High-mannose antibody-specific affinity material, preparation method therefor and use thereof

Abstract

A high-mannose antibody-specific affinity material, a preparation method therefor and a use thereof. Provided are a lectin variant comprising (a) a histidine tag and (b) a cysteine-containing segment, added to the C-terminus of a reference lectin; a nucleic acid molecule encoding the lectin variant, and a vector and a cell containing the nucleic acid molecule; a composition and a matrix comprising the lectin variant; and a use of the lectin variant for reducing or removing a high-mannose antibody from a sample. The lectin variant has a specific affinity for high-mannose antibodies, and can effectively bind to high-mannose antibodies formed during antibody production, to remove high-mannose byproducts contained in recombinant antibodies, thereby effectively promoting and achieving required antibody purification in downstream stages of production.

Description

High mannose antibody-specific affinity material and its preparation method and application Technical Field

[0001] The present invention generally relates to materials for antibody purification and methods for preparing the same; in particular, to high-mannose antibody-specific affinity materials for antibody purification and methods for preparing and applying the same. Background Art

[0002] Antibody glycosylation is a complex post-translational modification that has a significant impact on the efficacy, stability, immunogenicity, and pharmacokinetic properties of antibody drugs. The glycosylation form of therapeutic antibodies is mainly N-glycosylation modification of the Fc fragment.

[0003] N-glycosylation of antibody Fc fragments mainly includes three types: high mannose type, hybrid type, and sialic acid complex glycotype. Among them, the type with 5 to 9 mannose groups (Man5-Man9) remaining due to incomplete terminal mannose cleavage during the N-glycosylation process is called high mannose type (HM). Among monoclonal antibodies expressed in mammalian CHO cell culture, high mannose type antibodies account for approximately 1%-20%, which is much higher than the high mannose type antibody level of less than 0.1% in human serum.

[0004] High-mannose antibodies have been reported to bind with high affinity to the FcγRIIIa receptor, leading to enhanced ADCC effects. In some treatments requiring only antibody antagonist function, high-mannose modifications can potentially lead to unintended, high ADCC effects. Furthermore, high-mannose antibodies have a reduced affinity for complement protein C1q, leading to reduced CDC effects. Due to the presence of receptors for high-mannose antibodies in the liver, high-mannose antibodies are rapidly metabolized in the body and have a short half-life, potentially reducing their actual therapeutic efficacy. Due to the presence of high-mannose receptors in the body, high-mannose antibodies have the potential for off-target toxicity in ADC drug therapy. Furthermore, high mannose is highly immunogenic. Given these factors, controlling the levels of high-mannose antibodies in therapeutic antibody production is crucial.

[0005] Mannose modification levels vary significantly between different hosts. Even expression from different batches of the same cell line can exhibit variability. Currently, regulation and control of high-mannose antibody levels are typically performed during the upstream expression stage, and effective methods for reducing high-mannose antibodies during downstream purification are lacking.

[0006] Currently, only ConA fillers from different manufacturers on the market have the function of capturing high-mannose antibodies. Because ConA captures high-mannose antibodies by binding to mannose monomers, in addition to high-mannose antibodies, ConA can also bind to other glycosylated antibodies (such as G0F-GN and G1F-GN glycoforms). Therefore, ConA cannot provide specific affinity for high-mannose antibodies. In addition, the ConA protein exists in the form of a tetramer with a large molecular weight, which is not easy to recombinantly express and purify in prokaryotic cells. In addition, the binding to mannose requires the participation of divalent metal ions, which also limits its application in antibody production.

[0007] Therefore, there remains a need for materials and methods for effectively reducing high mannose antibodies in antibody purification. Summary of the Invention

[0008] After extensive research, the inventors developed a lectin variant that effectively reduces high-mannose antibodies. This protein has a specific affinity for high-mannose antibodies and can effectively bind to high-mannose antibodies formed during antibody production. This allows for the removal of high-mannose byproducts mixed with recombinant antibodies, effectively facilitating and achieving the desired antibody purification in downstream production stages.

[0009] In one aspect, provided herein is a lectin variant comprising (a) a histidine tag and (b) a cysteine-containing segment added to the C-terminus of a reference lectin.

[0010] In another aspect, provided herein is a composition comprising a lectin variant described herein.

[0011] In another aspect, provided herein is a nucleic acid molecule encoding a lectin variant described herein.

[0012] In another aspect, provided herein is a vector comprising a nucleic acid molecule encoding a lectin variant described herein.

[0013] In another aspect, provided herein is a cell comprising a nucleic acid molecule or vector described herein.

[0014] In another aspect, provided herein is a matrix for affinity purification, which is loaded with the lectin variant described herein.

[0015] In another aspect, provided herein is a method for preparing a matrix for affinity purification, comprising:

[0016] (1) providing a lectin variant as described herein;

[0017] (2) mixing the lectin variant with a filler to couple the lectin variant to the filler.

[0018] In another aspect, provided herein is a use of the lectin variant described herein for reducing or removing high mannose antibodies in a sample. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The present invention will be further described below with reference to the accompanying drawings, wherein these drawings are only for illustrating the embodiments of the present invention and are not intended to limit the scope of the present invention.

[0020] Figure 1A: Cationic SP HP purification chromatogram.

[0021] Figure 1B: Cationic SP HP purification SDS-PAGE. M: Marker, L: Loading; FT: Flow through; GE: Gradient elution.

[0022] Figure 2: LC / MS mass spectrometry identification results of lectin ESA-2v.

[0023] Figure 3A: Reduced mass spectrum of adalimumab produced by adding kifunensine. 51059.8 is the molecular weight of Man9 modification of the Fc heavy chain.

[0024] Figure 3B: Reduced mass spectrum of adalimumab produced by the normal process. 50638.1 is the molecular weight of the G0F-modified Fc heavy chain.

[0025] FIG4A : Schematic diagram of pull-down of lectin ESA-2v binding to adalimumab.

[0026] Figure 4B: Chromatograms of high-mannose adalimumab binding to conjugated and unconjugated ESA-2v lectin nickel columns. Yellow (lighter) represents the elution of high-mannose adalimumab bound to the nickel column conjugated to ESA-2v; blue (darker) represents the elution of high-mannose adalimumab bound to the nickel column unconjugated to ESA-2v.

[0027] FIG4C : SDS-PAGE images of the binding of high-mannose adalimumab to unconjugated (left panel) and conjugated (right panel) lectin ESA-2v nickel columns.

[0028] Figure 4D: Chromatograms of normal glycoform adalimumab binding to conjugated and unconjugated ESA-2v lectin nickel columns. Purple (dark) represents the elution of normal glycoform adalimumab bound to the nickel column with conjugated ESA-2v lectin; green (light) represents the elution of normal glycoform adalimumab bound to the nickel column with unconjugated ESA-2v lectin.

[0029] FIG4E : SDS-PAGE images of normal glycoform adalimumab bound to unconjugated (left panel) and conjugated (right panel) lectin ESA-2v nickel column.

[0030] Figure 4F: Identification of a small amount of normally expressed adalimumab bound by reduced mass spectrometry (sample in lane 8 of Figure 4E). 50411.8 is the molecular weight of the Man5 modification of the Fc heavy chain.

[0031] Figure 5A: Blank control group experiment, binding and dissociation diagrams of the blank sensor and two groups of adalimumab antibodies.

[0032] Figure 5B: Experimental group, binding and dissociation plots of the coupled lectin ESA-2v sensor and two groups of adalimumab antibodies. Yellow (light) represents the dissociation interference spectral shift curves of the binding of a series of high-mannose adalimumab to the lectin ESA-2v sensor, and purple (dark) represents the dissociation interference spectral shift curves of the binding of a series of normal glycosylated adalimumab to the lectin ESA-2v sensor.

[0033] Figure 6A: Reduced mass spectra of two sets of adalimumab antibodies. The left panel shows the antibody expressed using the standard process, with the molecular weight of the G0F-modified Fc heavy chain at 50638. The right panel shows the antibody expressed using the kifnobase-added process, with the molecular weight of the Man9-modified Fc heavy chain at 51058.3.

[0034] Figure 6B: Reduced mass spectra of two sets of trastuzumab antibodies. The left panel shows the antibody expressed using the standard process, with the molecular weight of the G0F-modified Fc heavy chain at 50595.2. The right panel shows the antibody expressed using the kifnobase-added process, with the molecular weight of the Man9-modified Fc heavy chain at 51015.5.

[0035] Figure 6C: Reduced mass spectra of two sets of rituximab antibodies. The left panel shows the antibody expressed using the standard process, with the molecular weight of the G0F-modified Fc heavy chain at 50508.5; the right panel shows the antibody expressed using the kifnobase-added process, with the molecular weight of the Man9-modified Fc heavy chain at 50928.9.

[0036] Figure 6D: Reduced mass spectra of two groups of denosumab antibodies. The left panel shows the antibody expressed using the standard process, with the molecular weight of the G0F-modified Fc heavy chain at 50421.8; the right panel shows the antibody expressed using the kifnobase-added process, with the molecular weight of the Man9-modified Fc heavy chain at 50842.2.

[0037] Figure 6E: Reduced mass spectra of two groups of pembrolizumab antibodies. The left panel shows the antibody expressed using the standard process, with the molecular weight of the G0F-modified Fc heavy chain at 50709.3. The right panel shows the antibody expressed using the kifnobase-added process, with the molecular weight of the Man9-modified Fc heavy chain at 51129.6.

[0038] Figure 7A: Schematic diagram of IgG antibody binding to lectin ESA-2v affinity medium

[0039] Figure 7B: Chromatograms of two groups of adalimumab antibodies bound to the ESA-2v lectin column. Blue (dark) represents the high-mannose form of the antibody; yellow (light) represents the normal process glycoform of the antibody.

[0040] Figure 7C: Chromatograms of two groups of trastuzumab antibodies bound to the ESA-2v column. Blue (dark) represents high-mannose antibodies; yellow (light) represents normal process glycoform antibodies.

[0041] Figure 7D: Chromatograms of two groups of rituximab antibodies bound to the ESA-2v lectin column. Blue (dark) represents high-mannose antibodies; yellow (light) represents normal process glycoform antibodies.

[0042] Figure 7E: Chromatograms of two groups of denosumab antibodies bound to the ESA-2v lectin column. Blue (dark) represents high-mannose antibodies; yellow (light) represents normal process glycoform antibodies.

[0043] Figure 7F: Chromatograms of two groups of pembrolizumab antibodies bound to the ESA-2v lectin column. Blue (dark) represents the high-mannose form of the antibody; yellow (light) represents the normal process glycoform of the antibody.

[0044] Figure 7G: SDS-PAGE of five high-mannose IgG antibodies binding to the ESA-2v lectin column and their elution. M: Marker, L: Loading, FT: Flow through, E: Elution.

[0045] Figure 7H: SDS-PAGE of five normal glycoform IgG antibodies binding to an ESA-2v lectin column and their elution. M: Marker, L: Loading, FT: Flow through, E: Elution. 20X concentration indicates 20-fold concentration of the eluted sample.

[0046] Figure 8: Reduction mass spectrometry results of affinity elution samples of antibodies of five IgG subtypes expressed using the normal process and lectin ESA-2v. DETAILED DESCRIPTION

[0047] The meaning of scientific and technological terms in this application is consistent with the general understanding of those skilled in the art, unless otherwise specified. In this application, "one" or its combination with various quantifiers includes both singular and plural meanings, unless otherwise specified. In this application, for the same parameter or variable, when multiple numerical values, numerical ranges, or combinations thereof are given for description, it is equivalent to specifically revealing these numerical values, range end values, and numerical ranges formed by any combination thereof. In this application, any numerical value, whether or not it is accompanied by a modifier such as "about", covers an approximate range that can be understood by those skilled in the art, such as plus or minus 10%, 5%, etc. In this article, each "implementation method" refers equally to and covers the implementation methods of various methods and systems of this application. In this application, one or more technical features in any implementation method can be freely combined with one or more technical features in any one or more other implementation methods, and the implementation methods obtained thereby also belong to the content disclosed in this application.

[0048] Lectins are sugar-binding proteins or glycoproteins of non-immune origin that have the ability to cause cell agglutination or precipitate glycoconjugates. Lectins are widely found in animals and plants. Their most significant characteristic is their ability to recognize complex carbohydrate structures in glycoproteins and glycolipids, particularly those found in cell membranes, namely the sugar groups on the cell membrane surface.

[0049] After extensive research, the inventors developed a lectin variant that effectively reduces high-mannose antibodies. Specifically, this lectin variant is optimized based on known lectin proteins to facilitate filler coupling and affinity binding to high-mannose antibodies.

[0050] In one aspect, provided herein is a lectin variant comprising (a) a histidine tag and (b) a cysteine-containing segment added to the C-terminus of a reference lectin.

[0051] The lectin variants described herein are optimized based on a reference lectin. Compared to the reference lectin, the optimized lectin variants have higher binding specificity for high-mannose antibodies and are more conducive to the purification and preparation of high-mannose antibody binding matrices.

[0052] As used herein, the term "reference lectin" generally refers to a lectin that is naturally occurring or known in the art.

[0053] In some embodiments, the reference lectin is selected from the group consisting of plant lectins, animal lectins, and microbial lectins. In some embodiments, the reference lectin is selected from eukaryotic lectins. In some embodiments, the reference lectin is selected from plant lectins. In some embodiments, the reference lectin is a lectin from red algae (Rhodophyta). In some embodiments, the reference lectin is a lectin from the Gigartinaceae. In some embodiments, the reference lectin is a lectin from the Solieriaceae. In some embodiments, the reference lectin is a lectin from the genus Eucheuma. In some embodiments, the reference lectin is a lectin from Eucheuma serra. In some embodiments, the reference lectin is a mannose-binding lectin. In a specific embodiment, the reference lectin is ESA-2 protein (Uniprot P84331).

[0054] In an exemplary embodiment, the reference lectin has the amino acid sequence shown in SEQ ID NO: 1:

[0055] Amino acid sequence of an exemplary reference lectin (SEQ ID NO: 1):

[0056] As used herein, the term "lectin variant" refers to a polypeptide that is substantially homologous to a reference (eg, native) lectin but differs from the reference (eg, native) lectin amino acid sequence by one or more deletions, insertions, or substitutions.

[0057] In some embodiments, the lectin variant has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity to the reference lectin.

[0058] In some embodiments, the lectin variant differs from the reference lectin by at most 30, at most 28, at most 26, at most 24, at most 22, at most 20, at most 18, at most 16, or at most 14 amino acid residues. In some embodiments, the lectin variant differs from the reference lectin by at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, or at least 12 amino acid residues. In some embodiments, the lectin variant comprises the amino acid sequence of the reference lectin. In some embodiments, the lectin variant comprises the complete amino acid sequence of the reference lectin, i.e., the lectin variant does not contain additions, substitutions, or deletions of amino acid residues within the amino acid sequence of the reference lectin (e.g., the amino acid sequence set forth in SEQ ID NO: 1).

[0059] In some embodiments, the lectin variant comprises at most 6, at most 5, at most 4, at most 3, at most 2, or at most 1 additional amino acid residues at the N-terminus of the reference lectin. In some embodiments, the lectin variant comprises 1 to 6, 2 to 6, 3 to 6, 1 to 4, 2 to 4, 3 to 4, 1 to 3, or 1 to 2 additional amino acid residues at the N-terminus of the reference lectin. In some embodiments, the lectin variant comprises at most 30, e.g., at most 28, at most 26, at most 24, at most 22, at most 20, at most 18, at most 16, or at most 14 additional amino acid residues at the C-terminus of the reference lectin. In some embodiments, the lectin variant comprises at most 6, at most 5, at most 4, at most 3, at most 2, or at most 1 additional neutral amino acid residues at the N-terminus of the reference lectin. In some embodiments, the lectin variant comprises at most 30, e.g., at most 28, at most 26, at most 24, at most 22, at most 20, at most 18, at most 16, or at most 14 additional neutral amino acid residues at the C-terminus of the reference lectin. In some embodiments, the neutral amino acid residue comprises methionine.

[0060] In some embodiments, the lectin variant comprises 7 to 30, 7 to 28, 7 to 26, 7 to 22, 7 to 20, 7 to 18, 7 to 16, 7 to 14, 8 to 30, 8 to 28, 8 to 26, 8 to 22, 8 to 20, 8 to 18, 8 to 16, 8 to 14, 9 to 30, 9 to 28, 9 to 26, 9 to 22, 9 to 20, 9 to 18, 9 to 16, 9 to 14 added amino acid residues at the C-terminus of the reference lectin.

[0061] In some embodiments, the lectin variant comprises the added amino acid residues at the C-terminus of the reference lectin comprising the (a) histidine tag and (b) cysteine-containing segment.

[0062] The lectin variants described herein comprise: (a) a histidine tag added to the C-terminus of a reference lectin.

[0063] In some embodiments, the (a) histidine tag is located at the N-terminus of the (b) cysteine-containing segment.

[0064] In some embodiments, the histidine tag is a polyhistidine tag. In some embodiments, the histidine tag comprises 4 to 20, 6 to 20, 6 to 18, 6 to 16, 6 to 14, 6 to 12, or 6 to 10 consecutive histidine residues. Preferably, the histidine tag comprises 2-20, 4-18, 6-16, 6-14, or 6-10 consecutive histidine residues. In some specific embodiments, the histidine tag comprises 6 to 10 consecutive histidine residues.

[0065] In some embodiments, the histidine tag can be linked to the C-terminus of the reference lectin via one or more linker regions. In some embodiments, the linker region linking the histidine tag to the C-terminus of the reference lectin comprises 1 to 15 amino acid residues, e.g., 1 to 15, 1 to 14, 1 to 13, 1 to 12, 1 to 11, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2 amino acid residues. In some exemplary embodiments, the linker region comprises: GGGGS, GGGS, GGS, GS, or any combination thereof. In some exemplary embodiments, the linker region comprises: GGGGS, GS, or any combination thereof.

[0066] The lectin variants described herein comprise: (b) a cysteine-containing segment added to the C-terminus of a reference lectin.

[0067] In some embodiments, the cysteine-containing segment comprises 1 to 16, 1 to 14, 1 to 12, 1 to 10, 1 to 8, 1 to 6, 1 to 5, or 1 to 4 amino acid residues. In some embodiments, the cysteine-containing segment comprises at most 5, at most 4, at most 3, at most 2, or 1 cysteine ​​residue. In some embodiments, the C-terminus of the cysteine-containing segment is a cysteine ​​residue.

[0068] In some embodiments, the cysteine-containing segment can be connected to the C-terminus of the histidine tag via one or more connecting regions. In some embodiments, the connecting region comprises 1 to 15 amino acid residues. In some embodiments, the total length of the one or more connecting regions connecting the cysteine-containing segment to the histidine tag can be at most 10, at most 9, at most 8, at most 7, at most 6, at most 5, at most 4, at most 3, at most 2, at most 1 amino acid residue. In some embodiments, the connecting region comprises GGGGS, GGGS, GGS, GS, or any combination thereof. In some embodiments, the connecting region comprises 1-3 amino acid residues. In some exemplary embodiments, the one or more connecting regions connecting the cysteine-containing segment to the histidine tag have 1 amino acid residue (e.g., G).

[0069] In some embodiments, any two adjacent cysteine ​​residues in the cysteine-containing segment are separated from each other by at most 2, at most 1, or 0 amino acid residues other than cysteine ​​and histidine. In some embodiments, any two adjacent cysteine ​​residues in the cysteine-containing segment are separated from each other by 0, 1, or 2 amino acid residues other than cysteine ​​and histidine.

[0070] In an exemplary embodiment, the lectin variant has the amino acid sequence shown in SEQ ID NO: 2.

[0071] Amino acid sequence of an exemplary lectin variant (SEQ ID NO: 2):

[0072] In some embodiments, the lectin variant has at least 80% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 2, for example, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity.

[0073] In some embodiments, the lectin variant differs from the amino acid sequence of SEQ ID NO: 2 by no more than 10, no more than 9, no more than 8, no more than 7, no more than 6, no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 amino acid residue.

[0074] In another aspect, provided herein is a composition comprising a lectin variant described herein.

[0075] In another aspect, provided herein is a nucleic acid molecule encoding a lectin variant described herein.

[0076] In another aspect, provided herein is a vector comprising a nucleic acid molecule described herein.

[0077] In some embodiments, the vector is a recombinant vector.

[0078] In some embodiments, the vector is selected from a prokaryotic expression plasmid vector and a eukaryotic expression plasmid vector. In some embodiments, the vector is a prokaryotic expression plasmid vector. In some exemplary embodiments, the prokaryotic expression plasmid vector may include pET28a(+), pET23a, pET30a, pColdII, pMAL-c2X, pGEX, etc.

[0079] In some embodiments, the vector comprises a nucleic acid molecule encoding a lectin variant described herein and a regulatory sequence operably linked to the nucleic acid molecule for expression thereof.

[0080] In some exemplary embodiments, the vector can be produced by incorporating or linking a nucleic acid molecule encoding the lectin variant described herein into the vector.

[0081] In another aspect, provided herein is a cell comprising a nucleic acid molecule or vector described herein.

[0082] In some embodiments, the cell is selected from a prokaryotic cell and a eukaryotic cell. In some embodiments, the cell is not a plant cell. In some embodiments, the cell is a prokaryotic cell. In some exemplary embodiments, the cell is an Escherichia coli cell.

[0083] In some exemplary embodiments, the cell can be produced by introducing a nucleic acid molecule encoding a lectin variant described herein into a cell. In some exemplary embodiments, the cell can be produced by incorporating or linking a nucleic acid molecule encoding a lectin variant described herein into a vector and transforming the vector into a cell.

[0084] In another aspect, provided herein is a matrix for affinity purification, which is loaded with the lectin variant described herein.

[0085] In some embodiments, the matrix comprises a filler and a lectin variant described herein.

[0086] In some embodiments, the filler is a chromatography filler. In some embodiments, the filler is an agarose-based resin filler. In some exemplary embodiments, the filler includes SulfoLink coupling resin, Sepharose-based resin, and Superflow resin.

[0087] In some embodiments, the matrix is ​​used to specifically bind to high mannose antibodies contained in a sample, thereby separating them from the sample. In some embodiments, the matrix is ​​used to reduce or remove high mannose antibodies contained in a sample.

[0088] In some exemplary embodiments, the high mannose antibodies may include, but are not limited to, high mannose IgG antibodies (eg, IgG1, IgG2, and IgG4 subtypes), high mannose Fc fusion proteins, and the like.

[0089] In another aspect, provided herein is a method for preparing a matrix for affinity purification, comprising:

[0090] (1) providing a lectin variant as described herein;

[0091] (2) mixing the lectin variant with a filler to couple the lectin variant to the filler.

[0092] In some embodiments, the filler is a chromatography filler. In some embodiments, the filler is an agarose-based resin filler. In some exemplary embodiments, the filler includes SulfoLink coupling resin, Sepharose-based resin, and Superflow resin. In some embodiments, the filler is nickel-chelated.

[0093] In some embodiments, the lectin variant in step (1) is purified. In some exemplary embodiments, the lectin variant is purified by nickel column, molecular sieve and ion exchange chromatography.

[0094] In some embodiments, before step (2), the method further comprises:

[0095] exchanging the lectin variant into a conjugate binding buffer; and

[0096] The medium was equilibrated with coupling binding buffer.

[0097] In some exemplary embodiments, the conjugate binding buffer comprises 50 mM Tris, 5 mM EDTA, pH 8.5.

[0098] In some embodiments, the mixing of step (2) is performed at room temperature (eg, 20° C.-25° C.).

[0099] In some embodiments, step (2) comprises: thoroughly mixing the lectin variant and the filler, and incubating at room temperature (e.g., incubating for 20 to 60 minutes, such as 30 to 60 minutes, 30 to 40 minutes, or 30 minutes), and then removing the supernatant.

[0100] In some embodiments, after step (2), the method further comprises:

[0101] Wash the packing with 1 M NaCl buffer;

[0102] Adding blocking buffer to the medium, mixing thoroughly and incubating at room temperature (e.g., incubating for 20 to 60 minutes, such as 30 to 60 minutes, 30 to 40 minutes, or 30 minutes), and then removing the supernatant; and

[0103] The medium was equilibrated with coupling binding buffer.

[0104] In some exemplary embodiments, the blocking buffer comprises 25 mM Tris, 150 mM NaCl, 5 mM EDTA, 50 mM cysteine, pH 8.5.

[0105] In an exemplary embodiment, the method for preparing a matrix for affinity purification comprises:

[0106] Step 1: Recombinant expression of lectin variants in E. coli.

[0107] Step 2: Purify the lectin variant protein using nickel column, molecular sieve and ion exchange chromatography to obtain high-purity lectin variant protein.

[0108] Step 3: Lectin variants were exchanged into the coupling binding buffer 50 mM Tris, 5 mM EDTA, pH 8.5.

[0109] Step 4: Equilibrate SulfoLink coupling resin (Thermo 20404) using coupling binding buffer 50 mM Tris, 5 mM EDTA, pH 8.5.

[0110] Step 5: Mix the lectin variant and SulfoLink coupled resin thoroughly and incubate for 30 minutes. Remove the supernatant.

[0111] Step 6: Rinse the SulfoLink coupling resin with 1 M NaCl buffer.

[0112] Step 7: Add blocking buffer (25 mM Tris, 150 mM NaCl, 5 mM EDTA, 50 mM cysteine, pH 8.5) to the SulfoLink coupled resin. Mix thoroughly and incubate for 30 minutes. Remove the supernatant.

[0113] Step 8: Equilibrate SulfoLink coupling resin with binding buffer 50 mM Tris, 5 mM EDTA, pH 8.5.

[0114] In some embodiments, the matrix is ​​used to specifically bind to high mannose antibodies contained in a sample, thereby separating them from the sample. In some embodiments, the matrix is ​​used to reduce or remove high mannose antibodies in a sample.

[0115] In some exemplary embodiments, the high mannose antibodies may include, but are not limited to, high mannose IgG antibodies (eg, IgG1, IgG2, and IgG4 subtypes), high mannose Fc fusion proteins, and the like.

[0116] In another aspect, provided herein is a use of the lectin variant described herein for reducing or removing high mannose antibodies in a sample.

[0117] Example

[0118] The present invention will be described in further detail below with reference to the embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0119] Example 1: Design and Generation of Exemplary Lectin Variants

[0120] Based on the gene sequence of the lectin protein ESA-2 (Uniprot P84331), the target gene was optimized by comprehensively considering expression, purification, and matrix coupling. The target gene was synthesized and inserted into the pET28a(+) (Novagen) expression vector. The amino acid sequence of the protein encoded by the optimized target gene (lectin ESA-2v) is shown in SEQ ID NO: 2.

[0121] The pET28a(+) plasmid containing the target gene was transformed into Escherichia coli BL21 (DE3) competent cells. The transformed competent cells were transferred to LB solid medium containing kanamycin and cultured at 37 degrees Celsius with the plate inverted overnight. Single colonies were picked from the culture plate, and fresh LB liquid medium containing kanamycin was added. The cells were cultured at 30 degrees Celsius and 200 rpm to prepare overnight seeds. On the third day, the seed solution was transferred to fresh LB liquid medium containing antibiotics at a 1% inoculum volume and cultured at 37 degrees Celsius and 200 rpm. When the OD600 of the bacterial solution was in the range of 0.6-0.8, 0.2 mM IPTG was added, the temperature was lowered to 18 degrees Celsius, and the culture was continued at 200 rpm overnight. On the fourth day, the cells were collected by centrifugation at 8000g for 15 minutes.

[0122] The cells were resuspended in equilibration buffer (25 mM Tris-HCl, pH 8.0, 200 mM NaCl, 10 mM imidazole, 0.1 mM PMSF, 0.5 mM TCEP) at a ratio of 1:5 (W / V). The resuspended solution was broken by a high-pressure homogenizer at 1000 bar, with three cycles. The supernatant was collected by centrifugation at 30,000 g for 30 minutes. Before chromatographic purification, the supernatant was filtered through a 0.22 μm filter membrane. An AKTA pure chromatography instrument was used to purify the clarified sample. The clarified sample was loaded onto a pre-equilibrated 5 mL Histrap HP chromatography column via a sample pump. A bubble sensor was used to detect complete sample loading. Upon detection of bubbles, the sample buffer line equilibrated the column until the UV280 baseline was completely stable. The target protein was eluted from the nickel column using elution buffer (25mM Tris-HCl, pH 8.0, 200mM NaCl, 250mM imidazole, 0.5mM TCEP). The eluted high-concentration sample was stored in the Loop ring, and then the sample was immediately transferred from the Loop ring to another column valve SEC (75pg, 120mL) column using SEC buffer and continued to elute for one column volume. Based on the purity of the protein, the target protein was further purified using a cation exchange chromatography column SP HP, and the target component was eluted using a salt ion concentration gradient (30mM-1000mM). SDS-PAGE showed that the peak 1 protein of SP HP had a higher purity, and peak 2 contained a small amount of low molecular weight impurities. The peak 1 component was merged as the final target protein (as shown in Figure 1A-Figure 1B). LC / MS was used to identify the molecular weight of the final protein. After calculation, the experimental molecular weight obtained by LC / MS and the theoretical molecular weight (theoretical molecular weight is 29249.78Da) completely matched (as shown in Figure 2).

[0123] Example 2: Exemplary Experimental Monoclonal Antibody Preparation

[0124] Two adalimumab production runs were produced in parallel using the WuXi Transient expression platform. One set was expressed normally according to the platform process; the other set was expressed in the expression medium supplemented with the mannosidase inhibitor kifuncinine to produce a high-mannose antibody. LC / MS analysis of the antibody's glycoforms revealed that the antibody produced using the normal process was primarily G0F, while the antibody produced with kifuncinine had a predominant glycoform of Man9 (Figures 3A and 3B).

[0125] Example 3: Preparation of lectin variant-containing filler and its enrichment effect on high-mannose adalimumab

[0126] Two 1 mL nickel columns (Cytiva-17524701) were connected to different column valves on an AKTA pure (Cytiva) instrument. One nickel column was washed with equilibration buffer (25 mM Tris-HCl, pH 8.0, 200 mM NaCl, 10 mM imidazole) until baseline equilibrium was achieved as a control group. The other nickel column was loaded with an excess of the lectin ESA-2v as an experimental group and then washed with equilibration buffer until baseline equilibrium was achieved. Adalimumab, produced in parallel, was loaded onto the ESA-2v-bound nickel column and washed with equilibration buffer (25 mM Tris-HCl, pH 8.0, 200 mM NaCl, 10 mM imidazole) until baseline stabilization was achieved. Bound proteins were then eluted with elution buffer (25 mM Tris-HCl, pH 8.0, 200 mM NaCl, 250 mM imidazole). To prevent the bound adalimumab from binding to the nickel column untreated with ESA-2v, two sets of adalimumab produced in parallel were loaded onto a control nickel column untreated with ESA-2v and eluted using the same elution buffer. Chromatographic and SDS-PAGE analysis showed that the nickel column untreated with ESA-2v did not bind to either set of adalimumab. The nickel column coupled with ESA-2v only bound the mannose-form of adalimumab, but also bound a small amount of normally expressed adalimumab. Mass spectrometry analysis of the small amount of normally expressed adalimumab detected the high-mannose form of Man5, demonstrating that ESA-2v can enrich the high-mannose form of adalimumab.

[0127] Figure 4A shows a schematic diagram of the pull-down of the binding of ESA-2v lectin to adalimumab. Figure 4B shows chromatograms of the binding of high-mannose adalimumab to conjugated and unconjugated ESA-2v lectin nickel columns. Yellow (light) indicates the elution of the binding of high-mannose adalimumab to the nickel column of conjugated ESA-2v lectin; blue (dark) indicates the elution of the binding of unconjugated ESA-2v lectin to the nickel column of high-mannose adalimumab. Figure 4C shows SDS-PAGE images of the binding of high-mannose adalimumab to unconjugated (left) and conjugated (right) ESA-2v lectin nickel columns. Comparison of the results revealed that ESA-2v lectin bound to high-mannose adalimumab. Figure 4D shows chromatograms of the binding of normal glycoform adalimumab to conjugated and unconjugated ESA-2v lectin nickel columns. Purple (dark) represents the elution of the normal glycoform of adalimumab bound to a nickel column conjugated with lectin ESA-2v; green (light) represents the elution of the normal glycoform of adalimumab bound to a nickel column unconjugated with lectin ESA-2v. Figure 4E shows SDS-PAGE images of the normal glycoform of adalimumab bound to an unconjugated (left) and conjugated (right) lectin ESA-2v nickel column. Comparison of the results revealed that lectin ESA-2v bound to a small amount of normally expressed adalimumab (lane 8). Figure 4F shows the reduced mass spectrometry analysis of a small amount of bound normally expressed adalimumab (sample from lane 8 in Figure 4E). The molecular weight of the Man5 modification on the Fc heavy chain is 50411.8. These results demonstrate that lectin ESA-2v can enrich high-mannose-modified adalimumab from adalimumab expressed using a conventional process.

[0128] The affinity of the lectin ESA-2v for the high-mannose adalimumab was determined using biolayer interferometry (BLI): The lectin ESA-2v was conjugated to an Anti-His sensor (Gator Prime) via a C-terminal histidine tag and then bound and dissociated with two sets of adalimumab at a gradient concentration of 0, 11nM, 33nM, 100nM, 300nM, and 900nM in a well plate. Calculations showed that the binding affinity of the lectin ESA-2v for the high-mannose adalimumab was 364nM (K on :2.23E+04,K off : 8.13E-03); lectin ESA-2v weakly binds to normally expressed adalimumab.

[0129] The results are shown in Figures 5A and 5B. Figure 5A shows the binding and dissociation curves of the blank sensor and two groups of adalimumab in the blank control experiment. The results show that neither group of adalimumab binds to the blank sensor. Figure 5B shows the binding and dissociation curves of the ESA-2v lectin-coupled sensor and two groups of adalimumab in the experimental group. The yellow (light) color represents the shift curves of the interference spectrum of the binding and dissociation of a series of high-mannose adalimumab with the ESA-2v lectin sensor, and the purple color represents the shift curves of the interference spectrum of the binding and dissociation of a series of normal glycosylated adalimumab with the ESA-2v lectin sensor.

[0130] The comparison results showed that the ESA-2v lectin bound to the high mannose adalimumab, while the binding signal with the normal glycoform adalimumab was weaker. The calculation showed that the binding affinity Kd of the ESA-2v lectin to the high mannose adalimumab was 364nM (K on :2.23E+04,K off :8.13E-03).

[0131] Example 4: Coupling rate of lectin variants to fillers

[0132] The sample of SP peak 1 was replaced with coupling buffer (50mM Tris, 5mM EDTA, pH 8.5). An appropriate amount of SulfoLink coupling resin (Thermo 20404) was added to the gravity column. After the filler gradually settled to the bottom of the gravity column, the binding buffer was slowly added from the top of the gravity column to balance the filler. ESA-2v lectin protein was added to the filler at a ratio of 1mL filler to 25mg ESA-2v lectin. The two were thoroughly mixed using a suspension instrument, and after incubation at room temperature for 30 minutes, the cover at the bottom of the gravity column was removed to allow the liquid to flow out from the bottom. 1M high salt buffer was then added to wash away nonspecifically bound impurities. Blocking buffer (25mM Tris, 150mM NaCl, 5mM EDTA, 50mM cysteine, pH 8.5) was then added. After mixing and incubating at room temperature for 30 minutes, the liquid was allowed to flow out from the bottom of the gravity column. Finally, the coupled filler was balanced with coupling buffer. Aliquot the filler and calculate the final coupling rate. Calculations show that 20 mg of ESA-2v protein can be coupled per ml of filler.

[0133] Example 5: Broad spectrum and specificity of binding of ESA-2v to IgG antibodies

[0134] To validate the broad spectrum of IgG binding of the lectin ESA-2v, multiple antibodies of different IgG subtypes were produced in parallel using the WuXi Trasient expression platform. For each IgG antibody, expression was divided into two groups: one group was expressed normally according to the platform process; the other group supplemented the expression medium with the mannosidase inhibitor kifnoside to generate high-mannose antibodies. Glycoforms were then characterized by mass spectrometry.

[0135] The results are shown in Figures 6A-6E. Figure 6A shows the reduced mass spectra of two groups of Adalimumab antibodies. The left figure shows the antibody expressed by the normal process, 50638 is the molecular weight of the G0F modification of the Fc heavy chain; the right figure shows the antibody expressed by the kifunicine process, 51058.3 is the molecular weight of the Man9 modification of the Fc heavy chain. Figure 6B shows the reduced mass spectra of two groups of Trastuzumab antibodies. The left figure shows the antibody expressed by the normal process, 50595.2 is the molecular weight of the G0F modification of the Fc heavy chain; the right figure shows the antibody expressed by the kifunicine process, 51015.5 is the molecular weight of the Man9 modification of the Fc heavy chain. Figure 6C shows the reduced mass spectra of two groups of Rituximb antibodies. The left figure shows the antibody expressed using the normal process, with 50508.5 being the molecular weight of the G0F modification of the Fc heavy chain; the right figure shows the antibody expressed using the kifunine addition process, with 50928.9 being the molecular weight of the Man9 modification of the Fc heavy chain. Figure 6D shows the reduced mass spectra of two groups of Denosumab antibodies. The left figure shows the antibody expressed using the normal process, with 50421.8 being the molecular weight of the G0F modification of the Fc heavy chain; the right figure shows the antibody expressed using the kifunine addition process, with 50842.2 being the molecular weight of the Man9 modification of the Fc heavy chain. Figure 6E shows the reduced mass spectra of two groups of Pembrolizumab antibodies. The left figure shows the antibody expressed using the normal process, with 50709.3 being the molecular weight of the G0F modification of the Fc heavy chain; the right figure shows the antibody expressed using the kifunine addition process, with 51129.6 being the molecular weight of the Man9 modification of the Fc heavy chain.

[0136] Among them, IgG1 antibodies include: Adalimumab, Trastuzumab, Rituximab; IgG2 antibodies include Denosumab; IgG4 antibodies include Pembrolizumab.

[0137] To verify the specific affinity of the filler for high-mannose antibodies, the IgG antibody was replaced with an equilibration buffer (20 mM Tris, 200 mM NaCl, pH 8.5). The IgG was then loaded onto a lectin ESA-2v affinity chromatography column. The column was rinsed with the equilibration buffer until the baseline was balanced and stable. Finally, the bound target protein was eluted with 0.1 M glycine-HCl, pH 1.0. SDS-PAGE and LC / MS analysis of the eluted fractions demonstrated that the lectin ESA-2v filler specifically bound only to high-mannose IgG antibodies and exhibited broad affinity for IgG antibodies.

[0138] The results are shown in Figures 7A-7H. Figure 7A shows a schematic diagram of IgG antibody binding to the ESA-2v affinity matrix. Figure 7B shows chromatograms of two groups of adalimumab antibodies binding to the ESA-2v column. Blue (dark) indicates a high-mannose antibody; yellow (light) indicates a normal-process glycoform antibody. Figure 7C shows chromatograms of two groups of trastuzumab antibodies binding to the ESA-2v column. Blue (dark) indicates a high-mannose antibody; yellow (light) indicates a normal-process glycoform antibody. Figure 7D shows chromatograms of two groups of rituximab antibodies binding to the ESA-2v column. Blue (dark) indicates a high-mannose antibody; yellow (light) indicates a normal-process glycoform antibody. Figure 7E shows chromatograms of two groups of denosumab antibodies binding to the ESA-2v column. Blue (dark) indicates a high-mannose antibody; yellow (light) indicates a normal process glycoform antibody. Figure 7F shows the chromatograms of two groups of pembrolizumab antibodies bound to the ESA-2v lectin column. Blue (dark) indicates a high-mannose antibody; yellow (light) indicates a normal process glycoform antibody. Figure 7G shows the SDS-PAGE of five high-mannose IgG antibodies bound to the ESA-2v lectin column and eluted. M: marker, L: load, FT: flow-through, E: eluate. Figure 7H shows the SDS-PAGE of five normal glycoform IgG antibodies bound to the ESA-2v lectin column and eluted. M: marker, L: load, FT: flow-through, E: eluate. 20X concentration means the eluted sample was concentrated 20 times.

[0139] The small amount of bound eluted IgG antibodies of the five normal glycoforms was concentrated, and the antibody glycoforms were then verified by LC / MS (as shown in Figure 8). The results showed that the bound antibody was the Man5 high-mannose form. The reason for the capture of the G0F glycoform is presumably due to the heterogeneity of the glycoforms of the two heavy chains of the antibody.

[0140] The lectin variants provided herein have specific affinity for high mannose antibodies and can effectively bind to high mannose antibodies (such as intact antibodies) formed during antibody production, so as to be used to effectively remove high mannose byproducts contained in recombinant antibodies, thereby promoting and achieving the desired antibody purification in the downstream stage of production.

Claims

1. A lectin variant comprising (a) a histidine tag and (b) a cysteine-containing segment added to the C-terminus of a reference lectin.

2. The lectin variant according to claim 1, wherein The reference lectin is selected from plant lectins, animal lectins and microbial lectins; preferably, the reference lectin is selected from plant lectins; preferably, the reference lectin is a lectin from red algae (Rhodophyta); preferably, the reference lectin has the amino acid sequence shown in SEQ ID NO:

1.

3. The lectin variant according to claim 1, wherein The cysteine-containing segment comprises 1-16 amino acid residues, preferably the cysteine-containing segment comprises 1-5 cysteine ​​residues, preferably any two adjacent cysteine ​​residues in the cysteine-containing segment are separated from each other by 0, 1, or 2 amino acid residues other than cysteine ​​and histidine; and / or The histidine tag is a polyhistidine tag, preferably the histidine tag comprises 2-20, 4-18, 6-16, 6-14 or 6-10 consecutive histidine residues.

4. The lectin variant according to claim 1, wherein The histidine tag is linked to the C-terminus of the reference lectin via one or more linker regions, preferably the linker region comprises 1 to 15 amino acid residues, preferably the linker region comprises GGGGS, GGGS, GGS, GS, or any combination thereof, preferably the linker region is GGGGS, GS, or any combination thereof; and / or The cysteine-containing segment is connected to the C-terminus of the histidine tag via one or more linker regions, preferably the linker region comprises 1-3 amino acid residues, preferably the linker region is G.

5. The lectin variant according to claim 1, wherein The lectin variant comprises 0-6 added amino acid residues at the N-terminus of the reference lectin; preferably, the amino acid residues are neutral amino acid residues; preferably, the neutral amino acid residues include methionine.

6. The lectin variant according to claim 1, wherein The lectin variant has the amino acid sequence shown in SEQ ID NO:

2.

7. A composition comprising the lectin variant according to any one of claims 1 to 6.

8. A nucleic acid molecule encoding the lectin variant according to any one of claims 1 to 6.

9. A vector comprising the nucleic acid molecule according to claim 8.

10. A cell comprising the nucleic acid molecule according to claim 8 or the vector according to claim 9.

11. A matrix for affinity purification, which is loaded with the lectin variant according to any one of claims 1 to 6.

12. A method for preparing a matrix for affinity purification, comprising: (1) providing a lectin variant according to any one of claims 1 to 6; (2) mixing the lectin variant with a filler to couple the lectin variant to the filler.

13. Use of the lectin variant according to any one of claims 1 to 6 for reducing or removing high mannose antibodies in a sample.

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