WO2022041390A1 - Low viscosity liquid formulation comprising high concentration anti-human interleukin 23 monoclonal antibody, and preparation method therefor - Google Patents

Abstract

A low viscosity liquid formulation comprising a high concentration anti-human interleukin 23 monoclonal antibody, and a preparation method therefor. The liquid formulation can be used as an injection, particularly a subcutaneous injection. The liquid formulation comprises 100 mg/ml or more of an anti-human interleukin 23 monoclonal antibody and 10-500 mM of a basic amino acid. The subcutaneous injection administration concentration of the anti-human interleukin 23 monoclonal antibody can reach at least 100-150 mg/mL. The preparation method for the liquid formulation comprises a concentration and purification method and a host cell protein removal method.

Description

Low-viscosity liquid preparation containing high-concentration anti-human interleukin-23 monoclonal antibody and preparation method thereof technical field

The present invention relates to the field of antibody pharmaceutical preparations. Specifically, the present invention relates to a monoclonal antibody against human interleukin 23 (hIL-23), a low-viscosity liquid preparation comprising a high concentration of the monoclonal antibody, and a preparation method thereof, and the liquid preparation can be used as an injection, especially a subcutaneous injection .

Background technique

IL-23 is mainly produced by activated dendritic cells, macrophages and monocytes, and is a member of the IL-12 heterodimeric cytokine family, mainly composed of IL-23p19 and IL-12/IL-23p40. composed of subunits. IL-23 receptor includes IL-12 receptor β1 and IL-23 receptor 2 subunits. IL-23 expresses receptor IL-23 on the surface of T cells, NK cells, monocyte macrophages/dendritic cells. 23R and IL-12Rβ1 act to activate downstream signaling pathways to exert biological functions. IL-23 mainly acts on Th17 cells, inducing them to produce IL-17A, IL-17F, IL-21, IL-22 and other pro-inflammatory cytokines. It plays an important role in autoimmune and inflammatory diseases such as encephalopathy and inflammatory bowel disease.

IL-23-mediated signaling and biological effects are associated with many types of diseases, including rheumatoid arthritis, juvenile rheumatoid arthritis, systemic juvenile rheumatoid arthritis, psoriatic arthritis, ankylosis Spondylitis, osteoarthritis, gastric ulcer, inflammatory bowel disease, ulcerative colitis, acute pancreatitis, primary biliary sclerosis, Hashimoto's thyroiditis, systemic lupus erythematosus, iridocyclitis, grapevine Meningitis, optic neuritis, idiopathic pulmonary fibrosis, systemic vasculitis/Wegener's granulomatosis, allergic/atopic disease, asthma, allergic rhinitis, eczema, adult respiratory distress syndrome, allergic exposure Atopic dermatitis, vitiligo, psoriasis, alopecia areata, pemphigus, scleroderma, allergic/atopic conjunctivitis, hypersensitivity pneumonitis, organ transplant rejection, graft-versus-host disease, systemic inflammatory response syndrome, Graves' disease disease, Raynaud's disease, type B insulin resistance diabetes, myasthenia gravis, nephrotic syndrome, nephritis, glomerulonephritis, acute renal failure, etc.

At present, some monoclonal antibody drugs targeting hIL-23 have been put into the market, such as Guselkumab (trade name Tremfya) developed by Johnson & Johnson and Risankizumab (trade name SKYRIZI) developed by AbbVie, which have been approved by the US FDA to treat psoriasis Crohn's disease, psoriatic arthritis, and phase III clinical studies for Crohn's disease and inflammatory bowel disease.

From the perspective of reducing the cost of clinical use of biologics and improving patient compliance, the preferred dosage form of anti-hIL-23 monoclonal antibody drugs is subcutaneous injection. Since the dose of subcutaneous injection is usually in the range of 100 mg to 600 mg, and the maximum subcutaneous injection volume is generally limited to less than 2 ml, the concentration of hIL-23 monoclonal antibody in hIL-23 monoclonal antibody injection administered by subcutaneous injection is usually It needs to reach more than 100 mg/mL, for example, 100-150 mg/mL.

A high concentration of a monoclonal antibody usually causes an increase in the viscosity of the solution containing the monoclonal antibody, and too high a viscosity will make it impossible to manually push the needle plunger to inject the drug subcutaneously. Therefore, usually for different monoclonal antibody molecules, it is necessary to specially adjust the components of the solution, pH and other conditions to obtain a low-viscosity liquid preparation containing a high concentration of the monoclonal antibody molecule, which can be used as injections, Especially subcutaneous injections.

Furthermore, high concentrations of monoclonal antibody injections present many challenges to the manufacturability of the production process, process scale-up, and ultimately patient administration. One of the main challenges is the ultra-high viscosity, which tends to form viscous solutions due to the biopolymeric nature of antibodies and the intermolecular interactions (such as hydrophobicity, charge interactions, etc.) that are enhanced at high concentrations. In some extreme cases, it will even bring a lot of challenges to the ultrafiltration membrane and ultrafiltration equipment itself. For example, the tangential flow rate is reduced due to the rapid rise of the pressure difference during final concentration, and the concentration polarization is gradually out of control until the occurrence of The phenomenon that protein precipitation blocks the membrane, which will inevitably lead to reduced recovery or process failure. On the other hand, even if the final high-concentration protein solution is obtained by improving the equipment or the type of membrane package, it is difficult to put it into practical clinical application, because the subcutaneous administration requires the use of a disposable sterile syringe for aspiration or the use of a prefilled needle In the final packaging form, too high a viscosity will make it impossible to manually push the needle plunger to inject the drug subcutaneously. Another difficulty in concentrating high-concentration monoclonal antibody liquids by ultrafiltration is that protein samples tend to aggregate to form soluble aggregates when highly concentrated, which further aggregates to form protein precipitates. Therefore, for the preparation of high-concentration anti-human IL-23 monoclonal antibody subcutaneous injections, it is necessary to develop an ultrafiltration concentration preparation method that can effectively reduce the viscosity of the ultrafiltration concentrate, reduce the aggregation of monoclonal antibodies, and improve its stability.

On the other hand, affinity purification is a very critical process step in the production of antibody drugs. This process captures and concentrates the antibodies in the fermentation broth to realize the first step of crude antibody purification. In the process of mass fermentation of genetically engineered cell lines such as Chinese hamster ovary cells (CHO), cells undergo apoptosis and cleavage in different physiological cycles, releasing host cell protein (HCP). HCP refers to protein components derived from host cells, including host cell structural proteins and transforming proteins (growth-promoting proteins secreted by cells). HCP may not only induce the body to produce anti-HCP antibodies and cause allergic reactions, but also may have an "adjuvant effect" that causes the body to produce antibodies to protein drugs, which affects the therapeutic effect of drugs. Quantitative determination of residual HCP in genetically engineered drugs is for quality control. An important means to help maintain the efficiency and consistency of the purification process. In antibody affinity purification, while ensuring high-efficiency antibody recovery, it is necessary to effectively remove HCP produced by engineered cells during the fermentation process.

Therefore, the development of an economical and feasible antibody affinity purification process that can effectively remove HCP in the large-scale antibody fermentation preparation is extremely meaningful for the further industrialization of antibody drugs.

At present, there are many methods that can remove HCP residues, each with its own characteristics: 1) Chromatography methods: including Protein A affinity chromatography, anion and cation chromatography, the ability to remove HCP in three chromatography processes, Protein A affinity chromatography Based on and chromatography, the removal ability is strong, and it is the main step of HCP removal. Anion and cation chromatography are mainly used as a further re-removal process of subsequent HCP. In the process of continuous improvement of the process of removing HCP by chromatography, various chromatography processes are continuously proposed and applied in actual production, which is the main means of HCP removal; 2) Tangential flow ultrafiltration method: the removal capacity of HCP is limited, and It is not easy to control the residual amount, and the process control coefficient is not high, so it can only be used as an auxiliary process to remove HCP; 3) Polymer precipitation method: PEG, polyacrylic acid and other high polymers are positively charged in a wide pH range, and the same The antibody binds to form a precipitate, while HCP is less prone to precipitation due to its low isoelectric point. The HCP content of the precipitated antibody samples was significantly reduced, but this process was not suitable for process scale-up, and the precipitation may have a certain impact on the activity of the antibody. Therefore, there are many process methods for removing HCP. Due to the differences in the fermentation process of the antibody samples and the properties of the antibodies, the selection process needs to be comprehensively considered.

SUMMARY OF THE INVENTION

In view of the deficiencies in the above-mentioned prior art, the object of the present invention is to provide a monoclonal antibody against human interleukin 23 (hIL-23), a low-viscosity liquid preparation comprising a high concentration of the monoclonal antibody and a preparation method thereof, the liquid The formulations can be used as injections, especially subcutaneous injections.

That is, the first aspect of the present invention includes:

1. A liquid formulation comprising:

100 mg/mL or more (preferably 300 mg/mL or less, more preferably 200 mg/mL or less, more preferably 150 mg/mL or less) anti-human interleukin-23 monoclonal antibody, and

10～500mM basic amino acid;

The anti-human interleukin-23 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) ),in:

(a) the amino acid sequence of CDR-H1 (in this specification CDR-H1 represents heavy chain CDR1) is shown in SEQ ID NO: 1 (NHEMS);

(b) the amino acid sequence of CDR-H2 (in this specification CDR-H2 represents heavy chain CDR2) is shown in SEQ ID NO: 2 (IITTSDTTYYATWAKG);

(c) the amino acid sequence of CDR-H3 (in this specification CDR-H3 represents heavy chain CDR3) is shown in SEQ ID NO: 3 (VDIVLLSVTSRI);

(d) the amino acid sequence of CDR-L1 (in this specification CDR-L1 represents light chain CDR1) is shown in SEQ ID NO: 4 (QASQSVSTYLS);

(e) the amino acid sequence of CDR-L2 (in this specification CDR-L2 represents light chain CDR2) is shown in SEQ ID NO: 5 (GASNLES); and

(f) The amino acid sequence of CDR-L3 (in this specification CDR-L3 represents light chain CDR3) is shown in SEQ ID NO: 6 (QSGYVFAGLT).

Here, the basic amino acid refers to one or two selected from arginine (Arg, R), lysine (Lys, L), histidine (His, H) and proline (Pro, P). kind or three or four.

Preferably, the above-mentioned liquid preparation is substantially free of impurity proteins (other proteins other than the anti-human interleukin-23 monoclonal antibody, the impurity protein content is less than 1%)

2. The liquid preparation according to item 1, wherein the anti-human interleukin-23 monoclonal antibody comprises a heavy chain variable region and a light chain variable region,

The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 7, and its amino acid sequence is EVQLVESGGGLVQPGGSLRLSCAASGFSLSNHEMSWVRQAPGKGLEWIGIITTSDTTYYATWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVDIVLLSVTSRIWGQGTLVTVSS; and,

The amino acid sequence of the light chain variable region is shown in SEQ ID NO: 8, and its amino acid sequence is DVVMTQSPSSLSASVGDRVTITCQASQSVSTYLSWYQQKPGKAPKLLIYGASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQSGYVFAGLTFGGGTKVEIK.

3. The liquid preparation according to the foregoing, wherein the basic amino acid is arginine, histidine, lysine, proline or a combination thereof.

4. The aforementioned liquid preparation, wherein the 10-500 mM basic amino acid is a combination of 50-300 mM arginine and 5-50 mM histidine.

5. The aforementioned liquid preparation, wherein the 10-500 mM basic amino acid is a combination of 100-200 mM (eg 150 mM) arginine and 10-30 mM (eg 20 mM) histidine.

6. The aforementioned liquid preparation, wherein the 10-500 mM basic amino acid is a combination of 50-300 mM arginine and 10-100 mM lysine.

7. The aforementioned liquid preparation, wherein the 10-500 mM basic amino acid is a combination of 100-200 mM (eg 150 mM) arginine and 40-60 mM (eg 50 mM) lysine.

8. The aforementioned liquid preparation, wherein the 10-500 mM basic amino acid is 10-100 mM (eg, 10-30 mM histidine).

9. The aforementioned liquid formulation further comprising 20-150 mg/mL (eg 50-100 mg/mL) of sucrose.

10. The aforementioned liquid preparation, comprising 130 mg/mL or more of anti-human interleukin-23 monoclonal antibody, and having a viscosity of 15 cP or less.

11. The aforementioned liquid preparation, comprising 150 mg/mL or more of anti-human interleukin-23 monoclonal antibody, and having a viscosity of 30 cP or less.

12. The aforementioned liquid preparation, comprising 100 mg/mL or more of an anti-human interleukin-23 monoclonal antibody and having a viscosity of 10 cP or less.

13. The aforementioned liquid preparation, which has a pH value of 5.5 to 6.5, preferably 6.0.

已先后在日本、美国和欧盟获批上市，且其剂型为皮下注射剂。临床试验结果显示，其在中度至重度斑块型银屑病方面的治疗效果优于强生重磅抗炎药

(ustekinumab)以及艾伯维畅销抗炎药

(adalimumab)。鉴于本发明的液体制剂同样为皮下注射剂、且其中包含的单克隆抗体在细胞水平显示出与Risankizumab相当的拮抗活性，本发明的液体制剂有望在预防和治疗相关疾病方面展现出良好的临床效果。 A first aspect of the present invention provides a liquid preparation comprising a novel anti-human interleukin 23 (hIL-23) monoclonal antibody, which comprises a high concentration (above 100 mg/mL) of the anti-human interleukin 23 monoclonal antibody and has a low Viscosity (less than 30cP), it can be easily injected with a syringe, so it is suitable for injection, especially subcutaneous injection. Moreover, the novel anti-human interleukin 23 (hIL-23) monoclonal antibody, compared with the existing anti-human interleukin 23 monoclonal antibodies (Guselkumab and Risankizumab), binds hIL-23 with comparable affinity, but at the cellular level The antagonistic activity was superior to that of Guselkumab and comparable to that of Risankizumab. To be clear, Risankizumab

It has been approved for marketing in Japan, the United States and the European Union, and its dosage form is subcutaneous injection. Clinical trial results show it outperforms Johnson &Johnson's blockbuster anti-inflammatory drug in moderate-to-severe plaque psoriasis

(ustekinumab) and AbbVie's best-selling anti-inflammatory drug

(adalimumab). Considering that the liquid preparation of the present invention is also a subcutaneous injection, and the monoclonal antibody contained therein shows an antagonistic activity comparable to that of Risankizumab at the cellular level, the liquid preparation of the present invention is expected to show good clinical effects in the prevention and treatment of related diseases.

Furthermore, a second aspect of the present invention includes:

1. a preparation method of anti-human IL-23 monoclonal antibody concentrated solution, it comprises the following steps:

Step A. The first ultrafiltration concentration step: for the solution containing the anti-human IL-23 monoclonal antibody, the flow rate is 110-320 L/m ² ·h, and the transmembrane pressure difference (TMP) is maintained at 0.7-1.4 bar. Concentrating the solution containing the anti-human IL-23 monoclonal antibody to a protein concentration of 40-50 mg/mL to obtain a first ultrafiltration concentrate;

Step B. Equal volume buffer replacement step: use a peristaltic pump to pump the buffer to be replaced into the first ultrafiltration concentrate, continue ultrafiltration, and adjust the pumping speed and permeability of the buffer to be replaced. The flow rate is consistent, and when the pumping amount of the buffer to be replaced reaches 6 to 10 times the weight of the first ultrafiltration concentrate, the equal volume buffer replacement is completed, and the ultrafiltration after the equal volume buffer replacement is obtained. Concentrate; wherein, the buffer to be replaced is selected as 10-30 mM histidine-hydrochloric acid buffer, and the pH range is between 5.5 and 6.0;

Step C. The second ultrafiltration concentration step: adjust the arginine concentration in the ultrafiltration concentrated solution after the equal volume of buffer replacement, so that the arginine concentration is 100mM to 200mM, and then perform ultrafiltration concentration, so that the protein concentration 100～200mg/mL, obtain the second ultrafiltration concentrated solution (that is, the concentrated solution of anti-human IL-23 monoclonal antibody); wherein, add the concentration of 1M to the ultrafiltration concentrated solution after the equal volume buffer replacement ~2M arginine stock solution, so that the final concentration of arginine is 100mM ~ 200mM;

The anti-human interleukin-23 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) ),in:

(a) the amino acid sequence of CDR-H1 (in this specification CDR-H1 represents heavy chain CDR1) is shown in SEQ ID NO: 1 (NHEMS);

(b) the amino acid sequence of CDR-H2 (in this specification CDR-H2 represents heavy chain CDR2) is shown in SEQ ID NO: 2 (IITTSDTTYYATWAKG);

(c) the amino acid sequence of CDR-H3 (in this specification CDR-H3 represents heavy chain CDR3) is shown in SEQ ID NO: 3 (VDIVLLSVTSRI);

(d) the amino acid sequence of CDR-L1 (in this specification CDR-L1 represents light chain CDR1) is shown in SEQ ID NO: 4 (QASQSVSTYLS);

(e) the amino acid sequence of CDR-L2 (in this specification CDR-L2 represents light chain CDR2) is shown in SEQ ID NO: 5 (GASNLES); and

(f) The amino acid sequence of CDR-L3 (in this specification CDR-L3 represents light chain CDR3) is shown in SEQ ID NO: 6 (QSGYVFAGLT).

2. the aforementioned preparation method, wherein, the anti-human interleukin-23 monoclonal antibody comprises a heavy chain variable region and a light chain variable region,

The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 7, and its amino acid sequence is EVQLVESGGGLVQPGGSLRLSCAASGFSLSNHEMSWVRQAPGKGLEWIGIITTSDTTYYATWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVDIVLLSVTSRIWGQGTLVTVSS; and,

The amino acid sequence of the light chain variable region is shown in SEQ ID NO: 8, and its amino acid sequence is DVVMTQSPSSLSASVGDRVTITCQASQSVSTYLSWYQQKPGKAPKLLIYGASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQSGYVFAGLTFGGGTKVEIK.

3. According to the aforementioned preparation method, wherein the protein concentration of the solution comprising the anti-human IL-23 monoclonal antibody in the step A is less than 15 mg/mL.

4. According to the aforementioned preparation method, wherein the protein concentration of the solution comprising the anti-human IL-23 monoclonal antibody in the step A is less than 10 mg/mL.

5. According to the aforementioned preparation method, wherein the buffer to be replaced in the step B is a 20 mM histidine-hydrochloric acid buffer with a pH value of 6.0.

6. According to the aforementioned preparation method, wherein the arginine concentration in the ultrafiltration concentrate after the equal volume of buffer replacement in the step C is 130 mM to 160 mM.

7. The aforementioned preparation method, wherein the protein concentration of the second ultrafiltration concentrate in the step C is 130 mg/mL or more, and the viscosity thereof is 15 cP or less.

8. According to the aforementioned preparation method, wherein the protein concentration of the second ultrafiltration concentrate in the step C is 150 mg/mL or more, and the viscosity thereof is 30 cP or less.

9. According to the aforementioned preparation method, the preparation method is suitable for ultrafiltration concentration of 200L pilot scale.

10. According to the aforementioned preparation method, wherein, the solution comprising the anti-human IL-23 monoclonal antibody in the step A is carried out by affinity chromatography, low pH inactivation, anion chromatography, cation chromatography and nanofiltration.

The method of reducing the viscosity of the high-concentration antibody liquid and improving the stability in the ultrafiltration liquid exchange process is simple and feasible, can be scaled up, can ensure the high purity of the sample and obtain a high recovery rate. According to the present invention, a concentrated solution of high-concentration and low-viscosity anti-human IL-23 monoclonal antibody with a protein concentration of 130 mg/mL or more and a viscosity of 10 cP or less can be prepared on a 200L pilot scale. Antibody concentrate solution can be used to prepare anti-human IL-23 monoclonal antibody subcutaneous injection.

Furthermore, a third aspect of the present invention includes:

1. A purification method of a recombinant humanized anti-human interleukin-23 monoclonal antibody, the method comprising carrying out affinity chromatography on a cell fermentation broth supernatant comprising a recombinant humanized anti-human interleukin-23 monoclonal antibody, the And chromatography includes the following steps:

Step A: Before sample loading, use equilibration buffer A to equilibrate the affinity chromatography column;

Step B: Load the cell fermentation broth supernatant containing the recombinant humanized anti-human interleukin-23 monoclonal antibody onto the affinity chromatography column; (Is the cell fermentation broth supernatant directly loaded?)

Step C: after loading, use the equilibration buffer A to equilibrate the affinity chromatography column again;

Step D: pre-eluting the affinity column with a pre-elution buffer;

Step E: Equilibrate the affinity chromatography column with Equilibration Buffer B; and

Step F: final elution of the affinity column with elution buffer, and sample collection;

The anti-human interleukin-23 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) ),in:

(a) the amino acid sequence of CDR-H1 (in this specification CDR-H1 represents heavy chain CDR1) is shown in SEQ ID NO: 1 (NHEMS);

(b) the amino acid sequence of CDR-H2 (in this specification CDR-H2 represents heavy chain CDR2) is shown in SEQ ID NO: 2 (IITTSDTTYYATWAKG);

(c) the amino acid sequence of CDR-H3 (in this specification CDR-H3 represents heavy chain CDR3) is shown in SEQ ID NO: 3 (VDIVLLSVTSRI);

(d) the amino acid sequence of CDR-L1 (in this specification CDR-L1 represents light chain CDR1) is shown in SEQ ID NO: 4 (QASQSVSTYLS);

(e) the amino acid sequence of CDR-L2 (in this specification CDR-L2 represents light chain CDR2) is shown in SEQ ID NO: 5 (GASNLES); and

(f) The amino acid sequence of CDR-L3 (in this specification CDR-L3 represents light chain CDR3) is shown in SEQ ID NO: 6 (QSGYVFAGLT).

2. According to the aforementioned purification method, wherein, the anti-human interleukin-23 monoclonal antibody comprises a heavy chain variable region and a light chain variable region,

The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 7, and its amino acid sequence is EVQLVESGGGLVQPGGSLRLSCAASGFSLSNHEMSWVRQAPGKGLEWIGIITTSDTTYYATWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVDIVLLSVTSRIWGQGTLVTVSS; and,

The amino acid sequence of the light chain variable region is shown in SEQ ID NO: 8, and its amino acid sequence is DVVMTQSPSSLSASVGDRVTITCQASQSVSTYLSWYQQKPGKAPKLLIYGASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQSGYVFAGLTFGGGTKVEIK.

3. According to the aforementioned purification method, wherein, the affinity chromatography column is a Protein A column.

4. According to the aforementioned purification method, wherein, the equilibration buffer A is phosphate buffer, Tris-HCl buffer or boric acid-borax buffer, the salt concentration is 5mM-0.25M, and the pH is 5.5-8.0.

The equilibration buffer A may contain NaCl or Na ₂ SO ₄ to reduce the non-specific adsorption between non-antibody proteins and Protein A filler, and the concentration of NaCl and/or Na ₂ SO ₄ is preferably below 250mM. The equilibration buffer A is preferably a phosphate buffer. The salt concentration of the equilibration buffer A is preferably 5mM-0.15M, more preferably 10mM-50mM; its pH is preferably pH6.5-7.5.

5. According to the aforementioned purification method, wherein, the pre-eluting buffer is phosphate buffer, citrate buffer, Tris-HCl buffer or sodium acetate-acetate buffer, and its salt concentration is 0.001M-0.5 M, its pH is 5.0-7.5.

The salt concentration of the pre-elution buffer is preferably 0.05M to 0.2M. The pH of the pre-elution buffer is preferably 5.5-6.5.

6. The purification method according to the foregoing, wherein the pre-elution buffer comprises 0.01M-1.0M guanidine hydrochloride. The concentration of guanidine hydrochloride is preferably between 0.05M and 0.3M.

7. According to the aforementioned purification method, wherein, the elution buffer is a citric acid-disodium hydrogen phosphate buffer, an acetate buffer, a glycine-HCl buffer or a citric acid-sodium citrate buffer, and its salt concentration is 5～100mM, its pH is 2.5-4.0.

The salt concentration of the elution buffer is preferably 20-50 mM, and its pH is preferably 2.9-3.8.

8. According to the aforementioned purification method, wherein, the equilibration buffer B is a phosphate buffer Tris-HCl buffer or a boric acid-borax buffer, and its salt concentration is 5mM-0.15M, and its pH is 5.5-8.0.

The equilibration buffer B may contain NaCl or Na ₂ SO ₄ to maintain conductivity while maintaining partial buffering capacity, and the concentration of NaCl and/or Na ₂ SO ₄ is preferably below 250 mM, for example, may be 10-100 mM.

9. A method for reducing host cell protein content in a recombinant humanized anti-human interleukin-23 monoclonal antibody preparation, comprising using the purification method described in any one of the preceding paragraphs to quantify a recombinant humanized anti-human interleukin-23 monoclonal antibody. The antibody is purified from the cell fermentation supernatant.

The antibody Protein A affinity purification process in the present invention is simple and feasible, and can be amplified and purified for production, the cell fermentation supernatant does not need to be pre-treated, the elution sample yield is high, and the HCP residue is also kept at a low level. (residual control amount is not higher than 0.5%), thereby reducing the pressure of removing HCP in subsequent purification steps, thereby ensuring that the residual amount of HCP in the final antibody sample is at a very low level. At the same time, the verification of the affinity purification process for different batches of fermentation supernatant in the invention proves that the affinity purification process in the invention has good stability.

The possible mechanism that the present invention can achieve the above technical effect is to use the pre-elution of affinity chromatography to reduce the interaction between HCP and the antibody and the filler matrix, that is, the active reagent guanidine hydrochloride can weaken the HCP with the antibody protein and the filler to varying degrees. The force between the media plays a role in removing HCP.

Description of drawings

FIG. 1 is a graph showing the results of nucleic acid electrophoresis for the construction of the QX004N (HZD90-32) transient expression plasmid. Wherein, M: Marker; Band 1: PCR product 90VH-Hu18; Band 2: pHZDCH, HindIII/NheI; Band 3: PCR product 90VK-Hu9; Band 4: pHZDCK, HindIII/BsiWI.

Figure 2 is a flow chart of transient expression.

Figure 3 is the electrophoresis detection chart of QX004N (HZD90-32).

detailed description

In the present specification, human interleukin 23 (Human interleukin 23, hIL-23) refers to a human-derived protein, which is a heterodimer composed of two subunits, p19 and p40. The amino acid sequence of p19 is shown in SEQ ID NO: 9, and the amino acid sequence of p40 is shown in SEQ ID NO: 10, wherein the underlined part represents the signal peptide.

In the present specification, "anti-human interleukin-23 monoclonal antibody" refers to a monoclonal antibody capable of binding human interleukin-23 with sufficient affinity such that the monoclonal antibody can be used as a diagnostic agent targeting human interleukin-23 and /or therapeutic agent.

The experimental results show that the novel anti-human interleukin 23 (IL-23) monoclonal antibody can specifically bind to the p19 subunit of human interleukin 23 (IL-23). The new anti-human interleukin 23 (IL-23) monoclonal antibody is comparable to or superior to the same type of monoclonal antibody products on the market in many biological activities. The biological activities include, for example, inhibition of IL-23-induced STAT3 phosphorylation in cells, inhibition of IL23-induced IL-17A release from mouse splenocytes, and IL-23-induced IL-23-induced IFN-γ release from human NK cells.

In one embodiment, the amino acid sequence of the heavy chain of the novel anti-human interleukin 23 (IL-23) monoclonal antibody is shown in SEQ ID NO: 11; the amino acid sequence of the light chain is shown in SEQ ID NO: 12 .

Wherein, SEQ ID NOs: 11 and 12 are both humanized sequences.

Example

Hereinafter, the present invention will be described in more detail by way of examples. It should be understood that the present invention is not limited to these examples.

It should be noted that the anti-human interleukin-23 monoclonal antibody QX004N in the following examples is the new anti-human interleukin-23 monoclonal antibody.

Example 1 Preparation of anti-human interleukin-23 monoclonal antibody QX004N

Procured human interleukin 23 (IL-23) from Shanghai Nearshore Technology Co., Ltd., used to immunize New Zealand rabbits, used B cell cloning technology to obtain antigen-binding specific antibody clones, and then screened for binding to IL-23 and having IL-23 inhibitory activity of monoclonal antibodies. First, the cell supernatant was detected by Binding ELISA to select clones that bind to IL-23; then, Blocking ELISA was used to detect the clones with IL-23 inhibitory activity. The above immunization and screening processes are entrusted to commercial companies.

Five clones were selected for recombinant expression and sequenced. The 90# clone was humanized. The homology alignment of human IgG germline sequence (Germline) was performed using NCBI IgBlast, IGHV3-66*01 was selected as the heavy chain CDR transplantation template, and the CDR region of the 90# cloned heavy chain (ie CDR-H1 (SEQ ID No: 1), CDR-H2 (SEQ ID No: 2) and CDR-H3 (SEQ ID No: 3)) were transplanted into the framework region of IGHV3-66*01; IGKV1-39*01 was selected as the light chain CDR transplant template, and the 90# CDR regions of cloned light chain (i.e. CDR-L1 (SEQ ID No: 4), CDR-L2 (SEQ ID No: 5) and CDR-L3 (SEQ ID No: 6)) were grafted into IGKV1-39*01 The framework region of the skeleton region; the specific site of the framework region is back-mutated, and the 103rd methionine (Met, M) in the heavy chain CDR-H3 is mutated to leucine (Leu, L) to obtain the monoclonal antibody of the present invention. QX004N variable region. Finally, the humanized heavy chain variable region sequence is shown in SEQ ID NO: 7; the humanized light chain variable region amino acid sequence is shown in SEQ ID NO: 8.

The gene of the above-mentioned heavy chain variable region (SEQ ID NO:7) is obtained by PCR amplification; the gene of the light chain variable region (SEQ ID NO:8) is obtained by PCR amplification. The heavy chain expression plasmid pHZDCH was digested with HindIII and NheI; the light chain expression plasmid pHZDCK was digested with HindIII and BsiWI; the PCR amplified genes were inserted into the corresponding expression plasmids with Infusion recombinase to construct the heavy chain expression plasmid pHZDCH- 90VH-Hu18 and light chain expression plasmid pHZDCK-90VK-Hu9.

Figure 1 shows the results of double-enzyme digestion of plasmids detected by nucleic acid electrophoresis. According to the results in Figure 1, it can be seen that the PCR amplification results of the variable region of the heavy chain and the variable region of the light chain of the antibody and the results of double-enzyme digestion of the heavy chain and light chain expression plasmids, wherein the plasmid sizes of the heavy chain and light chain are about 10000bp, the light chain variable region is about 438bp, and the heavy chain variable region is about 459bp.

ExpiCHO-S cells were co-transfected with the correct heavy chain expression plasmid and light chain expression plasmid. One day before transfection, ExpiCHO-S cells were diluted to 3×10 ⁶ cells/mL for pre-transfection passage. On the day of transfection, the cell density was diluted to 6×10 ⁶ cells/mL, and 25 mL of cells were placed in 125 mL shake flasks, waiting for transfection. The transfection and expression process is shown in Figure 2.

On days 4-8 after transfection, the culture supernatant was harvested for one-step purification with ProteinA. The purified antibody was detected by SDS-PAGE electrophoresis and named as QX004N (HZD90-32). The results of detection of the antibody by protein electrophoresis are shown in FIG. 3 . The protein electrophoresis was detected by denaturing reducing gel, and the results in Figure 3 showed that there were two bands, the sizes of the two bands were about 50kDa and 25kDa respectively, which were consistent with the theoretical molecular weights of the heavy chain (49.1kDa) and the light chain (23.1kDa).

Example 2 Determination of Equilibrium Dissociation Constant (K _D )

The affinity of QX004N (HZD90-32) to IL-23 was detected by BiacoreT200, and all procedures were performed at 25°C. A commercial Protein A chip was used, and an appropriate amount of antibody was immobilized by the capture method, so that the Rmax was around 50RU, and the capture flow rate was 10 μl/min. The antigen was serially diluted, the flow rate of the instrument was switched to 30 μl/min, and the concentration flowed through the reference channel and the immobilized antibody channel in order of concentration from low to high, and the buffer was used as a negative control. The chip was regenerated with pH 1.5 glycine after each binding and dissociation was completed. The 1:1 binding model in the Kinetics option was selected by the instrument's own analysis software for fitting, and the on-rate constant _ka , the dissociation rate constant k _d and the dissociation equilibrium constant K _D value of the antibody were calculated.

In addition, the affinity of QX004N (HZD90-32) and the currently commercialized monoclonal antibodies against IL-23, namely Guselkumab and Risankizumab, were compared, and the detection methods for known antibodies were compared with those for QX004N. The same, the results are shown in Table 1. Among them, Guselkumab and Risankizumab were obtained by purchasing commercially available medicines.

Table 1 Affinity of antibody binding to human IL-23

sample name k _a (10 ⁵ M ^-1 S ^-1 ) k _d (10 ^-5 S ^-1 ) K _D (10 ^-10 M) Guselkumab 5.01 3.19 0.63 Risankizumab 7.06 3.70 0.52 QX004N(HZD90-32) 3.66 3.50 1.01

The data in the table are: each sample was tested twice and the average value was calculated.

Example 3 Determination of cellular level biological activities of QX004N, Guselkumab and Risankizumab

The cellular-level biological activities of QX004N, Guselkumab and Risankizumab were determined under the same experimental conditions, and the experimental results showed that:

a. QX004N can inhibit IL-23-induced STAT3 phosphorylation activity in HEK Blue ^TM IL-23 cells with IC ₅₀ of 3.21ng/mL; Guselkumab and Risankizumab can also inhibit IL-23-induced HEK Blue ^TM IL-23 cells The phosphorylation activity of STAT3 in IL-23 and its IC ₅₀ were 6.18ng/mL and 3.51ng/mL, respectively, indicating that QX004N inhibited the signal transduction activity induced by IL-23 and the currently commercialized monoclonal antibody against IL-23, Risankizumab. Activity was comparable and superior to Guselkumab.

b. QX004N can inhibit IL-23-induced IL-17A release from mouse spleen cells with an IC ₅₀ of 11.7ng/mL; Guselkumab and Risankizumab can also inhibit IL-23-induced IL-17A release from mouse spleen cells, Its IC _50s were 13.5ng/mL and 8.43ng/mL, respectively, indicating that QX004N has a strong inhibitory activity against IL-23-induced IL-17A release from mouse splenocytes, and its ability to compete with existing commercial products (Guselkumab and Risankizumab) quite.

c. QX004N can inhibit IL-23-induced release of IFN-γ from human NK cells with an IC ₅₀ of 10.4ng/mL; Guselkumab and Risankizumab can also inhibit IL-23-induced release of IFN-γ from human NK cells with an IC 50 of 10.4 ng/mL; ₅₀ were 16.8ng/mL and 11.1ng/mL, respectively, indicating that the activity of QX004N in inhibiting the release of IFN-γ from human NK cells induced by IL-23 was stronger than that of the currently commercialized product Guselkumab, and was comparable to that of Risankizumab.

已在临床试验中被证实对中度至重度斑块型银屑病的治疗效果显著，QX004N亦有望在预防和治疗相关疾病方面展现出良好的临床效果。 It can be seen from the above that QX004N is superior to Guselkumab in the three biological activities measured at the cellular level, but it is incomparable with Risankizumab. Given Risankizumab

It has been confirmed in clinical trials that it has a significant therapeutic effect on moderate to severe plaque psoriasis, and QX004N is also expected to show good clinical effects in the prevention and treatment of related diseases.

Example 4 Preparation of Low Viscosity Liquid Formulation Comprising High Concentration QX004N 1

By performing the operations of affinity chromatography, low pH inactivation, anion chromatography, cation chromatography, ultrafiltration concentration and other operations on the fermentation broth of cells expressing anti-human IL-23 monoclonal antibody, an ultrafiltration concentrate (containing 20 mM) was obtained. His-HCl, pH 6.0). The SEC-HPLC method was used to analyze, and it was determined that the QX004N monomer in the ultrafiltration concentrate was more than 99%, the polymer was less than 1%, and basically did not contain impurity proteins. The concentration of QX004N in the ultrafiltration concentrate was determined by UV spectrophotometry to be 170 mg/mL. The viscosity of the ultrafiltration concentrate was determined to be 63.4 cP using a Riosen's μVISC viscometer.

Use a 0.2 μm filter membrane to filter the ultrafiltration concentrate, and then mix or replace it with different buffer solutions or additive stock solutions, so that the buffer system of the sample and the QX004N protein concentration are shown in Table 3. The same method was used to determine the concentration of the sample. viscosity.

table 3

It is generally considered that liquid formulations with a viscosity of less than 30 cP are suitable for use as subcutaneous injections. It can be seen from Table 3 that the subcutaneous administration concentration of QX004N can reach at least 100-150 mg/mL through the above-mentioned liquid preparation.

Example 5 Preparation 2 of Low Viscosity Liquid Formulation Comprising High Concentration QX004N

By performing the operations of affinity chromatography, low pH inactivation, anion chromatography, cation chromatography, ultrafiltration concentration and other operations on the fermentation broth of cells expressing anti-human IL-23 monoclonal antibody, an ultrafiltration concentrate (containing 20 mM) was obtained. His-HCl, pH 6.0). The SEC-HPLC method was used to analyze, and it was determined that the QX004N monomer in the ultrafiltration concentrate was more than 99%, the polymer was less than 1%, and basically did not contain impurity proteins. The concentration of QX004N in the ultrafiltration concentrate was determined by UV spectrophotometry to be 130 mg/mL. The viscosity of the ultrafiltration concentrate was determined to be 63.4 cP using a Riosen's μVISC viscometer.

Use a 0.2 μm filter membrane to filter the ultrafiltration concentrate, and then mix or replace it with different buffer solutions or additive stock solutions, so that the buffer system of the sample and the QX004N protein concentration are shown in Table 4. The same method was used to determine the concentration of the sample. viscosity.

Table 4

The high concentration ultrafiltration concentration of embodiment 6 QX004N

CHO cells are used as host cells to ferment QX004N in a 2L-scale bioreactor. The fermentation broth is obtained by centrifuge or deep membrane filtration, and Protein A chromatography is used to capture the target protein, and then undergo low pH virus inactivation. Clarification, anion and cation chromatography remove impurities and obtain intermediate samples to be ultrafiltered.

The above-mentioned intermediate was used to investigate the ultrafiltration process, and the ultrafiltration equipment was selected from the Labscale small ultrafiltration apparatus of Merck Millipore (holding two 50cm ² Pellicon XL membrane packs, the interception amount was 30KD). At a flow rate of 120 to 300 L/m ² ·h, and TMP maintained at 0.6 to 1.5 bar, the above intermediate sample was concentrated to about 40 to 50 mg/mL, and then replaced with an equal volume of buffer solution. Amino acid-hydrochloric acid buffer, pH 6.0. After the replacement volume reaches 7 times, the liquid exchange step is completed. The intermediate sample after liquid exchange is concentrated to 170 mg/mL using a 30kDa ultrafiltration centrifuge tube of Merck Millipore, and the sample is filtered with a 0.2 μm filter membrane. The viscosity value was measured after the buffer solution or additive mother solution was mixed, and the specific preparation scheme and viscosity value results are shown in Table 2 below.

Table 2

It can be seen from the results in Table 2 that with the increase of protein concentration, the viscosity of QX004N solution increases exponentially. If no additives are added, the viscosity of the monoclonal antibody will reach 63cP when the concentration is 170mg/mL. Such a high viscosity value cannot be used. The concentration process is achieved using conventional ultrafiltration membranes. Taking 150mg/mL protein concentration as an example, by adding 100mM-200mM basic amino acid or its combination, the antibody viscosity value under the same protein concentration condition can be reduced to less than 50% of the original, less than 20cP. Based on the above experimental results, the administration concentration of QX004N solution can be realized to be 100-150 mg/mL.

It was further scaled up to a 200L pilot scale, and the Merck Millipore Pellicon manual ultrafiltration system was used to hold five 0.5m ² Pellicon 2 type membrane cartridges. The nanofiltered sample (UF0) was subjected to the first step concentration (UF1) at a flow rate of 120 to 300 L/m ² ·h, and the TMP was controlled at 0.6 to 1.5 bar. The sample was concentrated to 40 to 50 mg/mL; then UA was used. Perform 7-fold equal volume replacement (DF), replace the sample to 20mmol/L histidine, pH 6.0; after the replacement, add arginine stock solution to a final concentration of 150mM, and further concentrate the sample (UF2) to recover the sample , the experimental results are summarized in Table 3 below.

table 3

The above results show that by adding arginine with a final concentration of 150mM, QX004N can successfully achieve high-concentration ultrafiltration concentration at the pilot level. will reach a viscosity value of 19.4 cP (Table 2). In addition, the purity of the sample did not change during the entire ultrafiltration process, and arginine played a better protective role.

Example 7 Comparison of HCP scavenging effects of different pre-eluting buffers on recombinant humanized anti-IL-23 monoclonal antibody (QX004N) fermentation broth

Fermentation of antibody QX004N: CHO cells were used as host cells, Dynamis was used as fermentation basal medium, and cells were cultured by conventional cell culture process.

When the cell viability was lower than 80% or cultured for 14 days, the harvest was started, and the harvested liquid was subjected to deep filtration using the primary filter MDOHC10FS1 and the secondary filter MX0HC10FS1, and the clarified cell culture supernatant was collected and recorded as the fermentation broth intermediate.

0.15L)，上样载量设定45mg/ml。上样前，用平衡缓冲液A：12mmol/L Na ₂HPO ₄，8mmol/L NaH ₂PO ₄，0.15mol/L NaCl平衡；上样后，再次用平衡缓冲液A平衡，至发酵液完全流过层析柱；之后采用不同的预洗脱缓冲液淋洗，预洗脱缓冲液组成如下表1所示；接着用平衡缓冲液B：6mmol/L Na ₂HPO ₄，4mmol/L NaH ₂PO ₄，pH7.2平衡；随后用洗脱缓冲液PB：7mmol/L Na ₂HPO ₄，15mmol/L枸橼酸，pH3.1洗脱并收集洗脱样品，对收集的洗脱样品进行抗体浓度及HCP残留含量测定。 The fermentation broth intermediate was subjected to affinity chromatography and loaded on a Protein A column (Merck

0.15L), and the loading capacity was set to 45mg/ml. Before loading, equilibrate with equilibration buffer A: 12 mmol/L Na ₂ HPO ₄ , 8 mmol/L NaH ₂ PO ₄ , 0.15 mol/L NaCl; after loading, equilibrate with equilibration buffer A again until the fermentation broth flows completely Then use different pre-elution buffers to rinse, the composition of pre-elution buffers is shown in Table 1 below; then use equilibration buffer B: 6mmol/L Na ₂ HPO ₄ , 4mmol/L NaH ₂ PO ₄ , equilibrated at pH 7.2; then eluted with elution buffer PB: 7mmol/L Na ₂ HPO ₄ , 15mmol/L citric acid, pH 3.1 and collected the elution samples, and the collected elution samples were subjected to antibody concentration and HCP residual content determination.

Antibody concentration determination method:

1. Adjust the wavelength of the spectrophotometer to 280nm, and use the elution-flush PB as a control to perform zero calibration.

2. Dilute the sample to be tested with elution buffer PB, measure the absorbance value of the sample at 280 nm (the absorbance value is guaranteed to be between 0.5 and 1.5), and calculate the sample concentration according to the following formula (QX004N extinction coefficient is 1.408).

HCP content determination method:

Sample dilution: Generally, the dilution factor is selected according to the estimated value of HCP in the sample, so that the final concentration of HCP falls within the range of the standard curve (generally 10ng/ml～80ng/ml). The sample dilution in one step should not exceed 10 times, and the minimum sampling amount should not be less than 5 μl.

Add 100 μl of Anti-CHO HRP to each well of the removed strip.

Loading: Add standard, sample, and spiked samples in a certain arrangement (two duplicate wells for each, standard products do not need duplicate wells), 50 μl/well, and seal the plate. Place in a horizontal shaker at room temperature, 180 rpm, for 2 hours, protected from light.

Wash the plate: discard the liquid in the well, add 300 μl/well of washing solution with a multi-channel 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 plate wash, try to pat dry the residual wash solution in the wells.

Color development and termination reading: add 100 μl/well of TMB reagent (TMB Substrate), let stand for color development for 30 minutes, and protect from light. After 30 minutes, 100 μl/well of Stop Solution was added, and the microplate reader was read at 450 nm, with 650 nm as the reference.

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

CHO cell protein residue (%)=average measured value of sample (ng/ml)×dilution multiple/protein content of undiluted sample (mg/ml)×10 ^-4 (%).

Table 4: Pre-Elution Buffer Composition

It can be seen from the results in Table 5 that the residual amount of HCP in the fermentation broth intermediate is greater than 15%, and after the treatment of the affinity purification process, the yield is greater than 95%; for the removal of HCP, the experimental group containing guanidine hydrochloride was used. The HCP residue of the sample is less than 0.05%, the load of removing HCP in the subsequent purification process steps is significantly reduced, and the sample is adjusted by pH. When the sample is loaded in the subsequent chromatography process step (anion exchange chromatography), the sample remains extremely clear, and no other treatment is required. Direct sample injection enhances the simplicity of the process and saves process time; in the experimental group using polysorbate 80 or NaCl, the HCP residue in the sample after affinity chromatography is still greater than 0.2%, and the removal effect of HCP residue is worse than that of guanidine hydrochloride.

Table 5: QX004N affinity purification yield and HCP residue results

Example 8 Comparison of HCP scavenging effects of different batches of recombinant humanized anti-IL-23 monoclonal antibody (QX004N) fermentation broth

The preparation of the fermentation broth intermediate and the method of affinity chromatography are the same as those in Example 1. A total of three batches of fermentation broth intermediates were prepared, and the pre-eluting buffer solution containing guanidine hydrochloride was used for pre-eluting and washing: 0.1 mol/L Sodium citrate, 0.1 mol/L guanidine hydrochloride, 11 mmol/L citric acid, pH 5.8 for washing. The test methods for the yield of the affinity process and the residual HCP content are as described in Example 1.

As shown by the results in Table 6, the HCP residues of the intermediate QX004N fermentation broth of different batches were all above 15%, and the cell culture process was stable; the affinity process yields of the three batches of samples were all greater than 95%, and the yields met the process requirements; The residues of HCP in the samples after and chromatography were all lower than 0.05%, and the removal of HCP by guanidine hydrochloride and elution eluting was stable.

Table 6: Different batches of QX004N affinity purification yield and HCP residue results

Industrial Applicability

According to the present invention, there are provided a monoclonal antibody against human interleukin 23 (hIL-23), a low-viscosity liquid preparation comprising a high concentration of the monoclonal antibody, and a preparation method thereof, and the liquid preparation can be used as an injection, especially subcutaneously injection.

Claims (6)

A liquid formulation comprising: 100 mg/mL or more (preferably 300 mg/mL or less, more preferably 200 mg/mL or less, more preferably 150 mg/mL or less) anti-human interleukin-23 monoclonal antibody, and 10～500mM basic amino acid; The anti-human interleukin-23 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) ),in: (a) The amino acid sequence of CDR-H1 is shown in SEQ ID NO: 1; (b) the amino acid sequence of CDR-H2 is shown in SEQ ID NO: 2; (c) the amino acid sequence of CDR-H3 is shown in SEQ ID NO: 3; (d) the amino acid sequence of CDR-L1 is shown in SEQ ID NO: 4; (e) the amino acid sequence of CDR-L2 is shown in SEQ ID NO: 5; and (f) the amino acid sequence of CDR-L3 is shown in SEQ ID NO: 6; Preferably, the liquid formulation is substantially free of contaminants. The liquid preparation according to claim 1, wherein the anti-human interleukin-23 monoclonal antibody comprises a heavy chain variable region and a light chain variable region, The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 7; and, The amino acid sequence of the light chain variable region is shown in SEQ ID NO: 8. A method for preparing an anti-human IL-23 monoclonal antibody concentrated solution, comprising the following steps: Step A is the first ultrafiltration concentration step: for the solution containing the anti-human IL-23 monoclonal antibody, the flow rate is 110-320 L/m ² ·h, and the transmembrane pressure difference (TMP) is maintained at 0.7-1.4 bar. Concentrating the solution containing the anti-human IL-23 monoclonal antibody to a protein concentration of 40-50 mg/mL to obtain a first ultrafiltration concentrate; Step B is an equal volume buffer replacement step: use a peristaltic pump to pump the buffer to be replaced into the first ultrafiltration concentrate, continue ultrafiltration, and adjust the pumping speed and permeability of the buffer to be replaced. The flow rate is consistent, and when the pumping amount of the buffer to be replaced reaches 6 to 10 times the weight of the first ultrafiltration concentrate, the equal volume buffer replacement is completed, and the ultrafiltration after the equal volume buffer replacement is obtained. Concentrate; wherein, the buffer to be replaced is selected as 10-30 mM histidine-hydrochloric acid buffer, and the pH range is between 5.5 and 6.0; Step C is the second ultrafiltration concentration step: adjust the arginine concentration in the ultrafiltration concentrated solution replaced by the equal volume buffer so that the arginine concentration is 100 mM to 200 mM, and then perform ultrafiltration concentration to make the protein concentration. It is 100～200mg/mL, and obtains the second ultrafiltration concentrated solution (that is, the concentrated solution of anti-human IL-23 monoclonal antibody); wherein, the concentration of 1M is added to the ultrafiltration concentrated solution after the equal volume buffer replacement ~2M arginine stock solution, so that the final concentration of arginine is 100mM ~ 200mM; The anti-human interleukin-23 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) ),in: (a) The amino acid sequence of CDR-H1 is shown in SEQ ID NO: 1; (b) the amino acid sequence of CDR-H2 is shown in SEQ ID NO: 2; (c) the amino acid sequence of CDR-H3 is shown in SEQ ID NO: 3; (d) The amino acid sequence of CDR-L1 is shown in SEQ ID NO: 4; (e) the amino acid sequence of CDR-L2 is shown in SEQ ID NO: 5; and (f) The amino acid sequence of CDR-L3 is shown in SEQ ID NO:6. The preparation method according to claim 3, wherein the anti-human interleukin-23 monoclonal antibody comprises a heavy chain variable region and a light chain variable region, The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 7; and, The amino acid sequence of the light chain variable region is shown in SEQ ID NO: 8. A method for purifying recombinant humanized anti-human interleukin-23 monoclonal antibody, the method comprising performing affinity chromatography on the supernatant of cell fermentation broth comprising the recombinant humanized anti-human interleukin-23 monoclonal antibody, the affinity layer The analysis includes the following steps: Step A: Before sample loading, use equilibration buffer A to equilibrate the affinity chromatography column; Step B: Load the cell fermentation broth supernatant containing the recombinant humanized anti-human interleukin-23 monoclonal antibody onto the affinity chromatography column; (Is the cell fermentation broth supernatant directly loaded?) Step C: after loading, use the equilibration buffer A to equilibrate the affinity chromatography column again; Step D: pre-eluting the affinity column with a pre-elution buffer; Step E: Equilibrate the affinity chromatography column with Equilibration Buffer B; and Step F: final elution of the affinity column with elution buffer, and sample collection; The anti-human interleukin-23 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) ),in: (a) The amino acid sequence of CDR-H1 is shown in SEQ ID NO: 1; (b) the amino acid sequence of CDR-H2 is shown in SEQ ID NO: 2; (c) the amino acid sequence of CDR-H3 is shown in SEQ ID NO: 3; (d) The amino acid sequence of CDR-L1 is shown in SEQ ID NO: 4; (e) the amino acid sequence of CDR-L2 is shown in SEQ ID NO: 5; and (f) The amino acid sequence of CDR-L3 is shown in SEQ ID NO:6. The purification method according to claim 5, wherein the anti-human interleukin-23 monoclonal antibody comprises a heavy chain variable region and a light chain variable region, The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 7; and, The amino acid sequence of the light chain variable region is shown in SEQ ID NO: 8.

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