CN113527490B

CN113527490B - Preparation method of anti-human IFNAR1 monoclonal antibody concentrated solution

Info

Publication number: CN113527490B
Application number: CN202110790532.XA
Authority: CN
Inventors: 薛刚; 朱华杰; 王云霞; 黄文俊; 戴长松; 李帅; 张秋月; 吴亦亮
Original assignee: Jiangsu Quanxin Biomedical Co ltd
Current assignee: Jiangsu Quanxin Biomedical Co ltd
Priority date: 2021-07-13
Filing date: 2021-07-13
Publication date: 2022-03-01
Anticipated expiration: 2041-07-13
Also published as: CN113527490A

Abstract

The invention discloses a preparation method of an antihuman IFNAR1 monoclonal antibody concentrated solution, which comprises the following steps: first ultrafiltration concentration: at a flow rate of 120-300L/m²H and transmembrane pressure (TMP) of 0.6 to 1.5bar, concentrating the solution containing the anti-human interferon alpha receptor 1(IFNAR1) monoclonal antibody to a protein concentration of 20 to 60mg/ml, to obtain a concentrated sample; ultrafiltration and replacement: replacing the concentrated sample with a replacement buffer solution to obtain an ultrafiltration replacement solution when the usage amount of the replacement buffer solution is 6-10 times of the weight of the concentrated sample; second ultrafiltration concentration: the ultrafiltration replacement solution and the basic amino acid mother liquor are mixed uniformly so that the concentration of the amino acid is 100-. The preparation method can reduce the viscosity of the high-concentration antibody liquid medicine and improve the stability, is simple and feasible, can carry out amplification production, can ensure the high purity of the sample and can obtain higher recovery rate.

Description

Preparation method of anti-human IFNAR1 monoclonal antibody concentrated solution

Technical Field

The invention relates to the technical field of biology, in particular to a preparation method of a concentrated solution of an anti-human IFNAR1 monoclonal antibody.

Background

The biological agent market is a rapidly growing market, has a plurality of research and development pipelines, brings more innovative therapies to patients, and provides more choices for biological agent targeting drugs for diseases which are difficult to treat by traditional chemical drugs. On the other hand, in order to reduce the clinical use cost of the biological preparation and improve the compliance of patients, the dosage form of the biological preparation is gradually changed from a freeze-dried dosage form to a water injection dosage form, and the dosage form is changed from an intravenous administration mode to a subcutaneous injection dosage form. Since the administration dose of the monoclonal antibody injection is usually in the range of 100mg to 600mg, and the volume of the subcutaneous injection solution is generally limited to 2ml or less, in such a case, it is necessary to prepare a highly concentrated protein preparation, and usually the protein content can be up to 100mg/ml or more.

High concentrations of monoclonal antibody injections present many challenges for manufacturing processes, process scaling, and ultimately patient administration. The most important challenge is the ultra-high viscosity, which tends to form highly viscous solutions due to the biopolymer properties of monoclonal antibodies, and the increased interaction forces (e.g., hydrophobicity, charge interactions, etc.) between protein molecules at high concentrations. In some extreme cases, gel-like substances are even formed, which brings about not less challenges to the ultrafiltration membrane and the ultrafiltration equipment, such as reduction of tangential flow rate due to rapid rise of pressure difference during final concentration, gradual runaway of concentration polarization until the phenomenon that protein precipitation blocks the membrane occurs, and thus necessarily causes reduction of recovery rate or process failure. On the other hand, even if the final high concentration protein solution is obtained by modifying the type of device or membrane package, it is difficult to put it into practical clinical use because it is necessary to aspirate it with a disposable sterile syringe or to use a final packaged form of a prefilled needle for subcutaneous administration, and excessively high viscosity causes a decrease in the sliding property of the filled syringe, thereby making it impossible to manually push it under the skin. Another difficulty in concentrating high concentration monoclonal antibody solutions by ultrafiltration is that protein samples tend to aggregate to form soluble aggregates during high concentration, further aggregating to form protein precipitates.

Therefore, for the preparation of high-concentration subcutaneous injection of anti-human IFNAR1 monoclonal antibody, the development of an ultrafiltration concentration preparation method capable of effectively reducing the viscosity of ultrafiltration concentrate, reducing aggregation of monoclonal antibody and improving the stability of monoclonal antibody is needed.

Disclosure of Invention

In order to solve the problems in the prior art, the invention provides a preparation method of an anti-human interferon alpha receptor 1(IFNAR1) monoclonal antibody concentrated solution, which prepares an ultrafiltration concentrated solution containing an anti-human IFNAR1 monoclonal antibody by ultrafiltration concentration, can effectively reduce the viscosity of the ultrafiltration concentrated solution, reduce the aggregation of the anti-human IFNAR1 monoclonal antibody and improve the stability of the ultrafiltration concentrated solution.

The anti-human interferon alpha receptor 1(IFNAR1) monoclonal antibody (QX006N) is a recombinant humanized monoclonal antibody which is self-developed and targets human type I interferon alpha/beta receptor 1, blocks the activity of all type I interferons including IFN alpha, IFN beta and IFN-omega, is intended to be used for treating Systemic Lupus Erythematosus (SLE), and is intended to be administered by intravenous injection or subcutaneous injection. Currently, no monoclonal antibody drug targeting human interferon alpha/beta receptor 1(IFNAR1) on the market exists at home and abroad, the Anifluumab developed by Aslicon has the fastest progress, the Anifluumab is currently subjected to phase 3 clinical research, the preparation specification is 150mg/1ml, and the intended administration mode is intravenous injection or subcutaneous injection. In vitro pharmacodynamic studies showed that QX006N biological activity was comparable to anifroumab. When the novel anti-human type I interferon alpha/beta receptor 1 monoclonal antibody is prepared into a subcutaneous injection, the protein content in the subcutaneous injection is as high as 100-150 mg/mL.

The specific technical scheme of the invention is as follows:

1. a method for preparing a concentrated solution of an anti-human interferon alpha receptor 1(IFNAR1) monoclonal antibody, which comprises the following steps:

first ultrafiltration concentration: at a flow rate of 120-300L/m²H and transmembrane pressure (TMP) of 0.6 to 1.5bar, concentrating the solution containing the anti-human interferon alpha receptor 1(IFNAR1) monoclonal antibody to a protein concentration of 20 to 60mg/ml, to obtain a concentrated sample;

ultrafiltration and replacement: replacing the concentrated sample with a replacement buffer solution to obtain an ultrafiltration replacement solution when the usage amount of the replacement buffer solution is 6-10 times of the weight of the concentrated sample;

second ultrafiltration concentration: mixing the ultrafiltration replacement solution and the alkaline amino acid mother solution uniformly to ensure that the concentration of the amino acid is 100-;

the anti-IFNAR 1 monoclonal antibody comprises three heavy chain complementarity determining regions (CDR-H1, CDR-H2 and CDR-H3) and three light chain complementarity determining regions (CDR-L1, CDR-L2 and CDR-L3), wherein:

(a) the amino acid sequence of CDR-H1 is shown in SEQ ID NO:1 is shown in the specification;

(b) the amino acid sequence of CDR-H2 is shown in SEQ ID NO:2 is shown in the specification;

(c) the amino acid sequence of CDR-H3 is shown in SEQ ID NO:3 is shown in the specification;

(d) the amino acid sequence of CDR-L1 is shown in SEQ ID NO:4 is shown in the specification;

(e) the amino acid sequence of CDR-L2 is shown in SEQ ID NO:5 is shown in the specification; and is

(f) The amino acid sequence of CDR-L3 is shown in SEQ ID NO: and 6.

2. The preparation method according to item 1, wherein the anti-human interferon alpha receptor 1(IFNAR1) monoclonal antibody comprises a heavy chain variable region and a light chain variable region, wherein,

the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO: 7 is shown in the specification; and the number of the first and second electrodes,

the amino acid sequence of the light chain variable region is shown as SEQ ID NO: shown in fig. 8.

3. The method of claim 2, wherein the amino acid sequence of the heavy chain of the anti-human interferon alpha receptor 1(IFNAR1) monoclonal antibody is as shown in SEQ ID NO: 10 is shown in the figure; the amino acid sequence of the light chain is shown as SEQ ID NO: shown at 11.

4. The process according to any one of claims 1 to 3, wherein, in the first ultrafiltration concentration, the solution containing the anti-human interferon alpha receptor 1(IFNAR1) monoclonal antibody is concentrated to a protein concentration of 30 to 50 mg/ml.

5. The production method according to any one of claims 1 to 4, wherein, in the ultrafiltration substitution, the concentration of the substitution buffer is 5 to 50mM, preferably 20 mM; preferably, the pH of the displacement buffer is between 6.0 and 7.0, preferably between 6.2 and 6.8.

6. The production method according to claim 5, wherein the substitution buffer is a histidine-hydrochloric acid buffer, a citric acid buffer, a phosphate buffer, or a sodium acetate-acetic acid buffer, preferably a histidine-hydrochloric acid buffer.

7. The production method according to any one of claims 1 to 6, wherein, in the second ultrafiltration concentration, the basic amino acid is arginine hydrochloride, lysine and/or proline, preferably arginine hydrochloride.

8. The production process according to any one of items 1 to 7, wherein the concentration of the basic amino acid mother liquor is 0.5 to 2.0mol/L, preferably 1 to 1.5 mol/L.

9. The production method according to any one of items 1 to 8, wherein the viscosity of the concentrated solution is 20cP or less.

10. The production method according to any one of items 1 to 9, wherein the solution containing an anti-human interferon alpha receptor 1(IFNAR1) monoclonal antibody is obtained by subjecting a cell fermentation broth expressing an anti-human interferon alpha receptor 1(IFNAR1) monoclonal antibody to affinity chromatography, low pH inactivation, anion chromatography, cation chromatography and nanofiltration.

ADVANTAGEOUS EFFECTS OF INVENTION

The preparation method of the anti-human interferon alpha receptor 1(IFNAR1) monoclonal antibody concentrated solution can reduce the viscosity of high-concentration antibody liquid medicine and improve the stability, is simple and easy to implement, can carry out amplification production, can ensure the high purity of a sample and obtain higher recovery rate. Meanwhile, the verification of high-concentration antibodies of different subtypes proves that the method for reducing the viscosity by adding the basic amino acid has stronger applicability and good stability.

Compared with the existing human interferon alpha receptor 1 monoclonal antibody (Aniflumab), the anti-human interferon alpha receptor 1(IFNAR1) monoclonal antibody has the advantages that the binding affinity of the anti-human interferon alpha receptor 1 monoclonal antibody to IFNAR1 is equivalent, and the neutralizing activity at a cellular level is equivalent to that of the Aniflumab.

The monoclonal antibody shows a neutralizing activity at a cellular level comparable to that of Aniflumab (prepared by expression of a patent publication sequence), and is expected to exhibit a good clinical effect in the prevention and treatment of related diseases.

Drawings

FIG. 1 is a diagram showing the results of nucleic acid electrophoresis for constructing transient expression plasmids of HZD 1203-45. Wherein, M: marker; strip 1: PCR product 362VH-Hu 6; strip 2: pHZDCH, HindIII/NheI; the strip 3: PCR product 362VK-Hu 20; the strip 4: pHZDCK, HindIII/BsiWI.

Fig. 2 is a transient expression flow diagram.

FIG. 3 is an electrophoretic image of QX006N (HZD 1203-45-IgG4.1).

Detailed Description

The present invention is described in detail in the following description of embodiments with reference to the figures, in which like numbers represent like features throughout the figures. While specific embodiments of the invention are shown in the drawings, it should be understood that the invention may be embodied in various forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

It should be noted that certain terms are used throughout the description and claims to refer to particular components. As one skilled in the art will appreciate, various names may be used to refer to a component. This specification and claims do not intend to distinguish between components that differ in name but not function. In the following description and in the claims, the terms "include" and "comprise" are used in an open-ended fashion, and thus should be interpreted to mean "include, but not limited to. The description which follows is a preferred embodiment of the invention, however, the description is given for the purpose of illustrating the general principles of the invention and not for the purpose of limiting the scope of the invention. The scope of the present invention is defined by the appended claims.

The invention provides a preparation method of a monoclonal antibody concentrated solution for resisting human interferon alpha receptor 1(IFNAR1), which comprises the following steps:

ultrafiltration and replacement: replacing the concentrated sample with a replacement buffer solution to obtain an ultrafiltration replacement solution when the usage amount of the buffer solution to be replaced is 6-10 times of the weight of the concentrated sample;

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

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

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

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

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

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

Wherein, SEQ ID NO:1 is as follows:

SYYMT

SEQ ID NO:2 is as follows:

VINVYGGTYYASWAKG

SEQ ID NO:3 is as follows:

EDVAVYMAIDL

SEQ ID NO:4 is as follows:

QASQSISNQLS

SEQ ID NO:5 has the following amino acid sequence:

DASSLAS

SEQ ID NO:6 is as follows:

LGIYGDGADDGIA

for example, the flow rate may be 120L/m²·h、150L/m²·h、180L/m²·h、200L/m²·h、250L/m²·h、300L/m²H, etc.;

the transmembrane pressure difference (TMP) may be 0.6bar, 0.7bar, 0.8bar, 0.9bar, 1.0bar, 1.1bar, 1.2bar, 1.3bar, 1.4bar, 1.5bar, etc.;

in the first ultrafiltration concentration, the protein concentration of the concentrated sample may be, for example, 20mg/ml, 30mg/ml, 40mg/ml, 50mg/ml, 60mg/ml, or the like;

in the ultrafiltration displacement, an ultrafiltration displacement solution is obtained when the amount of the displacement buffer used is 6 times, 7 times, 8 times, 9 times, 10 times, or the like of the weight of the concentrated sample, the amount being the volume usage amount.

In the second ultrafiltration concentration, the ultrafiltration substitution solution and the basic amino acid mother liquor are mixed so that the concentration of the amino acid may be, for example, 100mM, 110mM, 120mM, 130mM, 140mM, 150mM, etc., and the ultrafiltration concentration is performed to obtain a concentrated solution having a protein concentration of, for example, 100mg/ml, 110mg/ml, 120mg/ml, 130mg/ml, 140mg/ml, 150mg/ml, 160mg/ml, 170mg/ml, 180mg/ml, 190mg/ml, 200mg/ml, etc.

The monoclonal antibodies represent antibodies from a population of substantially homologous antibodies, i.e., the individual antibodies comprising the population are identical and/or bind the same epitope, with such variants typically being present in minor amounts, except for possible variant antibodies (e.g., containing naturally occurring mutations or produced during the production of monoclonal antibody preparations). Unlike polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on the antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies to be used in accordance with the present invention can be prepared by a variety of techniques including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods using transgenic animals comprising all or part of a human immunoglobulin locus, such methods and other exemplary methods of preparing monoclonal antibodies being described herein.

The anti-human interferon alpha receptor 1(IFNAR1) monoclonal antibody represents such a monoclonal antibody: which is capable of binding to human interferon alpha receptor 1 with sufficient affinity such that the monoclonal antibody is useful as a diagnostic and/or therapeutic agent targeting human interferon alpha receptor 1.

The anti-human interferon alpha receptor 1(IFNAR1) monoclonal antibody of the invention does not bind to target-independent proteins. Here, "irrelevant protein" means a protein other than human interferon alpha receptor 1 as a target; here, "not to bind" means: the invention of the anti human interferon alpha receptor 1(IFNAR1) monoclonal antibodies and its target human interferon alpha receptor 1 binding capacity as 100%, the invention of the anti human interferon alpha receptor 1 monoclonal antibodies and the unrelated protein binding capacity of less than 10%, such as 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or 0.

The monoclonal antibody of the human interferon alpha receptor 1(IFNAR1) can not be combined with the interferon alpha receptor 1 of other animal species. Here, "other animal species" refers to other animal species than humans, such as marmoset, cynomolgus, pig, dog, rabbit, rat, mouse, guinea pig, and the like; here, "not to bind" means: the invention of the anti human interferon alpha receptor 1(IFNAR1) monoclonal antibodies with other animal species of interferon alpha receptor 1 binding capacity of less than 10%, such as 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or 0 under the condition of the invention of the human interferon alpha receptor 1(IFNAR1) monoclonal antibodies and its target of human interferon alpha receptor 1 binding capacity as 100%.

The anti-human interferon alpha receptor 1 monoclonal antibody has an equilibrium dissociation constant (KD) of less than or equal to 1 mu M, less than or equal to 100nM, less than or equal to 50nM and less than or equal to 40 nM.

The Human Interferon alpha Receptor 1(Human Interferon alpha/beta Receptor 1, IFNAR1) represents a membrane protein derived from Human, and the amino acid sequence of the extracellular region of the membrane protein is shown as SEQ ID NO: 9, wherein the underlined part indicates the signal peptide.

SEQ ID NO：9：

MMVVLLGATTLVLVAVAPWVLSAAAGGKNLKSPQKVEVDIIDDNFILRWNRSDESVGNVTFSFDYQKTGMDNWIKLSGCQNITSTKCNFSSLKLNVYEEIKLRIRAEKENTSSWYEVDSFTPFRKAQIGPPEVHLEAEDKAIVIHISPGTKDSVMWALDGLSFTYSLVIWKNSSGVEERIENIYSRHKIYKLSPETTYCLKVKAALLTSWKIGVYSPVHCIKTTVENELPPPENIEVSVQNQNYVLKWDYTYANMTFQVQWLHAFLKRNPGNHLYKWKQIPDCENVKTTQCVFPQNVFQKGIYLLRVQASDGNNTSFWSEEIKFDTEIQAFLLPPVFNIRSLSDSFHIYIGAPKQSGNTPVIQDYPLIYEIIFWENTSNAERKIIEKKTDVTVPNLKPLTVYCVKARAHTMDEKLNKSSVFSDAVCEKTKPGNTSK

In one embodiment, the anti-human interferon alpha receptor 1(IFNAR1) monoclonal antibody comprises a heavy chain variable region and a light chain variable region, wherein,

Wherein, SEQ ID NO: 7 has the following amino acid sequence:

EVQLVESGGGLVQPGGSLRLSCAASGFSLSSYYMTWVRQAPGKGLEWVSVINVYGGTYYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREDVAVYMAIDLWGQGTLVTVSS

SEQ ID NO: the amino acid sequence of 8 is as follows:

AIQMTQSPSSLSASVGDRVTITCQASQSISNQLSWYQQKPGKAPKLLIYDASSLASGVPSRFSGSRSGTKFTLTISSLQPEDFATYYCLGIYGDGADDGIAFGGGTKVEIK

in one embodiment, the amino acid sequence of the heavy chain of the anti-human interferon alpha receptor 1(IFNAR1) monoclonal antibody is as set forth in SEQ ID NO: 10 is shown in the figure; the amino acid sequence of the light chain is shown as SEQ ID NO: shown at 11.

Wherein, SEQ ID NO: 10 is as follows:

EVQLVESGGGLVQPGGSLRLSCAASGFSLSSYYMTWVRQAPGKGLEWVSVINVYGGTYYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREDVAVYMAIDLWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

SEQ ID NO: 11 is as follows:

AIQMTQSPSSLSASVGDRVTITCQASQSISNQLSWYQQKPGKAPKLLIYDASSLASGVPSRFSGSRSGTKFTLTISSLQPEDFATYYCLGIYGDGADDGIAFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

wherein, SEQ ID NO: 10 and 11 are both humanized sequences.

In one embodiment, in the first ultrafiltration concentration, containing anti human interferon alpha receptor 1(IFNAR1) monoclonal antibody solution is concentrated to the protein concentration of 30-50 mg/ml.

In one embodiment, the present invention is not limited in any way as to the concentration of the substitution buffer in the ultrafiltration substitution, and the concentration of the substitution buffer can be selected as desired by those skilled in the art, for example, from 5 to 50mM, preferably 20 mM.

The pH of the substitution buffer is not particularly limited in the present invention, as long as it is slightly acidic, and for example, the pH of the substitution buffer is 6.0 to 7.0, preferably 6.2 to 6.8.

For example, the concentration of the displacement buffer may be 5mM, 10mM, 20mM, 30mM, 40mM, 50mM, etc.; the pH of the displacement solution may be 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, and the like.

In one embodiment, the replacement buffer is a histidine-hydrochloric acid buffer, a citric acid buffer, a phosphate buffer, or a sodium acetate-acetic acid buffer, preferably a histidine-hydrochloric acid buffer.

The citric acid buffer is, for example, a citric acid-sodium citrate buffer.

The phosphate buffer is, for example, disodium hydrogen phosphate-sodium dihydrogen phosphate buffer.

Preferably, the displacement buffer is pumped into the concentrated sample using a peristaltic pump, and the ultrafiltration is continued while adjusting the pumping rate of the displacement buffer to correspond to the permeation flow rate, and the displacement is completed when the pumping amount of the displacement buffer is 6 to 10 times the weight of the concentrated sample.

In one embodiment, in the second ultrafiltration concentration, the basic amino acid is arginine hydrochloride, lysine and/or proline, preferably arginine hydrochloride.

The concentration of the basic amino acid mother liquor is not subject to any restriction by the present invention and can be selected as desired by the person skilled in the art, for example in one embodiment the concentration of the basic amino acid mother liquor is from 0.5 to 2.0mol/L, preferably from 1 to 1.5 mol/L.

For example, the concentration of the basic amino acid mother liquor may be 0.5mol/L, 1.0mol/L, 1.5mol/L, 2.0mol/L, and the like.

In one embodiment, the concentrated solution has a viscosity of 20cP or less.

For example, the concentrated solution can have a viscosity of, for example, 20cP, 18cP, 15cP, 12cP, 10cP, 8cP, 5cP, 3cP, 1cP, and the like.

Preferably, in the second ultrafiltration concentration, calculating the protein concentration in the ultrafiltration replacement solution, calculating the theoretical volume of the ultrafiltration concentrated solution, adding the basic amino acid mother solution, and mixing, preferably, the volume of the basic amino acid mother solution is 1/9 of the theoretical volume of the ultrafiltration concentrated solution, so that the concentration of the amino acid is 100-150mM, and performing ultrafiltration concentration to obtain a concentrated solution with the protein concentration of 100-200mg/ml, wherein the viscosity of the concentrated solution is less than or equal to 20 cP.

In one embodiment, the solution containing an anti-human interferon alpha receptor 1(IFNAR1) monoclonal antibody is obtained by subjecting a cell fermentation broth expressing an anti-human interferon alpha receptor 1(IFNAR1) monoclonal antibody to affinity chromatography, low pH inactivation, anion chromatography, cation chromatography and nanofiltration.

The mammal used for producing the cell fermentation liquid includes but is not limited to various hybridoma cells, Chinese Hamster Ovary (CHO) cells, preferably CHO cells, which are currently used.

The affinity chromatography adopts Protein A to carry out affinity chromatography.

In one embodiment, in the first ultrafiltration concentration and ultrafiltration displacement, the materials of the ultrafiltration membrane used include, but are not limited to, modified Polyethersulfone (PES), polyvinylidene fluoride (PVDF), Cellulose Acetate (CA), etc., the pore size of which is usually 30kDa or 50kDa, and preferably, the ultrafiltration membrane is Pellicon2/Pellicon3 (type a screen, 30kDa), and the filtration membranes of sydows and pall.

In one embodiment, the anti-human interferon alpha receptor 1(IFNAR1) monoclonal antibody is an IgG4 type antibody that blocks the activity of all type I interferons including IFN alpha, IFN beta, and IFN-omega, intended for treatment of Systemic Lupus Erythematosus (SLE), intended for intravenous or subcutaneous administration. The protein concentration of the injection is as high as 150 mg/ml.

The invention uses the method to prepare the anti-human interferon alpha receptor 1(IFNAR1) monoclonal antibody concentrated solution, can reduce the viscosity, avoid the blocking phenomenon, and simultaneously inhibit protein aggregation and precipitation, thereby playing a role of a protective agent.

The method is simple and easy to implement, can be used for carrying out amplification production, and can ensure the high purity of the sample and obtain higher recovery rate.

Examples

The invention is described generally and/or specifically for the materials used in the tests and the test methods, in the following examples,% means wt%, i.e. percent by weight, unless otherwise specified. The reagents or instruments used are not indicated by manufacturers, and are all conventional reagent products which can be obtained commercially.

Example 1 preparation of anti-human Interferon alpha receptor 1 monoclonal antibody QX006N

Human interferon alpha receptor 1(IFNAR1) is purchased from Shanghai near-shore science and technology Limited company, is used for immunizing New Zealand rabbits, and an antigen binding specificity antibody clone is obtained by applying a B cell cloning technology, so that a monoclonal antibody which is bound with human IFNAR1 and has human IFNAR1 inhibitory activity is screened. First, cell supernatants were examined by Binding ELISA, and clones Binding to human IFNAR1 were selected; then HEK Blue IFN alpha/beta reporter gene cell method is used for detection, and the clone with human IFNAR1 inhibitory activity is selected. The immunization and screening process is entrusted to a commercial company for completion.

37 clones were selected in sequence for recombinant expression and sequencing. 362# and 1203# were determined to have the best cell neutralizing activity and the sequences of the two clones were very similar. Therefore, 362# was humanized and transformed, and when 1203# was obtained by screening and 1203# activity was found to be better, 1203# clone was humanized and transformed on the basis of 362# humanization. Carrying out homology alignment of human IgG germ line sequences (Germine) by using NCBI Igblast, selecting IGHV3-66 a 01 as a heavy chain CDR grafting template, and grafting CDR regions (namely CDR-H1(SEQ ID No:1), CDR-H2(SEQ ID No:2) and CDR-H3(SEQ ID No:3)) of 1203# clone heavy chain into framework regions of IGHV3-66 a 01; selecting IGKV 1-6X 01 as light chain CDR grafting template, grafting the CDR regions of 1203# clone light chain (namely CDR-L1(SEQ ID No:4), CDR-L2(SEQ ID No:5) and CDR-L3(SEQ ID No:6)) into the framework region of IGKV 1-6X 01; and (3) carrying out back mutation on a specific site of the framework region to obtain the variable region of the monoclonal antibody QX 006N. Finally, the humanized heavy chain variable region sequence is shown in SEQ ID NO: 7 is shown in the specification; the amino acid sequence of the humanized light chain variable region is shown as SEQ ID NO: shown in fig. 8.

The gene for the heavy chain variable region (SEQ ID NO: 7) and the gene for the light chain variable region (SEQ ID NO: 8) were obtained by PCR amplification using the gene sequence of the 362# humanized antibody as a template. The HindIII and NheI are used for double enzyme digestion of the heavy chain expression plasmid pHZDCH; HindIII and BsiWI are used for double digestion of the light chain expression plasmid pHZDCK; the PCR amplified genes were inserted into the corresponding expression plasmids using Infusion recombinase, respectively, to construct a heavy chain expression plasmid pHZDDCH-362 VH-Hu6 and a light chain expression plasmid pHZDCK-362VK-Hu 20. In the humanization modification process, the gene of the 1203# humanized antibody was numbered 362 and the protein was numbered 1203.

The results of double restriction by electrophoresis of nucleic acids are shown in FIG. 1. As can be seen from the results shown in FIG. 1, the PCR amplification results of the heavy chain variable region and the light chain variable region of the antibody and the results of double digestion of the heavy chain and light chain expression plasmids are shown, wherein the plasmid size of the heavy chain and the light chain is about 10000bp, the plasmid size of the light chain variable region is about 447bp, and the plasmid size of the heavy chain variable region is about 471 bp.

And obtaining a humanized antibody HZD1203-45 by performing humanized transformation on 1203 #. In order to reduce the ADCC effect of the antibody, the human IgG1 constant region of HZD1203-45 heavy chain expression plasmid pHZDCH-362VH-Hu6 was replaced by human IgG4 to obtain the heavy chain expression plasmid pHZDCH-362VH-Hu 6-IgG4.1.

ExpicHO-S cells were co-transfected with the correct sequence of the heavy chain expression plasmid pHZDDCH-362 VH-Hu6-IgG4.1 and the light chain expression plasmid pHZDCK-362VK-Hu 20. One day before transfection, ExpCHO-S cells were diluted to 3X 10⁶Individual cells/ml were passaged before transfection. On the day of transfection, cell density was diluted to 6X 10⁶Individual cells/ml, 125ml shake flasks with 25ml cells, waiting for transfection. The transfection and expression process is shown in FIG. 2.

Culture supernatants were harvested 4-8 days after transfection and purified in one step with ProteinA. The purified antibody was detected by SDS-PAGE and designated as QX006N (HZD1203-45-IgG4.1), and the results of detection of the antibody by protein electrophoresis are shown in FIG. 3. The protein electrophoresis was performed using denatured reducing gel, and the result of FIG. 3 shows two bands having sizes of about 50kDa and 25kDa, respectively, which are consistent with the theoretical molecular weights of the heavy chain (48.9kDa) and the light chain (23.4 kDa).

Example 2 equilibrium dissociation constant (K)_D) Measurement of (2)

BiacoreT200 was used to detect the affinity of QX006N (HZD1203-45-IgG4.1) to human IFNAR1, all at 25 ℃. A commercial Protein A chip is adopted, and a proper amount of antibody is fixed by a capture method, so that Rmax is about 50RU, and the capture flow rate is 10 mul/min. The antigen is subjected to gradient dilution, the flow rate of the instrument is switched to 30 mul/min, the antigen sequentially flows through a reference channel and a channel for fixing the antibody according to the sequence of the concentration from low to high, and the antigen flows through a buffer solution to serve as a negative control. Each binding and dissociationAfter completion, the chip was regenerated with glycine of pH 1.5. Selecting a 1:1 binding model in Kinetics options by using self-contained analysis software of an instrument for fitting, and calculating a binding rate constant k of the antibody_aDissociation rate constant k_dAnd dissociation equilibrium constant K_DThe value is obtained.

In addition, QX006N (HZD1203-45-IgG4.1) was compared with the affinity of a monoclonal antibody against human IFNAR1, that is, Anifrolumab, which has entered clinical stage III, and the detection method for the known antibody was the same as that for QX006N, and the results are shown in Table 1. Wherein the Anifrolumab is obtained by constructing an expression plasmid according to a 9D4 sequence provided by WO2009100309A2 and transforming an ExpicHO-S cell.

TABLE 1 affinity of antibodies for binding to human IFNAR1

Sample name	k_a(10⁵M^-1S^-1)	k_d(10^-5S^-1)	K_D(10^-10M)
				HZD1203-45-IgG4.1	3.47	3.76	1.08
Anifrolumab	18.67	12.40	0.67

The data in the table are: each sample was tested in triplicate and the data for the mean was calculated.

Example 3QX006N and Anifrolumab neutralize human Interferon-induced HEK Blue IFN α/β cell STAT1/2 phosphorylation Activity

HEK Blue IFN alpha/beta reporter cell line is used for measuring the phosphorylation activity of intracellular signaling molecule STAT1/2 mediated by IFNAR1 of QX006N antagonistic interferon: cells in culture were plated at 4X 10 per well⁴Cells were added to 96 wells, followed by incubation at 37 ℃ and 5% CO₂Incubated under conditions overnight. To the cells were added serial dilutions of antibody in the range of 0 to 5. mu.g/ml, and 0.2ng/ml IFN α.2b. Then at 37 ℃ and 5% CO₂Culturing for 24 hr, collecting cell culture supernatant, adding 10% QUANTI-Blue^TMThe detection reagent is at 37 ℃ and 5% CO₂The reaction is carried out for 1 hour under the condition, then the OD630nm value is detected and a dose-effect curve is drawn, and then the antagonistic activity of the antibody is analyzed, and the experimental result shows that QX006N can inhibit STAT1/2 phosphorylation in interferon-induced HEK Blue IFN alpha/beta cells and inhibit IC of 1/2 phosphorylation activity in interferon-induced HEK Blue IFN alpha/beta cells₅₀5.23ng/ml, while Aniflumab inhibited the IC of 1/2 phosphorylation activity in interferon-induced HEK Blue IFN alpha/beta cells₅₀It was 4.43 ng/ml.

Example 4QX006N and Anifrolumab Activity to neutralize human Interferon to inhibit Daudi cell proliferation

Determination of the cell proliferation activity of QX006N antagonistic interferon induced by IFNAR1 using Daudi human lymphoma cell line: cells in culture were plated at 4X 10 per well⁴Cells were added to 96 wells, followed by incubation at 37 ℃ and 5% CO₂Incubated under conditions overnight. To the cells were added serial dilutions of antibody ranging from 0 to 20. mu.g/ml, and 0.8ng/ml IFN α.2 b. Then at 37 ℃ and 5% CO₂Culturing for 72 hours under the condition, collecting cell culture, detecting cell proliferation condition by adopting CellTiter-Glo, drawing a dose effect curve, further analyzing antagonistic activity of the antibody, and the experimental result shows that QX006N can inhibit interferon inductionThe cell proliferation of Daudi of (1), EC inhibiting interferon-induced cell proliferation activity of Daudi₅₀29.9ng/ml, and the EC of Aniflumab for inhibiting interferon-induced cell proliferation activity of Daudi₅₀31.7 ng/ml.

Example 5QX006N and Anifrolumab neutralize human interferon to induce CXCL10/IP10 activity in whole blood.

Determination of the activity of QX006N in antagonizing interferon release by IFNAR1 at CXCL10/IP10 using human whole blood: whole blood was added to a 96-well plate at 100. mu.l/well and stored temporarily at 37 ℃ and 5% CO₂Under these conditions, serial dilutions of antibody ranging in concentration from 0 to 40. mu.g/ml were added to whole blood, with IFN α, 2b, 40ng/ml TNF α being added. Then at 37 ℃ and 5% CO₂Culturing for 48 hours under the condition, collecting cell culture supernatant, detecting the expression of CXCL10/IP10 in the supernatant by adopting a sandwich ELISA method, drawing a dose effect curve, and further analyzing the antagonistic activity of the antibody, wherein the experimental result shows that QX006N can inhibit interferon-induced whole blood from releasing CXCL10/IP10 and inhibit interferon-induced whole blood from releasing CXCL10/IP10 active IC₅₀698ng/ml, while Aniflumab inhibited interferon-induced whole blood release of CXCL10/IP10 Activity IC₅₀562 ng/ml.

Example 6 anti-human IFNAR1 monoclonal antibody high concentration ultrafiltration concentration viscosity comparison

The antibody QX006N obtained in example 1 was produced by fermentation in a 2L-scale bioreactor using CHO cells as host cells. Obtaining clarified fermentation liquor by a centrifugal machine or a deep membrane filtration, adopting Protein A chromatography to capture target Protein, then removing impurities by low pH value virus inactivation and anion and cation chromatography, carrying out nanofiltration to obtain a solution containing an anti-human interferon alpha receptor 1(IFNAR1) monoclonal antibody, and concentrating the solution by the following method:

(1) first ultrafiltration concentration: the ultrafiltration equipment is Labscale small-sized ultrafiltration instrument (clamping two 50cm pieces)²Pellicon XL membrane package with 30KD cut-off). At 120-²H flow rate, TMP is maintained at 0.6-1.5bar and the solution containing anti-human interferon alpha receptor 1(IFNAR1) monoclonal antibody is addedConcentrating to obtain a concentrated sample, wherein the concentration of the protein is about 30-50 mg/ml.

(2) Ultrafiltration and replacement: and the replacement solution is 20mM histidine-hydrochloric acid buffer solution, the pH value is 6.5, the histidine-hydrochloric acid buffer solution is pumped into the concentrated sample by using a peristaltic pump, ultrafiltration is continued, the pumping speed of the histidine-hydrochloric acid buffer solution is adjusted to be consistent with the permeation flow rate, namely, the weight of the sample is kept constant, and the replacement is completed when the volume of the histidine-hydrochloric acid buffer solution is 7 times of the weight of the concentrated sample to obtain the ultrafiltration replacement solution.

(3) Second ultrafiltration concentration: sampling and measuring the concentration of protein in the ultrafiltration displacement solution, calculating the theoretical volume of the ultrafiltration displacement solution at the moment, adding 1/9 theoretical volume of alkaline amino acid mother liquor, uniformly mixing to ensure that a buffer system is shown in table 1, then concentrating by using a merckmicrobio 30kDa ultrafiltration centrifugal tube to the concentration of the protein in table 1 to obtain concentrated solution, and respectively measuring the viscosity value of the concentrated solution by the following method:

1. taking a disposable special syringe, extracting 200-400 microliters of concentrated solution, exhausting bubbles in the syringe, and lightly wiping residual liquid with dust-free paper;

2. placing the injector into a pipe groove for fixation, clicking an instrument host interface, setting a shearing rate, clicking 'Run' for detection after the setting is finished, recording a viscosity value, and generally measuring for multiple times and taking an average value;

3. for the detection of a plurality of samples, after the previous sample is detected, the injector is taken out, the liquid in the tube is emptied, and after the tube opening is lightly wiped by the dust-free paper, the next sample can be sucked and measured.

The viscosity number results are shown in Table 2.

TABLE 2 viscosity number results

Numbering	Buffer system	Protein concentration mg/mL	Viscosity value cP
				A-1	20mM His-HCL	200	42.1
A-2	20mM His-HCL	180	29.3
				A-3	20mM His-HCL	150	22.5
A-4	20mM His-HCL	120	14.4
				A-5	20mM His-HCL +70mg/ml sucrose	150	25.2
A-6	20mM His-HCL +45mg/ml sorbitol	150	25.6
				A-7	20mM His-HCL+150mM NaCl	150	7.1
A-8	20mM His-HCL +150mM arginine hydrochloride	150	7.2
				A-9	20mM His-HCL +150mM lysine	150	7.3
A-10	20mM His-HCL +150mM proline	150	7.2
				A-11	20mM His-HCL +50mg/ml sucrose +50mM arginine hydrochloride	150	11.5

As shown in the results of Table 2, the viscosity of the anti-human IFNAR1 antibody increased exponentially with the increase of protein concentration, and the viscosity of the monoclonal antibody reached 42.1cP at a concentration of 200mg/ml without the addition of additives, so that the concentration process could not be realized by using a conventional ultrafiltration membrane. Taking 150mg/ml protein concentration as an example, the viscosity of the antibody under the same protein concentration condition can be reduced to below 50% of the original value and less than 20cP by adding 150mM basic amino acid or a combination thereof. Based on the above experimental results, the administration concentration of the anti-human IFNAR1 monoclonal antibody is 100-150 mg/ml.

Example 7 comparison of the different methods

The preparation of a solution (UF0) containing the anti-human interferon alpha receptor 1(IFNAR1) monoclonal antibody QX006N, the first ultrafiltration concentration and the ultrafiltration displacement were performed in the same manner as in example 6;

second ultrafiltration concentration: dividing the ultrafiltration displacement solution into two parts, and respectively carrying out the second-step concentration process:

A. sampling from the first part, determining the concentration of protein in the ultrafiltration displacement solution, calculating the theoretical volume of the ultrafiltration displacement solution at the moment, adding 1/9 theoretical volume of arginine hydrochloride mother liquor (1.5mol/L), mixing uniformly, and concentrating by using a merck Millipore 30kDa ultrafiltration centrifugal tube until the concentration of protein is 150mg/ml, and marking as concentrated solution 1(U1F 2);

B. concentrating the second part directly by the first ultrafiltration concentration method to obtain a concentrated solution 2(U2F2) with protein concentration of 150 mg/ml;

the concentrated solutions 1(U1F2) and 2(U2F2) were each tested for viscosity and purity, wherein viscosity was determined in the same manner as in example 6 and purity was determined as follows:

1. high performance liquid chromatography (Agilent 1260 or equivalent instrument), chromatography column (Waters,

BEH

specification: 3.5 μm, 7.8 × 300 mm; or an equivalent chromatographic column), wherein the mobile phase is PBS buffer solution;

2. unscrewing a quaternary pump pipeline, flushing the pipeline for 3 minutes by 100% mobile phase at the flow rate of 5ml/min, screwing a quaternary pump switch, connecting a chromatographic column, and flushing the chromatographic column by 100% mobile phase at the flow rate of 1.0ml/min for 30 minutes;

3. sample analysis method settings: the flow rate is 1.0ml/min, the analysis time is 15min, and the sample amount is 50 mg; the detection wavelength is 280 nm; before sample injection, the system adaptability is checked (continuous sample injection is carried out by using a reference substance, the sample injection frequency is not less than 5 times, and the result is calculated after 5 times), then 1-needle blank sample is analyzed, and then the sample is analyzed.

4. And (3) sequentially integrating the chromatographic peaks of each sample according to the sequence of polymers, main peaks, degradation products and the like, integrating all chromatographic peaks which are inconsistent with the retention time of the blank sample, and calculating the proportion of each peak by an area normalization method.

The measurement results are shown in Table 3.

TABLE 3 viscosity and purity results for different process treatments

From the results in Table 3, it is clear that the viscosity of the second concentration step of the ultrafiltration process was reduced from the original 22.5cP to 7.2cP after arginine hydrochloride addition, and arginine hydrochloride also acts as a protectant to inhibit the formation of the aggregates to some extent. After the high-concentration anti-human IFNAR1 monoclonal antibody is added with arginine hydrochloride, the viscosity value is obviously reduced, excessive back pressure is not caused, the control of the flow rate and the membrane pressure of the ultrafiltration process is facilitated, and the operability of the process is improved.

EXAMPLE 8 amplification experiment

Based on the above experimental results, example 6 was further scaled up to a 200L pilot scale using a mercuric bopellicon manual ultrafiltration system, sandwiching two 0.5m2 Pellicon type 2 membrane packages. Nanofiltration of the anti-human interferon alpha receptor 1(IFNAR1) monoclonal antibody containing solution (UF0) at a flow rate of 120-²H, controlling TMP at 0.6-1.5bar, firstly performing first ultrafiltration concentration (UF1) according to the method of the embodiment 6, and concentrating the sample to 30-50 mg/ml; then, substitution was carried out by the ultrafiltration substitution method of example 6, and after the substitution was completed, the arginine hydrochloride mother liquor was added and concentrated to a protein concentration of 150mg/ml, and further concentrated to a protein concentration of 193mg/ml (UF2), and the viscosity and purity of the collected samples were as shown in Table 4.

Table 4: summary of pilot scale confirmation results of anti-human IFNAR1 monoclonal antibody ultrafiltration process

As can be seen from the results in Table 4, after the ultrafiltration process is amplified, the control parameters and yield of the flow rate and the membrane pressure in the ultrafiltration process are basically consistent with those of the small scale, and the amplification is smoothly realized. The purity of the sample after ultrafiltration is basically consistent with that before ultrafiltration by the added arginine hydrochloride inhibitor polymer, the viscosity value is controlled to be below 20cP in the range of 190-200 mg/ml, and the arginine inhibitor polymer cannot be concentrated to such high concentration by the ultrafiltration system without the arginine additive.

In conclusion, the method of the invention can prepare the high-concentration low-viscosity concentrated solution of the anti-human interferon alpha receptor 1(IFNAR1) monoclonal antibody, the viscosity is controlled below 20cP when the protein concentration is 100-200mg/ml, and the obtained concentrated solution can be used for preparing the anti-human interferon alpha receptor 1(IFNAR1) monoclonal antibody subcutaneous injection.

The foregoing is directed to preferred embodiments of the present invention, other and further embodiments of the invention may be devised without departing from the basic scope thereof, and the scope thereof is determined by the claims that follow. However, any simple modification, equivalent change and modification of the above embodiments according to the technical essence of the present invention are within the protection scope of the technical solution of the present invention.

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Claims

(f) The amino acid sequence of CDR-L3 is shown in SEQ ID NO: and 6.

2. The method of claim 1, wherein, the anti human interferon alpha receptor 1(IFNAR1) monoclonal antibody contains heavy chain variable region and light chain variable region, wherein,

5. The production method according to any one of claims 1 to 3, wherein, in the ultrafiltration substitution, the concentration of the substitution buffer is 5 to 50 mM.

6. The production method according to any one of claims 1 to 3, wherein, in the ultrafiltration substitution, the concentration of the substitution buffer is 20 mM.

7. The production method according to any one of claims 1 to 3, wherein the pH of the substitution buffer is 6.0 to 7.0.

8. The production method according to any one of claims 1 to 3, wherein the pH of the substitution buffer is 6.2 to 6.8.

9. The method according to claim 5, wherein the substitution buffer is a histidine-hydrochloric acid buffer, a citric acid buffer, a phosphate buffer, or a sodium acetate-acetic acid buffer.

10. The method according to claim 6, wherein the substitution buffer is a histidine-hydrochloric acid buffer, a citric acid buffer, a phosphate buffer, or a sodium acetate-acetic acid buffer.

11. The method according to claim 7, wherein the substitution buffer is a histidine-hydrochloric acid buffer, a citric acid buffer, a phosphate buffer, or a sodium acetate-acetic acid buffer.

12. The method according to claim 8, wherein the substitution buffer is a histidine-hydrochloric acid buffer, a citric acid buffer, a phosphate buffer, or a sodium acetate-acetic acid buffer.

13. The method according to claim 5, wherein the substitution buffer is a histidine-hydrochloric acid buffer.

14. The method according to claim 6, wherein the substitution buffer is a histidine-hydrochloric acid buffer.

15. The method according to claim 7, wherein the substitution buffer is a histidine-hydrochloric acid buffer.

16. The method according to claim 8, wherein the substitution buffer is a histidine-hydrochloric acid buffer.

17. The production method according to any one of claims 1 to 3, wherein in the second ultrafiltration concentration, the basic amino acid is arginine hydrochloride, lysine and/or proline.

18. The production method according to any one of claims 1 to 3, wherein, in the second ultrafiltration concentration, the basic amino acid is arginine hydrochloride.

19. The production method according to any one of claims 1 to 3, wherein the concentration of the basic amino acid mother liquor is 0.5 to 2.0 mol/L.

20. The production method according to any one of claims 1 to 3, wherein the concentration of the basic amino acid mother liquor is 1 to 1.5 mol/L.

21. The production method according to any one of claims 1 to 3, wherein the viscosity of the concentrated solution is 20cP or less.

22. The process according to any one of claims 1 to 3, wherein the solution containing the anti-human interferon alpha receptor 1(IFNAR1) monoclonal antibody is obtained by subjecting a cell fermentation broth expressing the anti-human interferon alpha receptor 1(IFNAR1) monoclonal antibody to affinity chromatography, low pH inactivation, anion chromatography, cation chromatography and nanofiltration.