CN113521276A

CN113521276A - Liquid formulations comprising anti-human interferon alpha receptor 1(IFNAR1) monoclonal antibodies

Info

Publication number: CN113521276A
Application number: CN202110790531.5A
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 Qyuns Therapeutics Co Ltd
Current assignee: Jiangsu Qyuns Therapeutics Co Ltd
Priority date: 2021-07-13
Filing date: 2021-07-13
Publication date: 2021-10-22
Anticipated expiration: 2041-07-13
Also published as: CN113521276B

Abstract

The invention discloses a liquid preparation containing an anti-human interferon alpha receptor 1(IFNAR1) monoclonal antibody, which comprises an anti-human interferon alpha receptor 1(IFNAR1) monoclonal antibody with the protein concentration of 100-200mg/ml and basic amino acid with the amino acid concentration of 5-300 mM; 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 (f) the amino acid sequence of CDR-L3 is as shown in SEQ ID NO: and 6. The liquid preparation has low viscosity and can be easily injected by a syringe, and thus can be used as an injection, particularly a subcutaneous injection.

Description

Liquid formulations comprising anti-human interferon alpha receptor 1(IFNAR1) monoclonal antibodies

Technical Field

The invention relates to the technical field of biology, in particular to a liquid preparation containing an anti-human interferon alpha receptor 1(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 of anti-human IFNAR1 monoclonal antibody subcutaneous injection, it is required to develop a liquid preparation which can effectively reduce the viscosity of ultrafiltration concentrate, reduce aggregation of monoclonal antibody and improve its stability.

Disclosure of Invention

In order to solve the problems in the prior art, the invention provides a liquid preparation containing an anti-human interferon alpha receptor 1(IFNAR1) monoclonal antibody, the liquid preparation contains the anti-human interferon alpha receptor 1(IFNAR1) monoclonal antibody with the protein concentration of 100-200mg/ml and basic amino acid with the amino acid concentration of 5-300mM, the liquid preparation can effectively reduce the viscosity of ultrafiltration concentrated solution, reduce the aggregation of the anti-human IFNAR1 monoclonal antibody and improve the stability of the anti-human interferon alpha receptor 1(IFNAR1), and can be used for injections, particularly subcutaneous injections.

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. The new anti-human type I interferon alpha/beta receptor 1 monoclonal antibody is prepared into subcutaneous injection, and 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 liquid preparation comprising an anti-human interferon alpha receptor 1(IFNAR1) monoclonal antibody, said liquid preparation comprising an anti-human interferon alpha receptor 1(IFNAR1) monoclonal antibody having a protein concentration of 100-200mg/ml and a basic amino acid having an amino acid concentration of 5-300 mM;

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 liquid formulation of 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 liquid formulation 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 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.

4. The liquid preparation according to any one of claims 1 to 3, wherein the basic amino acid is one or more selected from arginine, lysine, histidine and proline.

5. The liquid preparation according to item 4, wherein the basic amino acid comprises 100-200mM arginine and 5-50mM histidine.

6. The liquid preparation according to item 4, wherein the basic amino acid comprises 100-200mM lysine and 5-50mM histidine.

7. The liquid preparation according to item 4, wherein the basic amino acid comprises 100-200mM proline and 5-50mM histidine.

8. The liquid formulation according to any one of claims 1 to 7, wherein the liquid formulation further comprises 20 to 150mg/ml of sucrose, preferably 50 to 100mg/ml of sucrose.

9. The liquid formulation according to any one of claims 1 to 4, wherein the liquid formulation further comprises 0.5 to 50mg/ml sorbitol, preferably 1 to 20mg/ml sorbitol.

10. The liquid preparation according to any of items 1 to 4, wherein the liquid preparation further comprises 20 to 300mg/ml of sodium chloride, preferably 100 to 200mg/ml of sodium chloride.

11. The liquid preparation according to any one of claims 1 to 10, wherein the pH of the liquid preparation is 5.5 to 6.5.

ADVANTAGEOUS EFFECTS OF INVENTION

The liquid preparation comprises an anti-human interferon alpha receptor 1(IFNAR1) monoclonal antibody, has low viscosity, can be injected by a syringe easily, and can be used as an injection, particularly a subcutaneous injection.

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 liquid preparation containing an anti-human interferon alpha receptor 1(IFNAR1) monoclonal antibody, which comprises the anti-human interferon alpha receptor 1(IFNAR1) monoclonal antibody with the protein concentration of 100-200mg/ml and basic amino acid with the amino acid concentration of 5-300 mM;

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

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

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

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

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

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

Wherein, the amino acid sequence of SEQ ID NO. 1 is as follows:

SYYMT

the amino acid sequence of SEQ ID NO 2 is as follows:

VINVYGGTYYASWAKG

the amino acid sequence of SEQ ID NO 3 is shown below:

EDVAVYMAIDL

the amino acid sequence of SEQ ID NO. 4 is as follows:

QASQSISNQLS

the amino acid sequence of SEQ ID NO 5 is shown below:

DASSLAS

the amino acid sequence of SEQ ID NO 6 is shown below:

LGIYGDGADDGIA

for example, the protein concentration may be 100mg/ml, 110mg/ml, 120mg/ml, 130mg/ml, 140mg/ml, 150mg/ml, 160mg/ml, 170mg/ml, 180mg/ml, 190mg/ml, 200mg/ml, etc.;

the amino acid concentration may be, for example, 5mM, 10mM, 20mM, 30mM, 40mM, 50mM, 60mM, 70mM, 80mM, 90mM, 100mM, 150mM, 200mM, 250mM, 300mM, or the like.

The use of the basic amino acid and the protein at the specific concentrations enables the viscosity of the liquid preparation to be controlled to 10cP or less, and the liquid preparation can be injected with a syringe with ease, and therefore, the liquid preparation can be used as an injection, particularly subcutaneous injection.

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, the basic amino acid is selected from one or more of arginine, lysine, histidine and proline.

The present invention is not limited in any way with respect to the concentration of the various basic amino acids, which can be selected as desired by the person skilled in the art, for example, in one embodiment, the basic amino acids comprise 100-200mM arginine and 5-50mM histidine.

For example, the basic amino acid can comprise 100mM, 110mM, 120mM, 130mM, 140mM, 150mM, 160mM, 170mM, 180mM, 190mM, 200mM, etc. arginine and 5mM, 10mM, 15mM, 20mM, 25mM, 30mM, 35mM, 40mM, 45mM, 50mM, etc. histidine.

For example, the basic amino acids are 20mM histidine and 150mM arginine.

The use of the above-mentioned basic amino acid composition enables the viscosity of a liquid preparation to be greatly reduced, for example, to 10cp or less.

In one embodiment, wherein the basic amino acid comprises 100-200mM lysine and 5-50mM histidine.

For example, the basic amino acid may comprise 100mM, 110mM, 120mM, 130mM, 140mM, 150mM, 160mM, 170mM, 180mM, 190mM, 200mM, etc. lysine and 5mM, 10mM, 15mM, 20mM, 25mM, 30mM, 35mM, 40mM, 45mM, 50mM, etc. histidine.

For example, the basic amino acids are 20mM histidine and 150mM lysine.

In one embodiment, the basic amino acids comprise 100-200mM proline and 5-50mM histidine.

For example, the basic amino acid may comprise proline at 100mM, 110mM, 120mM, 130mM, 140mM, 150mM, 160mM, 170mM, 180mM, 190mM, 200mM, etc., and histidine at 5mM, 10mM, 15mM, 20mM, 25mM, 30mM, 35mM, 40mM, 45mM, 50mM, etc.

For example, the basic amino acids are 20mM histidine and 150mM proline.

In one embodiment, the liquid formulation further comprises 20-150mg/ml sucrose, preferably 50-100mg/ml sucrose.

For example, the liquid formulation may further comprise 20mg/ml, 30mg/ml, 40mg/ml, 50mg/ml, 60mg/ml, 70mg/ml, 80mg/ml, 90mg/ml, 100mg/ml, 110mg/ml, 120mg/ml, 130mg/ml, 140mg/ml, 150mg/ml, and the like of sucrose.

In one embodiment, the liquid formulation comprises 50mg/ml sucrose.

In one embodiment, the liquid formulation further comprises from 0.5 to 50mg/ml sorbitol, preferably from 1 to 20mg/ml sorbitol.

For example, the liquid formulation may also contain 0.5mg/ml, 1mg/ml, 5mg/ml, 10mg/ml, 15mg/ml, 20mg/ml, 30mg/ml, 40mg/ml, 50mg/ml, and the like of sorbitol.

In one embodiment, for example, the liquid formulation comprises 70mg/ml sucrose.

In one embodiment, the liquid formulation further comprises 20-300mg/ml sodium chloride, preferably 100-200mg/ml sodium chloride.

For example, the liquid formulation may also contain 20mg/ml, 30mg/ml, 40mg/ml, 50mg/ml, 60mg/ml, 70mg/ml, 80mg/ml, 90mg/ml, 100mg/ml, 110mg/ml, 120mg/ml, 130mg/ml, 140mg/ml, 150mg/ml, 160mg/ml, 170mg/ml, 180mg/ml, 190mg/ml, 200mg/ml, 250mg/ml, 300mg/ml, and the like of sodium chloride.

In one embodiment, for example, the liquid formulation comprises 150mg/mL of sodium chloride, which in combination with histidine enables the viscosity of the liquid formulation to be controlled below 10 cP.

The present invention is not limited in any way as to the pH of the liquid formulation, which can be selected as desired by one skilled in the art, for example, in one embodiment, the pH of the liquid formulation is 5.5 to 6.5.

In one embodiment, the liquid preparation 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 ultrafiltration concentration, and then mixing a concentrated solution obtained by ultrafiltration concentration with a buffer solution or an additive mother liquor.

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, 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 liquid preparation of the present invention has a low viscosity, and can be easily injected by a syringe, and thus is suitable for use as an injection, particularly a subcutaneous injection.

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 assayed by BindingELISA 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. After each binding and dissociation, the chip was regenerated with glycine of pH 1.5. Selection of 1:1 node in Kinetics options by instrumental self-contained analysis softwareFitting the model, and calculating the 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 been currently in 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	3.47E+05	3.76E-05	1.08E-10
Anifrolumab	1.87E+06	1.24E-04	6.65E-11

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

Example 3 QX006N 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% CO₂Reacting for 1 hour under the condition, detecting an OD630nm value and drawing a dose-effect curve, and further analyzing the antagonistic activity of the antibody to obtain that QX006N can inhibit STAT1/2 phosphorylation in interferon-induced HEK Blue IFN alpha/beta cells, IC50 of QX006N for inhibiting 1/2 phosphorylation activity in interferon-induced HEK Blue IFN alpha/beta cells is 5.23ng/ml, and IC50 of anifroumab for inhibiting 1/2 phosphorylation activity in interferon-induced HEK Blue IFN alpha/beta cells is 4.43 ng/ml.

Example 4 QX006N 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, obtaining that QX006N can inhibit interferon-induced Daudi cell proliferation, EC50 of QX006N for inhibiting interferon-induced Daudi cell proliferation activity is 29.9ng/ml, and Aniflumab EC50 inhibiting interferon-induced cell proliferation activity was 31.7 ng/ml.

Example 5 QX006N 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 response curve, and further analyzing the antagonistic activity of the antibody to obtain that QX006N can inhibit the interferon-induced whole blood to release CXCL10/IP10, IC50 of QX006N to inhibit the interferon-induced whole blood to release CXCL10/IP10 activity is 698ng/ml, and IC50 of Aniflumab to inhibit the interferon-induced whole blood to release CXCL10/IP10 activity is 562 ng/ml.

Example 6 comparison of the viscosity of liquid formulations containing anti-human IFNAR1 monoclonal antibody and different basic amino acids

The antibody QX006N described in example 1 was produced by fermentation using CHO cells as host cells in a 2L scale bioreactor, clarified broth was obtained by centrifugation or submerged membrane filtration, Protein A chromatography was used to capture the target Protein, low pH viral inactivation and anion and cation chromatography were used to remove impurities, and ultrafiltration and concentration were carried out to obtain a concentrate containing the anti-human interferon alpha receptor 1(IFNAR1) monoclonal antibody, the concentrate containing 20mM His-HCL.

The concentrated solution obtained is analyzed by SEC-HPLC, and the operation steps are 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 sequentially integrating 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, calculating the proportion of each peak by an area normalization method, and determining that the QX006N monomer in the concentrated solution is more than 99 percent, the polymer is less than 1 percent and the concentrated solution does not contain heteroproteins basically.

The concentration of QX006N in the concentrated solution was measured by UV spectrophotometry as follows:

1. the wavelength of the spectrophotometer was adjusted to 280nm and the calibration was performed using blank buffer or water as a control.

2. The sample to be tested is diluted by blank buffer solution or water, the absorbance value of the sample at 280nm is measured (the absorbance value is ensured to be between 0.5 and 1.5), and the sample concentration is calculated according to the following formula (the extinction coefficient of QX006N is 1.469).

The concentration of QX006N in this concentrate was determined to be 200 mg/mL.

The viscosity of the concentrate was measured using a μ VISC viscometer from Sharp, and the procedure was as follows:

1. taking a disposable special syringe, extracting 200-400 microliters of a sample to be detected, 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 syringe is taken out, the liquid in the tube is emptied, the non-dust paper is used for slightly wiping the tube orifice and then the next sample can be sucked and measured, and the viscosity of the concentrated solution is measured to be 42.1 cP.

After filtering the samples with a 0.2 μm filter, the samples were mixed with different buffers or additive stocks of Table 2 so that the protein concentration is shown in Table 2, and then the viscosity values were determined as described above, and the results are shown in Table 2.

TABLE 2 viscosity results for the addition of different basic amino acid mother liquors

It is considered that the liquid preparation having a viscosity of less than 30cP is suitable for subcutaneous injection, and the results of Table 2 show that the liquid preparation of the present invention, QX006N, can be administered at a concentration of at least 100-180mg/mL or 150-180mg/mL by subcutaneous injection.

In conclusion, the liquid preparation of the invention has low viscosity, is suitable for injection, particularly subcutaneous injection, and has the advantages that the anti-human interferon alpha receptor 1(IFNAR1) monoclonal antibody has the same affinity with IFNAR1 and the neutralizing activity at a cellular level as that of the existing anti-human interferon alpha receptor 1 monoclonal antibody (Aniflumab).

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.

Sequence listing

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Claims

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

2. The liquid formulation of claim 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,

5. The liquid preparation as claimed in claim 4, wherein the basic amino acid comprises 100-200mM arginine and 5-50mM histidine.

6. The liquid preparation as claimed in claim 4, wherein the basic amino acid comprises 100-200mM lysine and 5-50mM histidine.

7. The liquid preparation as claimed in claim 4, wherein the basic amino acid comprises proline at 100-200mM and histidine at 5-50 mM.

8. The liquid formulation according to any one of claims 1 to 7, wherein the liquid formulation further comprises 20-150mg/ml sucrose, preferably 50-100mg/ml sucrose.

9. The liquid formulation according to any one of claims 1 to 4, wherein the liquid formulation further comprises 0.5-50mg/ml sorbitol, preferably 1-20mg/ml sorbitol.

10. The liquid formulation according to any one of claims 1 to 4, wherein the liquid formulation further comprises 20 to 300mg/ml of sodium chloride, preferably 100 and 200mg/ml of sodium chloride;

preferably, the pH of the liquid preparation is 5.5-6.5.