NO:6;

[0021] The Nanobody comprises a heavy chain variable region, which comprises the following CDR regions: CDR1 having an amino acid sequence as shown in SEQ ID NO: 1, CDR2 having an amino acid sequence as shown in SEQ ID NO: 2, and CDR3 having an amino acid sequence as shown in SEQ ID NO: 3.

[0022] Preferably, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:4.

[0023] Among them, the amino acid sequence shown in SEQ ID NO:1 is as shown in amino acid residues 31-35 in the amino acid sequence shown in SEQ ID NO:4; the amino acid sequence shown in SEQ ID NO:2 is as shown in amino acid residues 50-66 in the amino acid sequence shown in SEQ ID NO:4; and the amino acid sequence shown in SEQ ID NO:3 is as shown in amino acid residues 99-111 in the amino acid sequence shown in SEQ ID NO:4.

[0024] In the third aspect of the present invention, a nucleic acid encoding the anti-IgG1-CH1 Nanobody as described in the second aspect of the present invention is provided.

[0025] In the fourth aspect of the present invention, a recombinant expression vector is provided, wherein the vector contains the nucleic acid as described in the third aspect of the present invention.

[0026] Preferably, the vector is obtained by cloning the coding gene as described in the third aspect of the present invention into the NdeI / XhoI restriction sites in the pET28a plasmid.

[0027] In the fifth aspect of the present invention, a host cell is provided, wherein the host cell contains the recombinant expression vector as described in the fourth aspect of the present invention.

[0028] In the sixth aspect of the present invention, there is provided the use of the nanobody as described in the second aspect, the nucleic acid as described in the third aspect, the recombinant expression vector as described in the fourth aspect or the host cell as described in the fifth aspect in the preparation of IgG1, Fab or F(ab')2 fragment purification reagents.

[0029] The Fab segment is the active part of the antibody. The Fc segment cannot bind to the antigen, but it has antigenic epitopes of various Igs and also performs other biological activities of Ig. The IgG heavy chain can be cut near the carboxyl end of the interchain disulfide bond by using pepsin to obtain a large fragment F(ab')2 and several small molecular polypeptides (pFc'), the latter of which has no biological activity. F(ab')2 is composed of two Fabs and a hinge region, and can bind to two antigenic epitopes at the same time, so it can form an agglutination reaction or a precipitation reaction. The antibody is enzymatically cleaved with a protease to obtain a F(ab')2 fragment or a Fab fragment.

[0030] In the seventh aspect of the present invention, an affinity chromatography medium is provided, wherein the affinity chromatography medium uses the anti-human IgG1-CH1 domain nanobody as described in the second aspect as a ligand.

[0031] The present invention has the following technical effects: The present invention provides a new anti-IgG1-CH1 nano antibody, which can achieve high-efficiency purification of F(ab')2 and IgG1 monoclonal antibodies, can help improve the purification efficiency of these two fragments, and accelerate the application of IgG1 in the field of gene therapy. The anti-IgG1-CH1 nano antibody of the present invention can more specifically bind to monoclonal antibodies and antibody fragments (including Fab, F(ab')2, etc.) containing humanIgG-CH1. In the process of antibody purification, the non-specific binding of foreign proteins can be effectively reduced, and the purity of the purified antibodies can be improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a diagram showing the binding of phages enriched in each round of panning to BSA detected by ELISA;

[0033] Figure 2This is a diagram showing the binding of monoclonal phage to antigen protein detected by ELISA;

[0034] Figure 3 Schematic diagram of prokaryotic expression vector;

[0035] Figure 4 This is the result of the Anti IgG1-CH1 VHH pilot test;

[0036] Figure 5 , 6 The SDS-PAGE electrophoresis results for purification and purity verification;

[0037] Figure 7 ELISA results for the binding of IgG1-CH1 VHH to Human IgG1 and F(ab')2 fragments;

[0038] Figure 8 , 9 , 10 are the SDS-PAGE results of IgG monoclonal antibody, F(ab')2 and Fab purification using IgG1-CH1 VHH. DETAILED DESCRIPTION

[0039] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.

[0040] Example 1. Screening of anti-IgG1-CH1 nanobodies

[0041] 1. Experimental instruments and materials: ELISA instrument, 96-well ELISA plate, shaker

[0042] 2. Experimental methods:

[0043] Using human IgG1-CH1 as antigen, phage display technology was used to obtain the natural nanoantibody phage library of alpaca (library size was 1.47x10 9 ) was used to screen the nanobodies against IgG1-CH1. The nucleotide sequence of IgG1-CH1 is SEQ ID NO: 5, and the amino acid sequence is SEQ ID NO: 6.

[0044] Using the solid phase panning method, 100ug / mL IgG-CH1 was coated onto the ELISA strips, 100uL per well, and coated overnight at 4°C. Washed three times with PBST, 200uL 3% casein was added to each well, and blocked at 37°C for 2 hours. After washing three times with PBST, the phage display library (about 1x10 12CFU), incubate at 37℃ for 1 hour. Aspirate unbound phages and wash 10 times with PBST. Add 100uL of glycine-HCl (PH=2.2) solution to each well, react at 37℃ for 7 minutes, gently blow the plate wells to elute the adsorbed phages, and then add Tris-HCl (PH=8.8) solution to neutralize to neutral. The eluted phages infect TG1 cells in the logarithmic growth phase, amplify the recovered phages, and use them for the next round of panning.

[0045] After three rounds of panning, Phage-ELISA was used to verify specific enrichment. 2ug / mL IgG1-CH1 was coated on the ELISA strips and coated overnight at 4℃. After washing three times with PBST, the plates were blocked with 3% casein at 37℃ for 2 hours. After washing five times with PBST, the phage display library after three rounds of panning was added, and the first well was about 1x10 12 CFU was diluted 2 times, the second well was diluted 100 times, and the third well was a blank control, and the cells were combined at 37°C for 1 hour. After washing 5 times with PBST, HRP-labeled mouse anti-M13 secondary antibody was added and incubated at 37°C for 1 hour. After washing 5 times with PBST, TMB colorimetric solution was added and the cells were colorimetric at room temperature in the dark for 5-10 minutes. Finally, 2M sulfuric acid was used to terminate the colorimetric development. The absorbance at a wavelength of 450nm was read with an enzyme reader and a Phage-ELISA binding curve was made.

[0046] ELISA test results Figure 1 As shown, after three rounds of enrichment, the affinity of the phage population to IgG-CH1 increased in each round.

[0047] The phage monoclonal clones enriched in the third round were subjected to antigen binding analysis. The specific process was as follows:

[0048] The TG1 cells were infected with the third round of enriched phage library, and 440 monoclonal clones were randomly selected from them, amplified and recovered phages. 2ug / mL IgG1-CH1 was coated on the ELISA strips and coated overnight at 4℃. After washing three times with PBST, it was blocked with 3% casein at 37℃ for 2 hours. The 440 amplified monoclonal phages and the negative control auxiliary phage were incubated with PBST solution containing 3% casein at room temperature for 1 hour at a ratio of 1:1, and the incubated phages were added to the blocked ELISA plate and incubated at 37℃ for 1 hour. After washing 5 times with PBST, HRP-labeled mouse anti-M13 secondary antibody was added and incubated at 37℃ for 1 hour. After washing 5 times with PBST, TMB was added to develop color at room temperature in the dark for 5-10 minutes, and finally 2M sulfuric acid was used to stop the color development. The absorbance value at a wavelength of 450nm was read with an ELISA reader. The positive clones were those with absorbance values ​​more than twice that of the negative control. Analyze the binding ability of monoclonal phage to IgG-CH1. The test results are as follows Figure 2 As shown, 13 positive clones recognizing IgG-CH1 were found among 88 cloned phages.

[0049] The 13 positive clones were sequenced and analyzed, and one dominant enriched clone was screened out, whose antibody amino acid sequence was SEQ ID NO: 4.

[0050] In the amino acid sequence, amino acid residues 31-35 (ie, SEQ ID NO: 1) are heavy chain CDR1, amino acid residues 50-66 (ie, SEQ ID NO: 2) are heavy chain CDR2, and amino acid residues 99-111 (ie, SEQ ID NO: 3) are heavy chain CDR3.

[0051] Example 2 Expression and purification of IgG1-CH1 VHH

[0052] 1. Experimental materials: water bath, shaker, centrifuge, electrophoresis instrument, protein purification system, high-speed refrigerated centrifuge, Kan resistance, imidazole, Ni affinity chromatography column;

[0053] 2. Test methods:

[0054] 2.1 Plasmid transformation:

[0055] (1) Take out a piece of BL21 (DE3) competent cells stored at -80°C and place it on ice for 15 minutes;

[0056] (2) Add 5 μL of the plasmid obtained in Example 1, place on ice for 30 min, heat shock in a 42°C water bath for 90 s (accurate timing), and place on ice for 5 min;

[0057] (3) Aspirate all the bacterial liquid and spread it on a Kan-resistant plate. Incubate at 37°C for 18 h to obtain a single colony.

[0058] 2.2 Expression Verification

[0059] (1) Pick a single colony after transformation and inoculate it into 4 mL of 2*YT medium containing Kan resistance;

[0060] (2) After culturing at 37°C until OD600 = 0.6, IPTG was added to induce expression. The induction conditions were: 37°C, 220 rpm, 1 mM IPTG, 4 h;

[0061] (3) After induction, the whole bacterial solution was taken for 12% SDS-PAGE electrophoresis.

[0062] 2.3 Protein expression

[0063] (1) Activation of bacterial strains: Pick a single colony after transformation and inoculate it into 100 mL of 2*YT medium containing Kan resistance, and culture it at 37°C and 220 rpm for 18 h;

[0064] (2) Bacterial culture: 1% of the activated bacterial solution was transferred to 2*YT medium containing Kan resistance, and cultured at 37°C, 220 rpm until OD600 = 0.6 to induce expression. The induction conditions were: 30°C, 220 rpm, 1 mM IPTG, and cultured for 16 h.

[0065] (3) After the induction expression is completed, the bacterial solution is divided and centrifuged at 7000 rpm for 5 min at 4°C to collect the bacteria;

[0066] (4) Add 1× PBS solution at a ratio of 1 g of bacteria to 10 mL of lysis buffer and perform ultrasonic disruption. Centrifuge at 11,000 rpm for 20 min and the supernatant is purified.

[0067] 2.4 Protein purification: Ni affinity chromatography column purification

[0068] (1) Column loading: After the filler is mixed, use a pipette to draw the filler suspension and add it to the chromatography column. Connect the pump, connecting pipes, and chromatography column, and label the column.

[0069] (2) Equilibrate the column with 1× PBS solution and load the sample after equilibration;

[0070] (3) After loading, the sample was balanced with 1× PBS solution, and then washed with 20 mM PB + 1 M NaCl pH 7.5 buffer;

[0071] (4) After washing, the sample was equilibrated with 1× PBS solution and eluted with 100 mM imidazole. The detection wavelength was 280 nm. The protein peaks were collected separately. The protein concentration was measured using a Nano-300 instrument. 2 μg was loaded for electrophoresis detection.

[0072] (5) Dialysis: After combining the proteins according to the electrophoresis results, aspirate them into a dialysis bag, tie the dialysis bag tightly, mark it, and place it in a buffer containing 1× PBS (for the convenience of preparing affinity chromatography media) for dialysis;

[0073] (6) After dialysis, 2 μg was loaded for electrophoresis detection.

[0074] 3. Experimental results: SDS-PAGE electrophoresis results are shown in Figure 4 ,Depend on Figure 4 It can be seen that the estimated molecular weight of the target protein is 14kDa. The electrophoresis test results after measuring the protein concentration are as follows Figure 5 As shown, it can be seen that the protein is expressed, that is, the target protein is obtained by purification through Ni affinity chromatography column, and the electrophoresis results after dialysis are as follows Figure 6 As shown by Figure 6 The estimated expression level of the protein is 12 mg / L.

[0075] Example 3 ELISA verification

[0076] 1. Detection instruments and reagents: microplate reader, plate washer, TMB colorimetric solution, TMB colorimetric stop solution, F(ab')2 (HuamnIgG1-Kappa-Ides enzyme digestion)

[0077] 2. Test methods:

[0078] 2.1 Coating: Coat the ELISA plate according to the required amount of the experiment, dilute the protein F(ab')2 (Huamn IgG1-Kappa-Ides digestion) and Huamn IgG1 to 2 μg / mL, add 100 μL of the diluted antibody to each well of the ELISA plate, gently shake the ELISA plate to allow the antibody to cover the bottom of the ELISA plate wells, and incubate at 4°C overnight.

[0079] 2.2 Blocking: After incubation at 4°C overnight, place the ELISA plate on a plate washer, wash it 5 times with 0.05% PBST buffer, add 100 μL of casein blocking solution, incubate it at 37°C for 2 hours, place the ELISA plate on a plate washer, and wash it 5 times with 0.05% PBST buffer.

[0080] 2.3 Protein biotin labeling: Take 1 μL of the prepared biotin ester solution and add it to a solution containing 1 mg of anti-human IgG1-CH1 VHH and react at room temperature for 1 hour.

[0081] 2.4 Add primary antibody: Use biotin-labeled anti-human IgG1-CH1 VHH as primary antibody, dilute to 1000nM with PBS, start at 1000nM, dilute in 1 / 2 gradient, add 100μL to each well, incubate at 37℃ for 1h, place the ELISA plate on a plate washer, and wash 5 times with 0.05% PBST buffer.

[0082] 2.5 Adding secondary antibody: Add 100 μL of diluted horseradish peroxidase-labeled avidin (SA-HRP) to each well, react at 25°C for 30 min, and then wash five times with 0.05% PBST buffer.

[0083] 2.6 Substrate color development: Add TMB color development solution to each of the above reaction wells, develop the color at room temperature in the dark for 5 minutes, then add 50 μL TMB color stop solution to terminate the reaction, and read the optical density value at 450 nm using a microplate reader.

[0084] 3. Experimental results: Specific test results are as follows Figure 7 As shown, it can be seen that the coated F(ab')2 (Huamn IgG1-Kappa-Ides digestion) and the detected OD450 indicate that the IgG1-CH1VHH obtained in Example 2 can bind to Human IgG1 and F(ab')2 fragments.

[0085] Example 4 Filler Purification Application

[0086] 4.1 Purification of human IgG1 antibodies using IgG1-CH1 VHH coupled affinity media

[0087] Experimental verification: Whether the affinity chromatography medium using human IgG1-CH1 VHH obtained in Example 2 as a ligand can purify IgG1 antibodies

[0088] 1. Experimental instruments and materials: electrophoresis instrument, protein purification system, Human IgG1-Kappa antibody cell culture supernatant

[0089] 2. Experimental methods:

[0090] 2.1 Column loading: Human IgG1-CH1 VHH was coupled to a solid phase agarose filler to prepare an affinity medium, wherein the mass volume ratio of the solid phase agarose filler to human IgG1-CH1 VHH was 1 ml:10 mg, and the column was loaded into a 1 mL pre-loaded column; 1) The column was equilibrated with a 1×PBS solution at a flow rate of 1 ml / min and 5 times the column volume; 2) The sample was loaded after equilibration; 3) After loading, the column was equilibrated with a 1×PBS solution at a flow rate of 1 ml / min and 5 times the column volume; 4) The column was washed with a washing buffer 1 composed of 20 mM PB and 1 M NaCl, pH 7.5, and a washing buffer 2 composed of 50 mM NaAC and 150 mM NaCl, pH 5.5, at a flow rate of 1 ml / min and 5 times the column volume; 5) After the protein baseline was stable, the column was washed with 100 mM glycine-HCl pH 2.5 buffer was used for elution, flow rate was 1 ml / min, 5 column volumes, detection wavelength was 280 nm, and protein peaks were collected by separate tubes. After the protein concentration was measured using Nano-300 instrument, 2 μg was loaded for electrophoresis detection.

[0091] 3. Experimental results: See Figure 8 The human IgG1-CH1 VHH-coupled affinity medium can purify IgG1 antibodies from cell supernatants. It is estimated that the dynamic loading capacity of this affinity chromatography medium for IgG1 antibodies is about 10 mg / mL.

[0092] 4.2 Purification of F(ab')2 Antibody Fragments by IgG1-CH1 VHH Coupled Affinity Media

[0093] Experimental verification: Whether the affinity chromatography medium using human IgG1-CH1 VHH prepared in Example 2 as a ligand can purify F(ab')2 antibody fragments

[0094] 1. Experimental instruments and materials: electrophoresis instrument, protein purification system, F(ab')2 (Huamn IgG1-Kappa-Ides enzyme digestion)

[0095] 2. Experimental method: Column loading: Human IgG1-CH1 VHH was coupled to solid phase agarose filler to prepare affinity medium, the mass volume ratio of solid phase agarose filler to human IgG1-CH1 VHH was 1ml:10mg, and it was filled into a pre-loaded column; 1) The chromatography column was equilibrated with 1×PBS solution at a flow rate of 1ml / min and 5 times the column volume; 2) The sample was loaded after equilibration; 3) After loading, the column was equilibrated with 1×PBS solution at a flow rate of 1ml / min and 5 times the column volume; 4) The washing buffer 1 was composed of 20mM PB and 1M NaCl, pH 7.5, and the washing buffer 2 was composed of 50mM NaAC and 150mM NaCl, pH 5.5, at a flow rate of 1ml / min and 5 times the column volume for washing; 5) After the protein baseline was stable, the column was washed with 100mM glycine-HCl pH 2.5 buffer was used for elution, flow rate was 1 ml / min, 5 column volumes, detection wavelength was 280 nm, and protein peaks were collected by separate tubes. After the protein concentration was measured using Nano-300 instrument, 2 μg was loaded for electrophoresis detection.

[0096] 3. Experimental results: See Fig. 9 The affinity medium coupled to human IgG1-CH1 VHH can bind to F(ab')2 antibody fragments, and the dynamic loading capacity of the affinity chromatography medium for F(ab')2 is estimated to be about 8 mg / mL.

[0097] 4.3 Purification of Fab antibody fragments using IgG1-CH1 VHH coupled affinity medium

[0098] Experimental verification: Whether the affinity chromatography medium using human IgG1-CH1 VHH prepared in Example 2 as a ligand can purify Fab antibody fragments

[0099] 1. Experimental instruments and materials: electrophoresis instrument (manufacturer: Tianneng model: EPS-600), protein purification system (manufacturer: Saipu model: SCG100-V4), Fab (Huamn IgG1-Kappa type)

[0100] 2. Experimental method: Column loading: Human IgG1-CH1 VHH was coupled to solid phase agarose filler to prepare affinity medium, the mass volume ratio of solid phase agarose filler to human IgG1-CH1 VHH was 1ml:10mg, and it was filled into a 1mL pre-loaded column; 1) The chromatography column was equilibrated with 1×PBS solution at a flow rate of 1ml / min and 5 times the column volume; 2) The sample was loaded after equilibration; 3) After loading, the column was equilibrated with 1×PBS solution at a flow rate of 1ml / min and 5 times the column volume; 4) The washing buffer 1 was composed of 20mM PB and 1M NaCl, pH 7.5, and the washing buffer 2 was composed of 50mM NaAC and 150mM NaCl, pH 5.5, at a flow rate of 1ml / min and 5 times the column volume for washing; 5) After the protein baseline was stable, the column was washed with 100mM glycine-HCl pH 3.2 Buffer was used for elution, flow rate was 1 ml / min, 5 column volumes, detection wavelength was 280 nm, and protein peaks were collected by separate tubes. After the protein concentration was measured using Nano-300 instrument, 2 μg was loaded for electrophoresis detection.

[0101] 3. Experimental results: See Fig.10 The affinity medium coupled with human IgG1-CH1 VHH can bind to Fab antibody fragments, and the dynamic loading capacity of the affinity chromatography medium for Fab is estimated to be about 7 mg / mL.

[0102] Finally, it should be noted that the above embodiments are only illustrative of the principles, performance and efficacy of the present invention, and are not intended to limit the present invention. Anyone familiar with the technology may modify or change the above embodiments without violating the spirit and scope of the present invention. Therefore, all equivalent modifications or changes made by those with ordinary knowledge in the technical field without departing from the spirit and technical ideas disclosed by the present invention should still be covered by the claims of the present invention.

Claims

1. A method for purifying human IgG1 or its Fab or F(ab')2 fragments, characterized in that: The purification method comprises the following steps: 1) The nanobody against the human IgG1-CH1 domain is coupled to a solid phase NHS-activated agarose filler to prepare an affinity medium, which is then packed into a prepacked column; 2) using an equilibrium buffer to equilibrate the chromatography column coupled with the anti-human IgG1-CH1 VHH protein, and loading the sample solution onto the column; 3) Equilibrate the chromatography column using an equilibration buffer; 4) Wash with wash buffer 1 and wash buffer 2 in sequence; 5) Eluting with an elution buffer to collect a purified antibody solution; The anti-human IgG1-CH1 domain Nanobody comprises a heavy chain variable region, which comprises the following CDR regions: CDR1 with an amino acid sequence as shown in SEQ ID NO: 1, CDR2 with an amino acid sequence as shown in SEQ ID NO: 2, and CDR3 with an amino acid sequence as shown in SEQ ID NO:

3.

2. The method according to claim 1, characterized in that In the step 1), the mass volume ratio of the solid phase agarose filler to the human IgG1-CH1 VHH is 1 ml:10 mg.

3. The method according to claim 1, characterized in that In the steps 2) and 3), the equilibrium buffer is 1×PBS solution, the flow rate is 0.5-2 ml / min, and the volume of the column is 5-10 times.

4. The method according to claim 1, characterized in that: In the step 4), the washing buffer 1 comprises 20 mM PB and 1 M NaCl, with a pH of 7.5, and the washing buffer 2 comprises 50 mM NaAC and 150 mM NaCl, with a pH of 5.5, a flow rate of 1-5 ml / min, and 3-10 column volumes.

5. The method according to claim 1, characterized in that In the step 5), the elution buffer composition is 100 mM glycine-HCl, pH is 3.2-2.5, flow rate is 0.5-3 ml / min, and 3-5 times column volume.

6. A nanobody against the human IgG1-CH1 domain, characterized in that The Nanobody comprises a heavy chain variable region, which comprises the following CDR regions: CDR1 having an amino acid sequence as shown in SEQ ID NO: 1, CDR2 having an amino acid sequence as shown in SEQ ID NO: 2, and CDR3 having an amino acid sequence as shown in SEQ ID NO:

3.

7. The anti-human IgG1-CH1 domain Nanobody according to claim 6, characterized in that The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:

4.

8. A nucleic acid encoding the anti-human IgG1-CH1 Nanobody according to claim 6 or 7.

9. A recombinant expression vector, characterized in that: The vector contains the nucleic acid according to claim 8.

10. The recombinant expression vector according to claim 9, characterized in that: The vector is obtained by cloning the coding gene into the NdeI / XhoI restriction site in the pET28a plasmid.

11. A host cell, characterized in that The host cell contains the recombinant expression vector according to claim 9.

12. Use of the Nanobody according to claim 6, the nucleic acid according to claim 8, the recombinant expression vector according to claim 9 or the host cell according to claim 11 in purifying human IgG1 and its Fab or F(ab')2 fragments.

13. An affinity chromatography medium, characterized in that The affinity chromatography medium uses the anti-human IgG1-CH1 domain nanobody as claimed in claim 6 or 7 as a ligand.

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