Affinity purification method for reducing content of host cell protein in production of anti-human CD117 monoclonal antibody

Abstract

The invention discloses an affinity purification method for reducing the content of host cell protein in the production of an anti-human CD117 monoclonal antibody, which comprises the following steps: mixing a first buffer solution with an affinity chromatography medium to obtain a balanced affinity chromatography medium, then mixing the fermentation liquor of the anti-human CD117 monoclonal antibody with the balanced affinity chromatography medium to obtain a second buffer solution, and mixing the fermentation liquor of the anti-human CD117 monoclonal antibody with the balanced affinity chromatography medium to obtain the affinity purification method for reducing the content of host cell protein in the production of the anti-human CD117 monoclonal antibody. Obtaining a mixture; and carrying out first elution on the mixture, and then carrying out second elution so as to remove host cell protein, thereby obtaining the anti-human CD117 monoclonal antibody. The method is simple and easy to implement, amplification purification production can be carried out, the cell fermentation supernate does not need to be pretreated, the elution sample yield is high, meanwhile, the HCP residual quantity is kept at a low level, and therefore the pressure for removing HCP in the subsequent purification step is relieved.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to an affinity purification method for reducing the content of host cell protein in the production of anti-human CD117 monoclonal antibodies. Background Art

[0002] Antibody drugs are a type of protein drugs produced after host cell culture. In the process of producing recombinant proteins, antibodies, vaccines and other biological drugs, host cells will produce host cell proteins (HCPs, Host Cell Proteins) due to cell secretion, stress, lysis, etc. Therefore, HCP is a key process-related impurity produced in the production process of antibody drugs. As an exogenous protein, HCP may cause allergic reactions or other adverse reactions, resulting in unstable efficacy of drug products. Antibody drugs usually control the content of impurities such as residual host cell proteins to one ten-thousandth to one millionth of the drug amount to ensure the safety and effectiveness of the drug. At present, there are a series of basic purification modes to choose from for the purification of antibody drugs, such as gel filtration chromatography, ion exchange chromatography, affinity chromatography, etc. However, in the process of antibody drug process development, due to the large number of molecular types and the complicated upstream cell culture process (including the optimization of various process parameters such as inoculation density, culture temperature, rotation speed, dissolved oxygen, feeding time, feeding amount, etc.), it is necessary to purify each sample separately to remove impurities such as HCP, DNA, and polymers; at the same time, in the downstream purification process development stage, it is necessary to analyze and optimize multiple process parameters such as column height, loading capacity, buffer system pH, and buffer system conductivity. The number of experiments is extremely large, so it is necessary to develop a fast and efficient purification process to purify the samples separately in a short time, obtain high-purity samples, and measure the sample quality to ensure that the high-purity samples can be used for other experimental studies. At present, a small number of instruments have been launched at home and abroad for high-throughput protein purification, such as TECAN Resolvex A200, JANUSG3 BioTx Pro, etc. The instrument price is relatively expensive, and the sample volume it processes is small, and it cannot process large-volume samples. At the same time, the sample harvest is small, which cannot meet a large number of detection and application needs. Summary of the invention

[0003] Anti-human CD117 monoclonal antibodies are drugs that can be used to treat autoimmune diseases such as chronic urticaria and eosinophilic esophagitis. The applicant has developed a new recombinant humanized anti-human CD117 monoclonal antibody, which has a comparable affinity for binding to CD117 compared to the anti-human CD117 monoclonal antibody Barzolvolimab in the prior art, and its neutralization activity at the cellular level is slightly better than Barzolvolimab. In order to solve the problem of residual host cell protein in the production of the recombinant humanized anti-human CD117 monoclonal antibody, the present application provides a method for effectively reducing CHO host cell protein (HCP) during the production of the monoclonal antibody, which has low material cost, high protein purity, and is easy to scale up. In the present application, the HCP content is effectively reduced mainly by antibody affinity chromatography pre-elution elution, which meets the large-scale high-quality purification preparation requirements of antibody drugs and ensures the safety of clinical use of antibody drugs.

[0004] The specific technical solutions of this application are as follows:

[0005] The present application provides an affinity purification method for reducing the content of host cell protein in the production of anti-human CD117 monoclonal antibodies, characterized in that it comprises the following steps:

[0006] Mixing the first buffer solution and the affinity chromatography medium to obtain a balanced affinity chromatography medium, and then mixing the fermentation broth of the anti-human CD117 monoclonal antibody with the balanced affinity chromatography medium to obtain a mixture;

[0007] The mixture is subjected to a first elution, followed by a second elution, thereby removing host cell proteins and obtaining an anti-human CD117 monoclonal antibody;

[0008] The anti-human CD117 monoclonal antibody comprises three heavy chain complementary determining regions (CDR-H1, CDR-H2 and CDR-H3) and three light chain complementary determining regions (CDR-L1, CDR-L2 and CDR-L3), wherein:

[0009] The amino acid sequence of CDR-H1 is shown in SEQ ID NO: 1;

[0010] The amino acid sequence of CDR-H2 is shown in SEQ ID NO: 2;

[0011] The amino acid sequence of CDR-H3 is shown in SEQ ID NO: 3;

[0012] The amino acid sequence of CDR-L1 is shown in SEQ ID NO: 4;

[0013] The amino acid sequence of CDR-L2 is shown in SEQ ID NO: 5;

[0014] The amino acid sequence of CDR-L3 is shown in SEQ ID NO:6.

[0015] Furthermore, the anti-human CD117 monoclonal antibody comprises a heavy chain variable region and a light chain variable region, wherein:

[0016] the amino acid sequence of the heavy chain variable region is as set forth in SEQ ID NO:7, or an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO:7;

[0017] The amino acid sequence of the light chain variable region is as shown in SEQ ID NO:8 or an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO:8.

[0018] Further, the affinity chromatography medium is selected from a chromatography medium in which the ligand is cross-linked to agarose, polyvinyl ether, hydroxylated polyether resin, polyacrylic acid resin, polystyrene divinyl phenyl resin, polymethacrylic acid resin, polystyrene resin, hydroxyapatite or glass matrix, preferably a chromatography medium in which the ligand is cross-linked to agarose;

[0019] Preferably, the ligand is Protein A, Protein G or Protein L, more preferably Protein A.

[0020] Furthermore, the first buffer solution is selected from one of phosphate buffer, Tris-HCl buffer or boric acid-borax buffer, and the salt concentration in the first buffer solution is 10mM-0.25M, and the pH is 5.5-8.0.

[0021] Furthermore, when the mixture is eluted for the first time, the first elution solution is used for elution;

[0022] The first elution solution is a phosphate buffer, a Tris-HCl buffer or a citric acid-sodium citrate buffer, the salt concentration of the first elution solution is 50 mM-1.0 M, and the pH is 5.0-7.5.

[0023] Further, a pre-elution active agent is added to the first elution solution;

[0024] The pre-elution active agent is selected from one of guanidine hydrochloride, polysorbate 80 or sodium chloride, preferably guanidine hydrochloride;

[0025] Preferably, the concentration of the guanidine hydrochloride is 10 mM-1.0 M.

[0026] Furthermore, after the first elution and before the second elution, the method further comprises using a second buffer solution to balance the chromatography medium after the first elution;

[0027] Preferably, the second buffer solution is selected from one of phosphate buffer, Tris-HCl buffer or boric acid-borax buffer;

[0028] The salt concentration of the second buffer solution is 5 mM-0.15 M, and the pH is 5.5-8.0.

[0029] Further, when performing the second elution, the second elution solution is used for elution;

[0030] The second elution solution is selected from one of citric acid-disodium hydrogen phosphate buffer, acetate buffer or glycine-HCl buffer, and the salt concentration of the second elution solution is 5mM-0.1M, and the pH is 2.5-4.0.

[0031] Further, after the second elution, the pH of the anti-human CD117 monoclonal antibody was adjusted using a pH adjustment buffer.

[0032] The pH adjusting buffer is selected from one of phosphate buffer, Tris-HCl buffer or boric acid-borax buffer, the salt concentration in the pH adjusting buffer is 0.5M-5M, and the pH is 7.0-10.0.

[0033] The present application also provides a method for reducing the content of host cell protein in a recombinant humanized anti-human CD117 monoclonal antibody preparation, which comprises the step of purifying the supernatant of cell fermentation broth containing the recombinant humanized anti-human CD117 monoclonal antibody using the aforementioned purification method.

[0034] The affinity process described in the present application is simple and easy, and can be scaled up for purification production. The cell fermentation supernatant does not need to be pre-treated, and the elution sample yield is high. At the same time, the HCP residue is also kept at a low level (the residual control amount is not higher than 5%), thereby reducing the pressure of removing HCP in the subsequent purification step, thereby ensuring that the HCP residue in the final sample of the anti-human CD117 monoclonal antibody is at an extremely low level. The possible mechanism by which the present application can achieve the above-mentioned technical effect is to reduce the effect of HCP on the antibody and the filler matrix in the pre-elution of affinity chromatography, that is, the active reagent guanidine hydrochloride can reduce the force between HCP and the antibody protein and the filler medium to varying degrees, thereby removing HCP. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1This is a diagram showing the results of nucleic acid electrophoresis for constructing the HZD524-61 transient expression plasmid, where M: Marker; Band 1: 524VH-Hu35, HindIII / EcoRI; Band 2: pQXHC, HindIII / EcoRI; Band 3: PCR product 524VK-Hu17; Band 4: pQX2.3, HindIII / BsiWI.

[0036] Figure 2 It is a flowchart of transient expression.

[0037] Figure 3 This is the electrophoresis detection diagram of QX013N (HZD524-61).

[0038] Figure 4 QX013N neutralization activity analysis (M07e cell proliferation assay). DETAILED DESCRIPTION

[0039] The following is a description of the exemplary embodiments of the present application, including various details of the embodiments of the present application to facilitate understanding, which should be considered as merely exemplary. Therefore, it should be recognized by those of ordinary skill in the art that various changes and modifications can be made to the embodiments described herein without departing from the scope and spirit of the present application. Similarly, for clarity and conciseness, the description of well-known functions and structures is omitted in the following description.

[0040] It should be noted that certain words are used in the specification and claims to refer to specific components. Those skilled in the art should understand that technicians may use different nouns to refer to the same component. This specification and claims do not use the difference in nouns as a way to distinguish components, but use the functional differences of components as the criterion for distinction. As mentioned throughout the specification and claims, "including" or "comprising" are open-ended terms and should be interpreted as "including but not limited to". The subsequent description of the specification is a preferred embodiment of the present application, but the description is based on the general principles of the specification and is not intended to limit the scope of the present application. The scope of protection of the present application shall be determined by the attached claims.

[0041] In the present application, "monoclonal antibody" means an antibody derived from a population of substantially homologous antibodies, that is, each antibody constituting the population is identical and / or binds to the same epitope, except for possible variant antibodies (e.g., containing naturally occurring mutations or produced during the production of monoclonal antibody products), such variants are usually present in trace amounts. Different from polyclonal antibody products that generally include different antibodies for different determinants (epitopes), each monoclonal antibody of a monoclonal antibody product is directed to a single determinant on an antigen. Thus, the modifier "monoclonal" indicates that the antibody is derived from a substantially homologous antibody population, and should not be interpreted as requiring the antibody to be produced by any ad hoc method. For example, the monoclonal antibody to be used according to the present 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, and such methods and other exemplary methods for preparing monoclonal antibodies are described herein.

[0042] In this 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 in this specification refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be expressed by the equilibrium dissociation constant (K D Affinity can be measured by common methods known in the art.

[0043] Mammalian cells

[0044] The mammalian cells used in the in vitro fermentation production of the monoclonal antibodies described in the present application include, but are not limited to, various hybridoma cells currently in common use and Chinese hamster ovary cells (CHO), preferably CHO cells.

[0045] The present application provides an affinity purification method for reducing the host cell protein content in the production of anti-human CD117 monoclonal antibodies, comprising the following steps:

[0046] Step 1: preparing a fermentation broth of anti-human CD117 monoclonal antibody;

[0047] Step 2: mixing the first buffer solution and the affinity chromatography medium to obtain a balanced affinity chromatography medium, and then mixing the fermentation broth of the anti-human CD117 monoclonal antibody with the balanced affinity chromatography medium to obtain a mixture;

[0048] Step 3: The mixture is subjected to a first elution, followed by a second elution, thereby removing host cell proteins and obtaining anti-human CD117 monoclonal antibodies.

[0049] The anti-human CD117 monoclonal antibody comprises three heavy chain complementary determining regions (CDR-H1, CDR-H2 and CDR-H3) and three light chain complementary determining regions (CDR-L1, CDR-L2 and CDR-L3), wherein:

[0050] The amino acid sequence of CDR-H1 (in this specification, CDR-H1 means heavy chain CDR1) is shown in SEQ ID NO: 1 (NKDVMG);

[0051] The amino acid sequence of CDR-H2 (in this specification, CDR-H2 means heavy chain CDR2) is shown in SEQ ID NO: 2 (GIYTGSGSTYYASWAKG);

[0052] The amino acid sequence of CDR-H3 (in this specification, CDR-H3 means heavy chain CDR3) is shown in SEQ ID NO: 3 (DLFYSNYYNL);

[0053] The amino acid sequence of CDR-L1 (in this specification, CDR-L1 means light chain CDR1) is shown in SEQ ID NO: 4 (QASESISNYLS);

[0054] The amino acid sequence of CDR-L2 (in this specification, CDR-L2 means light chain CDR2) is shown in SEQ ID NO: 5 (KASTLAS);

[0055] The amino acid sequence of CDR-L3 (in the present specification, CDR-L3 refers to light chain CDR3) is shown in SEQ ID NO: 6 (QNTYVSSGSIT).

[0056] The anti-human CD117 monoclonal antibody comprises a heavy chain variable region and a light chain variable region, wherein

[0057] In the present application, the anti-human CD117 monoclonal antibody comprises a heavy chain variable region and a light chain variable region, wherein:

[0058] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 7, which has an amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAASGFSFSNKDVMGWVRQAPGKGLEWIAGIYTGSGSTYYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLFYSNYYNLWGQGTLVTVSS;

[0059] The amino acid sequence of the heavy chain variable region is an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:7.

[0060] The amino acid sequence of the light chain variable region is shown in SEQ ID NO: 8, and its amino acid sequence is DIQMTQSPSSVSASVGDRVTITCQASESISNYLSWYQQKPGKAPERLIYKA STLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQNTYVSSGSITFGGG TKVEIK.

[0061] The amino acid sequence of the light chain variable region is an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:8.

[0062] In the present application, the anti-human CD117 monoclonal antibody refers to a monoclonal antibody that can bind to human CD117 with sufficient affinity so that the monoclonal antibody can be used as a diagnostic agent and / or therapeutic agent targeting human CD117.

[0063] The anti-human CD117 monoclonal antibody of the present application does not bind to proteins unrelated to the target. Here, "unrelated protein" refers to other proteins other than human CD117 as a target; Here, "unbound" means: when the binding ability of the anti-human CD117 monoclonal antibody of the present invention to human CD117 as its target is taken as 100%, the binding ability of the anti-human CD117 monoclonal antibody of the present invention to the unrelated protein is less than 10%, for example 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or 0.

[0064] The anti-CD117 monoclonal antibodies described in the present application have an equilibrium dissociation constant (KD) of ≤1 μM, ≤100 nM, ≤50 nM, or ≤40 nM.

[0065] The human CD117 described in the present application represents a membrane protein derived from humans, and the amino acid sequence of its extracellular region is shown in SEQ ID NO: 9, wherein the underlined portion represents a signal peptide.

[0066] SEQ ID NO:9:

[0067] MRGARGAWDFLCVLLLLLRVQTGSS QPSVSPGEPSPPSIHPGKSDLIVRVGDEIRLLCTDPGFVKWTFEILDETNENKQNEWITEKAEATNTGKYTCTNKHGLSNSIYVFVRDPAKLFLVDRSLYGKEDNDTLVRCPLTDPEVTNYSLKG CQGKPLPKDLRFIPDPKAGIMIKSVKRAYHRLCLHCSVDQEGKSVLSEKFILKVRPAFKAVPVVSVSKASYLLREGEEFTVTCTIKDVSSSVYSTWKRENSQTKLQEKYNSWHHGDFNYERQATL TISSARVNDSGVFMCYANNTFGSANVTTTLEVVDKGFINIFPMINTTVFVNDGENVDLIVEYEAFPKPEHQQWIYMNRFTTDKWEDYPKSENESNIRYVSELHLTRLKGTEGGTYTFLVSNSDVN AAIAFNVYVNTKPEILTYDRLVNGMLQCVAAGFPEPTIDWYFCPGTEQRCSASVLPVDVQTLNSSGPPFGKLVVQSSIDSSAFKHNGTVECKAYNDVGKTSAYFNFAFKGNNKEQIHPHTLFTP.

[0068] In a specific embodiment, the amino acid sequence of the heavy chain of the anti-human CD117 monoclonal antibody of the present application is as shown in SEQ ID NO: 10, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence of SEQ ID NO: 10;

[0069] The amino acid sequence of the light chain is as shown in SEQ ID NO:11 or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:11.

[0070] SEQ ID NO: 10

[0071] EVQLVESGGGLVQPGGSLRLSCAASGFSFSNKDVMGWVRQAPGKGLEW

[0072] IAGIYTGSGSTYYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC

[0073] ARDLFYSNYYNLWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGC

[0074] LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG

[0075] TQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFP

[0076] PKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE

[0077] EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ

[0078] PREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK

[0079] TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL

[0080] SLSPGK

[0081] SEQ ID NO: 11

[0082] DIQMTQSPSSVSASVGDRVTITCQASESISNYLSWYQQKPGKAPERLIYKA

[0083] STLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQNTYVSSGSITFGGG

[0084] TKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD

[0085] NALQSGNSQESVTEQDSKDSTYLSSTLTLSKADYEKHKVYACEVTHQG

[0086] LSSPVTKSFNRGEC

[0087] Among them, SEQ ID NOs: 10 and 11 are both humanized sequences.

[0088] In this application, "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from the components of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in a cell that normally contains the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0089] In the present application, "isolated nucleic acid encoding anti-human CD117 monoclonal antibody" means one or more nucleic acid molecules encoding antibody heavy and light chains, including such nucleic acid molecules in a single vector or separate vectors, and such nucleic acid molecules present in one or more locations in a host cell.

[0090] In this application, "vector" means a nucleic acid molecule capable of amplifying another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that integrate into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors."

[0091] In this application, "host cell", "host cell line" and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acids have been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells", which include the primary transformed cells and the progeny derived therefrom (regardless of the number of passages). Progeny may not be completely identical to the parental cells in terms of nucleic acid content, but may contain mutations. Mutant progeny with the same function or biological activity screened or selected for the initially transformed cells are included in this specification.

[0092] As used herein, a "pharmaceutical composition" means a preparation which is in such a form as to permit the biological activity of the active ingredients contained therein to exert its effect, and which contains no additional components which are unacceptably toxic to the subject to which the formulation is to be administered.

[0093] In the present application, "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical composition other than an active ingredient, which is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives.

[0094] In this application, "monoclonal antibody" is generally a human antibody, which can be prepared using techniques known to those skilled in the art. For example, human antibodies are generally described in van Dijk, MA and van de Winkel, JG, Curr. Opin. Pharmacol. 5:368-374 (2001) and Lonberg, N., Curr. Opin. Immunol. 20:450-459 (2008).

[0095] Antibodies can be prepared by administering immunogens to transgenic animals that have been modified to stimulate the production of complete human antibodies or complete antibodies with human variable regions in response to antigenic attack. These animals typically contain a portion or all of the human immunoglobulin loci, which replace the endogenous immunoglobulin loci, or are present outside the chromosomes or randomly integrated in the animal. In such transgenic mice, the endogenous immunoglobulin loci are generally inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, N., Nat. Biotech. (Nature Biotechnology) 23: 1117-1125 (2005). See also, for example, the XENOMOUSE described in U.S. Patent Nos. 6,075,181 and 6,150,584. TM Technology; US Patent No. 5,770,429 describes Technology; US Patent No. 7,041,870 describes Technology, and U.S. Patent Application Publication No. US2007 / 0061900 described The human variable regions from intact antibodies generated by such animals can be further modified, for example, by combining with a different human constant region.

[0096] Human antibodies can also be prepared by hybridoma-based methods. Human myeloma and mouse-human hybrid myeloma cells for producing human monoclonal antibodies have been described (see, e.g., Kozbor, D., J. Immunol. 133: 3001-3005 (1984); Brodeur, BR et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York (1987), pp. 51-63; 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. USA 103: 3557-3562 (2006). Other methods include those described in, for example, U.S. Pat. No. 7,189,826 (which describes the production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26 (4); 265-268 (which describes human-human hybridomas). Human hybridoma technology (Trioma technology) is also described in Vollmers, HP and Brandlein, S., Histology and Histopathology 20: 927-937 (2005); Vollmers, HP and Brandlein, S., Methods and Findings in Experimental and Clinical Pharmacology 27: 185-191 (2005).

[0097] Human antibodies can also be made by isolating Fv clone variable domain sequences selected from human-derived phage display libraries, and such variable domain sequences can then be combined with the desired human constant domains.

[0098] Human antibodies can also be selected based on self-antibody libraries, that is, human antibodies can be isolated by screening combinatorial libraries for antibodies with desired one or more activities. For example, various methods for producing phage display libraries and screening such libraries for antibodies with desired binding characteristics are known in the art. This method is reviewed in, for example, Hoogenboom, HR, et al., Methods in Molecular Biology 178: 1-37 (2001), and is further described in, for example, McCafferty, J., et al., Nature 348: 552-554 (1990); Clackson, T., et al., Nature 352: 624-628 (1991); Marks, JD, et al., J. Mol. Biol. 222: 581-597 (1992); Marks, JD and Bradbury, A., Methods in Molecular Biology 248:161-175(2003); Sidhu, SS et al., J. Mol. Biol. 338: 299-310 (2004); Lee, CV et al., J. Mol. Biol. 340: 1073-1093 (2004); Fellouse, FA, Proc. Natl. Acad. Sci. USA 101:12467-12472 (2004); and Lee, CV et al., J. Immunol. Methods 284:119-132 (2004).

[0099] In certain phage display methods, the repertoires of VH and VL genes are cloned separately by polymerase chain reaction (PCR) and randomly recombined in a phage library, which is then screened for antigen-binding phage, 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 the immunogen without the need to construct hybridomas. Alternatively, non-immunized repertoires can be cloned (e.g., from humans) to provide a single source of antibodies to a large number of non-self and also self-antigens without any immunization, as described by Griffiths, AD et al., EMBO J, 12: 725-734 (1993). Finally, unimmunized libraries can also be generated synthetically by cloning unrearranged V gene segments from stem cells and using PCR primers containing random sequences to encode highly variable CDR3 regions and achieve rearrangement in vitro, as described by Hoogenboom, HR 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 Publication Nos. 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936 and 2009 / 0002360.

[0100] The antibody can also be a multispecific antibody, such as a bispecific antibody. Bispecific antibodies are monoclonal antibodies with binding specificity to at least two different sites. The techniques for generating multispecific antibodies include, but are not limited to, the recombinant co-expression of two pairs of immunoglobulin heavy chains-light chains with different specificities (see Milstein, C. and Cuello, AC, Nature 305: 537-540 (1983); WO 93 / 08829; and Traunecker, A. et al., EMBO J. 10: 3655-3659 (1991)) and "node-into-hole" engineering (see, e.g., U.S. Pat. No. 5,731,168). It can also be achieved by engineering electrostatic manipulation effects for generating antibody Fc-heterodimer molecules (WO 2009 / 089004); cross-linking 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)); using leucine zippers to generate bispecific antibodies (see, e.g., Kostelny, SA et al., J. Immunol. 148: 1547-1553 (1992)); using "diabody" technology for generating bispecific antibody fragments (see, e.g., Holliger, P. et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993)); and using single-chain Fv (scFv) dimers (see, e.g., Gruber, M. et al., J. Immunol. 152:5368-5374 (1994)); and preparing trispecific antibodies (as described, e.g., in Tutt, A. et al., J. Immunol. 147:60-69 (1991)) to generate multispecific antibodies.

[0101] The monoclonal antibodies described in this application also include engineered antibodies having three or more functional antigen binding sites, including "octopus antibodies" (see, e.g., US 2006 / 0025576).

[0102] The antibodies in the present application may also include the multispecific antibodies described in WO 2009 / 080251, WO 2009 / 080252, WO 2009 / 080253, WO 2009 / 080254, WO 2010 / 112193, WO 2010 / 115589, WO 2010 / 136172, WO 2010 / 145792, and WO 2010 / 145793, WO 2011 / 117330, WO 2012 / 025525, WO 2012 / 025530, WO 2013 / 026835, WO 2013 / 026831, WO 2013 / 164325, or WO 2013 / 174873.

[0103] The monoclonal antibodies described in the present application may also be antibody variants, for example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. The amino acid sequence variants of the antibody can be prepared by introducing suitable modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletion, and / or insertion and / or substitution of residues in the amino acid sequence of the antibody. Any combination of deletion, insertion, and substitution can be performed to obtain the final construct, as long as the final construct has the desired characteristics, such as antigen binding. Therefore, in certain embodiments, there are provided antibody variants with one or more amino acid substitutions, and the sites of interest for substitution mutations include HVR and FR, for example, amino acid substitutions can be introduced into the antibody of interest and screened for products with desired activity, for example, retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

[0104] In the present application, the affinity chromatography medium is selected from a chromatography medium with a ligand cross-linked to agarose, polyvinyl ether, hydroxylated polyether resin, polyacrylic acid resin, polystyrene divinyl phenyl resin, polymethacrylic acid resin, polystyrene resin, hydroxyapatite or glass matrix, preferably a chromatography medium with a ligand cross-linked to agarose;

[0105] Preferably, the ligand is Protein A, Protein G or Protein L, more preferably Protein A.

[0106] The ligand can specifically bind to the monoclonal antibody.

[0107] 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 and Mabselect Sure of GE Healthcare, NMab Pro of Suzhou Nanovita Technology Co., Ltd., and A

[0108] In the present application, the first buffer solution is selected from one of phosphate buffer, Tris-HCl buffer or boric acid-borax buffer. The present application does not impose any restrictions on the salt concentration in the first buffer solution, and those skilled in the art can select it as needed. For example, the salt concentration in the first buffer solution is 10mM-0.25M, and the pH is 5.5-8.0.

[0109] Specifically, the salt concentration in the first buffer solution may be 10 mM, 20 mM, 50 mM, 0.1 M, 0.15 M, 0.2 M or 0.25 M, etc.

[0110] Specifically, the pH may be 5.5, 6, 7 or 8, etc.

[0111] In the present application, the first buffer solution is one of phosphate buffer, Tris-hydrochloric acid buffer or boric acid-borax buffer. More preferably, NaCl or Na2SO4 is added to the first buffer solution to reduce nonspecific adsorption between non-antibody proteins and fillers.

[0112] Specifically, the salt concentration in the first buffer solution is 10 mM-0.25 M, preferably 10 mM-50 mM, and more preferably 20 mM.

[0113] The present application does not impose any limitation on the pH value of the first buffer solution, and those skilled in the art may select the pH value as needed. For example, the pH value of the first buffer solution is 5.5-8.0, preferably 6.8.

[0114] The amount of NaCl or Na2SO4 added can be determined according to the needs of those skilled in the art. The concentration of NaCl or Na2SO4 can be selected by those skilled in the art as needed. For example, the concentration of NaCl or Na2SO4 can be 0-250 mM, preferably 150 mM.

[0115] Specifically, the phosphate buffer may be a buffer of disodium hydrogen phosphate and sodium dihydrogen phosphate.

[0116] In the present application, when the anti-human CD117 monoclonal antibody fermentation broth is combined with the equilibrated affinity chromatography medium, the first buffer solution is used for equilibration.

[0117] In the present application, when the mixture is eluted for the first time, a first elution solution is used for elution. The first elution solution is selected from phosphate buffer, Tris-HCl buffer or citric acid-sodium citrate buffer.

[0118] The salt concentration in the first elution solution is 50mM-1.0M, for example, it can be 50mM, 60mM, 70mM, 80mM, 90mM, 100mM, 110mM, 120mM, 130mM, 140mM, 150mM, 160mM, 170mM, 180mM, 190mM, 200mM, 250mM, 300mM, 350mM, 400mM, 450mM, 500mM, 550mM, 600mM, 650mM, 700mM, 750mM, 800mM, 850mM, 900mM, 950mM, 1000mM.

[0119] The present application does not impose any limitation on the pH value of the first elution solution, and those skilled in the art can select it as needed. For example, the pH value of the first elution solution is 5.0-7.5, preferably 5.8.

[0120] Specifically, the pH of the first elution solution may be 5.0, 5.5, 6.0, 6.5, 7.0 or 7.5, etc.

[0121] In the present application, a pre-elution active agent is added to the first elution solution.

[0122] Specifically, the pre-elution active agent is selected from one of guanidine hydrochloride, polysorbate 80 or sodium chloride. Preferably, the pre-elution active agent is guanidine hydrochloride.

[0123] The present application does not impose any limitation on the concentration of guanidine hydrochloride, and those skilled in the art can select it as needed. For example, the concentration of guanidine hydrochloride is 10 mM-1 M, preferably 50 mM-0.15 M, and more preferably 0.1 M.

[0124] Specifically, the concentration of the guanidine hydrochloride can be 10 mM, 50 mM, 0.1 M, 0.15 M, 0.2 M, 0.3 M, 0.4 M, 0.5 M, 0.6 M, 0.7 M, 0.8 M, 0.9 M or 1 M.

[0125] In some embodiments, the first elution solution consists of sodium citrate, NaCl and citric acid. In the first elution solution, the concentration of sodium citrate is 0.1 M, the concentration of NaCl is 0.5 M, the concentration of citric acid is 7 mM, and the pH of the first elution solution is 5.8.

[0126] In some embodiments, the first elution solution consists of sodium citrate, guanidine hydrochloride and citric acid. In the first eluent, the concentration of sodium citrate is 0.1 M, the concentration of guanidine hydrochloride is 0.1 M, the concentration of citric acid is 11 mM, and the pH of the first eluent is 5.8.

[0127] The present application does not impose any limitation on the salt concentration in the first elution solution, and those skilled in the art may select the salt concentration as needed. For example, in one embodiment, the salt concentration in the first elution solution is 0-0.5M, preferably 0.1M.

[0128] For example, the salt concentration in the first elution solution may be 0, 0.1 M, 0.2 M, 0.3 M, 0.4 M, 0.5 M, etc.

[0129] In the present application, after the first elution, before the second elution, the method further includes using a second buffer solution to equilibrate the chromatography medium after the first elution.

[0130] The second buffer solution is a phosphate buffer, a Tris-HCl buffer or a boric acid-borax buffer.

[0131] Preferably, the second buffer solution is one of phosphate buffer, Tris-HCl buffer or boric acid-borax buffer.

[0132] Preferably, NaCl or Na2SO4 is added to the second buffer solution to maintain conductivity while retaining partial buffering capacity.

[0133] The present application does not impose any limitation on the salt concentration in the second buffer solution, the pH of the second buffer solution, and the concentration of NaCl or Na2SO4, and those skilled in the art may select them as needed.

[0134] Specifically, the salt concentration of the second buffer solution is 5mM-150mM, preferably 10mM-50mM, for example, 5mM, 6mM, 7mM, 8mM, 9mM, 10mM, 11mM, 12mM, 13mM, 14mM, 15mM, 16mM, 17mM, 18mM, 19mM, 20mM, 25mM, 30mM, 35mM, 40mM , 45mM, 50mM, 55mM, 60mM, 65mM, 70mM, 75mM, 80mM, 85mM, 90mM, 95mM, 100mM, 110mM, 120mM, 130mM, 140mM, 150mM etc., more preferably 10mM; preferably, the pH value is 5.5-8.0, preferably 6.5-7.5, further preferably 7.2.

[0135] The phosphate buffer may be, for example, a buffer of disodium hydrogen phosphate and sodium dihydrogen phosphate.

[0136] In the present application, when the second elution is performed, the second elution solution is used for elution;

[0137] The second elution solution is selected from one of citric acid-disodium hydrogen phosphate buffer, acetate buffer or glycine-HCl buffer, preferably citric acid-disodium hydrogen phosphate buffer.

[0138] The present application does not impose any limitation on the pH of the second elution solution, and those skilled in the art may select the pH as needed. For example, in one embodiment, the pH of the second elution solution is 2.9-4.0.

[0139] Specifically, the pH of the second elution solution can be 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9 or 4, etc.

[0140] The present application does not impose any limitation on the salt concentration in the second elution solution, and those skilled in the art can select it as needed. For example, in one embodiment, the salt concentration in the second elution solution is 5-100 mM, preferably 5-50 mM.

[0141] For example, the salt concentration in the second elution solution can be 5 mM, 7 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, etc.

[0142] In some embodiments, the second elution solution consists of disodium hydrogen phosphate and citric acid, the concentration of the disodium hydrogen phosphate is 7 mM, the concentration of the citric acid is 15 mM, and the pH of the second eluent is 3.1.

[0143] In the present application, the affinity medium can be cleaned after the second elution. Preferably, the cleaning buffer includes but is not limited to citric acid-disodium hydrogen phosphate buffer, hydrochloric acid, glycine, NaOH, preferably citric acid-disodium hydrogen phosphate buffer and NaOH solution.

[0144] In the present application, after the second elution, the pH of the anti-human CD117 monoclonal antibody is adjusted using a pH adjustment buffer.

[0145] The pH adjustment buffer is selected from one of a phosphate buffer, a Tris-HCl buffer or a boric acid-borax buffer.

[0146] The salt concentration in the pH adjustment buffer is 0.5M-5M, and the pH is 7.0-10.0.

[0147] Specifically, the salt concentration in the pH adjustment buffer can be 0.5M, 1M, 1.5M, 2M, 2.5M, 3M, 3.5M, 4M, 4.5M, 5M, etc.

[0148] Specifically, the pH of the pH adjustment buffer may be 7.0, 7.5, 8, 8.5, 9, 9.5, or 10.0.

[0149] The present application also provides a method for reducing the content of host cell protein in a recombinant humanized anti-human CD117 monoclonal antibody preparation, which comprises the step of purifying the supernatant of cell fermentation broth containing the recombinant humanized anti-human CD117 monoclonal antibody by the aforementioned purification method.

[0150] The Protein A affinity purification process used in the present application is simple and easy, and can be scaled up for purification production. The cell fermentation supernatant does not need to be pre-treated, and the elution sample yield is high. At the same time, the HCP residue is also maintained at a low level (the residual control amount is not higher than 5%), thereby reducing the pressure of removing HCP in the subsequent purification steps, thereby ensuring that the HCP residue in the final antibody sample is at an extremely low level.

[0151] In one embodiment, the mammals used for the in vitro fermentation production of the monoclonal antibody include, but are not limited to, various hybridoma cells currently used and Chinese hamster ovary cells (CHO), preferably CHO cells.

[0152] Example

[0153] The present application provides a general and / or specific description of the materials and test methods used in the experiments. In the following examples, unless otherwise specified, % means wt%, i.e., weight percentage. The reagents or instruments used without indicating the manufacturer are all conventional reagent products that can be obtained commercially.

[0154] Example 1 Preparation of anti-human CD117 monoclonal antibody QX013N

[0155] The human CD117 antigen produced by Quanxin Biotechnology was used as an immunogen to immunize New Zealand rabbits, and monoclonal antibodies with antigen binding specificity were obtained using B cell cloning technology, and then monoclonal antibodies that bind to human CD117 and have the ability to inhibit human CD117 activity were screened. The target clones were selected through analysis and screening by Binding ELISA and Blocking ELISA. The above immunization and screening processes were entrusted to commercial companies.

[0156] Five clones were selected for recombinant expression and sequencing. It was determined that clone 524 had the best cell neutralization activity. Therefore, clone 524 was humanized. NCBI IgBlast was used to perform homology alignment of human IgG germline sequences, and IGHV3-66*01 was selected as a heavy chain CDR transplantation template, and the CDR region of the heavy chain of clone #524 (i.e., CDR-H1 (SEQ ID No: 1), CDR-H2 (SEQ ID No: 2), and CDR-H3 (SEQ ID No: 3)) was transplanted into the framework region of IGHV3-66*01; IGKV1-12*01 was selected as a light chain CDR transplantation template, and the CDR region of the light chain of clone #524 (i.e., CDR-L1 (SEQ ID No: 4), CDR-L2 (SEQ ID No: 5), and CDR-L3 (SEQ ID No: 6)) was transplanted into the framework region of IGKV1-12*01; back mutations were performed on specific sites in the framework region and site-directed mutations were performed in the CDR region to obtain the variable region of the monoclonal antibody QX013N of the present application. Finally, the sequence of the humanized heavy chain variable region is shown in SEQ ID NO: 7; the amino acid sequence of the humanized light chain variable region is shown in SEQ ID NO: 8.

[0157] The gene of the heavy chain (SEQ ID NO: 10) was synthesized by GenScript, and the heavy chain gene was obtained by double digestion with HindIII and EcoRI, and the gene of the light chain variable region (SEQ ID NO: 8) was obtained by PCR amplification. The heavy chain expression plasmid pQXHC was double digested with HindIII and EcoRI; the light chain expression plasmid pQX2.3 was double digested with HindIII and BsiWI; the heavy chain gene was inserted into the heavy chain expression plasmid using T4 ligase to construct the heavy chain expression plasmid pQXHC-524VH-Hu35; the light chain PCR amplified gene was inserted into the light chain expression plasmid using Infusion recombinase to construct the light chain expression plasmid pQX2.3-524VK-Hu17.

[0158] The results of nucleic acid electrophoresis detection of the double-enzyme-digested heavy chain gene fragment, PCR-amplified light chain variable region gene fragment and double-enzyme-digested plasmid are as follows: Figure 1 As shown. Figure 1 From the results, we can see that the double enzyme digestion results of the antibody heavy chain and the PCR amplification results of the light chain variable region and the results of double enzyme digestion of the heavy chain and light chain expression plasmids show that the plasmid size of the heavy chain and light chain is about 5000bp, the full length of the heavy chain is about 1410bp, and the light chain variable region is about 447bp.

[0159] The heavy chain expression plasmid pQXHC-524VH-Hu35 (the amino acid sequence of the expressed heavy chain is shown in SEQ ID NO: 10) and the light chain expression plasmid pQX2.3-524VK-Hu17 (the amino acid sequence of the expressed light chain is shown in SEQ ID NO: 11) with the correct sequence were co-transfected into ExpiCHO-S cells. One day before transfection, ExpiCHO-S cells were diluted to 3×10 6 On the day of transfection, the cell density was diluted to 6×10 6 cells / ml, 25ml cells were placed in a 125ml shake flask and waited for transfection. The transfection and expression process is as follows Figure 2 shown.

[0160] On the 6th day after transfection, the culture supernatant was harvested and purified by Protein A. The purified antibody was detected by SDS-PAGE electrophoresis and named QX013N (HZD524-61). The results of protein electrophoresis for detecting the antibody were as follows: Figure 3 As shown. Protein electrophoresis was detected using denaturing reducing gel. Figure 3 The results showed that there were two bands with sizes of approximately 50 kDa and 25 kDa, respectively, which were consistent with the theoretical molecular weights of the heavy chain (49.2 kDa) and the light chain (23.4 kDa).

[0161] Example 2 Equilibrium dissociation constant (K D )

[0162] Biacore T200 was used to detect the affinity of QX013N (HZD524-61) to human KIT, and all processes were carried out at 25°C. The commercial Sensor chip CM5 chip was used to fix an appropriate amount of antibodies by capture method, so that Rmax was around 50RU, and the capture flow rate was 10μl / min. The antigen was gradient diluted, and the instrument flow rate was switched to 30μl / min. The reference channel and the channel of the fixed antibody were flowed in order from low to high concentration, and the buffer was flowed as a negative control. After each binding and dissociation, the chip was regenerated with pH1.5 glycine. Use the instrument's own analysis software to select the 1:1 binding model in the Kinetics option for fitting, and calculate the antibody binding rate constant ka, dissociation rate constant kd, and dissociation equilibrium constant KD value.

[0163] In addition, the affinity of QX013N (HZD524-61) was compared with that of CDX-0159, a monoclonal antibody against human CD117 that is currently in Phase 2 clinical trials. The detection method for known antibodies was the same as that for QX013N, and the results are shown in Table 1. CDX-0159 was constructed based on the sequence provided by patent NZ630363B, and was transiently transfected into ExpiCHO-S cells to obtain the product.

[0164] Table 1 Affinity of anti-human CD117 monoclonal antibodies binding to human CD117

[0165] Sample name <![CDATA[K a (10 5 M -1 S -1 )]]> <![CDATA[K d (10 -4 S -1 )]]> <![CDATA[K D (10 -9 M)]]> CDX-0159 2.51 5.94 2.49 QX013N(HZD524-61) 2.02 2.51 1.40

[0166] The data in the table are: each sample was tested three times and the average value was calculated.

[0167] Example 3 QX013N and CDX-0159 neutralize the proliferation activity of recombinant human SCF-induced M07E cells

[0168] M07E cells were used to determine the activity of QX013N and CDX-0159 in neutralizing the cell proliferation induced by recombinant human SCF. M07E cells were plated at 4 × 10 4 Cells were plated into 96-well plates, and the antibody was diluted to a concentration range of 0 to 10 ug / ml. After adding the antibody to the cells and incubating for 1 hour, 50 ng / ml of recombinant human SCF was added and mixed. The cells were cultured at 37°C and 5% CO2 for 24 hours. 100 μL of the cell suspension was collected and added to a black 96-well plate. 100 μL of Cell Counting-Lite 2.0 Luminescent Cell Viability Assay reagent (Vazyme, DD1101-02) was taken and mixed with the cell suspension. The cell proliferation was detected using a multifunctional microplate reader and a dose-effect curve was drawn ( Figure 4 The neutralizing activity of the antibodies was then analyzed.

[0169] Figure 4 The results shown in the figure show that QX013N has the activity of neutralizing the proliferation of M07e cells induced by recombinant human SCF, with an IC50 value of 50.55 ng / ml, while the IC50 value of the control antibody CDX-0159 is 55.11 ng / ml. The activity of QX013N in neutralizing the proliferation of M07e cells induced by human SCF is slightly better than that of the control antibody CDX-0159.

[0170] Example 4 Comparison of the removal effects of different pre-elution buffers on HCP in the fermentation broth of recombinant humanized anti-CD117 monoclonal antibody (QX013N)

[0171] The antibody QX013N obtained in Example 1 was produced using CHO cells as host cells and EB600 culture medium solution as fermentation basal medium. The cells were cultured using conventional cell culture techniques. When the cell viability was lower than 60% or the cells were cultured for 14-18 days, the harvest was started. The harvest solution was deep filtered using a primary filter CSCMD4070PCP and a secondary filter CSCMDH0HPPCP, and the clarified cell culture supernatant was collected and recorded as a fermentation broth intermediate (CH harvest supernatant). The fermentation broth intermediate was subjected to affinity chromatography as follows:

[0172] A Protein A chromatography column (NMab Pro, Suzhou Nanovita Technology Co., Ltd., 18 mL) was equilibrated with a first equilibration buffer (20 mM phosphate buffer and 0.15 M NaCl), and the QX013N fermentation broth intermediate was loaded onto the equilibrated Protein A chromatography column to bind thereto. After the injection, the column was re-equilibrated with the first equilibration buffer;

[0173] Then, an intermediate pre-elution was performed, and the composition of the intermediate pre-elution buffer was shown in Table 2. After the elution, a second equilibration buffer (10 mM phosphate buffer, pH 7.2) was used for equilibration;

[0174] Then, a final elution was performed to collect the sample, and the antibody concentration of the sample was tested. Subsequently, the pH of the sample was adjusted to 7.5 using a pH adjustment buffer, and the residual content of the host protein (HCP) was measured; wherein, the final elution buffer was 7mM Na2HPO4, 15mM citric acid, pH 3.1; the pH adjustment buffer was 2M Tris-HCl, pH 9.5; and the cleaning buffer 0.1MNaOH, 1MNaCl was used to clean the chromatography column.

[0175] The antibody concentration was determined as follows:

[0176] 1. Adjust the spectrophotometer wavelength to 280 nm and use the second equilibrium buffer as a control to perform zero calibration.

[0177] 2. Dilute the sample to be tested with the second equilibrium buffer, measure the absorbance of the sample at 280nm (the absorbance value is guaranteed to be between 0.5 and 1.5), and calculate the sample concentration according to the following formula (the extinction coefficient of QX013N is 1.505).

[0178]

[0179] The results obtained are shown in Table 3.

[0180] Determination method of HCP content:

[0181] 1. Sample dilution: Generally, the dilution factor is selected based on the estimated value of HCP in the sample, so that the final HCP concentration falls within the range of the standard curve (0ng / ml to 100ng / ml). The sample dilution factor should not exceed 20 times, and the minimum sampling volume should not be less than 10μl.

[0182] 2. Add 100 μl of Anti-CHO HRP to each well of the removed strips.

[0183] 3. Sample loading: Add standard, sample, and spiked sample (two replicates each) in a certain arrangement, 50 μl / well, seal the plate, and place on a horizontal shaker at room temperature, 180 rpm, for 2 hours, away from light.

[0184] 4. Wash the plate: discard the liquid in the wells, add 300μl / well of washing solution with a multichannel pipette, let it stand for 30 seconds, shake off the liquid, pat dry on absorbent paper, and wash the plate 4 times. After the last wash, try to pat dry the remaining washing solution in the wells.

[0185] 5. Color development and stop reading: Add TMB reagent (TMB Substrate) at 100μl / well, let it stand for 30 minutes, and protect from light. After 30 minutes, add 100μl / well of stop solution (Stop Solution), read at 450nm on a microplate reader, and use 650nm as a reference.

[0186] 6. Select analysis software for data analysis, and use the OD value of the standard as the ordinate and the concentration as the abscissa to make a four-parameter standard curve. Substitute the OD value of the sample into the standard curve to obtain the actual measured value of the HCP of the added sample.

[0187] 7. Residual CHO cell protein content (%) = average measured value of sample (mg / ml) × dilution factor / undiluted sample protein content (mg / ml) (%), the results are shown in Table 3.

[0188] Table 2 Composition of intermediate pre-elution buffer

[0189] Serial number Pre-elution buffer 1 0.1M sodium citrate, 0.5M sodium chloride, 7mM citric acid, pH 5.8 2 0.1M sodium citrate, 0.1M guanidine hydrochloride, 11mM citric acid, pH 5.8

[0190] Table 3 QX013N concentration and HCP residual content results

[0191]

[0192] From the results in Table 3, it can be seen that the residual HCP content in the fermentation broth intermediate is greater than 15%, and the yield is greater than 99% after the affinity purification process. For the removal of HCP, the above-mentioned method is adopted, and the residual HCP of the sample after affinity chromatography is less than 0.5%, and the HCP load removed in the subsequent purification process step is significantly reduced. After the pH of the sample is adjusted, when the subsequent chromatography process step (anion exchange chromatography) is loaded, the sample remains extremely clear and can be directly injected without other treatment, which enhances the simplicity of the process and saves process time.

[0193] The above is only the preferred embodiment of the present application, and does not limit the present application in other forms. Any technician familiar with the profession may use the above disclosed technical content to change or modify it into an equivalent embodiment with equivalent changes. However, any simple modification, equivalent change and modification made to the above embodiment based on the technical essence of the present application without departing from the technical solution of the present application still falls within the protection scope of the technical solution of the present application.

Claims

1. An affinity purification method for reducing the content of host cell protein in the production of anti-human CD117 monoclonal antibodies, characterized in that: The steps include: Mixing the first buffer solution and the affinity chromatography medium to obtain a balanced affinity chromatography medium, and then mixing the fermentation broth of the anti-human CD117 monoclonal antibody with the balanced affinity chromatography medium to obtain a mixture; The mixture is subjected to a first elution, followed by a second elution, thereby removing host cell proteins and obtaining an anti-human CD117 monoclonal antibody; The anti-human CD117 monoclonal antibody comprises three heavy chain complementary determining regions (CDR-H1, CDR-H2 and CDR-H3) and three light chain complementary determining regions (CDR-L1, CDR-L2 and CDR-L3), wherein: The amino acid sequence of CDR-H1 is shown in SEQ ID NO: 1; The amino acid sequence of CDR-H2 is shown in SEQ ID NO: 2; The amino acid sequence of CDR-H3 is shown in SEQ ID NO: 3; The amino acid sequence of CDR-L1 is shown in SEQ ID NO: 4; The amino acid sequence of CDR-L2 is shown in SEQ ID NO: 5; The amino acid sequence of CDR-L3 is shown in SEQ ID NO:

6.

2. The affinity purification method according to claim 1, characterized in that The anti-human CD117 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 as set forth in SEQ ID NO:7, or an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO:7; The amino acid sequence of the light chain variable region is as shown in SEQ ID NO:8 or an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO:

8.

3. The affinity purification method according to any one of claims 1 to 3, characterized in that The affinity chromatography medium is selected from a chromatography medium with a ligand cross-linked to agarose, polyvinyl ether, hydroxylated polyether resin, polyacrylic acid resin, polystyrene divinyl phenyl resin, polymethacrylic acid resin, polystyrene resin, hydroxyapatite or glass matrix, preferably a chromatography medium with a ligand cross-linked to agarose; Preferably, the ligand is Protein A, Protein G or Protein L, more preferably Protein A.

4. The affinity purification method according to any one of claims 1 to 3, characterized in that The first buffer solution is selected from one of phosphate buffer, Tris-HCl buffer or boric acid-borax buffer, and the salt concentration in the first buffer solution is 10mM-0.25M, and the pH is 5.5-8.

0.

5. The affinity purification method according to any one of claims 1 to 4, characterized in that: When the mixture is eluted for the first time, the first elution solution is used for elution; The first elution solution is selected from one of phosphate buffer, Tris-HCl buffer or citric acid-sodium citrate buffer, and the salt concentration of the first elution solution is 50mM-1.0M, and the pH is 5.0-7.

5.

6. The affinity purification method according to claim 5, characterized in that adding a pre-elution active agent to the first elution solution; The pre-elution active agent is selected from one of guanidine hydrochloride, polysorbate 80 or sodium chloride, preferably guanidine hydrochloride; Preferably, the concentration of the guanidine hydrochloride is 10 mM-1.0 M.

7. The affinity purification method according to any one of claims 1 to 6, characterized in that After the first elution, before the second elution, the method further comprises using a second buffer solution to equilibrate the chromatography medium after the first elution; Preferably, the second buffer solution is selected from one of phosphate buffer, Tris-HCl buffer or boric acid-borax buffer; The salt concentration of the second buffer solution is 5 mM-0.15 M, and the pH is 5.5-8.

0.

8. The affinity purification method according to any one of claims 1 to 7, characterized in that: When performing the second elution, the second elution solution is used for elution; The second elution solution is selected from one of citric acid-disodium hydrogen phosphate buffer, acetate buffer or glycine-HCl buffer, and the salt concentration of the second elution solution is 5mM-0.1M, and the pH is 2.5-4.

0.

9. The affinity purification method according to any one of claims 1 to 8, characterized in that After the second elution, the pH of the anti-human CD117 monoclonal antibody was adjusted using a pH adjustment buffer. The pH adjusting buffer is selected from one of phosphate buffer, Tris-HCl buffer or boric acid-borax buffer, the salt concentration in the pH adjusting buffer is 0.5M-5M, and the pH is 7.0-10.

0.

10. A method for reducing the content of host cell proteins in a recombinant humanized anti-human CD117 monoclonal antibody preparation, comprising the step of purifying the supernatant of a cell fermentation broth containing the recombinant humanized anti-human CD117 monoclonal antibody using the purification method described in any one of claims 1 to 9.

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