CN113912728B - Affinity purification method for reducing host cell protein content in anti-human interleukin-33 monoclonal antibody production - Google Patents
Affinity purification method for reducing host cell protein content in anti-human interleukin-33 monoclonal antibody production Download PDFInfo
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Abstract
The application discloses an affinity purification method for reducing host cell protein content in anti-human interleukin-33 monoclonal antibody production, which comprises the following steps: equilibrium before loading: balancing the affinity chromatography medium by using a buffer solution, thereby obtaining a balanced affinity chromatography medium; loading: combining the anti-human interleukin-33 monoclonal antibody fermentation broth with a well-balanced affinity chromatography medium; eluting: pre-eluting and final eluting the mixed solution to remove host cell proteins and obtain a purified anti-human interleukin-33 monoclonal antibody; the anti-human interleukin-33 monoclonal antibody comprises three heavy chain complementarity determining regions, CDR-H1, CDR-H2 and CDR-H3, and three light chain complementarity determining regions, CDR-L1, CDR-L2 and CDR-L3. The method is simple and easy to implement, can carry out amplified purification production, does not need pretreatment on cell fermentation supernatant, has high eluted sample yield, and keeps the residual quantity of HCP at a lower level, thereby reducing the pressure for removing HCP in the subsequent purification step.
Description
Technical Field
The present application relates to the field of biotechnology. In particular, the application relates to an affinity purification method for reducing the protein content of host cells in the production of anti-human interleukin-33 (IL-33) monoclonal antibodies.
Background
Affinity purification is a very critical process step in the antibody drug production process, which captures and concentrates the antibodies in the fermentation broth to realize the first step of crude purification of the antibodies. In the process of mass fermentation of genetically engineered cell lines such as chinese hamster ovary Cells (CHO), cells undergo apoptosis and lysis in different physiological cycles, releasing host cell proteins (Host cell protein, HCP). HCP refers to protein components derived from host cells, including host cell structural proteins and transforming proteins (cell secreted growth-promoting proteins). The HCP can induce the organism to produce anti-HCP antibody, cause anaphylactic reaction, and can also cause the organism to produce antibody to protein medicine by 'adjuvant effect', so as to influence the therapeutic effect of the medicine, and the quantitative determination of residual HCP in the genetic engineering medicine is an important means for quality control, thereby being beneficial to maintaining the effectiveness and consistency of the purification process. In the affinity purification of antibodies, HCPs produced by engineering cells during fermentation are effectively removed while ensuring high antibody recovery. Therefore, the application provides an antibody affinity purification process which can be widely applied to large-scale fermentation of antibodies and is economical and feasible, and is of great significance for further industrialized popularization of antibody medicaments.
At present, more methods for removing HCP residues can be carried out, and each method has the characteristics that: 1) Chromatography mode: the method comprises Protein A affinity chromatography, anion and cation chromatography, wherein the three chromatography processes are based on HCP removal capability, the Protein A affinity chromatography is high in removal capability, the method is a main step of HCP removal, and the anion and cation chromatography is mainly used as a further HCP removal process in the follow-up. In the continuous perfection process of the process for removing the HCP by the chromatography, various chromatography processes are continuously put forward for practical production, and are the main means for removing the HCP; 2) Tangential flow ultrafiltration mode: the removal capability of HCP is limited, the residual quantity is not easy to control, the process control coefficient is not high, and the method can only be used as an auxiliary process for removing HCP; 3) Polymer precipitation mode: the high polymers such as PEG, polyacrylic acid and the like have positive charges in a wider pH range, and are combined with the antibody to form precipitation through the action of charges, while HCP is not easy to precipitate due to lower isoelectric point. The HCP content of the precipitated antibody sample is significantly reduced, but the process is not suitable for process scale-up and precipitation may have some effect on the activity of the antibody. Therefore, the process for removing HCP is numerous, and the selection process needs to be comprehensively considered due to the differences of the sample fermentation process and the antibody properties of each target antibody.
Disclosure of Invention
In order to solve the problem of Host Cell Protein (HCP) residues in antibody production, the application discloses a novel method for effectively reducing CHO host cell protein in antibody purification production, which can be widely applied to an antibody affinity purification process, and the used materials are low in cost and easy to process amplification. In the application, the effect between HCP and antibody and filler matrix is reduced in the pre-elution of affinity chromatography, namely the acting force between HCP and antibody protein and filler medium can be weakened to different degrees through the active reagent, so that the effect of removing HCP is achieved, the requirements of large-scale high-quality purification preparation of antibody medicines are met, and the safety of clinical use of antibody medicines is ensured.
The specific technical scheme of the application is as follows:
the application provides an affinity purification method for reducing the protein content of host cells in the production of anti-human interleukin-33 monoclonal antibody, which comprises the following steps:
equilibrium before loading: balancing the affinity chromatography medium by using a buffer solution, thereby obtaining a balanced affinity chromatography medium;
loading: combining the anti-human interleukin-33 monoclonal antibody fermentation liquor with the balanced affinity chromatography medium for loading;
Eluting: then, pre-eluting and final eluting are carried out successively, so that host cell proteins are removed, and a purified anti-human interleukin-33 monoclonal antibody is obtained;
the anti-human interleukin-33 monoclonal antibody comprises three heavy chain complementarity determining regions, CDR-H1, CDR-H2 and CDR-H3, and three light chain complementarity determining regions, CDR-L1, CDR-L2 and CDR-L3, wherein:
the amino acid sequence of CDR-H1 is shown in SEQ ID NO:1 is shown in the specification;
the amino acid sequence of CDR-H2 is shown in SEQ ID NO:2 is shown in the figure;
the amino acid sequence of CDR-H3 is shown in SEQ ID NO:3 is shown in the figure;
the amino acid sequence of CDR-L1 is shown in SEQ ID NO:4 is shown in the figure;
the amino acid sequence of CDR-L2 is shown in SEQ ID NO:5 is shown in the figure;
the amino acid sequence of CDR-L3 is shown in SEQ ID NO: shown at 6.
Preferably, the anti-human interleukin-33 monoclonal antibody comprises a heavy chain variable region and a light chain variable region, wherein,
the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: shown in figure 7;
the amino acid sequence of the light chain variable region is shown in SEQ ID NO: shown at 8.
Preferably, the affinity chromatography medium is selected from one of chromatography media with ligands crosslinked to agarose, polyvinyl ether, hydroxylated polyether resin, polyacrylic resin, polystyrene divinylbenzene resin, polymethacrylic resin, polystyrene resin, hydroxyapatite, glass matrix, preferably chromatography media with ligands crosslinked to polyvinyl ether;
Preferably, the ligand is Protein A, protein G or Protein L, more preferably Protein A.
Preferably, the buffer one is selected from one of phosphate buffer, tris-HCl buffer and boric acid-borax buffer; in the buffer solution I, the salt concentration is 5 mM-0.25M, and the pH is 5.5-8.0.
Preferably, pre-eluting is performed with a pre-eluting buffer, wherein the pre-eluting buffer is a neutral buffer and/or an acidic buffer;
preferably, the neutral buffer is selected from one of phosphate buffer, tris buffer and glycine buffer;
preferably, the acidic buffer is selected from one of a citric acid-disodium hydrogen phosphate buffer, an acetic acid-sodium acetate buffer, and a citric acid-trisodium citrate buffer;
preferably, the pH of the pre-elution buffer is from 5.0 to 7.5, more preferably from 5.5 to 6.5.
Preferably, when pre-eluting is performed with a pre-elution buffer, a pre-elution active agent is also added to the pre-elution buffer;
preferably, the pre-eluting active agent is selected from one or more of guanidine hydrochloride, polysorbate 80 and sodium chloride;
preferably, the pre-elution active agent is guanidine hydrochloride;
Further preferably, the concentration of guanidine hydrochloride is 0.01 to 1M.
Preferably, the final elution is performed with a final elution buffer;
the final elution buffer is one or two or more than three selected from a citric acid-disodium hydrogen phosphate buffer, an acetic acid buffer, a glycine-HCl buffer and a citric acid-sodium citrate buffer, and is preferably a citric acid-disodium hydrogen phosphate buffer;
preferably, the pH of the citric acid-disodium hydrogen phosphate buffer is 2.9-3.8.
Preferably, the equilibration is performed using buffer one when anti-human interleukin-33 monoclonal antibody fermentation broth is combined with the equilibrated affinity chromatography medium.
Preferably, in the eluting step, after pre-eluting, before final eluting, the pre-eluted solution is further equilibrated with a buffer.
Preferably, the buffer II is selected from one of phosphate buffer, tris-HCl buffer and boric acid-borax buffer;
preferably, the buffer II is phosphate buffer or Tris-HCl buffer;
preferably, the pH of the buffer II is 5.5-8.0.
ADVANTAGEOUS EFFECTS OF INVENTION
The affinity purification process is simple and feasible, can carry out amplified purification production, does not need pre-treatment of cell fermentation supernatant, has higher elution sample yield, and simultaneously maintains the residual HCP at a lower level (the residual control amount is not higher than 0.1 percent), thereby reducing the pressure of removing HCP in the subsequent purification step, and further ensuring that the residual HCP of the final sample of the human interleukin-33 (IL-33) monoclonal antibody is at an extremely low level. Meanwhile, in the application, the affinity purification process verification is carried out on fermentation supernatants of different batches, so that the affinity purification process has good stability.
The anti-human interleukin-33 (IL-33) monoclonal antibody of the present application has a comparable affinity for binding human interleukin-33 to the existing anti-human interleukin-33 (IL-33) monoclonal antibody (Etkimab/ANB 020), and has a comparable neutralizing activity at the cellular level to Etkimab/ANB 020.
Interleukin-33 targeting monoclonal antibody drug (itemekimab/REGN 3500) developed by samnofei corporation is intended for the treatment of inflammatory diseases such as chronic obstructive pulmonary disease (clinical stage III), asthma (clinical stage II) and the like, and Etokimab/ANB020 developed by AnaptysBio corporation is intended for chronic sinusitis (clinical stage II).
The monoclonal antibody of the present application shows a neutralizing activity at a cellular level comparable to that of Etokimab/ANB020 (prepared according to the expression of the sequence disclosed in the patent publication), which is expected to exhibit a good clinical effect in the prevention and treatment of related diseases.
Drawings
FIG. 1 is a diagram showing the results of nucleic acid electrophoresis for constructing a QX007N (HZD 78-70) transient expression plasmid.
Wherein M: a Marker; strip 1: PCR product 78VH-Hu25; strip 2: pQX2.1, hindIII/NheI; strip 3: PCR product 78VK-Hu3-CK; strip 4: pQX1 HindIII/BamHI.
FIG. 2 is a transient expression flow chart.
FIG. 3 is an electrophoretically detected pattern of QX007N (HZD 78-70).
FIG. 4 is a graph showing that neutralization of recombinant human interleukin-33 by QX007N (HZD 78-70) and Etkimab/ANB 020 induces HEK Blue TM Activity pattern of NF-. Kappa.B/AP-1 signaling in IL-33 cells.
FIG. 5 is a graph showing that neutralization of native human interleukin-33 by QX007N (HZD 78-70) and Etkimab/ANB 020 induces HEK Blue TM Activity pattern of NF-. Kappa.B/AP-1 signaling in IL-33 cells.
FIG. 6 is a graph showing the activity of QX007N (HZD 78-70) and EtOkimab/ANB020 in neutralizing recombinant human interleukin-33 to induce KU812 cells to release IL-5.
FIG. 7 is a graph showing the activity of QX007N (HZD 78-70) and EtOkimab/ANB020 in neutralizing recombinant human interleukin-33 to induce IFN-gamma release from human whole blood.
Detailed Description
Exemplary embodiments of the application are described below, including various details of embodiments of the application to facilitate understanding, which should be considered as merely exemplary. Accordingly, those of ordinary skill in the art will recognize that various changes and modifications of the embodiments described herein can be made without departing from the scope and spirit of the application. Also, descriptions of well-known functions and constructions are omitted in the following description for clarity and conciseness.
The application provides an affinity purification method for reducing the protein content of host cells in the production of anti-human interleukin-33 monoclonal antibody, which comprises the following steps:
Equilibrium before loading: balancing the affinity chromatography medium by using a buffer solution, thereby obtaining a balanced affinity chromatography medium;
loading: combining the anti-human interleukin-33 monoclonal antibody fermentation liquor with the balanced affinity chromatography medium for loading;
eluting: then, pre-eluting and final eluting are carried out successively, so that host cell proteins are removed, and a purified anti-human interleukin-33 monoclonal antibody is obtained;
the anti-human interleukin-33 monoclonal antibody comprises three heavy chain complementarity determining regions, CDR-H1, CDR-H2 and CDR-H3, and three light chain complementarity determining regions, CDR-L1, CDR-L2 and CDR-L3, wherein:
CDR-H1 has the sequence as set forth in SEQ ID NO:1 (SYHMI);
CDR-H2 has the amino acid sequence as set forth in SEQ ID NO:2 (VIYPNSNIYYATWAKG);
CDR-H3 has the sequence as set forth in SEQ ID NO:3 (TIYVHVYSALSI);
CDR-L1 has the sequence as set forth in SEQ ID NO:4 (QASESVLNEVS);
CDR-L2 has the sequence as set forth in SEQ ID NO:5 (FASKLAS);
CDR-L3 has the sequence as set forth in SEQ ID NO:6 (QQDWSMDNIDNA).
In the present application, CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, CDR-L3 represent a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, a light chain CDR1, a light chain CDR2, a light chain CDR3, respectively.
In the present application, "monoclonal antibody" means an antibody obtained from a population of substantially homologous antibodies, i.e., the individual antibodies comprising the population are identical and/or bind to the same epitope, except for possible variant antibodies (e.g., containing naturally occurring mutations or produced during production of monoclonal antibody preparations), which are typically present in minor amounts. Unlike polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being derived from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies to be used according to the application can be prepared by a variety of techniques including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods using transgenic animals comprising all or part of a human immunoglobulin locus, such methods and other exemplary methods of preparing monoclonal antibodies are described herein.
In the present application, a "monoclonal antibody" is generally a human antibody, which can be prepared using techniques well known to those skilled in the art, e.g., the human antibody is generally described in van Dijk, m.a. and van de Winkel, J.G., curr.Opin.Pharmacol.5:368-374 (2001) and Lonberg, n., curr.opin.immunol.20:450-459 (2008).
Antibodies can be prepared by administering an immunogen to transgenic animals that have been modified to stimulate production of intact human antibodies or intact antibodies with human variable regions upon challenge with the antigen, typically containing a portion or all of the human immunoglobulin loci that replace endogenous immunoglobulin loci, either extrachromosomally or randomly integrated within the animal. In such transgenic mice, the endogenous immunoglobulin loci have generally been inactivated, see Lonberg, n., nat. Biotech (natural biotechnology) 23:1117-1125 (2005) for a review of methods for obtaining human antibodies from transgenic animals. See also XENOMOUSE, e.g., as described in U.S. Pat. Nos. 6,075,181 and 6,150,584 TM A technique; described in U.S. Pat. No.5,770,429A technique; U.S. Pat. No.7,041,870 describes +.>Technology, and U.S. patent application publication No. US 2007/0061900 describes +. >Techniques. Human variable regions from whole antibodies produced by such animals may be further modified, for example, by combining with different human constant regions.
Human antibodies can also be prepared by hybridoma-based methods. Human myeloma and mouse-human hybridoma cells for the production of human monoclonal antibodies have been described (see, e.g., kozbor, d., j. Immunol.133:3001-3005 (1984); brodeur, b. R. Et al, monoclonal Antibody Production Techniques and Applications, marcel Dekker, inc., new York (1987), pp.51-63; and Boerner, p. Et al., j. Immunol.147:86-95 (1991)). Human antibodies produced via human B cell hybridoma technology are also described in Li, j.et al, proc.Natl. Acad.Sci.USA103:3557-3562 (2006). Other methods include those described, for example, in U.S. Pat. No.7,189,826 (which describes the generation of monoclonal human IgM antibodies from hybridoma cell lines), ni, xiandai Mianyixue,26 (4); 265-268 (which describe human-human hybridomas). Human hybridoma technology (Trioma technology) is also described in Vollmers, h.p. and Brandlein, s. Histology and Histopathology 20:927-937 (2005), h.p. and Brandlein, s. Methods and Findings in Experimentaland Clinical Pharmacology 27:185-191 (2005).
Human antibodies can also be generated by isolating Fv clone variable domain sequences selected from phage display libraries derived from humans, and then such variable domain sequences can be combined with the desired human constant domain.
Human antibodies may also be selected based on libraries of antibodies, i.e., they may be isolated by screening combinatorial libraries for antibodies having the desired activity or activities. For example, a variety of methods for producing phage display libraries and screening such libraries for antibodies possessing desired binding characteristics are known in the art. Such methods are reviewed in, for example, hoogenboom, H.R. et al, methods in Molecular Biology 178:178:1-37 (2001), and further described in, for example, mcCafferty, J.et al, nature 348:552-554 (1990); clackson, T.et al, nature 352:624-628 (1991); marks, J.D.et al, J.mol.biol.222:581-597 (1992); marks, J.D. and Bradbury, A., methods in Molecular Biology 248:161-175 (2003); sidhu, S.S. et al, J.mol.biol.338:299-310 (2004); lee, C.V.et al, J.mol.biol.340:1073-1093 (2004); felloise, F.A., proc.Natl.Acad.Sci.USA 101:12467-12472 (2004); and Lee, C.V.et al, J.Immunol.methods 284:119-132 (2004).
In some phage display methods, a complete set of VH and VL genes are cloned separately by Polymerase Chain Reaction (PCR) and randomly recombined in a phage library, and antigen-binding phages are then screened in the phage library, as described in Winter, g.et al, ann.rev.immunol.12:433-455 (1994). Phages typically display antibody fragments as single chain Fv (scFv) fragments or as Fab fragments. Libraries from immunized sources provide high affinity antibodies to immunogens without the need to construct hybridomas. Alternatively, a repertoire of non-immunity can be cloned (e.g., from humans) to provide a single source of antibodies to a large array of non-self and also self-antigens in the absence of any immunization, as described by Griffiths, a.d. et al, EMBO J,12:725-734 (1993). Finally, non-immune libraries can also be generated synthetically by cloning unrearranged V gene segments from stem cells, and encoding highly variable CDR3 regions using PCR primers containing random sequences and effecting rearrangement in vitro, as described by Hoogenboom, H.R.and Winter, G.J.mol.biol.227:381-388 (1992). Patent publications describing human antibody phage libraries include, for example: U.S. patent No.5,750,373 and U.S. patent publications nos. 2005/007574, 2005/019455, 2005/0266000, 2007/017126, 2007/0160598, 2007/027764, 2007/0292936 and 2009/0002360.
The antibody may also be a multispecific antibody, e.g., a bispecific antibody. Bispecific antibodies are monoclonal antibodies that have binding specificities for at least two different sites. Techniques for generating multispecific antibodies include, but are not limited to, recombinant co-expression of two pairs of immunoglobulin heavy-light chains with different specificities (see Milstein, c.and Cuello, a.c., nature305:537-540 (1983); WO 93/08829; and Traunecker, a.et al., EMBO j.10:3655-3659 (1991)), and "section-in-hole" engineering (see, e.g., U.S. Pat. No.5,731,168). It is also possible to generate engineered electrostatic steering effects of antibody Fc-heterodimer molecules (WO 2009/089004); crosslinking two or more antibodies or fragments (see, e.g., U.S. Pat. No.4,676,980 and Brennan, M.et al., science 229:81-83 (1985)); bispecific antibodies were generated using leucine zippers (see, e.g., kostelny, s.a.et al, j.immunol.148:1547-1553 (1992)); the "diabody" technique for the generation of bispecific antibody fragments is used (see, e.g., holliger, p.et al., proc. Natl. Acad. Sci. USA90:6444-6448 (1993)); and the use of single chain Fv (scFv) dimers (see, e.g., gruber, M.et al, J.Immunol.152:5368-5374 (1994)); and preparing a trispecific antibody (as described, for example, in Tutt, A.et al, J.Immunol.147:60-69 (1991)) to produce a multispecific antibody.
Monoclonal antibodies described herein also include engineered antibodies having three or more functional antigen binding sites, including "octopus antibodies" (see, e.g., US 2006/0025576).
Antibodies herein also include multispecific antibodies described in WO 2009/080251, WO 2009/080252, WO2009/080253, WO 2009/080254, WO 2010/112193, WO 2010/115589, WO2010/136172, WO 2010/145792, WO 2010/145793, WO 2011/117330, WO 2012/025525, WO 2012/025530, WO 2013/026835, WO2013/026831, WO 2013/164325, or WO 2013/174873.
The monoclonal antibodies described herein may also be antibody variants, e.g., where it may be desirable to improve the binding affinity and/or other biological properties of the antibodies. Amino acid sequence variants of antibodies can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of, residues within the amino acid sequence of an antibody. Any combination of deletions, insertions, and substitutions may be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, such as antigen binding. Thus, in certain embodiments, antibody variants having one or more amino acid substitutions are provided, the sites of interest for substitution mutations include HVRs and FRs, e.g., amino acid substitutions can be introduced into an antibody of interest and products selected for desired activity, e.g., retained/improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.
In the present application, the mammalian cells used for the in vitro fermentation production of the monoclonal antibodies include, but are not limited to, various hybridoma cells, chinese hamster ovary Cells (CHO), preferably CHO cells, which are currently in common use.
In the present application, "host cell", "host cell line" and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include primary transformed cells and the progeny derived therefrom (regardless of the number of passages). The progeny may not be exactly identical in nucleic acid content to the parent cell, but may contain a mutation. Mutant progeny screened or selected for the same function or biological activity for the originally transformed cells are included in the specification.
In the present application, "affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, "binding affinity" as used herein refers to an inherent binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can generally be determined by the equilibrium dissociation constant (K D ) And (3) representing. Affinity can be measured by common methods known in the art.
In the application, "ultrafiltration" refers to tangential flow ultrafiltration (Tangential Flow Filtration, TFF) which refers to a filtration mode in which the flow direction of liquid is tangential (parallel) to the surface of a filter membrane, and compared with the traditional filtration mode (NFF) in which the flow direction is perpendicular to the filter membrane, the tangential flow filter membrane has less particle accumulation on the surface, stable filtration speed and is suitable for separating a large-volume sample. Wherein, molecules larger than the aperture of the membrane are intercepted and concentrated gradually, substances smaller than the aperture of the membrane penetrate the membrane and are separated from the macromolecular solution, thus realizing the separation of macromolecules and small molecules. Tangential flow ultrafiltration is thus commonly used for concentration of biological products, dialysis, displacement of buffer solutions, separation of molecules of different sizes, etc.
In the present application, human interleukin-33 means that human interleukin-33 located in the nucleus is hydrolyzed by protease to form mature human interleukin-33, which is secreted to the outside of the cell, exerts the biological activity of human interleukin-33, and has the amino acid sequence as shown in SEQ ID NO: 9.
SEQ ID NO：9：
SITGISPITEYLASLSTYNDQSITFALEDESYEIYVEDLKKDEKKDKVLLSYYESQHPSNESGDGVDGKMLMVTLSPTKDFWLHANNKEHSVELHKCEKPLPDQAFFVLHNMHSNCVSFECKTDPGVFIGVKDNHLALIKVDSSENLCTENILFKLSET
In the present application, the expression "anti-human interleukin-33 monoclonal antibody" means a monoclonal antibody as follows: which is capable of binding human interleukin-33 with sufficient affinity such that the monoclonal antibody is useful as a diagnostic and/or therapeutic agent for targeting human interleukin-33.
In the present application, the anti-human interleukin-33 monoclonal antibody does not bind to a target-independent protein. Herein, "unrelated protein" refers to a protein other than human interleukin-33 as a target; here, "not bonded" means: in the case where the binding capacity of the anti-human IL-33 monoclonal antibody of the application to human interleukin-33 as its target is taken as 100%, the binding capacity of the anti-human IL-33 monoclonal antibody of the application to the unrelated protein is less than 10%, for example 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or 0.
The anti-human IL-33 monoclonal antibody of the application may bind to interleukin-33 of a human, cynomolgus monkey, or may not bind to interleukin-33 of other animal species. Herein, "other animal species" refers to animal species other than humans, cynomolgus monkeys, such as pigs, dogs, rabbits, rats, mice, guinea pigs, etc.; here, "unbound" when determining the species specificity of the anti-human IL-33 monoclonal antibody of the application means: in the case where the binding capacity of the anti-human IL-33 of the present application to human interleukin-33 as its target is taken as 100%, the binding capacity of the anti-human IL-33 monoclonal antibody of the present application to interleukin-33 of other animal species is less than 5%, for example, 4%, 3%, 2%, 1% or 0.
The anti-human IL-33 monoclonal antibody of the present application has an equilibrium dissociation constant (K) of 1. Mu.M, 100nM, 50nM, 40nM or less D )。
Experimental results show that the anti-human IL-33 monoclonal antibody can specifically bind to human interleukin-33.
The anti-human IL-33 monoclonal antibody provided by the application is equivalent to or better than the same kind of monoclonal antibody products on the market in terms of various biological activities. Such as neutralizing the activity of recombinant/native human interleukin-33 to induce NF- κB/AP-1 signaling in cells, neutralizing the activity of interleukin-33 to induce KU812 cells to release IL-5, neutralizing interleukin-33 to induce human whole blood to release IFN- γ, etc.
In one embodiment, the heavy chain of the anti-human IL-33 monoclonal antibody of the application has an amino acid sequence as set forth in SEQ ID NO:10 is shown in the figure; the amino acid sequence of the light chain is shown in SEQ ID NO: 11.
SEQ ID NO：10：
EVQLVESGGGLVQPGGSLRLSCAASGFSLSSYHMIWVRQAPGKGLEWVGVIYPNSNIYYATWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARTIYVHVYSALSIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID NO：11：
AFQMTQSPSSVSASVGDRVTITCQASESVLNEVSWYQQKPGKAPKLLIYFASKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQDWSMDNIDNAFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
Wherein, SEQ ID NO:10 and 11 are both humanized sequences.
In one embodiment, the anti-human interleukin-33 monoclonal antibody comprises a heavy chain variable region and a light chain variable region, wherein,
the heavy chain variable region has the amino acid sequence as set forth in SEQ ID NO:7 (EVQLVESGGGLVQPGGSLRLSCAASGFSLSSYHMIWVRQAPGKGLEW VGVIYPNSNIYYATWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA RTIYVHVYSALSIWGQGTLVTVSS);
The light chain variable region has the amino acid sequence as set forth in SEQ ID NO:8 (AFQMTQSPSSVSASVGDRVTITCQASESVLNEVSWYQQKPGKAPKLLI YFASKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQDWSMDNID NAFGGGTKVEIK).
In a specific embodiment, the affinity chromatography medium is selected from one of chromatography media with ligands crosslinked to agarose, polyvinyl ether, hydroxylated polyether resin, polyacrylic resin, polystyrene divinylbenzene resin, polymethacrylic resin, polystyrene resin, hydroxyapatite, glass matrix, preferably chromatography media with ligands crosslinked to polyvinyl ether;
preferably, the ligand is Protein A, protein G or Protein L, more preferably Protein A.
The ligands of the application may specifically bind to monoclonal antibodies.
In the present application, there is no limitation on the affinity filler, which can be confirmed according to the needs of those skilled in the art, for example, the affinity filler can be MabSelect, mabSelect Sure, botryn Biotechnology Co., ltd., protein A Diamond, MERCK of GE healthcareA。
In one embodiment, buffer one includes, but is not limited to, one of phosphate buffer, tris-HCl buffer, and boric acid-borax buffer. The salt concentration in the buffer one is not limited in any way, and one skilled in the art can select the salt concentration according to the need, for example, in one embodiment, the salt concentration in the buffer one is 5 mM-0.25M, for example, 5mM, 10mM, 20mM, 50mM, 0.1M, 0.15M, 0.2M, 0.25M, etc.; the pH of the buffer solution is 5.5 to 8.0, and may be, for example, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, etc.
In one embodiment, the buffer one is a phosphate buffer or a Tris-HCl buffer.
In a preferred embodiment, naCl or Na is added to the buffer one 2 SO 4 To reduce non-specific adsorption between non-antibody proteins and fillers.
In one embodiment, the buffer one is phosphate buffer, and NaCl or Na is added into the buffer one 2 SO 4 To reduce non-specific adsorption between the non-antibody protein and the filler, the salt concentration of the phosphate buffer is between 5mM and 0.15M, preferably between 10 and 50mM, more preferably 20mM.
The pH of the buffer I is not limited in any way, and may be selected by one skilled in the art as desired, for example, in one embodiment, the pH of the buffer I is from 6.5 to 7.5, preferably 6.9.
For NaCl or Na 2 SO 4 The amount of (C) added, which can be determined according to the needs of those skilled in the art, for NaCl or Na 2 SO 4 The concentration of (2) may be selected as desired by one skilled in the art, and in one embodiment, naCl or Na 2 SO 4 The concentration of (C) may be 0 to 250mM, preferably 150mM.
In one embodiment, the phosphate buffer may be one or both of disodium hydrogen phosphate buffer and sodium dihydrogen phosphate buffer.
In one embodiment, the equilibration is performed using buffer one when anti-human interleukin-33 monoclonal antibody fermentation broth is combined with the equilibrated affinity chromatography medium.
In one embodiment, the pre-elution is performed with a pre-elution buffer, which is a neutral buffer and/or an acidic buffer.
In a specific embodiment, the neutral buffer is selected from one of a phosphate buffer, a tris buffer and a glycine buffer, and the acidic buffer is selected from one of a citric acid-disodium hydrogen phosphate buffer, an acetic acid-sodium acetate buffer and a citric acid-trisodium citrate buffer.
The pH of the pre-elution buffer is not limited in any way, and may be selected by those skilled in the art as desired, for example, in one embodiment, the pH of the pre-elution buffer is 5.0 to 7.5, for example, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, etc., and more preferably 5.5 to 6.5.
In one embodiment, when pre-eluting with a pre-elution buffer, a pre-elution active agent is also added to the pre-elution buffer; the pre-elution active agent includes, but is not limited to, one or more of guanidine hydrochloride, polysorbate 80 and sodium chloride, and preferably, the pre-elution active agent is guanidine hydrochloride.
The concentration of guanidine hydrochloride is not limited in any way, and may be selected by those skilled in the art according to the need, and for example, in one embodiment, the concentration of guanidine hydrochloride may be, for example, 0.01M, 0.05M, 0.1M, 0.15M, 0.2M, 0.3M, 0.4M, 0.5M, 0.6M, 0.7M, 0.8M, 0.9M, 1M, etc., preferably 0.05 to 0.15M, and more preferably 0.1M.
The salt concentration in the pre-elution buffer is not limited in any way, and may be selected by those skilled in the art as desired, for example, in one embodiment, the salt concentration in the pre-elution buffer is 0 to 0.5M, preferably 0.1M.
In one embodiment, the final elution is performed with a final elution buffer; the final elution buffer includes, but is not limited to, one or two or more of citric acid-disodium hydrogen phosphate buffer, acetic acid buffer, glycine-HCl buffer, and citric acid-sodium citrate buffer, preferably citric acid-disodium hydrogen phosphate buffer.
The pH of the final elution buffer is not limited in any way and may be selected by one skilled in the art as desired, for example, in one embodiment, the pH of the final elution buffer is 2.9 to 3.8, e.g., 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, etc.
The salt concentration in the final elution buffer is not limited in any way, and one skilled in the art can select the salt concentration as desired, for example, in one embodiment, the salt concentration in the second elution solution is 5 to 100mM, for example, 5mM, 10mM, 20mM, 30mM, 40mM, 50mM, 60mM, 70mM, 80mM, 90mM, 100mM, etc., preferably 10 to 50mM.
For example, the salt concentration in the second elution solution may be 5mM, 10mM, 20mM, 30mM, 40mM, 50mM, 60mM, 70mM, 80mM, 90mM, 100mM, etc.
In one embodiment, the affinity medium may be regenerated after final elution using a regeneration buffer, preferably including, but not limited to, citric acid-disodium hydrogen phosphate buffer, hydrochloric acid, glycine, naOH, preferably citric acid-disodium hydrogen phosphate buffer and NaOH solution.
In one embodiment, the eluting step, after pre-eluting, before final eluting, further comprises equilibration of the pre-eluted solution with a buffer.
In a specific embodiment, the buffer II is phosphate buffer, tris-HCl buffer or boric acid-borax buffer.
In a preferred embodiment, the buffer II is one of a phosphate buffer, a Tris-HCl buffer and a boric acid-borax buffer.
In a preferred embodiment, naCl or Na is added to the buffer II 2 SO 4 To maintain conductance while maintaining partial buffering capacity.
The salt concentration in the buffer II and the pH of the buffer II are not limited in any way, and may be selected as desired by those skilled in the art. For example, in one embodiment, the salt concentration in the buffer II is 5 mM-0.15M, preferably 10-50 mM, more preferably 20mM; the pH of the buffer II is 5.5 to 8.0, preferably 6.5 to 7.5, and more preferably 7.2.
For NaCl or Na 2 SO 4 The amount of (C) added, which can be determined according to the needs of those skilled in the art, for NaCl or Na 2 SO 4 The concentration of (2) may be selected as desired by one skilled in the art, and in one embodiment, naCl or Na 2 SO 4 The concentration of (C) may be 0 to 250mM, preferably 10mM.
In a specific embodiment, the phosphate buffer may be, for example, a buffer of disodium hydrogen phosphate and sodium dihydrogen phosphate.
The affinity purification process is simple and feasible, can carry out amplified purification production, does not need pre-treatment of cell fermentation supernatant, has higher elution sample yield, and simultaneously maintains the residual HCP at a lower level (the residual control amount is not higher than 0.1 percent), thereby reducing the pressure of removing HCP in the subsequent purification step, and further ensuring that the residual HCP of the final sample of the antibody is at an extremely low level. Meanwhile, the affinity purification process verification of fermentation supernatants of different batches proves that the affinity purification process has good stability.
Examples
Hereinafter, the present application will be described more specifically with reference to examples. It should be understood that the present application is not limited to these embodiments.
Example 1 preparation of anti-human Interleukin-33 monoclonal antibody QX007N
Human interleukin-33 (hIL-33) is purchased from Shanghai offshore technology Co., ltd for immunization of New Zealand rabbits, and antigen-binding specific antibody clones are obtained by using B cell cloning technology, so that monoclonal antibodies which bind to human interleukin-33 and have human interleukin-33 inhibitory activity are screened out. Firstly, detecting cell supernatant by using Binding ELISA, and selecting clones combined with human interleukin-33; reuse of HEK Blue TM IL-33 reporter gene cell method is used for detection, and clones with human interleukin-33 inhibiting activity are selected. The above immunization and screening process is delegated to commercial companies.
12 clones were picked up successively for recombinant expression and sequenced. The cell neutralization activity of the 78# is determined to be optimal, and the 78# is subjected to humanized modification. Human IgG germ line sequence (Germline) homology alignment was performed using NCBI IgBlast, IGHV 3-66.01 was selected as a heavy chain CDR-grafting template, and CDR regions of the 78# cloned heavy chain (i.e., CDR-H1 (SEQ ID No: 1), CDR-H2 (SEQ ID No: 2) and CDR-H3 (SEQ ID No: 3)) were grafted into the framework regions of IGHV 3-66.01; IGKV1-12 x 01 is selected as a light chain CDR grafting template, and CDR regions (namely CDR-L1 (SEQ ID No: 4), CDR-L2 (SEQ ID No: 5) and CDR-L3 (SEQ ID No: 6)) of a 78# cloned light chain are grafted into a framework region of the IGKV1-12 x 01; and carrying out back mutation on the specific site of the framework region to obtain the variable region of the monoclonal antibody QX 007N. Finally, the amino acid sequence of the humanized heavy chain variable region is shown in SEQ ID NO: shown in figure 7; the amino acid sequence of the humanized light chain variable region is shown as SEQ ID NO: shown at 8.
The heavy chain variable region (SEQ ID NO: 7) gene and the light chain full length (SEQ ID NO: 11) gene were obtained by PCR amplification. Double cleavage of the heavy chain expression plasmid pQX2.1 with HindIII and NheI; transient expression plasmid pQX1 was digested with HindIII and BamHI; the PCR amplified genes are respectively inserted into corresponding expression plasmids by using an information recombinase to construct a heavy chain expression plasmid pQX2.1-78VH-Hu25 and a light chain expression plasmid pQX2.2-78VK-Hu3. Wherein pQX2.2 refers to the pQX1 plasmid expressing the light chain.
The results of double digestion of the plasmid detected by nucleic acid electrophoresis are shown in FIG. 1. From the results of FIG. 1, it can be seen that the results of the full length PCR amplification of the heavy and light chain of the antibody and the results of double cleavage of the heavy and light chain expression plasmids, wherein the plasmid size of the heavy and light chain was about 5000bp, the heavy chain variable region was about 480bp, and the full length of the light chain was about 781bp.
The correct sequence heavy chain expression plasmid pQX2.1-78VH-Hu25 (the full-length amino acid sequence of the expressed heavy chain is shown as SEQ ID NO: 10) and the light chain expression plasmid pQX2.2-78VK-Hu3 (the full-length amino acid sequence of the expressed light chain is shown as SEQ ID NO: 11) are co-transfected into an ExpiCHO-S cell. The day before transfection, the ExpiCHO-S cells were diluted to 3X 10 6 Each cell/ml was passaged before transfection. On the day of transfection, cell densities were diluted to 6X 10 6 25ml cells were shake-bottled at 125ml per cell/ml and waited for transfection. The transfection and expression process is shown in FIG. 2.
On day 5 post transfection, culture supernatants were harvested and purified in one step using Protein A. Purified antibodies were detected by SDS-PAGE, designated QX007N (HZD 78-70), and the results of detection using protein electrophoresis are shown in FIG. 3. The results of FIG. 3 show that there are two bands of about 50kDa and 25kDa in size, respectively, consistent with the theoretical molecular weights of the heavy (49.3 kDa) and light (23.4 kDa) chains, as measured by denaturing reducing gels.
EXAMPLE 2 equilibrium dissociation constant (K D ) Is (are) determined by
The affinity of QX007N (HZD 78-70) for human interleukin-33 was measured with Biacore T200 and all procedures were performed at 25 ℃. A commercial Protein A chip is adopted, and a proper amount of antibody is fixed by a capturing method, so that Rmax is about 50RU, and the capturing flow rate is 10 mu l/min. The antigen was subjected to gradient dilution, the instrument flow rate was switched to 30. Mu.l/min, and the flow was sequentially from low to high concentration through the reference channel and the channel for immobilized antibody, and through the buffer as a negative control. After completion of each binding and dissociation, the chip was regenerated with glycine at pH 1.5. The 1:1 binding model in the Kinetics option is selected for fitting by using the self-contained analysis software of the instrument, and the binding rate constant k of the antibody is calculated a Dissociation rate constant k d Equilibrium dissociation constant K D Values.
In addition, the affinity of QX007N (HZD 78-70) to Eokimab/ANB 020, a monoclonal antibody against human interleukin-33 developed by AnaptysBio, was compared, and the detection method for the known antibody was the same as that for QX007N, and the results are shown in Table 1. Wherein Etrokimab/ANB 020 is prepared by constructing an expression plasmid and transiently transferring an ExpiCHO-S cell according to an APE4909 sequence provided by patent WO2015106080A 2.
TABLE 1 affinity of anti-human interleukin-33 antibodies for binding human interleukin-33
| Sample name | k a (10 5 M -1 S -1 ) | k d (10 -4 S -1 ) | K D (10 -10 M) |
| QX007N | 3.92 | 4.81 | 12.28 |
| ANB 020 | 4.24 | 3.69 | 8.69 |
Furthermore, based on the same detection method as described above, we have also found that Etokimab/ANB020 can bind to interleukin-33 of cynomolgus monkey, rhesus monkey, while QX007N (HZD 78-70) can bind to interleukin-33 of cynomolgus monkey, but not interleukin-33 of rhesus monkey.
Example 3 neutralization of human interleukin-33 induced HEK Blue TM Activity detection of IL-33 cell NF- κB/AP-1 Signal transduction
HEK Blue TM IL-33 cells are produced by stable transfection of human embryonic kidney cells HEK 293 with the human IL1RL1 gene and the response of TNF-alpha and IL-1β is blocked, therefore HEK-Blue TM IL-33 cells respond specifically to IL-33. Interleukin-33 binds to cell surface IL-1RL1/IL-1RAcP triggering a signaling cascade leading to NF-. Kappa.B/AP-1 signaling and production of secreted alkaline phosphatase (secreted alkaline phosphatase, SEAP), thereby detecting the biological activity of interleukin-33 or performing antibody screening.
By HEK Blue TM IL-33 cells assayed for neutralizing activity of QX007N (HZD 78-70) on human interleukin-33. HEK Blue TM IL-33 cells at 4X 10 per well 4 The individual cells were seeded into 96 wells at 37℃and 5% CO 2 The culture was carried out overnight under the conditions. Diluting the antibody to a concentration ranging from 0 to 500ng/ml, mixing the diluted solution with 2ng/ml recombinant human interleukin-33, incubating for 1h, adding into cells at 37deg.C and 5% CO after incubation 2 Culturing under the condition for 24 hours, collecting cell culture supernatant, and adding QUANTI-Blue at a ratio of 1:10 TM In detection reagent (InvivoGen, rep-qbs 2) and reacted at 37℃for 1 hour using VariOD detection by oskan LUX multifunctional enzyme labeling instrument 630nm Values, data were analyzed using a four parameter curve fit using softMaxPro software (fig. 4), and the antagonistic activity of the antibodies was analyzed.
The results of fig. 4 show that: QX007N (HZD 78-70) can inhibit HEK Blue induction by recombinant human interleukin-33 TM NF-. Kappa.B/AP-1 Signal transduction in IL-33 cells, IC thereof 50 6.67ng/ml; in contrast, for Eokimab/ANB 020, IC was measured by the same method 50 6.05ng/ml.
Example 4 neutralization of native human interleukin-33 induced HEK Blue TM Activity detection of IL-33 cell NF- κB/AP-1 Signal transduction
Natural human interleukin-33 was prepared and the neutralizing activity of QX007N (HZD 78-70) on natural human interleukin-33 was verified. Culturing HFL-1 cells, inducing with 200ng/ml TNF-alpha for 24 hr, collecting cells, lysing cells by repeated freeze thawing method, collecting supernatant containing human interleukin-33, and culturing with HEK Blue TM IL-33 cells demonstrated activity.
HEK Blue TM IL-33 cells at 4X 10 per well 4 The individual cells were seeded into 96 wells at 37℃and 5% CO 2 Culturing overnight under condition, diluting antibody to a concentration ranging from 0 to 1000ng/ml, adding diluted solution and natural human interleukin-33, mixing, adding into cells at 37deg.C and 5% CO 2 Culturing under the condition for 24 hours, collecting cell culture supernatant, and adding QUANTI-Blue at a ratio of 1:10 TM In the detection reagent, the reaction is carried out for 1 hour at 37 ℃, and the OD is detected by using a Varioskan LUX multifunctional enzyme-labeled instrument 630nm Values, data were analyzed using a 4 parameter curve fit using SoftMax Pro software (fig. 5), and the neutralizing activity of the antibodies was analyzed.
The results of fig. 5 show that: QX007N (HZD 78-70) can inhibit natural human interleukin-33 from inducing HEK Blue TM NF-. Kappa.B/AP-1 Signal transduction in IL-33 cells, IC thereof 50 3.91ng/ml; in contrast, for Eokimab/ANB 020, IC was measured by the same method 50 2.5ng/ml.
EXAMPLE 5 Activity assay for neutralizing human interleukin-33-induced KU812 (human peripheral blood basophilic leukemia cells) to release IL-5
The activity of neutralizing human interleukin-33 was evaluated using human interleukin-33-induced KU812 (human peripheral blood basophilic leukemia cells) to release IL-5 as an index, and QX007N (HZD 78-70). KU812 cells (2X 10) were seeded in 96-well plates 5 Individual cells/well), followed by addition of antibody and recombinant human interleukin-33 (final concentration 4 ng/ml) at 37℃and 5% CO 2 Culturing under conditions for 24 hours, collecting cell culture supernatant, and performing Human IL-5DuoSet ELISA (R&D, DY 205) to detect the expression level of IL-5 in the supernatant, and OD was detected using a Varioskan LUX multifunctional microplate reader 450nm Values, data were analyzed using a 4 parameter curve fit using SoftMax Pro software (fig. 6), and the neutralizing activity of the antibodies was analyzed.
The results of fig. 6 show that: QX007N (HZD 78-70) is capable of neutralizing IL-5 releasing activity of human interleukin-33 induced KU812 (human peripheral blood basophilic leukemia cells), IC thereof 50 5.87ng/ml; in contrast, for Eokimab/ANB 020, IC was measured by the same method 50 44ng/ml.
Example 6 Activity assay for neutralizing human interleukin-33 to induce human Whole blood to release IFN-y
The neutralization activity of QX007N (HZD 78-70) was further characterized by using mononuclear cells from human whole blood as the basis of the assay and IFN-gamma as the assay index. Whole blood plates (100. Mu.L/well) from healthy volunteers were used followed by the addition of antibody and recombinant human interleukin-33 (final concentration 4 ng/ml) at 37℃and 5% CO 2 Culturing under the condition for 24 hours, detecting OD by using Varioskan LUX multifunctional enzyme labeling instrument 450nm Values, data were analyzed using a 4 parameter curve fit using SoftMax Pro software (fig. 7), and the neutralizing activity of the antibodies was analyzed.
The results of fig. 7 show that: QX007N (HZD 78-70) can neutralize the activity of human interleukin-33 to induce human whole blood to release IFN-gamma, and the IC50 is 16ng/ml; in contrast, for Eokimab/ANB 020, IC was measured by the same method 50 31.9ng/ml.
EXAMPLE 7 comparison of the effect of different buffers on the removal of HCP from recombinant humanized anti-human Interleukin-33 monoclonal antibody (QX 007N) fermentation broths
Preparing fermentation liquid of an anti-human interleukin-33 monoclonal antibody:
using CHO cells as host cells and Eden-B600 as a fermentation basal medium, the antibody QX007N obtained in example 1 was produced. Cell culture was performed using a conventional cell culture process, harvesting was started when the cell viability was lower than 80% or when the culture was up to 18 days, the harvest was subjected to depth filtration using a primary filter MD0HC10FS1 and a secondary filter MX0HC10FS1, and clarified cell culture supernatant was collected, thereby obtaining a fermentation broth of anti-human interleukin-33 monoclonal antibody (referred to as fermentation broth intermediate for short).
Balancing the affinity chromatography medium by using a buffer solution, thereby obtaining a balanced affinity chromatography medium; then combining the anti-human interleukin-33 monoclonal antibody fermentation broth with the balanced affinity chromatography medium, specifically as follows:
Buffer one (12 mmol/L Na) 2 HPO 4 ，8mmol/L NaH 2 PO 4 Balancing Protein A chromatographic column (AT Protein A Diamond Plus, 0.15L) with 0.15mol/L NaCl), loading the intermediate of the QX007N fermentation liquor on the balanced Protein A chromatographic column to combine with the intermediate of the QX007N fermentation liquor, setting the loading capacity to 55mg/mL, and balancing by using buffer solution I until the fermentation liquor completely flows through the chromatographic column;
then pre-elution was performed using a pre-elution buffer with a composition as shown in Table 2 followed by buffer two (6 mmol/L Na 2 HPO 4 、4mmol/L NaH 2 PO 4 pH 7.2) to equilibrate;
next, a final elution is performed to collect the sample and the collected sample is subjected to an antibody concentration and a residual content of host protein (HCP), wherein the final elution is performed using a final elution buffer of 7mmol/L Na 2 HPO 4 15mmol/L citric acid, pH 3.1. And the chromatographic column was regenerated with regeneration buffer (100 mmol/L NaOH,1mol/L NaCl). And collecting an eluted sample for measuring the concentration of the antibody and the residual content of HCP.
TABLE 2 pre-elution buffer composition
The method for measuring the concentration of the antibody is as follows:
1. the spectrophotometer wavelength was tuned to 280nm and zeroing was performed using final elution buffer as a control.
2. The sample to be measured was diluted with a final elution buffer, the absorbance of the sample at 280nm was measured (absorbance was guaranteed to be between 0.5 and 1.5), and the concentration of the sample was calculated according to the following formula (QX 007N extinction coefficient of 1.513).
The results obtained are shown in Table 3.
Method for determining HCP content:
1. sample dilution: the dilution ratio is generally selected based on the estimated HCP concentration in the sample, so that the final HCP concentration falls within the standard curve range (generally 10 ng/mL-80 ng/mL). The one-step dilution of the sample is not more than 10 times, and the minimum sampling amount is not less than 5 mu L.
2. 100. Mu.L of Anti-CHO HRP was added to each well of the strip.
3. Loading: adding standard substance, sample and standard sample (each two compound holes, standard substance does not need compound holes) respectively according to a certain arrangement, 50 μl/hole, and sealing plate. The mixture was placed on a horizontal shaker at 180rpm for 2 hours and protected from light.
4. Washing the plate: the liquid in the hole is discarded, 300 mu L/hole of washing liquid is added by a multichannel pipettor, the liquid is thrown off after standing for 30 seconds, the liquid is beaten dry on absorbent paper, and the plate is washed for 4 times. After the last plate washing is completed, residual washing liquid in the holes needs to be dried as much as possible.
5. Color development and termination reading: TMB reagent (TMB Substrate) was added at 100. Mu.L/well, and the mixture was allowed to stand and develop for 30 minutes, and protected from light. After 30 minutes, 100. Mu.l/well of Stop Solution (Stop Solution) was added, and the microplate reader was read at 450nm with 650nm as reference.
6. And selecting analysis software to perform data analysis, and taking the OD value of the standard substance as an ordinate and the concentration as an abscissa to make a four-parameter standard curve. Substituting the OD value measured by the sample into a standard curve to obtain an actual measurement value of the HCP of the added sample.
Cho cell protein residual (%) =average measured value of sample (ng/mL) ×dilution factor/undiluted sample protein content (mg/mL) amount -4 (%) and the results are shown in Table 3.
TABLE 3QX007N affinity purification yield and HCP residual results
As can be seen from the results in Table 3, the residual HCP in the fermentation broth intermediate is more than 25%, and the yield is more than 95% after the treatment of the affinity purification process; for HCP removal, a guanidine hydrochloride-containing experimental group is adopted, the HCP residue of a sample after affinity chromatography is lower than 0.1%, the HCP load is obviously reduced after the subsequent purification process step, and the sample is kept clear when the sample is subjected to the subsequent chromatography process step (anion exchange chromatography) loading after the pH adjustment, so that the sample can be directly injected without other treatment, the process simplicity is enhanced, and the process time is saved; and the HCP residue of the sample after the affinity chromatography is still more than 0.1% by adopting polysorbate 80 or NaCl experimental group, and the HCP residue removal effect is inferior to that of guanidine hydrochloride.
Example 8 comparison of the clearance of HCP from different batches of recombinant humanized anti-IL-33 monoclonal antibody (QX 007N) fermentation broths
Preparation of fermentation liquor intermediate and affinity chromatography method the same as in example 7, three batches of fermentation liquor intermediate were prepared altogether, and the pre-elution was performed using a pre-elution buffer containing guanidine hydrochloride: 0.1mol/L sodium citrate, 0.1mol/L guanidine hydrochloride, 11mmol/L citric acid, and pH 5.8. The yields of the affinity process and the HCP residual content were measured as described in example 7, and the results are shown in Table 4.
TABLE 4 affinity purification yields and HCP residue results for different batches of QX007N
As shown by the results in Table 4, the HCP residues of the intermediates of the QX007N fermentation broths of different batches are all more than 20%, and the cell culture process is stable; the affinity process yields of the three batches of samples are all more than 95%, and the yields meet the process requirements; the HCP residue of the sample after the affinity chromatography is lower than 0.1%, and the removal of HCP is kept stable by adopting guanidine hydrochloride and elution.
In summary, the present application adopts the above-mentioned affinity purification method, and the residual amount of HCP in the obtained monoclonal antibody is kept at a low level, and the residual control amount is not higher than 0.1%, so that the pressure for removing HCP in the subsequent purification step is reduced, and the affinity purification process of the present application has good stability.
The above description is only a preferred embodiment of the present application, and is not intended to limit the application in any way, and any person skilled in the art may make modifications or alterations to the disclosed technical content to the equivalent embodiments. However, any simple modification, equivalent variation and variation of the above embodiments according to the technical substance of the present application still fall within the protection scope of the technical solution of the present application.
Sequence listing
<110> Jiangsu Xin biomedical stock Co., ltd
<120> affinity purification method for reducing host cell protein content in production of anti-human interleukin-33 monoclonal antibody
<130> TPE01862
<150> 2021113006928
<151> 2021-11-4
<160> 11
<170> PatentIn version 3.5
<210> 1
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> Synthesis by hand
<400> 1
Ser Tyr His Met Ile
1 5
<210> 2
<211> 16
<212> PRT
<213> artificial sequence
<220>
<223> Synthesis by hand
<400> 2
Val Ile Tyr Pro Asn Ser Asn Ile Tyr Tyr Ala Thr Trp Ala Lys Gly
1 5 10 15
<210> 3
<211> 12
<212> PRT
<213> artificial sequence
<220>
<223> Synthesis by hand
<400> 3
Thr Ile Tyr Val His Val Tyr Ser Ala Leu Ser Ile
1 5 10
<210> 4
<211> 11
<212> PRT
<213> artificial sequence
<220>
<223> Synthesis by hand
<400> 4
Gln Ala Ser Glu Ser Val Leu Asn Glu Val Ser
1 5 10
<210> 5
<211> 7
<212> PRT
<213> artificial sequence
<220>
<223> Synthesis by hand
<400> 5
Phe Ala Ser Lys Leu Ala Ser
1 5
<210> 6
<211> 12
<212> PRT
<213> artificial sequence
<220>
<223> Synthesis by hand
<400> 6
Gln Gln Asp Trp Ser Met Asp Asn Ile Asp Asn Ala
1 5 10
<210> 7
<211> 120
<212> PRT
<213> artificial sequence
<220>
<223> Synthesis by hand
<400> 7
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Leu Ser Ser Tyr
20 25 30
His Met Ile Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Gly Val Ile Tyr Pro Asn Ser Asn Ile Tyr Tyr Ala Thr Trp Ala Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Thr Ile Tyr Val His Val Tyr Ser Ala Leu Ser Ile Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 8
<211> 110
<212> PRT
<213> artificial sequence
<220>
<223> Synthesis by hand
<400> 8
Ala Phe Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Glu Ser Val Leu Asn Glu
20 25 30
Val Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Phe Ala Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Asp Trp Ser Met Asp Asn
85 90 95
Ile Asp Asn Ala Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105 110
<210> 9
<211> 159
<212> PRT
<213> artificial sequence
<220>
<223> Synthesis by hand
<400> 9
Ser Ile Thr Gly Ile Ser Pro Ile Thr Glu Tyr Leu Ala Ser Leu Ser
1 5 10 15
Thr Tyr Asn Asp Gln Ser Ile Thr Phe Ala Leu Glu Asp Glu Ser Tyr
20 25 30
Glu Ile Tyr Val Glu Asp Leu Lys Lys Asp Glu Lys Lys Asp Lys Val
35 40 45
Leu Leu Ser Tyr Tyr Glu Ser Gln His Pro Ser Asn Glu Ser Gly Asp
50 55 60
Gly Val Asp Gly Lys Met Leu Met Val Thr Leu Ser Pro Thr Lys Asp
65 70 75 80
Phe Trp Leu His Ala Asn Asn Lys Glu His Ser Val Glu Leu His Lys
85 90 95
Cys Glu Lys Pro Leu Pro Asp Gln Ala Phe Phe Val Leu His Asn Met
100 105 110
His Ser Asn Cys Val Ser Phe Glu Cys Lys Thr Asp Pro Gly Val Phe
115 120 125
Ile Gly Val Lys Asp Asn His Leu Ala Leu Ile Lys Val Asp Ser Ser
130 135 140
Glu Asn Leu Cys Thr Glu Asn Ile Leu Phe Lys Leu Ser Glu Thr
145 150 155
<210> 10
<211> 450
<212> PRT
<213> artificial sequence
<220>
<223> Synthesis by hand
<400> 10
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Leu Ser Ser Tyr
20 25 30
His Met Ile Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Gly Val Ile Tyr Pro Asn Ser Asn Ile Tyr Tyr Ala Thr Trp Ala Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Thr Ile Tyr Val His Val Tyr Ser Ala Leu Ser Ile Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
115 120 125
Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
130 135 140
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
145 150 155 160
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
165 170 175
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
180 185 190
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
195 200 205
Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
210 215 220
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
225 230 235 240
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
245 250 255
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
260 265 270
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
275 280 285
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
290 295 300
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
305 310 315 320
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
325 330 335
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
340 345 350
Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu
355 360 365
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
370 375 380
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
385 390 395 400
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
405 410 415
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
420 425 430
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
435 440 445
Gly Lys
450
<210> 11
<211> 217
<212> PRT
<213> artificial sequence
<220>
<223> Synthesis by hand
<400> 11
Ala Phe Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Glu Ser Val Leu Asn Glu
20 25 30
Val Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Phe Ala Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Asp Trp Ser Met Asp Asn
85 90 95
Ile Asp Asn Ala Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr
100 105 110
Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu
115 120 125
Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro
130 135 140
Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly
145 150 155 160
Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr
165 170 175
Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His
180 185 190
Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val
195 200 205
Thr Lys Ser Phe Asn Arg Gly Glu Cys
210 215
Claims (19)
1. An affinity purification method for reducing the protein content of a host cell in the production of an anti-human interleukin-33 monoclonal antibody, comprising the steps of:
equilibrium before loading: balancing the affinity chromatography medium by using a buffer solution, thereby obtaining a balanced affinity chromatography medium;
loading: combining an anti-human interleukin-33 monoclonal antibody fermentation broth with the equilibrated affinity chromatography medium;
eluting: then, pre-eluting and final eluting are carried out successively, so that host cell proteins are removed, and a purified anti-human interleukin-33 monoclonal antibody is obtained;
The anti-human interleukin-33 monoclonal antibody comprises three heavy chain complementarity determining regions, CDR-H1, CDR-H2 and CDR-H3, and three light chain complementarity determining regions, CDR-L1, CDR-L2 and CDR-L3, wherein:
the amino acid sequence of CDR-H1 is shown in SEQ ID NO:1 is shown in the specification;
the amino acid sequence of CDR-H2 is shown in SEQ ID NO:2 is shown in the figure;
the amino acid sequence of CDR-H3 is shown in SEQ ID NO:3 is shown in the figure;
the amino acid sequence of CDR-L1 is shown in SEQ ID NO:4 is shown in the figure;
the amino acid sequence of CDR-L2 is shown in SEQ ID NO:5 is shown in the figure;
the amino acid sequence of CDR-L3 is shown in SEQ ID NO: shown at 6.
2. The affinity purification method according to claim 1, wherein the anti-human interleukin-33 monoclonal antibody comprises a heavy chain variable region and a light chain variable region, wherein,
the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: shown in figure 7;
the amino acid sequence of the light chain variable region is shown in SEQ ID NO: shown at 8.
3. The affinity purification method according to claim 1 or 2, wherein the affinity chromatography medium is selected from one of chromatography media with ligands cross-linked to agarose, polyvinyl ether, hydroxylated polyether resin, polyacrylic resin, polystyrene-divinylbenzene resin, polymethacrylic resin, polystyrene resin, hydroxyapatite, glass matrix.
4. The affinity purification method of claim 3, wherein the affinity chromatography medium is a chromatography medium with ligands crosslinked to polyvinyl ether.
5. The affinity purification method according to claim 4, wherein the ligand is Protein A, protein G or Protein L.
6. The affinity purification process according to claim 4, wherein said ligand is Protein A.
7. The affinity purification method according to claim 1 or 2, wherein the buffer one is selected from one of phosphate buffer, tris-HCl buffer and boric acid-borax buffer; in the buffer solution I, the salt concentration is 5 mM-0.25M, and the pH is 5.5-8.0.
8. The affinity purification method according to claim 1 or 2, wherein the pre-elution is performed with a pre-elution buffer, which is a neutral buffer or an acidic buffer.
9. The affinity purification method of claim 8, wherein the neutral buffer is selected from one of a phosphate buffer, a tris buffer and a glycine buffer.
10. The affinity purification method of claim 8, wherein the acidic buffer is selected from one of a citric acid-disodium hydrogen phosphate buffer, an acetic acid-sodium acetate buffer, and a citric acid-trisodium citrate buffer.
11. The affinity purification method according to claim 8, wherein the pH of the pre-elution buffer is 5.0 to 7.5.
12. The affinity purification method according to claim 8, wherein the pH of the pre-elution buffer is 5.5 to 6.5.
13. The affinity purification method of claim 8, wherein a pre-elution active agent is further added to the pre-elution buffer when pre-elution is performed with the pre-elution buffer.
14. The affinity purification method according to claim 13, wherein the pre-eluting active is one or more selected from the group consisting of guanidine hydrochloride, polysorbate 80 and sodium chloride.
15. The affinity purification method of claim 13, wherein the pre-elution activator is guanidine hydrochloride.
16. The affinity purification method according to claim 15, wherein the concentration of guanidine hydrochloride is 0.01-1 m.
17. The affinity purification method according to claim 1 or 2, wherein the final elution is performed with a final elution buffer;
the final elution buffer is one or two or more than three of citric acid-disodium hydrogen phosphate buffer, acetic acid buffer, glycine-HCl buffer and citric acid-sodium citrate buffer.
18. The affinity purification method of claim 17, wherein the final elution buffer is a citric acid-disodium hydrogen phosphate buffer.
19. The affinity purification method according to claim 18, wherein the pH of the citric acid-disodium hydrogen phosphate buffer is 2.9-3.8.
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| CN113272448A (en) * | 2018-11-09 | 2021-08-17 | 深圳华大智造科技股份有限公司 | Massively parallel sequencing Using unlabeled nucleotides |
| CN113603775A (en) * | 2021-09-03 | 2021-11-05 | 江苏荃信生物医药有限公司 | Anti-human interleukin-33 monoclonal antibody and application thereof |
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| CN113234157A (en) * | 2021-07-09 | 2021-08-10 | 上海普铭生物科技有限公司 | Affinity-mature humanized anti-human IL-33 monoclonal antibody and application thereof |
| CN113603775A (en) * | 2021-09-03 | 2021-11-05 | 江苏荃信生物医药有限公司 | Anti-human interleukin-33 monoclonal antibody and application thereof |
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