阅读说明：本技术 免疫球蛋白结合蛋白及其应用 (Immunoglobulin binding proteins and uses thereof ) 是由 江必旺 程雷 于 2020-07-30 设计创作，主要内容包括：本发明涉及免疫球蛋白分离纯化技术领域,具体而言,涉及一种免疫球蛋白结合蛋白及其应用。所述免疫球蛋白结合蛋白由葡萄球菌蛋白A的E、C和Z-domain拼接得到,并意外获得了抗体载量的提升和耐碱特性,进而可用于免疫球蛋白的亲和层析。(The invention relates to the technical field of immunoglobulin separation and purification, and particularly relates to an immunoglobulin binding protein and application thereof. The immunoglobulin binding protein is spliced by E, C and Z-domain of staphylococcal protein A, and the improvement of antibody carrying capacity and alkali resistance are unexpectedly obtained, so that the immunoglobulin binding protein can be used for affinity chromatography of immunoglobulin.)

1. An immunoglobulin-binding protein having an amino acid sequence shown by SEQ ID NO. 1.

2. The immunoglobulin-binding protein of claim 1, wherein amino acid E at position 25 is replaced with a and amino acid R at position 27 is replaced with K.

3. The immunoglobulin-binding protein of claim 1 or 2, wherein amino acid G at position 46 is replaced with A.

4. A protein multimer comprising two or more immunoglobulin-binding protein repeating units of claim 1.

5. The protein multimer of claim 4, comprising 4, 5, 6, 7, or 8 of said repeating units.

6. The protein multimer according to claim 4 or 5, further comprising a terminal coupling group at its N-terminus.

7. The protein multimer according to claim 6, wherein the terminal coupling group comprises arginine and/or cysteine.

8. Nucleic acid encoding the immunoglobulin-binding protein of any one of claims 1 to 3 or the protein multimer of any one of claims 4 to 7.

9. Vector, characterized in that it comprises a nucleic acid according to claim 8.

10. Host cell, characterized in that it comprises a nucleic acid according to claim 8 or a vector according to claim 9.

11. A method for producing the immunoglobulin-binding protein of any one of claims 1 to 3 or the protein multimer of any one of claims 4 to 7, comprising:

culturing the host cell of claim 10 under suitable culture conditions; and

recovering the immunoglobulin-binding protein or protein multimer thus produced from the culture medium or from the cultured cells.

12. An affinity chromatography medium comprising a solid support and a ligand grafted to said solid support;

the ligand is an immunoglobulin-binding protein according to any one of claims 1 to 3 or a protein multimer according to any one of claims 4 to 7.

13. The affinity chromatography media of claim 12, wherein the solid support is selected from any one of bentonite, glass microspheres, quartz microspheres, magnetic beads, calcium hydroxy phosphate, alumina, polyacrylamide gel, starch gel, dextran gel, cellulose, agarose, silicon, ceramic, cyclodextrin, chitosan, carrageenan, guar gum, gum arabic, gum ghatti, gum tragacanth, karaya gum, locust bean gum, xanthan gum, pectin, mucin, liver thioesters, gelatin, polyurethane, polystyrene divinyl benzene, polymethyl methacrylate, polyacrylamide, polyethylene glycol terephthalate, polyvinyl acetate, polyethylene, polypropylene, polyvinyl chloride, polyvinyl pyrrolidone, or copolymers of any of the foregoing.

14. An affinity chromatography separation apparatus comprising the affinity chromatography medium according to any one of claims 12 to 13.

15. Use of the affinity chromatography medium of any one of claims 12 to 13, or the affinity chromatography separation device of claim 14, for separating or enriching an immunoglobulin from a liquid medium.

16. Use according to claim 15, wherein the liquid medium is selected from cell cultures, ascites or blood extracts, or diluted solutions thereof.

Technical Field

The invention relates to the technical field of immunoglobulin separation and purification, and particularly relates to an immunoglobulin binding protein and application thereof.

Background

With the advent and development of biotechnology, the prevention and treatment of diseases has revolutionized. Today's biotechnology penetrates almost every corner of our lives, where research directed at antibodies has also performed prominently. From polyclonal antibodies, monoclonal antibodies to recombinant antibodies, every technological leap will give people unlimited surprise. However, like all other protein drugs, antibody production technology, production scale, and purification technology are important technical links that restrict antibody production. The antibody purification technology becomes the key, and the good and bad of the purification technology and the large and small scale often determine the vitality of the antibody drug production.

The affinity molecule with special structure is made into solid phase adsorbent and placed in chromatographic column, and when the protein mixture liquid to be separated passes through the chromatographic column, the protein with affinity to the adsorbent is adsorbed and retained in the chromatographic column. The protein without affinity is separated from the separated protein by directly flowing out without being adsorbed, and then the bound protein is eluted by changing the binding condition by using a proper eluent, and the method for separating and purifying the protein is called affinity chromatography. Certain structural sites on some of the biomolecules recognize and bind to other molecules, such as enzyme-substrate recognition binding, receptor-ligand recognition binding, and antibody-antigen recognition binding, which is both specific and reversible, and which can be released by changing conditions. This binding capacity between biomolecules is called affinity. Affinity chromatography is a protein separation and purification method designed according to the principle, and is a chromatographic technique for separating target proteins or other molecules capable of specifically binding to ligands in a protein mixture by using a chromatographic medium covalently linked with the specific ligands.

Affinity chromatography is an adsorption chromatography in which an antigen (or antibody) and a corresponding antibody (or antigen) are specifically bound, and the binding is reversible under certain conditions. Therefore, after the antigen (or antibody) is solid-phased, the corresponding antibody (or antigen) in the liquid phase can be selectively bound on the solid phase carrier, thereby separating from other proteins in the liquid phase and achieving the purpose of separation and purification. The method has the advantages of high efficiency, rapidness, simplicity and the like.

Staphylococcus Protein A (SPA) contains 5 domains capable of specifically binding to Fc region of antibody IgG molecule, and is Staphylococcus aureusStaphylococcus aureus) Staphylococcus aureus, a constituent protein in the cell wall of leatherThe Lancefield's positive bacteria are common suppurative infectious bacteria in cross infection in hospitals, the diameter of the bacteria is about 0.8 mu m, the bacteria are in a small ball shape, and the bacteria are named after grape bunch shape due to accumulation. SPA is capable of binding to many mammalian IgG antibodies (often the Fc fragment) and is therefore one of the earliest proteins used for affinity chromatography and antibody purification.

However, since SPA is also a protein, it requires high physicochemical properties for affinity adsorption and elution, and is particularly resistant to alkaline reagents, and the amount of antibody bound is often not optimal.

Disclosure of Invention

The invention relates to an immunoglobulin binding protein, which has an amino acid sequence shown in SEQ ID NO. 1.

According to a further aspect, the invention also relates to a protein multimer comprising two or more repeating units represented by SEQ ID NO. 1.

The invention also relates to nucleic acids, vectors, host cells and methods of production relating to the immunoglobulin-binding proteins and protein multimers.

According to a further aspect of the invention, the invention also relates to an affinity chromatography medium and an affinity chromatography separation device containing the affinity chromatography medium, wherein the chromatography medium comprises a solid phase carrier and a ligand grafted on the solid phase carrier;

the ligand is an immunoglobulin-binding protein as described above or a protein multimer as described above.

The invention also relates to the use of the above product for the isolation or enrichment of immunoglobulins from a liquid medium.

The immunoglobulin binding protein is spliced by E, C and Z-domain of staphylococcal protein A, and the improvement of antibody carrying capacity and alkali resistance are unexpectedly obtained, so that the immunoglobulin binding protein can be used for affinity chromatography of immunoglobulin.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is an electrophoretogram of a protein multimer purified in one embodiment of the present invention;

FIG. 2 is a chromatogram of G-Z-E-1 before mutation in one embodiment of the present invention;

FIG. 3 is a chromatogram of G-Z-E-2 after mutation in one embodiment of the present invention.

Detailed Description

Reference will now be made in detail to embodiments of the invention, one or more examples of which are described below. Each example is provided by way of explanation, not limitation, of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. For instance, features illustrated or described as part of one embodiment, can be used on another embodiment to yield a still further embodiment.

It is therefore intended that the present invention cover the modifications and variations of this invention provided they come within the scope of the appended claims and their equivalents. Other objects, features and aspects of the present invention are disclosed in or are apparent from the following detailed description. It is to be understood by one of ordinary skill in the art that the present discussion is a description of exemplary embodiments only, and is not intended as limiting the broader aspects of the present invention.

The invention relates to an immunoglobulin binding protein, which has an amino acid sequence shown in SEQ ID NO. 1.

The immunoglobulin-binding protein may bind to the Fc region of an immunoglobulin.

In the present invention, the term "immunoglobulin" is a protein that binds to a specific antigen, and broadly refers to all proteins and protein fragments comprising complementarity determining regions (CDR regions), particularly full-length antibodies comprising an Fc fragment or variants thereof. The term "full length antibody" includes polyclonal antibodies and monoclonal antibodies. Variants comprising an Fc segment are well known in the art, e.g., scFv-Fc and the like. The type of antibody can be selected from IgG, IgA, IgM, IgE, IgD. Preferably, the antibody is at least one of IgG (IgG 1, IgG2, IgG3, or IgG 4), IgA, and IgM. Furthermore, the term "immunoglobulin" includes naturally occurring antibodies as well as non-naturally occurring antibodies, including, for example, chimeric (chimeric), bifunctional (bifunctional), humanized (humanized) antibodies and human antibodies, as well as related synthetic isomeric forms (isofoms).

The Fc portion of an "immunoglobulin" may be from animals including humans and all animal husbandry (e.g., livestock and pets) and wild, preferably mammals, more preferably: pig, dog, rabbit, human, monkey, mouse, and cow.

In some embodiments, an "immunoglobulin" may also be a fusion protein.

The "immunoglobulin" is preferably an antibody drug, in particular a monoclonal antibody drug, examples of which may be selected from the group consisting of antibodies as shown below:

anti-GD 2 antibody 3F8, abamectin (Abagonomab), Abciximab (Abciximab), ACZ885 (canakinumab), Adalimumab (Adalilimumab), Addenamumab (Adetalimumab), Addenamumab (Adecatuzumab), Aframomumab (Afelimomab), Atubuzumab (Affutuzumab), Pezidozumab (Alizezumab pegol), Alemtuzumab (Allituzumab), pentoxydumumab (Altuzumab pentate), Maumomab (Anatomamafenox), Anluzumab (Anlunuzumab) (IMA-638), Adelizumab (Apotuzumab), Acitumomab (Artuzumab (Artumomab), Aselizumab (Aselizumab), Atuzumab (Atolizumab), Abelizumab (Abelizumab), Abelizumab (Bezizumab), Abelizumab (Bezizumab (Abelizumab), Abelizumab (Abelizumab), Abelix (Abelix), Abelizumab (Abelix), Abelix (Abelix), Abelix (Abelix), Abelizumab) and Abelix (Abelix, bivatuzumab-DMl (Bivatuzumab mertansine), Lantuzumab (Blinatumomab), Brentuximab vedotin, Briakin, canamycin (Canakin), Memantib (Canuzumab mertansine), Carocumab Pendule (Capromab pendend), Rituzumab (Catuzaxomaxomab), Celizumab (Cedellizumab), Petzuzumab (Certifizumab (Ceruzumab), Cetuximab (Cetuximab), Poxitezumab (Ectatuzumab), Cetuzumab (Citutuzumab), Cetuzumab (Cixutuzumab), Clexituzumab (Clenidoximab), Clevelituzumab (Clevelitumab) tetatan, CNTO148 (Googlucab), Metalizumab (Cetuzumab), Cetuzumab (Ctenolizumab), Cetuzumab (C, Efuzumab (Efugulimuab), exemestab (Elsilimumab), peggolimumab (Enlimomab pegumab pegol), Ceipilimumab (Epitumomabetuzutan), Epratuzumab (Epratuzumab), Erlizumab (Erlizumab), Eimazezumab (Ertumaxomab), Elastazumab (Etarazeumab), Ebivimumab (Exbivizumab), Fanolisomab (Fanolisomab), Faramumab (Faralmomab), Novizumab (Felvizumab), Nozazinuzumab (Fezakinumab), Figituzumab (Figituzumab), Artuzumab (Fotuzumab), Furazumamab (Fuvizumab), Furazumab (Rituzumab), Gaolizumab (Gelatizumab), Iguzumab (Iguratimab (Igmumab), Fumazumab (Freumacumumab), Gaolizumab (Inumumab), Gaolizumab (Golomavizumab), Ezimab (Golomavitezomib (Golomab), Golomavizumab ozimab (Golomiki), Golomavituzumab (Golomab (Golomavimex), Evoximab), Golomab (Golomab), Golomab (Iumumab), Golomab (Golomab) and Evomex, Evomex (Golomab), Golomab (Golomab) and Izepinx (Golomab) and Ivu (Golomab) and Izepinx (Golomab, Izezumab ozolomide (Inotuzumab), Ipilimumab (Iilimumab), itumumab (Iratumab), Kaliximab (Keliximab), Rabezumab (Labetuzumab), Lebrizumab (Lebrikizumab), Lemazumab pegol (Lemalezumab), Ledellimumab (Lerdelimumab), Lexalimumab (Lexatuzumab), Ribizumab (Libivirumab), Lintuzumab (Lintuzumab), Lucatazumab (Lucatumab), Luximab (Lumiliximab), Mapatummab (Mapatummab), Mastemab (Mashimumab), Matuzumab (Matuzumab), Melizumab (Melizumab), Metiumumab (Metalizumab), Metalizumab (Metalizumab) (Metelmovatuzumab), Mutamumab (Mutamumab), Mutamumab (Mutamiflumuzumab), Mutamumab (Mutamiflumab (Mutamumab), Mutamiflumab (Mutamiflumab), Mutamumab (Mutamiflumab), Mutamiflamotuzumab (Mutamumab), Mutamiflumab (McSt, Mutamumab), Mutamimumab (Mutamiflamotuzumab), Mutamab (Mutamiflamotuzumab), Mutam-24, Mutamab), Mutamiflumab (Mutamab), Mutamab (Mutamiflamotuzumab), Mutamab (Mutamiflamotuzumab), Mutam-E (Mutamiflamotuzumab), Mutame (M), Mutame, Mutamiflamot, Nebacaumab (nebamumab), nemuximab (Necitumumab), nerrimumab (Nerelimomab), nimesulide (Nimotuzumab), Nivolumab (Nivolumab), mercaptomomab (nofectmomab merutan), Ocrelizumab (Ocrelizumab), orimomab (odumab), Ofatumumab (Ofatumumab), Omalizumab (ofatumab), Omalizumab (Omalizumab), moelcuzumab (opolimumab) (oportumab), agovacizumab (orinomab), oxizumab (oteliximab), palexizumab (pagiximab) JQ lizumab (Palivizumab), Panitumumab (Panitumumab), pemirocumumab (Pembrolizumab), pemirolimumab (pemirolimumab), pemirolimumab (pezimab), pemirolimumab (140 (pezimab), Ranibizumab), pemirotuzumab (pezimab), pemirotuzumab (pezimab), pemiro (pezimab), pemirotuzumab), pemiro (e (pezimab), pemiro (e), pemphigovix (e (pezimab), pemphilizumab), pemphiliz, Rigaviruzumab (Regavirumab), Raylelizumab (Reslizumab), Rituximab (Rilutummab), Rituximab (Rituximab), Rotuzumab (Robatuzumab), rolizumab (Rotaluzumab), rolizumab (Rotallizumab), rovellizumab (Rovellizumab), lurizumab (Ruplizumab), sartomozumab (Satumomab), Sevivimab (Sevirumab), Celulizumab (Sibrolizumab), Sifalizumab (Sifalimab), Sirtuzumab (Siltuximab), Celizumab (Siplizumab), Sulizumab (Solanazemab), Southlizumab (Soneplizumab), Sonotuzumab (Soneuzumab), Sontuzumab (Southuzumab), strelizumab), Thiazazumab (Suveluzumab), Raylelizumab (Tanellizumab), Tanellizumab (Tanellizumab), Tanellizumab, TGN1412, tiximumab (ticolimumab), Tremelimumab (Tremelimumab), tiuqiuzumab (Tigatuzumab), TNX-355 (ibalizumab), TNX-650, TNX-901 (talizumab), tollizumab (Tocilizumab), Tositumomab (Tositumomab), Trastuzumab (Trastuzumab), Tremelimumab (tremelizumab), simukulzumab (tuzumab), tuviruzumab (Tuvirumab), uralizumab (Urtoxazumab), usteklizumab (usekinumab), vallizumab (validoximab), vedolumab (velizumab), vemuralizumab (vemuralizumab), vemuralizumab (vemuralizumab), and the like (vemuralizumab).

In some embodiments, the immunoglobulin-binding protein is substituted with a at amino acid E at position 25 and K at amino acid R at position 27.

The mutated amino acids are more easily eluted. The elution pH before mutagenesis was about 3.0 and after mutagenesis was about 3.5.

In some embodiments, the immunoglobulin-binding protein has a substitution of amino acid G at position 46 to a.

The mutation of glycine, which is not alkali-resistant, to alanine is more advantageous for maintaining the alkali-resistant properties of the immunoglobulin-binding protein.

According to a further aspect, the invention also relates to a protein multimer comprising two or more repeating units represented by SEQ ID NO. 1.

In some embodiments, the protein multimer contains 4, 5, 6, 7, or 8 of the repeating units.

In some embodiments, the protein multimer also has a terminal coupling group at the N-terminus.

In some embodiments, the terminal coupling group comprises arginine and/or cysteine.

It will be readily understood that the invention also claims proteins substantially identical to said immunoglobulin-binding protein or said protein multimer, such proteins being substantially identical to the immunoglobulin-binding protein of SEQ ID NO:1, and retains the function of specifically binding to an immunoglobulin, and a sequence having at least about 80% identity, at least about 90% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, or at least about 99% identity; furthermore, such proteins are still capable of specifically binding immunoglobulins and subsequently eluting them after soaking in 0.5mol/L NaOH for 24h at 25 ℃ and can maintain a loading of about 56 mg/ml.

The invention also relates to nucleic acids encoding the immunoglobulin-binding proteins as described above or the protein multimers as described above.

The invention also relates to a vector comprising a nucleic acid as described above.

The term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector is capable of expressing a protein encoded by an inserted polynucleotide, the vector is referred to as an expression vector. The vector may be introduced into a host cell by transformation, transduction, or transfection, and the genetic material elements carried thereby are expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to: a plasmid; phagemid; a cosmid; artificial chromosomes such as Yeast Artificial Chromosomes (YACs), Bacterial Artificial Chromosomes (BACs), or artificial chromosomes (PACs) derived from P1; bacteriophage such as lambda phage or M13 phage, animal virus, etc. Animal viruses that may be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papilloma viruses, papilloma polyoma vacuolatum viruses (e.g., SV 40). In some embodiments, regulatory elements commonly used in genetic engineering, such as enhancers, promoters, Internal Ribosome Entry Sites (IRES), and other expression control elements (e.g., transcription termination signals, or polyadenylation signals and poly-U sequences, etc.) are included in the vectors of the present invention.

In some embodiments, the vector of the present invention may further comprise a gene used for screening (e.g., an antibiotic resistance gene), a nucleic acid for producing a fluorescent protein, or the like. The fluorescent protein can be selected from green fluorescent protein, blue fluorescent protein, yellow fluorescent protein, orange fluorescent protein or red fluorescent protein.

The invention also relates to a host cell comprising a nucleic acid as described above or a vector as described above.

The term "host cell" refers to a cell which can be used for introducing a vector, and includes, but is not limited to, prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblast, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells. The host cell is preferably a prokaryotic cell, more preferably E.coli, e.g.E.coli BL (DE 3).

The present invention also relates to a method of producing an immunoglobulin-binding protein as described above or a protein multimer as described above, comprising:

culturing a host cell as described above under suitable culture conditions; and

recovering the immunoglobulin-binding protein or protein multimer thus produced from the culture medium or from the cultured cells.

The invention also relates to an affinity chromatography medium, which comprises a solid phase carrier and a ligand grafted on the solid phase carrier;

the ligand is an immunoglobulin-binding protein as described above or a protein multimer as described above.

In some embodiments, the solid support is selected from any one of bentonite, glass microspheres, quartz microspheres, magnetic beads, calcium hydroxy phosphate, alumina, polyacrylamide gel, starch gel, dextran gel, cellulose, agarose, silicon, ceramic, cyclodextrin, chitosan, carrageenan, guar gum, gum arabic, gum ghatti, gum tragacanth, karaya gum, locust bean gum, xanthan gum, pectin, mucin, liver thioesters, gelatin, polyurethane, polystyrene divinyl benzene, polymethyl methacrylate, polyacrylamide, polyethylene glycol terephthalate, polyvinyl acetate, polyethylene, polypropylene, polyvinyl chloride, polyvinyl pyrrolidone, or copolymers formed of any of several.

The solid phase carrier may be selected from suitable shapes, forms, materials and modifications depending on the application. The solid support surface can be substantially flat or planar. Or may be circular or the like. The shape of the surface of the solid support includes, but is not limited to, pores, depressions, pillars, ridges, channels, membranes, or the like. The solid support is preferably a particulate material having a substantially planar surface. The shape of the particulate matter is preferably spherical or substantially spherical.

Further, the solid support may be non-porous, but preferably comprises one or more pores, more preferably a porous network, allowing free passage of macromolecules. In some embodiments, the pore diameter is 300-5000A; in the present invention, "non-porous" means that the matrix support has no pores that are appreciably measurable, for example, a pore diameter ≦ 20A.

In some embodiments, the solid support is activated by covering certain groups (e.g., hydroxyl groups, etc.) on the surface of the solid support with an intermediate compound (e.g., CNBr) and by providing new chemical groups (e.g., -CN) on the surface of the solid support, such that the new groups can covalently react with the affinity ligand to achieve covalent attachment.

Thus, it will be readily understood that the surface of the solid support may also be modified with groups for activation, such as at least one of epoxy, amino, aldehyde, hydroxyl, carboxyl, oxo, and thiol groups. The modification sites typically also comprise derivatizing groups with these corresponding chemical functionalities.

The invention also relates to an affinity chromatography separation device containing the affinity chromatography medium.

The affinity chromatography separation device can be SPE solid phase extraction column, centrifuge tube with separation membrane, separation membrane (membranes), fast detection biochip (bio-chips), fiber bundle column, monolithic column and conventional analytical grade or preparative chromatographic column, etc.

The invention also relates to the use of an affinity chromatography medium as described above, or an affinity chromatography separation device as described above, for separating or enriching immunoglobulins from a liquid medium.

In some embodiments, the use comprises at least one step of treating the affinity chromatography media or the affinity chromatography separation device with basic conditions.

In some embodiments, the alkaline conditions should be in the vicinity of the pH provided by 0.5mol/L NaOH, such as 7 to14, or 10 to14, or 12 to14, or 13 to14, or 13.5 to 14. The alkaline treatment time may be up to 24 hours, for example 8 hours, 16 hours or 20 hours.

In some embodiments, the liquid medium is a sample comprising immunoglobulins or a diluted solution thereof. Any sample comprising immunoglobulins may be used in the present invention. The immunoglobulin-containing sample may comprise, for example, a cell culture (particularly a cell culture supernatant), a blood extract (particularly a serum extract), and ascites (e.g., ascites of an animal such as a rat, a mouse, a rabbit, an sheep, a horse, a cow, a camel, etc.). As an example, antibodies can be expressed in Chinese Hamster Ovary (CHO) cells in a stirred tank bioreactor.

Embodiments of the present invention will be described in detail with reference to examples.