A vh3 binding molecule and uses thereof

Abstract

The present application relates to a kind of VH3 binding molecules and its application, the VH3 binding molecule includes polypeptide unit, the amino acid sequence of the polypeptide unit is selected from one or more of SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9 or SEQ ID NO.11.The VH3 binding molecule provided by the present application can be specifically combined with the antibody containing VH3 type heavy chain, not combined with Fc, and the chromatography medium prepared by using it as ligand has good purification efficiency, load and alkali resistance.The chromatography medium prepared by using the VH3 binding molecule described in the present application is the supplement of conventional Protein A chromatography medium combined with antibody Fc fragment in the field of antibody purification, and it has wide application prospect in the field of antibody engineering and purification process.

Description

TECHNICAL FIELD

[0001] The present application relates to a binding molecule, in particular to a VH3 binding molecule and its application. BACKGROUND

[0002] Antibodies are key molecules in the immune system, which are composed of heavy and light chains, and the heavy chain variable region (VH) and light chain variable region (VL) together constitute the antigen binding site of the antibody. The heavy chain variable region containing VH3 domain (referred to as VH3 type heavy chain) is the most common and important member in the VH family, and dominates in many commercial biological antibody preparations.

[0003] Protein A is a cell wall protein isolated from Staphylococcus aureus, which is composed of multiple domains, such as E, D, A, B, and C domains. These domains can specifically recognize and bind to the Fc fragment of immunoglobulin IgG, while the binding ability to the Fab fragment is relatively weak. Therefore, protein A is mainly used for the isolation and purification of immunoglobulin IgG containing Fc fragments. This highly specific binding ability makes protein A a powerful tool for antibody purification in the field of biotechnology.

[0004] However, for some special types of antibody fragments, traditional protein A is difficult to achieve efficient and specific purification, such as antibody fragments with VH3 type heavy chain variable region but without Fc fragment (such as Fab, scFv, domain antibody, nanobody, and BiTe). Currently, the purification of this type of antibody still uses a relatively complex scheme.

[0005] Therefore, it is of great significance to develop a polypeptide molecule that can specifically bind to antibodies containing VH3 type heavy chain without binding to Fc, which can improve the purification efficiency and resolution of antibodies containing VH3 type heavy chain, and has a broad application prospect in the field of biopharmaceuticals, especially in antibody engineering and purification process. SUMMARY

[0006] Based on this, the purpose of the present application is to provide a VH3 binding molecule which can specifically bind to VH3 type heavy chain variable region without binding to Fc fragment, thereby improving the separation and purification performance of this type of antibody.

[0007] In order to achieve the above purpose, the present application provides the following technical solutions:

[0008] In a first aspect, the present application provides a VH3 binding molecule, wherein the VH3 binding molecule comprises one or more polypeptide units, and the amino acid sequence of the polypeptide unit is selected from the group consisting of SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9 and SEQ ID NO. 11.

[0009] Further, in the VH3 binding molecule, the VH3 binding molecule comprises 3-6 polypeptide units.

[0010] Preferably, in the VH3 binding molecule, the amino acid sequences of the polypeptide units are different from each other or at least two of them are the same.

[0011] Preferably, in the VH3 binding molecule, the amino acid sequences of the polypeptide units are the same.

[0012] Further, in the VH3 binding molecule, the first amino acid at the N-terminus is methionine and the amino acid at the C-terminus is cysteine.

[0013] In a second aspect, the present application provides a nucleic acid molecule, wherein the nucleic acid molecule comprises a nucleotide sequence encoding the VH3 binding molecule.

[0014] In a third aspect, the present application provides an expression vector, wherein the expression vector comprises the nucleic acid molecule.

[0015] In a fourth aspect, the present application provides a chromatography medium, wherein the chromatography medium comprises the VH3 binding molecule as a ligand.

[0016] In a fifth aspect, the present application provides the use of the chromatography medium in separating and purifying an antibody comprising a VH3 type heavy chain.

[0017] In a sixth aspect, the present application provides a method for separating and purifying an antibody comprising a VH3 type heavy chain, wherein the method comprises: contacting a sample to be purified with the chromatography medium, and collecting the purified target after removing impurities by elution.

[0018] Compared with the prior art, the present application has the following advantages:

[0019] The present application provides a VH3 binding molecule and its application, which can specifically bind to VH3 type heavy chain variable region and does not bind to Fc fragment. The chromatography medium prepared by using the VH3 binding molecule as ligand has good purification effect, load and alkali resistance on antibodies containing VH3 type heavy chain. The chromatography medium prepared by using the VH3 binding molecule of the present application is a supplement to the conventional Protein A chromatography medium which binds to the Fc fragment of antibody in the field of antibody purification, and has a wide application prospect in the field of antibody engineering and purification process. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 SDS-PAGE electrophoretogram of VH3-2 binding molecule corresponding to SEQ ID NO. 2.

[0021] Figure 2 SDS-PAGE electrophoretogram of VH3-3 binding molecule corresponding to SEQ ID NO. 3.

[0022] Figure 3 SDS-PAGE electrophoretogram of VH3-4 binding molecule corresponding to SEQ ID NO. 4.

[0023] Figure 4 SDS-PAGE electrophoretogram of VH3-5 binding molecule corresponding to SEQ ID NO. 5.

[0024] Figure 5 SDS-PAGE electrophoretogram of VH3-6 binding molecule corresponding to SEQ ID NO. 6.

[0025] Figure 6 SDS-PAGE electrophoretogram of VH3-(2-6) binding molecule corresponding to SEQ ID NO. 2-6 after expression and purification.

[0026] Figure 7 SDS-PAGE electrophoretogram of VH3-7 binding molecule corresponding to SEQ ID NO. 7 after expression and purification.

[0027] Figure 8 SDS-PAGE electrophoretogram of VH3-8 binding molecule corresponding to SEQ ID NO. 8 after expression and purification.

[0028] Figure 9 SDS-PAGE electrophoretogram of VH3-(9-11) binding molecule corresponding to SEQ ID NO. 9-11 after expression and purification.

[0029] Figure 10 Chromatogram of Mab1 sample purified by using chromatography medium 1.

[0030] Figure 11 Chromatogram for purification of Mabl sample using chromatography medium 5.

[0031] Figure 12 Chromatogram for purification of Mab2 sample using chromatography medium 1.

[0032] Figure 13 Chromatogram for purification of Mab2 sample using chromatography medium 5.

[0033] Figure 14 Electropherogram of samples collected at different stages of purification of Mabl, Mab2 samples using chromatography medium 1 and chromatography medium 5. DETAILED DESCRIPTION

[0034] In order to make the above objectives, features and advantages of the present application more clear and comprehensible, the specific embodiments of the present application will be described in detail below with reference to the accompanying drawings. In the following description, a large number of specific details are set forth in order to provide a thorough understanding of the present application. However, the present application can be implemented in many different ways other than those described herein, and one of ordinary skill in the art can make similar improvements without departing from the spirit of the present application, and therefore the present application is not limited to the specific embodiments disclosed below.

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. The materials, reagents used in the following examples, as well as other materials used, were obtained from commercial vendors, unless otherwise stated.

[0036] The present application will be described in detail below with reference to specific embodiments, which are used to understand but not to limit the present application.

[0037] In a first aspect, the present application provides a VH3 binding molecule comprising a polypeptide unit, the amino acid sequence of the polypeptide unit is selected from one or more of SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9 or SEQ ID NO. 11.

[0038] In certain embodiments, the VH3 binding molecule comprises 3-6 polypeptide units.

[0039] In certain embodiments, the amino acid sequences of the plurality of polypeptide units in the VH3 binding molecule are different from each other or at least two are the same.

[0040] In some embodiments, the amino acid sequences of the polypeptide units in the VH3 binding molecule are identical.

[0041] In some embodiments, the first amino acid at the N-terminus of the VH3 binding molecule is methionine, and the amino acid at the C-terminus is cysteine.

[0042] In a second aspect, the present application provides a nucleic acid molecule comprising a nucleotide sequence encoding the VH3 binding molecule of the present application.

[0043] In a third aspect, the present application provides an expression vector comprising the nucleic acid molecule of the present application.

[0044] In a fourth aspect, the present application provides a chromatography medium having the VH3 binding molecule of the present application as a ligand.

[0045] In a fifth aspect, the present application provides use of the chromatography medium of the present application in separating and purifying an antibody comprising a VH3 type heavy chain.

[0046] In a sixth aspect, the present application provides a method for separating and purifying an antibody comprising a VH3 type heavy chain, the method comprising: contacting a sample to be purified with the chromatography medium of the present application, and collecting the purified target after elution.

[0047] Example 1 Design of VH3 binding molecule

[0048] SEQ ID NO. 1 is a polypeptide unit of a conventional Protein A known to be capable of binding to the Fc fragment of an antibody but not to the VH fragment. Ten polypeptide units having amino acid sequences shown in SEQ ID NO. 2 to SEQ ID NO. 11 were obtained by performing amino acid mutations based on the sequence of SEQ ID NO. 1.

[0049] First, ten VH3 binding molecules were designed based on the ten polypeptide units, each VH3 binding molecule being designed to comprise a tetramer of four identical repeated polypeptide units, and the first amino acid at the N-terminus being methionine (M) and the amino acid at the C-terminus being cysteine (C). The optimal VH3 binding molecule that can be used for the purification of an antibody comprising a VH3 type heavy chain was further selected by experiments, thereby determining the optimal polypeptide unit sequence; in the experiments, four sequences of SEQ ID NO. 1 were designed into a tetramer as a control molecule. An example of the naming of the VH3 binding molecule: the binding molecule corresponding to SEQ ID NO. 2 is named VH3-2, and the naming of other VH3 binding molecules is similarly derived.

[0050] Example 2 Molecular weight and isoelectric point of VH3 binding molecule

[0051] The molecular weight and isoelectric point (PI) of the 10 VH3 binding molecules designed in Example 1 are shown in Table 1.

[0052] Table 1 Molecular weight and isoelectric point of VH3 binding molecules

[0053]

[0054] Expression and purification of VH3 binding molecules in Example 3

[0055] The amino acid sequences of the control molecule described in Example 1 and the 10 VH3 binding molecules in Table 1 are respectively designed into corresponding gene sequences (if multiple sequences are designed due to codon degeneracy, any one of them can be selected), and a biological company is commissioned to synthesize them. The synthesized genes are constructed into the expression vector pET28a to construct recombinant plasmids, which are verified by sequencing and then transformed into E. coli BL21 (DE3) competent cells. The transformed single colonies are inoculated into LB medium containing kanamycin, and cultured overnight at 37°C on a shaker; then the bacterial cells are inoculated into LB medium containing kanamycin at an inoculation amount of 1%, and cultured at 37°C under shaking until the OD 600 is about 0.6-0.8, and the expression is carried out without induction and under the condition of 1 mM IPTG, respectively. After the expression is completed, the bacterial solution is subjected to ultrasonic disruption, and the sample is collected for SDS-PAGE electrophoresis detection to verify the expression of the target protein.

[0056] The single colonies of recombinant plasmids transformed into E. coli BL21 are expanded in large-volume medium according to the above operation method, and after induction expression, the induced bacterial cells are collected by centrifugation, then 1% Triton X-100 is added to the PBS at a mass-volume ratio (g / mL) of 1:10 to resuspend the bacterial cells and perform ultrasonic disruption to lyse the bacterial cells. The disrupted and lysed bacterial cells are centrifuged to obtain the supernatant to be purified. The supernatant is purified using a Ni affinity chromatography column, and the purification conditions are shown in Table 2. Then the purified sample is dialyzed with pH 7.0 PBS, and after dialysis, SDS-PAGE electrophoresis detection is performed.

[0057] Table 2 Conditions for purification of target protein using Ni affinity chromatography column

[0058]

[0059] Figures 1-5The figure of SDS-PAGE verification result of protein expression of VH3 binding molecules corresponding to polypeptide units of SEQ ID NO. 2~6 by electrophoresis. The position of target protein was estimated according to protein molecular weight on the electrophoresis map. According to the result of electrophoresis map, it was found that the target protein was displayed at the estimated position under non-induced and induced conditions, and the protein content was relatively more under induced condition. The result showed that the recombinant plasmid was successfully constructed and the VH3 binding molecule protein was successfully expressed. Based on the same SDS-PAGE verification result, the VH3 binding molecules formed by polypeptide units of SEQ ID NO. 1, SEQ ID NO. 7~11 were also successfully expressed under the expression system.

[0060] The expressed target protein was subjected to electrophoresis detection after Ni affinity column purification and dialysis, Figures 6-9 The SDS-PAGE electrophoresis map of VH3 binding molecules corresponding to polypeptide units of SEQ ID NO. 2~11. It can be seen from the figure that the purified protein showed high purity.

[0061] Example 4: Verification of binding affinity of VH3 binding molecules

[0062] The binding affinity of the control molecule and 10 kinds of VH3 binding molecules expressed and purified in Example 3 to VH fragment (known to be VH3 type heavy chain) and Fc fragment of Trastuzumab was detected by ELISA. The sequences of VH fragment and Fc fragment were from the heavy chain of Trastuzumab, wherein the sequence of VH fragment was as shown in SEQ ID NO. 12 (including the sequence of heavy chain variable region and heavy chain constant region CH1), and the sequence of Fc fragment was as shown in SEQ ID NO. 13. Both fragments were prepared by a biological company.

[0063] 1. Biotin labeling: biotin NHS ester was reacted with the control molecule and each VH3 binding molecule at a molar ratio of 20:1 at room temperature for 1-2 h, and then dialyzed to remove excess unreacted biotin NHS ester to obtain biotin-labeled molecules.

[0064] 2. ELISA detection: The VH fragment and the Fc fragment of trastuzumab were diluted to 2 μg / mL respectively, and 100 μL / well was added to each well of the enzyme-labeled plate, which was incubated at 4°C overnight for coating; the coating solution was discarded, and the enzyme-labeled plate was washed with 0.05% PBST, 100 μL of blocking solution was added to each well, which was incubated at 37°C for 2 h, and then washed; 100 nM of biotin-labeled molecules was used as a starting dose, which was diluted by 1 / 4 gradient, and 11 concentration gradients were set, 100 μL of which was added to each well, which was incubated at 37°C for 1 h, and then washed; 100 μL of avidin-labeled horseradish peroxidase was added to each well, which was reacted at 25°C for 30 min, and then washed; 50 μL of TMB color developing solution was added, which was colored at room temperature for 5 min in the dark, and then the stop solution was added, and the optical density value at 450 nm was read by an enzyme-labeled instrument.

[0065] According to the fitting of the concentration of the control molecule and each VH3 binding molecule with the measured OD450 value, the EC50 value of each molecule with the VH fragment and the Fc fragment of trastuzumab was calculated by fitting the curve, and the test results are shown in Tables 3 and 4, respectively.

[0066] From Table 3, it can be seen that the EC50 values of the control molecule and the VH3 binding molecules with different sequences with the VH fragment of trastuzumab are different, and based on the size of the EC50 value, the affinity of each molecule with the VH fragment of trastuzumab is evaluated. Among them, the control molecule has no binding capacity for the VH fragment of trastuzumab, and the size of the binding affinity of the remaining VH3 binding molecules with the VH fragment of trastuzumab is: VH3-6 > VH3-4 > VH3-3 > VH3-5 > VH3-11 > VH3-8 > VH3-7 > VH3-9 > VH3-10 > VH3-2.

[0067] From Table 4, it can be seen that the VH3-(7-11) binding molecules corresponding to SEQ ID NO. 7-SEQ ID NO. 11 have no binding capacity for the Fc fragment of trastuzumab, and the size of the binding affinity of each molecule with the Fc fragment of trastuzumab is: VH3-2 > VH3-3 > control molecule > VH3-4 > VH3-5 > VH3-6.

[0068] Table 3 Binding affinity of each molecule with the VH fragment of trastuzumab

[0069]

[0070] Table 4 Binding affinity of each molecule with the Fc fragment of trastuzumab

[0071]

[0072] Example 5 Test of purification effect of chromatography medium

[0073] Further verify the purification effect of the chromatography medium prepared by using the control molecule and 10 VH3 binding molecules as ligands respectively on the actual sample. The matrix microspheres used in the chromatography medium described in this embodiment are Focurose 6FF agarose microspheres (Suzhou Fudoo Biotechnology Co., Ltd.), and other types of matrix microspheres in the art can also be used.

[0074] 1. Preparation of chromatography medium

[0075] (1) Activation of matrix microspheres: 100 g of Focurose 6FF matrix microspheres was washed with deionized water and added to a 250 mL reaction bottle, 20 g of 50 wt% sodium hydroxide solution was added, stirred and mixed, then 0.1 g of sodium borohydride was added, stirred for 10 minutes, 30 g of epichlorohydrin was added, the temperature was raised to 35°C, and stirred for 4 h. Filtration, washing with deionized water, to obtain intermediate microspheres with a large number of epoxy groups on the surface.

[0076] (2) Ligand coupling: 100 g of activated matrix microspheres from step (1) was added to a 250 mL reaction bottle, 100 mL of 15 mg / mL ligand solution (control molecule or VH3 binding molecule) was added and stirred and mixed, the temperature was raised to 30-40°C, anhydrous sodium sulfate was added and stirred for 2-4 h, then filtered, and washed with phosphate or carbonate buffer.

[0077] (3) Blocking: 100 g of microspheres prepared in step (2) was added to a 250 mL reaction bottle, ethanolamine blocking solution was added and stirred and mixed, and after stirring at room temperature for 20-24 h, it was filtered; washed with an acidic solution and an alkaline solution, and washed with deionized water until neutral, to obtain a chromatography medium coupled with a ligand, wherein the acidic solution has a pH of 4.0 and contains 0.1 M sodium acetate and 0.5 M sodium chloride, and the alkaline solution has a pH of 8.5 and contains 0.1 M Tris and 0.5 M sodium chloride.

[0078] 2. Chromatography purification test

[0079] (1) Sample preparation

[0080] The Mab1 sample was a commercially available trastuzumab powder (complete antibody, containing Fc fragment and VH3 domain heavy chain), which was dissolved using PB buffer (pH 7.2, 20 mM PB, 120 mM NaCl) to make its concentration 3.5 mg / mL.

[0081] The Mab2 sample was a culture solution of rituximab expressed by CHO cells (complete antibody, containing Fc fragment and VH1 domain heavy chain, the sequence including the heavy chain shown in SEQ ID NO. 14 and the light chain shown in SEQ ID NO. 15), which was prepared by a biological company on commission, and the protein concentration was adjusted to 3.8 mg / mL using PB buffer.

[0082] (2) Buffer and column preparation

[0083] Equilibration buffer, elution buffer 1, and wash buffer: 20 mM PB, 120 mM NaCl, pH 7.2;

[0084] Elution buffer 2: 20 mM citric acid-sodium citrate, 1 M NaCl, pH 6.0;

[0085] Elution buffer 3: 20 mM citric acid-sodium citrate, pH 6.0;

[0086] Elution buffer: 20 mM citric acid-sodium citrate, pH 3.6;

[0087] Regeneration buffer: 200 mM citric acid;

[0088] Sanitization buffer: 0.5 M NaOH.

[0089] Column: 0.8 cm in diameter, 10 cm in height, 5 mL in column volume. The column specifications of the two types of chromatography media were kept the same.

[0090] (3) Chromatography process

[0091] The two samples were purified using the aforementioned chromatography media prepared with the control molecule and the VH3-5 binding molecule as ligands (named as chromatography medium 1 and chromatography medium 5, respectively), and the chromatography methods, samples, sample loadings, and buffers used in the two types of chromatography media were the same. Table 5 shows the specific purification conditions.

[0092] Table 5. Purification conditions

[0093]

[0094] The chromatograms of the purification of the two samples using the two types of chromatography media are shown in FIG. 1, and the results of the SDS-PAGE electrophoresis detection of the samples collected at different stages of purification are shown in FIG. 2. From the chromatograms and the electrophoresis results, the following conclusions can be drawn: Figures 10-13 Figure 14 Figures 10-14

[0095] a. When the Mab1 sample was purified, the chromatogram and the electrophoresis showed that there was no target antibody fragment in the flow-through of the two types of chromatography media, indicating that the two types of chromatography media could bind well to the Mab1 sample. The use of a solution of 20 mM citric acid-sodium citrate, pH 3.6, could cause the dissociation of the Mab1 sample; the chromatography medium 5 had a relatively long tail during the elution and dissociation process, and the peak area was larger than that of the chromatography medium 1 when using 200 mM citric acid for regeneration, indicating that the binding force of the chromatography medium 5 to the Mab1 sample was stronger.

[0096] ​​​b. When the Mab2 sample was purified, the chromatogram and electrophoresis of the chromatography medium 1 showed no flow-through of the target antibody fragment ( Figure 12 The first broad peak in the chromatogram represents an impurity peak from the cell culture fluid, indicating that Resin 1 binds well to the Mab2 sample. A 20mM citric acid-sodium citrate solution, pH 3.6, dissociates the Mab2 sample. When testing Resin 5, the UV value of the flowthrough sample in the chromatogram of Resin 5 was significantly higher than that of Resin 1, indicating that more material had flowed through. The electropherogram revealed that the load flowthrough contained a large amount of target antibody fragments, while no distinct peaks were observed during elution and regeneration, indicating that Resin 5 did not effectively bind to the Mab2 sample.

[0097] Through the above chromatography purification tests, it can be concluded that chromatography media 1 and 5 have good binding and desorption capabilities for Mab1 samples and can be used for the purification of Mab1 samples; chromatography media 1 can bind Mab2 samples well, while chromatography media 5 cannot bind Mab2 samples well.

[0098] Using the same chromatography purification test method, the purification efficacy of chromatographic media prepared with ligands containing VH3 binding molecules other than the control molecule and VH3-5 was further verified for Mab1 and Mab2 samples. The test results showed that the chromatographic media corresponding to the binding molecules VH3-6, 7, 8, 9, and 11 exhibited comparable binding to the target proteins of Mab1 and Mab2 samples as chromatographic media 5. However, the binding of the other VH3 binding molecules to the two samples failed to achieve the expected results.

[0099] 3. Purity testing

[0100] Mab3 sample preparation: Mab3 sample consisted of trastuzumab culture medium expressed in CHO cells (intact antibody, comprising the Fc fragment and VH3 heavy chain domain, with the heavy chain sequence represented by SEQ ID NO. 16 and the light chain represented by SEQ ID NO. 17). This was prepared by a biotechnology company and the protein concentration was adjusted to 3.8 mg / mL using PB buffer. Prior to purification, both samples were adjusted to pH 6.0 using 1M Tris and then filtered through 0.22μm for later use.

[0101] Using the same chromatography purification test method as above, Mab3 samples were purified using the chromatography media corresponding to VH3-5, 6, 7, 8, 9, and 11 (three parallel tests were set up for each chromatography medium). The average purity of the target molecules measured is shown in Table 6. It can be concluded from Table 6 that the purity of the target molecules was above 95%.

[0102] Table 6 Purity test results of target molecules

[0103]

[0104] Further, the sequences of SEQ ID NO. 5, 6, 7, 8, 9, 11 are designed into trimer and hexamer protein molecules respectively, and the purification effect on Mab1 sample and Mab2 sample is further tested. From the test results, the trimer and hexamer protein molecules can still bind to Mab1 sample and not bind to Mab2 sample, therefore, the trimer and hexamer protein molecules corresponding to these polypeptide units can still be used for the purification of antibodies containing VH3 domain, and the pentamer protein molecules are also applicable by analogy.

[0105] In summary, the comparative experiments of different antibody samples purified by using the control molecules and different VH3 binding molecules show that the chromatography medium 5-9 and the chromatography medium 11 can effectively and specifically recognize the VH3 type heavy chain of trastuzumab, and do not recognize the Fc fragment of the antibody and the VH1 type heavy chain, and the purity of the target molecule after purification reaches more than 95%, which can meet the purity requirements. Therefore, the polypeptide units SEQ ID NO. 5, 6, 7, 8, 9, 11 provide a new solution for the purification of antibody fragments with the same or higher homology (such as nanobodies) as the VH3 type heavy chain of trastuzumab.

[0106] Example 6 and Example 7 further test the load and alkali resistance of the chromatography medium in Table 6.

[0107] Example 6 Load of chromatography medium

[0108] According to the 10% DBC (i.e. the load when the concentration of the target product in the sample flow reaches 10% of the initial target product concentration) method, the load test is carried out (3 parallel tests are set for each chromatography medium).

[0109] The test procedure is as follows: the VH fragment of trastuzumab in Example 4 is dissolved in 50mM PB, pH7.0 to make its concentration 3.0mg / mL, and then filtered with 0.22μm as the sample for load test. A 5mL chromatography column (diameter 0.8cm, height 10cm) is used, the retention time is 5min, 50mM PB, pH7.0 is used to equilibrate the chromatography column, after equilibration, the sample is loaded, and the load test is carried out according to the 10% DBC method.

[0110] Determination of 10% DBC end point: according to the test of 5min retention time, continue to load, and start collecting a fixed volume when the UV starts to rise, collect one tube every 0.5CV or 1CV. Determine the concentration corresponding to each tube, determine the collection position when the concentration reaches 0.3mg / mL, and calculate the total loading volume V according to the chromatogram. 上样 .

[0111] Calculated loading:

[0112] Loading (10% DBC) = C 初始 * (V 上样 - V 滞留体积 - 1 / 3 V 柱体积 ) / V 柱体积 ,

[0113] Where: C 初始 is the initial concentration of the sample loaded,

[0114] V 上样 is the volume of sample loaded at which the concentration of the sample in the elution profile reaches 10% of the initial concentration,

[0115] V 滞留体积 is the volume of sample retained by the chromatography system (other than the column),

[0116] V 柱体积 is the volume of the column.

[0117] The average values of the loadings tested are shown in Table 7, from which it can be concluded that the loadings meet the requirements for the loadings of the chromatography media.

[0118] Table 7 Loadings

[0119]

[0120] Example 7 Alkali resistance of the chromatography media

[0121] The loadings tested in Example 6 were taken as the initial loadings at 0h of soaking; then the alkali resistance performance test was started after the elution-regeneration-equilibrium steps. The chromatography instrument was used to fill 5 mL chromatography columns (3 parallel tests for each chromatography medium) with 0.5M NaOH, then the machine was stopped, and the NaOH solution in the chromatography media was completely replaced with the equilibration solution 50mM PB, pH7.0 after different soaking times (15.5h, 23.5h, 39h, 55h) respectively, and the average loading of the chromatography media was tested after each alkali soaking, and the alkali resistance performance of the chromatography media was determined by the changes in the loadings corresponding to different soaking times.

[0122] From the alkali resistance test results in Table 8 (the unit of the loadings in Table 8 is: mg VH fragment / mL chromatography medium), the change in the loading was within 10% during the soaking time of 0.5M NaOH, 55h, which represented good alkali resistance performance.

[0123] Table 8 Alkali resistance test results

[0124]

[0125] The above embodiments only express several implementation manners of the present application, and the description is relatively specific and detailed, but it should not be understood as a limitation on the patent scope of the application. It should be pointed out that for ordinary skilled persons in the art, several modifications and improvements can be made without departing from the concept of the present application, which are all within the protection scope of the present application. Therefore, the protection scope of the patent of the present application should be subject to the appended claims, and the description and drawings can be used to explain the content of the claims.

Claims

1. A VH3 binding molecule, characterized in that The VH3 binding molecule comprises a polypeptide unit, the amino acid sequence of which is selected from one or more of SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9 or SEQ ID NO.11; when the VH3 binding molecule comprises no less than two polypeptide units, the amino acid sequences of the multiple polypeptide units are the same.

2. A VH3 binding molecule according to claim 1, characterized in that The VH3 binding molecule further comprises a first amino acid methionine at the N-terminus and an amino acid cysteine ​​at the C-terminus.

3. A VH3 binding molecule according to claim 1 or 2, characterized in that The VH3 binding molecule comprises a multimer consisting of 3-6 polypeptide units.

4. A nucleic acid molecule, characterized in that The nucleic acid molecule comprises a nucleotide sequence encoding the VH3 binding molecule according to any one of claims 1 to 3.

5. An expression vector, characterized in that The expression vector comprises the nucleic acid molecule of claim 4.

6. A chromatography medium, characterized in that The chromatography medium uses the VH3 binding molecule according to claim 3 as a ligand.

7. Use of the chromatography medium according to claim 6 in separating and purifying antibodies comprising a VH3 heavy chain.

8. A method for separating and purifying an antibody comprising a VH3 heavy chain, characterized in that: The method comprises: contacting the sample to be purified with the chromatographic medium according to claim 6, and collecting the purified target substance after elution and impurity removal.

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