Stable formulations containing anti-IL-4R antibodiesTechnical FieldThe present invention relates to subcutaneous formulations of anti-IL-4R antibodies that are stable at room temperature for at least 6 months, which formulations can be used for the treatment of related diseases suitable for treatment with anti-IL-4R antibodies by subcutaneous administration.
BackgroundMonoclonal antibody drugs are the most rapidly developed industry in the field of biopharmaceuticals in recent years, and compared with chemical small molecule drugs, monoclonal antibody drugs have more complex physicochemical properties, lower stability and easy formation of aggregates during storage. The change of physicochemical properties may cause the immunogenicity of the pharmaceutically active agent to be changed, so that a prescription needs to be screened according to the characteristics of the monoclonal antibody medicament, and a proper preparation is developed to ensure the safety, the effectiveness and the stability of the protein medicament (see the role of the prescription of the recombinant monoclonal antibody medicament preparation and relevant evaluation points, xiao, luo Jianhui, volume 28 and 16 of the journal of new drugs in China 2019).
Protein formulations should help maintain the stability and bioactivity of the biopolymer during production, packaging, storage and shipment until final delivery to the target site in the patient.
Conventional antibody drug delivery methods include intravenous delivery, subcutaneous injection and intramuscular injection. Intravenous delivery is typically high-dose or multi-dose, takes about 90 minutes or more for injection, and requires assistance from medical-knowledgeable personnel and prior preparation procedures, thus making it inconvenient and costly for patients, doctors, and medical personnel. Intramuscular injections generally have small injection points, which can cause side effects such as tissue hardening, edema, etc., so that the injection volume cannot be too large, and it is generally considered that the injection volume cannot exceed 20 ml, and the injection time cannot be too long. In contrast, subcutaneous injection has the advantage of being immediately administered and is convenient for the patient to self-administer, but the absorption rate is relatively low compared to intravenous injection, and when the injection amount is 3-5mL or more, it may cause swelling and pain at the injection site due to slow absorption. For this reason, subcutaneous injection of protein therapeutics is typically limited to small 2mL or less solution injections.
The anti-IL-4R antibodies that have been marketed at present are mainly Dupilumab (Dupilumab) from Sainophenanthrene, and AZD-1402 (Pieris Pharmaceuticals Inc, astraZeneca plc), AK-120 (Akeso Biopharma Inc), CM-310 (KeyMed Biosciences Co Ltd, CSPC Pharmaceuticals Group Limited) are also in clinical settings. Dipirumab is an interleukin 4 receptor alpha antagonist for use in the treatment or prevention of diseases such as atopic dermatitis, allergic asthma, chronic rhinosinusitis and other conditions in which nasal polyps are associated. Currently, when the anti-IL-4R antibody preparation on the market is suitable for subcutaneous injection, 2mL can be injected at most, and the administration is carried out once a week, the single administration amount of the medicine is limited by the existing preparation form, and the administration period is short. Therefore, there is a need to develop a new formulation that can reduce the number of administrations to patients and reduce the frequency of subcutaneous injections.
Disclosure of Invention
The problem to be solved by the present invention is to provide a stable pharmaceutical formulation comprising a high concentration of anti-IL-4R antibody that can be subcutaneously injected, with a single injection volume of more than 2ml, e.g. a single injection volume of 3ml,4ml,5ml,6ml,7ml,8ml, or more.
Subcutaneous injection is a classical micro-volume injection mode, the injection must enter an action target point through a skin matrix, and the skin matrix composed of structural macromolecules such as collagen, elastin, fibronectin and Hyaluronic Acid (HA) HAs a complex three-dimensional structure, so that the diffusion rate and the infusion amount of subcutaneous injection are limited. The inventors have unexpectedly found that the anti-IL-4R antibodies and hyaluronidases of the invention can form stable formulations, and that the presence of hyaluronidase does not affect the activity of the antibodies of the invention, which stable formulations enable subcutaneous injections at higher volumes. First, the preparation of high concentration antibody formulations is challenging, higher concentrations of protein can be very viscous, increasing the risk of aggregation; secondly, it is very difficult to add hyaluronidase and maintain a high concentration of antibody and achieve a certain stability. Also, although antibodies have very similar overall structures, antibodies differ in amino acid composition and glycosylation, and each antibody has different aggregation behavior, so that a stable preparation is obtained for a certain antibody, particularly a preparation containing hyaluronidase at the same time is not regularly circulated.
The present invention unexpectedly results in a stable pharmaceutical formulation comprising a high concentration of anti-IL-4R antibody that can be used for subcutaneous injection in single doses exceeding 2ml.
In a first aspect the invention provides a pharmaceutical formulation comprising an anti-IL-4R antibody comprising the following components:
i. an anti-IL-4R antibody, preferably, the antibody comprises a heavy chain variable region comprising SEQ ID NO. 1 and a light chain variable region comprising SEQ ID NO. 2; and
hyaluronidase;
optionally, the pharmaceutical formulation further comprises
i. An antioxidant; and/or
A first buffer; and/or
Heat stabilizer; and/or
iv, an organic cosolvent; and/or
v. viscosity reducing agent; and/or
Salt ion enhancers or second buffers; and/or
Wherein the formulation has a pH of about 4.5 to about 7.5.
Optionally, the pharmaceutical formulation further comprises:
i. an antioxidant;
a first buffer;
iii a heat stabilizer;
iv, an organic cosolvent;
v. viscosity reducing agent; and/or
Salt ion enhancers or second buffers;
wherein the formulation has a pH of about 4.5 to about 7.5.
Alternatively, the concentration of the antibody is from about 100mg/ml to about 200mg/ml, preferably from about 115mg/ml to about 185mg/ml, preferably from about 135mg/ml to about 175mg/ml, preferably from 150mg/ml to about 175mg/ml, and more preferably about 150mg/ml or 175mg/ml.
The term "mM" as used herein means "mmol/L".
Hyaluronidase is an enzyme that degrades hyaluronic acid and reduces the viscosity of hyaluronic acid in the extracellular matrix, thereby increasing tissue permeability. The enzymatic activity of hyaluronidases (including rHuPH 20) can be defined by units/ml (U/ml) or by total enzymatic activity (U) in a particular formulation, as further explained below.
A standard definition of one unit of enzyme activity (U) is the amount of enzyme per unit time that catalyzes a defined amount of substrate reaction, e.g., one mole or one nanomole of substrate per minute. Techniques for determining hyaluronidase preparation activity are known in the art, and hyaluronidase activity is generally expressed in USP units or units, hereinafter referred to as "U/ml" for hyaluronidase activity.
Hyaluronidase activity refers to the ability of the enzyme to catalyze cleavage of hyaluronic acid. The United States Pharmacopeia (USP) provides a measurement of hyaluronidase, wherein hyaluronidase activity can be determined indirectly by: after allowing the enzyme to react with HA at 37℃for 30 minutes, the amount of higher molecular weight hyaluronic acid, or hyaluronan substrate, remaining was measured (see USP XXII-NFXVII (1990) 644-645United States Pharmacopeia Convention,Inc,Rockville,Md.). A reference standard solution can be used in the assay to determine the relative activity (in units) of any hyaluronidase. In vitro assays for assaying hyaluronidase activity of hyaluronidase (e.g., rHuPH 20) are known in the art and are described herein.
Alternatively, the hyaluronidase is produced by recombinant DNA technology, derived from human samples, or extracted from animal tissue.
Preferably a humanized recombinant hyaluronidase rHuPH20 (SEQ ID NO: 5) manufactured by recombinant DNA technology.
Alternatively, the hyaluronidase rHuPH20 is present at a concentration of about 1000U/ml to about 12000U/ml, preferably about 1000U/ml to about 10000U/ml, preferably about 2000U/ml to about 6000U/ml, preferably about 2000U/ml to about 4000U/ml. The amount of hyaluronidase should be such that an increased dispersion and absorption of the dipivluzumab for use is possible.
Optionally, the antioxidant is selected from at least one of methionine, ascorbic acid, citric acid, tartaric acid, or any other excipient for minimizing oxidation. Preferably, the antioxidant is methionine. The antioxidant effectively reduces free radicals, so that the concentration of the oxidant is reduced, the oxidation reaction of the monoclonal antibody is reduced, and the function of stabilizing the activity of the hyaluronidase is also realized.
Alternatively, the antioxidant is present at a concentration of about 1mM to about 50mM, preferably about 5mM to about 25mM, preferably about 10mM to about 20mM, preferably about 10mM, about 15mM or about 20mM.
Alternatively, the formulation has a pH of about 5.0 to about 7.5, preferably about 5.5 to about 7.0, preferably about 5.5 to about 6.5, preferably about 5.5 to 6.0, preferably about 5.5 to about 5.9.
The buffer capacity of the buffer is critical, ensuring the stability of the antibody. Optionally, the first buffer is selected from at least one of acetate buffer, histidine buffer, phosphate, citrate, boric acid, tris buffer, HEPES, preferably histidine buffer; and/or
The second buffer is at least one selected from acetate buffer, histidine buffer, phosphate, citrate, boric acid, tris buffer, HEPES, preferably acetate buffer;
further preferably, the first buffer is different from the second buffer.
Alternatively, the concentration of the first buffer is from about 1mM to about 100mM, preferably from about 10mM to about 50mM, preferably from about 15mM to about 35mM, preferably about 20mM; and/or
The concentration of the second buffer is from about 1mM to about 100mM, preferably from about 5mM to about 50mM, preferably from about 10mM to about 20mM, preferably about 12.5mM.
Alternatively, the first buffer is histidine buffer at a concentration of about 15mM to about 35mM, preferably about 20mM, and/or
The second buffer is acetate buffer at a concentration of about 10mM to about 20mM, preferably about 12.5mM.
Alternatively, the concentration of the salt ion enhancing agent is about 5mM-100mM, preferably about 5mM-50mM, more preferably about 5mM-25mM, and even more preferably about 12.5mM.
Optionally, the salt ion enhancer is sodium chloride or potassium chloride.
Alternatively, the heat stabilizer is present at a concentration of about 100mM to about 300mM, preferably about 100mM to about 250mM, preferably about 150mM to about 250mM, preferably about 155mM to about 210mM.
Optionally, the heat stabilizer is at least one selected from sucrose, trehalose and mannitol, preferably sucrose or trehalose.
Optionally, the organic cosolvent is selected from at least one of polysorbate 20, polysorbate 80, poloxamer, propylene glycol, triton (polyethylene glycol octylphenyl ether), polyethylene glycol 3350 and sodium dodecyl sulfate, preferably, the organic cosolvent is polysorbate 20 or polysorbate 80, and further preferably, the organic cosolvent is polysorbate 80. Among them, polysorbate 20 and polysorbate 80 are mainly distinguished by the structure of fatty acid side chains, and the difference in fatty acid side chain length and saturation degree leads to the difference in affinity of polysorbate with proteins. The addition of the organic cosolvent reduces aggregation of the protein in the processes of stirring, shaking, freeze thawing and freeze drying, and plays a role in preventing the protein from being adsorbed to the surface of the container.
For the same polysorbate, the ability to stabilize the protein is related to its concentration, and the inventors found that polysorbate (e.g., polysorbate 20) in a specific formulation was able to inhibit protein aggregation caused by shaking when a concentration was reached below which polysorbate 20 was unable to reduce protein instability caused by shaking (see recombinant monoclonal antibody drug formulation action and related review points, xiao, luo Jianhui, journal of new drugs, vol.28, 16, of China 2019).
Alternatively, the organic co-solvent is polysorbate 20 or polysorbate 80 at a concentration of about 0.05% w/v to about 1.2% w/v, preferably about 0.05% w/v to about 0.5% w/v, preferably about 0.05% w/v to about 0.25% w/v, further preferably 0.05% w/v to about 0.2% w/v.
Alternatively, the viscosity-lowering agent is an amino acid selected from at least one of arginine, lysine and proline, preferably, the viscosity-lowering agent is arginine.
Alternatively, the viscosity reducing agent is present at a concentration of from 1mM to about 100mM, preferably from about 5mM to about 80mM, preferably from 10mM to about 50mM, preferably from about 10mM to about 30mM, preferably from about 12.5mM to about 25mM.
The invention also provides a stable pharmaceutical formulation wherein at least 96.0% of the antibody is present in monomeric form after 20 weeks of storage at 4 ℃ as determined by size exclusion chromatography.
The invention also provides a stable liquid pharmaceutical formulation wherein at least 95.5% of the antibody is present in monomeric form after 20 weeks of storage at 25 ℃ as determined by size exclusion chromatography;
the invention also provides a stable pharmaceutical formulation wherein at least 95.0% of the antibody is present in monomeric form after 12 weeks of storage at 37 ℃ as determined by size exclusion chromatography.
The present invention also provides a stable pharmaceutical formulation comprising: i. about 150mg/ml to about 175mg/ml of an anti-IL-4R antibody, wherein the antibody comprises a heavy chain variable region comprising SEQ ID No. 1 and a light chain variable region comprising SEQ ID No. 2; about 2000U/ml to about 4000U/ml rHuPH20; about 10mM to about 20mM methionine; about 10mM to about 15mM acetate buffer and/or about 18mM to about 22mM histidine buffer; about 150mM to about 160mM sucrose; about 0.05% w/v to about 0.25% w/v polysorbate 80; about 23mM to about 27mM arginine; wherein the formulation has a pH of about 5.5 to about 6.5, preferably about 5.5 to 5.9.
The present invention also provides a stable liquid pharmaceutical formulation comprising: i. about 150mg/ml to about 175mg/ml of an anti-IL-4R antibody, wherein the antibody comprises a heavy chain variable region comprising SEQ ID No. 1 and a light chain variable region comprising SEQ ID No. 2; about 2000U/ml to about 4000U/ml rHuPH20; about 10mM to about 20mM methionine; about 10mM to about 15mM acetate buffer and/or about 18mM to about 22mM histidine buffer; v. about 190mM to about 230mM trehalose; about 0.05% w/v to about 0.2% w/v polysorbate 80; about 12.5mM to about 25mM arginine; wherein the formulation has a pH of about 5.5 to about 6.5, preferably about 5.5 to 5.9.
The present invention also provides a stable liquid pharmaceutical formulation comprising: i. about 150mg/ml to about 175mg/ml of an anti-IL-4R antibody, wherein the antibody comprises a heavy chain variable region comprising SEQ ID No. 1 and a light chain variable region comprising SEQ ID No. 2; about 2000U/ml to about 4000U/ml rHuPH20; about 10mM to about 20mM methionine; about 18mM to about 22mM histidine buffer; v. about 190mM to about 230mM trehalose; about 0.05% w/v to about 0.2% w/v polysorbate 80; about 23mM to about 27mM arginine; about 5mM to about 12.5mM sodium chloride; wherein the formulation has a pH of about 5.5 to about 6.5.
A stable pharmaceutical formulation comprising: i. about 150mg/ml to about 175mg/ml of an anti-IL-4R antibody, wherein the antibody comprises a heavy chain variable region comprising SEQ ID NO. 1 and a light chain variable region comprising SEQ ID NO. 2; about 2000U/ml to about 4000U/ml rHuPH20; about 10mM to about 20mM methionine; about 12.5mM to about 20mM histidine buffer and about 12.5mM to about 15mM acetate buffer; about 150mM to about 210mM trehalose; about 0.05% w/v to about 0.2% w/v polysorbate 80; about 12.5mM to about 25mM arginine; wherein the formulation has a pH of about 5.9 to about 6.5.
A stable pharmaceutical formulation comprising: i. about 150mg/ml to about 175mg/ml of an anti-IL-4R antibody, wherein the antibody comprises a heavy chain variable region comprising SEQ ID NO. 1 and a light chain variable region comprising SEQ ID NO. 2; about 2000U/ml to about 4000U/ml rHuPH20; about 10mM to about 20mM methionine; about 12.5mM to about 20mM histidine buffer and about 12.5mM to about 15mM acetate buffer; v. about 155mM to about 210mM sucrose; about 0.05% w/v to about 0.2% w/v polysorbate 80; about 12.5mM to about 25mM arginine; wherein the formulation has a pH of about 5.9 to about 6.5.
The invention also provides a freeze-dried preparation prepared by the preparation of any one of the above.
In a second aspect the present invention provides a container comprising a stable liquid pharmaceutical formulation according to any one of the first aspects.
Optionally, the container is a tube, bottle, vial or syringe.
In a third aspect the invention provides a kit comprising a container according to the second aspect and a device for infusing the formulation.
In a fourth aspect, the invention provides the use of a pharmaceutical formulation according to any one of the first aspects for the manufacture of a medicament for the treatment of a disease associated with an anti-IL-4 antibody.
The invention also provides a pharmaceutical formulation according to any one of the first aspects for use in the treatment of a related disorder suitable for treatment with an anti-IL-4 antibody.
The invention also provides a method of treating an IL-4-associated disorder comprising administering a therapeutically effective amount of the formulation of any one of the first aspects.
DrawingsFig. 1: AD model construction: 0,6,7,9,11,14,16,18,21,23,25 days from left to right, i.e. the experiment was run for 26 days in total; mice were sensitized by applying 25 μl of 0.8% oxazolone solution evenly to the right ear and back of the mice on day 0, followed by 25 μl of 0.4% oxazolone solution applied to the same sites on day 7,9,11,14,16,18,21,23,25 of the experiment for challenge.
Fig. 2: the weight of the mice in the different groups was varied over 0-25 days, with the horizontal axis being the number of days of the experiment (in days) and the vertical axis being the weight (in grams).
Fig. 3: the variation of the ear thickness of the mice during the experimental period, the horizontal axis is the number of days of the experiment (unit: day), and the vertical axis is the ear thickness of the mice (unit: mm).
Fig. 4: total lgE content in serum from mice of different groups on day 26 of the experiment, with the vertical axis being the total lgE content in serum from mice (unit: ng/ml).
Detailed DescriptionThe following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the pharmaceutical formulations of the present invention, and are not intended to limit the scope of what is claimed.
Terminology
In this application, when referring to a specifically recited value or range of values, the term "about" or "approximately" as used herein generally means within 20%, preferably within 10%, more preferably within 5% of the given value or range.
The term "IL-4R" means a human cytokine receptor that specifically binds to interleukin-4 (IL-4), IL-4Rα.
The term "anti-IL-4R antibody" means a human antibody or antigen-binding fragment thereof that specifically binds to human IL-4R.
The term "antibody" generally refers to immunoglobulin molecules comprising four polypeptide chains, two heavy (H) chains and two light (L) chains, interconnected by disulfide bonds, and multimers (e.g., igM) thereof; however, immunoglobulin molecules consisting of heavy chains only (i.e., lacking light chains) are also included within the definition of the term "antibody". Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region comprises three domains: CH1, CH2 and CH3. Each light chain comprises a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region comprises one domain (CL 1). VH and VL regions can be further subdivided into regions of hypervariability, termed Complementarity Determining Regions (CDRs), alternating with regions of more conservation termed Framework Regions (FR). Each VH and VL comprises 3 CDRs and 4 FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
The term "antibody" as used herein is used in the broadest sense and includes immunoglobulin molecules, specifically including monoclonal antibodies, antigen binding fragments, bispecific, multispecific antibodies, dimeric, tetrameric or multimeric antibodies, single chain antibodies, domain antibodies, and any other modified configuration of immunoglobulin molecules comprising an antigen binding site having the desired specificity, so long as they exhibit the desired biological activity, unless specifically indicated otherwise.
The term "monoclonal antibody" is shorthand for monoclonal antibodies and refers to antibodies obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical and/or bind to the same epitope, except for possible variants that may be generated during the production of the monoclonal antibody, which variants are typically present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. Monoclonal antibodies may be monovalent, bivalent or multivalent.
Hyaluronidases fall into three broad classes, mammalian hyaluronidases, bacterial hyaluronidases, hyaluronidases from leeches, other parasites and crustaceans, which can be manufactured from recombinant DNA technology, derived from human samples or extracted from animal tissue. The term "rHuPH20" refers to recombinant human hyaluronidase rHuPH20 (SEQ ID NO: 5), a humanized recombinant protein.
The term "pharmaceutical formulation" refers to a combination comprising one or more pharmaceutically active ingredients and a plurality of excipients, which achieves a safe and effective effect in treating a patient and stably storing. The pharmaceutically active ingredient refers to antibodies, chemical molecules, natural compounds, etc. that exert therapeutic effects.
The term "excipient" refers broadly to any ingredient other than the active therapeutic ingredient. Excipients may be inert, inactive and/or non-pharmaceutically active.
Excipients may be used for a variety of purposes, for example as carriers, vehicles, diluents, tablet aids, and/or for improved administration, and/or absorption of the active substance.
Formulation of pharmaceutically active ingredients with different excipients is known in the art, see for example Remington: the Science and Practice of Pharmacy (e.g., 19 th edition (1995), and any updated versions).
Non-limiting examples of excipients are: solvents, diluents, organic co-solvents, viscosity reducing agents, antioxidants, buffers, preservatives, isotonic agents, chelating agents and stabilizers.
The term "stable" means that all proteins therein retain substantially their physical, chemical, biological activity after storage at the intended storage temperature, e.g. 0-40 ℃. The antibody in the preparation does not maintain 100% of physical, chemical and biological activity after being stored for a certain period of time, and the preparation can be stable. After storage for a period of time, the formulation may be considered "stable" in that more than about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the antibody structure and function is maintained.
The stability of the formulation can be determined by a variety of parameter indicators. For example, it can be characterized by determining the percentage of monomer remaining in the formulation after storage at a given temperature for a period of time. The percentage of monomer may be determined by size exclusion chromatography. The given temperature may be selected from 4 ℃, 25 ℃, 37 ℃, and the storage time for stability may be selected from 2 weeks, 6 weeks, 10 weeks, 12 weeks, 14 weeks, 16 weeks, etc. The formulation was considered "stable" with a percentage of monomer greater than 90%, 92%, 95%, 96%, 97%, 98%, 99% as measured by size exclusion chromatography.
The present invention provides a container comprising a stable pharmaceutical formulation provided herein. The term "container" may be a tube, bottle, vial or syringe. In embodiments wherein the container is a syringe, the container further comprises an injection needle. Thus, the present invention provides containers comprising stable liquid formulations for single or multiple dose administration.
The term "kit" may comprise the pharmaceutical formulation described herein and a device for administration, e.g., the pharmaceutical formulation may be packaged with a device for administration, e.g., a syringe, inhaler, graduated cup, dropper, or applicator. The pharmaceutical formulation may be filled in a container as defined above. The kit optionally contains instructions for use, including instructions for dosage, dosing regimen, and mode of administration.
The term "effective amount" refers to an amount that provides the desired effect. In the case of pharmaceutical preparations, it is the amount of active ingredient that is effective to treat the disease in the patient. An effective amount may prolong progression free survival, produce an objective response, increase overall survival time, and/or ameliorate one or more IL-4 related symptoms.
The term "related diseases to which anti-IL-4R (interleukin-4 receptor) antibodies are therapeutic" includes, for example, arthritis (including septic arthritis), herpes, chronic primary urticaria, scleroderma, hypertrophic scars, whapple's disease, benign prostatic hyperplasia, lung diseases such as asthma (mild, moderate and severe), inflammatory diseases such as inflammatory bowel disease, allergic reactions, kawasaki disease, sickle cell disease, churg-Strauss syndrome, graves' disease, preeclampsia, sjogren's syndrome, autoimmune lymphoproliferative syndrome, autoimmune hemolytic anemia, barrett's esophagus, autoimmune uveitis, tuberculosis (disease), atopic dermatitis, ulcerative colitis, fibrosis, and kidney disease, etc. (see patent CN 201410018529.6).
The stable pharmaceutical formulation obtained by the present invention unexpectedly has the following beneficial technical effects: the pharmaceutical formulation obtained in one aspect is stable at 25 ℃ for 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months or more, and at 37 ℃ or 40 ℃ for 3 months, 4 months, 5 months, 6 months or more; on the other hand, the obtained pharmaceutical preparation is verified by an animal model, can realize single injection of at least 4ml, and obtains better pharmacokinetic data.
The anti-IL-4R antibody used in the following embodiments is dipivumab.
The reagents or apparatus used in the present invention are conventional products commercially available without reference to the manufacturer or source. The sources of the Toluzumab and rHuPH20 enzymes used therein are as follows:
the dollopirox: the dolaprizumab used in the embodiment of the invention is the dolaprizumab self-made by a conventional antibody preparation method, and the prepared dolaprizumab sequence (the heavy chain is SEQ ID NO:3, the light chain is SEQ ID NO: 4) is consistent with the dolaprizumab sequence sold in the market. The production of dulluzumab by generally known recombinant protein techniques, in particular by known methods, the preparation of genetically engineered chinese hamster ovary cell line (CHO), expanded in cell culture. The spent pilumab was harvested from the cell culture fluid and purified using immobilized protein a affinity chromatography, cation exchange chromatography, filtration step to remove viral contaminants, followed by anion exchange chromatography and ultrafiltration/diafiltration steps.
rHuPH20 enzyme: the rHuPH20 enzyme used in the examples of the present invention was produced by a generally known recombinant protein production technique. The process begins with thawing cells from a working cell bank or a master cell bank and expanding through cell culture in a series of roller bottles. The rHuPH20 enzyme was secreted into the culture fluid. The harvest was clarified by filtration and then treated with solvent, detergent to inactivate the virus. The protein is then purified by a series of column chromatography processes to remove relevant impurities from the process and product. The rHuPH20 standard used in the detection of the rHuPH20 enzyme activity is purchased from the national food and drug institute (original name: national institute of biological products of medicine) and has the product number YZ-140603.
The pharmaceutically active ingredient is formulated with various excipients by formulation methods known in the art.
Preparation of liquid preparation of dolaprizumab
To prepare a liquid formulation, purified dulluzumab is buffer exchanged against diafiltration buffer containing the desired buffer composition and, if desired, concentrated to an antibody concentration greater than the target concentration. After the target concentration is reached, excipients (e.g., trehalose, rHuPH20, polysorbate, etc.) are added to the antibody solution as a stock solution. Finally, the protein concentration is adjusted to the target concentration with the final configuration buffer.
All the formulated solutions were filtered (e.g., optionally with a 0.22 μm sterile filter) and filled under sterile conditions into sterile glass vials (e.g., 4ml glass vials), stoppered (e.g., with a fluororesin layered butyl rubber stopper), and capped (e.g., with an aluminum/plastic peel seal). The fill volume may be about 2ml. The formulated solutions were stored at different temperatures (4 ℃, 25 ℃, 37 ℃ or 40 ℃) for different times.
Preparation of freeze-dried preparation of dolapruzumab
The preparation of the dipivoxilizumab freeze-dried preparation of the invention is not particularly limited, and can be carried out by adopting a known freeze-dried preparation method. The lyophilized formulation can be prepared, for example, by the following method: the liquid formulation was prepared first, filtered, and the filtered liquid formulation was aliquoted into sterile glass vials as described above for the dipivumab liquid formulation. The vials were partially sealed with an ETFE (copolymer of ethylene and tetrafluoroethylene) coated rubber stopper suitable for use in the lyophilization process, followed by a freeze-drying cycle.
Lyophilization is performed in, for example, a LyoStar II freeze dryer (FTS Systems, stone Ridge, NY, USA): the product is first cooled from room temperature to, for example, about 5 ℃ (pre-cooling), then subjected to a freezing step at, for example, about-40 ℃ at a cooling rate of, for example, about 1 ℃/minute, and then subjected to a holding step at, for example, about-40 ℃ for, for example, about 2 hours. The first drying step is carried out at a temperature of, for example, about-25 c and a chamber pressure of, for example, about 80 μbar for, for example, about 72 hours. Subsequently, a second drying step is started from-25 ℃ with a temperature ramp of 0.2 ℃/minute up to 25 ℃, followed by a holding step at e.g. about 25 ℃ for at least 8 hours with a chamber pressure of e.g. about 80 μbar. Storing the lyophilized powder in a refrigerator at-80deg.C.
The various resulting formulations were evaluated to determine the overall physicochemical stability of the resulting formulation of moderately pilumab and rHuPH 20. The resulting formulations were analyzed for formulation stability by the following method:
(1) SEC-HPLC was used to detect soluble high molecular weight species (aggregates, HMW) and low molecular weight hydrolysis products (LMW) in the formulation. Wherein the SEC-HPLC refers to size exclusion chromatography for quantification of aggregates and fragments of proteins. Monomers, aggregates and fragments eluted from the column at different times and were detected by a standard UV detector. For example, shimadzu LC-20AT high performance liquid chromatography, and Waters columns XBridge Protein BEH SEC,7.8 x 300mm,3.5 μm,The method is carried out. Intact monomers, aggregates and hydrolysates are separated by isocratic elution using, for example, about 200mM trisodium phosphate solution (e.g., about ph=6.8) as mobile phase and detected at a wavelength of 280 nm.
(2) Antibody concentration was detected using BCA (biquinolinecarboxylic acid) protein concentration detection method, specifically, protein-to-Cu under alkaline conditions2+ Reduction to Cu+ ，Cu+ Complex forming purple color with BCA reagent, two molecules of BCA chelating one Cu+ . And comparing the absorption value of the complex at 562nm with a standard curve to calculate the concentration of the protein to be detected. The rate of antibody degradation in the formulation was assessed by detecting the antibody concentration, reflecting the stability of the formulation.
(3) The determination of rHuPH20 enzymatic activity is based on the formation of a precipitate upon binding of Hyaluronic Acid (HA) to acidified serum. The activity was measured by incubating hyaluronidase with HA in 96-well plate format at 37 ℃ for 30min, followed by adding acidified serum to precipitate undigested HA. The resulting turbidity was measured at 640nm and the turbidity decrease caused by enzymatic cleavage of the HA substrate was a measure of hyaluronidase activity. Sample activity was read from a standard curve generated by rHuPH20 assay of dilutions of the reference standard. Specifically, rHuPH20 activity can be determined, for example, as follows: firstly, preparing an enzyme activity standard curve, adding 30 mu L of the standard curve into a 96-well plate per well, and preheating the standard curve and hyaluronic acid working solution in a water bath at 37 ℃ for 5min; secondly, diluting the sample to be within the range of the standard curve; adding 30 mu L/hole hyaluronic acid working solution into a preheated 96-well plate, uniformly mixing, and carrying out water bath at 37 ℃ for 6min; finally, 240. Mu.L/well serum working solution was added to terminate the reaction of the substrate with the enzyme, and the absorbance at OD640nm was measured after 30min at room temperature.
Results of the co-formulation of dolaprimab and hyaluronidase and stability test are provided in the examples below, wherein the smaller the difference between each index and each parameter measured under the initial conditions, the more stable the formulation.
Example Co-formulation and stability data
ND =not determined
Example 1. Co-formulation of dipivoxil and hyaluronidase 1.
The formula comprises the following components: 150mg/ml of dolaprimab, 20mM histidine hydrochloride/histidine hydrochloride buffer, 12.5mM sodium acetate/acetic acid, 210mM trehalose, 0.05% (w/v) polysorbate 80, 25mM arginine, 20mM methionine, rHuPH20 2000U/ml, pH 5.9.
Table 1: stability data for coformulation 1
Example 2. Co-formulation of dipivoxil with hyaluronidase 2.
The formula comprises the following components: 150mg/ml of dolaprizumab, 20mM histidine hydrochloride/histidine hydrochloride buffer; 12.5mM sodium acetate/acetic acid, 210mM trehalose, 0.2% w/v polysorbate 80, 25mM arginine, 20mM methionine, rHuPH20 2000U/ml, pH 5.9.
Table 2: stability data for coformulation 2
Example 3 Co-formulation of Dedipivoxil and hyaluronidase 3
The formula comprises the following components: 150mg/ml of dolaprizumab, 20mM histidine hydrochloride/histidine hydrochloride buffer; 12.5mM sodium acetate/acetic acid, 155mM sucrose, 0.05% w/v polysorbate 80, 25mM arginine, 20mM methionine, rHuPH20 2000U/ml, pH 5.9.
Table 3: stability data for coformulation 3
Example 4. Co-formulation of dipivoxil with hyaluronidase 4.
The formula comprises the following components: 150mg/ml of dolaprimab, 20mM histidine hydrochloride/histidine hydrochloride buffer, 12.5mM sodium acetate/acetic acid, 155mM sucrose, 0.2% w/v polysorbate 80, 25mM arginine, 20mM methionine, rHuPH20 2000U/ml, pH 5.9.
Table 4: stability data for coformulation 4
Example 5 Co-formulation of Dupiruzumab and hyaluronidase 5
The formula comprises the following components: 150mg/ml of dolaprizumab, 20mM histidine hydrochloride/histidine hydrochloride buffer; 12.5mM sodium acetate/acetic acid, 155mM sucrose, 0.2% w/v polysorbate 80, 12.5mM arginine, 20mM methionine, rHuPH20 2000U/ml, pH 5.9.
Table 5: stability data for coformulation 5
Example 6 Co-formulation of Dedipivoxil and hyaluronidase 6
The formula comprises the following components: 150mg/ml of dolaprizumab, 20mM histidine hydrochloride/histidine hydrochloride buffer; 12.5mM sodium acetate/acetic acid, 155mM sucrose, 0.2% w/v polysorbate 80, 25mM arginine, 10mM methionine, rHuPH20 2000U/ml, pH 5.9.
Table 6: stability data for coformulation 6
Example 7 Co-formulation of Dedipivoxil and hyaluronidase 7
The formula comprises the following components: 175mg/ml of dolaprizumab, 20mM histidine hydrochloride/histidine hydrochloride buffer; 12.5mM sodium acetate/acetic acid, 155mM sucrose, 0.2% w/v polysorbate 80, 25mM arginine, 20mM methionine, rHuPH20 2000U/ml, pH 5.7.
Table 7: stability data for coformulation 7
Example 8 Co-formulation of Dedipivoxil and hyaluronidase 8
The formula comprises the following components: 150mg/ml of dolaprizumab, 20mM histidine hydrochloride/histidine hydrochloride buffer; 12.5mM sodium acetate/acetic acid, 155mM sucrose, 0.2% w/v polysorbate 80, 25mM arginine, 20mM methionine, rHuPH20 2000U/ml, pH 6.4.
Table 8: stability data for coformulation 8
Example 9 Co-formulation of Dedipivoxil and hyaluronidase 9
The formula comprises the following components: 175mg/ml of dolaprizumab, 20mM histidine hydrochloride/histidine hydrochloride buffer; 12.5mM sodium chloride, 155mM sucrose, 0.2% w/v polysorbate 80, 25mM arginine, 20mM methionine, rHuPH20 2000U/ml, pH 5.9.
Table 9: stability data for coformulation 9
Example 10 Co-formulation of Dedipivoxil and hyaluronidase 10
The formula comprises the following components: 150mg/ml of dolaprizumab, 20mM histidine hydrochloride/histidine hydrochloride buffer; 12.5mM sodium chloride, 155mM sucrose, 0.2% w/v polysorbate 80, 25mM arginine, 20mM methionine, rHuPH20 2000U/ml, pH 5.7.
Table 10: stability data for coformulation 10
Example 11 Co-formulation of Dedipivoxil and hyaluronidase 11
The formula comprises the following components: 150mg/ml of dolaprizumab, 20mM histidine hydrochloride/histidine hydrochloride buffer; 12.5mM sodium chloride, 155mM sucrose, 0.2% w/v polysorbate 80, 25mM arginine, 20mM methionine, rHuPH20 2000U/ml, pH 6.4.
Table 11: stability data for coformulation 11
Example 12 Co-formulation of Dedipivoxil and hyaluronidase 12
The formula comprises the following components: 160mg/ml of dolaprimab, 20mM histidine hydrochloride/histidine hydrochloride buffer; 155mM sucrose, 0.2% w/v polysorbate 80, 25mM arginine, 20mM methionine, rHuPH20 2000U/ml, pH 5.9.
Table 12: stability data for coformulation 12
Example 13 Co-formulation of Dedipivoxil and hyaluronidase 13
The formula comprises the following components: 150mg/ml of dolaprizumab, 20mM histidine hydrochloride/histidine hydrochloride buffer; 155mM sucrose, 0.2% w/v polysorbate 80, 25mM arginine, 20mM methionine, rHuPH20 2000U/ml, pH 5.7.
Table 13: stability data for coformulation 13
Example 14 Co-formulation of Dedipirstuzumab and hyaluronidase 14
The formula comprises the following components: 150mg/ml of dolaprizumab, 20mM histidine hydrochloride/histidine hydrochloride buffer; 155mM sucrose, 0.2% w/v polysorbate 80, 25mM arginine, 20mM methionine, rHuPH20 2000U/ml, pH 6.4.
Table 14: stability data for coformulation 14
Example 15 Co-formulation of Dedipirstuzumab and hyaluronidase 15
The formula comprises the following components: 150mg/ml of dolaprizumab, 20mM histidine hydrochloride/histidine hydrochloride buffer; 210mM trehalose, 0.2% w/v polysorbate 80, 25mM arginine, 20mM methionine, rHuPH20 2000U/ml, pH5.9.
Table 15: stability data for coformulation 15
Example 16 Co-formulation of Dedipivoxil and hyaluronidase 16
The formula comprises the following components: 150mg/ml of dolaprizumab, 20mM histidine hydrochloride/histidine hydrochloride buffer; 12.5mM sodium acetate/acetic acid, 210mM trehalose, 0.2% w/v polysorbate 80, 25mM arginine, 20mM methionine, rHuPH20 2000U/ml, pH 7.8.
Table 16: stability data for coformulation 16
Example 17 Co-formulation of Dedipirstuzumab and hyaluronidase 17
The formula comprises the following components: 150mg/ml of dolaprizumab, 20mM histidine hydrochloride/histidine hydrochloride buffer; 12.5mM sodium acetate/acetic acid, 210mM trehalose, 0.2% w/v polysorbate 80, 25mM arginine, rHuPH20 2000U/ml, pH5.9
Table 17: stability data for coformulation 17
Example 18 Co-formulation of Dedipivoxil and hyaluronidase 18
The formula comprises the following components: 150mg/ml of dolaprizumab, 20mM histidine hydrochloride/histidine hydrochloride buffer; 12.5mM sodium acetate/acetic acid, 210mM trehalose, 0.2% w/v polysorbate 80, 25mM arginine, 55mM methionine, rHuPH20 2000U/ml, pH5.9.
Table 18: stability data for coformulation 18
Example 19 Co-formulation of Dedipivoxil and hyaluronidase 19
The formula comprises the following components: 150mg/ml of dolaprizumab, 20mM histidine hydrochloride/histidine hydrochloride buffer; 12.5mM sodium acetate/acetic acid, 310mM trehalose, 0.2% w/v polysorbate 80, 25mM arginine, 20mM methionine, rHuPH20 2000U/ml, pH 5.9.
Table 19: stability data for coformulation 19
Co-formulation formulations of Toluzumab and hyaluronidase of examples 12-15 appeared as macroscopic particles when left at 37℃for 2 weeks. It was shown that the buffer system of the two buffers of examples 1-11 of the present invention, or a combination of one buffer with an ionic enhancer (e.g., sodium chloride, potassium chloride solution), provides a more stable formulation than the buffer system of a single buffer.
The co-formulation of the dipivoxil and hyaluronidase of example 16 showed poor stability despite the two buffers, indicating that pH also affected the stability of the formulation.
Formulation formulations of dulcamab and hyaluronidase of examples 17-18, wherein methionine is absent in example 17, the stability of the formulation is poor compared to examples 1-11; the formulation of example 18 contained 55mM methionine and was also less stable than the formulations of examples 1-11.
The formulation of the duloxetab and hyaluronidase of example 19 contained 310mM trehalose as compared to examples 1-11, and it was shown by comparison with other examples that too high a heat stabilizer such as trehalose could also affect the stability of the formulation.
The above stability test results show that the co-formulation of the present invention is stable for 5 months or more at 4 ℃, 25 ℃, 37 ℃ and/or 40 ℃. At least 96.0% of the antibody in monomeric form is present after 20 weeks of storage at 4 ℃ as determined by size exclusion chromatography; at least 95.5% of the antibody in monomeric form is present after 20 weeks of storage at 25 ℃; at least 95.0% of the antibody is present in monomeric form after 12 weeks of storage at 37 ℃ as determined by size exclusion chromatography. The formulations of examples 1-11 of the present invention meet formulation stability requirements.
Animal efficacy test
Description of animals
Species: a mouse
Variety: B-hIL4hIL4RA micro
Age: for 6-8 weeks
Weight: 18-20g
The source is as follows: baioersty chart
Raising of
Mice were housed in IVC (independent ventilated cage) and provided with sterile water for access. Mice were fed twice daily (AM and PM) except for study day. Prior to starting the study, animals were assessed for overall health and body weight was recorded. The study was performed after determining the health of the mice.
AD (Atopic dermatitis ) model construction: according to FIG. 1, mice were sensitized by uniformly applying 25. Mu.L of 0.8% Oxazolone solution to the right ear and back of the mice on day 0, followed by 25. Mu.L of 0.4% Oxazolone solution to the same sites on day 7,9,11,14,16,18,21,23,25 for excitation. The mice were weighed twice a week after the start of the experiment and ear thickness was measured, and subcutaneous administration was started from day 6, and the dosing schedule, dosing frequency, dosing dose and volume of the blank (Mock Control) and experimental groups a-C are shown in table 20, and blood was collected from the orbital venous plexus at day 26, and IgE levels in serum were detected using kit MOUSE IgE ELISA KIT (ek).
Table 20 dosing regimen, dosing frequency, dosing dose and volume for each experimental group
The total IgE in serum was found by mice weight change, ear thickness: compared with a blank control group (MC), the weight change of mice in each of the A group, the B group and the C group is basically consistent, so that the mice are free from toxic and side effects when being dosed in each experimental group, the ear thickness and the total IgE concentration in serum are in negative correlation with the dosage, and A, B, C groups have obvious drug effects and are equivalent in drug effects, so that the dosage can be prolonged in animal bodies by increasing the subcutaneous dosage through being combined with PH20, the dosing frequency is reduced, and the drug effects equivalent to those of a common preparation (non-hyaluronidase combined preparation) are achieved. The co-formulation of the present invention can achieve a single injection volume of greater than 2ml, such as 3ml,4ml,5ml,6ml,7m,8ml, or more, and obtain better pharmacokinetic data.
The present invention has been illustrated by the above-described embodiments, but it should be understood that the above-described embodiments are for purposes of illustration and description only and are not intended to limit the invention to the embodiments described. In addition, those skilled in the art will appreciate that the present invention is not limited to the embodiments described above, and that many variations and modifications are possible in light of the teachings of the invention, which variations and modifications are within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.